5,6-dihydro-1h-pyridin-2-one compounds

The invention relates to 5,6-dihydro-1Hpyridin-2-one compounds and pharmaceutical compositions containing such compounds that are useful in the treatment of hepatitis C virus infections.

Background of the Invention

Hepatitis C is one of the major health problems worldwide. The World Health Organization estimates that 170 million people are chronic carriers of the hepatitis C virus (HCV), with 4 million carriers in the United States alone. In the United States, HCV infection is the cause of 40% of chronic liver disease, and HCV disease is the most common cause of liver transplantation. HCV infection leads to a chronic infection, and approximately 70% of infected individuals will develop chronic histological changes in the liver (chronic hepatitis), with a 10-40% risk of cirrhosis and an estimated 4% lifetime risk of hepatocellular carcinoma. The CDC estimates that each year in the United States there are 35,000 new cases of HCV infection and approximately ten thousand deaths attributed to HCV disease.

The current standard of care is a combination of pegylated interferon/ribavirin at a cost of approximately US$31,000/year. These drugs have problems with difficult dosages and side effects that preclude their use in almost half of diagnosed patients. Treatment with pegylated interferon is associated

2/317 with life-threatening flu-like symptoms, irritability, inability to concentrate, suicidal fixation, and leukocytopenia. Ribavirin is associated with hemolytic anemia and birth defects.

The overall response to this standard therapy is low; approximately one-third of patients do not respond. Of those who do respond, a large fraction relapse within six months of completing 6–12 months of therapy. As a consequence, the long-term response rate for all patients who start treatment is only about 50%. The relatively low response rate and significant side effects of current anti-HCV drug treatments, along with the long-term negative effects of chronic HCV infection, result in a continued medical need for improved therapy. Antiviral medications to treat RNA virus diseases such as HCV are few and, as described above, are frequently associated with multiple adverse effects.

Several publications have described NS5B inhibitors as useful in the treatment of hepatitis C infection. See, for example, the publication of U.S. Patent Application 2008/0031852 (describing [1,2-b]pyridazinone compounds); the publication of U.S. Patent Application 2006/0189602 (disclosing certain pyridazinones); the publication of U.S. Patent Application 2006/0252785 (disclosing selected heterocycles); and the international publications WO 03/059356, WO 2002/098424 and WO 01/85172 (each describing a particular class of substituted thiadiazines).

Although medications are available in some cases to reduce disease symptoms, few drugs effectively inhibit essential viral replication. The importance and prevalence of RNA virus diseases, including but not limited to chronic hepatitis C virus infection, coupled with the limited availability and effectiveness of antiviral medications, highlight this issue.

The current 3/317 [of these diseases] has created a mandatory and ongoing need for new medications to treat them.

Summary of the Invention

The present invention describes novel 5,6-dihydro-1H-pyridin-2-one compounds and pharmaceutically acceptable salts thereof, which are useful in the treatment or prevention of hepatitis C virus infection in a patient in need thereof, comprising administering to the patient a therapeutically or prophylactically effective amount of a 5,6-dihydro-1H-pyridin-2-one compound.

In general terms, the invention relates to compounds of

Formula I where

X is N or

CR³or

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<img file="BRPI0809685A2_D0001.tif" />

The β ring is a 6-membered aryl or heterocycline, optionally replaced by 1-3 R segments.¹where R¹ It's H, halo, nitro, -CHR⁴S(O2) R⁵, -C(S(O)2R⁵)=CHR% -NR5R6, -NR⁴S(O)2R⁵, or -NR⁴S(O)2NR5R6, where R⁴R⁵and R⁶ are independently H, C1-C1 alkyl, C3-C8 cycloalkyl, C(O)O-(C1-C1 alkyl), aryl or heterocyclyl, or R⁴ and R⁵ or R⁵ and R⁶ They combine with the atom(s) to which they are attached to form a 5- or 6-membered heterocyclyl ring.

R² is H, Ci-Cõ alkyl, C3-C8 cycloalkyl, -Ci-Có alkylene (C3-C8 cycloalkyl), -Ci-Cô alkylene (aryl), -Ci-Cô alkylene (heterocyclyl), aryl or heterocyclyl,

R³ It is H, halo, or C1-C1e alkyl.

Z is -(CR¹³R¹⁴)_n- or O, n is 1 or 2,

R⁷ and R⁸ are independently H or C1-C1 alkyl,

R⁹R¹⁰R¹¹R¹²R¹³R¹⁴R¹⁵ and R¹⁶ are independently H, C1-C1e alkyl, hydroxyl or halo, where the alkyl, alkylene, aryl, cycloalkyl or heterocyclyl segments given above in R¹R²R³R⁴R⁵R⁶R⁷R⁸R⁹R¹⁰R¹¹R¹²R¹³R¹⁴R¹⁵ and R¹⁶ are each optionally and independently replaced by 1-3 substitutes selected from:

Alkylamine,

5/317

Amino,

Aryl, cycloalkyl, heterocyclyl,

Ci-Cõ alkyl, Ci-Cõ haloalkyl, C1-C0 hydroxyalkyl, Ci-Cõ alkoxy, Ci-Cõ alkylamine, Ci-Cõ dialkylamine, C₂-Cõ alkenyl, or C2-C0 alkynyl, where each of which may be interrupted by one or more heteroatoms,

Carboxyl,

Cyan

Halo,

Hydroxy

Ceto,

Nitro,

-C(O)OH, -C(O)NH₂, -C(O)(Ci-C₆ alkylamine), -C(O)(C1-C₆ dialkylamine), -C(O)2-(Ci-Có alkyl), -C(O)₂-(C3-C8 cycloalkyl), -C(O)2-(aryl), -C(O)2-(heterocyclyl), -C(O)₂-(Ci-Cõ alkylene) aryl, -C(O)₂-(C1-C6 alkylene)heterocyclyl, -C(O)₂-(Ci-C6 alkylene)cycloalkyl, -C(O)(Ci-Cõ alkyl), -C(O)(C3-Cs cycloalkyl), -C(O)(aryl), -C(O) (heterocyclyl), C(O)(Ci-Cõ alkylene) aryl, -C(O)(Ci-Cõ alkylene) heterocyclyl, and C(O)(Ci-Cõ alkylene)cycloalkyl,

Where each of the above optional substituents may be further optionally replaced by 1-5 substituents selected from amino, cyano, halo, hydroxy, nitro, C1-C6 alkylamine, C1-C6 dialkylamine, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkenyl, and C1-C6 hydroxyalkyl, where each alkyl is optionally replaced by one or more halo substituents,

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Or a pharmaceutically acceptable salt, hydrate, solvate, tautomer or stereoisomer thereof.

In one embodiment, the invention relates to compounds of Formula I where Ring B is selected from:

<img file="BRPI0809685A2_D0002.tif" />

In another embodiment, Ring B is:

<img file="BRPI0809685A2_D0003.tif" />

Where R¹ It's H, halo, nitro, -CHR⁴-S(O)2R⁵, -C(S(O)2R⁵)=CHR⁴-, NR5R6, -NR⁴S(O)2R⁵, or -NR⁴S(O)2NR5R6.

In one embodiment, the invention relates to compounds of Formula I where R¹ It's -NR⁴ONLY)₂R⁵where R⁴ and R⁵ They are independently H, C1-C8 alkyl or C3-C8 cycloalkyl.

In another embodiment, the invention relates to compounds of

Formula I where R¹ is selected from:

í»io^NV° oT ° V ° Me . S

In one embodiment, the invention relates to compounds of

Formula I where R² is selected from:

7/317

<img file="BRPI0809685A2_D0004.tif" />

In another embodiment, the invention relates to compounds of

Formula I where R² is selected from:

<img file="BRPI0809685A2_D0005.tif" />

In another embodiment, the invention relates to compounds of

Formula I where R² is selected from:

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<img file="BRPI0809685A2_D0006.tif" />

<img file="BRPI0809685A2_D0007.tif" />

<img file="BRPI0809685A2_D0008.tif" />

<img file="BRPI0809685A2_D0009.tif" />

In one embodiment, the invention relates to compounds of Formula I where R³ It is selected from hydrogen or C1-C1 alkyl.

In another embodiment, the invention relates to compounds of

Formula I where R³ is selected from:

H§-₅Me-f

In one embodiment, the invention relates to compounds of Formula I where R⁷ and R⁸ They are FI.

In one embodiment, the invention relates to compounds of

Formula I where R⁹R¹⁰R¹¹R¹²R¹³R¹⁴R¹⁵and R^iõ They are independently selected from:

H, MeX,_{( s}

X, X.^H°-|· ,: and H .

In a further embodiment, the invention relates to Formula 1 compounds where R⁹R¹⁰R¹¹R¹²R¹³R¹⁴R¹⁵ and R¹⁶ They are independently selected from:

H, Me-^-_} HO-|- , í e .

In another embodiment, the invention relates to compounds of Formula I where R⁹R¹⁰R¹¹R¹²R¹³R¹⁴R¹⁵ and R¹⁶ They are H or hydroxyl.

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In yet another embodiment, the invention relates to compounds of Formula I where R⁹R¹⁰R¹¹R¹²R¹³R¹⁴R¹⁵ and R¹⁶ They are H.

In a modality n is 1.

In another embodiment, the invention relates to compounds selected from:

(rac-di-exo)-7V-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1, 1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide, (rac-di-endoFlV-fS-tS-^-fluoro-benzylJ-õ-hydroxy-T-oxo-S-aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide, (rac-di-endo)-lV-{3-[3-(5-fluoro-pyridin-2-ylmethyl)-6-hydroxy-4-oxo-3aza-tricyclo[6.2.1.0²⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo [1,2,4]thiadiazin-7-yl}-methanesulfonamide,

7V-{3-[( IS,2 S,7R,8Rj-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide,

7V-{3-[(1R,2R,7S,8S)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide,

7V-{3-[(1R,2S,7R,8S)-3-/4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyl [6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide,

N-{3-[(1S,2R,7 8.8Rj-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide,

10/317 (rac-di-exo)-A-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-1 l-oxa-3-azatricycle [6.2.1.0²’⁷]undec-5-en-5-yl]~ 1, 1-dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide, (rac-di-exo)-7V-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²’⁷]undeca-5,9-dien-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide, (rac-di-endo)-7V-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²’⁷]undeca-5,9-dien-5-yl]-1,1-dioxo- l,4-dihydro-lÀ⁶-benzo[1,2,4] thiadiazin-7-yl}-methanesulfonamide,

7V-{3-[(1 S,2R,7 S,81?)-3-(4-fluoro-3-methyl-benzyl)-6-hydroxy-4-oxo-1 1oxa-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide, (rac-di-exo)-7V-{3-[3-(4-fluoro-3-methyl-benzyl)-6-hydroxy-4-oxo-1loxa3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide, (rac-di-exo)-7V-{3-[3-(3,3-Dimethyl-butyl)-6-hydroxy-4-oxo-1 l-oxa-3aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-l, 1-dioxo- 1,4-dihydro-lA⁶-benzo [1,2,4]thiadiazin-7-yl}-methanesulfonamide,

7V-{3-[(l S,27?,7S,87?)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-l-oxa-3aza-tricycle [6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo [1,2,4]thiadiazin-7-yl}-methanesynlfonamide,

7V-{3-[(17?,2S,77?,8S)-3-(4-fluoro-3-methyl-benzyl)-6-hydroxy-4-oxo-3aza-tricycle [6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-1A⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide, lV-{3-[(1/?,2 S,77?, 88)-3-(3,4-Difluoro-benzyl)-6-hydroxy-4-oxo-3-aza~ tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lA⁶-benzo

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[1,2,4]thiadiazin-7-yl}-methanesulfonamide,

7V-{3-[(17?,2S,77?,8S)-3-f3,4-Difluoro-benzyl)-6-hydroxy-4-oxo-3-azatricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-l, l-dioxo-l,4-dihydro-lÀ⁶-benzo l,2,4]thiadiazin-7-yl}-N-methyl-methanesulfonamide,

7V-{3-[(17?,2S,77?,8S)-3-(4-fluoro-3-methyl-benzyl)-6-hydroxy-4-oxo-3aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lA⁶-benzo[1,2,4]thiadiazin-7-yl}-7V-methyl-methanesulfonamide, (17?,2S,77?,8S)-3-(4-fluoro-benzyl)-6-hydroxy-5-(7-iodo-1,1-dioxo-1,4dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-aza-tricyclo[6.2.1.0²>⁷]undec-5en-4-one, (17?,2S,77?,8S)-5-[7-(l_;l-Dioxo-4,5-dihydro-lH-lÀ⁶-thiophen-2-yl)-l,ldioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl]-3-(4-fluoro-benzyl)-6hydroxy-3-aza-tricycle [6.2.1.0²’⁷]undec-5-en-4-one, (17?,2S,77?,8S)-5-[7-(l,l-Dioxo-tetrahydro-lÀ⁶-thiophen-2-yl)-l,l-dioxol,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl]-3-(4-fluoro-benzyl)-6-hydroxy-3aza-tricycle [6.2.1.0²>⁷]undec-5-en-4-one, (17?,2S,77?,8S)-7V-{3-[6-Hydroxy-3-(3-methylbutyl)-4-oxo-3-azatricyclo [6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide, (rac-di-exo)-7V-{3-[3-(2-Cyclopropyl-ethyl)-6-hydroxy-4-oxo-l l-oxa-3aza-tricycle [6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo [1,2,4]thiadiazin-7-yl}-methanesulfonamide, (17?,2S,77?,8S)-7V-{3-[6-Hydroxy-3-(3-methyl-butyl)-4-oxo-3-aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1, 1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-7V-methyl-methanesulfonamide, (rac-di-exo)-7V-{3-[6-Hydroxy-3-(3-methyl-butyl)-4-oxo-1 l-oxa-3-aza

12/317 tricycle [6.2.1.0²>⁷]undec-5-en-5-yl]-1,1 -dioxo- 1,4-dihydro-lÀ⁶-benzo [ 1,2,4]thiadiazin-7-yl}-methanesulfonamide, (11?,2S,71?,8S)-5-[ 7-( 1, l-Dioxo-lÀ⁶-isothiazolidin-2-yl)-l, 1-dioxo- 1,4dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl]-3-(4-fluoro-benzyl)-6-hydroxy-3-azatricycle [6.2.1.0²>⁷]undec-5-en-4-one, (1R, 2S, 7R, 8S) -N- [3- (6-Hydroxy-4-oxo-3-aza-tricyclo [6.2.1.0²’⁷]undec5-en-5-yl)-l, 1-dioxo- 1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl]methanesulfonamide, (1R,2S/7R,8S)-5-(1,1-Dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²>⁷]undec-5-en-4one, lV-{3-[( 11?,2 S,71?,8S)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,4]thiazin-7yl}-methanesulfonamide, (11?, 2S, 7R, 8S)-5-(7-Amino-1, 1-dioxo-1,4-dihydro-lÀ^&-benzo[1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²>⁷]un-dec5-en-4-one, (rac-di-exo)-7V-{3-[6-Hydroxy-3-(3-methyl-butyl)-4-oxo-3-aza-tricycle [6.2.1.0²>⁷]undeca-5,9-dien-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide, (rac-di-exo)-lV-{3-[3-(3,3-Dimethyl-butyl)-6-hydroxy-4-oxo-3-azatricycle [6.2.1.0²>⁷]undeca-5,9-dien-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo [1,2,4]thiadiazin-7-yl}-methanesulfonamide, (rac-di-exo)-6-Hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-3-yl)-3-(3-methyl-butyl)-1-oxa-3-aza-tricycle

[6.2.1,0²7]undec-5-en-4-ona,

13/317 (177.2 8,777.8S)-6-Hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-3-yl)-3-(3-methyl-butyl)-3-aza-tricyclo[6.2.1.0²>⁷] undec-5-en-4-one, {3-[(177.2 S, 777.8 S)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo [1,2,4]thiadiazin-7-yl}-cyclopropane sulfonic acid amide, (rac-di-exo)-7V43-[3-(3,3-Dimethyl-butyl)-6-hydroxy-4-oxo-3-azatricyclo [6.2.1.0²’⁷]undeca-5,9-dien-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo [1,4] thiazin-7-yl}-methanesulfonamide, (rac-di-exo)-/V-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²’⁷]undeca-5,9-dien-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,4]thiazin-7-yl}-methanesulfonamide, (rac-di-exo)-7V-{3-[6-Hydroxy-3-(3-methyl-butyl)-4-oxo-3-aza-tricycle [6.2.1.0²’⁷]undeca-5,9-dien-5-yl]-1,1-dioxo- 1,4-dihydro- lÀ⁶-benzo[1,4]thiazin-7-yl}-methanes sulfonamide, (rac-di-exo)-7V-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²>⁷]undeca-5,9-dien-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-N-methyl-methanesulfonamide, f3-(17?,2S,777.8S)-[6-Hydroxy-3-(3-methyl-butyl)-4-oxo-3-aza-tricycle [6.2.1.0²-⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-cyclopropane sulfonic acid amide, (177.2S,777.8S)-7V-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-benzene-sulfonamide, {3-[6-Hydroxy-3-(3-methyl-butyl)-4-oxo-l-oxa-3-aza-tricycle [6.2.1.0²·⁷] undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro- 1λ⁶- benzo [1,2,4]thia14/317 diazin-7-yl}-amide of (rac-di-exo)-cyclopropanesulfonic acid, {3-[3-(3,3-Dimethyl-butyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²-⁷] undeca-5,9-dien-5-yl]-1,1 -dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7yl}-amide of (rac-di-exo)cyclopropanesulfonic acid,

7V-[3-(1R,2S,71?,8S)-3-Cyclopentyl-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²>⁷]undec-5-en-5-yl)-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl]-methanesulfonamide, {3-[6-Hydroxy-3-(3-methyl-butyl)-4-oxo-3-aza-tricycle [6.2.1.0²-⁷]undeca-5,9-dien-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶(rac-di-exo)-cyclopropanesulfonic acid-benzo[1,2,4]thiadiazin-7-yl}amide, {3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undeca-5,9-dien-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thidiazin-7-yl}amide (rac-di-exo)-cyclopropanesulfonic acid,

7V-[3-(1R,2S,7R,8S)-(3-Cyclopentyl-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²'⁷]undec-5-en-5-yl)-l,l-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl]-methanesulfonamide-7V-isopropyl carbamate, (rac-di-exo)-7V-[3-(3-Cyclopentyl-6-hydroxy-4-oxo-l-oxa-3-azatricyclo [6.2.1.0²>⁷]undec-5-en-5-yl)-1, 1-dioxo-1,4-dihydro-lÀ⁶-benzo [1,2,4]thiadiazin-7-yl]-methanesulfonamide,

N-{3-[f2S,7R)-3-(4-flLior-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.2.0²·⁷] dodec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide, cisdV-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.2.0²>⁷] dodec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide, /2.R,7S)-.ZV-{3-[3-(4-fl·uor-benzyl)-6-hydroxy-4-oxo-3-aza-tetracycle

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[6.3.2.0²’⁷.0⁹>ⁿ]tridec-5-en-5-yl]-l,l-dioxo-l,4-dihydro-lÀ⁶-benzo[1,2,4] thiadiazin-7-yl}-methanesulfonamide, f2S,7R)-.ZV-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tetracycle [6.3.2.0²’⁷.0⁹>¹¹]tridec-5-en-5-yl]-l,l-dioxo-1,4-dihydro-lA⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide, (1R,2S,7R,8S)-5-(1,1-Dioxo-7-pyrrolidin-1-yl-1,4-dihydro-lÀ⁶-benzo [1,2,4]thiadiazin-3-yl)-6-hydroxy-3-(3-methyl-butyl)-3-aza-tricycle [6.2.1.0²’⁷] undec-5-en-4-one, {3-[(1R,2S,7R,8S)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²-⁷]undec-5-en-5-yl]-l, l-dioxo-l,4-dihydro-lÀ⁶-benzo[1,2,4] thiadiazin-7-yl}-pyridine-3-sulfonic acid amide, (lR,2S,7R,8S)-A-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lA⁶-benzo[1,2,4]thiadiazin-7-yl}-sulfamide, (IR, 2S,7R,8S)-77-[3-(3-Benzyl-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1. 0²’⁷]undec-5-en-5-yl)-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadia-zin-7yl]-methanesulfonamide, (1R,2S,7R,8S)-IV-[3-(6-Hydroxy-3-isobutyl-4-oxo-3-aza-tricycle [6.2.1. 0²>⁷]undec-5-en-5-yl)-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl]-methanesulfonamide, (1R,2S,7R,8S)-7V-[3-[3-(3-Chloro-4-ylfluoro-benzyl)-6-hydroxy-4-oxo-3aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo 1,2,4] thiadiazin-7-yl}-methanesulfonamide, (rac-di-exo)-7V-[{3-[6-Hydroxy-3-(3-methyl-butyl)-4-oxo-1 l-oxa-3-azatricyclo[6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÃ⁶-benzo[1,2,4] thiadiazin-7-yl} - A-methyl-methanesulfonamide,

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IV-{3-[(1J?,2S,71?,8S)-3-(3-fluoro-4-methyl-benzyl)-6-hydroxy-4-oxo-3aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-l, 1-dioxo- 1,4-dihydro- lÀ⁶-benzo [1,2,4]thiadiazin-7-yl}-methanesulfonamide, (1J?,2S,7.R,8>S)-5-(7-Bromo-1,1-dioxo-1,4-dihydro-lÀ⁶-pyrido[2,3-e] [1,2,4] thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricycle [6.2.1. 0²’⁷undec-5-en-4-one,

7V-{3-[(II?, 2S, 7R, 8S)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-l, 1 -dioxo- 1,4-dihydro- lÀ⁶-pyrido[2,3-e][1,2,4] thiadiazin-7-yl}-methanesulfonamide, (1 R,2 S,7R,8 S)-5-( 1,1 -Dioxo-1,4-dihydro-lÀ^the-pyrido[2,3-e] [1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²’⁷]undec-5en-4-one, (1R,2S,7I?,8S)-5-(1,1-Dioxo-1,4-dihydro-U6-pyrido[4,3-e][1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²’⁷]undec-5en-4-one, (1R,2 S,7R,8S)-5-( 1,1 -Dioxo-1,4-dihydro- lÀ⁶-pyrido[3,2-e] [ 1,2,4] thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[ 6.2.1.0²’⁷undec-5en-4-ona,

IV-{3-[(1R,2S,7.R,8S)-3-(2-Cyclopropyl-ethyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²>⁷]undec-5-en-5-yl]-l, 1-dioxo-1,4-dihydro- Ιλθ-benzo [1,2,4] thiadiazin-7-yl}-methanesulfonamide,

IV-{3-[(1R,2S,7.R,8S)-3-(4-fluor-benzyl)-6,9-dihydroxy-4-oxo-3-aza-tricycle [6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide,

IV-{3-[(1R,2S,7R,8S)-3-4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0] L-arginine salt²’⁷]undec-5-en-5-yl]-l, 1-dioxo-1,4-dihydro17/317 lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide,

7743-((17^,28,7.R,8S)-3-(4-fluoro-benzyl)-6-hydroxy4-oxo-3-aza-tricyclo[6.2.1.0] L-Lysine Salt²’⁷]undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro- 1λ^όbenzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide,

N-{3-[(17?,2S,77?,8S)-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0] hemimagnesium salt²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide,

Sodium salt of 7V-{3-[(1A,2S,7A,8S)-3-/4-fluoro-benzyl)-6-hydroxy-4oxo-3-aza-tricyclo[6.2.1.0²-⁷]undec-5-en-5-yl]-1, 1-dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide, and

Potassium salt of N-{3-[(17?,2S,77?,8S)-3-(4-fluoro-benzyl)-6-hydroxy4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide.

The invention is also directed to pharmaceutically acceptable salts and pharmaceutically acceptable solvates of the compounds of Formula I. Advantageous methods of manufacturing the compounds of Formula I are also described.

In one aspect, the invention embodies a method for treating or preventing hepatitis C virus infection in a mammal in need thereof, preferably in a human in need thereof, comprising administering to the patient a therapeutically effective amount or prophylactically of a compound of Formula I. In one embodiment, the invention encompasses a method for treating or preventing hepatitis C virus infection by administering to a patient in need thereof a therapeutically effective amount or prophylactically of a compound of Formula I, which is an NS5B HCV polymerase inhibitor.

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In another aspect, the invention encompasses a method for treating or preventing hepatitis C virus infection in a patient in need thereof, comprising administering to the patient a therapeutically or prophylactically effective amount of a compound of Formula I and a pharmaceutically acceptable excipient, carrier or vehicle.

In another aspect, the invention encompasses a method for treating or preventing hepatitis C virus infection in a patient in need thereof, comprising administering to the patient a therapeutically or prophylactically effective amount of a compound of Formula I and an additional therapeutic agent, preferably an antiviral agent or an additional immunomodulatory agent.

Detailed Description of the Invention

Where the following terms are used in this Descriptive Report, they are used as defined below:

The terms “comprising”, “having” and “including” are used here in this document in their broad, not limited, senses.

The term “alkyl,” as used herein unless otherwise indicated, includes saturated monovalent C1-C12 hydrocarbon radicals having straight, branched, or cyclic segments (including fused and bicyclic and spirocyclic bridging segments) or a combination of the preceding segments. For an alkyl group to have cyclic segments, the group must have at least three carbon atoms.

The term “alkylene”, as used herein unless otherwise indicated, includes a C1-C12 divalent alkyl radical, as exemplified by -CH2CH2CH2CH2-.

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The term “alkenyl”, as used herein unless otherwise indicated, includes C1-C12 alkyl segments having at least one carbon-carbon double bond where alkyl is as defined above and including the E and Z isomers of said alkenyl segment.

The term “alkynyl”, as used herein unless otherwise indicated, includes C1-C12 alkyl segments having at least one carbon-carbon triple bond where alkyl is as defined above.

The term “alkoxy”, as used herein unless otherwise indicated, includes O-alkyl groups where alkyl is as defined above.

The term “Me” means methyl, “Et” means ethyl, and “Ac” means acetyl.

The term “cycloalkyl”, as used herein in this document, unless otherwise indicated, refers to a fused, spiro- or non-fused, or monocyclic, saturated or partially saturated, non-aromatic bicyclic or tricyclic hydrocarbon, referred to herein in this document as containing a total of 3 to 10 carbon atoms, preferably 5-8 carbon atoms in the ring. Exemplary cycloalkyl groups include monocyclic rings having 3-7, preferably 3-6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. Illustrative examples of cycloalkyl groups are derived from, but not limited to, the following:

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<img file="BRPI0809685A2_D0010.tif" />

The term “aryl”, as used herein unless otherwise indicated, includes an organic radical derived from an aromatic hydrocarbon by the removal of a hydrogen atom, such as phenyl or naphthyl, containing a total of 6 to 10 carbon atoms.

The term “heterocyclic” or “heterocyclyl”, as used herein unless otherwise indicated, includes aromatic (e.g., heteroaryl) and non-aromatic heterocyclic groups containing from one to four heteroatoms each selected from O, S, and N, wherein each heterocyclic group has from 4 to 10 atoms in its ring system and on the condition that the ring of said group does not contain two adjacent O atoms. Non-aromatic heterocyclic groups include groups having only 3 atoms in their ring systems, but aromatic heterocyclic groups must have at least 5 atoms in their ring systems. Heterocyclic groups include benzo-fused ring systems. An example of a 4-membered heterocyclic group is azetidinyl (derived from azetidine). An example of a 5-membered heterocyclic group is thiazolyl, and an example of a 10-membered heterocyclic group is quinolinyl. Examples of non-aromatic heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidine, morpholine, thiomorpholine, thioxanyl.

21/317 piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolidinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-aza-bicyclo[3.1.0]hexanyl, 3-aza-bicyclo[4,1.0] heptanyl, 3H-indolyl and quinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The preceding groups, as derivatives of the groups listed above, may be C-linked or N-linked where possible. For example, a pyrrole-derived group may be a pyrrole-1-yl (N-linked) or pyrrole-2-yl (C-linked) or pyrrole-3-yl (C-linked). Furthermore, an imidazole-derived group may be imidazole-1-yl (N-linked) or imidazole-2-yl (C-linked). The 4-10 membered heterocycle can optionally be substituted at any carbon, sulfur, or nitrogen atom(s) of the ring by one to two oxo groups per ring. An example of a heterocyclic group where 2 carbon atoms in the ring are substituted with oxo segments is 1,1-dioxo-thiomorpholinyl. Other illustrative examples of 4-10 membered heterocycles are derived from, but not limited to, the following:

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<img file="BRPI0809685A2_D0011.tif" />

Unless otherwise defined, “alkyl”, “alkylene”, “alkenyl”, “alkynyl”, “aryl”, “cycloalkyl” or “heterocyclyl” are each optionally and independently substituted by 1-3 substituents selected from alkylamine, amino, aryl, cycloalkyl, heterocyclyl, C1-C1 alkyl, C1-C1 halo alkyl, C1-C1 hydroxyalkyl, C1-C1 alkoxy, C1-C1 alkylamine, C1-C1 dialkylamine, C2-C1 alkenyl or C1₂Cõ alkinyl, where each of which may be interrupted by one or more heteroatoms, carboxyl, cyano, halo, hydroxyl, nitro, -C(O)OH, -C(O)₂-(Ci-C₆ alkyl), -C(O)₂-(W₃-W₈ cycloalkyl), -C(O)₂- (arila), -C(O)₂23/317 (heterocyclyl), -C(O)2-(C1-C6 alkylene)aryl, -C(O)2-(C1-C6 alkylene)heterocyclyl, -C(O)2-(C1-C6 alkylene)cycloalkyl, -C(O)(C1-C6 alkyl), -C(O)(C3-C5 cycloalkyl), -C(O)(aryl), -C(O)(heterocyclyl), -C(O)(C1-C6 alkylene)aryl, -C(O)(C1-C6 alkylene)heterocyclyl, and -C(O)(C1-C6 alkylene)cycloalkyl, wherein each of these optional substituents may be further optionally substituted by 15 substituents selected from amino, cyano, halo, hydroxy, nitro, C1-C1 alkylamine, C1-C1 dialkylamine, C1-C1 alkyl, C1-C1 alkoxy, C1-C1 alkenyl and C1-C1 hydroxyalkyl, where each alkyl is optionally replaced by one or more halo substituents, for example, CF3.

The term "immunomodulator" refers to natural or synthetic products capable of modifying the normal or abnormal immune system through stimulation or suppression.

The term “prevention” refers to the ability of a compound or composition of the invention to prevent a disease identified herein in patients diagnosed as having the disease or who are at risk of developing such disease. The term also encompasses the prevention of further disease progression in patients who are already suffering from or experiencing symptoms of such disease.

The term “patient” or “subject” means an animal (e.g., cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, guinea pig, etc.) or a mammal, including chimeric and transgenic animals and mammals. In the treatment or prevention of HCV infection, the term patient or “subject” preferably means a monkey or a human, with greater preference given to a human. In one specific modality, the patient or subject is infected with or exposed to the hepatitis C virus. In certain modalities, the patient is a human infant (age 0-2), child (age 2-17), adolescent (age 12-17), or adult (age 18-19).

24/317 and above) or geriatric (age 70 and above). Additionally, the patient includes immunocompromised patients such as HIV-positive patients, cancer patients, patients undergoing immunotherapy or chemotherapy. In one particular modality, the patient is a healthy individual, that is, not presenting symptoms of other viral infections.

The term “therapeutically effective amount” refers to an amount of the compound of the invention sufficient to provide a benefit in the treatment or prevention of viral disease, to delay or minimize symptoms associated with viral infection or viral-induced disease, or to cure or improve the disease or infection, or its cause. In particular, a therapeutically effective amount means an amount sufficient to provide a therapeutic benefit in vivo. Used in connection with a quantity of a compound of the invention, the term preferably encompasses a non-toxic quantity that enhances therapy overall, reduces or prevents symptoms or causes of disease, or increases therapeutic efficacy or synergies with another therapeutic agent.

The term “prophylactically effective amount” refers to an amount of a compound of the invention or other active ingredient sufficient to result in the prevention of infection, recurrence, or spread of viral infection. A prophylactically effective amount may refer to an amount sufficient to prevent initial infection or recurrence or spread of infection or an infection-associated illness. When used in connection with a quantity of a compound of the invention, the term preferably encompasses a non-toxic quantity that enhances general prophylaxis or increases prophylactic efficacy or synergies with another prophylactic or therapeutic agent.

The term "in combination" refers to the use of more than one prophylactic and/or therapeutic agent simultaneously or sequentially.

25/317 and in such a way that their respective effects are additive or synergistic.

The term "treatment" refers to:

(i) to prevent a disease, disorder or condition from occurring in an animal that may be predisposed to the disease, disorder and/or condition, but has not yet been diagnosed as having it;

(ii) inhibition of the disease, disorder or condition, that is, stopping its development; and (iii) alleviating the disease, disorder or condition, that is, causing regression of the disease, disorder and/or condition.

The terms “R” and “S” indicate the specific stereochemical configuration of a substituent on an asymmetric carbon atom in a chemical structure as drawn.

The term “rac” indicates that a compound is a racemate, which is defined as an equimolar mixture of a pair of enantiomers. A “rac” compound does not exhibit optical activity. The chemical name or formula of a racemate is distinguished from those of enantiomers by the prefix (±)- or rac- (or racem-) or by the symbols RS and SR.

The terms “endo” and “exo” describe the relative orientation of substituents attached to non-bridgehead atoms in a bicyclic [xyz] alkane (x > y > z > 0).

The terms “syn” and “anti” describe the relative orientation of substituents attached to bridgehead atoms in a bicyclic [xyz] alkane (x > y > z > 0).

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<img file="BRPI0809685A2_D0012.tif" />

endo

The term “exo” is given to a substituent (e.g., Br bonded to C-2 in the example below) that is oriented toward the highest numbered bridge (z-bridge, e.g., C-7 in the example below); if the substituent is oriented away from the highest numbered bridge, it is given the description “endo”.

The term “sin” is given to a substituent attached to the highest numbered bridge (bridge z, for example, F attached to C-7 in the example below) and oriented towards the lowest numbered bridge (bridge x, for example, C-2 and C-3 in the example below); if the substituent is oriented away from the lowest numbered bridge, it is described as “anti.”

<img file="BRPI0809685A2_D0013.tif" />

2-exo-bromo-7-syn-fluorobicycle [2.2.1] heptane

2-endo-bromo-7-anti-fluorobiciclo[2.2. l]heptane

The terms “cis” and “trans” are descriptors that show the relationship between two ligands bonded to separate atoms that are connected by a double bond or are contained within a ring. The two

27/317 ligands are said to be located in cis relative to each other if they are on the same side of a plane. If they are on opposite sides, their relative position is described as trans. The appropriate reference plane of a double bond is perpendicular to that of the relevant σ bonds and passes through the double bond. For a ring, this means the mid-plane of the ring(s).

The compounds of the invention may exhibit the phenomenon of tautomerism. Although Formula I cannot expressly describe all possible tautomeric forms, it is to be understood that Formula I is intended to represent any tautomeric form of the compound described and is not to be limited merely to a specific compound form described by the formula drawings. For illustrative purposes, and by no means limiting the range of tautomers, compounds of Formula 1 may exist as follows:

When X = N:

r¹³„_tom . RV; I

O. o

R'⁵ ,OH HN

When X = CR³:

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R

R

<img file="BRPI0809685A2_D0014.tif" />

R¹

<img file="BRPI0809685A2_D0015.tif" />

Or the compounds of Formula I can exist as follows:

When X = N:

oh, oh_r9 fan¹ b ^_Ri jA 1⁸^R > Â8^M THE

R¹⁵ ,O ^TlaN fA r^: oo O. O o S

R»_r7OHHH'_(Ibm)1 HN

W“'· — r¹³ À'hN'--o R¹² oh r1«^R To laugh¹¹' R²

When X = CR³:

R³

OO, S.

R^s

R¹² g ' r -b - n

ZI_L H ÃàA' °_R1?^K_R2 r9 a.^rAA á 114 II h^R’^{2 OH}r18^K_R2

Some of the inventive compounds may exist as single stereoisomers (i.e., essentially free of other stereoisomers), racemates, and/or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates, and mixtures thereof are intended to be within the scope of the present invention. Preferably, the optically active inventive compounds are

29/317 used in optically pure form.

As generally understood by those skilled in the art, an optically pure compound having a chiral center (i.e., an asymmetric carbon atom) is one that consists essentially of one of two possible enantiomers (i.e., it is enantiomerically pure), and an optically pure compound having more than one chiral center is one that is both diastereomerically pure and enantiomerically pure. Preferably, the compounds of the present invention are used in a form that is at least 90% free of other enantiomers or diastereomers of the compounds, that is, a form that contains at least 90% of a single isomer (80% enantiomeric excess (“ee”) or diastereomeric excess (“de”), more preferably at least 95% (90% ee or de), even more preferably at least 97.5% (95% ee or de), and most preferably at least 99% (98% ee or de).

Furthermore, Formula I is intended to cover solvated as well as non-solvated forms of the identified structures. For example, Formula I includes compounds of the indicated structure in both hydrated and non-hydrated forms. Other examples of solvates include the structures in combination with isopropanol, ethanol, methanol, DMSO, ethyl acetate, pentyl acetate, acetic acid, or ethanolamine.

In addition to the compounds of Formula I, the invention includes pharmaceutically acceptable prodrugs, pharmaceutically acceptable metabolites, and pharmaceutically acceptable salts of such compounds and metabolites.

A pharmaceutically acceptable prodrug is a compound that can be converted under physiological conditions or by solvolysis to the specified compound or to a pharmaceutically acceptable salt of such compound before exhibiting its pharmacological effects.

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Typically, the prodrug is formulated with the objective(s) of increasing chemical stability, increased patient acceptance and consent, increased bioavailability, prolonged duration of action, increased organ selectivity, enhanced formulation (e.g., increased water solubility), and/or decreased side effects (e.g., toxicity). The prodrug can be readily prepared from the compounds of Formula I using methods known in the art, such as those described in Burger's Medicinal Chemistry and Drug Chemistry, 1, 172-178, 949-982 (1995). See also Bertolini et al., J. Med. Chern., 40, 2011-2016 (1997); Shan et al., J. Pharm. Sci., 86 (7), 765-767; Bagshawe, Drug Dev. Res., 34, 220-230 (1995); Bodor, Advances in Drug Res., 13, 224-331 (1984); Bundgaard, Design of Prodrugs (Elsevier Press 1985); Larsen, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen et al., eds., Harwood Academic Publishers, 1991); Dear et al., J. Chromatogr. B, 748, 281-293 (2000); Spraul et al., J. Pharmaceutical &amp; Biomedical Analysis, 10, 601-605 (1992); and Prox et al., Xenobiol., 3, 103-112 (1992).

“A pharmaceutically active metabolite” is intended to mean a pharmacologically active product produced through metabolism in the body of a specified compound or salt thereof. After entering the body, many drugs are substrates for chemical reactions that can change their physical properties and biological effects. These metabolic conversions, which usually affect the polarity of the compounds of Formula I, alter the way in which drugs are distributed and excreted from the body. However, in some cases, drug metabolism is required for a therapeutic effect. For example, anti-cancer drugs from the anti-metabolite class can be converted into their active forms after they have been transported into a cancer cell.

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Since many drugs undergo metabolic transformations of some kind, the biochemical reactions that play a role in drug metabolism can be numerous and diverse. The primary site of drug metabolism is the liver, although other tissues may also participate.

A distinctive feature of many of these transformations is that the metabolic products or “metabolites” are more polar than the original drugs, although a polar drug sometimes produces a less polar product. Substances with high lipid/water partition coefficients, which readily pass through membranes, also readily diffuse back from the tubular urine through the renal tubular cells into the plasma. Thus, these substances tend to have low renal clearance and a long residence time in the body. If a drug is metabolized to a more polar compound, one with a lower partition coefficient, its tubular reabsorption will be greatly reduced. Furthermore, the secretion mechanisms specific to anions and cations in the proximal renal tubules and parenchymal liver cells operate under highly polar substances.

As a specific example, phenacetin (acetophenethidine) and acetanilide are both mild analgesics and antipyretic agents, but they are transformed within the body into a more effective and more polar metabolite, p-hydroxyacetanilide (acetaminophen), which is widely used today. When a dose of acetanilide is given to a person, successive metabolites peak and decay sequentially in the plasma. During the first hour, acetanilide is the main component of the plasma. In the second hour, once the acetanilide level drops, the concentration of the metabolite acetaminophen reaches a peak. Finally, after a few hours, the main component of the plasma is an additional metabolite that is inert and can be excreted from the body.

32/317 body. Thus, the plasma concentrations of one or more metabolites, as well as the drug itself, can be pharmacologically important.

"A pharmaceutically acceptable salt" is intended to mean a salt that retains the biological effectiveness of the free acids and bases of a specified compound and that is not biologically or otherwise undesirable. A compound of the invention may possess sufficient acidity, sufficient basicity, or both functional groups and accordingly react with any number of inorganic or organic bases and inorganic or organic acids to form a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include those salts prepared by reacting the compounds of the present invention with a mineral or organic acid or an inorganic base, such as salts including sulfates, pyrosulfates, bisulfates, sulfides, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyn-1,4-dioates, hexyn-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates and mandelates.

If the inventive compound is a base, the desired pharmaceutically acceptable salt can be prepared by any method appropriate in the art, for example, by treating the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid,

33/317 mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, and an α-hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid or the like.

If the inventive compound is an acid, the desired pharmaceutically acceptable salt can be prepared by any appropriate method, for example, by treating the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide or the like. Illustrative examples of suitable salts include organic salts derived from amino acids, such as glycine and arginine, ammonia, primary, secondary and tertiary amines and cyclic amines, such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

In the case of agents that are solids, it is to be understood by those skilled in the art that the inventive compounds and salts may exist in different crystalline, co-crystal, or polymorphic forms, all of which are intended to be within the scope of the present invention and specified formulas.

Treatment and Prevention Methods for Hepatitis C Viral Infections

The present invention provides methods for treating or preventing a hepatitis C virus infection in a patient in need thereof.

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The present invention further provides methods for introducing a therapeutically effective amount of the compound of Formula I or a combination of such compounds into the bloodstream of a patient for the treatment and/or prevention of hepatitis C viral infections.

The magnitude of a prophylactic or pharmaceutical dosage of a compound of Formula I of the invention or a pharmaceutically acceptable salt, solvate, or hydrate thereof in the treatment or prevention of an acute or chronic infection will, however, vary with the nature and severity of the infection and the route by which the active ingredient is administered. Dosage, and in some cases frequency of dosing, will also vary according to the infection being treated, age, body weight, and individual patient response. Appropriate dosage regimens can be readily selected by those skilled in the art with due consideration of such factors.

The methods of the present invention are particularly well suited for human patients. In particular, the methods and dosages of the present invention may be useful for immunocompromised patients including, but not limited to, cancer patients, HIV-infected patients, and patients with immunodegenerative disease. Furthermore, the methods may be useful for immunocompromised patients currently in a state of remission. The methods and dosages of the present invention are also useful for patients undergoing other antiviral treatments. The prevention methods of the present invention are particularly useful for patients at risk of viral infection. These patients include, but are not limited to, healthcare workers, for example, doctors, nurses, hospice volunteers; military personnel; teachers; childcare workers; patients traveling to or living in overseas locations, particularly in developing countries.

35/317 of social aid workers, missionaries and foreign diplomats. Finally, the methods and compositions include the treatment of refractory or treatment-resistant patients such as those resistant to reverse transcriptase inhibitors, protease inhibitors, etc.

Doses

The toxicity and efficacy of the compounds of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the lethal dose for 50% of the population) and the ED50 (the therapeutically effective dose in 50% of the population). The dosage ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the LD50/ED50 ratio.

Data obtained from cell culture tests and animal studies can be used in formulating a dosage range of compounds for human use. The dosage of such compounds is preferably within the range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration used. For any compound used in the method of the invention, the therapeutically effective dosage can be initially estimated from cell culture tests. Dosage can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves half the maximum inhibition of symptoms) as determined in cell culture; Alternatively, the dosage of the Formula I compound can be formulated in animal models to achieve a circulating plasma concentration range of the compound that corresponds to the concentration required to achieve a fixed magnitude of response. This information

36/317 Calcium can be used to more precisely determine useful dosages in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

The protocols and compositions of the invention are preferably tested in vitro, and then in vivo, for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro tests that can be used to determine whether administration of a specific therapeutic protocol is indicated include in vitro cell culture tests in which cells that react to the effects of Formula I compounds are exposed to the ligand and the magnitude of the response is measured by an appropriate technique. The contribution of the compound of Formula I is then evaluated with respect to the potency of the compound of Formula I and the degree of conversion of the prodrug of the compound of Formula I. Compounds for use in methods of the invention can be tested in appropriate animal model systems prior to testing in humans, including, but not limited to, rats, mice, chickens, cows, monkeys, rabbits, hamsters, etc. The compounds can then be used in appropriate clinical trials.

The magnitude of a prophylactic or therapeutic dosage of a prodrug of a compound of Formula I of the invention or a pharmaceutically acceptable salt, solvate, or hydrate thereof in the treatment or prevention of an acute or chronic infection or condition will vary with the nature and severity of the infection, and the route by which the active ingredient is administered. Dosage, and perhaps frequency of dosing, will also vary according to the infection being treated, age, body weight, and the individual patient's response. Appropriate dosage regimens can be readily selected by those skilled in the art with due consideration of such factors.

In one modality, the dosage administered depends on the specific compound being used, and the patient's weight and condition.

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Also, dosages may differ for various particular Formula I compounds; appropriate dosages can be determined based on the in vitro measurements cited previously and on animal studies, such that lower dosages will be suitable for those Formula I compounds that show effectiveness at lower concentrations than other Formula I compounds when measured in the systems described or referenced herein. In general, the daily dosage is in the range of about 0.001 to 100 mg/kg, preferably about 1 to 25 mg/kg, with a greater preference for about 5 to 15 mg/kg. For the treatment of humans infected with the hepatitis C virus, about 0.1 mg to about 15 g per day is administered in about one to four divided doses per day, preferably 100 mg to 12 g per day, with a greater preference for 100 mg to 8,000 mg per day.

Furthermore, the recommended daily dosage course can be administered in cycles as single agents or in combination with other therapeutic agents. In one embodiment, the daily dosage is administered as a single dose or in equally divided doses. In a related embodiment, the recommended daily dosage can be administered once a week, twice a week, three times a week, four times a week, or five times a week.

In one embodiment, the compounds of the invention are administered to provide systemic distribution of the compound within the patient. In a related embodiment, the compounds of the invention are administered to produce a systemic effect on the body.

In another embodiment, the compounds of the invention are administered orally, mucosally (including sublingual, buccal, rectal, nasal, or vaginal), parenterally (including subcutaneous, intramuscular, single-dose injection, intra-arterial, or intravenous), transdermally, or topically. In a specific embodiment, the compounds of the invention are...

38/317 administered via mucosal (including sublingual, buccal, rectal, nasal or vaginal), parenteral (including subcutaneous, intramuscular, single-dose injection, intra-arterial or intravenous), transdermal or topical administration. In a specific further embodiment, the compounds of the invention are administered via oral administration. In a specific further embodiment, the compounds of the invention are not administered via oral administration.

Different therapeutically effective amounts may be applied to different infections, as will be readily known to those with common knowledge of the art. Similarly, amounts sufficient to treat or prevent such infections, but insufficient to cause, or sufficient to reduce, adverse effects associated with conventional therapies are also encompassed by the dosage amounts and dosing frequency schedule described above.

Combination Therapy

Specific methods of the invention further comprise the administration of an additional therapeutic agent (i.e., a therapeutic agent other than a compound of the invention). In certain embodiments of the present invention, the compounds of the invention may be used in combination with at least one other therapeutic agent. Therapeutic agents include, but are not limited to, antibiotics, antiemetic agents, antidepressants and antifungal agents, anti-inflammatory agents, antiviral agents, anticancer agents, immunomodulatory agents, α-interferons, β-interferons, ribavirin, alkylating agents, hormones, cytokines or toll receptor modulators. In one embodiment, the invention encompasses the administration of an additional therapeutic agent that is specific for HCV or demonstrates anti-HCV activity.

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The compounds of Formula I of the invention can be administered or formulated in combination with antibiotics. For example, they may be formulated with a macrolide (e.g., tobramycin (Tobi®)), a cephalosporin (e.g., cephalexin (Keilex®), cefradine (Velosef®), cefuroxime (Ceftin®), cefprozil (Cefzil®), cefaclor (Ceclor®), cefixime (Suprax®) or cefadroxil (Duricef®)), a clarithromycin (e.g., clarithromycin (Biaxin®)), an erythromycin (e.g., erythromycin (EMicina®)), a penicillin (e.g., penicillin V (V-Cillin K® or Pen Vee K®)) or a quinolone (e.g., ofloxacin (Floxin®), ciproiloxacin (Cipro®) or norfloxacin (Noroxin®)), aminoglycoside antibiotics (e.g., apramycin, arbecacin, bambermycins, butyrosine, dibecacin, neomycin, neomycin undecylenate, netilmicin, paromomycin, ribostamycin, sisomycin, and spectinomycin), amphenicol antibiotics (e.g., azidanphenicol, chloramphenicol, ylorphenicol, and thiamphenicol), antibiotics: ansamycins (e.g., rifamide and rifampin), carbacephems (e.g., loracarbe), carbapenems (e.g., biapenem and imipenem), cephalosporins (e.g., cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefozopran, cefpimizole, cefpyramide, and cefpirome), cefamycins (e.g., cefbuperazone, cefmetazole, and cefminox), monobactams (e.g., aztreonam, carumonam, and tigemonam), oxacephems (e.g., flomoxef and moxalactam), penicillins (e.g., andinocillin, andinocillin pivoxil, amoxicillin, bacampicillin, benzylpenicillin acid, sodium benzylpenicillin, epicillin, phenbenicillin, floxacili25 na, penicillin, penetamate hydrodide, o-benetamine penicillin, penicillin O, penicillin V, benzathine penicillin V, hydrabamine penicillin V, penimepicillin and potassium phencyclidine), lincosamides (e.g., clindamycin and lincomycin), amphomycin, bacitracin, capreomycin, colistin, enduracidin, enviomycin, Tetracyclines (e.g., apicicline, chlortetracycline, clomocycline, and demeclocycline), 2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans (e.g., furaltadone and furazolium chloride), quinolones, and analogues thereof.

40/317 (e.g., cinoxacin, clinafloxacin, flumequine, and grepagloxacin), sulfonamides (e.g., acetyl sulfamethoxypyrazine, benzylsulfamide, noprilsulfamide, phthalylsulfacetamide, sulfacrisoidine, and sulfacitin), sulfones (e.g., diathymosulfone, sodium glucosulfone, and solasulfone), cycloserine, mupirocin, and tuberin.

The compounds of Formula I of the invention can also be administered or formulated in combination with an anti-emetic agent. Suitable antiemetic agents and mixtures thereof include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acetyl leucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautin, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, metalatal, metopimazine, nabilone, oxiperndil, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine, thioproperazine, tropisetron.

The compounds of Formula I of the invention can be administered or formulated in combination with an antidepressant. Suitable antidepressants include, but are not limited to, binedaline, caroxazone, citalopram, dimethazen, fencanamine, indalpine, indeloxazine chloride, nefopam, nomifensine, oxitriptan, oxipertine, paroxetine, sertraline, tiazemine, trazodone, bemoxine, iproclozide, iproniazid, isocarboxazid, nialamide, octamoxine, phenelzine, cotinine, rolicipine, rolipram, maprotiline, metralindole, mianserin, mirtazepine, adinazolam, amitriptyline, amitriptyline oxide, amoxapine, butriptyline, clomipramine, demexiptiline, desipramine, dibenzepine, dimethacrine, dothiepin, doxepin, fluacizine, imipramine, imipramine N-oxide, iprindol, lofepramine, melitracen, metapramine, nortriptyline, noxiptiline, opipramol, pizotiline, propizepine, protriptyline, quinupramine, tianeptine, trimipramine, adrafinil, benactizine, bupropion, butaceti

41/317 na, dioxadrol, duloxetine, etoperidone, febarbamate, femoxetine, fenpentadiol, fluoxetine, fluvoxamine, hematoporphyrin, hypericin, levofacetoperane, medifoxamine, milnacipran, minaprine, moclobemide, nefazodone, oxaflozane, piberaline, prolintane, pirisuccideanol, ritanserin, roxindole, rubidium chloride, sulpiride, tandospirone, tozalinone, tofenacin, toloxatone, tranylcypromine, L-tryptophan, venlafaxine, viloxazine, and zimeldine.

The compounds of Formula I of the invention may be administered or formulated in combination with an antifungal agent. Suitable antifungal agents include, but are not limited to, amphotericin B, itraconazole, ketoconazole, fluconazole, intrathecal, flucytosine, miconazole, butoconazole, clotrimazole, nystatin, terconazole, tioconazole, ciclopirox, econazole, haloprogrin, naftifine, terbinafine, undecylenate, and griseofulvin.

The compounds of Formula I of the invention can be administered or formulated in combination with an anti-inflammatory agent. Useful anti-inflammatory agents include, but are not limited to, nonsteroidal anti-inflammatory drugs such as salicylic acid, acetylsalicylic acid, methyl salicylate, diflunisal, salsalate, olsalazine, sulfasalazine, acetaminophen, indomethacin, sulindac, etodolac, mefenamic acid, meclofenamate sodium, tolmetin, ketorolac, diclofenac, ibuprofen, naproxen, naproxen sodium, fenoprofen, ketoprofen, flurbinprofen, oxaprozin, piroxicam, meloxicam, ampiroxicam, droxicam, Pivoxicam, tenoxicam, nabumetom, phenylbutazone, oxyphenbutazone, antipyrine, aminopyrine, apazone, and nimesulide; leukotriene antagonists including, but not limited to, zileuton, aurothioglucose, sodium gold thiomalate, and auranofin; Steroids including, but not limited to, alclometasone dipropionate, ancinonide, beclometasone dipropionate, betamethasone, betamethasone benzoate, betamethasone dipropionate, betamethasone sodium phosphate, betamethasone valerate, clobetasol propionate, clocortolone pivalate, hydrocortisone, hydrocortisone derivatives, desonide, desoximetasone, dexamethasone, flunisolide, flucoxinolide, flurandrenolide, halcinocid, medrisone, methylprednisolone, Methyl prednisolone acetate, methyl prednisolone succinate sodium, mometasone furoate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone phosphate sodium, prednisolone tebuate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate and triamcinolone hexacetonide; and other anti-inflammatory agents including, but not limited to, methotrexate, colchicine, allopurinol, probenecid, sulfinpyrazone and benzbromarone.

The compounds of Formula I of the invention can be administered or formulated in combination with another antiviral agent. Useful antiviral agents include, but are not limited to, protease inhibitors, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and nucleoside analogs. Antiviral agents include, but are not limited to, zidovudine, acyclovir, gangciclovir, vidarabine, idoxuridine, trifluridine, levovirine, viramidine and ribavirin, as well as foscarnet, amantadine, rimantadine, saquinavir, indinavir, amprenavir, lopinavir, ritonavir, α-interferons, β-interferons, adefovir, clevadine, entecavir, pleconaril.

The compounds of Formula I of the invention can be administered or formulated in combination with an immunomodulatory agent. Immunomodulatory agents include, but are not limited to, methotrexate, leflunomide, cyclophosphamide, cyclosporine A, mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualine, brequinar, malononitrileamides (e.g., leflunomide), T-cell receptor modulators and cytokine receptor modulators, peptide mimetics and antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs fragments, etc.).

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ScFvs, Fab or F(ab)2 or epitope linkage fragments), nucleic acid molecules (e.g., antisense and triple helix nucleic acid molecules), small molecules, organic compounds, and inorganic compounds. Examples of T-cell receptor modulators include, but are not limited to, anti-T-cell receptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412 (Boehringer), IDEC-CE9.1® (IDEC and SKB), mAB 4162W94, Orthoclone, and OKTcdr4a (Janssen-Cilag))), anti-CD3 antibodies (e.g., Nuvion (Product Design Labs), OKT3 (Johnson &amp; Johnson), or Rituxan (IDEC)), anti-CD5 antibodies (e.g., a ricin-linked anti-CD5 immune conjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies, anti-CD40 binding monoclonal antibodies (e.g., IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH 1H (Ilex)), anti-CD2 antibodies, anti-CD1a antibodies (e.g., Xanelim (Genentech)), anti-B7 antibodies (e.g., IDEC-114 (IDEC)), CTLA4 immunoglobulin, and toll receptor-like modulators (TLRs). Examples of cytokine receptor modulators include, but are not limited to, soluble cytokine receptors (e.g., the extracellular domain of a TNF-α receptor or a fragment thereof, the extracellular domain of an IL-β receptor or a fragment thereof, and the extracellular domain of an IL-6 receptor or a fragment thereof), cytokines or fragments thereof (e.g., interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, TNF-α, interferon (IFN)-α, IFN-β, IFN-γ, and GM-CSF), anti-cytokine receptor antibodies (e.g., anti-IFN receptor antibodies, anti-IL-2 receptor antibodies (e.g., Zenapax (Protein Design Labs)), anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies, anti-IL-10 receptor antibodies, anti-IL-12 receptor antibodies), anti-cytokine antibodies (e.g., anti-IFN antibodies, anti-TNF-α antibodies, anti-IL-Ιβ, anti-IL-6 antibodies, anti-IL-8 antibodies (e.g. ABX-IL-8 (Abgenix)) and anti-IL-12 antibodies).

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The compounds of Formula I of the invention can be administered or formulated in combination with an agent which inhibits viral enzymes including, but not limited to, HCV protease inhibitors such as BILN 2061, SCH-503034, ITMN-191 or VX-950; and NS5B polymerase inhibitors such as NM107 (and its prodrug NM283), R1626, R7078, BILN1941, GSK625433, GILD9128 or HCV796.

The compounds of Formula I of the invention can be administered or formulated in combination with an agent which inhibits HCV polymerase, such as those described in Wu, Curr Drug Targets Infect Disord. 2003, 3(3), 207-19 or in combination with compounds that inhibit the viral helicase function, such as those described in Bretner M, et al Nucleosides Nucleotides Nucleic Acids. 2003, 22(5-8), 1531, or with inhibitors of other specific HCV targets, such as those described in Zhang X, IDrugs 2002, 5(2), 154-8.

The compounds of Formula I of the invention can be administered or formulated in combination with an agent that inhibits viral replication.

The compounds of Formula I of the invention can be administered or formulated in combination with cytokines. Examples of cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), platelet-derived growth factor (PDGF), erythropoietin (Epo), epidermal growth factor (EGF), fibroblast growth factor (FGF), granulocyte macrophage-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), prolactin and interferon (IFN), for example, IFN-α and IFN-γ).

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The compounds of Formula I of the invention can be administered or formulated in combination with hormones. Examples of hormones include, but are not limited to, luteinizing hormone-releasing hormone (LHRH), growth hormone (GH), growth hormone-releasing hormone, ACTH, somatostatin, somatotropin, somatomedin, parathyroid hormone, hypothalamic releasing factors, insulin, glucagon, enkephalins, vasopressin, calcitonin, heparin, low molecular weight heparins, heparinoids, synthetic and natural opioids, thyroid-stimulating hormones 10 insulin and endorphins.

The compounds of Formula I of the invention can be administered or formulated in combination with β-interferons, including, but not limited to, interferon β-Ia and interferon β-Ib.

The compounds of Formula I of the invention may be administered or formulated in combination with α-interferons which include, but are not limited to, interferon α-1, interferon α-2a (roferone), interferon α-2b, introna, Peg-Introna, Pegasys, consensus interferon (infergen) and albuferone.

The compounds of Formula I of the invention may be administered or formulated in combination with an absorption enhancer, particularly those that are targeted to the lymphatic system, including, but not limited to, sodium glycolate; sodium caprate; 7V-Iauryl-β-D-maltopyranoside; EDTA; mixed micelle and those reported in Muranishi Crit. Rev. Ther. Drug Carrier Syst., 7, 1-33, which is hereby incorporated by reference in its entirety. Other absorption enhancers may also be used. Thus, the invention also encompasses a pharmaceutical composition comprising one or more compounds of Formula I of the invention and one or more absorption enhancers.

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The compounds of Formula I of the invention can be administered or formulated in combination with a cytochrome P450 monooxygenase inhibitor, such as, but not limited to, ritonavir or a pharmaceutically acceptable salt, ester and prodrug thereof to enhance the pharmacokinetics (e.g., increased half-life, increased time to reach peak plasma concentration, increased blood levels) of a compound of Formula I that is metabolized by cytochrome P450 monooxygenase. Thus, the invention also encompasses a pharmaceutical composition comprising compounds of Formula I of the invention and one or more cytochrome P450 monooxygenase inhibitors.

The compounds of Formula I of the invention can be administered in combination with food to increase the absorption of the compounds of Formula I in the gastrointestinal tract and to increase the bioavailability of the compounds of Formula I.

The compounds of Formula I of the invention may be administered or formulated in combination with an alkylating agent. Examples of alkylating agents include, but are not limited to, nitrogen mustards, ethyleneimines, methylmelamines, alkyl sulfonates, nitrosoureas, triazenes, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine, streptozocin, dacarbazine, and temozolomide.

The compounds of the invention and other therapeutic agents may act additively or, more preferably, synergistically. In one embodiment, a composition comprising a compound of the invention is administered concurrently with the administration of another therapeutic agent, which may be part of the same composition or in a composition different from that comprising the compounds of the invention. In another embodiment, a compound of the invention is administered before or subsequent to the administration of another therapeutic agent.

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In a separate embodiment, a compound of the invention is administered to a patient who has not previously received or is not currently receiving treatment with another therapeutic agent, particularly an antiviral agent.

In one embodiment, the methods of the invention comprise the administration of one or more compounds of Formula I of the invention without an additional therapeutic agent.

Pharmaceutical Compositions and Dosage Forms

Pharmaceutical compositions and unit dosage forms comprising a compound of Formula I of the invention or a pharmaceutically acceptable salt or hydrate thereof are also encompassed by the invention. Individual dosage forms of the invention may be suitable for oral, mucosal (including sublingual, buccal, rectal, nasal or vaginal), parenteral (including subcutaneous, intramuscular, single-dose injection, intra-arterial or intravenous), transdermal or topical administration. Pharmaceutical compositions and dosage forms of the invention typically also comprise one or more pharmaceutically acceptable excipients. Sterile dosage forms are also contemplated.

In an alternative embodiment, the pharmaceutical composition encompassed by this embodiment includes a compound of Formula I of the invention or a pharmaceutically acceptable salt or hydrate thereof and at least one additional therapeutic agent. Examples of additional therapeutic agents include, but are not limited to, those listed above.

The composition, form, and type of dosage forms of the invention will typically vary depending on their uses. For example,

48/317 A dosage form used in the acute treatment of a disease or a related disease may contain larger amounts of one or more of the active ingredients it contains than a dosage form used in the chronic treatment of the same disease. Similarly, a parenteral dosage form may contain smaller amounts of one or more of the active ingredients it comprises than an oral dosage form used to treat the same disease or disorder. These and other ways in which specific dosage forms encompassed by this invention will vary from one another will be readily apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990). Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; packets; tablets; expectorant lozenges; dispersions; suppositories; liniments; poultices; pastes; powders; dressings; creams; plasters; solutions; plasters; aerosols (e.g., nasal sprays or inhalers); gels; Liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions or water-in-oil emulsions), solutions and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

Typical pharmaceutical compositions and dosage forms comprise one or more carriers, excipients, or diluents. Suitable excipients are well known to those skilled in the art of pharmacy, and non-limiting examples of suitable excipients are provided herein. If a particular excipient is suitable for incorporation into a pharmaceutical composition or

49/317 The dosage form depends on a variety of factors well known in the art, including, but not limited to, the manner in which the dosage form will be administered to the patient. For example, oral dosage forms such as tablets may contain excipients not appropriate for use in parenteral dosage forms. The suitability of a particular excipient may also depend on the specific active ingredients in the dosage form.

This invention also encompasses anhydrous pharmaceutical compositions and dosage forms comprising active ingredients, since water can facilitate the degradation of some compounds. For example, the addition of water (e.g., 5%) is widely accepted in pharmaceutical techniques as a means of simulating long-term storage in order to determine characteristics such as shelf life or the stability of formulations over time. See, for example, Carstensen, Drug Stability: Principles &amp; Practice, 2nd ed., Marcel Dekker, NY, NY, 1995, pp. 379-80. Indeed, water and heat accelerate the decomposition of some compounds. Thus, the effect of water on the formulation can be of great significance since dew and/or moisture are commonly encountered during the manufacture, handling, packaging, storage, transport, and use of formulations.

Pharmaceutical compositions and anhydrous dosage forms of the invention can be prepared using ingredients containing low moisture or anhydrous substances and under conditions of low dew and low humidity.

An anhydrous pharmaceutical composition could be prepared and stored in such a way that its anhydrous nature is maintained. Accordingly, anhydrous compositions are preferably packaged using materials known to prevent exposure to water so that they can be included in appropriate formulation kits. Examples of appropriate packaging include, but are not limited to,

50/317 hermetically sealed plates, plastics , unit dose containers (e.g., bottles), blister packs and strip packaging.

The invention also encompasses pharmaceutical compositions and dosage forms comprising one or more compounds that reduce the rate at which an active ingredient will decompose. Such compounds, which are referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or saline buffers.

Like the quantities and types of excipients, the specific quantities and types of active ingredients in a dosage form may differ depending on factors such as, but not limited to, the route by which it is to be administered to patients. However, typical dosage forms of the invention comprise the compounds of Formula I of the invention or a pharmaceutically acceptable salt or hydrate thereof comprising from 0.1 mg to 1500 mg per unit to provide doses of about 0.01 to 200 mg/kg per day.

Oral Dosage Forms

Pharmaceutical compositions of the invention that are suitable for oral administration may be presented as discrete dosage forms, such as, but not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). These dosage forms contain predetermined amounts of active ingredients and may be prepared by pharmacy methods well known to those skilled in the art. See generally, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).

Typical oral dosage forms of the invention are prepared by combining the active ingredient(s) in a mixture.

51/317 intimate with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients may have a wide variety of forms depending on the desired form of preparation for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in oral solid dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.

Due to their ease of administration, tablets and capsules represent the most advantageous unit dosage forms for oral administration when solid excipients are used. If desired, tablets can be coated using standard aqueous or non-aqueous techniques. These dosage forms can be prepared by any pharmacy method. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately mixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then molding the product into the desired presentation if necessary.

For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing the active ingredients in a free-flowing form such as powder or granules, optionally mixed with an excipient, in a suitable machine. Molded tablets can be made by molding a mixture of a powdered compound moistened with an inert liquid diluent in a suitable machine.

Examples of excipients that can be used in oral dosage forms of the invention include, but are not limited to, binders,

52/317 Fillers, disintegrants and lubricants. Suitable binders for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, tragacanth powder, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pregelatinized starch, Hydroxypropyl methylcellulose (e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof. The binder or filler in the pharmaceutical compositions of the invention is typically present from about 50 to about 99 percent by weight of the pharmaceutical composition or dosage form.

Suitable forms of microcrystalline cellulose include, but are not limited to, materials sold as AVICEL-PH-101, AVICEL-PH-103, AVICEL RC-581, AVICEL-PH-105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, PA) and mixtures thereof. A specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581. Anhydrous or low-moisture excipients or additives include AVICEL-PH-103™ and Starch 1500 LM.

Disintegrants are used in the compositions of the invention to provide tablets that disintegrate when exposed to an aqueous environment. Tablets containing too much disintegrant may...

53/317 disintegrate during storage, while those containing too little may not disintegrate at a desired rate or under the desired conditions. Thus, a sufficient amount of disintegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients should be used to form oral solid dosage forms of the invention. The amount of disintegrant used varies based on the type of formulation and is readily discernible by those with common knowledge of the art. Typical pharmaceutical compositions comprise from about 0.5 to about 15 percent by weight of disintegrant, specifically from about 1 to about 5 percent by weight of disintegrant.

Disintegrants that can be used in the pharmaceutical compositions and dosage forms of the invention include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, potassium polacrilin, sodium starch glycolate, potato or tapioca starch, pregelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used in the pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil). Zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, for example, a silica gel siloid (AEROSIL 200, manufactured by WR Grace Co. of Baltimore, MD), a coagulated synthetic silica aerosol (sold by Degussa Co. of Plano, TX), CAB-O-SIL (a pyrogenic silicon dioxide product).

54/317 sold by Cabot Co. of Boston, MA) and mixtures thereof. If used in any way, lubricants are typically used in an amount of less than about 1 percent by weight of the pharmaceutical compositions or dosage forms in which they are incorporated.

Delayed-Release Dosage Forms

The active ingredients of the invention can be administered by controlled-release means or by delivery devices that are well known to those in the common art. Examples include, but are not limited to, those described in U.S. Patents Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled release of one or more active ingredients using, for example, hydropropyl methylcellulose, other polymeric matrices, gels, permeable membranes, osmotic systems, multi-layer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those with common knowledge of the art, including those described herein, can be readily selected for use with the active ingredients of the invention. The invention thus encompasses simple unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gel capsules and caplets that are adapted for controlled release.

All controlled-release pharmaceutical products

55/317 have a common goal of increasing drug therapy relative to that achieved by its uncontrolled counterpart. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum amount of the drug substance being employed to cure or control the condition in a minimum period of time. The advantages of controlled-release formulations include extended drug activity, reduced dosing frequency, and increased patient acceptance. Furthermore, controlled-release formulations can be used to alter the onset time of action or other characteristics, such as drug levels in the blood, and can thus alter the occurrence of side effects (e.g., adverse effects).

Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that readily produces the desired therapeutic effect and gradually and continuously release other amounts of the drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of the drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of the drug being metabolized and excreted from the body. The controlled release of an active ingredient can be stimulated by various conditions, including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds.

Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients via various routes, including but not limited to subcutaneous, intravenous (including single-dose injection), intramuscular, and intra-arterial. Because their administration typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are used.

56/317 Parenteral dosage forms are preferably sterile or capable of being sterilized before administration to a patient. Examples of parenteral dosage forms include, but are not limited to, ready-to-inject solutions, dry and/or lyophilized products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection (reconstitutable powders), ready-to-inject suspensions, and emulsions.

Suitable vehicles that can be used to deliver parenteral dosage forms of the invention are well known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate and benzyl benzoate.

Compounds that increase the solubility of one or more of the active ingredients disclosed herein may also be incorporated into parenteral dosage forms of the invention.

Transdermal Dosage Forms

Transdermal dosage forms include "reservoir-type" or "matrix-type" patches, which can be applied to the skin and worn for a specific period of time to allow the penetration of a desired amount of the active ingredients.

Appropriate excipients (e.g., carriers and diluents) and other materials that can be used to provide forms of

57/317 Transdermal and topical dosages encompassed by this invention are well known to those skilled in pharmaceutical techniques and depend on the particular tissue to which a given pharmaceutical composition or dosage form will be applied. With this fact in mind, typical excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof.

Depending on the specific tissue being treated, additional components may be used before, in conjunction with, or subsequent to treatment with the active ingredients of the invention. For example, penetration enhancers may be used to aid in the delivery of the active ingredients to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl and tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polividone); urea; and various water-soluble or insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied, can also be adjusted to improve the delivery of one or more active ingredients. Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients in order to improve delivery.

In this regard, stearates can serve as a lipid carrier for the formulation, as an emulsifying agent, or as a surfactant.

58/317 te, and as a delivery enhancer or penetration enhancer. Salts, hydrates or solvates other than the active ingredients may be used to further adjust the properties of the resulting composition.

Topical Dosage Forms

The topical dosage forms of the invention include, but are not limited to, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other forms known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 18th 10th ed., Mack Publishing, Easton, PA (1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea &amp; Febiger, Philadelphia (1985).

The appropriate excipients (e.g., carriers and diluents) and other materials that can be used to provide 15 transdermal and topical dosage forms encompassed by this invention are well known to those skilled in pharmaceutical techniques and depend on the particular tissue to which a given pharmaceutical composition or dosage form will be applied. With this fact in mind, typical excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof.

Depending on the specific tissue to be treated, additional components may be used before, in conjunction with, or subsequent to treatment with the active ingredients of the invention. For example, penetration enhancers may be used to aid in the delivery of the active ingredients to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl and tetrahydrofuryl alcohols; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide;

59/317 polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polividone); urea and various water-soluble or insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).

Mucosal Dosage Forms

Mucosal dosage forms of the invention include, but are not limited to, ophthalmic solutions, sprays and aerosols, or other forms known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, PA (1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea &amp; Febiger, Philadelphia (1985). Dosage forms suitable for treating mucosal tissues within the oral cavity may be formulated as mouthwashes or as oral gels. In one embodiment, the aerosol comprises a carrier. In another embodiment, the aerosol is carrier-free.

The compounds of Formula I of the invention can be administered directly to the lungs by inhalation. For inhalation administration, a compound of Formula I can be conveniently delivered to the lungs by a number of different devices. For example, a Metered Dose Inhaler (“MDI”) that uses containers containing an appropriate low-boiling-point propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or another appropriate gas, can be used to deliver a Formula I compound directly to the lung. MDI devices are available from a number of suppliers such as 3M Corporation, Aventis, Boehringer Ingelheim, Forest Laboratories, Glaxo-Wellcome, Schering-Plough and Vectura.

Alternatively, a Dry Powder Inhaler (DPI) device can be used to deliver a Formula I compound to the lungs.

60/317 (See, for example, Raleigh et al., Proc. Amer. Assoc. Cancer Research Annual Meeting, 1999, 40, 397, which is incorporated herein by reference). DPI devices typically use a mechanism such as a gas explosion to create a cloud of dry powder inside a container, which can then be inhaled by the patient. DPI devices are also well known in the art and can be purchased from a number of vendors which include, for example, Fisons, Glaxo-Wellcome, Inhale Therapeutic Systems, ML Laboratories, Qdose and Vectura. A popular variation is the multi-dose DPI system (“MDDPI”), which allows the delivery of more than one therapeutic dose. MDDPI devices are available from companies such as AstraZeneca, GlaxoWellcome, IVAX, Schering Plough, SkyePharma and Vectura. For example, gelatin capsules and cartridges for use in an inhaler or insufflator may be formulated containing a mixed powder of a compound and a suitable powder base such as lactose or starch for these systems.

Another type of device that can be used to deliver a Formula I compound to the lung is a liquid spray device supplied, for example, by Aradigm Corporation. Liquid spray systems use extremely small nozzle orifices to aerosolize liquid drug formulations that can be directly inhaled into the lung.

In one embodiment, a nebulizer device is used to deliver a Formula I compound to the lung. Nebulizers create aerosols from liquid drug formulations using, for example, ultrasonic energy to form fine particles that can be readily inhaled (See, for example, Verschoyle et al., British J. Cancer, 1999, 80, Suppl 2, 96, which is incorporated herein by reference). Examples of nebulizers include devices supplied by Sheffield/Systemic Pulmonary Delivery Ltd.

61/317 (See Armer et al., U.S. Patent 5,954,047; van der Linden et al., U.S. Patent 5,950,619; van der Linden et al., U.S. Patent 5,970,974, which are incorporated herein by reference), Aventis and Batelle Pulmonary Therapeutics.

In one embodiment, an electrohydrodynamic (“EHD”) aerosol device is used to deliver Formula I compounds to the lungs. EHD aerosol devices use electrical energy to aerolyze liquid drug solutions or suspensions (See, for example, Noakes et al., U.S. Patent 4,765,539; Coffee, U.S. Patent). 4,962,885; Coffee, PCT Application WO 94/12285; Coffee, PCT Application WO 94/14543; Coffee, PCT Application WO 95/26234, Coffee, PCT Application WO 95/26235, Coffee, PCT Application WO 95/32807, which are incorporated herein by reference). The electrochemical properties of the Formula I compound formulations may be important parameters for optimizing the delivery of this drug to the lungs using an EHD aerosol device, and such optimization is routinely performed by a person skilled in the art. EHD aerosol devices can deliver drugs to the lungs more efficiently than existing pulmonary delivery technologies. Other methods of intrapulmonary delivery of the compounds of Formula I will be well known to those skilled in the art and are within the scope of the invention.

Liquid drug formulations suitable for use with nebulizer and liquid spray devices and EHD aerosol devices will typically include a compound of Formula I with a pharmaceutically acceptable carrier. Preferably, the pharmaceutically acceptable carrier is a liquid such as alcohol, water, polyethylene glycol, or a perfluorocarbon. Optionally, other material may be added to alter the aerosol properties of the solution or suspension of the compound of Formula I. Preferably, this material is a liquid such as an alcohol, glycol, polyglycol, or a fatty acid. Other methods

62/317 formulations of solutions or suspensions of liquid drugs for use in aerosol devices are known to those skilled in the art (See, for example, Biesalski, US patent 5,112,598; Biesalski, US patent 5,556,611, which are incorporated herein by reference). A compound of Formula I may also be formulated into rectal or vaginal compositions, such as suppositories or retention enemas, for example, containing bases for conventional suppositories such as cocoa butter or other glycerides.

In addition to the formulations described previously, a compound of Formula I may also be formulated as a depot preparation. Such long-acting formulations may be administered by implant (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or an ion-exchange resin, or as economical soluble derivatives, for example, as an economical soluble salt.

Alternatively, other pharmaceutical delivery systems may be employed. Liposomes and emulsions are well-known examples of delivery vehicles that can be used to deliver the compounds of Formula I. Certain organic solvents such as dimethyl sulfoxide may also be employed, although usually at the cost of increased toxicity. A compound of Formula I may also be delivered in a controlled-release system. In one embodiment, a pump may be used (Sefton, CRC Crit. Ref Biomed Eng., 1987, 14, 201; Buchwald et al., Surgery, 1980, 88, 507; Saudek et al., N. Engl. J. Med., 1989, 321, 574). In another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and

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Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chern., 1983, 23, 61; see also Levy et al., Science, 1985, 228, 190; During et al., Ann. Neurol., 1989, 25, 351; Howard et al., J. Neurosurg., 71, 105 (1989). In yet another embodiment, a controlled-release system may be placed in close proximity to the target of the compounds of the invention, for example, the lung, thus requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115 (1984)). Another controlled-release system may be used (see, for example, Langer, Science, 1990, 249, 1527).

Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide mucosally encapsulated dosage forms by this invention are well known to those skilled in pharmaceutical techniques and depend on the particular method or site to which a given pharmaceutical composition or dosage form will be administered. With this fact in mind, typical excipients include, but are not limited to, water, ethanol, ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof, which are non-toxic and pharmaceutically acceptable. Examples of such additional ingredients are well known in the art. See, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, PA (1990).

The pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied, can also be adjusted to improve the delivery of one or more active ingredients. Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve release. Compounds such as stearates

64/317 can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients in order to improve delivery. In this respect, stearates can serve as a lipid carrier for the formulation, as an emulsifying or surfactant agent, and as a delivery enhancer or penetration enhancer. Different salts, hydrates, or solvates of the active ingredients can be used to further adjust the properties of the resulting composition.

Kits

The invention provides a pharmaceutical package or kit comprising one or more containers containing a compound of Formula I useful for the treatment or prevention of a Hepatitis C virus infection. In other embodiments, the invention provides a pharmaceutical package or kit comprising one or more containers comprising a compound of Formula I useful for the treatment or prevention of a Hepatitis C virus infection and one or more containers comprising an additional therapeutic agent, including, but not limited to, those listed above, in particular an antiviral agent, an interferon, an agent that inhibits viral enzymes or an agent that inhibits viral replication. Preferably, the additional therapeutic agent is HCV-specific or demonstrates anti-HCV activity.

The invention also provides a pharmaceutical package or kit comprising one or more containers comprising one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) may be a warning in the form prescribed by a government agency regulating the production, use and sale of pharmaceutical or biological products, such warning reflecting the agency's approval for the manufacture, use or sale for human administration.

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The inventive agents can be prepared using the reaction routes and synthesis schemes as described below, employing general techniques known in the art and using readily available starting materials. The synthesis of compounds not 5 exemplified according to the invention can be successfully effected by modifications obvious to those skilled in the art, for example, by appropriate protection of interfering groups, by changing other suitable reagents known in the art, or by making routine modifications of the reaction conditions. Alternatively, other reactions disclosed herein or generally known in the art will be recognized as having applicability in the preparation of other compounds of the invention.

Preparation of Compounds

In the synthetic schemes described below, unless otherwise indicated, all temperatures are given in degrees Celsius and all parts and percentages are by weight.

The reagents were purchased from commercial suppliers such as Aldrich Chemical Company or Lancaster Synthesis Ltd and were used without further purification unless otherwise indicated. All solvents were purchased from commercial suppliers such as Aldrich, EMD Chemicals or Fisher and used as received.

The reactions described below were generally carried out under positive argon or nitrogen pressure at ambient temperature (unless otherwise indicated) in anhydrous solvents, and the reaction flasks were fitted with rubber septa for the introduction of substrates and reagents via syringe. The glassware was oven-dried and/or heat-dried.

66/317

The reactions were analyzed by TLC and/or LC-MS or HPLC and terminated when judged by the consumption of the starting material. Analytical thin-layer chromatography (TLC) was performed on glass plates pre-coated with silica gel on 60 F254 0.25 mm plates (EMD Chemicals) and visualized with UV light (254 nm) and/or iodine on silica gel and/or heating with TLC staining such as ethanolic phosphomolybdic acid, ninhydrin solution, potassium permanganate solution or ceric sulfate solution. Preparative thin-layer chromatography (prep-TLC) was performed on silica gel-precoated glass plates in 60 F254 0.5 mm (20 x 20 cm, Thomson Instrument Company) plates and visualized with UV light (254 nm).

The plans were typically made by doubling the volume of the reaction with the solvent or solvent extraction and then washing with the indicated aqueous solutions using 25% by volume of the extraction volume unless otherwise indicated. The product solutions were dried over anhydrous Na2SO4 and/or MgSO4 before filtration and evaporation of the solvents under reduced pressure in a rotary evaporator and recorded as solvents removed under vacuum. Column chromatography was completed under positive pressure using Merck silica gel 60, 230-400 mesh, or neutral alumina 50-200 mesh; ISCO flash chromatography was performed using pre-packed RediSep silica gel columns; or Analogix flash chromatography was performed using pre-packed SuperFlash silica gel columns. Hydrogenolysis was carried out at the pressure indicated in the examples or at ambient pressure.

The Ή-NMR spectra and¹³C-NMR spectra were recorded on a Varian Mercury-VX400 instrument operating at 400 MHz. NMR spectra were obtained as CDCl3 solutions (reported in ppm), using chloroform as a reference standard (7.27 ppm for the proton and 77.00 ppm for the carbon), CD3OD (3.4 and 4.8 ppm for the protons and

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49.3 ppm for carbon), DMSO-de (2.49 ppm for the proton) or tetramethylsilane internally (0.00 ppm) when appropriate. Other NMR solvents were used when necessary. When peak multiplicities are reported, the following abbreviations are used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), bs (broad singlet), dd (doublet of doublets), dt (doublet of triplets). Coupling constants, when given, are reported in Hertz (Hz).

Infrared (IR) spectra were recorded on an ATR FT-IR spectrometer when pure or solid oils were measured, and data are reported in wavenumbers (cm-¹Reported mass spectra are (+)-ES or APCI (+) LC/MS conducted by the Analytical Chemistry Department of Anadys Pharmaceuticals, Inc. Elemental analyses were conducted by Atlantic Microlab, Inc. in Norcross, GA. Melting points (mp) were determined on an open capillary apparatus and are uncorrected.

Enantiomeric excess (ee) values ​​were determined by HPLC analysis using Chiralpak (Chiral Technologies Inc.) AS-RH columns, 2.1 x 150 mm, 5 microns, λ = 312 nm or AS-RH columns, 4.6 x 250 mm, 5 microns, λ = 310 nm.

AS-RH, 2.1 x 150 mm, 5 microns: HPLC separation by binary gradient. Solvent A: 0.1% Formic Acid in Water, Solvent B: 0.1% Formic Acid in Acetonitrile. 10 g/L of sample dissolved in 50% methanol - 50% water [0.1 mg/mL] were injected.

<td>Time (min)</td><td>%B</td><td>Flow rate (mL/min)</td>

<td> 0,0</td><td> 55</td><td> 0,3</td>

<td> 5,0</td><td> 95</td><td> 0,3</td>

<td> 5,5</td><td> 95</td><td> 0,3</td>

<td> 6,0</td><td> 55</td><td> 0,3</td>

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12,0 55

0,3

AS-RH, 4.6 x 250 mm, 5 microns: HPLC separation by binary gradient. Solvent A: 0.05% TFA in Water, Solvent B: 0.05% TFA in Acetonitrile. 3-5 μL of sample dissolved in acetonitrile [1 mg/mL] were injected.

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<td>Time (min)</td><td>%B</td><td>Flow rate (mL/min)</td>

<td> 0,0</td><td> 50</td><td> 0,8</td>

<td> 8,0</td><td> 95</td><td> 0,8</td>

<td> 10,0</td><td> 95</td><td> 0,8</td>

<td> 11,0</td><td> 50</td><td> 0,8</td>

<td> 13,0</td><td> 50</td><td> 0,8</td>

The synthetic pathways described and experimental procedures utilize many common chemical abbreviations, 2,2-DMP (2,2-dimethoxypropane), Ac (acetyl), ACN (acetonitrile), Bn (benzyl), BnOH (benzyl alcohol), Boc (tert-butoxycarbonyl), BocçiO (di-tert-butyl dicarbonate), Bz (benzoyl), CSI (chlorosulfonyl isocyanate), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), DCC (Alkicyclohexylcarbodiimide), DCE (1,2-dichloroethane), DCM (dichloromethane), DEAD (diethylazodicarboxylate), DIEA (diisopropylethylamine), DMA /AN-dimethylacetamide), DMAP (4-(N,Ndimethylamino)pyridine), DMF /A^-dimethylformamide), DMSO (dimethyl sulfoxide), EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride). Et (ethyl), EtOAc (ethyl acetate), EtOII (ethanol), I-IATU (O-(7-aza-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), IIBTU (O-benzotriazol-1-yl-AAA',7V-tetramethyluronium hexafluorophosphate), HB' (hydrogen fluoride), HOAc (acetic acid), HOBT (l-hydroxybenzotriazole hydrate), HPLC (high-performance liquid chromatography), IPI (isopropyl alcohol), KHMDS (potassium bis(trimethylsilyl)amide), KN(TMS)2 (potassium bis(trimethylsilyl)amide), KO^{Bu (potassium tert-butoxide), LDA (lithium diisopropylamine), MCPBA (3-chloroperbenzoic acid), Me (methyl), MeCN (acetonitrile), MeOH (methanol), NaBH(OAc)s (sodium triacetoxyborohydride), NaCNBHs (sodium cyanoborohydride), NaH (sodium hydride), NaN(TMS)2 (sodium bis(trimethylsilyl)amide), NaOAc (sodium acetate), NaOEt (sodium ethoxide), Phc (phenylalanine), PPTS (pyridinium p-toluenesulfonate), PS (supported polymer), l^y (pyridine), pyBOP (hexafluorophosphate of (benzotriazol-l-yloxy)tripyrrolidmophosphonium), TEA (triethylamine), TFA (trifluoroacetic acid), TFAA (trifluoroacetic anhydride), THE (tetrahydrofuran), TLC (thin layer chromatography), Tol (toluene), Vai (valine) and the like.

Scheme 1 provides a general procedure that can be used to prepare saturated 5,6-dihydro-1H-pyridin-2-one compounds of Formula 1.

Scheme 1

<td>The RX^theR^g [RJ] R¹²R^,b ' R²R¹¹the¹^RX^R I zX^r7I 0' Or R R³ 1 r¹⁰MR¹¹X R¹² 7 R</td><td>0, .0 X. jr. ox V h jj || B γ— R¹HO'CHT'N H R = is a starting group DCC, DMI- aliphatic or aromatic R^go&gt; -^RJL I^B&gt;^R1R^{2 H}Net₃.dmf_; dcm 60°C %°^H T^sTbL_r1ψνν_N--·· 1 [ H jfiÁ⁰'° r₂</td>

The intermediates of saturated iV-substituted β-amino acid esters, which can be obtained as described by one of the methods in Schemes 3, 4, 6, 7 or 8, can be condensed with a carboxylic acid intermediate (or a salt thereof, for example, sodium salt) using standard peptide coupling conditions used for the formation of amide linkages, such as DCC, to produce the amide shown. This intermediate can be cyclized with or without isolation in the presence of a base (e.g., triethylamine) to give the desired saturated 5,6-dihydro-1H-pyridin-2-one compounds.

Scheme 2 provides a general procedure that can be used to prepare unsaturated 5,6-dihydro-1W-pyridin-2-one compounds of Formula I.

Scheme 2

72/317

<img file="BRPI0809685A2_D0016.tif" />

Net₃DMF, DCM

60°C

<img file="BRPI0809685A2_D0017.tif" />

The intermediates of unsaturated cyclic JV-substituted β-amino acid esters (with R as defined in Scheme 1), which can be obtained as described by one of the methods in Schemes 5 or 9, can be condensed with a carboxylic acid intermediate (or a salt thereof, for example, sodium salt) using standard peptide coupling conditions used for the formation of amide linkages, such as DCC, to produce the amide shown. This intermediate can be cyclized with or without isolation in the presence of a base (e.g., triethylamine) to give the desired unsaturated 5,6-dihydro-1Hpyridin-2-one compounds.

73/317

Scheme 3 provides a general procedure that can be used to prepare intermediates of saturated cyclic β-amino acid/V-substituted esters from saturated anhydrides.

Scheme 3

ROH

Quinine or Quinidine^R,°1 7 R'Ã .

R'² ' _rw^r

CCI₄Toluene - 55°C

R¹⁰ - 'OR

R” &lt;OH

I7¹ -r>6

CICO_?Et. Et₃N, THE. 0 =C

2. NaN₃, THF/HjO, 0-^25 °C

3. Benzene, reflux

4. BnOH, Et₃N, CH₂CI₂reflux_Rw

R^{r Z}

R¹² R»h

OR

Cbz

R^-CHO or

R^C(O)-R-_R’_6r7THE

Pd-C, H₂ 0 ahrv_R,_QR NaCNBH₃. NaOAc_Riu_{

EtOAc, 25 “C^R 12 ^The · NH- Molten sieve 4Â^L ..

^R ,_andR⁹ WleOH, 25°C

Commercially available saturated cyclic meso-anhydrides can be made asymmetric with the aid of enzymes or chiral reagents, such as cinchona alkaloids (e.g., quinine or quinidine) as described in the literature to provide optically active saturated cyclic dicarboxylic acid monoesters (with R as defined in Scheme 1). See J. Org. Chem., 65, 6984-6991 (2000); Synthesis, 11, 1719-1730 (2001), and references cited herein in this document.

These intermediates can be further elaborated into esters of optically active saturated β-amino acids (e.g., Cbz-protected) via a rearrangement reaction, such as the Curtius rearrangement (shown) or the Hofmann degradation. Hydrogenation of protected saturated β-amino acid esters under standard conditions can be used to remove the protecting group 20 and provide optically active saturated β-amino acid esters, which can be isolated (and used) either as free bases or

74/317 and their corresponding salts. Optically active saturated β-amino acid esters (or their salts) can then be treated with aldehydes or ketones, where R^x and R^w are independently C1-C5 alkyl, C3-C8 cycloalkyl, -C1-C5 alkylene(C₃-Cs cycloalkyl), -C1-C5 5 alkylene(aryl), -C1-C5 alkylene(heterocyclyl), aryl, or heterocyclyl, or

R^w can be combined with R^x to form a 3- to 8-membered ring, in the presence of a reducing agent (such as sodium cyanoborohydride) to provide optically active saturated cyclic β-amino acid N-substituted ester intermediates. Alternatively, the reaction sequence described above can be carried out without enzymes or chiral reagents leading to corresponding achiral intermediates and products.

Scheme 4 provides a general procedure that can be used to prepare intermediates of saturated cyclic N15 substituted β-amino acid esters from unsaturated anhydrides.

Scheme 4

ROH

<img file="BRPI0809685A2_D0018.tif" />

z) o

Quinine or Quinidine

<img file="BRPI0809685A2_D0019.tif" />

CCI4, Toluene - 55°C^R V? 1 CICO_?EtEi_sN.THF, 0'C^RU · % 2 THRH₂O, 0—»25 °CR⁹ . · i. ' “ OH

R¹3. Benzene, reflux._{w Ci}.

_R16^R 4. BnOH, Et₃N, CH₂CI₂reflux_Ri_andthe^BH

Pd-C, H₂(1 aim)

EtOAc, 25 °C

R'-CHO or

NaCNBH₃. NaOAc

4A soft sieve

MeOH, 25°C

OR

NH

R²

Commercially available unsaturated cyclic meso-anhydrides can be made asymmetric as described above (Scheme 20 3) to provide optically active unsaturated cyclic dicarboxylic acid monoesters (with R as defined in Scheme 1). These

75/317 intermediates can be further elaborated into optically active protected cyclic β-amino acid esters (e.g., protected Cbz) via a rearrangement reaction, such as the Curtius rearrangement (shown) or a Hofmann degradation. The 5-hydrogenation of optically active protected cyclic β-amino acid esters under standard conditions removes the protecting group and reduces the olefin to provide optically active saturated cyclic β-amino acid esters, which can be isolated (and used) either as free bases or their corresponding salts.

Optically active saturated β-amino acid esters (or their salts) can then be treated with aldehydes or ketones (with R^x and R^w as defined in Scheme 3) in the presence of a reducing agent (such as sodium cyanoborohydride) to provide optically active saturated β-amino acid ester intermediates.15 Alternatively, the reaction sequence described above can be carried out without enzymes or chiral agents leading to the corresponding achiral intermediates and products.

Scheme 5 provides a general procedure that can be used to prepare intermediates of unsaturated cyclic N20 substituted β-amino acid esters from unsaturated anhydrides.

Scheme 5

76/317

ROH

Quinine or Quinidine

R^y

R¹²^R'>⁰

Z^OR

OH

i. CICOjEt. EÇN, THE 0'C

Nak₃THF/H₂O, 0 >25 CR³

CCI₄Toluene - 55°C

3. Benzene, reflux r'-ng_0(;

4. tBuOH, Et₃N, CH₂CI₂, reflux R^l;R°^H

R^X-CHO

<td></td><td colspan="4">air</td>

<td>HCl, dioxane</td><td rowspan="2">R^1Sp₇THE R⁹z R¹² ' _₈NH₂.|ICI r^1bR</td><td>R^X-COLOR' NaCNBH₃NaOAc</td><td>R⁹</td><td>R'⁵_r7THE OR</td>

<td>And₂THE</td><td>4-mol MeOH sieve, 25°C</td><td>R'^?</td><td>_TR^ RR²</td>

Commercially available unsaturated cyclic meso-anhydrides can be made asymmetric as described above (Scheme 4) to provide optically active unsaturated cyclic dicarboxylic acid monoesters (with R as defined in Scheme 1). These intermediates can be further elaborated into optically active protected cyclic β-amino acid esters (e.g., Boc-protected) via a rearrangement reaction, such as the Curtius rearrangement (shown) or a Hofmann degradation. The Boc protecting group can then be selectively removed in the presence of the olefin, thus leading to optically active unsaturated β-amino acid ester intermediates, which can be isolated (and used) either as salts or their corresponding free bases.

Optically active unsaturated β-amino acid esters (or their salts) can then be treated with aldehydes or ketones (with R^x and R^w as defined in Scheme 3) in the presence of a reducing agent (such as sodium cyanoborohydride) to provide optically active cyclic unsaturated β-amino acid substituted ester intermediates. Alternatively, the reaction sequence described above can be carried out without enzymes or chiral reagents leading to the corresponding achiral intermediates and products.

Scheme 6 provides a general alternative procedure that

77/317 can be used to prepare intermediates of saturated cyclic TV-substituted amino acid esters.

Scheme 6

<img file="BRPI0809685A2_D0020.tif" />

1. CSI Et₂0, 25°C

2. In₂ONLY₃ h₂o, o -c

<img file="BRPI0809685A2_D0021.tif" />

aq. HCl

<img file="BRPI0809685A2_D0022.tif" />

SOCI₂

ROH

<img file="BRPI0809685A2_D0023.tif" />

R^X-CHO or

R^X-COLOR^W

NaCNBH_s. NaOAc

4-mol MeOH sieve, 25°C

<img file="BRPI0809685A2_D0024.tif" />

Bicyclic olefins, such as norbornene, can be reacted with chlorosulfonyl isocyanate to produce the β-lactams shown. These intermediates can be hydrolyzed in the presence of a strong acid (such as hydrochloric acid) to give saturated cyclic β-amino acids (or their salts), which can then be further elaborated into their corresponding esters using standard conditions (with R as defined in Scheme 1). Esters of saturated cyclic β-amino acids can then be treated with aldehydes or ketones (with R^x and R^w as defined in Scheme 3) in the presence of a reducing agent, such as sodium cyanoborohydride, to provide the intermediates of saturated cyclic TV-substituted β-amino acid esters.

Scheme 7 provides a general scheme describing a method that can be used to resolve di-exo enantiomers by diastereomeric crystallization.

Scheme 7

78/317

<img file="BRPI0809685A2_D0025.tif" />

R^H = MO, El

NaHCOj

<img file="BRPI0809685A2_D0026.tif" />

rac-di-exo

EtOAc (1S)-10-CSA

<img file="BRPI0809685A2_D0027.tif" />

In₂CO₃

<img file="BRPI0809685A2_D0028.tif" />

R^X-CHO or R^X-COLOR^W

NaCNBH₃NaOAc

4-mol MeOH sieve, 25°C

<img file="BRPI0809685A2_D0029.tif" />

The derivatives of racemic di-exo-β-amino acid esters obtained from norbornene as described above can be resolved by forming diastereomeric salts with an optically pure acid, such as (1S)-(+)-10-camphorsulfonic acid. The ester derivatives of (1/?,2/?,3S,4S'/-β-amino acids) form a crystalline salt with (1S)(+)-10-camphorsulfonic acid that can be selectively isolated by filtration from a suitable solvent (e.g., ethyl acetate) and treated with a base, such as sodium carbonate, to provide the pure enantiomerically free cyclic (11?,27?,3S,4S)-β-amino acid esters. Optically pure (1/?,21?,3S,4S)-P-Gt cyclic amino acid esters (or their salts) can then be treated with aldehydes or ketones (with R^x and R^w as defined in Scheme 3) in the presence of a reducing agent, such as sodium cyanoborohydride, to provide the desired optically pure saturated cyclic 77-substituted β-amino acid ester intermediates.

Scheme 8 provides an alternative procedure that can be used to prepare enantiomerically pure saturated 7V-substituted cyclic β20 amino acid ester intermediates.

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Scheme 8

<img file="BRPI0809685A2_D0030.tif" />

rac-di-βχΌ (IS)-ÍO-CSA

EtOAc, EIO1I

50-75 °C

<img file="BRPI0809685A2_D0031.tif" />

K₂CO₃, EtOAc then R^X-CHO or R^X-COLOR^W

R^X-COLOR^W

NaCNBH₃, AcOH

EtOH, 25 O

<img file="BRPI0809685A2_D0032.tif" />

A β-lactam (prepared as described in Scheme 6) can be opened and resolved by forming diastereomeric salts with an optically pure acid, such as (15)-(+)-10-camphorsulfonic acid (as described in Scheme 7) in the presence of an alcohol (e.g., ethanol) to directly provide the diastereomerically pure (1R,2R,3S,4^^-amino acid) ester as a salt with (15)-(+)-10-camphorsulfonic acid. Treatment with a base, such as potassium carbonate, followed by reductive alkylation with aldehydes or ketones (with R^x and R^w as defined in Scheme 3) in the presence of a reducing agent, such as sodium cyanoborohydride, provides the desired enantiomerically pure saturated cyclic α-substituted aznino acid ester intermediates.

Scheme 9 provides a general procedure that can be used to prepare intermediates of saturated cyclic N-substituted β-amino acid esters.

<img file="BRPI0809685A2_D0033.tif" />

Scheme 9

R^X-CHO or

R*-C(O)-R”'

NaCNBH₃NaOAc

4-mol MeOH sieve, 25°C

<img file="BRPI0809685A2_D0034.tif" />

Esters of saturated cyclic β-amino acids (or their

80/317 salts, with R as defined in Scheme 1) can be purchased, prepared from the corresponding commercially available saturated β-amino acids, or can be prepared by methods described in Schemes 3, 4, 6, or 7. The esters of saturated β-amino acids can then be treated with aldehydes or ketones (with R^x and R^w as defined in Scheme 3) in the presence of a reducing agent, such as sodium cyanoborohydride, to provide the desired saturated cyclic TV-substituted β-amino acid ester intermediates. In each case, the desired saturated cyclic β-amino acid esters or saturated cyclic IV-substituted β-amino acid ester intermediates may be optically active.

Scheme 10 provides an alternative general procedure that can be used to prepare intermediates of saturated cyclic TV-substituted β-amino acid esters.

Scheme 10

<img file="BRPI0809685A2_D0035.tif" />

Esters of saturated β-cyclic amino acids (or their salts, with R as defined in Scheme 1) can be purchased, prepared from the corresponding commercially available saturated β-cyclic amino acids, or prepared by methods described in Schemes 3, 4, 6, or 7. Esters of saturated β-cyclic amino acids can then be treated with halides or pseudohalides X.^the (for example, bromides, iodides or triflates), where R^v It is an aryl or heterocycline, in the presence of a metallic catalyst such as copper (e.g., under Ullmann reaction conditions) or palladium (e.g., under Buchwald-Hartwig reaction conditions), for

81/317

<img file="BRPI0809685A2_D0036.tif" />

to provide intermediates of saturated cyclic β-amino acid substituted TV esters. In each case, the desired saturated cyclic β-amino acid esters or intermediates of saturated cyclic β-amino acid substituted 7V esters may be optically active.

Scheme 11 provides a general procedure that can be used to prepare intermediates of unsaturated cyclic N-substituted β-amino acid esters.

Scheme 11

R*-CHO Of R¹⁵₇OR*-C(O>R^w

NaCNBHv NaOAc ||^ OK

R^&lt;2 Molten sieve 4A p16R^k MeOH, 25°C

Esters of unsaturated cyclic β-amino acids (or their salts, with R as defined in Scheme 1) can be purchased, prepared from the corresponding commercially available unsaturated cyclic β-amino acids, or prepared by methods described in Scheme 5. Esters of unsaturated cyclic β-amino acids can then be treated with aldehydes or ketones (with R^x and R^was defined in Scheme 3) in the presence of a reducing agent, such as sodium cyanoborohydride, to provide the desired unsaturated cyclic N-substituted β-amino acid ester intermediates. In each case, the desired unsaturated cyclic β-amino acid esters or unsaturated cyclic IV-substituted β-amino acid ester intermediates may be optically active.

Scheme 12 provides an alternative general procedure that can be used to prepare intermediates of unsaturated cyclic IV-substituted β-amino acid esters.

82/317

Scheme 12

<img file="BRPI0809685A2_D0037.tif" />

R^v-X^the

<img file="BRPI0809685A2_D0038.tif" />

Esters of unsaturated cyclic β-amino acids (or their salts, with R as defined in Scheme 1) can be purchased, prepared from the corresponding commercially available unsaturated cyclic β-amino acids, or prepared by methods described in Scheme 5. Esters of unsaturated cyclic β-amino acids can then be treated with halides or pseudohalides X.^the (for example, bromides, iodides or triflates), where R^v It is aryl or heteroethyl, in the presence of a metallic catalyst such as copper (for example, under Ullmann reaction conditions), to provide the intermediates of unsaturated cyclic TV-substituted β-amino acid esters. In each case, the desired unsaturated cyclic JV15-substituted β-amino acid esters or intermediates of unsaturated cyclic JV15-substituted β-amino acid esters can be optically active.

Scheme 13 provides a general procedure that can be used to prepare 7-substituted-1,1-dioxo-1,4-dihydro-1-acid intermediates.⁶-benzo[1,4]t.iazin-3-yl-acetic.

Scheme 13

83/317

<img file="BRPI0809685A2_D0039.tif" />

NH2-NH2OH2O

EtOH. Λ

<img file="BRPI0809685A2_D0040.tif" />

Sn(ll)CI

EtOH, Λ

R = NO?

R = NH?

<td>MeSO^CI</td><td>s., . .</td><td>R N_the</td><td>MCPBA</td><td>0 0 0 I^S-</td><td>R^N s</td>

<td></td><td>Kl</td><td>' OO</td><td> -----------------------</td><td></td><td> 0 0</td>

<td></td><td>IN</td><td></td><td>CH₂CI₂</td><td>EtO ' N</td><td></td>

<td>EtO ' C</td><td></td><td></td><td></td><td>R</td><td></td>

<td></td><td></td><td>R - H</td><td></td><td></td><td>R - BOC</td>

<td colspan="2">(BOC)jO, dmap</td><td></td><td></td><td>TF/VCH₂Ci?</td><td></td>

<td>CH?Cl</td><td> 2 —</td><td>R = BOC</td><td></td><td>i. 1</td><td>- R = H</td>

aq. UOH

MeOH

0

<img file="BRPI0809685A2_D0041.tif" />

N

H

Commercially available 6-Nitrobenzothiazole can be treated with hydrazine to obtain 2-amino-5-nitrobenzenethiol, which can subsequently be reacted with chloroacetoacetate to give the ethyl ester of (7-nitro-4H-benzo[1,4]thiazin-3-yl)acetic acid. The reduction of the nitro group to the amino group can be effected by reaction with tin(II) chloride. The subsequent reaction with sulfonyl chloride, such as methylsulfonyl chloride, can be used to obtain the corresponding sulfonamides. Protection of both nitrogens with an appropriate protecting group such as a Boc group can be achieved by using standard amino group protection methods. The sulfides can be oxidized using an appropriate oxidizing reagent (e.g., MCPBA) to give the sulfones. Finally, deprotection of the amino groups using trifluoroacetic acid, followed by hydrolysis of the esters, can be used to provide the desired acid intermediates.

84/317

Scheme 14 provides a general procedure that can be used to prepare 7-substituted-l,l-dioxo-1,4-dihydro-1-acid intermediates.⁶-benzo[1,2,4]thiadiazin-3-yl-acetic acid.

Scheme 14 oo

THE

Cl

<td>N₂</td><td>1.) SOCl₂DMF reflux, 4 h</td><td>0 0 h₂n^the</td>

<td></td><td>2 INHAOH. THF</td><td>Cl</td>

O o

Pd-C, Hj(50psi) S

MeQIVTHF h_?N

NO. (^NH4^O₃. GuSOa

Cone, aq NH,₍OH

118 ^S'W

OOS

H_ZN

H₂N

O.1 RO

THE

Cl

THF, or

DMA-EljO (1.Ί) 0 - >25 ”C

R Mo. Et

THE

HN

THE

RO O

MeSOjCI

O. O

H_ZN-pyridine, DCM

Η OO s _ _ 3.8 wt% aq NaOH. ONOO ----- . . _ _ '

HO NH

H^Ns

O o

Commercially available 2-chloro-5-nitrobenzenesulfonic acid can be treated with thionyl chloride to give sulfonyl chloride, which can be further treated with ammonia to provide the sulfonamide intermediate. The chloride can be displaced with ammonia by treatment with ammonium hydroxide and ammonium carbonate in the presence of copper(II) sulfate. Reduction of the nitro group under standard hydrogenation conditions yields the aniline intermediate, which can be treated with sulfonyl chloride, such as methylsulfonyl chloride, to produce the corresponding sulfonamide. Acylation of the 215 amino segment with malonyl chlorides, for example, ethyl 3-chloro-3-oxopropionate, gives the corresponding amide, which can simultaneously be cyclized to thiadiazine dioxide and hydrolyzed to the desired acid intermediate.

Scheme 15 provides an alternative procedure that

85/317 can be used to prepare the intermediate 2-amino-5-nitrobenzenesulfonamide.

Scheme 15

<img file="BRPI0809685A2_D0042.tif" />

i.)poci₃Q

120 °C^Lo

2.) NH₃ (g) or NH₄OH

<img file="BRPI0809685A2_D0043.tif" />

Commercially available 2-amino-5-nitrobenzenesulfonic acid can be converted to the corresponding sulfbnyl chloride with phosphoryl chloride in the presence of a suitable co-solvent, such as sulfolan. Treatment with ammonia, for example, aqueous ammonium hydroxide solution or ammonia gas, provides the desired 210-amino-5-nitrobenzenesulfonamide intermediate.

Scheme 16 provides an alternative procedure that can be used to prepare the intermediate 2-amino-5-methanesulfonylaminobenzenesulfonamide.

Scheme 16

86/317

<img file="BRPI0809685A2_D0044.tif" />

NE1_SACN, 92 °C

<img file="BRPI0809685A2_D0045.tif" />

<img file="BRPI0809685A2_D0046.tif" />

Pd-C, H_z (50 psi)

MeSO₃H

THF. EtOH. HjO

OO Pc!-C, H₂ (1 atm) O .0

N^S h^N°² H₂N^S ii 7*

H_SN EtOH, H;O, 55^UCH₂N

NH_£ • MgSO₃H!

I

MeS0₂CI pyridine, ACN

I o .o

H_?N &lt;

H?N

The intermediate 2-chloro-5-nitrobenzenesulfonamide (prepared as described in Scheme 14) can be treated with a benzyl amine, such as benzylamine, to displace the chlorine segment. 5-Hydrogenation under standard conditions in the presence of an acid (e.g., methanesulfonic acid) can be used to remove the benzyl group and to reduce the nitro group at the same time, allowing the intermediate 2,5-diaminobenzenesulfonamide to be formed as a salt. Alternatively, the 2,5-diaminobenzenesulfonamide salt can be prepared by reducing 2-amino-5-nitrobenzenesulfonamide (prepared as described in Schemes 14 and 15) under standard hydrogenation conditions in the presence of an acid (e.g., methanesulfonic acid). The subsequent reaction with methanesulfonyl chloride provides the desired 2-amino-5-methanesulfonylaminobenzenesulfonamide intermediate.

Scheme 17 provides a procedure that was used.

87/317 to prepare the acid intermediate (7-iodo-1,1-dioxo-1,4-dihydro-1λ⁶benzo[1,2,4]thiadiazin-3-yl)-acetic acid.

Scheme 17 oos H_ZN h₂n

THE

RO X

R = Mb, Et

X = OR. F. cr Cl

OO

HjN 7

HN '' “ O

8% aq NaOH 100 °C

RO O

Commercially available 2-Aminobenzenesulfonamide can be treated with TV-iodosuccinimide (NIS) to provide 2-amino5-iodobenzenesulfonamide. Acylation with a malonyl halide monoester, such as ethyl 3-chloro-3-oxopropionate, or with a dialkyl malonate, such as diethyl malonate, provides the corresponding amide 10, which can be simultaneously cyclized to thiadiazine dioxide and hydrolyzed to the desired acid intermediate (or a salt thereof, for example, sodium salt).

Scheme 18 provides a procedure that was used to prepare the acid intermediate (l,l-dioxo-l,4-dihydro-lA⁶-benzo[1,2,4] 15 thiadiazin-3-yl)-acetic.

Scheme 18

88/317

O, ,,O h₂n''

R = Me, Et

X = OR, F, or Cl

0 is:

h₂n· jHN^ ' kf^THE

RO'' 0

8% aqueous NaOH

100 °C

THE

HO' '

<img file="BRPI0809685A2_D0047.tif" />

Commercially available 2-Aminobenzenesulfonamide can be acylated with a malonyl halide monoester, such as ethyl 3-chloro-3-oxopropionate, or with a dialkyl malonate, such as diethyl malonate, to yield the corresponding amide, which can be simultaneously cyclized to thiadiazine dioxide and hydrolyzed to the desired acid intermediate (or a salt thereof, for example, sodium salt).

Scheme 19 provides a procedure that was used to prepare the amide intermediate of 3-aminopyridine-4-sulfonic acid.

Scheme 19

<img file="BRPI0809685A2_D0048.tif" />

NaSH

MeOH 60 °C

<img file="BRPI0809685A2_D0049.tif" />

chloramine

I NaOH

NeOH

<img file="BRPI0809685A2_D0050.tif" />

MCPBA

DCM

<img file="BRPI0809685A2_D0051.tif" />

SnCI₂ cone. HCI (aq.) 0°C to 25°C

<img file="BRPI0809685A2_D0052.tif" />

The amide intermediate of 3-aminopyridine-415 sulfonic acid can be prepared following the procedures described in Tetrahedron 1998, 54, 13645-13654 (steps 2-4). Commercially available 4-chloro-3-nitropyridine can be treated with hydrate of

89/317 sodium sulfide to displace the chlorine segment. Treatment with chloramine followed by oxidation of the sulfur with an oxidizing agent, such as MCPBA, provides the nitro-sulfonamide intermediate. Reduction of the nitro group using standard conditions (e.g., tin(II) chloride) provides the desired 3-aminopyridine-4-sulfonic acid amide intermediate.

Scheme 20 provides a procedure that was used to prepare the amide intermediate of 4-aminopyridine-3-sulfonic acid.

Scheme 20

i) clause₃h Qa,zp i)pci₅, poci₃ q^o^{145 W}’^{C 130} °^W_____

P II j I j^h2ⁿ 2} SOCI₂ 2)NH₄OH

120 °C 10

Commercially available pyridin-4-ylamine can be treated with chlorosulfonic acid in the presence of thionyl chloride to give the sulfonyl chloride intermediate. Treatment with aqueous ammonium solution gives the desired amide intermediate of 15 4-amino-pyridino-3-sulfonic acid.

Scheme 21 provides a procedure that was used to prepare the amide intermediate of 3-aminopyridine-2-sulfonic acid.

Scheme 21

90/317

<img file="BRPI0809685A2_D0053.tif" />

BnSH K₂CO₃

EtOH, H₂THE

<img file="BRPI0809685A2_D0054.tif" />

<img file="BRPI0809685A2_D0055.tif" />

AcOH, H₂O, DCM

0°C to 25°C

<img file="BRPI0809685A2_D0056.tif" />

nh₄oh

DCM

<img file="BRPI0809685A2_D0057.tif" />

Fe, NH|Cl (aq.)

EtOH, 105 °C

<img file="BRPI0809685A2_D0058.tif" />

Commercially available 2-Chloro-3-nitropyridine can be treated with benzyl mercaptan in the presence of a base (e.g., potassium carbonate) to provide the sulfide intermediate.

Oxidation with 1,3-dichloro-5,5-dimethylhydantoin yields sulfonyl chloride, which can be treated with aqueous ammonium hydroxide solution to produce the sulfonamide intermediate. Reduction of the nitro group using standard hydrogenation conditions yields the desired amide intermediate of 3-aminopyridine-2-sulfonic acid.

Scheme 22 provides a procedure that was used to prepare the amide intermediate of 4-aminopyridine-3-sulfonic acid.

Scheme 22

<img file="BRPI0809685A2_D0059.tif" />

NaN₃

DMF, 90 °C

<img file="BRPI0809685A2_D0060.tif" />

NaBH₄

MeOH

<img file="BRPI0809685A2_D0061.tif" />

Commercially available 4-chloropyridine-3-sulfonic acid amide can be treated with sodium azide to provide the azide intermediate, which can be reduced with a reducing agent (e.g., sodium borohydride) to allow the desired 4-aminopyridine-3-sulfonic acid amide intermediate.

91/317

Scheme 23 provides a procedure that was used to prepare the acid intermediates derived from the amide intermediates of aminopyridine sulfonic acid.

Scheme 23

<td>0 o</td><td>0 L</td><td> 0</td><td>0 OS</td><td>CO</td>

<td>s q</td><td>RO</td><td>X</td><td>HoN V 1! b</td><td>8%&lt;iq NaOH 0 N^S</td>

<td>H_ZN</td><td>R^k Ms X - OR</td><td>El F, or Cl</td><td>HN 0</td><td>100 °C HO N H</td>

RO O

The aminopyridine sulfonic acid amide intermediates described in Schemes 19-21 can be acylated with a malonyl halide monoester, such as ethyl 3-chloro-3-oxo-propionate, or with a dialkyl malonate, such as diethyl malonate, to allow the corresponding amide, which can be simultaneously cyclized to thiadiazine dioxide and hydrolyzed to the desired acid intermediate (or a salt thereof, for example, sodium salt).

Scheme 24 provides a general procedure that was used to prepare the 5,6-dihydro-1H-pyridin-2-one compounds of Formula I 15 from the corresponding iodine precursors.

Scheme 24

R⁴

<img file="BRPI0809685A2_D0062.tif" />

Cul, Sarcosine K₃DUST₄

DMF, 100 °C

<img file="BRPI0809685A2_D0063.tif" />

Optionally substituted 5,6-Dihydro-1H-pyridin-2-ones

92/317 can be treated with substituted sulfonamides in a copper-mediated displacement reaction to allow the desired 5,6-dihydro-1H-pyridin-2-one compounds of Formula I.

Scheme 25 provides a general procedure that was used to prepare the 5,6-dihydro-1H-pyridin-2-one compounds of Formula I having a sulfonamide segment from the corresponding iodine precursors.

Scheme 25

<img file="BRPI0809685A2_D0064.tif" />

N3N3,

<img file="BRPI0809685A2_D0065.tif" />

DMSO-H₇THE

Cul, Na-ascorbato

<img file="BRPI0809685A2_D0066.tif" />

H₂Pd-C

MeOH-ElOAc

<img file="BRPI0809685A2_D0067.tif" />

SUN

<img file="BRPI0809685A2_D0068.tif" />

H

Optionally substituted 5,6-Dihydro-1H-pyridin-2-ones can be treated with sodium azide to displace the iodine segment. Reduction of the azide intermediate using standard conditions, such as catalytic hydrogenation, yields the aniline intermediate. Further reaction with an optionally substituted sulfamoyl chloride in the presence of a base (e.g., triethylamine) yields the desired 5,6-dihydro-1H-pyridin-2-one compounds of Formula I.

Scheme 26 provides a general procedure that was used to prepare the TV-substituted 5,6-dihydro-1H-pyridin-2-one compounds of Formula I.

Scheme 26

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<img file="BRPI0809685A2_D0069.tif" />

k₂co₃

DMF

<img file="BRPI0809685A2_D0070.tif" />

The sulfonamide segment of optionally substituted 5,6-dihydro-1H-pyridin-2-ones can be α-alkylated by treatment with an alkylating agent, such as alkyl halides or X-hybrid pseudohalides.^the (e.g., chlorides, bromides, iodides, mesylates, tosylates, triflates, or chloroformates), in the presence of a base (e.g., potassium carbonate) to provide the desired A-substituted 5,6-dihydro-1Hpyridin-2-one compounds of Formula I.

Scheme 27 provides a procedure that was used to prepare the 5,6-dihydro-1H-pyridin-2-one compounds of Formula I from the corresponding iodine precursors.

Scheme 27

<img file="BRPI0809685A2_D0071.tif" />

<img file="BRPI0809685A2_D0072.tif" />

H_2s Pd-C

DMF

<img file="BRPI0809685A2_D0073.tif" />

Optionally substituted 5,6-Dihydro-1H-pyridin-2-ones can be treated with stananes, such as the unsaturated cyclic sulfone shown above, in a Stille-type palladium-catalyzed reaction to yield the unsaturated intermediates shown. The reduction of

94/317 alkene using standard hydrogenation conditions yields the desired 5,6-dihydro-1H-pyridin-2-one compounds of Formula I.

Scheme 28 provides a general procedure that was used to prepare the 5,6-dihydro-1H-pyridin-2-one compounds of Formula 1 from the corresponding iodine.

Scheme 28

<img file="BRPI0809685A2_D0074.tif" />

H

Cul, L-Proline

K3PO4 -----------------------------------------------------------J

DMSO. 90 °C

<img file="BRPI0809685A2_D0075.tif" />

Optionally substituted 5,6-Dihydro-1H-pyridin-2-ones can be treated with amines in a copper-mediated displacement reaction to provide the desired 5,6-dihydro-1//-pyridin2-one compounds of Formula I.

Example 1 (rac-di-exo)-7V-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.I.0²-⁷]undec-5-cn-5-yl-l, 1-dioxo-l,4-dihydro-lÀ⁶-benzo[1,2,4] thiadiazin -7-yl} -methanesulfonamide

<img file="BRPI0809685A2_D0076.tif" />

a) 2-Chloro-5-nitrobenzenesulfonamide

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<img file="BRPI0809685A2_D0077.tif" />

To a solution of thionyl chloride (11 mL) and 2-chloro-5-nitrobenzenesulfonic acid (4.78 g, 20.1 mmol) was added N,N-dimethylformamide (0.92 g/L) and the reaction mixture was heated under reflux for 4 h. The reaction mixture was then carefully stopped by pouring it into water and the product was isolated by vacuum filtration. Sulfonyl chloride was dissolved in a minimal amount of toluene and then added to a mixture of concentrated aqueous ammonium hydroxide solution (25 mL) and tetrahydrofuran (25 mL) at -10 °C. After stirring for 2 h, the reaction was stopped by adding 6.0 M aqueous hydrochloric acid solution until pH 4 was reached. The layers were separated and the organic layer was concentrated under vacuum until it formed sludge. Pentane was added and the product was isolated by vacuum filtration to give 2-chloro-5-nitrobenzenesulfonamide (2.0 g, 8.48 mmol, 42.4%) as a solid. Ή NMR (400 MHz, DMSO-d₆) δ: 7.94 (d, 1H, J = 8.8 Hz), 7.97 (bs, 2H), 8.40 (dd, III, J, = 8.6 Hz, J₂ = 3.1 Hz), 8.64 (d, 1H, J = 3.1 Hz).

the. 2-Amino-5-nitrobenzenesulfonamide

<img file="BRPI0809685A2_D0078.tif" />

2-Chloro-5-nitrobenzenesulfonamide (1.95 kg, 8.30 mol), ammonium carbonate (1.983 kg, 20.64 mol), and copper(II) sulfate (394 g, 2.47 mol) were loaded into an autoclave and diluted with a 30% aqueous ammonium hydroxide solution (11.7 L, 330 mol).

The 96/317 mixture was heated to 118 °C for 3 days and then cooled to 23 °C. The mixture was filtered and the solids were then washed with water (20 L). This solid was dissolved in heated methanol (20 mL/g), and the mixture was filtered to remove undissolved solids. The filtrate was stored at 4 °C overnight, and the resulting solid product was then filtered. The filtrate was partially concentrated by vacuum distillation and, when the concentrate was cooled to 23 °C, the solid product was then filtered. The two solid results were combined and further dried under vacuum at 45 °C to give the desired product, 2-amino-5-nitrobenzenesulfonamide (1.10 kg, 5.06 mol, 61%), as a solid. UI NMR (400 MHz, DMSO-d₆/ δ-6.89 (d, J = 9.3 Hz, 1H), 7.12 (bs, 2H), 7.57 (bs, 2H), 8.07 (dd, Ji = 9.0 Hz, J₂ = 2.6 Hz, 1H), 8.43 (d, J = 3.0 Hz, 1H).

Alternatively, 2-amino-5-nitrobenzenesulfonamide can be prepared as follows:

2-Amino-5-nitrobenzenesulfonic acid (200.00 g, 0.917 mol) was suspended in heated sulfolan (250 mL) and the suspension was heated to 80 °C. Phosphorus oxychloride (126 mL, 1.375 mol) was added and the resulting mixture was heated to 110-120 °C and stirred for 4 h. The resulting solution was cooled to 60 °C and added dropwise to a concentrated aqueous solution of ammonium hydroxide (800 mL, 11.9 mol) at &lt;10 °C. The flask was rinsed with heated sulfolan (50 mL) and the wash was added to the reaction mixture above. The resulting suspension was stirred at 25°C for 1 h, heated to 95°C and stirred for 1 hour. The mixture was cooled to 80°C and the pH was adjusted to 6-8 with 3.0 M aqueous hydrochloric acid solution (-600 mL) and allowed to cool to 25°C. The dark green suspension was filtered, the wet filter cake was washed with water (300 mL) and dried at 60°C overnight to give the crude product (140 g) as a yellow-green solid. The crude product was dissolved in a 0.5 M aqueous solution of hydroxide.

97/317 sodium (1.4 L, 0.7 mol). Charcoal (14 g) was added and the mixture was heated under reflux and stirred for 15 min. The mixture was filtered through Celite and washed with 0.5 M aqueous sodium hydroxide solution (100 mL). The pH of the filtrate was adjusted to 6-8 with concentrated aqueous hydrochloric acid solution (-60 mL) and the yellow suspension was allowed to cool to 25°C. The mixture was filtered and the wet filter cake was washed with water (200 mL) and dried at 60°C overnight to give the desired product, 2-amino-5-nitrobenzenesulfonamide (130 g, 0.599 mol, 65%) as a bright yellow powder.

b) 2,5-Diaminobenzenesulfonamide

<img file="BRPI0809685A2_D0079.tif" />

2-Amino-5-nitrobenzenesulfonamide (5.00 kg, 23.0 mol), methanol (65 L), tetrahydrofuran (65 L), and 10% palladium on carbon (250 g) were loaded into an autoclave. The mixture was cyclized with nitrogen and hydrogen purges (3 times), and the mixture was then stirred under hydrogen (50 psi) at 23°C overnight. The catalyst was removed by filtration, and the filtrate was then concentrated under vacuum to give a brown solid. The solid was further dried under vacuum at 45°C to give the desired product, 2,5-diaminobenzenesulfonamide (4.21 kg, 22.4 mol, 98%), as a solid. NMR (400 MHz, DMSO-d/λ): 4.54 (2H, bs), 4.98 (2H, bs), 6.55 - 6.60 (2H, m), 6.87 (1H, d, J = 2.2 Hz), 6.99 (2H, bs). LC-MS (ESI) calculated for C6H9N3O2S 187.04, found 188.3 [M+H⁺].

d) 2-Amino-5-methanesulfonylamino-benzenesulfonamide

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<img file="BRPI0809685A2_D0080.tif" />

H

<img file="BRPI0809685A2_D0081.tif" />

2,5-Diaminobenzenesulfonamide (4.20 kg, 22.4 mol) was dissolved in dichloromethane (120 L) and pyridine (8.00 kg, 89.9 mol), and the resulting solution was cooled to 0°C. Methanesulfonyl chloride (2.80 kg, 24.4 mol) was added slowly, and the resulting mixture was allowed to heat to 23°C and stirred for 2 days. The mixture was filtered, and the resulting solid was washed with dichloromethane (2 x 20 L). The solid was diluted with water (100 L) and 1.0 M aqueous hydrochloric acid solution (25 L), and then stirred at 23°C for 1 h. The mixture was filtered and the resulting solid was washed with water (20 L) and then with methyl tert-butyl ether (2 x 10 L). The solid was further dried under vacuum at 45°C to give the desired product, 2-amino-5-methanesulfonylaminobenzenesulfonamide (4.39 kg, 16.5 mol, 73%) as a pale pink solid, ill NMR (400 MHz, CD₃OD) 6=2.89 (3H, s), 6.82 (III, d, J = 8.5 Hz), 7.20 (III, dd, Ji = 8.5Hz, J₂ = 2.5IIz), 7.58 (III, d, J = 2.5 Hz). LC-MS (ESI) calcd for C7H11N3O4S2 265.02, found 266.0 [M+H⁺],

Alternatively, 2-amino-5-methanesulfonylaminobenzenesulfonamide can be prepared as follows:

aj - Benzylamino-5-nitro-benzenesulfonamide

<img file="BRPI0809685A2_D0082.tif" />

H

A mixture of 2-chloro-5-nitro-benzenesulfonamide (2.20 kg, 9.30 mol), benzylamine (1.5 L, 13.9 mol), triethylamine (2.5 L, 18.1

A mixture of 99/317 mol and acetonitrile (22.0 L) was heated to 92°C for 20 h. The mixture was then cooled to 40°C and partially concentrated under vacuum. The residue was added to water at 0°C (22.0 L) and the resulting suspension was heated to 23°C and stirred for 2 h. The suspension was filtered and the solid was washed with water (5 L). The washed solid was suspended in absolute ethanol (11 L) and then filtered and washed with absolute ethanol (5 L). The solid was further dried under vacuum at 45°C to give the desired product, 2-benzylamino-5-nitrobenzenesulfonamide (2.40 kg, 7.81 mol, 84%), as a yellow solid. Ή NMR (400 MHz, DMSO-dg) LA 4.64 (2H, d, J = 4.6 Hz), 6.81 (1H, d, J = 9.4 Hz), 7.23 -7.44 (6H, m), 7.77 (2H, bs), 8.11 (1H, dd, J = 9.4 Hz, J₂ = 2.3 Hz), 8.49 (1H, d, J = 3.1 Hz). LC-MS (ESI) calcd for C13H13N3O4S 307.06, found 308.2 [M+H⁺] (100%), 615.2 [2M+IP] (81%).

bj 2,5 - Diaminobenzenesulfonamide methanesulfonate

<img file="BRPI0809685A2_D0083.tif" />

Methanesulfonic acid (465 mL, 7.16 mol) was slowly added to a solution of 2-benzylamino-5-nitrobenzenesulfonamide (2.20 kg, 7.16 mol) and tetrahydrofuran (11.0 L). The resulting solution was added to a mixture of 10% palladium in carbon (220 g of 50% water-wettable catalyst) and water (1.1 L) in a hydrogenation reactor. The mixture was further diluted with absolute ethanol (21.0 L) and then hydrogenated with hydrogen at 55 psi at 50 °C for 21 h. An additional 10% palladium in carbon (55 g of 50% water-wettable catalyst) was added, and hydrogenation at 55 psi and 50 °C was continued for 22 h. The resulting suspension was diluted with water (1.1 L) and then filtered through a Celite pad.

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The filtrate was partially concentrated under vacuum and then diluted with acetonitrile (15.4 L). The solution was again partially concentrated under vacuum and diluted with acetonitrile (15.4 L). The resulting suspension was partially concentrated under vacuum and stirred at 23 °C for 2 h. The suspension was filtered and the solid was then washed with acetonitrile (3 L). The solid was further dried under vacuum at 45 °C to give the desired product, 2,5-diaminobenzenesulfonamide methanesulfonate (1.88 kg, 6.64 mol, 93%), as a purple solid. HI NMR (400 MHz, DMSO-de) 2.34 (311, s), 6.05 (2H, b), 6.87 (III, d, J = 8.6 Hz), 7.20 (III, dd, Ji = 8.6 Hz, J₂ = 2.3 Hz), 7.38 (2H, s), 7.53 (1H, d, J = 2.3 Hz), 9.62 (3H, b). LC-MS (ESI) calcd for C6H9NO2S 187.04, found 187.9 [M+H⁺],

Alternatively, 2,5-diaminobenzenesulfonamide methanesulfonate can be prepared as follows:

2-Amino-5-nitrobenzenesulfonamide (prepared as described in Example lb, 100.00 g, 0.460 mol) and palladium in 5% carbon (wet, 5.00 g) were suspended in ethanol (2 L) and water (100 mL). Methanesulfonic acid (33 mL, 0.51 mol) was added, and the resulting mixture was heated to 55 °C and stirred under atmospheric hydrogen for 8 h. The mixture was filtered, and the filtrate was concentrated under vacuum to a volume of approximately 450 mL. Acetonitrile (1 L) was added to the concentrate and the resulting mixture was stirred at 25 °C overnight. The suspension was filtered to give the desired product, 2,5-diaminobenzenesulfonamide methanesulfonate (122.36 g, 0.432 mol, 93.8%) as a purple solid.

cj 2-Amino-5-methanesulfonylamino-benzenesulfonamide

<img file="BRPI0809685A2_D0084.tif" />

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2,5-Diaminobenzenesulfonamide methanesulfonate (1.80 kg, 6.35 mol) was suspended in acetonitrile (24 L). Pyridine (1.55 L, 19.1 mol) was added, followed by a slow and careful addition of methanesulfonyl chloride (517 mL, 6.68 mol). After stirring at 23°C for 20 h, the mixture was partially concentrated under vacuum at 55°C. Water (18 L) was added to the concentrate, and the resulting suspension was stirred at 23°C for 2 h. The solid was filtered and then washed with water (4 L) and air-dried on the filter. The solid was suspended in absolute ethanol (9 L), stirred at 23°C for 9 h, and then filtered. The solid was washed with absolute ethanol (2 x 2 L) and then further dried under vacuum at 50°C to give the desired product, 2-amino-5-methanesulfonylaminobenzenesulfonamide (1.45 kg, 5.48 mol, 86%), as a purple solid.

e) Ethyl ester of 7V-(4-methanesulfonylamino-2-sulfamoylphenyl)-malonamic acid

<img file="BRPI0809685A2_D0085.tif" />

2-Amino-5-methanesulfonylaminobenzenesulfonamide (23.27 g, 87.81 mmol) was dissolved in /V,/V-dimethylacetamide (100 mL) and diethyl ether (100 mL). Ethyl 3-chloro-3-oxopropionate (13.88 g, 92.20 mmol) was added and the reaction mixture was stirred at 25°C for 1 h. The reaction mixture was diluted with ethyl acetate (400 mL) and extracted with water (400 mL). The aqueous layer was then extracted back with ethyl acetate (2 x 200 mL). The combined organic layers were dried over sodium sulfate, filtered, and most of the solvent was removed under vacuum to a volume of -100 mL.

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Hexanes (-100 mL) were added to the stirred solution, resulting in the formation of a precipitate. The precipitate was collected by vacuum filtration, washed with hexanes, and dried under high vacuum to provide the analytically pure product, N-(4-methanesulfonylamino-2-sulfamoylphenyl)-malonamic acid ethyl ester (31.22 g, 85.53 mmol, 97.4%), as a light brown solid.^THEH NMR (400 MHz, CD₃OD) □: 1.31 (3H, t, J = 7.0 Hz), 3.00 (3H, s), 3.59 (2H, s), 4.25 (2H, quartet, J = 6.9 Hz), 7.42 - 7.45 (1H, m), 7.86 (1H, m), 7.92 (1H, d, J = 8.8 Hz).

f) 7V-(4-Methanesulfonylamino-2sulfamoyl-phenyl)-malonamic acid methyl ester

<img file="BRPI0809685A2_D0086.tif" />

2-Amino-5-methanesulfonylaminobenzenesulfonamide (prepared as described in Example Id, 1.70 kg, 6.40 mol) was dissolved in tetrahydrofuran (35 L) and then cooled to 0°C. Methyl 3-chloro-3-oxopropionate (792 mL, 7.40 mol) was added slowly, and the resulting mixture was then heated to 23°C and stirred for 2 days. The solvent was removed under vacuum, and the residue was then diluted with water (4 L) and saturated aqueous sodium bicarbonate solution (2 L).

The resulting solid was filtered and then washed with water (5 L). The solid was suspended in hot methanol (15 mL/g) and then cooled to 23°C and filtered to give the desired product, 7V-(4-methanesulfonylamino-2-sulfamoylphenyl) methyl ester.

103/317 malonamic (1.68 kg, 4.61 mol, 72%), as a brown solid. HI NMR (400 MHz, DMSO-do) □: 3.02 (3H, s), 3.60 (2H, s), 3.66 (3H, s), 7.38 (1H, dd, Ji = 2.3 Hz, J₂ = 8.6 Hz), 7.53 (2H, bs), 7.73 (1H, d, J= 2.4 Hz), 7.83 (1H, d, 8.7 Hz), 9.43 (1H, s), 9.99 (1H, s).

g) Acid (7-methanesulfonylammo-1,1-dioxo-1,4-dihydrolÀ⁶-benzo[1,2,4] thiadiazin-3-yl)-acetic acid

<img file="BRPI0809685A2_D0087.tif" />

Ethyl ester of 7V-/4-methanesulfonylamino-2sulfamoylphenyl)-malonamic acid (prepared as described in Example 1e, 9.55 g, 26.16 mmol) was dissolved in an 8% aqueous sodium hydroxide solution (262 mL) and heated to 100°C for 1.5 h. The reaction mixture was cooled to 0°C and the solution was slowly acidified by adding 12.0 M aqueous hydrochloric acid solution until pH 1-2 was reached. A precipitate began to form and the suspension was stirred for 30 min at 0°C. The precipitate was collected by vacuum filtration, washed with cold water, and dried under high vacuum to provide the desired product, (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1) acid.⁶benzo[1,2,4]thiadiazin-3-yl)-acetic acid (7.20 g, 21.621 mmol, 82.6%), as a pinkish solid. Ή NMR (400 MHz, DMSO-d₆) δ: 3.03 (3H, s), 3.56 (2H, s), 7.33 (1H, d, J = 9.1 Hz), 7.52 -7.54 (2H, m), 10.09 (1H, s), 12.24 (1H, s), 13.02 (1H, bs). LC-MS (ESI) calcd for Ci₀HiiN₃THE_theS2 333.01, found 334.1 [M+H⁺].

Alternatively, acid (7-methanesulfonylamino-1,1-dioxol,4-dihydro-lÀ⁶-benzo[l,2,4]thiadiazin-3-yl)acetic acid can be prepared as follows:

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Methyl ester of 7T-/4-methanesulfonylamino-2sulfamoylphenyl)-malonamic acid (prepared as described in Example 1g, 1.35 kg, 3.69 mol) was added to a 3.8% by weight aqueous solution of sodium hydroxide (14.0 kg). The resulting mixture was stirred at 23°C for 30 h and then cooled to 0°C. A 2.0 M aqueous solution of hydrochloric acid (9.72 L) was slowly added, stirring at 0°C was continued for 30 min, and the mixture was then filtered. The solid was washed with water (1.4 L) and then pasted in a mixture of methanol (1.4 L) and diethyl ether (2.7 L). After filtration, the solid was washed with diethyl ether (2 x 1.4 L) and then dried under vacuum at 23°C to give the desired product, acid (7-methanesulfonylamino-1,1-dioxol,4-dihydro-1⁶-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (1.07 kg, 3.21 mol, 87%), as a light brown solid.

h) Ethyl ester of (rac-di-exo)-3-(4-fluorobenzylamino)bicyclo[2.2.1]heptane-2-carboxylic acid

THE

<img file="BRPI0809685A2_D0088.tif" />

XX

Ethyl ester chloride of (racUi-nxo)-3-Ammobicyclo[2.2.l]heptane-2-carboxylic acid (1 g, 4.6 mmol) was suspended in methanol (23 mL). Sodium acetate (0.755 g, 9.2 mmol) was added followed by powdered molecular sieves of 4A (1 g) and 4-fluorobenzaldehyde (0.571 g, 4.6 mmol). Sodium cyanoborohydride (0.578 g, 9.2 mmol) was added and the mixture was stirred at 25°C for 16 h. The mixture was poured into a 1:1 mixture of saturated aqueous sodium bicarbonate solution (200 mL) and ethyl acetate (200 mL). After stirring, both layers were passed through a plug.

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Celite. The organic layer was further washed with saturated aqueous brine solution (50 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to give the crude product, ethyl ester of (rac-di-exo)-3-(4-fluoro-benzylammo)-bicyclo[2.2.1]heptane-2-carboxylic acid (0.993 g, 3.41 mmol, 74%), as a clear oil. LC-MS (ESI) calcd for C17H22FNO2 291.16, found 292.1 [M+H⁺],

i) (rac-di-exo)-IV-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-1 λ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0089.tif" />

F

Acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1À⁶Benzo[1,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 0.1 g, 0.299 mmol) was dissolved in anhydrous A,A-dimethylmethionate (1.5 mL). Ethyl ester of (rac-di-exo)-3-(4-fluoro-benzyl-amino)bicyclo[2.2.1]heptane-2-carboxylic acid (0.087 g, 0.3 mmol) was added followed by a solution of 7V,A-dicyclohexylcarbodiimide in 1.0 M dichloromethane (0.315 mL, 0.315 mmol). The mixture was stirred at 25°C for 1.5 h. Triethylamine (0.124 mL, 0.9 mmol) was added and the mixture was stirred at 50°C for 16 h. Upon cooling, the mixture was poured into a 1.0 M aqueous hydrochloric acid solution (100 mL). The product was extracted over ethyl acetate (100 mL). The organic layer was washed with a 1.0 M aqueous hydrochloric acid solution (50 mL), a saturated aqueous brine solution (25 mL), and dried over magnesium sulfate. Purification was performed by flash column chromatography (Merck silica gel 60, 40

106/317 μπι, 0 to 1% methanol in dichloromethane) followed by crystallization with methanol yielded the desired product, (rac-di-exo)-A-{3-[3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]- 1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[l,2,4]thiadiazin-7-yl}-methanesulfonamide (0.08 g, 0.141 mmol, 47%), as a white powder. Ή NMR (400 MHz, DMSO-cA) δ: 1.18 - 1.21 (2H, m), 1.39 - 1.59 (5H, m), 2.61 - 2.64 (1H, m), 3.03 (1H, d, J = 14.2 Hz), 3.05 (3H, s), 3.53 (1H, d, J= 9.3 Hz), 4.41 (1H, d, J = 14.8 Hz), 4.96 (1H, d, J = 15.5 Hz), 7.14 (2H, t, J = 9.0 Hz), 7.32 (2H, dd, Ji = 8.7 Hz, J₂ = 6.2 Hz), 7.50 (1H, dd, Ji = 9.3 Hz, J₂ ~ 2.4 Hz), 7.55 - 7.57 (2H, m), 10.17 (1H, s). LC-MS (ESI) calcd for C25H25FN4O6S2 560.12, found 561.3 [M+H⁺HPLC analysis: Chiralpak AS-RH 4.6 x 250 mm, 5 microns at room temperature, Solvent A - Solvent B3 (see table for gradient), 0.8 mL/min, 310 nm, t1 = 7.72 min, t2 = 9.00 min.

Example 2 (rac-di-endo)-N-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²'⁷]undec-5-en-5-il]-1,1-dioxo-1,4-dihydro-lÃ⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

OH N

a) Ethyl ester of (ruc-di-endo)-3-(4-fluoro-benzylamino)-bicyclo[2.2.1]heptane-2-carboxylic acid

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<img file="BRPI0809685A2_D0090.tif" />

<img file="BRPI0809685A2_D0091.tif" />

Ethyl ester chloride of (rac-di-endo)-3-ammobicyclo[2.2.1]heptane-2-carboxylic acid (1 g, 4.6 mmol) was suspended in methanol (23 mL). Sodium acetate (0.755 g, 9.2 mmol) was added followed by powdered molecular sieves of 4 Å (1 g) and 4-fluorobenzaldehyde (0.571 g, 4.6 mmol). Sodium cyanoborohydride (0.578 g, 9.2 mmol) was added and the mixture was stirred at 25°C for 16 h. The mixture was poured into a 1:1 mixture of saturated aqueous sodium bicarbonate solution (200 mL) and ethyl acetate (200 mL). After stirring, both layers were passed through a Celite plug. The organic layer was further washed with saturated aqueous brine solution (50 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the crude product, ethyl ester of (diendo)-3-(4-fluoro-benzylamino)-bicyclo[2.2.1]heptane-2-carboxylic acid (1.096 g, 3.77 mmol, 82%), as a clear oil. LC-MS (ESI) calcd for C17II22FNO2 291.16, found 292.1 [M+H⁺].

b) (rac-di-en.do)-IV-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3aza-tricycle [6.2.1.0²-⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-bcnzo [1,2,4]thiadiazin-7-yl}-methanesulfonamide

108/317

<img file="BRPI0809685A2_D0092.tif" />

Acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- 1λ⁶Benzo[1,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 0.1 g, 0.299 mmol) was dissolved in anhydrous A,A-dimethylformamide (1.5 mL). Ethyl ester of (rac-iii-encio)-3-(4-fluoro-benzylamino)bicyclo[2.2.l]heptane-2-carboxylic acid (0.087 g, 0.3 mmol) was added followed by a solution of AA-dicyclohexylcarbodiimide in 1.0 M dichloromethane (0.315 mL, 0.315 mmol). The mixture was stirred at 25°C for 1.5 h. Triethylamine (0.124 mL, 0.9 mmol) was added and the mixture was stirred at 50°C for 16 h. Upon cooling, the mixture was poured into a 1.0 M aqueous hydrochloric acid solution (100 mL). The product was extracted over ethyl acetate (100 mL). The organic layer was washed with a 1.0 M aqueous hydrochloric acid solution (50 mL), a saturated aqueous brine solution (25 mL), and dried over magnesium sulfate. Purification by flash column chromatography (Merck silica gel 60, 4063 pm, 0 to 1% methanol in dichloromethane) followed by crystallization with methanol yielded the desired product, (rac-di-endo)-A-{3-[3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1dioxo- l,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (0.092 g, 0.164 mmol, 55%), as a white powder. Ή NMR (400 MHz, DMSO-cAj 2: 1.25 - 1.48 (6H, m), 2.64 - 2.73 (2H, m), 3.06 (3H, s), 3.24 (1H, d, J = 23.8 Hz), 3.72 (1H, d, J = 11.6 Hz), 4.07 (III, d, J = 14.8 Hz), 5.12 (1H, d, J = 15.3 Hz), 7.14 (2H, t, J = 8.6 Hz), 7.39 (2H, dd, Ji = 8.1 Hz, J₂ = 5.7 Hz), 7.51 (1H, dd, Ji = 8.5 Hz, J₂ = 2.4 Hz), 7.57 - 7.60 (2H, m), 10.18 (1H, s). LC-MS (ESI) calcd for C25H25FN4O6S2 560.12,

109/317 found 561.4 [M+H⁺], HPLC Analysis: Chiralpak AS-RH 4.6 x 250 mm, 5 microns a rt., Solvent A - Solvent B (see table for gradient), 0.8 mL/min, 310 nm, tl = 7.58 min, /2 = 10.08 min.

Example 3 (rac-di-endo 1-7V43-[3-15-fluoro-pyridin-2-ylmethyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²-⁷]undec-5-en-5-yl]-1, 1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0093.tif" />

a) Ethyl ester of (rac-di-encZo)-3-[(5-fluoro-pyridin-210 ylmethyl)-amino]-bicyclo[2.2.1]heptane-2-carboxylic acid

<img file="BRPI0809685A2_D0094.tif" />

<img file="BRPI0809685A2_D0095.tif" />

Ethyl ester chloride of (rac-di-endo)-3-Aminobicyclo[2.2.1]heptane-2-carboxylic acid (1 g, 4.6 mmol) was suspended in methanol (23 mL). Sodium acetate (0.755 g, 9.2 mmol) was added, followed by 4A molecular sieve powder (1 g), followed by 5-fluorpyridine-2-carbaldehyde (0.576 g, 4.6 mmol). Sodium cyanoborohydride (0.578 g, 9.2 mmol) was added, and the mixture was stirred at 25°C for 16 h. The mixture was poured into a 1:1 mixture of saturated aqueous solution.

110/317 of sodium bicarbonate (200 mL) and ethyl acetate (200 mL). After stirring, both layers were passed through a Celite plug. The organic layer was further washed with saturated aqueous brine solution (50 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to give the crude product, Ethyl ester of (rac-di-(?ndo)-3[(5-fluoropyridin-2-ylmctyl)-amino]bicyclo[2.2.l]heptane-2-carboxylic acid (1.247 g, 4.27 mmol, 93%), as a clear oil. LC-MS (ESI) calcd for C16H21FN2O2 292.16, found 293.1 [M+II⁺],

b) (rac-di-endo)-7V-{3-[3-(5-fluoro-pyridin-2-ylmethyl)-6-hydroxy-4oxo-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]~ 1,1-dioxo- 1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0096.tif" />

Acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- 1λ⁶15 benzo[l,2,4] thiadiazin-3-yl)-acetic acid (prepared as described in Example

1g, 0.1 g, 0.299 mmol) was dissolved in anhydrous 7V-dimethylformamide (1.5 mL). Ethyl ester of (rac-di-endo)-3-[(5-fluoro-pyridin-2-ylmethyl)amino]-bicyclo[2.2.1]heptane-2-carboxylic acid (0.088 g, 0.3 mmol) was added followed by a 1.0 M solution of 7V,7V-dicyclohexylcarbo20 diimide in dichloromethane (0.315 mL, 0.315 mmol). The mixture was stirred at 25°C for 1.5 h. Triethylamine (0.124 mL, 0.9 mmol) was added and the mixture was stirred at 50°C for 16 h. Upon cooling, the mixture was poured into a 1.0 M aqueous hydrochloric acid solution (100 mL). The product was extracted with ethyl acetate (100 mL). The layer

111/317 organic material was washed with 1.0 M aqueous hydrochloric acid solution (50 mL), saturated aqueous brine solution (25 mL), dried over magnesium sulfate, and concentrated under vacuum. Crystallization with methanol yielded the desired product, (rac-di-endo)-/V-{3-[3-(5-fluoro-pyridin-2-ylmethyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²>⁷] undec-5-en-5-yl]-l, 1dioxo-1,4-dihydro-lÀ^the-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (0.087 g, 0.156 mmol, 52%), as a light yellow powder.

UI NMR (400 MHz, DMSO-d₆) 7: 1.24 - 1.50 (6H, m), 2.64 2.73 (2H, m), 3.05 (3H, s), 3.28 (1H, d, J = 12.2 Hz), 3.89 (1H, d, J = 14.9 Hz), 4.25 (III, d, J = 15.7 Hz), 5.10 (III, d, J = 15.5 Hz), 7.46 - 7.51 (2H, m), 7.55 - 7.57 (211, m), 7.66 - 7.71 (III, m), 8.49 (III, d, J = 2.3 Hz), 10.17 (1H, s). LC-MS (ESI) calcd for C24II24FN5O6S2 561.12, found 562.4 [M+H⁺].

Example 4

7V-{3-[(1S,2S,77?, 8/?F3-/4-fluoro-benzyl)6-hydroxy4oxo3-aza tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro- 1λ⁶benzoph 1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0097.tif" />

a) (1R,2S,3R,4S)-3-(methoxycarbonyl)bicyclo[2.2.1]hept5-ene-2-carboxylic acid

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<img file="BRPI0809685A2_D0098.tif" />

The starting material (a) was prepared as described in J. Org. Chem. 2000, 65, 6984-6991. cis-5-Norbornene-endo-2,3-dicarboxylic anhydride (4.104 g, 25 mmol) was suspended in a 1:1 mixture of toluene and carbon tetrachloride (500 mL). The mixture was stirred for 20 min. Quinine (8.92 g, 27.5 mmol) was added and the flask was degassed and backfilled with nitrogen. The solution was cooled to -55 °C. While stirring, methanol (3.04 mL, 75 mmol) was added. The mixture was stirred at -55 °C for 20 h. Under heating to 25 °C, the mixture was concentrated under vacuum to a thick oil. The oil was dissolved in ethyl acetate (400 mL), washed with 1.0 M aqueous hydrochloric acid solution (2 x 400 mL), saturated aqueous brine solution (100 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the desired product, (1R,2S,3R,48)-3-(methoxycarbonyl)bicyclo acid [2.2. [l]hept-5-ene-2-carboxylic acid (4.8 g, 24.5 mmol, 98%), as a clear wax solid. NMR (400 MHz, DMSO)₆) Π: 1.26 (1H, d, J = 8.5 Hz), 1.33 (1H, d, J = 8.8 Hz), 3.00 (1H, s), 3.03 (1H, s), 3.21 - 3.30 (2H, m), 3.45 (3H, s), 6.02 - 6.04 (1H, m), 6.14 - 6.16 (1H, m), 11.86 (1H, s).

b) (1 S,2R,3S,4R)-3-{[(benzyloxy)carbonyl]amino}bicyclo[2.2.1] hept-5-ene-2-carboxylate of media

<img file="BRPI0809685A2_D0099.tif" />

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(1S, 2R, 3S, 41?)-3-(methoxycarbonyl)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (4.61 g, 23.5 mmol) was dissolved in anhydrous tetrahydrofuran (40 mL). The flask was degassed and back-filled with nitrogen, and the mixture was cooled to 0 °C. Triethylamine (9.9 mL, 70.5 mmol) was added, followed by the dropwise addition of ethyl chloroformate (4.48 mL, 47 mmol) with vigorous stirring. Immediate precipitation was observed. Additional tetrahydrofuran (60 mL) was added. The mixture was stirred at 0 °C for 1 h. Sodium azide (4.58 g, 70.5 mmol) was dissolved in water (30 mL) and added to the reaction mixture at 0 °C. The mixture was stirred at 0 °C for 5 min. The ice bath was removed. The mixture was heated to 25 °C and stirred for 2 h. The mixture was poured into water (300 mL) and the product extracted in ethyl acetate (300 mL). The organic layer was further washed with a half-saturated aqueous solution of sodium bicarbonate (2 x 100 mL), a saturated aqueous solution of brine (100 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a clear oil. The oil was dissolved in anhydrous benzene (50 mL) and refluxed while stirring under nitrogen for 2 h. Upon cooling to 25 °C, the solution was concentrated under vacuum to provide a slightly yellow oil. The oil was dissolved in dichloromethane (30 mL) and benzyl alcohol (2.68 mL, 25.9 mmol) was added followed by triethylamine (6.61 mL, 47 mmol). The mixture was refluxed under nitrogen for 16 h. Upon cooling to 25 °C, the solution was concentrated under vacuum to provide a golden oil. Purification by flash column chromatography (Merck silica gel 60, 40-63 gm, 15% ethyl acetate in hexanes) yielded the desired product, (1S,2R,3S,4R)-3-{[(benzyloxy)carbonyl]amino}bicyclo[2.2. [l]hept-5-ene-2-carboxylate methyl (5.51 g, 18.31 mmol, 78%), as a clear oil.¹H NMR (400 MHz, CDCI3) 7: 1.38 (1H, d, J = 9.1 Hz), 1.50 (1H, d, J = 9.4 Hz), 3.10 (2H, s), 3.21 (1H, dd, Ji = 9.2 Hz, J₂ = 2.3 Hz), 3.53 (3H, s), 4.62 (1H, dt, Ji = 9.4 Hz, J₂ = 2.9 Hz), 5.07 (2H, q, J = 13.0 Hz), 5.29 (1H, d, J = 8.6 Hz), 6.15 - 6.17 (1H, m), 6.37 - 6.38 (1H, m), 7.29 - 7.35 (5H, m). LC-MS (ESI) calcd for

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C17II19NO4 301.13, found 258.1 (100%), 302.2 [M+H⁺] (70%), 603.5 [2M+H⁺] (20%).

c) Methyl (1R,2R,3S,4S)-3-aminobicyclo[2.2.1]heptane-2-carboxylate chloride

<img file="BRPI0809685A2_D0100.tif" />

(1S,2R,3S,4R)-3-{[(Benzyloxy)carbonyl]amino}bicyclo[2.2.1]hept-5-ene-2-carboxylate methyl (5.5 g, 18.27 mmol) was dissolved in ethyl acetate (75 mL). Palladium in carbon 5% (650 mg) was added. The flask was degassed and fed back with hydrogen gas via a balloon. The mixture was stirred at 25 °C for 16 h. The mixture was passed through a Celite plug and the filtrate was concentrated under vacuum to provide a thick, clear oil. The oil was dissolved in ethyl acetate (15 mL) and added dropwise, with vigorous stirring, to a mixture of 4.0 M hydrochloric acid solution in 1,4-dioxane (10 mL, 40 mmol) in diethyl ether (90 mL). The desired product began to precipitate as a white solid. The mixture was stirred for 20 min. The precipitate was collected by vacuum filtration and washed with additional diethyl ether (15 mL). The solid was further dried under vacuum for 1 h to give the desired product, methyl (1R,2R,3S,4S)-3-aminobicyclo[2.2.1]heptane-2-carboxylate chloride (2.61 g, 12.69 mmol, 69%), as a white powder. NMR (400 MHz, DMSO-d) □ : 1.34 - 1.43 (411, m), 1.54 (1H, d, J = 9.5 Hz), 1.68 (11I, d, J = 11.4 Hz), 2.47 - 2.48 (2H, m), 3.03 (1H, d, J = 11.0 Hz, J₂ = 4.0 Hz), 3.49 - 3.53 (1H, m), 3.62 (3H, s), 8.07 (3H, bs). LC-MS (ESI) calcd for C9H15NO2 (free amine) 169.11, found 170.1 [M+H⁺] (100%),339,2 [2M+H⁺] (50%).

d) (1R,2R,3S,4S)-3-[(4-fluoro-benzyl)amino]bicyclo-[2.2.1] hep115/317 methyl tane-2-carboxylate

THE

<img file="BRPI0809685A2_D0101.tif" />

Methyl (I/?,2I?,3S,4S)-3-aminobicyclo[2.2.1]heptane-2-carboxylate chloride (1 g, 4.86 mmol) was dissolved in methanol (23 mL). Sodium acetate (0.755 g, 9.2 mmol) was added followed by powdered molecular sieves of 4A (1 g) and 4-fluorobenzaldehyde (0.571 g, 4.6 mmol). Sodium cyanoborohydride (0.578 g, 9.2 mmol) was added and the mixture was stirred at 25°C for 16 h. The mixture was poured into a mixture of saturated aqueous sodium bicarbonate solution (200 mL) and ethyl acetate (300 mL). After stirring, both layers were passed through a Celite plug. The organic layer was further washed with saturated aqueous sodium bicarbonate solution (100 mL), saturated aqueous brine solution (100 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the crude product, (lR,2R,3S,4S)._>)-3-[(4-iluor-benzyl)amino]bicyclo[2.2.1]heptane-2-carboxylate methyl (1.172 g, 4.23 mmol, 92%), as a clear oil. LC-MS (ESI) calcd for C16H20FNO2 277.15, found 278.2 [M+H⁺],

e) N-{3-[(1S,2S,7I?,8I?)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-320 aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- wool⁶-benzo

[1,2,4]thiadiazin-7-yl}-methanesulfonamide

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<img file="BRPI0809685A2_D0102.tif" />

/1R,2R,33/4 8/-3-[(4-fluoro-benzyl)amino]bicyclo[2.2. l]heptane

Methyl -2-carboxylate (0.087 g, 0.3 mmol) was dissolved in anhydrous N,N-dimethylformamide (2.8 mL). Acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1-⁶-benzo[1,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 0.1 g, 0.3 mmol) was added followed by IV-methylmorpholine (0.07 mL, 0.63 mmol). The mixture was stirred until completely dissolved, approximately 5 min. l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.061 g, 0.315 mmol) was added and the mixture was stirred at 25°C for 4 h. Triethylamine (0.126 mL, 0.9 mmol) was added and the mixture was stirred at 50°C for 16 h. Upon cooling to 25°C, the solution was diluted with ethyl acetate (25 mL) and washed with 1.0 M aqueous hydrochloric acid solution (2 x 25 mL), saturated aqueous brine solution (10 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a golden oil. The oil was dissolved in methanol (4 mL) and the product was precipitated by adding 1.0 M aqueous hydrochloric acid solution (4 mL) with stirring. The solid was collected by vacuum filtration and further dried under vacuum to provide the desired product, N-{3-[(1S,2S,7R,8R)-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1O²-⁷]undec-5-en-5-yl]1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (0.0805 g, 0.144 mmol, 48%), as a white powder. Ή NMR (400 MHz, DMSO-dô) □: 1.23 - 1.48 (6H, m), 2.67 - 2.68 (2H, m), 3.06 (3H, s), 3.24 (1H, d, J= 15.0 Hz), 3.72 (1H, d, J = 11.9 Hz), 4.07 (1H, d, J = 15.6 Hz), 5.12 (III, d, J = 15.7 Hz), 7.14 (2H, t, J = 8.4 Hz), 7.39 (211, dd, Ji =

117/317, 2 Hz, J₂ = 5.8 Hz), 7.51 (1H, dd, Ji = 8.4 Hz, J₂ = 2.3 Hz), 7.57 - 7.60 (2H, m), 10.18 (1H, s). LC-MS (ESI) calcd for C25H25FN4O6S2 560.12, found 561.3 [M+H⁺], ee = 99% [HPLC analysis: Chiralpak AS-RH 4.6 x 250 mm, 5 microns at rt, Solvent A - Solvent B (see table for gradient), 0.8 mL/min, 310 nm, tl = 7.58 min (main), i2 = 10.08 min].

Example 5

7V43-[(1R,2R,7 S,8S)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro- 1λ⁶10 benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0103.tif" />

a) (1S,2R,3S,4R)-3-(methoxycarbonyl)bicyclo[2.2.l]hept5-ene-2-carboxylic acid

<img file="BRPI0809685A2_D0104.tif" />

The starting material (a) was prepared as described in J.

Org. Chem. 2000, 65, 6984-6991. Cis-5-Norbornene-endo-2,3-dicarboxylic anhydride (8.21 g, 50 mmol) was suspended in a 1:1 mixture of toluene and carbon tetrachloride (250 mL). The mixture was stirred for 10

118/317 min. Quinidine (17.84 g, 55 mmol) was added, and the flask was degassed and backfilled with nitrogen. The solution was cooled to -55°C.

While stirring, methanol (6.08 mL, 150 mmol) was added. The mixture was stirred at -55°C for 18 h. Upon heating to 25°C, the mixture was concentrated under vacuum to a thick oil. The oil was dissolved in a mixture of ethyl acetate (400 mL) and 1.0 M aqueous hydrochloric acid solution (300 mL). After agitation, the layers were separated and the organic layer was further washed with 1.0 M aqueous hydrochloric acid solution (2 x 100 mL), saturated aqueous brine solution (100 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the desired product, (lS,2K,3S,41?)-3-(methoxycarbonyl)bicyclo[2.2.l]hept-5-ene-2-carboxylic acid (9.15 g, 46.6 mmol, 94%), as a clear oil.^]H NMR (400 MHz, DMSO-do) 7= 1.26 (1H, d, J = 8.4 Hz), 1.33 (1H, d, J = 8.4 Hz), 3.00 (1H, s), 3.03 (1H, s), 3.21 - 3.29 (2H, m), 3.45 (3H, s), 6.02 - 6.04 (1H,

m), 6.14 - 6.16 (1H, m), 11.86 (1H, s).

b) (1R/2 S,3R,4 8)-3-{[(Benzyloxy)carbonyl]amino}bicyclo[2.2.1] methyl hept-5-ene-2-carboxylate

H

H

The intermediate (b) was thesis 2001, 11, 1719-1730. Cyclo[2.2.l]hept-5-ene-2-carboxylic acid (8.94 g, 45.57 mmol) was dissolved in anhydrous tetrahydrofuran (200 mL). The flask was degassed and fed back with nitrogen, and the mixture was cooled to 0°C. Triethylamine (19.2 mL, 136.7 mmol) was added followed by dropwise addition of ethyl chloroformate (8.69 mL, 91.1 mmol) with stirring.

THE

ÁDMe

N-Cbz

H prepared as described in SynS, 2 R, 3 S, 41?) - 3 - (methoxycarbonyl) bi 119/317 vigorously. Immediate precipitation was observed. The mixture was stirred at 0°C for 1 h. Sodium azide (8.89 g, 136.7 mmol) was dissolved in water (60 mL) and added to the reaction mixture at 0°C. The mixture was stirred at 0°C for 1 h. The ice bath was removed. The mixture was heated to 25°C and continued to be stirred for 2 h. The mixture was poured into water (400 mL) and the product extracted in ethyl acetate (400 mL). The organic layer was further washed with a half-saturated aqueous solution of sodium bicarbonate (2 x 200 mL), a saturated aqueous solution of brine (2 x 200 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to yield a slightly brown oil. The oil was dissolved in anhydrous benzene (100 mL) and refluxed with stirring under nitrogen for 2 h. Upon cooling to 25°C, the solution was concentrated under vacuum to yield a slightly brown oil. The oil was dissolved in dichloromethane (60 mL) and benzyl alcohol (5.19 mL, 50.13 mmol) was added followed by triethylamine (12.81 mL, 91.14 mmol). The mixture was refluxed under nitrogen for 16 h. Upon cooling to 25°C, the solution was concentrated under vacuum to yield a golden oil. Purification by flash column chromatography (Merck silica gel 60, 4063 gm, 10% ethyl acetate in hexanes) gave the desired product, (lR,2S,3R,4S/-3-{[(benzyloxy)carbonyl]amino}bicyclo[2.2. l]methyl hept-5-ene-2-carboxylate (10.1 g, 33.55 mmol, 74%), as a clear oil. Ή NMR (400 MHz, CDC1₃) □: 1.38 (1H, d, J = 8.7 Hz), 1.50 (1H, d, J = 8.4 Hz), 3.10 (2H, s), 3.21 (1H, d, J = 8.8 Hz), 3.53 (3H, s), 4.59 - 4.64 (1H, m), 5.07 (2H, q, J = 13.0 Hz), 5.29 (1H, d, J = 8.3 Hz), 6.15 - 6.17 (1H, m), 6.37 - 6.38 (1H, m), 7.27 - 7.36 (5H, m). LC-MS (ESI) calcd for C17H19NO4 301.13, found 258.1 (100%), 302.2 [M+H⁺] (70%), 603.5 [2M+H⁺] (20%).

c) Methyl (1S,2S,3R,4R)-3-aminobicyclo[2.2.1]hepta-no-2-carboxylate chloride

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<img file="BRPI0809685A2_D0105.tif" />

Methyl /1 R,2S,3R,4S)-3-{[(Benzyloxy)carbonyl]amino}bicyclo[2.2.1]hept-5-ene-2-carboxylate (10 g, 33.22 mmol) was dissolved in ethyl acetate (150 mL). Palladium in carbon 5% (1.5 mg) was added. The flask was degassed and fed back with hydrogen gas via a balloon. The mixture was stirred at 25°C for 2 h. The mixture was passed through a Celite plug and the filtrate was concentrated under vacuum to a volume of 50 mL. The solution was added dropwise, with vigorous stirring, to a mixture of 4.0 M hydrochloric acid solution in 1,410 dioxane (20 mL) in diethyl ether (200 mL). The desired product began to precipitate as a white solid. The mixture was stirred for 10 min. The precipitate was collected by vacuum filtration and washed with additional diethyl ether (15 mL). The solid was further dried under vacuum for 1 h to give the desired product, methyl (1S,2S,3R,4R)-3-aminobicyclo[2.2.1]heptane-2-carboxylate chloride (5.21 g, 25.33 mmol, 76.3%), as a white powder.^TH NMR (400 MHz, DMSO-d₆) δ: 1.33 - 1.42 (4H, m), 1.54 (1H, d, J= 10.3 Hz), 1.69 (1H, d, J = 11.5 Hz), 2.46 - 2.48 (2H, m), 3.03 (1H, dd, Ji = 10.8 Hz, J₂ = 4.1 Hz), 3.46 - 3.55 (1H, m), 3.62 (3H, s), 8.09 (3H, bs). LC-MS (ESI) calcd for C9H15NO2 (free amine) 169.11, found 170.1 [M+H⁺] (100%), 339.2 [2M+H⁺] (50%).

d) (1S,2S,3R,4R)-3-[(4-fluoro-benzyl)amino]bicyclo[2.2.1] methyl heptane-2-carboxylate

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<img file="BRPI0809685A2_D0106.tif" />

Methyl (1S,2S,3R,4R)-3-aminobicyclo[2.2.1]heptane-2-carboxylate chloride (1 g, 4.86 mmol) was dissolved in methanol (23 mL). Sodium acetate (0.755 g, 9.2 mmol) was added followed by powdered molecular sieves of 4A (1 g) and 4-fluorobenzaldehyde (0.571 g, 4.6 mmol). Sodium cyanoborohydride (0.578 g, 9.2 mmol) was added and the mixture was stirred at 25°C for 16 h. The mixture was poured into a mixture of saturated aqueous sodium bicarbonate solution (200 mL) and ethyl acetate (300 mL). After stirring, both layers were passed through a Celite plug. The organic layer was further washed with saturated aqueous sodium bicarbonate solution (100 mL), saturated aqueous brine solution (100 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the crude product, (lS,2S,37?,41?)-3-[(4-fluoro-benzyl)amino]bicyclo[2.2. l]heptane-215 methyl carboxylate (1.11 g, 4.0 mmol, 87%), as a clear oil. LCMS (ESI) calcd for C16H20FNO2 277.15, found 278.2 [M+H⁺].

e) IV-{3-[(1R,2J?, 7S,8S/-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4thiadiazin-7-yl}-methanesulfonamide

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<img file="BRPI0809685A2_D0107.tif" />

(1S,2S.3/?,4R/-3-[(4-fluorobenzyl)amino]bicyclo[2.2.1]heptane-2-carboxylate methyl (0.087 g, 0.3 mmol) was dissolved in anhydrous N,N-dimethylformamide (2.8 mL). Acid (7-methanesulfonylamino-1,1dioxo-1,4-dihydro-1A⁶-benzo[1,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 0.1 g, 0.3 mmol) was added followed by 7V-methylmorpholine (0.07 mL, 0.63 mmol). The mixture was stirred until completely dissolved, approximately 5 min. l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.061 g, 0.315 mmol) was added and the mixture was stirred at 25°C for 4 h. Triethylamine (0.126 mL, 0.9 mmol) was added and the mixture was stirred at 50°C for 16 h. Upon cooling to 25 °C, the solution was diluted with ethyl acetate (25 mL) and washed with 1.0 M aqueous hydrochloric acid solution (2 x 25 mL), saturated aqueous brine solution (10 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a golden oil. The oil was dissolved in methanol (4 mL) and the product was precipitated by adding 1.0 M aqueous hydrochloric acid solution (4 mL) with stirring. The solid was collected by vacuum filtration and further dried under vacuum to provide the desired product, N- {3-[(lR,2R,7S,8SJ-3(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1,0²’⁷]undec-5-en-5-yl]1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (0.0781 g, 0.139 mmol, 46%), as a white powder.^THEH NMR (400 MHz, DMSO-dgj L: 1.23 - 1.48 (6H, m), 2.67 - 2.68 (2H, m), 3.06 (3H, s), 3.24 (1H, d, J= 15.0 Hz), 3.72 (1H, d, J= 11.9 Hz), 4.07 (1H, d, J = 15.6 Hz), 5.12 (1H, d, J= 15.7 Hz), 7.14 (2H, t, J= 8.4 Hz), 7.39 (2H, dd, Ji =

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8/2 Hz, J₂ = 5.8 Hz), 7.51 (1H, dd, Ji = 8.4 Hz, J₂ = 2.3 Hz), 7.57 - 7.60 (2H, m), 10.18 (1H, s). LC-MS (ESI) calcd for C25H25FN4O6S2 560.12, found 561.3 [M+H⁺]. ee = 99% [HPLC Analysis: Chiralpak AS-RH 4.6 x 250 mm, 5 microns at room temperature, Solvent A - Solvent B (see table for gradient), 0.8 mL/min, 310 nm, t1 = 7.58 min, t2 = 10.08 min (main)]

Example 6

7V-{3-[(17?, 2 8.71?, 8 8)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza10 tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0108.tif" />

a) (1S,2S,3R,4R)-3-(methoxycarbonyl)bicyclo[2.2.l]hept5-ene-2-carboxylic acid

<img file="BRPI0809685A2_D0109.tif" />

The starting material (a) was prepared as described in J.

Org. Chem. 2000, 65, 6984-6991. cis-5-Norbornene-exo-2,3-dicarboxylic anhydride (5 g, 30.45 mmol) was suspended in a 1:1 mixture of toluene and carbon tetrachloride (610 mL). The mixture was stirred for 10

124/317 min. Quinine (10.87 g, 33.5 mmol) was added, and the flask was degassed and backfilled with nitrogen. The solution was cooled to -55°C. While stirring, methanol (3.7 mL, 91.35 mmol) was added. The mixture was stirred at -55°C for 16 h. Upon heating to 25°C, the mixture was concentrated under vacuum until foam formed. The foam was dissolved in a mixture of ethyl acetate (400 mL) and 1.0 M aqueous hydrochloric acid solution (400 mL). The layers were separated and the organic layer was further washed with 1.0 M aqueous hydrochloric acid solution (2 x 200 mL), saturated aqueous brine solution (100 mL) and dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the desired product, (18,2S,3R,4R)-3(methoxycarbonyl)bicyclo[2.2.l]hept-5-ene-2-carboxylic acid (5.95 g, 30.3 mmol, 99%), as a clear oil. Ή NMR (400 MHz, DMSO-dg/δ: 1.31 (1H, d, J = 8.5 Hz), 1.98 (1H, d, J = 8.6 Hz), 2.51 (2H, d, J = 1.6 Hz), 2.95 (2H, bs), 3.52 (3H, s), 6.17 -6.21 (2H, m), 12.16 (1H, s).

b) flR,2R,3S,4S)-3-{[(benzyloxy)carbonyl]amino}bicyclo[2.2.1] hept-5-ene-2-carboxylate of media

<img file="BRPI0809685A2_D0110.tif" />

(1S,2S,31?,4i?/-3-(methoxycarbonyl)bicyclo[2.2.l]hept-5-ene-2-carboxylic acid (5.9 g, 30 mmol)) was dissolved in anhydrous tetrahydrofuran (133 mL). The flask was degassed and back-filled with nitrogen, and the mixture was cooled to 0 °C. Triethylamine (12.64 mL, 90 mmol) was added, followed by dropwise addition of ethyl chloroformate (5.72 mL, 60 mmol) with vigorous stirring. Immediate precipitation was observed. The mixture was stirred at 0 °C for 1 h. Sodium azide (5.86 g, 90 mmol) was dissolved in water (40 mL) and added to

125/317 reaction mixture at 0 °C. The mixture was stirred at 0 °C for 5 min. The ice bath was removed. The mixture was heated to 25 °C and continued to be stirred for 2 h. The mixture was poured into water (300 mL) and the product extracted in ethyl acetate (300 mL). The organic layer was further washed with a half-saturated aqueous solution of sodium bicarbonate (2 x 100 mL), a saturated aqueous solution of brine (100 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a light brown oil. The oil was dissolved in anhydrous benzene (66 mL) and refluxed with stirring under nitrogen for 2 h. Upon cooling to 25 °C, the solution was concentrated under vacuum to provide a light brown oil. The oil was dissolved in dichloromethane (40 mL) and benzyl alcohol (3.41 mL, 33 mmol) was added followed by triethylamine (8.44 mL, 60 mmol). The mixture was re-flushed under nitrogen for 16 h. Upon cooling to 25 °C, the solution was concentrated under vacuum to provide a thick oil. Purification by flash column chromatography (Merck silica gel 60, 4063 gm; I^the column: 3:1 hexanes/ethyl acetate; 2^the column: 2:4:1 dichloromethane/pentane/diethyl ether) yielded the desired product, /lR,2R,3S,4S/-3-{[(benzyloxy)carbonyl]amino}bicyclo[2.2.l]hept-5-ene-2-carboxylate methyl (6.95 g, 23.09 mmol, 77%), as a pale yellow oil, 1H NMR (400 MHz, CDC1₃) C: 1.59 (1H, d, J = 9.3 Hz), 1.96 (1H, d, J = 9.3 Hz), 2.66 (1H, d, J = 7.9 Hz), 2.75 (1H, s), 2.96 (III, s), 3.59 (311, s), 4.01 (1H, t, J = 8.5 Hz), 5.09 (2H, q, J = 10.4 Hz), 5.46 (III, d, J = 9.4 Hz), 6.17 - 6.22 (2H, m), 7.29 - 7.36 (5H, m). LC-MS (ESI) calcd for C17H19NO4 301.13, found 258.1 (100%), 302.2 [M+H⁺] (70%), 603.5 [2M+H+] (20%).

c) Methyl (1S,2R,3S,4R)-3-aminobicyclo[2.2.1]heptane-2-carboxylate chloride

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<img file="BRPI0809685A2_D0111.tif" />

(1 /?_J2/?₎3S_?4S)-3-{[(benzyloxy)carbonyl]amino}bicyclo[2.2.1]hept-5-ene-2-carboxylate methyl (1 g, 3.32 mmol) was dissolved in ethyl acetate (15 mL). Palladium in carbon 5% (120 mg) was added. The flask was degassed and fed back with hydrogen gas via a balloon. The mixture was stirred at 25°C for 16 h. The mixture was passed through a Celite plug and the filtrate was concentrated under vacuum to provide a thick, clear oil. The oil was dissolved in diethyl ether (10 mL) and added dropwise, with vigorous stirring, to a mixture of 4.0 mL and 1 mL.

M of hydrochloric acid solution in 1,4-dioxane (1.8 mL) in diethyl ether (18 mL). The desired product began to precipitate as a white solid. Additional diethyl ether (10 mL) was added and the mixture was stirred for 10 min. The precipitate was collected by vacuum filtration, washed with additional diethyl ether (2 x 8 mL). The solid was further dried under vacuum for 1 h to give the desired product, (lS,21?,3S,4R)-3-aminobicyclo[2.2] chloride. [l]heptane-2-carboxylate methyl (0.64 g, 3.11 mmol, 94%), as a white powder.^X1H (1H, d,

J = 7.8 Hz), 3.30 - 3.38 (1H, m), 3.61 (3H, s), 8.05 (3H, bs). LC-MS (ESI) calcd for C9H15NO2 (free amine) 169.11, found 170.3 [M+H⁺] (100%), 339.3 [2M+H⁺] (50%).

d) (1S,2/?,3S,4I?)-3-[(4-fluoro-benzyl)amino]bicyclo[2.2. l]methyl heptane-2-carboxylate

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<img file="BRPI0809685A2_D0112.tif" />

Methyl (15,2R,3S,4R)-3-aminobicyclo[2.2.1]heptano-2-carboxylate chloride (prepared as described in Example 6c, 0.5 g, 2.43 mmol) was dissolved in methanol (12 mL). Sodium acetate (0.4 g, 4.86 mmol) was added followed by powdered molecular sieves.

4-fluorobenzaldehyde (0.5 g) and 4-fluorobenzaldehyde (0.302 g, 2.43 mmol) were added. Sodium cyanoborohydride (0.305 g, 4.86 mmol) was added and the mixture was stirred at 25°C for 16 h. A mixture of saturated aqueous solution of sodium bicarbonate (200 mL) and ethyl acetate (300 mL) was then poured into the mixture.

After agitation, both layers were passed through a Celite plug. The organic layer was further washed with saturated aqueous sodium bicarbonate solution (100 mL), saturated aqueous brine solution (100 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the crude product, methyl 15(1S,2R,3S,4R)-3-[(4-fluoro-benzyl)amino]bicyclo[2.2.1]heptane-2-carboxylate (0.663 g, 2.39 mmol, 98%), as a clear oil. LC-MS (ESI) calcd for C16H20FNO2 277.15, found 278.2 [M+H⁺],

e) A-f3-[(H?,2S,7I?,8S)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3aza-tricycle [6.2.1.0²-⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- l-À⁶-benzo 20 [1,2,4]thiadiazin-7-yl}-methanesulfonamide

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<img file="BRPI0809685A2_D0113.tif" />

(1S,2R,3S,4R)-3-[(4-fluorobenzyl)amino]bicyclo[2.2.1]heptane-2-carboxylate methyl (0.6 g, 2.16 mmol) was dissolved in anhydrous N,N-dimethylformamide (20 mL). (7-methanesulfonylamino-1,1dioxo-1,4-dihydro-1)⁶-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 0.72 g, 2.16 mmol) was added followed by 7V-methylmorpholine (0.5 mL, 4.54 mmol). The mixture was stirred until completely dissolved, approximately 5 min. l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.435 g, 2.27 mmol) was added and the mixture was stirred at 25°C for 45 min. Triethylamine (0.91 mL, 6.48 mmol) was added and the mixture was stirred at 50°C for 16 h.

Upon cooling to 25°C, the solution was diluted with ethyl acetate (300 mL) and washed with 1.0 M aqueous hydrochloric acid solution (3 x 300 mL), saturated aqueous brine solution (100 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a golden oil. Purification by flash column chromatography (Merck silica gel 60, 40-63 pm, 0 to 0.75% methanol in dichloromethane) yielded the product as a white foam. The foam was dissolved in methanol (10 mL) and the product was precipitated by adding a 1.0 M aqueous hydrochloric acid solution (20 mL) with stirring. The solid was collected by vacuum filtration and further dried under vacuum to provide the desired product, 77-{3-[(17?,2S,7R,8S)-3~(4fluorobenzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfone

129/317 mide (0.573 g, 1.02 mmol, 47%), as a white powder.¹H NMR (400 MHz, DMSO-de) □: 1.16 - 1.22 (2H, m), 1.37 - 1.65 (4H, m), 2.49 2.53 (III, m), 2.63 (1H, d, J = 2.3 Hz), 3.02 (1H, d, J = 8.5 Hz), 3.05 (3H, s), 3.52 (1H, d, J = 9.4 Hz), 4.41 (1H, d, J = 15.6 Hz), 4.95 (1H, d, J = 15.6 Hz), 7.14 (2H, t, J = 9.0 Hz), 7.32 (2H, dd, Ji = 8.1 Hz, J₂= 5.7 Hz), 7.50 (1H, dd, Ji = 9.5 Hz, J₂ = 2.3 Hz), 7.55 - 7.57 (2H, m), 10.17 (1H, s). LC MS (ESI) calculated for C25H25FN4O6S2 560.12, found 561.3 [M+H+]. ee = 90% [Analysis-HPLC: Chiralpak AS-RH 2.1 x 150 mm, 5 microns at rt, Solvent A - Solvent B (see table for gradient), 0.3 mL/min, 312 nm, tl = 4.3 min (principal), t2 = 6.0 min].

Alternatively, lV-{3-[(1R,2S,7R;8S)-3-(4-fluoro-benzyl)-6hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lA⁶-benzo[l,2,4]thiadiazin-7-yl}-methanesulfonamide can be prepared as follows:

f) /rac-c/i-exo)-3-Aza-triciclo[4.2.1.0²>⁵]nonan-4-ona .0

Q/J— NH me-di-exo

I3-cyclo[2.2.l]hept-2-ene (1000 g, 10.6 mol) was dissolved in ethyl acetate (1.7 L) and the resulting solution was cooled to 0°C. Chlorosulfonyl isocyanate (969 mL, 11.1 mol) was added at 0-20°C for 30 min. The mixture was heated to 25°C and stirred for 4 h, then cooled to 0°C. A mixture of sodium sulfide (1500 g, 11.9 mol) in water (6 L) was added at 0-20°C. The milky suspension was stirred at 25°C for 30 min and cooled to 0°C. A 50% aqueous solution of sodium hydroxide (1.6 L, 30.3 mol) was added at 0-15°C to adjust the pH to 7. A saturated aqueous solution of sodium carbonate was then added.

130/317 (300 mL) was added to adjust the pH to 7.5-8.0. The mixture was filtered and the solid was washed with ethyl acetate (3 x 2 L) and the solid was discarded. The combined ethyl acetate extracts were washed with saturated aqueous brine solution (2 L), dried over magnesium sulfate and filtered. The solution was concentrated under vacuum to dryness to provide the desired product, (rac-di-exo)-3-aza-tricyclo[4.2.1 .(It⁵]nonan4-one (1220 g, 8.9 mol, 84%), as a white vitreous solid.^]H NMR (400 MHz, CDC1₃) □: 1.02 - 1.11 (2H, m), 1.24 (1H, dt, Ji = 10.9 Hz, J₂ = 1.6 Hz), 1.51 - 1.72 (3H, m), 2.37 - 2.37 (1H, m), 2.43-2.44 (1H, m), 2.99-3.00 (III, m), 3.40 (1H, d, J = 3.4 Hz), 5.73 (1H, bs).

g) Chloride of (rac-di-exo)-3-Amino-bicyclo[2,2,1]heptane-2-carboxylic acid

THE

<img file="BRPI0809685A2_D0114.tif" />

rac'd i-sxo

A (rac-di-exo)-3-aza-triciclo[4.2.1.0²-⁵]nonan-4-one (23.37 g, 170.4 mmol) was added to a 12.0 M aqueous hydrochloric acid solution (150 mL). The mixture was stirred at 25°C for 12 h. The solvent was evaporated under vacuum and the crude compound was dried under high vacuum for 0.5 h. The crude compound was triturated with acetone and filtered to give (rac-di-exo)-3-amino-bicyclo[2.2.1]heptane-2-carboxylic acid chloride (28.43 g, 148.3 mmol, 87%) as a white solid.^XH NMR (400 MHz, DMSO-d₆) □ : 1.15 - 1.26 (3H, m), 1.42 - 1.59 (2H, m), 1.87 (1H, d, J = 10.3 Hz), 2.33 (1H, d, J = 3.4 Hz), 2.45 (1H, d, J = 2.3 Hz), 2.67 (1H, d, J = 7.6 Hz), 3.23 - 3.26 (1H, m), 7.93 (3H, bs), 12.73 (1H, bs).

h) Ethyl ester chloride of (rac-di-exo)-3-aminobicyclo[2.2.l]heptane-2-carboxylic acid

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Ο rTv^oEt rac-di-exo

To absolute ethanol (75 mL) at -10°C, thionyl chloride (4.1 mL, 54.5 mmol) was added dropwise, followed by (racdi-exo)-3-amino-bicyclo[2.2.l]heptane-2-carboxylic acid chloride (9.60 g, 50.1 mmol).

The mixture was stirred at 0°C for 1 h, at 25°C for 4 h, and heated under reflux for 0.5 h. The solution was concentrated under vacuum and dried under high vacuum to give crude (rac-di-exo)-3-amino-bicyclo[2.2.1]heptane-2-carboxylic acid ethyl ester chloride (11.01 g, 50.1 mmol, 100%) as a dirty white solid. 44 NMR (400 MHz, DMSO-10) □□

1.17 - 1.27 (3H, m), 1.21 (3H, t, J= 7.0 Hz), 1.43 -1.57 (2H, m), 1.91 (1H, d, J = 10.0 Hz), 2.36 (1H, d, J = 3.9 Hz), 2.42 (1H, d, J = 3.0 Hz), 2.72 (1H, d, J = 7.6 Hz), 3.28 (1H, d, J = 8.3 Hz), 4.00 - 4.13 (2H, m), 8.06 (3H, bs).

i) (rac-di-exo)-3-amino-bicyclo acid ethyl ester [2.2.

l]heptane-2-carboxylic

<img file="BRPI0809685A2_D0115.tif" />

/aodi-exo

To the ethyl ester chloride of /rac-di-exo)-3-aminobicyclo[2.2.l]heptane-2-carboxylic acid (11.01 g, 50.1 mmol) was added a saturated aqueous solution of sodium bicarbonate (50 mL) and the mixture was stirred at 25 °C for 0.5 h. The crude product was extracted with ethyl acetate (3 x 100 mL). The solution was dried over magnesium sulfate, filtered, and concentrated under vacuum and dried under high vacuum for 2 h to

132/317 provide crude ethyl ester of (rctc-di-exo)-3-aminobicyclo[2.2.l]heptane-2-carboxylic acid (8.17 g, 44.6 mmol, 89%) as a brown oil. μ-I NMR (400 MHz, CDC1₃) □: 1.10 - 1.26 (3H, m), 1.29 (3H, t, J = 7.0 Hz), 1.45 - 1.62 (2H, m), 1.86 (211, bs), 1.95 (1H, dt, Ji = 10.3 Hz, J₂ = 1.9 Hz), 2.09 (1H, d, J = 4.5 Hz), 2.49 (1H, d, J = 4.2 Hz), 2.56 (1H, d, J = 9.0 Hz), 3.24 (1H, d, J = 7.7 Hz), 4.09 - 4.21 (2H, m).

j) (1R,2S,3R,4S)-3-Ethoxycarbonyl-bicyclo[2.2. l]hept-2-yl-amino (l'S)-(+)-10-camphorsulfonate

<img file="BRPI0809685A2_D0116.tif" />

To a solution of ethyl ester of (rac-di-exo)-3-amino-bicyclo[2.2.l]heptane-2-carboxylic acid (408.47 g, 2.98 mol) in ethyl acetate (500 mL) was added a solution of (lS)-(+)10-camphorsulfonic acid (691.70 g, 2.98 mol) in ethanol (800 mL) at 50-75 °C for 30 min. The resulting solution was stirred at 70 °C for 1 h. More ethyl acetate (2.7 L) was added at >55 °C. The solution was allowed to cool to 50 °C and seeded with (lR,2S,3R,4S)-3-ethoxycarbonylbicyclo[2.2.l]hept-2-yl-ammonium (l'S)-(+)-10-camphorsulfonate (ca. 20 mg). The mixture was allowed to cool to 25 °C and stirred for 16 h. The suspension was filtered and the wet filter cake was washed with ethyl acetate (2 x 500 mL). The crude salt was recrystallized with ethanol (600 mL) and ethyl acetate (3 L) to give the desired product, (lR,2S,3R,4S)-3-ethoxycarbonylbicyclo[2.2.l]hept-2-yl-ammonium (l'S)-(+)-10-camphorsulfonate. l]hept-2-yl-amino (l'S)-(+)-10-camphorsulfonate (334.84 g, 0.806 mol, 27%, >99.5%), as a white solid.^}H NMR (400 MHz, CDCI3) 5: 0.84 (3H, s), 1.08 (3H, s), 1.30 (3H, t, J = 6.9 Hz), 1.32 - 1.43 (4H, m), 1.58 - 1.75 (3H, m), 1.89 (1H, d, 17.7 Hz), 1.95 - 2.07 (3H, m), 2.33 (1H, dt, Jj = 18.4 Hz, J₂ = 3.9 Hz), 2.53 (1H, s), 2.58 - 2.65

133/317 (1H, m), 2.69 (1H, d, J = 2.9 Hz), 2.76 - 2.79 (2H, m), 3.26 (1H, d, J = 14.1 Hz), 3.60 (1H, d, J = 7.4 Hz), 4.14 - 4.27 (2H, m), 7.80 (3H, bs).

Alternatively, /lR,2S,3R,4S/-3-ethoxycarbonyl-bicyclo [2.2.1]hept-2-yl-amino (1'S)-(+)-10-camphorsulfonate can be prepared as follows:

(rac-di-exo)- 3-Aza-triciclo[4.2.1,0²’⁵]nonan-4-one (prepared as described in Example 6f, 1220 g, 8.9 mol) was dissolved in ethyl acetate (1.7 L). The solution was heated to 50 °C and the (18)(+)-10-camphorsulfonic acid solution (2066 g, 8.9 mol) in ethanol (2.5 L) was heated to 50–75 °C for 30 min. The resulting solution was stirred at 70 °C for 2 h. More ethyl acetate (8 L) was added, lowering the temperature to >55°C, and the solution was seeded with (lJ?,2S,3R,4S)-3-ethoxycarbonylbicyclo[2.2.l]hept-2-yl-ammonium (l'S)-(+)-10-camphorsulfonate (ca. 100 mg). The mixture was allowed to cool to 25°C and stirred for 16 h. The precipitate was collected by filtration, and the wet filter cake was washed with ethyl acetate (2 x 2 L). The crude salt was dried at 25°C for 48 h, then recrystallized with ethanol (2 L) and ethyl acetate (2.5 L) to give the desired product, fll?,2S,31?,4S'j-3-ethoxycarbonyl-bicyclo[2.2.l]hept-2-yl-ammonium(l'S)-(+)-10-camphorsulfonate (920 g, 2.21 mol, 25%, >99.9% de), as a white solid.

k) Ethyl ester of (1 S,2R,3S,41k)-3-Amino-bicyclo acid [2.2. 1 ]heptane-2-carboxylic acid

<img file="BRPI0809685A2_D0117.tif" />

Ao (1R,2S,3R,4S)-3-ethoxycarbonyl-bicyclo[2.2. l]hept-2-yl-amino (rS)-(+)-10-camphorsulfonate (2.76 g, 6.64 mmol) was added

134/317 ethyl acetate (28 mL) and saturated aqueous sodium carbonate solution (28 mL) were mixed and stirred at 25 °C for 0.5 h. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (2 x 50 mL). The solution was dried over magnesium sulfate, filtered, 5 and concentrated under vacuum and dried under high vacuum for 1 h to give ethyl ester of (1S,2R,3S,4R)-3-amino-bicyclo[2.2.1]heptane-2-carboxylic acid (1.15 g, 6.28 mmol, 95%), as a colorless oil. UI NMR (400 MHz, CDC1₃) □: 1.10 - 1.26 (3H, m), 1.29 (3H, t, J = 7.0 IIz), 1.45 - 1.62 (2H, m), 1.86 (2H, bs), 1.95 (1H, dt, Ji = 10.3 Hz, = 1.9 Hz), 2.09 (III, d, J = 4.5 Hz), 2.49 (1H, d, J = 4.2 Hz), 2.56 (1H, d, J = 9.0 Hz), 3.24 (III, d,

J = 7.7 Hz), 4.09 - 4.21 (2H, m).

In order to determine the enantiomeric excess, ethyl ester of (1S,211,3S,4R)-3-amino-bicyclo[2.2.1]heptane-2-carboxylic acid was derived to the (S)-mandelate salt as follows: To a solution of 15 ethyl ester of (1S,2R,3S,4R)-3-amino-bicyclo[2.2.1]heptane-2-carboxylic acid (34.2 mg, 0.187 mmol) in ethyl acetate (1 mL) was added (S)-α-hydroxyphenylacetic acid (28.7 mg, 0.187 mmol) and the mixture was stirred at 25°C for 0.5 h. The solid was filtered and dried under high vacuum to give (lR,2S,3R,4S)-3-ethoxycarbonyl-bicyclo[2.2.l]hept-220 yl-ammonium(S)-α-hydroxyphenylacetate (11.4 mg, 0.034 mmol, 18%, of =

97%), as a white solid. UI NMR (400 MHz, CDCI3) □ : 1.08 - 1.20 (3H, m), 1.28 (311, t, J = 7.1 Hz), 1.50 - 1.59 (211, m), 1.79 (III, d, J =

10.9 Hz), 2.23 (1H, s), 2.46 - 2.48 (2H, m), 3.04 (1H, d, J = 7.8 Hz), 4.05

- 4.18 (2H, m), 4.89 (1H, s), 5.49 (3H, bs), 7.22 -7.31 (3H, m), 7.43 (211, 25 d,J = 6.9Hz).

1) Ethyl ester of /1S,2R,3S,4R)-3-(4-fluorobenzylamino)bicyclo[2.2.1]heptane-2-carboxylic acid

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<img file="BRPI0809685A2_D0118.tif" />

To a solution of ethyl ester of (1S,2R,3S,4R)-3amino-bicyclo[2.2.1]heptane-2-carboxylic acid (1.15 g, 6.28 mmol) in ethanol (30 mL) was added 4-fluorobenzaldehyde (0.68 mL, 6.31 mmol), glacial acetic acid (0.4 mL, 6.99 mmol), and sodium cyanoborohydride (1.04 g, 15.7 mmol) at 25°C. After stirring for 3 h, the mixture was diluted with ethyl acetate (50 mL) and stopped with a saturated aqueous solution of sodium bicarbonate (50 mL) for 0.5 h. The mixture was filtered through Celite. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (2 x 50 mL). When all the solvent was removed, a solid was formed. The solid was filtered, washed with water, and dried under vacuum to give the desired product, Ethyl ester of (1S,2I?,3S,4R)-3-(4-fluoro-benzylamino)-bicyclo[2.2.1]heptane-2-carboxylic acid (1.74 g, 5.97 mmol, 95%), as a white solid. Ή NMR (400 MHz, CdCl₃) □: 1.05 - 1.16 (2H, m), 1.21 (1H, dt, Ji = 8.0 Hz, J₂ = 1.6 Hz), 1.27 (3H, t, J = 7.4 Hz), 1.45 -1.61 (2H, m), 1.94 (1H, dt, Ji = 10.1 Hz, J₂ = 1.9 Hz), 2.28 (1H, d, J = 3.9 Hz), 2.43 (1H, d, J = 3.3 Hz), 2.60 (1H, dd, Ji = 8.8 Hz, J₂ = 1.5 Hz), 2.94 (1H, d, J = 7.8 Hz), 3.66 (1H, d, J = 13.2 Hz), 3.80 (1H, d, J = 13.5 Hz), 4.13 (2H, q, J = 7.0 Hz), 6.97 (2H, t, J = 8.5 Hz), 7.26 (2H, t, J = 7.1 Hz).

Alternatively, the ethyl ester of (1S,2R,3S,4R)-3-(4-fluorobenzylamino)-bicyclo[2.2.1]heptane-2-carboxylic acid can be prepared as follows:

(1 R,2S,3R_; 4Sj-3-Ethoxycarbonyl-bicyclo[2.2.l]hept.-2-yl-ammonium (l'S)-(+)-10-camphorsulfonate (prepared as described in Example 6j,

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2000 Potassium carbonate powder (1320 g, 9.62 mol) was suspended in ethyl acetate (20 L). The suspension was stirred at 25°C for 16 hours and filtered. The ethyl acetate filtrate was concentrated under vacuum to provide free amine (1050 g) as a liquid. The liquid was dissolved in ethanol (10 L), and 4-fluorobenzaldehyde (558 mL, 5.3 mol) and acetic acid (362 mL, 6.3 mol) were added, raising the temperature to 28-30°C. The solution was allowed to cool to 25°C and stirred for 30 min. A cloudy solution of sodium cyanoborohydride (756 g, 12.03 mol) in ethanol (5 L) was added over 20 min, raising the temperature to 45-50°C. The mixture was allowed to cool to 25°C and stirred for 16 h. The mixture was concentrated under vacuum to a volume of approximately 13-14 L. Water (1-2 L) was added, and the resulting mixture was further concentrated under vacuum. A saturated aqueous solution of sodium bicarbonate (4 L) and water (4 L) were added with stirring. The pH was adjusted to 8.08.5 by adding an additional saturated aqueous solution of sodium bicarbonate (-500 mL). The mixture was stirred for 1 h before the solids were collected by filtration and the wet filter cake was washed with water (2 L). The solid was dried under vacuum at 35°C for 64 h to give the desired product, ethyl ester of (1S,2R,3S,4R)-3-/4-fluorobenzylamino)-bicyclo[2.2. l]heptane-2-carboxylic acid (1350 g, 4.63 mol, 96%), as a white solid.

m) Ethyl ester of acid (1S,2R,3S,4R)-3-{(4-fluorobenzyl)[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-acetyl]-amino}-bicyclo[2.2. l]heptane-2-carboxylic acid

<img file="BRPI0809685A2_D0119.tif" />

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To a solution of ethyl ester of (18,217,38,417)-3-(4-fluorobenzylamino)-bicyclo[2.2.1]heptane-2-carboxylic acid (100.6 mg, 0.345 mmol) in α,α-dimethylformamide (3.0 mL) was added (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1α⁶-benzo[1,2,4]thiadiazin3-yl)-acetic acid (prepared as described in Example 1g, 120.8 mg, 0.362 mmol), 4-dimethylaminopyridine (10.6 mg, 0.086 mmol), and l-[3(dimethylamino)propyl]-3-ethylcarbodiimide chloride (70.9 mg, 0.362 mmol). After stirring at 25°C for 12 h, the mixture was diluted with ethyl acetate and acidified with 1.0 M aqueous hydrochloric acid solution to pH 1. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (2 x 20 mL). The combined organic layer was dried over magnesium sulfate, filtered, and concentrated under vacuum, and dried under high vacuum to provide the crude product, ethyl ester of (1S,2R,3S,417)-3-{(4-fluorobenzyl)-[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1-⁶-benzo[1,2,4]thiadiazin-3-yl)-acetylj-amino}bicyclo[2.2.l]heptane-2-carboxylic acid, as a weak yellow oil. The crude product was used in a subsequent step without further purification. LC-MS (ESI) calcd for C27II31FN4O7S2 606.16, found 607.2 [M+H⁺]·

n) lV-{3-[(117.2S,717.8S)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo [1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0120.tif" />

A solution of ethyl ester of the acid (1S,2R,3S,4R)-3-{(4

138/317 fluoro-benzyl)-[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- 1λ⁶Crude benzo[l,2,4]thiadiazin-3-yl)-acetyl]-amino}-bicyclo[2.2.l]heptane-2-carboxylic acid in absolute ethanol (3 mL) was added to a 21 wt% sodium ethoxide solution in ethanol (0.51 mL, 1.37 mmol). After stirring at 60°C for 2 h, the mixture was diluted with ethyl acetate and acidified with 1.0 M aqueous hydrochloric acid solution at pH 1. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (2 x 20 mL). The combined organic layer was dried over magnesium sulfate, filtered, and concentrated under vacuum. The crude mixture was purified by flash column chromatography (Teledyne Isco RediSep column; 0 to 100% ethyl acetate in hexanes) to provide the desired product, A-[3-[(1R,2S,7R,8S)-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-l, 1-dioxo-l,4-dihydro-lÀ⁶-benzo[l,2,4]thiadiazin-7-yl}-methanesulfonamide (131.5 mg, 0.235 mmol, 68% in two steps), as a dirty white solid. Ή NMR (400 MHz, CDCh) □: 1.28 (2H, d, J = 11.0 Hz), 1.47 (1H, t, J = 10.8 Hz), 1.57 - 1.74 (3H, m), 2.56 (1H, d, J = 3.2 Hz), 2.75 (III, d, J = 2.3 Hz), 2.96 (HI, d, J = 9.2 Hz), 3.02 (311, s), 3.58 (1H, d, J = 9.2 Hz), 4.42 (III, d, J = 15.5 Hz), 5.03 (1H, d, J = 15.7 Hz), 7.04 (2H, t, J = 8.5 Hz), 7.31 (2H, dd, Ji = 7.9 Hz, J₂ = 5.5 Hz), 7.37 (III, d, J = 8.8 Hz), 7.54 (III, dd, Ji = 8.3 Hz, J₂ - 2.3 Hz), 7.69 (1H, d, J = 2.3 Hz). LC-MS (ESI) calcd for C25H25FN4O6S2 560.12, found 561.4 [M+H⁺]. ee = 98.5% [HPLC analysis: Chiralpak AS-RH 2.1 x 150 mm, 5 microns at rT, Solvent A Solvent B (see table for gradient), 0.3 mL/min, 312 nm, 11 = 7.58 min (main), 12 = 8.95 min].

Alternatively, A-{3-[(1R,2S,7R,8S)-3-/4-fluoro-benzyl)-6hydroxy-4-oxo -3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[l,2,4]thiadiazin-7-yl}-methanesulfonamide can be prepared as follows:

Acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- 1λ⁶

139/317 benzo[1,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 1.88 kg, 5.63 mol) and ethyl ester of (1S,2R,3S,4R)-3-(4-fluorobenzylamino)-bicyclo[2.2.1]heptane-2-carboxylic acid (prepared as described in Example 61, 1.72 kg, 5.91 mol) were dissolved in acetonitrile (18.8 L) at 23°C. TV-Methylmorpholine (1.25 kg, 12.4 mol) was added and the resulting suspension was stirred at 23°C for 1 h. The suspension was cooled to 0°C and l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide chloride (1.19 kg, 6.20 mol) was added in portions. The mixture was stirred at 0°C for 3 h, then allowed to warm to 23°C and stirred overnight. Triethylamine (1.88 kg, 18.6 mol) was added and the mixture was then heated to 50°C for 3 h. The mixture was partially concentrated under vacuum at 45°C, then diluted with ethyl acetate (22.5 L) and washed with 2.0 M aqueous hydrochloric acid solution (22.6 L). The resulting aqueous fraction was extracted with ethyl acetate (2 x 9.4 L). The combined organic extracts were washed with 1.0 M aqueous hydrochloric acid solution (10.4 L) and then with water (18.8 L). The resulting organic fraction was filtered through Celite (600 g), and the filtrate was then partially concentrated under vacuum at 45°C. Absolute ethanol (5.6 L) was added to the residue, and the mixture was then heated to 50°C with stirring. Dichloromethane (400 mL) was added in portions until crystallization began. Absolute ethanol (20.7 L) was added in portions over 1 h, and the resulting mixture was stirred at 23°C.^DC overnight. The mixture was filtered and the solid was then washed with absolute ethanol (1.9 L). The solid was further dried under vacuum at 45°C to give the desired product, A-{3-[(lR,2S,7R,8S)-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1,0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo-[l,2,4]-thiadiazin-7-yl} methanesulfonamide (2.46 kg, 4.39 mol, 78%), as a dirty white crystalline solid.

X-ray data - Graph 1 shows an X-ray diffraction of N-{3-[lR,2S,7R,8S/-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-azatricyclo[6. 2.1.0²7] undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin

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-7-yl}-methanesulfonamide (as prepared in Example 6 on a kg scale). In the X-ray plot, the diffraction angle 2θ is plotted on the x-axis and the peak intensity is plotted on the y-axis. The strongest lines in the X-ray diffraction plot are observed at angles of 6.2°, 17.9°, 19.7°, 20.5°.^the, 22.6°, and 24.8° ± 0.3°, with lines of lower intensity at 12.4°, 16.5°, 18.7°, 21.6°, 23.2°, 24.1°, 25.6°, 26.6°, 27.1°, 28.5°, and 29.3°.

Figure 1 - X-ray diffraction in powder

<img file="BRPI0809685A2_D0121.tif" />

IR Data - Graph 2 shows a PT-Raman spectrum of 7V-{3-[(1R,2S,7R,8S)-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²'⁷]-undec-5-en-5-yl]-l,l-dioxo-l,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (as prepared in Example 6 on a kg scale), which is characterized by the following principal bands at 1617, 1524, 1321, 1260, 1229, 1217, and 1163 cm⁻¹^-1, with secondary bands at 1498, 1465, 1147, 836, 727, and 406 cm-¹.

Graph 2. H'-Raman spectrum

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<img file="BRPI0809685A2_D0122.tif" />

Example 7 lV-{3-[(1S,2R,7S,8R)-3-(4-fluoro-benzyl)-6-hydroxy)xy-4-oxo-3-azatricyclo[6.2.1.0²-⁷]undec-5-en-5-yl]-1, 1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0123.tif" />

a) (1R,2R,3S,4S)-3-(methoxycarbonyl)bicyclo[2.2.1]hept- acid

5-ene-2-carboxylic acid

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<img file="BRPI0809685A2_D0124.tif" />

The starting material (a) was prepared as described in J. Org. Chem. 2000, 65, 6984-6991. cis-5-Norbonene-exo-2,3-dicarboxylic anhydride (5 g, 30.45 mmol) was suspended in a 1:1 mixture of 5-toluene and carbon tetrachloride (150 mL). The mixture was stirred for 10 min. Quinidine (10.9 g, 33.5 mmol) was added and the flask was degassed and back-fed with nitrogen. The solution was cooled to 55°C. While stirring, methanol (3.7 mL, 91.35 mmol) was added. The mixture was stirred at -55°C for 16 h. Upon heating to 25°C, the mixture was concentrated under vacuum to provide a foam. The foam was dissolved in a mixture of ethyl acetate (400 mL) and 1.0 M aqueous hydrochloric acid solution (400 mL). The layers were separated and the organic layer was further washed with 1.0 M aqueous hydrochloric acid solution (2 x 100 mL), saturated aqueous brine solution 15 (100 mL) and dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the desired product, (lR,2R,3S,4S)-3(methoxycarbonyl)bicyclo[2.2.l]hept-5-ene-2-carboxylic acid (5.92 g, 30.2 mmol, 99%), as a clear oil.^XH NMR (400 MHz, DMSO-dej δ: 1.29 (1 H, d, J= 10.2 Hz), 1.96 (1H, d, J= 8.6 Hz), 2.47 - 2.49 (2H, m), 2.93 20 2.94 (2H, m), 3.51 (3H, s), 6.15 - 6.20 (2H, m), 12.15 (1H, s).

b) (1S,2S, 3R, 4R)-3-{[(benzyloxy)carbonyl]amino}bicyclo[2.2.1] methyl hept-5-ene-2-carboxylate

<img file="BRPI0809685A2_D0125.tif" />

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Intermediate (b) was prepared as described in Synthesis 2001, 11, 1719-1730. (1R,2R,3S,4S)-3-(methoxycarbonyl)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (5.9 g, 30 mmol) was dissolved in anhydrous tetrahydrofuran (133 mL). The flask was degassed and fed back with nitrogen and the mixture was cooled to 0°C. Triethylamine (12.64 mL, 90 mmol) was added followed by the dropwise addition of ethyl chloroformate (5.72 mL, 60 mmol) with vigorous stirring. Immediate precipitation was observed. The mixture was stirred at 0°C for 1 h. Sodium azide (5.86 g, 90 mmol) was dissolved in water (40 mL) and added to the reaction mixture at 0°C. The mixture was stirred at 0°C for 5 min. The ice bath was removed. The mixture was heated to 25°C and continued to be stirred for 2 h. The mixture was poured into water (300 mL) and the product extracted in ethyl acetate (350 mL). The organic layer was further washed with a half-saturated aqueous sodium bicarbonate solution (2 x 100 mL), a saturated aqueous brine solution (100 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a light brown oil.

The oil was dissolved in anhydrous benzene (66 mL) and refluxed with stirring under nitrogen for 2 h. Upon cooling to 25°C, the solution was concentrated under vacuum to yield a light yellow oil. The oil was dissolved in dichloromethane (40 mL) and benzyl alcohol (3.41 mL, 33 mmol) was added followed by triethylamine (8.44 mL, 60 mmol). The mixture was refluxed under nitrogen for 16 h. Upon cooling to 25°C, the solution was concentrated under vacuum to yield a thick oil. Purification by flash column chromatography (Merck silica gel 60, 40-63 μm; I^the column: 3:1 hexanes/ethyl acetate; 2^the column: 2:4:1 dichloromethane/pentane/diethyl ether) yielded the desired product, methyl (1S,2S,3R,4R^3-{[(benzyloxy)carbonyl]amino}bicyclo[2.2.1]hept-5-ene-2-carboxylate (6.195 g, 20.58 mmol, 69%), as a pale yellow oil. 1H NMR (400 MHz, CDC1₃) δ: 1.60 (1H, d, J = 9.4 Hz), 1.97 (1H, d, J = 9.3 Hz), 2.66 (1H, d, J = 7.5 Hz), 2.75 (1H, s), 2.96 (1H, s), 3.60 (3H, s),

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4.02 (1H, t, J= 8.9 Hz), 5.09 (2H, q, J = 10.5 Hz), 5.47 (1H, d, J= 8.8 Hz), 6.18 - 6.23 (2H, m), 7.29 -7.37 (5H, m). LC-MS (ESI) calcd for C17II19NO4 301.13, found 258.1 (100%), 302.2 [M+H⁺] (70%), 603.4 [2M+H⁺] (20%).

c) Methyl chloride (1R,2S,3R,4S)-3-aminobicyclo[2.2.1] heptane-2-carboxylate

THE

<img file="BRPI0809685A2_D0126.tif" />

Methyl (1R,2S,3R,4S)-3-{[(benzyloxy)carbonyl]amino} bicyclo[2.

2.1] Hept-5-ene-2-carboxylate (1 g, 3.32 mmol) was dissolved in ethyl acetate (15 mL). Palladium in carbon 5% (120 mg) was added. The flask was degassed and fed back with hydrogen gas via a balloon. The mixture was stirred at 25°C for 16 h. The mixture was passed through a Celite plug and the filtrate was concentrated under vacuum to provide a thick, clear oil. The oil was dissolved in diethyl ether (10 mL) and added dropwise, with vigorous stirring, to a mixture of 4.0 M hydrochloric acid solution in 1,4-dioxane (1.8 mL, 7.2 mmol) in diethyl ether (18 mL). The desired product began to precipitate as a white solid. Additional diethyl ether (10 mL) was added and the mixture was stirred for 10 min. The precipitate was collected by vacuum filtration and washed with additional diethyl ether (2 x 8 mL). The solid was further dried under vacuum for 1 h to give the desired product, (lR,2S,3R,4S)-3-aminobicyclo[2.2.l]heptane-2-carboxylate methyl chloride (0.554 g, 2.7 mmol, 81%), as a soft powder.^]H NMR (400 MHz, DMSO-de) δ: 1.18 -1.27 (3H, m), 1.37- 1.61 (2H, m), 1.90 (1H, d, J = 11.0 Hz), 2.35 (1H, d, J= 3.8 Hz), 2.44 (1H, d, J = 3.1 Hz), 2.75 (1H, d, J = 8.7 Hz), 3.29 - 3.34 (1H, m), 3.61 (3H, s), 8.03 (3H, bs). LC-MS (ESI) calculated for C9II15NO2 (free amine) 169.11, found 170.3 [M+H⁺]

145/317 (100%), 339.3 [2M+H⁺] (50%).

d) (1R,2S,3R,4S)-3-[(4-fluoro-benzyl)amino]bicyclo[2.2.1] methyl heptane-2-carboxylate

<img file="BRPI0809685A2_D0127.tif" />

Methyl (lR,2S,3R,4S)-3-aminobicyclo[2.2.1]heptane-2-carboxylate chloride (0.5 g, 2.43 mmol) was dissolved in methanol (12 mL). Sodium acetate (0.4 g, 4.86 mmol) was added followed by powdered 4A molecular sieves (0.5 g) and 4-fluorobenzaldehyde (0.302 g, 2.43 mmol). Sodium cyanoborohydride (0.305 g, 4.86 mmol) was added and the mixture was stirred at 25°C for 3 h. The mixture was poured into ethyl acetate (300 mL) and stirred with saturated aqueous sodium bicarbonate solution (200 mL). Both layers were passed through a Celite plug. The organic layer was further washed with saturated aqueous sodium bicarbonate solution (100 mL), saturated aqueous brine solution (100 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the crude product, flR,2S,3R,4S)-3-[(4-fluoro-benzyl)amino]bicyclo[2.2. [l]heptane-2-carboxylate methyl (0.675 g, 2.43 mmol, 99%), as a clear oil. LC-MS (ESI) calcd for C16H20FNO2 277.15, found 278.2 [M+IF].

e) 7V-{3-[(15,2R,7S,8R/-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3aza-tricyclo-[6.2.1.0²>⁷]undec-5-en-5-yl]-1, 1-dioxo-1,4-dihydro-lÃ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

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<img file="BRPI0809685A2_D0128.tif" />

Methyl /1R,2S,3R,4S)-3-[(4-fluorobenzyl)amino]bicyclo[2.2.1]heptane-2-carboxylate (0.6 g, 2.16 mmol) was dissolved in anhydrous N,N-dimethylformamide (20 mL). (7-methanesulfonylamino-1,1dioxo-1,4-dihydro-1A⁶-benzo[1,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 0.72 g, 2.16 mmol) was added followed by α-methylmorpholine (0.5 mL, 4.54 mmol). The mixture was stirred until everything was dissolved, approximately 5 min. l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.435 g, 2.27 mmol) was added and the mixture was stirred at 25°C for 45 min. Triethylamine (0.91 mL, 6.48 mmol) was added and the mixture was stirred at 50°C for 16 h. Upon cooling to 25°C, the solution was diluted with ethyl acetate (300 mL) and washed with 1.0 M aqueous hydrochloric acid solution (3 x 300 mL), saturated aqueous brine solution (100 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a golden oil. Purification by flash column chromatography (Merck silica gel 60, 40-63 pm, 0 to 0.75% methanol in dichloromethane) yielded the product as a white foam. The foam was dissolved in methanol (10 mL) and the product was precipitated by adding a 1.0 M aqueous hydrochloric acid solution (20 mL) with stirring. The solid was collected by vacuum filtration and further dried under vacuum to yield the desired product, A-{3[(1S,2R,7S,8R)-3-/4-fluorobenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo{6.2.1.0²,⁷]undec-5-en-5-yl]-1, 1-dioxo-1,4-dihydro-lÀ^the-benzo[1,2,4]thiadiazin-7yl}-methanesulfonamide (0.592 g, 1.06 mmol, 49%), as a white powder. NMR (400 MHz, DMSO-d₆) δ: 1.15 - 1.22 (2H, m), 1.39 - 1.61

147/317 (4H, m), 2.49 - 2.55 (1H, m), 2.62 - 2.63 (1H, m), 3.02 (1H, d, J = 9.8 Hz), 3.05 (3H, s), 3.52 (1H, d, J = 9.3 Hz), 4.41 (1H, d, J= 15.5 Hz), 4.95 (1H, d, J = 15.5 Hz), 7.14 (2H, t, J = 8.7 Hz), 7.32 (2H, dd, Ji = 8.2 Hz, J₂ = 5.7 Hz), 7.50 (1H, dd, Ji = 8.4 Hz, J₂ = 2.4 Hz), 7.55 -7.57 (2H, m), 5 10.17 (1H, s). LC-MS (ESI) calcd for C25H25FN4O6S2 560.12, found 561.3 [M+H⁺], ee = 96% [HPLC analysis: Chiralpak AS-RH 2.1 x 150 mm, 5 microns at rt, Solvent A - Solvent B (see table for gradient), 0.3 mL/min, 312 nm, t1 = 4.3 min, t2 = 6.0 min (main)].

Example 8 (rac-di-exo)-A-í3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-11-oxa-3-azatricyclo[6.2.1.0²3]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lA⁶-benzo [l^Altiadiazin-T-iU-methanesulfonamide

<img file="BRPI0809685A2_D0129.tif" />

a) Methyl ester of (rac-di-exo)-3-amino-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid

<img file="BRPI0809685A2_D0130.tif" />

To a stirred solution of (rac-di-exo)-3-amino-7-oxabicyclo[2.2.l]heptane-2-carboxylic acid (1.0 g, 6.37 mmol) in methanol

148/317 anhydrous and benzene (1:1, 20 mL), a 2.0 M solution of (trimethylsilyl)diazomethane in diethyl ether (6.37 mL, 12.7 mmol) was added dropwise. The resulting mixture was stirred and concentrated under vacuum to provide the desired product, methyl ester of (rac-di-exo)-3amino-7 oxa-bicyclo[2.2.1]heptane 2-carboxylic acid (1.02 g, 5.85 mmol, 94%), as a colorless oil.^XH NMR (400 MHz, CDC1₃) δ: 1.53 (2H, m), 1.62 (2H, m), 2.68 (1H, d, J = 7.6 Hz), 3.27 (1H, d, J = 7.6 Hz), 3.59 (3H, s), 4.14 (1H, d, J = 6.0 Hz), 4.67 (1H, d, J = 4.8 Hz).

b) (rac-di-exo)-3-(4-fluoro-benzyl-amino)-7-oxa-bicyclo[2.2] acid methyl ester. l]heptane-2-carboxylic acid

THE

<img file="BRPI0809685A2_D0131.tif" />

4-Fluorobenzaldehyde (0.62 mL, 5.85 mmol) was added to a solution of (racdiexo)-3-amino 7-oxabicyclo[2.2.l]heptane-2-carboxylic acid methyl ester (1.02 g, 5.85 mmol) in anhydrous methanol (20 mL) at 25°C under a nitrogen atmosphere. After stirring for 10 min, glacial acetic acid (0.8 mL) and sodium cyanoborohydride (920 mg, 14.6 mmol) were added sequentially, and the resulting mixture was stirred at 25°C for 18 h. The reaction mixture was poured into a saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate. The combined organic layers were washed with a saturated aqueous brine solution, dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was dried under high vacuum to give the desired product, methyl ester of (rac-di-exo)-3-(4-fluorobenzylamino)-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid (1.40 g, 5.02 mmol, 86%), as a colorless oil.^XH NMR (400 MHz, CDCI3) δ:

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1.73 (4I-I, m), 2.86 (1H, d, &lt;7 = 8.0 Hz), 3.15 (1H, d, J = 8.0 Hz), 3.68 (1H, d, J = 13.6 Hz), 3.73 (3H, s), 3.84 (1H, d, J = 13.6 Hz), 4.46 (1H, d, J = 5.2 Hz), 4.73 (1H, d, J= 4.8 Hz), 6.99 (2H, m), 7.26 (2H, m).

c) (rac-di-exo)-3-{(4-fluoro-benzyl)-[2(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1λ) acid methyl ester⁶benzo[1,2,4]thiadiazin-3-yl)-acetyl]-amino}-7-oxa-bicyclo[2.2.l]heptane-2carboxylic acid

F

To a stirred solution of methyl ester of (rac-diexo)-3-(4-fluoro-benzylamino)-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid (190 mg, 0.68 mmol) in anhydrous α,α-dimethylformamide (4 mL) under a nitrogen atmosphere, (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1α) acid⁶-benzo[1,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 185 mg, 0.55 mmol), IV-methylmorpholine (149 gL, 1.36 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (130 mg, 0.68 mmol) were added sequentially. After stirring at 25°C for 3 h, the reaction mixture was poured into 1.0 M aqueous hydrochloric acid solution and extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous brine solution, dried over sodium sulfate, filtered, and concentrated under vacuum. The crude product, methyl ester of (rac-di-exo)-3-{(4-fluoro-benzyl)-[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1A⁶-benzo[1,2,4]thiadiazin3-yl)-acetyl]-amino}-7-oxa-bicyclo[2,2,1]heptane-2-carboxylic acid was used in the next step without further purification. LC-MS (ESI) calculations were performed to

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C25H27FN4O8S2 594.13, found 595.2 [M+H⁺].

d) (rac-di-exo)-A-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-1 1oxa-3-aza-tricyclo[6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo- 1,4-dihydro- 1À⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0132.tif" />

A stirred solution of the methyl ester of (rac-diexo)-3-{(4-fluorobenzyl)-[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-l^theCrude -benzo[1,2,4]thiadiazin-3-yl)-acetyl]-amino}-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid in absolute ethanol (8 mb) under a nitrogen atmosphere, a 21% by weight solution of sodium ethoxide in ethanol (0.81 mL, 1.10 mmol) was added. The mixture was stirred at 60°C for 30 min, and then cooled to 25°C. A 1.0 M aqueous solution of hydrochloric acid (4 mL, 4 mmol) was slowly added to the mixture, in which a white solid precipitated. The suspension was shaken for 15 min, filtered through a funnel filter and washed with water. The solid was collected, dried under high vacuum to provide (rac-di-exo)-7V-{3-[3-(4-fluorobenzyl)-6-hydroxy-4-oxo-l l-oxa-3-aza-tricyclo[6.2.1.0²>⁷]undec-5-en-5-yl]1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide analytically pure (165 mg, 0.29 mmol, 53% over two steps), as a dirty white solid. Ή NMR (400 MHz, DMSO-dg) δ: 1.67 (4H, m), 3.05 (3H, s), 3.32 (1H, m), 3.79 (1H, d, J = 9.2 Hz), 4.39 (1H, d, J = 15.6 Hz), 4.69 (1H, d, J = 4.4 Hz), 4.76 (1H, d, J = 3.6 Hz), 5.04 (HI, d, J = 15.6 Hz), 7.16 (2H, m), 7.33 (2H, m), 7.49 (1H, dd, J = 9.2 Hz), 7.56 (2H, m), 10.17 (1H, s), 13.89 (1H, s). LC-MS (ESI) calcd for

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C24H23FN4O7S2 562.1 O, found 563.4 [M+H⁺].

Example 9 (rac-di-exo)-A-f3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²’⁷]undeca-5,9-dien-5-yl]-l, l-dioxo-l,4-dihydro-lÀ⁶benzoph 1,2,4]thiadiazin-7-yl}-methanesulfonamide

OH N

F

a) Methyl ester chloride of (rac-di-exo)-3-aminobicyclo[2.2.l]hept-5-ene-2-carboxylic acid

THE

OMe

NH₂'HCI

(rac&lt;ü-exo)-3-amino-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid chloride (1 g, 5.27 mmol) was dissolved in methanol (7 mL). Benzene (10 mL) was added followed by dropwise addition of a 2.0 M solution of (trimethylsilyl)diazomethane in dichloromethane (5 mL, 10 mmol). The yellow solution was stirred at 25°C for 10 min. An additional 2.0 M solution of (trimethylsilyl)diazomethane in dichloromethane (2 mL, 4 mmol) was added. The yellow solution was stirred at 25°C for 10 min. The solution was concentrated under vacuum to give a yellow oil. The oil was dissolved in methanol (15 mL) and concentrated under vacuum to give the desired product, methyl ester chloride of (rac-diexo)-3-amino-bicyclo[2.2.l]hept-5-ene-2-carboxylic acid (1.07 g, 5.25 mmol,

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99%), as a yellow oil. LC-MS (ESI) calcd for C9I-I13NO2 (free amine) 167.09, found 168.2 [M+H⁺] (100%), 335.4 [2M+H⁺] (25%).

b) methyl ester of (rac-di-exo)-3-(4-fluorobenzylamino)-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid

THE

NH

Methyl ester chloride of (racMi-exo)-3-aminobicyclo[2.2.l]hept-5-ene-2-carboxylic acid (1.07 g, 5.25 mmol) was suspended in methanol (23 mL). Sodium acetate (0.865 g, 10.54 mmol) was added followed by 4-alpha molecular sieves (1 g) and 4-fluorobenzaldehyde (0.621 g, 5 mmol). Sodium cyanoborohydride (0.662 g, 10.54 mmol) was added and the mixture was stirred at 25°C for 16 h. The mixture was poured into a 1:1 mixture of saturated aqueous sodium bicarbonate solution (200 mL) and ethyl acetate (200 mL). The mixture was stirred and the layers separated. The organic layer was further washed with saturated aqueous brine solution (50 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to give the crude product, methyl ester of (rac-di-exo)-3-/4-fluoro-benzy-1amino)-bicyclo[2.2.1]hept5-ene-2-carboxylic acid (1.044 g, 3.79 mmol, 76%), as a clear oil. LCMS (ESI) calcd for CieHisFNOa 275.13, found 276.2 [M+H⁺].

c) (rac-di-exo)-IV-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricycle [6.2.1.0² ’⁷]undeca-5,9-dien-5-yl]-l, 1-dioxo-l,4-dihydro-lÀ^the-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

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<img file="BRPI0809685A2_D0133.tif" />

Methyl ester of (rac-di-exo)-3-(4-fluoro-benzylamino)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (0.083 g, 0.3 mmol) was dissolved in anhydrous 7V,7V-dimethylformamide (2.8 mL). (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1)⁶-benzo[1,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1, 0.1 g, 0.3 mmol) was added followed by N-methylmorpholine (0.07 mL, 0.63 mmol). The mixture was stirred until everything was dissolved, approximately 5 min. l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.061 g, 0.315 mmol) was added and the mixture was stirred at 25 °C for 45 min. Triethylamine (0.126 mL, 0.9 mmol) was added and the mixture was stirred at 50 °C for 16 h. After cooling to 25 °C, the solution was diluted with 1.0 M aqueous hydrochloric acid solution (8 mL). The resulting precipitate was collected by vacuum filtration, dissolved in methanol, and concentrated under vacuum to give the crude product as a beige powder. Purification by flash column chromatography (Merck silica gel 60, 40-63 μTn, 0 to 0.75% methanol in dichloromethane) yielded the product as a white foam. The foam was ground with a 1:1 mixture of diethyl ether and hexanes (6 mL) and the resulting solid was collected by vacuum filtration. The solid was vacuum dried for 16 h to give the desired product, (rac-di-exo)-7V-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²-⁷]undeca-5,9-dien-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (40.3 mg, 0.072 mmol, 24%), as a white powder. NI NMR (400 MHz, DMSO-d₆) δ: 1.37 (III, d, J = 9.5 Hz), 1.62 (1H, d, J = 9.4 Hz), 2.81 - 2.89 (1H, m), 3.05 (3H, s), 3.19 154/317

3.40 (4H, m), 4.52 (1H, d, J = 15.5 Hz), 5.04 (1H, d, J = 14.8 Hz), 6.13 (1H, dd, JI = 5.5 Hz, J₂ = 3.1 Hz), 6.35 (1H, dd, JI = 5.9 Hz, J₂ = 2.5 Hz), 7.14 (2H, t, J= 9.1 Hz), 7.34 (2H, dd, Ji - 7.8 Hz, J₂ = 5.6 Hz), 7.49 (1H, dd, Ji = 8.8 Hz, J₂ = 2.0 Hz), 7.54 - 7.59 (2H, m), 10.16 (1H, s). LC5 MS (ESI) calcd for C₂5H₂3FN4OqS₂ 558.10, found 559.1 [M+H⁺].

Example 10 (rac-di-endo)-/V-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²>⁷]undeca-5,9-dien-5-yl]-l,l-dioxo-l,4-dihydro-lÃ⁶-benzo[1,2,4]thiadiazin-7-ill-methanesulfonamide

<img file="BRPI0809685A2_D0134.tif" />

a) Ethyl ester of (rac-di-endo)-3-(4-fluorobenzylamino)-bicyclo[2.2. l]hept-5-ene-2-carboxylic acid

<img file="BRPI0809685A2_D0135.tif" />

<img file="BRPI0809685A2_D0136.tif" />

^v F

Ethyl ester chloride of /rcic-di-endo)-3-amino15 bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (1 g, 4.6 mmol) was suspended in methanol (23 mL). Sodium acetate (0.753 g, 9.18 mmol) was added followed by powdered molecular sieves of 4α (1 g) and 4-fluorobenzaldehyde (0.57 g, 4.59 mmol). Sodium cyanoborohydride (0.577 g,

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9.18 mmol) was added and the mixture was stirred at 25°C for 16 h. The mixture was poured into a 1:1 mixture of saturated aqueous sodium bicarbonate solution (200 mL) and ethyl acetate (200 mL). The mixture was stirred and the layers separated. The organic layer was further washed with saturated aqueous brine solution (50 mL), dried over magnesium sulfate, filtered and concentrated under vacuum to provide the crude product, methyl ester of (rac-di-endo)-3-(4-fluoro-benzylamino)bicyclo[2.2. l]hept-5-ene-2-carboxylic acid (1.18 g, 4.08 mmol, 88%), as a clear oil. LC-MS (ESI) calcd for C17H20FNO2 289.15, found

290.2 [M+H⁺],

b) (rac-di-endo)-A-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3aza-tricycle [6.2.1.0²’⁷]undeca-5,9-dien-5-yl]-1,1 -dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0137.tif" />

Methyl ester of (rac-di-endo)-3-(4-fluoro-benzylamino)bicyclic[2.2.1]hept-5-ene-2-carboxylic acid (0.087 g, 0.3 mmol) was dissolved in anhydrous α,α-dimethylformamide (2.8 mL). Acid (7methanesulfonylamino-1,1-dioxo-1,4-dihydro-1α⁶-benzo[1,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 0.1 g, 0.3 mmol) was added followed by N-methylmorpholine (0.07 mL, 0.63 mmol). The mixture was stirred until everything was dissolved, approximately 5 min. l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.061 g, 0.315 mmol) was added and the mixture was stirred at 25 °C for 45 min. Triethylamine (0.126 mL, 0.9 mmol) was added and the mixture was stirred.

156/317 at 50 °C for 16 h. After cooling to 25 °C, the solution was diluted with 1.0 M aqueous hydrochloric acid solution (8 mL, 8 mmol). The resulting precipitate was collected by vacuum filtration, dissolved in methanol, and concentrated under vacuum to give the crude product as a beige powder. Purification by flash column chromatography (Merck silica gel 60, 4063 pm, 0 to 0.75% methanol in dichloromethane) gave the product as a white foam. The foam was ground with a 1:1 mixture of diethyl ether and hexanes (6 mL) and the resulting solid was collected by vacuum filtration. The solid was dried under vacuum for 16 h to give the desired product, (rac-&lt;ii-endo)-A-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo3-aza-tricyclo[6.2.1.03'⁷]Undeca-5,9-dien-5-yl]~ 1,1-dioxo-1,4-dihydro-lÀ⁶benzo[l,2,4]thiadiazin-7-yl}-methanesulfonamide (69.3 mg, 0.124 mmol, 41%), as a white powder. NMR (400 MHz, DMSO-cZ6j δ: 1.28 - 1.28 (2H, m), 3.33 - 3.41 (3H, m), 3.92 - 4.00 (1H, m), 4.33 (1H, d, J = 14.7 Hz), 4.96 (1H, d, J= 15.6 Hz), 5.89 - 5.92 (1H, m), 6.11 - 6.13 (1H, m), 7.10 (2H, t, J = 9.1 Hz), 7.35 (2H, dd, Ji = 8.2 Hz, = 5.9 Hz), 7.43 (III, dd, Ji = 8.4 Hz, = 2.4 Hz), 7.48 - 7.50 (2H, m), 10.10 (1H, s). LC-MS (ESI) calcd for C25H23FN4O6S2 558.10, found 559.0 [M+H⁺],

Example 11

N-{3-[(1S,2R,7S,8R)-3-(4-fluor-3-methyl-benzyl)-6-hydroxy-4-oxo-1-oxa-3aza-tricycle [6.2,1.0²>⁷]undec-5-en-5-yl1-1,1-dioxo-1,4-dihydro- 1À⁶benzoph 1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0138.tif" />

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a) (1S,2/?,3S,4/?)-3-(methoxycarbonyl)-7-oxa-bicyclo[2 acid.

2.1] heptane-2-carboxylic

<img file="BRPI0809685A2_D0139.tif" />

exo-4,10-Dioxa-tricycle[5,2.1.0²’⁶Decane-3,5-dione (5.10 g, 30.3 mmol) was dissolved in a 1:1 mixture of toluene and carbon tetrachloride (600 mL). The mixture was cooled to -55°C under a nitrogen atmosphere, and then quinine (10.54 g, 32.5 mmol) was added. Methanol (3.59 mL, 90 mmol) in a 1:1 mixture of toluene and carbon tetrachloride (30 mL) was added slowly via an addition funnel. The suspension was stirred at -55°C for 60 h and then allowed to warm to 25°C. The mixture was concentrated under vacuum and the residue was dissolved in ethyl acetate (400 mL), washed with 1.0 M aqueous hydrochloric acid solution (2 x 300 mL) and saturated aqueous brine solution, dried over magnesium sulfate and filtered. The filtrate was concentrated under vacuum to give the desired product, (1S,2R,3S,4R)-3-(methoxycarbonyl)-7-oxa-bicyclo[2,2] acid. l]carboxylic heptane-2 (2.46 g, 12.3 mmol, 41 %), as a clear oil, IU NMR (400 MHz, DMSO-dgj δ: 1.49 - 1.53 (4H, m), 2.99 (2H, s), 3.50 (3H, s), 4.66 (211, m), 12.15 (1H, s).

b) (1R,2S,3R,4S)-3-{[(benzyloxy)carbonyl]amino}-7-oxa-bicyclo [2.2.1] methyl heptane-2-carboxylate

THE

OMe

<img file="BRPI0809685A2_D0140.tif" />

(1S,27?,3S,4J?)-3-(methoxycarbonyl)-7-oxa-bicyclo[2.2.1] acid

158/317 heptane-2-carboxylic acid (2.46 g, 12.3 mmol) was dissolved in anhydrous tetrahydrofuran (35 mL) and cooled to -10°C under a nitrogen atmosphere. Triethylamine (5.13 mL, 36.9 mmol) was added followed by the dropwise addition of ethyl chloroformate (2.35 mL, 24.6 mmol) with vigorous stirring. Immediate precipitation was observed. The mixture was stirred at -10°C for 1 h. Sodium azide (2.40 g, 36.9 mmol) was dissolved in water (17 mL) and added to the reaction mixture at -10°C. The mixture was stirred at -10°C for 15 min, then heated to 25°C and stirred for 2 h. The mixture was poured into water (100 mL) and extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium bicarbonate solution and saturated aqueous brine solution, dried over magnesium sulfate, and filtered. The filtrate was concentrated under vacuum to give the acyl azide intermediate as a clear oil. The oil was dissolved in anhydrous benzene (80 mL) and refluxed for 2 h under a nitrogen atmosphere. The solution was allowed to cool to 25°C and concentrated under vacuum to yield a yellow oil. The oil was dissolved in dichloromethane (45 mL). Triethylamine (3.46 mL, 24.6 mmol) and benzyl alcohol (1.27 mL, 12.3 mmol) were added sequentially. The resulting mixture was refluxed for 16 h under a nitrogen atmosphere. The mixture was allowed to cool to 25°C, concentrated under vacuum, and the residue was purified by flash column chromatography (Teledyne Isco RediSep column; 0 to 50% ethyl acetate in hexanes) to give the desired product, methyl (1R,2S,3R,4S)-3{[(benzyloxy)carbonyl]amino}-7-oxa-bicyclo[2.2.1]heptane-2-carboxylate (2.23 g, 7.30 mmol, 59%), as a clear oil.^XH NMR (400 MHz, CDC1)₃) δ: 1.51 (2H, m), 1.72 (1H, m), 1.79 (1H, m), 2.97 (III, d,J = 8.4 Hz), 3.56 (3H, s), 4.33 (1H, m), 4.37 (d, 1H, J = 5.6 Hz), 4.78 (1H, d, J = 4.4 Hz), 5.10 (2H, m), 5.42 (1H, d, J = 10.0 Hz), 7.35 (5H, m). LC-MS (ESI) calcd for C16H19NO5 305.1, found 306.5 [M+H⁺].

c) (TR,2S,3R,4S/-3-amino-7-oxa-bicyclo[2.2. l]methylheptane-2-carboxylate

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<img file="BRPI0809685A2_D0141.tif" />

To a solution of methyl (1R,2S,3R,4S)-3-{[(benzyloxy)carbonyl]amino}-7-oxa-bicyclo[2.2.1]heptane-2-carboxylate (2.23 g, 7.30 mmol) in ethyl acetate (60 mL), palladium in carbon 5% (0.5 g, 22% by weight) was added. The flask was degassed and fed back with hydrogen gas via a balloon. The mixture was stirred at 25°C for 16 h, passed through a Celite plug and washed with ethyl acetate. The filtrate was concentrated under vacuum to give the desired product, methyl (lR,2S,3R,4S)-3-amino-7-oxa-bicyclo[2.2.l]heptane-2-carboxylate (1.0 g, 5.84 mmol, 80%), as a clear oil.^JN NMR (400 MHz, CDCh) δ: 1.43 (211, m), 1.67 (1H, m), 1.76 (1H, m), 2.82 (1H, d, J = 7.6 Hz), 3.41 (1H, d, J = 7.6 Hz), 3.73 (3H, s), 4.28 (1H, d, J = 6.0 Hz), 4.81 (1H, d, J = 4.8 Hz).

d) (1R,2S,3R,4S)-3-[(4-fluoro-3-methylbenzyl)amino]-7-oxa-bicyclo[2.2.1] methyl heptane-2-carboxylate

<img file="BRPI0809685A2_D0142.tif" />

To a stirred solution of (1R,2S,3R,4S)-3-amino-7-oxabicyclo[2.2.1]heptane-2-carboxylate methyl (400 mg, 2.34 mmol) in methanol (8 mL) under a nitrogen atmosphere, 4-fluoro-3-methylbenzaldehyde (0.29 mL, 2.34 mmol) was added. The mixture was stirred for 10 min, and then acetic acid (0.4 mL) was added, followed by sodium cyanoborohydride (368 mg, 5.85 mmol). The resulting mixture

160/317 was stirred at 25°C for 16 h, and then poured into a mixture of saturated aqueous sodium bicarbonate solution (50 mL) and ethyl acetate (100 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous brine solution, dried over magnesium sulfate, and filtered. The filtrate was concentrated under vacuum to provide the desired product, (lR,2S,3R,4S)._>)-3-[(4-fluoro-3-methylbenzyl)amino]-7oxa-bicyclo[2.2.1]heptane-2-carboxylate methyl (610 mg, 2.08 mmol, 80%), as a clear oil.^THEN NMR (400 MHz, CDC1₃) δ: 1.40 (2H, m), 10 1.76 (2H, m), 2.27 (3H, d, J = 2.0 Hz), 2.86 (1H, d, J = 7.6 Hz), 3.16 (III, d, J = 8.4 Hz), 3.64 (1H, d, J = 13.2 Hz), 3.74 (311, s), 3.79 (1H, d, J =

13.6 Hz), 4.46 (1H, d, J = 5.2 Hz), 4.73 (1H, d, J = 4.8 Hz), 6.92 (2H, m), 7.07 (2H, m).

e) N-{3-[(1S,2R,7 S,8R)-3-(4-fluoro-3-methyl-benzyl)-6-hydroxy15 4-oxo-1-oxa-3-aza-tricyclo[6.2.1.0²-⁷]undec-5-en-5-yl]-l, 1-dioxo-1,4dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0143.tif" />

To a stirred solution of methyl /lR,2S,3R,4S/-3-[(4-fluoro-3-methylbenzyl)amino]-7-oxa-bicyclo[2.2.l]heptane-2-carboxylate (100 mg, 0.34 mmol) and acid (7-methanesulfonylamino-l, 1-dioxo-1,4-dihydro-l⁶benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 114 mg, 0.34 mmol) in A, anhydrous A-dimethylformamide (4 mL) under a nitrogen atmosphere, A-methylmorpholine (0.075 mL, 0.68 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (72 mg, 0.37

161/317 mmol) were added sequentially. The mixture was stirred at 25°C for 2.5 h, poured into a 1.0 M aqueous hydrochloric acid solution, and then extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous brine solution, dried over magnesium sulfate, and filtered. The filtrate was concentrated under vacuum to provide the amide intermediate, which was used in the next step without further purification.

The above intermediate was dissolved in ethanol (5 mL), a 21% by weight solution of sodium ethoxide in ethanol (0.5 mL, 1.36 mmol) was added, and the mixture was stirred at 60°C for 30 min. The reaction mixture was cooled to 0°C, and then a 0.3 M aqueous hydrochloric acid solution (10 mL) was added slowly. The product precipitated upon stirring. The solid was collected by filtration, washed with water, and further purified by prep-HPLC [Luna 5gm C18 column (2)

100 AXIA 150 x 21.2 mm, 5 microns, 30%-95% in 7 min @ 30 mL/min flow rate, 0.05% trifluoroacetic acid in acetonitrile / 0.05% trifluoroacetic acid in water] to provide the desired product, N-{3[(1S,2R,7S,8R/-3-(4-fluoro-3-methylbenzyl)-6-hydroxy-4-oxo-11-oxa-3-azatricyclo[6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[ 1,2,

4] thiadiazin-7-yl}-methanesulfonamide (60 mg, 0.10 mmol, 31%), as a dirty white solid. Ή NMR (400 MHz, DMSO-dg) δ: 1.50 - 1.71 (4H, m), 2.21 (3H, d, J = 2.0 Hz), 3.05 (3H, s), 3.40 (1H, m), 3.78 (1H, d, J = 9.2 Hz), 4.34 (1H, d, J = 15.2 Hz), 4.71 (1H, d, J = 4.8 Hz), 4.76 (1H, d, J = 3.6 Hz), 5.02 (1H, d, J = 15.6 Hz), 7.06 - 7.21 (3H, m), 7.49 (1H, dd,

J = 8.4, 2.4 Hz), 7.56 (2H, m). LC-MS (ESI) calcd for C25H25FN4O7S2 576.1, found 577.5 [M+H⁺]· Anal, calcd for C25H25FN4O7S2: C, 52.07; H, 4.37; N, 9.72; found: C, 51.75; H, 4.63; N, 9.77.

Example 12 (rac-di-exo)-A-{3-(3-(4-fluoro-3-methyl-benzyl)-6-hydroxy-4-oxo-1-oxa-330 aza-tricyclo[6.2.1.0²-⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- 1λ⁶162/317 benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0144.tif" />

a) Methyl ester of (racAl-exo)-3-(4-fluoro-3-methylbenzylamino)-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid

<img file="BRPI0809685A2_D0145.tif" />

4-Iluor-3-methylbenzaldehyde (0.14 mL, 1.10 mmol) was added to a solution of (rac-di-exo)-3-amino-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid methyl ester (prepared as described in Example 8a, 200 mg, 1.17 mmol) in anhydrous methanol (10 mL) at 25°C under a nitrogen atmosphere. After stirring for 20 min, glacial acetic acid (0.3 mL) and sodium cyanoborohydride (184 mg, 2.93 mmol) were added sequentially, and the resulting mixture was stirred at 25°C for 18 h. The reaction mixture was poured into a saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate.

The combined organic layers were washed with a saturated aqueous brine solution, dried over sodium sulfate, and filtered. The filtrate was concentrated under vacuum to provide the desired product, methyl ester of (rac-di-exo)-3-(4-fluoro-3-methylbenzylamino)-7-oxa acid.

163/317 bicyclo[2.2.l]heptane-2-carboxylic acid (167 mg, 0.57 mmol, 49%), as a yellow oil.^XH NMR (400 MHz, CDC1₃) δ: 1.38 -1.48 (2H, m), 1.65 - 1.82 (2H, m), 2.27 (3H, s), 2.89 (1H, d, J = 7.6 Hz), 3.26 (1H, d, J = 8.0 Hz), 3.67 (1H, d, J = 13.2 Hz), 3.75 (3H, s), 3.86 (1H, d, J = 13.2 Hz), 4.66 (1H, d, J = 5.2 Hz), 4.71 (1H, d, J = 4.4 Hz), 6.93 (1H, t, J = 9.6 Hz), 7.13 (III, m), 7.19 (III, m). LC-MS (ESI) calcd for C16H20FNO3 293.14, found 294.3 [M+H⁺).

b) (rac-di-exo)-7V-{3-[3-(4-fluoro-3-methyl-benzyl)-6-hydroxy-4oxo-1 l-oxa-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

OJ» h ,

To a stirred solution of methyl ester of (rac-diexo)-3-(4-fluoro-3-methylbenzylamino)-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid (26 mg, 0.08 mmol) in anhydrous N,A-dimethylformamide (2 mL) under a nitrogen atmosphere, (7-methanesulfonylamino-1,1dioxo-1,4-dihydro-1A⁶-benzo[1,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 30 mg, 0.09 mmol), N-methylmorpholine (22 pL, 0.2 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (19 mg, 0.10 mmol) were added sequentially. After stirring at 25°C for 1.5 h, the reaction mixture was diluted with ethyl acetate, washed with 1.0 M aqueous hydrochloric acid solution and saturated aqueous brine solution, dried over sodium sulfate and filtered. The filtrate was concentrated under vacuum to provide the crude intermediate. The crude amide intermediate was dissolved in absolute ethanol (5 mL), and a

164/317 A 21% by weight solution of sodium ethoxide in ethanol (0.13 mL, 0.35 mmol) was added. The mixture was stirred at 60°C for 1 h, and then allowed to cool to 25°C. A 1.0 M aqueous solution of hydrochloric acid (4 mL) was slowly added to the mixture, and a white solid 5 precipitated upon stirring. The solid was collected by filtration, washed with water, and vacuum dried to provide the desired product, (rac-di-exo)-N-{3[3-(4-fluoro-3-methyl-benzyl)-6-hydroxy-4-oxo-11-oxa-3-azatricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-l, 1-dioxo-1,4-dihydro-lÀ⁶-benzo[l,2,4]thiadiazin-7-yl}-methanesulfonamide (20 mg, 0.034 mmol, 43%), 10 as a dirty white solid. *H NMR (400 MHz, DMSO-dó) δ: 1.50 1.70 (4H, m), 2.21 (3H, s), 3.05 (3H, s), 3.78 (1H, d, J = 8.8 Hz), 4.33 (1H, d, J = 15.2 Hz), 4.70 (1H, d, J = 4.4 Hz), 4.76 (1H, d, J = 4.0 Hz), 5.03 (1H, d, J= 14.8 Hz), 7.04 -7.20 (3H, m), 7.49 (1H, m), 7.56 (211, m), 10.17 (1H, s). LC-MS (ESI) calcd for C25H25FN4O7S2 576.11, found at 577.3 [M+H⁺].

Example 13 (rac-di-exo)-7V43-[3-(3,3-dimethyl-butyl)-6-hydroxy-4-oxo-1 l-oxa-3-azatricyclo[6.2.10²’⁷]undec-5-en-5-yl]-1,1-dioxo-1.4-dihydro-lÀ⁶-benzo [1,2,4]thiadiazin-7-yl}-methanesulfonamide

a) Methyl ester of (rac-di-exo)-3-(3,3-dimethylbutylamino)-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid

165/317

<img file="BRPI0809685A2_D0146.tif" />

3,3-Dimethylbutyraldehyde (120 mg, 1.20 mmol) was added to a solution of methyl ester of (rac-di-exo)-3-amino-7-oxabicyclo[2.2.l]heptane-2-carboxylic acid (prepared as described in Example 8a, 200 mg, 1.17 mmol) in anhydrous methanol (10 mL) at 25°C under a nitrogen atmosphere. After stirring for 20 min, glacial acetic acid (0.3 mL) and sodium cyanoborohydride (150 mg, 2.38 mmol) were added sequentially, and the resulting mixture was stirred at 25°C for 18 h. The reaction mixture was poured into a saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate. The combined organic layers were washed with a saturated aqueous brine solution, dried over sodium sulfate, and filtered. The filtrate was concentrated under vacuum to give the desired product, methyl ester of (rac-di-exo)-3-(3,3-dimethylbutylamino)-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid (300 mg, 1.17 mmol, 100%), as a yellow oil. UI NMR (400 MHz, CDC1₃) δ: 0.91 (9H, s), 1.42 (4H, m), 1.70 1.80 (2H, m), 2.46 (1H, m), 2.74 (1H, m), 2.89 (1H, d, J = 8.4 Hz), 3.26 (1H, d, J= 8.0 Hz), 3.71 (3H, s), 4.61 (1H, s), 4.70 (1H, m). LC-MS (ESI) calculated for C14H25NO3 255.18, found 256.2 [M+H⁺].

b) (rac-di-exo)-3-(3,3-dimethyl-butyl)-6-hydroxy-5-(7-iodo-1,1dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-11-oxa-3-aza-tricyclo[6. 2.1.0^{2 7}undec-5-en-4-one

166/317

<img file="BRPI0809685A2_D0147.tif" />

To a stirred solution of methyl ester of (rac-diexo)-3-(3,3-dimethylbutylamino)-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid (200 mg, 0.78 mmol) in anhydrous α,α-dimethylformamide (4 mL) under a nitrogen atmosphere, (7-iodo-1,1-dioxo-1,4-dihydro-1A⁶Benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in US Patent Application 2008/0031852, 287 mg, 0.78 mmol), N-methylmorpholine (0.2 mL, 1.72 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (165 mg, 0.86 mmol) were added sequentially. After stirring at 25°C for 2.5 h, the reaction mixture was diluted with ethyl acetate, washed with 1.0 M aqueous hydrochloric acid solution and saturated aqueous brine solution, dried over sodium sulfate and filtered. The filtrate was concentrated under vacuum to provide the crude amide intermediate. The above intermediate was dissolved in absolute ethanol (10 mL), and a 21% by weight solution of sodium ethoxide in ethanol (1.17 mL, 3.16 mmol) was added. The mixture was stirred at 60°C for 1 h, and then allowed to cool to 25°C. A 1.0 M aqueous hydrochloric acid solution (4 mL) was slowly added to the mixture, and a white solid precipitated upon stirring. The solid was collected by filtration, washed with water, and dried under vacuum to provide the desired product, (rac-di-exo)-3-(3,3-dimethylbutyl)-6-hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro-lA⁶-benzo[1,2,4]thiadiazin-3-yl)-11 -oxa-3-azatricyclo[6.2.1.0²’⁷] undec-5-en-4-one (200 mg, 0.35 mmol, 45%), as a yellow solid. *H NMR (400 MHz, DMSO-de) δ: 0.93 (9H, s), 1.45 (2H, m), 1.63 (2H, m), 1.71 (2H, m), 1.95 (1H, m), 3.30 (1H, m), 3.84

167/317 (HI, m), 3.89 (1H, m), 4.74 (2H, bs), 7.35 (1H, d, J = 8.4 Hz), 7.98 (HI, dd, J = 8.4, 1.6 Hz), 8.07 (1H, d, J = 1.6 Hz). LC-MS (ESI) calcd for C22H26IN3O5S 571.06, found 572.3 [M+H⁺].

c) (rac-di-exo)-A-{3-[3-(3,3-dimethylbutyl)-6-hydroxy-4-oxo-1 1oxa-3-aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro- 1λ⁶benzoph 1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0148.tif" />

A reaction flask was loaded with copper(I) iodide (20 mg, 0.11 mmol), sarcosine (N-methylglycine) (10 mg, 0.11 mmol), methanesulfonamide (83 mg, 0.87 mmol), (rac&lt;ii-exo)-3-(3,3-dimethylbutyl)-6-hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-l l-oxa-3-aza-tricyclo[6.2.1.0²’⁷undec-5-en-4-one (100 mg, 0.17 mmol) and potassium phosphate (111 mg, 0.52 mmol). The flask was degassed and back-filled with nitrogen, and then anhydrous α,Adimethylformamide (3 mL) was added. The resulting suspension was vigorously shaken at 100°C for 16 h and then allowed to cool to 25°C. The mixture was passed through a Celite plug and washed with 10% methanol/dichloromethane. The filtrate was concentrated under vacuum, and the residue was purified by prep-HPLC [Luna 5pm C18 column (2) 100A AXIA 150 x 21.2 mm, 5 microns, 30%-95% in 7 min @ 30 mL/min flow rate, 0.05% trifluoroacetic acid in acetonitrile/ 0.05% trifluoroacetic acid in water] to provide the desired product, (rac-di-exo)-N-{3-[3-(3,3-dimethyl-butyl)-6-hydroxy-4-oxo-11-oxa-3-aza-t,ricyclo[6.2.1.0%⁷|undcc5 en-5-il]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanes

168/317 sulfonamide (25 mg, 0.046 mmol, 27%), as a dirty white solid.^XH NMR (400 MHz, DMSO-d₆) δ: 0.94 (9H, s), 1.48 (2H, m), 1.63 (2H, m), 1.70 (2H, m), 2.97 (1H, m), 3.30 (1H, m), 3.80 - 3.90 (2H, m), 4.75 (2H, s), 7.49 (1H, dd, J = 8.4, 2.4 Hz), 7.56 (2H, m), 10.17 (1H, s). LC-MS (ESI) calculated for C23H30N4O7S2 538.16, found 539.4 [M+H⁺].

Example 14

N-{3-\(1S,2R,7S,&R)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-1-oxa-3-azatricyclo[6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lA⁶-benzo [1,2,4]thiadiazin-7-iU-methanesulfonamide

<img file="BRPI0809685A2_D0149.tif" />

a) (rac-di-exo)-methyl-3-amino-7-oxa-bicyclo[2.2. l]heptane-2carboxylate

<img file="BRPI0809685A2_D0150.tif" />

A stirred solution of (rac-di-exo)-3-amino-7-oxa15 bicyclo[2.2.1]heptane-2-carboxylic acid (1.0 g, 6.37 mmol) in a 1:1 mixture of anhydrous methanol and benzene (20 mL) was added dropwise to a 2.0 M solution of (trimethylsilyl)diazomethane in hexanes (6.37 mL, 12.7 mmol). The resulting mixture was stirred for 1 h and concentrated under vacuum to give the desired product, {rac-di-exo)-methyl-3-amyrio-720 oxa-bicyclo[2.2.1]heptane-2-carboxylate (1.02 g, 5.96 mmol, 94%), as

169/317 a colorless oil. 1H NMR (400 MHz, CDC1₃) δ: 1.53 (2H, m), 1.62 (211, m), 2.68 (1H, d, J = 7.6 Hz), 3.27 (1H, d, J = 7.6 Hz), 3.59 (3H, s), 4.14 (1H, d, J = 6.0 Hz), 4.67 (1H, d, J = 4.8 Hz).

b) (rac-di-exo)-methyl-3-[(4-fluoro-benzyl)amino]-7-oxa-bicyclo [2.

2. l]heptane-2-carboxylate

<img file="BRPI0809685A2_D0151.tif" />

F

4-Fluorobenzaldehyde (0.62 mL, 5.85 mmol) was added to a solution of (rac-di-exo)-methyl-3-amino-7-oxa-bicyclo[2.2.1]heptane-2-carboxylate (1.02 g, 5.85 mmol) in anhydrous methanol (20 mL) at 25°C under a nitrogen atmosphere. After stirring for 10 min, glacial acetic acid (0.8 mL) and sodium cyanoborohydride (920 mg, 14.6 mmol) were added sequentially, and the resulting mixture was stirred at 25°C for 18 h. The reaction mixture was poured into saturated sodium bicarbonate solution and extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous brine solution, dried over sodium sulfate, and filtered. The filtrate was concentrated under vacuum, and the residue was dried under high vacuum to give the desired product, (rac-di-exo)-methyl-3-[(4-ylfluorobenzyl)amino]-7-oxabicyclo[2.2.1]heptane-2-carboxylate (1.40 g, 5.01 mmol, 86%), as a colorless oil. Ή NMR (400 MHz, CDCI3) δ: 1.30 - 1.42 (2H, m), 1.73 (2H, m), 2.86 (1H, d, J = 8.0 Hz), 3.15 (1H, d, J = 8.0 Hz), 3.68 (1H, d, J = 13.6 Hz), 3.73 (3H, s), 3.84 (1H, d, J = 13.6 Hz), 4.46 (1H, d, J = 5.2 Hz), 4.73 (1H, d, J =4.8 Hz), 6.99 (2H, m), 7.26 (2H, m).

c) /lR,2S,3R,4Sj-3-[(4-fluoro-benzyl)amino]-7-oxa-bicyclo[2.2.l]heptane-2-carboxylate methyl

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<img file="BRPI0809685A2_D0152.tif" />

To a stirred solution of (rac-di-exo)-methyl-3-[(4-fluorobenzyl)amino]-7-oxa-bicyclo[2.2.1]heptane-2-carboxylate (210 mg, 0.75 mmol) in ethyl acetate (6 mL), a solution of (S)-(+)-mandelic acid (57.2 mg, 0.38 mmol) in ethyl acetate (3 mL) was added dropwise. The clear solution became cloudy and became a suspension upon stirring for 15 min, and stirring was continued for 20 min. The solid was collected by filtration, washed with ethyl acetate, and dried under vacuum to provide the desired product as a salt form of mandelic acid (90 mg, 0.21 mmol, 56%) (>96% of, based on ΠΊ NMR analysis), as a white solid. ΠΊ NMR (400 MHz, CDCI3) δ: 1.37 - 1.49 (2H, m), 1.75 (2H, m), 2.88 (1H, d, J = 8.0 Hz), 3.78 (1H, d, J = 7.6 Hz), 3.65 (1H, d, J = 12.8 Hz), 3.72 (3H, s), 3.91 (1H, d, 13.2 Hz), 4.62 (1H, d, J = 5.2 Hz), 4.70 (1H, d, J = 4.8 Hz), 5.12 (1H, s), 6.94 (2H, m), 7.19 (2H, m), 7.34 (311, m), 7.46 (2H, m).

The intermediate obtained above (90 mg, 0.21 mmol) was suspended in a 1:1 mixture of saturated aqueous sodium bicarbonate solution (5 mL) and ethyl acetate (5 mL), and stirred for 30 min at 25°C. The layers were separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous brine solution, dried over magnesium sulfate, and filtered. The filtrate was concentrated under vacuum to provide the free amine, /IR,2S,3R,4S/-3-[(4-fluoro-benzyl)amino]-7-oxa-bicyclo[2.2.l]heptane-2-carboxylate methyl (56 mg, 0.21 mmol, 95%), as a clear oil.^JH NMR (400 MHz, CDCI3) δ: 1.25 - 1.44 (2H, m), 1.64 - 1.82 (2H, m), 2.86 (1H, d, J = 8.0 Hz), 3.15 (1H, d, J = 8.0 Hz), 3.67 (1H, d, J = 13.6 Hz),

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3.73 (3H, s), 3.84 (1H, d, J = 13.6 Hz), 4.46 (1H, d, J = 5.2 Hz), 4.72 (1H, d, J-4.8 Hz), 6.99 (2H, m), 7.26 (2H, m).

d) 7V-{3-[( 1 S,2R,7 S, 8/?)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-11oxa-3-aza-tricyclo[6.2.1.0²-⁷]undec-5-en-5-yl]-l, l-dioxo-l,4-dihydro-lλ⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0153.tif" />

To a stirred solution of (1R,2S,3R,4S)-3-[(4-fluorobenzyl)amino]-7-oxa-bicyclo[2.2.1]heptane-2-carboxylate methyl (0.056 g, 0.21 mmol) in anhydrous α,α-dimethylformamide (3 mL) under a nitrogen atmosphere, acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1α⁶-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 0.070 g, 0.21 mmol) was added followed by N-methylmorpholine (0.046 mL, 0.42 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (40 mg, 0.21 mmol). The mixture was stirred at 25°C for 1 h, triethylamine (0.88 mL, 0.63 mmol) was added, and the resulting mixture was stirred at 50°C for 16 h. The reaction mixture was allowed to cool to 25°C, diluted with ethyl acetate, washed with 1.0 M aqueous hydrochloric acid solution and saturated aqueous brine solution, dried over magnesium sulfate, and filtered. The filtrate was concentrated under vacuum and the residue was purified by pre-HPLC [Luna 5gm C 18 (2) 100A AXIA 150 x 21.2 mm column, 5 microns, 30%-95% in 7 min @ 30 mL/min flow rate, 0.05% trifluoroacetic acid in acetonitrile/ 0.05% trifluoroacetic acid in water] to provide the desired product, N-{3-[(1S,2R,7S,877)-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-11-oxa-3-aza-tricyclo[6.2.1,0²-⁷]undec-5-en-5172/317 il]-1,1-dioxo- 1,4-dihydro-1 λ⁶-benzo[1,2,4]thiadiazίn-7-yl}methanesulfonamίde (48 mg, 0.085 mmol, 41%) as an off-white solid. Π-I NMR (400 MHz, DMSO-de/ δ: 1.51 - 1.71 (4H, m), 3.05 (3H, s), 3.30 (1H, m), 3.79 (1H, d, J =8.4 Hz), 4.40 (1H, d, J = 15.6 Hz), 5 4.70 (1H, d, J = 4.8 Hz), 4.76 (1H, d, J = 4.8 Hz), 5.04 (1H, d, J = 14.8

Hz), 7.15 (2H, t, J = 8.4 Hz), 7.33 (2H, m), 7.49 (1H, dd, J = 8.4, 2.4 Hz), 7.55 (2H, m). LC-MS (ESI) calcd for C24H23FN4O7S2 562.1, found 563.5 [M+H⁺], Anal, calcd for C24H23FN4O7S2: C, 51.24; 11, 4.12; N, 9.96; found: C, 51.10; H, 4.51; N, 9.98. ee > 98% [10 HPLC analysis: Chiralpak AS-RH 4.6 x 250 mm, 5 microns, 0.8 mL/min, 310 nm].

Example 15

1R, 2S,7R, 8S)-3-/4-fluoro-3-methyl-benzyl)-6-hydroxyl·-4-oxo-3-azatricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lA⁶-benzo [1,2,4]thiadiazin-7-iI}-methanesulfonamide

<img file="BRPI0809685A2_D0154.tif" />

a) (1S,2R,3S,4R)-3-(4-fluoro-3-methylbenzylamino)-bicyclo[2.2.1] heptane-2-carboxylate methyl

<img file="BRPI0809685A2_D0155.tif" />

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Methyl (lS,2R,3S,41?)-3-aminobicyclo[2.2.1]heptane-2-carboxylate chloride (prepared as described in Example 6c, 0.32 g, 1.56 mmol) was dissolved in methanol (8 mL). Sodium acetate (0.26 g, 3.12 mmol) was added followed by powdered molecular sieves of 4A (0.32 g) and 4-fluoro-3-methylbenzaldehyde (0.19 mL, 1.56 mmol). Sodium cyanoborohydride (0.24 g, 3.12 mmol) was added and the mixture was stirred at 25°C for 16 h. The mixture was poured into a mixture of saturated aqueous sodium bicarbonate solution (20 mL) and ethyl acetate (30 mL). After stirring, both layers were passed through a Celite plug. The organic layer was further washed with saturated aqueous sodium bicarbonate solution (10 mL), saturated aqueous brine solution (10 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the crude product, (lS,27?,3S,4R)-3-(4-fluoro-3-methyl-benzylamino)-bicyclo[2.2. l]heptane-2-carboxylate methyl (0.35 g, 1.20 mmol, 77%), as a clear oil. LCMS (ESI) calcd for C17H22FNO2 291.36, found 292.5 [M+H⁺],

b) N-{3-[(l-/?,2S,7i?,8S)-3-(4-fluoro-3-methyl-benzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydrolÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0156.tif" />

(1S,2R,3S,4R)-3-(4-fluoro-3-methylbenzylamino)-bicyclo[2.2.1]heptane-2-carboxylate methyl (0.090 g, 0.31 mmol) was dissolved in anhydrous 7V,IV-dimethylformamide (3 mL). Acid (7-methanesulfonylamino1,1-dioxo-1,4-dihydro-1A⁶-benzo[1,2,4]thiadiazin-3-yl)-acetic acid (prepared

174/317 as described in Example 1g, 0.10 g, 0.31 mmol) was added followed by α-methylmorpholine (0.071 mL, 0.65 mmol). The mixture was stirred until everything was dissolved, approximately 5 min. l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.062 g, 0.32 mmol) was added and the mixture was stirred at 25°C for 4 h. Triethylamine (0.13 mL, 0.92 mmol) was added and the mixture was stirred at 50°C for 16 h. Upon cooling to 25°C, the solution was diluted with ethyl acetate (50 mL) and washed with 1.0 M aqueous hydrochloric acid solution (2 x 50 mL), saturated aqueous brine solution (20 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a golden oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 50 to 100% ethyl acetate in hexanes) yielded the desired product, A-{3-[(1R,2S,7R,8S)-3-(4-fluoro-3-methylbenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²>⁷]undec-5-en-5-yl]-l, l-dioxo-1,4dihydro-lÀ⁶-benzo[l,2,4] thiadiazin-7-yl}-methanesulfonamide (0.12 g, 0.21 mmol, 68%), as a white powder.^XH NMR (400 MHz, CDCh) δ: 1.43 - 2.85 (9H, m), 2.29 (3H, s), 3.07 (3H, s), 3.45 - 3.47 (1H, m), 5.17 - 5.21 (2H, m), 6.95 -7.05 (3H, m), 7.59 - 7.66 (311, m). LC-MS (ESI) calculated for C26H27FN4O6S2 574.64, found 575.3 [M+H⁺],

Example 16

Nd3-[(1R,2S, 7R,8S)-3-(3,4-difluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²’⁷]undec-5-en-5-yl1-1, 1-dioxo-1,4-dihydro- 1À⁶benzoph 1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0157.tif" />

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a) (15,2R,3S,4R)-3-(3,4-difluorobenzylamino)-bicyclo[2.2.1] heptane-2-carboxylate methyl

THE

<img file="BRPI0809685A2_D0158.tif" />

<img file="BRPI0809685A2_D0159.tif" />

FF

Methyl (1S,2R,3S,4R)-3-aminobicyclo[2.2.1]heptane-2-carboxylate chloride (prepared as described in Example 6c, 0.32 g, 1.58 mmol) was dissolved in methanol (8 mL). Sodium acetate (0.26 g, 3.16 mmol) was added followed by powdered molecular sieves of 4A (0.33 g) and 3,4-difluorobenzaldehyde (0.17 mL, 1.58 mmol). Sodium cyanoborohydride (0.22 g, 3.16 mmol) was added and the mixture was stirred at 25°C for 16 h. The mixture was poured into a mixture of saturated aqueous sodium bicarbonate solution (20 mL) and ethyl acetate (30 mL). After stirring, both layers were passed through a Celite plug. The organic layer was further washed with saturated aqueous sodium bicarbonate solution (10 mL), saturated aqueous brine solution (10 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the crude product, (1S,2R,3S,4R)-3-(3,4-difluorobenzylamino)-bicyclo[2.2. l]heptane-2-carboxylate methyl (0.36 g, 1.22 mmol, 78%), as a clear oil. LCMS (ESI) calcd for C16H19F2NO2 295.32, found 296.3 [M+H⁺].

b) A-{3-[( IR, 2S, 7R, 85)-3-(3,4-difluoro-benzyl)-6-hydroxy-4-oxo3-aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo [1,2,4]thiadiazin-7-yl}-methanesulfonamide

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<img file="BRPI0809685A2_D0160.tif" />

(1S,2R,3S,4R)-3-(4-fluoro-3-methylbenzylamino)-bicyclo[2.2.1] heptane-2-carboxylate methyl (0.088 g, 0.30 mmol) was dissolved in anhydrous α,α-dimethylformamide (3 mL). (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- α⁶-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 0.10 g, 0.30 mmol) was added followed by N-methylmorpholine (0.069 mL, 0.63 mmol). The mixture was stirred until everything was dissolved, approximately 5 min. l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.060 g, 0.32 mmol) was added and the mixture was stirred at 25°C for 4 h. Triethylamine (0.12 mL, 0.90 mmol) was added and the mixture was stirred at 50°C for 16 h. Upon cooling to 25°C, the solution was diluted with ethyl acetate (40 mL) and washed with 1.0 M aqueous hydrochloric acid solution (2 x 20 mL), saturated aqueous brine solution (20 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a golden oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 50 to 100% ethyl acetate in hexanes) yielded the desired product, A-{3-[(lR,2S,7R,88)-3-(3,4-difluorobenzyl)-6-hydroxy-4oxo-3-aza-tricyclo[6.2.1,0²,⁷]undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazine-7-yl}-methanesulfonamide (0.082 g, 0.14 mmol, 47%), as a white powder. NMR (400 MHz, CDC1₃), 7.60 - 7.66 (3H, m). LC-MS (ESI) calcd for C25FI24F2N4O6S2 578.61, found 579.2 [M+H⁺],

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Example 17

7V-{3-[( 1R,2 8,7R,881-3-(3,4-difluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo

[1,2,4]thiadiazin-7-yl}-N-methyl-methanesulfonamide

<img file="BRPI0809685A2_D0161.tif" />

7V-{3-[( IR, 2 S,7R,8 8)-3-(3,4-difluoro-benzyl)-6-hydroxy-4-oxo-3aza-tricycle [6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ^the-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (prepared as described in Example 16, 14 mg, 0.20 mmol) was dissolved in A1/V-dimethylformamide (7 mL). Potassium carbonate (55 mg, 0.40 mmol) and iodomethane (0.014 mL, 0.22 mmol) were added sequentially. The reaction was stirred at 25°C for 18 h. The reaction was stopped by adding 1.0 M aqueous hydrochloric acid solution (20 mL). The mixture was extracted with ethyl acetate (3 x 30 mL). The combined organic layers 15 were washed with saturated aqueous brine solution (20 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a golden oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 20% ethyl acetate in dichloromethane) yielded the desired product, 7V-{3-[(lR,2 S,7R,88)-3-(3,420 difluorobenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo [6.2.1,0²,⁷]undec-5-en-5-yl]1,1-dioxo-1,4-dihydro-lÃ⁶-benzo[1,2,4]thiadiazin-7-yl}-7V-methyl-methanesulfonamide (94 mg, 0.06 mmol, 77%) as a white powder. HI NMR (400 MHz, CDC1₃) δ: 1.42 - 1.76 (6H, m), 2.51 - 2.54 (III, m), 2.84 2.88 (2H, m), 3.07 (3H, s), 3.38 (3H, s), 3.47 - 3.49 (1H, m), 5.07 - 5.10

178/317 (2H, m), 6.96 - 7.16 (3I-I, m), 7.72 - 8.01 (3H, m). LC-MS (ESI) calcd for C26H26F2N4O6S2 592.63, found 593.4 [M+H⁺].

Example 18 /\M3-[(lR,2S,7R,8S)-3-(4-fluoro-3-methylbenzyl)6hydroxyl:i-4-oxo-3-azatricyclo[6.2.1.0²-⁷lundec-5-en-5-yl]-1, 1-dioxo-1,4-dihydro- lA⁶-benzo[1,2,4]thiadiazin-7-yl)-7V-methyl-methanesulfonamide

<img file="BRPI0809685A2_D0162.tif" />

7V-{3-[(1R,2S,7R,8S)-3-(4-fluoro-3-methylbenzyl)-6-hydroxy-4oxo-3-aza-tricyclo[6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- 1A⁶10 [Benzof 1,2,4] thiadiazin-7-yl}-methanesulfonamide (prepared as described in Example 15, 92 mg, 0.16 mmol) was dissolved in N,N-dimethylformamide (6 mL). Potassium carbonate (44 mg, 0.32 mmol) and iodomethane (0.011 mL, 0.18 mmol) were added sequentially. The reaction was stirred at 25°C for 18 h. The reaction was stopped by adding 1.0 M aqueous hydrochloric acid solution (20 mL). The mixture was extracted with ethyl acetate (3 x 30 mL). The combined organic layers were washed with saturated aqueous brine solution (20 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a golden oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 20% ethyl acetate in dichloromethane) yielded the desired product, 7V-{3-[(1R,2S,7R,8S)-3-(4-fluoro-3-methylbenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²>⁷]undec-5-en5-yl] -1,1 -dioxo-1,4-dihydro- lA⁶-benzo[1,2,4]thiadiazin-7-yl}-7V-methyl-meta-

179/317 nosulfonamide (91 mg, 0.15 mmol, 96%), as a white powder.¹H NMR (400 MHz, CDC1₃) δ: 1.18 - 2.85 (9H, m), 2.90 (311, s), 2.97 (311, s), 3.38 (3H, s), 3.45 - 3.47 (1H, m), 5.17 - 5.21 (2H, m), 6.96 - 7.08 (3H, m), 7.72 - 8.02 (3H, m). LC-MS (ESI) calcd for C27H29FN4O6S2 588.67, 5 found 589.2 [M+H⁺],

Example 19 (177.2S,7R,8S)-3-(4-fluoro-benzyl)-6-hydroxy-5-(7-iodo-1,1-dioxo-1,4dihydro-1A⁶-benzo[1,2,4]thiadiazin-3-yl)-3-aza-tricyclo[6.2.1.0²[F]undec-5en-4-one ΐν,ΐν-diisopropylethylamine (1.79 mL, 10.3 mmol) and (benzotriazol-1-yloxy)-tris(dimethylamino)-phosphoniohexafluorophosphate (1.52 g, 3.44 mmol) were added sequentially to an ethyl ester solution of (1S,2I?,3S_J47?)-3-(4-fluoro-benzylamino)-bicyclo[2.2.1] 15 heptane-2-carboxylic acid (prepared as described in Example 61, 1.0 g, 3.43 mmol) and (7-iodo-1,1-dioxo-1,4-dihydro-1A6-benzo[1,2,4] thiadiazin-3-yl)-acetic acid (prepared as described in US Patent Application 2008/0031852, 1.26 g, 3.44 mmol) in IV,JV-dimethylformamide (25 mL) at 25°C. The resulting solution was stirred at 25°C for 19-20 h, and then concentrated under vacuum. The residue was partitioned between a 1.0 M aqueous solution of hydrochloric acid (150 mL) and ethyl acetate (2 x 150 mL). The organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting orange oil was dissolved.

180/317 in ethanol (50 mL) at 25°C. A 21% by weight solution of sodium ethoxide in ethanol (3.33 mL, 10.3 mmol) was added and the reaction mixture was heated to 60°C for 3 h. After cooling to 25°C, the reaction mixture was concentrated under vacuum to approximately 5 mL and then partitioned between 1.0 M aqueous hydrochloric acid solution (150 mL) and ethyl acetate (2 x 150 mL). The organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by flash column chromatography (Teledyne Isco RediSep column; 0 to 60%) ethyl acetate in hexanes) to provide the desired product, (lR,2S,7R,8S)-3-(4-fluorobenzyl)-6-hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro-lA⁶-benzo[1,2,4]thiadiazin-3-yl)-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-4-one (1.0 g, 1.69 mmol, 49%), as a pale yellow foam.^lH NMR (400 MHz, DMSO-de) δ: 1.15 - 1.21 (3H, m), 1.38 - 1.41 (1H, m), 1.46 - 1.61 (3H, m), 2.62 (1H, d, J = 2.4 Hz), 2.98 (1H, d, J = 9.4 Hz), 3.52 (1H, d, J = 9.3 Hz), 4.40 (1H, d, J = 15.7 Hz), 4.95 (1H, d, J = 15.6 Hz), 7.14 (2H, d, J = 17.9 Hz), 7.11 -7.16 (2H, m), 7.30 -7.34 (3H, m), 7.97 (1H, dd, Ji = 2.3 Hz, J₂ = 8.6 Hz), 8.07 (1H, d, J = 1.5 Hz). LC-MS (ESI) calcd for C24H21FIN3O4S 593.03, found 594.2 [M+H⁺],

Example 20 (177,28,777,88)-5-(7-( 1,1 -dioxo-4,5-dihydro- 1H- IA⁶- thiophen-2-yl)-1, 1dioxo-1,4-dihydro- 1 A⁶-benzo[1,2,4]thiadiazin-3-yl]-3-(4-fluoro-benzyl)-6hydroxy-3-aza-tricyclo[6.2.1.0²4]undec-5-en-4-ona

<img file="BRPI0809685A2_D0163.tif" />

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A solution of (1/7,28,7R,8S)-3-(4-fluorobenzyl)-6-hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3aza-tricycle [6.2.1.0²>⁷undec-5-en-4-one (prepared as described in Example 19, 200 mg, 0.34 mmol) and tributyl-(1,1-dioxo-4,5-dihydro-1H1A)⁶-thiophen-2-yl)-stanane (prepared as described in US Patent Application 2008/0031852), 180 mg, 0.44 mmol) in anhydrous N,N-dimethylformamide (7 mL) under a nitrogen atmosphere, Pd(PPh3)4 (20 mg, 0.017 mmol) was added. The resulting mixture was stirred at 90°C for 22 h, and then allowed to cool to 25°C. The reaction mixture was concentrated under vacuum and the residue was purified by prep-HPLC [Luna 5pm C18 column (2) 100A AXIA 150 x 21.2 mm, 5 microns, 30%95% in 7 min @ 30 mL/min flow rate, 0.05% trifluoroacetic acid in acetonitrile/ 0.05% trifluoroacetic acid in water] to provide the desired product, (117.2S,717.8S)-5-[7-(1,1-dioxo-4,5-dihydro-1H-1A⁶thiophen-2-yl)-1,1-dioxo-1,4-dihydro-1A⁶-benzo[1,2,4]thiadiazin-3-yl]-3-(4ylfluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-4-one (38 mg, 0.065 mmol, 20%), as a dirty white solid. Ή NMR (400 MHz, DMSO-de) δ: 1.19 (2H, m), 1.42 - 1.61 (4H, m), 2.50 (1H, m), 2.65 (1H, m), 2.97 (2H, m), 3.05 (1H, m), 3.53 (311, m), 4.43 (III, d, J = 14.4 Hz), 4.96 (1H, d, J = 15.6 Hz), 7.15 (2H, m), 7.33 (2H, m), 7.41 (III, t, J = 3.6 Hz), 7.66 (1H, d, J = 9.2 Hz), 7.96 (1H, dd, J = 8.8, 2.4 Hz), 8.10 (1H, d, J- 2.4 Hz). LC-MS (ESI) calcd for C28H26FN3O6S2 583.12, found 584.2 [M+H⁺],

Example 21 (117,28,717,88)-5-(7-( 1, 1-dioxo-tetrahydro-lÀ⁶-thiophen-2-yl)-1,1-dioxo-1,4dihydro-lÀ⁶-benzo[1,2,4 lthiadiazin-3-yl]-3-(4-fluorobenzyl)-6-hydroxy-3aza-tricyclo[6.2.1.0²-⁷undec-5-en-4-one

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F (1R,2S,7R,8S)-5-[7-(1,1-dioxo-4,5-dihydro-1H-lÀ⁶-thiofen-2-yl)

-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-3-yl]-3-(4-fluoro-benzyl)6-hydroxy-3-aza-tricyclo[6.2.1.0²>⁷[Undec-5-en-4-one (prepared as described in Example 20, 30 mg, 0.05 mmol)] was dissolved in methanol (15 mL) and 5% palladium in charcoal (100 mg) was added. The flask was degassed and fed back with hydrogen gas via a flask. The mixture was stirred at 25°C for 16 h. The mixture was passed through a Celite plug, washed with 10% methanol/dichloromethane, and the filtrate was concentrated under vacuum. The residue was purified by prep-HPLC [Luna 5gm C18 column (2) 100A AXIA 150 x 21.2 mm, 5 microns, 30%-95% in 7 min @ 30 mL/min flow rate, 0.05% trifluoroacetic acid in acetonitrile/ 0.05% trifluoroacetic acid in water] to provide the desired product, (lR,2S,7R,8S)-5-[7-(l,1-dioxo-tetrahydro-l⁶-thiophen-2yl) -1,1 -dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-3-yl]-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²>⁷undec-5-en-4-one (26 mg, 0.044 mmol, 86%), as a white powder. NMR (400 MHz, DMSO-de) δ: 1.19 (2H, m), 1.40 - 1.60 (4H, m), 2.12 (1H, m), 2.67 (1H, m), 2.36 (III, m), 2.50 (1H, m), 2.65 (1H, m), 3.03 (1H, m), 3.23 (1H, m) 7.33 (2H, m), 7.58 (1H, d, J = 8.8 Hz), 7.69 (1H, dd, J = 8.8,2.4 Hz), 7.82 (1H, d, J = 2.4 Hz); LC-MS (ESI) calculated for C28H28FN3O6S2 585.14, found 586.3 [M+H⁺],

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Example 22 (lR2S,7R,8S)-N-{3-[6-hydroxy-3-(3-methylbutyl)-4-oxo-3-azatricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- 1Ã⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0164.tif" />

a) Ethyl ester of (1S,2R,3S,4R)-3-(3-methyl-butylamino)-bicyclo[2.2.1] heptane-2-carboxylic acid

<img file="BRPI0809685A2_D0165.tif" />

Ethyl ester of (1S,2R,3S,4R)-3-amino-bicyclo[2.2.1]heptane-2-carboxylic acid (prepared as described in Example 6k, 0.5 g, 2.7 mmol) was dissolved in methanol (25 mL). Isovaleraldehyde (0.233 g, 2.7 mmol) was added followed by acetic acid (1 mL). The solution continued to be stirred at 25°C for 10 min. Sodium cyanoborohydride (0.424 g, 6.75 mmol) was added and the mixture was stirred at 25°C for 5 min.

h. The mixture was poured into a half-saturated aqueous sodium bicarbonate solution (100 mL). The aqueous layer was extracted with ethyl acetate (2 x 100 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under vacuum to give the crude product, ethyl ester of (1S,21?,3S,4R)-3-(3-methylbutylamino)-bicyclo[2.2.1]hep acid.

184/317 ethane-2-carboxylic acid (0.43 g, 1.7 mmol, 63%), as a light yellow oil, was used in the next step without any further purification. LC-MS (ESI) calcd for C15H27NO2 253.2, found 254.1 [M+H⁺].

b) Ethyl ester of (1S,2R,3S) acid₎4R)-3[[2(7methanesulfonylamino-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-acetyl](3-methyl-butyl)-amino]-bicyclo[2.2. l]heptane-2-carboxylic acid

<img file="BRPI0809685A2_D0166.tif" />

Acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1À⁶Benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 0.216 g, 0.649 mmol) was dissolved in anhydrous α,α-dimethylformamide (5 mL). Ethyl ester of 2R,3S,4R)-3-(3-Methylbutylamino)-bicyclo[2.2.l]heptane-2-carboxylic acid (0.164 g, 0.649 mmol) was added followed by α-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.13 g, 0.681 mmol). Then α-methylmorpholine (0.138 g, 1.36 mmol) was added to the above reaction mixture. The mixture was stirred at 25°C for 4 h. The solution was poured into a 1.0 M aqueous hydrochloric acid solution (100 mL). The aqueous layer was extracted with ethyl acetate (2 x 100 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the crude product, ethyl ester of (1S,2R,3S,4A)-3-[[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1A⁶-benzo[1,2,4]thiadiazin-3-yl)-acetyl]-(3-methylbutyl)-amino]-bicyclo[2,2,1]heptane-2-carboxylic acid, as a light yellow oil, was used in the next step without any further purification. LC-MS (ESI) calcd for C25H36N4O7S2 568.2, found 569.5 [M+H⁺].

c) (1R,2S,7R,8S)-N-{3-[6-hydroxy-3-(3-methyl-butyl)-4-oxo-3

185/317 aza-tricycle [6.2.1,0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0167.tif" />

The acid ethyl ester (15,2R,3S,4R)-3-[[2-(7-methanesul5 phonylamino-1,1-dioxo-1,4-dihydro-lÀ⁶Crude -benzo[1,2,4]thiadiazin-3-yl)-acetyl](3-methylbutyl)-amino]-bicyclo[2.2.1]heptane-2-carboxylic acid was dissolved in ethanol (7 mL) and a 21% by weight solution of sodium ethoxide in ethanol (1.15 mL, 3.25 mmol) was added to the above solution. The mixture was stirred at 60°C for 4 hours and cooled to 25°C. The mixture was poured into 0.5 M aqueous hydrochloric acid solution (100 mL). The aqueous layer was extracted with ethyl acetate (2 x 100 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under vacuum to give the crude product, which was purified by flash column chromatography (Teledyne Isco RediSep column; 100% ethyl acetate) to give the desired product, (lR,2S,7R,8S)-7V-{3-[6-hydroxy-3(3-methylbutyl)-4-oxo-3-aza-tricyclo[6.2.1.0²⁷]undec-5-en-5-yl]-l,l-dioxol,4-dihydro-lÀ⁶-benzo[l,2,4]thiadiazin-7-yl}-methanesulfonamide (0.19 g, 0.364 mmol, 56.1% in two steps), as a dirty white solid.^XH NMR (400 MHz, DMSO-d₆) Ô: 0.89 - 0.93 (6H, m), 1.16 - 1.23 (2H, m),

1.28 - 1.32 (1H, m), 1.35 - 1.62 (7H, m), 1.99 - 1.99 (1H, m), 2.52 2.54 (1H, m), 2.63 (1H, bs), 3.06 (3H, s), 3.62 - 3.72 (2H, m), 7.50 (1H, dd, Ji = 8.9 Hz, J₂ = 2.2 Hz), 7.57 -7.59 (211, m), 10.17 (III, s). LC-MS (ESI) ealed to C23H30N4O5S2 522.16, found 523.6 [M+H⁺].

Example 23

186/317 (rac-di-exo)-A-í3-[3-(2-cyclonropy-ethyl)-6-hydroxy-4-oxo-1-oxa-3-azatricyclo[6,2.1.0²-⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-iU-methanesulfonamide

<img file="BRPI0809685A2_D0168.tif" />

a) Cyclopropylacetaldehyde

To a solution of oxalyl chloride in 2.0 M dichloromethane (9.8 mL, 9.6 mmol) at -78°C, dimethyl sulfoxide was added dropwise. After stirring for 15 min at -78°C, a solution of cyclopropyl ethyl alcohol (1.5 g, 17.4 mmol) in dichloromethane (3.5 mL) was added. After stirring for an additional 1 h, triethylamine (13.8 mL, 98.3 mmol) was added. The reaction mixture was allowed to warm to 25°C and diluted with water. The organic layer was separated and the aqueous layer was extracted with dichloromethane (3 x 50 mL). The combined organic layers were dried over anhydrous magnesium sulfate and concentrated under vacuum at 0°C to give the crude cyclopropylacetaldehyde, which was used in the next step without further purification.^XH NMR (400 MHz, CDCI3) δ: 0.19 (2H, dd, Ji = 10.3 Hz, J₂ = 5.2 Hz), 0.62 (2H, dd, Ji = 13.2 Hz, J₂ = 5.3 Hz), 1.03 - 0.97 (1H, m), 2.30 (2H, d, J = 5.1 Hz), 9.79 (1H, d, J = 1.7 Hz).

b) Methyl ester of (rac-di-exo)-3-(2-cyclopropylethylamino)-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid

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<img file="BRPI0809685A2_D0169.tif" />

Cyclopropylacetaldehyde (148 mg, 1.75 mmol) was added to a solution of (rac-di-exo)-3-amino-7-oxabicyclo[2.2.l]heptane-2-carboxylic acid methyl ester (prepared as described in Example 8a, 300 mg, 1.75 mmol) in anhydrous methanol (10 mL) at 25°C under a nitrogen atmosphere. After stirring for 20 min, glacial acetic acid (0.3 mL) and sodium cyanoborohydride (150 mg, 2.38 mmol) were added sequentially, and the resulting mixture was stirred at 25°C for 18 h. The reaction mixture was poured into a saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate. The combined organic layers were washed with a saturated aqueous brine solution, dried over sodium sulfate, and filtered. The filtrate was concentrated under vacuum to give the desired product, methyl ester of (rac-di-exo)-3-(2-cyclopropylethylamino)-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid (220 mg, 0.92 mmol, 53%), as a yellow oil. UI NMR (400 MHz, CDC1₃) δ: 0.19 (2H, m), 0.52 (211, m), 0.70 (1H, m), 1.65 - 2.00 (6H, m), 3.10 (1H, d, J = 8.4 Hz), 3.23 (2H, m), 3.82 (3H, s), 3.93 (1H, m), 4.90 (III, d, J = 4.8 Hz), 5.17 (1H, d, J = 5.2 Hz).

c) (rac-di exo)'M{3-[3-(2-cyclopropyl-ethyl) 6-hydroxy-4-oxo-11oxa-3-aza-tricyclo[6.2.1.0²>⁷]undec-5-en-5-yl]-l, 1-dioxo- 1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

188/317

<img file="BRPI0809685A2_D0170.tif" />

To a stirred solution of methyl ester of (rac-diexo)-3-(2-cyclopropylethylamino)-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid (120 mg, 0.50 mmol) in anhydrous α,7V-dimethylformamide (2 mL) under a nitrogen atmosphere, α(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1α⁶-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 107 mg, 0.32 mmol), TV-methylmorpholine (0.12 mL, 1.09 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (105 mg, 0.55 mmol) were added sequentially. After stirring at 25°C for 2 h, the reaction mixture was diluted with ethyl acetate, washed with 1.0 M aqueous hydrochloric acid solution and saturated aqueous brine solution, dried over sodium sulfate and filtered. The filtrate was concentrated under vacuum to provide the crude intermediate. The crude amide intermediate was dissolved in absolute ethanol (5 mL), and a 21% by weight sodium ethoxide solution in ethanol (0.8 mL, 2.16 mmol) was added. The mixture was stirred at 60°C for 2 h, and then allowed to cool to 25°C. A 0.5 M aqueous hydrochloric acid solution (10 mL) was added, and then the mixture was extracted with ethyl acetate, washed with saturated aqueous brine solution, dried over sodium sulfate, and filtered. The filtrate was concentrated under vacuum and the residue was purified by pre-HPLC [Luna 5gm C18 column (2) 100A AXIA 150 x 21.2 mm, 5 microns, 30%-95% in 7 min @ 30 mL/min flow rate, 0.05% trifluoroacetic acid in acetonitrile/ 0.05% trifluoroacetic acid in water] to provide the desired product, (rac-di-exo)-A-{3-[3-(2-cyclopropylethyl)6-hydroxy-4-oxo-1-oxa-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-l, 1dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

189/317 (25 mg, 0.048 mmol, 15%), as a yellow solid. HI NMR (400 MHz, DMSO-dõ) δ: 0.07 (2H, m), 0.41 (2H, m), 0.70 (1H, m), 1.37 - 1.72 (611, m), 3.05 (3H, s), 3.07 (1H, m), 3.30 (m, 1H), 3.92 (2H, m), 4.74 (2H, m), 7.49 (1H, m), 7.55 (2H, m), 10.16 (1H, s). LC-MS (ESI) calcd for 5 C22II26N4O7S2 522.12, found 523.4 [M+H⁺],

Example 24 (1R,2S,7R,8S)-7V-f3-[6-hydroxy-3-(3-methyl-butyl)-4-oxo-3-azatricyclo[6.2.1.0²-⁷]undec-5-en-5-yl|-1,1 -dioxo-1,4-dihydro- 1À⁶benzofl,2,4]thiadiazin-7-yl}-N-methyl-methanesulfonamide

<img file="BRPI0809685A2_D0171.tif" />

(lR,2S,7R,8S)-IV-{3-[6-Hydroxy-3-(3-methyl-butyl)-4-oxo-3-azatricycle [6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (prepared as described in Example 22, 90 mg, 0.172 mmol) was dissolved in anhydrous 7V,IV-dimethylformamide (2 mL). Potassium carbonate (0.04 g, 0.344 mmol) was added followed by iodomethane (0.027 g, 0.189 mmol). The mixture was stirred at 25°C for 5 h. The reaction mixture was extracted with ethyl acetate (2 x 100 mL) and water (100 mL). The organic layer was washed with saturated aqueous brine solution (50 pL), dried over sodium sulfate 20, and filtered. The filtrate was concentrated under vacuum to provide the crude product, which was purified by flash column chromatography (Teledyne Isco RediSep column; 40% ethyl acetate in hexanes) to provide the desired product, (lR,2S,7R,8S)-N-{3-[6-hydroxy-3-(3-methylbutyl)-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-il]-1,1-dioxo-1,4-dihi190/317 dro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-A-methyl-methanesulfonamide (0.052 g, 0.097 mmol, 56.4%), as a white solid. HI NMR (400 MHz, DMSO-de) δ; 0.92 (6H, d, J = 6.4 Hz), 1.21 - 1.26 (HI, m), 1.28 - 1.33 (1H, m), 1.39 - 1.63 (7H, m), 2.54 (1H, bs), 2.63 - 2.67 (1H, m), 3.00 5 (3H, s), 3.06 - 3.17 (1H, m), 3.29 (3H, s), 3.63 - 3.72 (2H, m), 7.62 (1H, d, J = 8.5 Hz), 7.70 -7.73 (1H, m), 7.85 (1H, s). LC-MS (ESI) calcd for C24H32N4O6S2 536.18, found 537.6.6 [M+H⁺],

Example 25 (rac-di-exo)-N-{3-[6-hydroxy-3-(3-methyl-butyl)-4-oxo-1-oxa-3-aza10 tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0172.tif" />

a) Methyl ester of (rac-di-exo)-3-(3-methylbutylamino)-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid

<img file="BRPI0809685A2_D0173.tif" />

Methyl ester of (rac-di-exo)3-amino-7-oxa bicyclo[2.2.1]heptane-2-carboxylic acid (prepared as described in Example 8a, 0.4 g, 2.34 mmol) was dissolved in methanol (20 mL). Isovaleraldehyde

191/317 (0.202 g, 2.34 mmol) was added followed by acetic acid (1 mL). The solution was continued to be stirred at 25°C for 10 min. Sodium cyanoborohydride (0.37 g, 5.85 mmol) was added and the mixture was stirred at 25°C for 16 h. The mixture was poured into a half-saturated aqueous sodium bicarbonate solution (150 mL). The aqueous layer was extracted with ethyl acetate (2 x 150 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under vacuum to give the crude product, methyl ester of (rac-di-exo)-3-(3-methylbutylamino)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (0.3 g, 2.34 mmol, 53.1%), as a clear oil. LC-MS (ESI) calcd for C13H23NO3 241.17, found 242.4 [M+H⁺]·

b) Methyl ester of (rac-di-exo)-3-[[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-acetyl]-(3-methyl-butyl)-amino]-7-oxa-bicyclo[2.2. l]heptane-2-carboxylCO

<img file="BRPI0809685A2_D0174.tif" />

Acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- 1λ⁶Benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 0.211 g, 0.696 mmol) was dissolved in anhydrous Α,Α-dimethylformamide (6 mL). Methyl ester of (rac-di-cxo)-3-(3-Methylbutylamino)-7-oxabicyclo[2.2.l]heptane-2-carboxylic acid (0.168 g, 0.696 mmol) was added followed by l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.14 g, 0.731 mmol). Then Α-methylmorpholine (0.148 g, 1.46 mmol) was added to the above reaction mixture. The mixture was stirred at 25°C for 5 h. The solution was then poured into a 1.0% aqueous solution of hydrochloric acid.

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M (100 mL). The aqueous layer was extracted with ethyl acetate (2 x 100 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under vacuum to provide the crude product, methyl ester of (rac-di-exo)-3-[[2-(7-mctanesulfonylamino-1,1-dioxo-1,4-dihydrol⁶-benzo[1,2,4]thiadiazin-3-yl)-acetyl]-(3-methylbutyl)-amino]-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid, as a light yellow oil, was used in the next step without any further purification. LC-MS (ESI) calcd for C23II32N4O8S2 556.17, found 557.4 [M+H⁺],

c) (rczc-di-exo)-7V-{3-[6-hydroxy-3-(3-methyl-butyl)-4-oxo-11-oxa3-aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- wool⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0175.tif" />

<img file="BRPI0809685A2_D0176.tif" />

The methyl ester of (rac-di-exo)-3-[[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- lÀ⁶Crude -benzo[1,2,4]thiadiazin-3-yl)-acetyl]-(3-methylbutyl)-amino]-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid was dissolved in ethanol (7 mL), and a 21% by weight solution of sodium ethoxide in ethanol (1.3 mL, 3.48 mmol) was added to the above solution. The mixture was stirred at 60°C for 4 hours and cooled to 25°C. The mixture was poured into 0.5 M aqueous hydrochloric acid solution (100 mL). The aqueous layer was extracted with ethyl acetate (2 x 100 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under vacuum to give the crude product, which was purified by flash column chromatography (Teledyne Isco RediSep column; 100% ethyl acetate) to give the desired product, (rac-di-exo)-R-{3-[6-hydroxy-3193/317 (3-methyl-butyl)-4-oxo-1 l-oxa-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]1, 1-dioxo-1,4-dihydro- lA⁶-benzo[1,2,4]thidiazin-7-yl}methanesulfonamide (0.07 g, 0.133 mmol, 19.2% in two steps), as a light yellow solid, hl NMR (400 MHz, DMSO-de) δ: 0.92 (6H, d, J= 5.5 Hz), 1.06 - 1.31 (2H, m), 1.40 - 1.72 (6H, m), 3.05 (3H, s), 3.17 - 3.22 (1H, m), 3.80 - 3.87 (2H, m), 4.72 - 4.73 (2H, m), 7.47 -7.55 (3H, m), 10.12 (1H, bs). LC-MS (ESI) calcd for C22H28N4O7S2 524.14, found 525.4 [M+H⁺].

Example 26 (1R,2 8,7R,8 8)-5-(7-( 1,1 -dioxo- lA⁶-isothiazolidin-2-iI)-1,1-dioxo-1,4dihydro- lA⁶-benzo[1,2,4]thiadiazin-3-yl]-3-(4-fluoro-benzyl)-6-hydroxy-3-azatricyclo[ 6.2. EQ²· ⁷undec-5-en-4-one

<img file="BRPI0809685A2_D0177.tif" />

A reaction flask was loaded with copper iodide (1) (8 mg, 0.042 mmol), sarcosine (Af-methyl glycine) (9 mg, 0.1 mmol), 1,1-isothiazolidine dioxide (204 mg, 1.685 mmol), (1R,2S,7R,88)-3-(4-fluorobenzyl)-6-hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro-lA⁶-benzo[1,2,4]thiadiazin-3-yl)-3-aza-tricycle [6.2.1.0²>⁷undec-5-en-4-one (prepared as described in Example 19, 100 mg, 0.168 mmol) and potassium phosphate (179 mg, 0.842 mmol). The flask was degassed and back-filled with nitrogen, and then R,R-dimethylformamide (3 mL) was added. The resulting suspension was vigorously shaken at 100°C for 17 h and then allowed to cool to 25°C. The mixture was diluted with acetate.

194/317 ethyl (30 mL) and washed with 1.0 M aqueous hydrochloric acid solution (2 x 20 mL) and saturated aqueous brine solution (40 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated under vacuum. Purification by flash column chromatography (Teledyne Isco RediSep column; I^the column: 100% dichloromethane, 2^the column: 5% hexanes in dichloromethane) to provide the desired product. The crude product was ground with absolute ethanol (3x) and vacuum dried at 60°C to provide the desired product, (lR,2S,7R,8S)-5-[7-(l,l-dioxo-lÀ⁶isothiazolidin-2-yl)-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl]-3(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²·⁷undec-5-en-4-one (70 mg, 0.119 mmol, 71%), as a solid. NMR (400 MHz, DMSO-d₆) δ: 1.17 - 1.24 (2H, m), 1.40 - 1.61 (4H, m), 2.39 - 2.46 (2H, m), 2.51 2.54 (1H, m), 2.64 - 2.65 (1H, m), 3.03 - 3.05 (1H, m), 3.53 - 3.60 (3H, m) m), 7.62 (1H, d, J = 8.5 Hz). LC-MS (ESI) calculated for C27H27FN4O6S2 586.14, found 587.4 [M+H⁺].

Example 27 (lR,2S,7R,8S)-7V-[3-(6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en5-il) -1,1 -dioxo-1.4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl]methanesulfonamide

<img file="BRPI0809685A2_D0178.tif" />

A suspension of (lR,2S,3R,4S)-3-ethoxycarbonyl-bicyclo[2.2.l]hept-2-yl-ammonium (l'S)-(+)-10-camphorsulfonate (prepared as described in Example 6j, 5.00 g, 12.0 mmol) and potassium carbonate

195/317 (4.16 g, 30.1 mmol) in ethyl acetate (80 mL) was stirred at 25°C for 5 h, then filtered through a frit medium. The filtrate was concentrated under vacuum to provide the crude ethyl ester of (1S,2R,3S,4R)-3amino-bicyclo[2.2.1]heptane-2-carboxylic acid (2.14 g). A portion of this material (1.00 g, 5.52 mmol) and (7-methanesulfonylamino-1,1dioxo-1,4-dihydro-1) acid were used to obtain the ethyl ester of crude (1S,2R,3S,4R)-3amino-bicyclo[2.2.1]heptane-2-carboxylic acid.⁶-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 1.84 g, 5.52 mmol) were dissolved in /V,N-dimethylformamide at 25°C and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (1.11 g, 5.79 mmol) and TV-methylmorpholine (1.27 mL, 11.6 mmol) were added sequentially. The resulting solution was stirred at 25°C for 22 h, and then concentrated under vacuum. The residue was partitioned between 1.0 M aqueous hydrochloric acid solution (200 mL) and ethyl acetate (2 x 150 mL). The organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting orange oil was dissolved in ethanol (70 mL) at 25°C. A 21% by weight solution of sodium ethoxide in ethanol (10.7 mL, 33.0 mmol) was added, and the reaction mixture was heated to 90°C for 3 days. After cooling to 25°C, the reaction mixture was concentrated under vacuum to a volume of approximately 5 mL and then partitioned between 1.0 M aqueous hydrochloric acid solution (200 mL) and ethyl acetate (2 x 150 mL). The organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by flash column chromatography (Teledyne Isco RediSep column; 0 to 7% methanol in dichloromethane) to provide the desired product, (1R,2S,7R,8S)-N-[3-(6hydroxy-4--oxo-3-aza-tricyclo[6.2.1.0²,⁷]undec-5-en-5-yl)-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[l,2,4]thiadiazin-7-yl]-methanesulfonamide (0.25 g, 0.552 mmol, 10%), as a white solid. Ή NMR (400 MHz, DMSO-de) δ: 1.17 - 1.22 (2H, m), 1.39 - 1.58 (3H, m), 2.24 (1H, bs), 2.32 (1H, bs), 2.64 - 2.66 (III, m), 2.84 - 2.87 (1H, m), 3.04 (311, s), 3.54 (1H, bs), 3.63 (1H, bs), 7.49 (211, bs), 7.55 (1H, bs), 8.11 (III, bs), 9.52 (1H, bs), 10.12 (1H, bs). LC-MS (ESI) calcd for C18H20N4O6S2 452.08, found

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453.2 [M+H⁺],

Example 28 (17?,25,777,88)-5-( 1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²-⁷undec-5-en-4-one

<img file="BRPI0809685A2_D0179.tif" />

a) 7V-(2-sulfamoyl-phenyl)-malonamic acid ethyl ester

<img file="BRPI0809685A2_D0180.tif" />

2-Aminobenzenesulfonamide (5 g, 29 mmol) was dissolved in 7V,7V-dimethylacetamide (25 mL) and diethyl ether (25 mL). Ethyl-3-chloro-310 oxopropionate (4.6 g, 30.45 mmol) was added to the above reaction solution. The reaction mixture was stirred at 25°C for 3 h. The product began to precipitate and was collected by vacuum filtration. The solid was dissolved in ethyl acetate (200 mL) and extracted with water (200 mL). The aqueous layer was extracted back with ethyl acetate (200 mL).

The combined organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum to give the crude product, 7V-(2-sulfamoylphenyl)-malonamic acid ethyl ester, as a white solid, which was used in the next step without further purification. UI NMR (400 MHz, DMSO-d₆) δ: 1.23 (3H, t, J = 7.0 Hz), 3.61 (2H, s), 4.14

197/317 (2H, quartet, J = 7.0 Hz), 7.29 - 7.33 (1H, m), 7.53 (2H, bs), 7.56 -7.60 (1H, m), 7.84 -7.86 (1H, m), 7.97 - 7.99 (1H, m), 9.54 (1H, bs). LC-MS (ESI) calcd for C11H14N2O5S 286.06, found 287.1 [M+H⁺].

b) Acid (1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[l,2,4]thiadiazin-35yl)-acetic

<img file="BRPI0809685A2_D0181.tif" />

Solid sodium hydroxide (3.48 g, 87 mmol) was dissolved in water to make a saturated solution. The ethyl ester of crude N-(2-sulfamoylphenyl)-malonamic acid was added to the sodium hydroxide solution. The reaction mixture was heated to 110°C for 2.5 h and then cooled to 25°C. The reaction mixture was acidified by the slow addition of 12.0 M aqueous hydrochloric acid solution (9.67 g, 116 mmol) while cooling in an ice-water bath. The product precipitated and was collected by vacuum filtration. The solid was washed with cold water and dried under high vacuum to give the crude product, acid (l,l-dioxo-l,4-dihydro-l⁶-benzo[1,2,4]thiadiazin-3-yl)-acetic acid (5 g, 20.8 mmol, 71.7% in two steps), as a white solid. HI NMR (400 MHz, DMSO-cfo) δ: 3.58 (2H, s), 7.31 (1H, d, J = 8.0 Hz), 7.44 (1H, dd, J1 = 7.8 Hz, J₂ = 7.8 Hz), 7.67 (1H, dd, J1 = 7.8 Hz, J₂ = 7.8 Hz), 7.79 20 (1H, d, J= 7.9 Hz), 12.18 (1H, bs), 13.03 (1H, bs). LC-MS (ESI) calcd for C9H8N2O4S 240.02, found 241.1 [M+H⁺].

c) Ethyl ester of acid (1S,2R,3S,4R)-3-[[2-(1,1-dioxo-1,4dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-acetyl]-(4-fluoro-benzyl)-amino]bicyclo[2.2.1]heptane-2-carboxylic acid

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<img file="BRPI0809685A2_D0182.tif" />

Acid (1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)acetic acid (0.2 g, 0.833 mmol) was dissolved in anhydrous A,A-dimethylformamide (8 mL). Ethyl ester of (1S,2R,3S,4R)-3-(4-fluorobenzylamino)-bicyclo[2.2.1]heptane-2-carboxylic acid (prepared as described in Example 61, 0.244 g, 0.833 mmol) was added followed by l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.168 g, 0.875 mmol). Then A-methylmorpholine (0.177 g, 1.75 mmol) was added to the above reaction mixture. The mixture was stirred at 25°C for 16 h. The solution was poured into a 1.0 M aqueous hydrochloric acid solution (100 mL). The aqueous layer was extracted with ethyl acetate (2 x 100 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under vacuum to provide the crude product, ethyl ester of (1S,2R,3S,4R)-3-[[2-(1,1-dioxo-1,4-dihydro-1A⁶-benzo[1,2,4]thiadiazin-3-yl)acetyl]-(4-fluorobenzyl)amino]-bicyclo[2,2,1]heptane-2-carboxylic acid, as an orange oil, which was used in the next step without any further purification. LC-MS (ESI) calcd for C26H28FN3O5S 513.58, found 514.4 [M+H⁺].

d) (lR,2S,7R,8S)-5-(l, l-dioxo-1,2-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²-⁷undec- 5-en-4-one

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<img file="BRPI0809685A2_D0183.tif" />

The acid ethyl ester (1S,2R,3S,4R)-3-[[2-(1,1-dioxo-1,4dihydro-lÀ⁶Crude -benzo[1,2,4]thiadiazin-3-yl)-acetyl]-(4-fluoro-benzyl)-ammo]-bicyclo[2.2.1]heptane-2-carboxylic acid was dissolved in ethanol (8 mL), and a 21% by weight solution of sodium ethoxide in ethanol (1.6 mL, 4.2 mmol) was added to the above solution. The mixture was stirred at 60°C for 4 hours and allowed to cool to 25°C. The mixture was poured into 0.5 M aqueous hydrochloric acid solution (100 mL). The product began to precipitate and was collected by vacuum filtration. The precipitate was purified by flash column chromatography (Teledyne Isco RediSep column; 100% ethyl acetate) to provide the desired product, (lR,2S,7R,8S)-5-(l,1dioxo-1,2-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6hydroxy-3-aza-tricyclo[6.2.1.0²’⁷undec-5-en-4-one (0.242 g, 0.517 mmol, 62.1% in two steps), as a white solid. NMR (400 MHz,

DMSO-dg) δ: 1.16 - 1.22 (2H, m), 1.40 - 1.60 (4H, m), 2.51 (1H, bs), 2.64 (1H, d, J = 2.1 Hz), 3.03 (1H, d, J = 8.0 Hz), 3.54 (1H, d, J = 9.3 Hz), 4.42 (1H, d, J = 15.6 Hz), 4.97 (1H, d, J = 15.7 Hz), 7.15 (2H, t, J = 8.8 Hz), 7.33 (2H, dd, J1 = 8.0 Hz, J₂ = 5.9 Hz), 7.45 -7.53 (2H, m), 7.67 -7.71 (1H, m), 7.85 (1H, d, J = 7.9 Hz). LC-MS (ESI) calcd for

C24H22FN3O4S 467.13, found 468.2 [M+H⁺]. Anal, calcd for C24H22FN3O4S: C, 61.66; H, 4.74; N, 8.99; found C, 61.96; H, 4.88; N, 8.99.

Example 29

7V-{3-[(1R,2S,7R,8S)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza

200/317 tricycle[6.2.1.0²-⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,41 thiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0184.tif" />

a) Acid (7-methanesulfonylamino-1, 1-dioxo-1,4-dihydro5 lÀ⁶-benzo[l,4] thiazin-3-yl)-acetic

<img file="BRPI0809685A2_D0185.tif" />

A solution of ethyl ester of (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-l) acid⁶-benzo[1,4]thiazin-3-yl)-acetic acid (prepared as described in US Patent Application 2008/0031852, 10,600 mg, 1.666 mmol) in methanol (30 mL) was cooled to 0°C in an ice-water bath and treated with 2.0 M aqueous lithium hydroxide solution (4.17 mL, 8.332 mmol). The reaction mixture was allowed to warm to 25°C and stirred for 1 h. The methanol was removed under vacuum and the reaction mixture was poured into 0.5 M aqueous hydrochloric acid solution (20 mL) on ice, extracted with ethyl acetate (3 x 50 mL), dried over sodium sulfate, filtered, and concentrated under vacuum to provide an orange solid. The crude solid was triturated with diethyl ether to provide the desired product, (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1λ)⁶benzo[l,4]thiazin-3-yl)-acetic acid (409 mg, 1.232 mmol, 73.9%), as a yellow solid. LC-MS calcd for C11H12N2O6S2 332.4, found 333.0 [M+H⁺].

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b) IV-{3-[(1 R,2S,7R, 8S)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo3-aza-tricycle [6.2.1.0²>⁷]undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro- 1λ⁶benzo[1,4] thiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0186.tif" />

(1S,2R,3S,4R)-3-[(4-fluorobenzyl)amino]bicyclo[2.2.1]heptane-2-carboxylate methyl (prepared as described in Example 6d, 0.20 g, 0.69 mmol) was dissolved in anhydrous A,A-dimethylformamide (7 mL). Acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[l,4]thiazin-3-yl)-acetic acid (0.23 g, 0.69 mmol) was added followed by N-methylmorpholine (0.17 mL, 1.52 mmol). The mixture was stirred until everything was dissolved, approximately 5 min. l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.15 g, 0.76 mmol) was added and the mixture was stirred at 25°C for 16 h. The reaction was stopped by adding a saturated aqueous sodium bicarbonate solution (20 mL). The mixture was extracted with ethyl acetate (3 x 30 mL). The organic layers were combined and washed with a saturated aqueous brine solution (20 mL). The resulting solution was dried over magnesium sulfate, filtered, and concentrated under vacuum to give a golden oil. The oil was dissolved in ethanol (10 mL). A 21% by weight solution of sodium ethoxide in ethanol (0.65 mL, 1.74 mmol) was added. The reaction was stirred at 60°C for 16 h. The reaction was stopped by adding a 1.0 M aqueous hydrochloric acid solution (10 mL). The mixture was extracted with ethyl acetate (3 x 20 mL). The organic layer was then washed with a saturated aqueous sodium bicarbonate solution (2 x 20 mL), a saturated aqueous brine solution (20 mL), and dried over magnesium sulfate.

202/317 filtered, and vacuum concentrated to provide a clear oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 22 to 75% ethyl acetate in hexanes) yielded the desired product, JV-{3[(lR,2S,7R,8S)-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²>⁷] undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro- lÀ⁶-benzo[1,4]thiazin-7-yl}-methanesulfonamide (0.020 g, 0.04 mmol, 5.3%), as a yellow powder.^XH NMR (400 MHz, CDC1₃) δ: 1.21 - 1.64 (6H, m), 2.52 - 2.71 (3H, m), 3.07 (3H, s), 3.39 - 3.52 (1H, m), 5.15 - 5.28 (2H, m), 6.60 (1H, s), 7.02 -7.06 (2H, m), 7.22 -7.26 (2H, m), 7.54 -7.66 (3H, m). LC-MS (ESI) calcd for

C26H26FN3O6S2 559.63, found 560.5 [M+H⁺],

Example 30 (1R,2S,7R,8S)-5-(7-amino-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo [1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-azatricyclo[6.2.10²’⁷undec-5-en-4-one

a) (1R,2S,7R,8S)-5-(7-azido-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²>⁷undec5-en-4-ona

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<img file="BRPI0809685A2_D0187.tif" />

(1R,2S,7R,8S)-3-(4-fluoro-benzyl)-6-hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro-lÀ^&amp;-benzo[1,2,4]thiadiazin-3-yl)-3-aza-tricyclo[6.2.IO²·⁷undec-5-en-4-one (prepared as described in Example 19, 0.513 g, 0.864 mmol), sodium azide (1.12 g, 17.2 mmol), sodium ascorbate (0.086 g, 0.43 mmol), copper(I) iodide (0.16 g, 0.84 mmol), and trans-/V,/V-dimethylcyclohexane-1,2-diamine (0.20 mL, 1.27 mmol) were dissolved in a 5:1 mixture of dimethyl sulfoxide and water (10 mL) at 25 °C. The reaction flask was degassed and back-fed with nitrogen (5 x). After stirring at 25 °C for 14 h, the reaction mixture was partitioned between water (150 mL) and ethyl acetate (2 x 150 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by flash column chromatography (Teledyne Isco RediSep column; 0 to 60% ethyl acetate in hexanes) to provide the desired product, (lR,2S,7R,8S)-5-(7-azido-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-4-one (0.348 g, 0.684 mmol, 79%), as a dark brown foam, which was used in the next step without any further purification, b) (lR,2S,7R,8S)-5-(7-Amino-l, 1dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6hydroxy-3-aza-tricyclo[6.2.1.0²,7]undec-5-en-4-ona

204/317 (1 R,2S,7 R,8S)-5-(7-Rzido-1,1-dioxo-1,4-dihydro-1À6-benzo[1, 2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²-⁷Undec-5-en-4-one (0.348 g, 0.684 mmol) was dissolved in a 1:1 mixture of methanol and ethyl acetate (15 mL) at 25 °C. Palladium on carbon (0.40 g, 5%, "wet") was added, resulting in a black suspension. The reaction was maintained under a hydrogen atmosphere (flask) at 25 °C for 6 h, and then filtered through Celite. The Celite was washed with ethyl acetate (2 x 30 mL) and the filtrate was concentrated under vacuum. The residue was purified by flash column chromatography (Teledyne Isco RediSep column; 50 to 100% ethyl acetate in hexanes) to provide the desired product, /lR,2S,7R,8S)-5-(7-amino-l,l-dioxo-l,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-azatricyclo[6.2.1.0²-⁷undec-5-en-4-one (0.159 g, 0.330 mmol, 48%), as a pale yellow solid. 1H NMR (400 MHz, CDC1₃) δ: 1.08 - 1.19 (3H, m), 1.40 - 1.57 (3H, m), 2.99 (1H, d, J = 7.2 Hz), 3.31 (3H, s), 3.36 3.37 (1H, m), 3.50 (1H, d, J = 7.8 Hz), 4.39 (1H, d, J = 14.6 Hz), 4.93 (1H, d, J = 14.5 Hz), 6.86 - 6.91 (3H, m), 7.13 - 7.15 (2H, m), 7.21 (1H, d, J = 8.8 Hz), 7.30 (2H, bs), 13.79 (1H, s). LC-MS (ESI) calcd for C₂₄H₂3FN₄THE₄S 482.14, found 483.4 [M+H⁺].

Example 31 (rac-di-exo)-7V-f3-[6-Hydroxy-3-(3-methyl-butyl)-4-oxo-3-azatricyclo[6.2.1.0²,7]undeca-5,9-dien-5-yl}-1,1-dioxo-1,4-dihydro- 1λ⁶benzof 1,2,4]thiadiazin-7-iIj-methanesulfonamide

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<img file="BRPI0809685A2_D0188.tif" />

a) Methyl ester chloride of (rac-di-exo)-3-aminobicyclo[2.2.l]hept-5-ene-2-carboxylic acid

O &lt;/ owe ^^^kνη₂· hci

(rac-di-exo)-3-Amino-bicyclo[2.2.l]hept-5-ene-2-carboxylic acid chloride (1.0 g, 5.27 mmol) was dissolved in methanol (7 mL). Benzene (10 mL) was added followed by the dropwise addition of a 2.0 M solution of (trimethylsilyl)diazomethane in diethyl ether (5 mL, 10 mmol). After stirring for 10 min, an additional 2.0 M solution of (trimethylsil10 lil)diazomethane in diethyl ether (3 mL, 6 mmol) was added and the reaction mixture was stirred for a further 20 min; after this time the solution remained yellow in color. The solvents were removed under vacuum, the residue was collected in methanol (15 mL) and the solvent was removed under vacuum to give the crude product, 15-methyl ester chloride of (rac-di-exo)-3-amino-bicyclo[2.2.l]hept-5-ene-2-carboxylic acid (0.98 g, 4.83 mmol, 91.6%), as a yellow oil, which was used in the next step without any further purification.

b) Methyl ester of (rac-di-exo)-3-(3-Methylbutylamino)-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid

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<img file="BRPI0809685A2_D0189.tif" />

Methyl ester chloride of (rac-di-exo)-3-aminobicyclo[2.2.1]hept-5-ene-2-carboxylic acid (0.955 g, 4.703 mmol) was suspended in methanol (10 mL). Sodium acetate (0.791 g, 9.652 mmol) was added followed by 4A powdered molecular sieves (1.0 g) and 3-methylbutyraldehyde (0.385 g, 4.468 mmol). Sodium cyanoborohydride (0.593 g, 9.406 mmol) was added and the mixture was stirred at 25°C for 18 h. The mixture was poured into a saturated aqueous sodium bicarbonate solution (100 mL) and extracted with ethyl acetate (100 mL). The layers were separated, and the organic layer was washed with a saturated aqueous brine solution, dried over sodium sulfate, filtered, and concentrated under vacuum. Further purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 10% methanol in dichloromethane) yielded the desired product, methyl ester of (rac-di-exo)-3(3-methyl-butylamino)-bicyclo[2.2.l]hept-5-ene-2-carboxylic acid (0.774 g, 3.14 mmol, 66.7%), as a yellow oil.^XH NMR (400 MHz, CDCI3) δ: 0.89 (3H, d, J= 1.6 Hz), 0.90 (311, d, J= 2.1 Hz), 1.27 - 1.39 (2H, m), 1.54 - 1.64 (2H, m), 2.07 (1H, d, J = 9.4 Hz), 2.42 -2.56 (2H, m), 2.66 2.73 (1H, m), 2.84 (1H, bs), 2.92 (1H, bs), 2.98 (1H, d, J = 7.7 Hz), 3.69 (3H, s), 6.14 (2H, s). LC-MS calculation for C14H23NO2 237.17, found 238.2 [M+H⁺],

c) (rac-di-exo)-3-[[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-lÀ) acid methyl ester⁶-benzo[1,2,4]thiadiazin-3-yl)-acetyl]-(3-methyl-butyl)-amino]-bicyclo[2.2. l]hept-5-ene-2-carboxylic acid

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<img file="BRPI0809685A2_D0190.tif" />

To the methyl ester solution of (rac-di-exo)-3-(3-methylbutylamino)-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (208.7 mg, 0.880 mmol) in A,A-dimethylformamide (3 mL) was added (7methanesulfonylamino-1,1-dioxo-1,4-dihydro-1A⁶-benzo[1,2,4]thiadiazin3-yl)-acetic acid (prepared as described in Example 1g, 351.7 mg, 1.056 mmol) and the mixture was vortexed until all the material had completely dissolved. To a 1.0 M solution of 7V,7V-dicyclohexylcarbodiimide in dichloromethane (1.144 mL, 1.144 mmol) was added the above solution and the mixture was stirred at 25°C for 18 h under a nitrogen atmosphere. The precipitated 1V,7V-dicyclohexylurea was removed by filtration over Celite, the filter cake was washed with dichloromethane (3 x 5 mL), and the solvent was removed under vacuum. The residue was dissolved in a 1:1 mixture of ethyl acetate and hexanes (20 mL), washed with saturated aqueous brine solution, saturated aqueous sodium bicarbonate solution, and water. The layers were separated, and the aqueous layers were extracted back with a 1:1 mixture of ethyl acetate and hexanes (20 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum to provide the crude product, methyl ester of (rac-diexo)-3-[[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1A⁶benzo[1,2,4]thiadiazine-3-yl)-acetyl]-(3-methylbutyl)-amino]-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid, as a yellow oil, which was used in the next step without any further purification. LC-MS calcd for C24H32N4O7S2 552.17, found 553.3 [M+H⁺],

d) (rac-di-exo)-A-{3-[6-Hydroxy-3-(3-methyl-butyl)-4

208/317 oxo-3-aza-triciclo[6.2.1.0²-⁷]undeca-5,9-dien-5-yl]-1, 1-dioxo-1, 4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazm-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0191.tif" />

The methyl ester of (rac-di-exo)-3-[[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-lÀ⁶Crude -benzo[1,2,4]thiadiazin-3-yl)-acet.yl](3-methyl-butyl)-amino]-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid was dissolved in ethanol (20 mL). To this solution was added a 21% by weight solution of sodium ethoxide in ethanol (1.141 g, 3.52 mmol) and the reaction mixture was stirred at 25°C for 2 h. Upon addition of a 1.0 M aqueous hydrochloric acid solution, the product precipitated and was collected by vacuum filtration. Further purification by trituration with dichloromethane and ethyl acetate yielded a cinnamon-colored solid, which was further washed with ethyl acetate and then vacuum-dried to provide the desired product, (rac-di-exo)-A-{3-[6-hydroxy-3-(3-methylbutyl)-4-oxo-3-aza-tricyclo[6.2.1.0²,⁷]undeca-5,9-dien-5-yl]-1,1-dioxo-1, 4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (116.6 mg, 0.224 mmol, 25.5%), as a dirty white solid.¹H NMR (400 MHz, DMSO-dõ) δ: 0.93 (6H, d, J = 5.3 Hz), 1.35 -1.70 (5H, m), 2.85 (1H, d, J = 8.7 Hz), 3.06 (3H, s), 3.13 - 3.31 (5H, m), 3.49 (1H, d, J = 8.6 Hz), 3.75 - 3.83 (1H, m), 6.23 (1H, s), 6.39 (1H, s), 7.50 - 7.58 (3H, m), 10.18 (1H, s). LC-MS calcd for C23H28N4O6S2 520.15, found 521.4 [M+H⁺],

Example 32 (rac-di-exo) - A4 3-13-(3,3 - Dimethyl-butyl) - 6 - hydroxy - 4 - oxo - 3 - aza

209/317 tricycle[6.2.1.0².⁷ lundeca-5,9-dien-5-yl]-1,1-dioxo-1,4-dihydro- 1À⁶benzoph 1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0192.tif" />

a) (rac-di-exo)-3-(3,3-dimethyl-butylamino)-bicyclo[2.2] acid methyl ester. l]hept-5-ene-2-carboxylic acid

<img file="BRPI0809685A2_D0193.tif" />

Methyl ester chloride of (rac-di-exo)-3-Aminobicyclo[2.2.l]hept-5-ene-2-carboxylic acid (0.984 g, 4.846 mmol) was suspended in methanol (10 mL). Sodium acetate (0.795 g, 9.692 mmol) was added followed by 4A powdered molecular sieves (1.0 g) and 3,3-dimethylbutyraldehyde (0.461 g, 4.604 mmol). Sodium cyanoborohydride (0.610 g, 9.692 mmol) was added and the mixture was stirred at 25°C for 18 h. The mixture was poured into a saturated aqueous sodium bicarbonate solution (100 mL) and extracted with ethyl acetate (100 mL). The 15 layers were separated, and the organic layer was washed with saturated aqueous brine solution, dried over sodium sulfate, filtered, and concentrated under vacuum. Further purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 10% methanol in dichloromethane) yielded the desired product, methyl ester of (rac-di-exo)-3-acid.

210/317 (3,3-dimethyl-butylamino)-bicyclo[2.2.l]hept-5-ene-2-carboxylic acid (0.773 g, 3.08 mmol, 63.5%), as a yellow oil, HI NMR (400 MHz, CDCI3) δ: 0.91 (9H, s), 1.31 - 1.40 (2H, m), 1.55 (1H, d, J = 9.5 Hz), 2.08 (1H, d, J = 8.8 Hz), 2.43 - 2.49 (1H, m), 2.55 (1H, d, J = 7.8 Hz), 2.64 - 2.74 (1H, m), 2.85 (III, bs), 2.93 (1H, bs), 2.99 (1H, d, J= 7.7 Hz), 3.69 (3H, s), 6.15 (2H, s). LC-MS calcd for C15H25NO2 251.19, found 252.2 [M+H⁺],

b) Methyl ester of (rac-di-exo)-3-{(3,3-Dimethylbutyl)-[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- 1A⁶-benzo [1, 2,4]thiadiazin-3-yl)-acetyl]-amino)-bicyclo[2.2. l]hept-5-ene-2-carboxylic acid

<img file="BRPI0809685A2_D0194.tif" />

To the methyl ester solution of (rnc-di-exo)-3-(3,3-dimethylbutylamino)-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (233.3 mg, 0.929 mmol) in A,lV-dimethylformamide (3 mL) was added (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1⁶-benzo[1,2,4]thiadiazin3-yl)-acetic acid (prepared as described in Example 1g, 371.2 mg, 1.115 mmol) and the mixture was vortexed until all the material had been completely dissolved. A 1.0 M solution of N,N-dicyclohexylcarbodiimide in dichloromethane (1.208 mL, 1.208 mmol) was added to the above solution and the mixture was stirred at 25°C for 18 h under a nitrogen atmosphere. The precipitated 7V,7V-dicyclohexylurea was removed by filtration over Celite, the filter cake was washed with

211/317 dichloromethane (3 x 5 mL), and the solvent was removed under vacuum. The residue was diluted in a 1:1 mixture of ethyl acetate and hexanes (20 mL), washed with saturated aqueous brine solution, saturated aqueous sodium bicarbonate solution, and water. The layers were separated, and the aqueous layers were extracted back with the 1:1 mixture of ethyl acetate and hexanes (20 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum to provide the crude product, methyl ester of (rac-di-exo)-3-{(3,3-dimethylbutyl)-[2-(7-methanesulfonylamino-1,1-dioxol,4-dihydro-1-⁶-benzo[1,2,4]thiadiazin-3-yl)-acetyl]-amino}-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid, as a yellow oil, which was used in the next step without any further purification. LC-MS calcd for C25H34N4O7S2 566.19, found 567.4 [M+H⁺].

c) (rac-di-exo)-7V-{3-[3-(3,3-Dimethyl-butyl)-6-hydroxy-4-oxo-3aza-tricyclo[6.2.1.0²⁷]undeca-5,9-dien-5-yl]-1,1 -dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0195.tif" />

The methyl ester of (rac-di-exo)-3-{(3,3-dimethylbutyl)[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1A^theCrude -benzo[1,2,4]thiadiazin-3-yl)-acetyl]-amino}-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (710.8 mg, 0.929 mmol) was dissolved in ethanol. To this solution was added a 21% by weight solution of sodium ethoxide in ethanol (1.204 g, 3.716 mmol) and the reaction mixture was stirred at 25°C for 2 h. By adding a 1.0 M aqueous solution of hydrochloric acid the product

212/317 precipitated and was collected by vacuum filtration. Further purification by trituration with dichloromethane and ethyl acetate gave a cinnamon-colored solid, which was further washed with ethyl acetate and then vacuum-dried to provide the desired product, (rac-di-exo)-N-{3-[3-(3,3-dimethylbutyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²>⁷]undeca-5,9-dien5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (210.7 mg, 0.394 mmol, 42.5% in two steps), as a dirty white solid. Ή NMR (400 MHz, DMSO-d₆) δ: 0.96 (9H, s), 1.39 1.56 (4H, m), 2.85 (1H, d, J = 9.3 Hz), 3.06 (3H, s), 3.12 - 3.30 (5H, m), 3.49 (1H, d, J = 9.1 Hz), 3.74 - 3.81 (1H, m), 6.23 (1H, s), 6.40 (III, s), 7.50 -7.60 (3H, m), 10.18 (1H, s). LC-MS calcd for C24H30N4O6S2 534.16, found 535.4 [M+H⁺],

Example 33 (rac-di-exo)-6-Hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro-lÀ⁶benzol 1,2,4]thiadiazin-3-yl)-3-(3-methyl-butyl)-1-oxa-3-azatricyclo[6.2.1.0²’⁷undec-5-en-4-one

<img file="BRPI0809685A2_D0196.tif" />

a) (rac-di-exo)-3-[[2-(7-Iodo-1,1-dioxo-) acid methyl ester

1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-acetyl]-(3-methyl-butyl)-amino]7-oxa-bicyclo[2.2.1] heptane -2-carboxylic acid

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<img file="BRPI0809685A2_D0197.tif" />

Acid (7-Iodo-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in US Patent Application 2008/0031852, 0.2 g, 0.546 mmol) was dissolved in anhydrous N,N5-dimethylformamide (5 mL). Methyl ester of (rac-di-exo)-3-(3-Methylbutylamino)-7-oxa-bicyclo[2.2.l]heptane-2-carboxylic acid (prepared as described in Example 25a, 0.132 g, 0.546 mmol) was added followed by l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.11 g, 0.573 mmol). Then A-methylmorpholine (0.116 g, 1.15 mmol) was added to the reaction mixture above. The mixture was stirred at 25°C for 16 h.

The solution was poured into a 1.0 M aqueous hydrochloric acid solution (100 mL). The aqueous layer was extracted with ethyl acetate (2 x 100 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under vacuum to provide the crude product, methyl ester of (rac-di15 exo)-3-[[2-(7-iodo-1,1-dioxo-1,4-dihydro- lÀ^the-benzo[1,2,4]thia-diazin-3-yl)acetyl]-(3-methylbutyl)amino]-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid, as a light yellow oil, which was used in the next step without any further purification. LC-MS (ESI) calcd for C22H28IN3O6S 589.07, found 590.5 [M+H⁺]

b) (rac-di-exo)-6-Hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro-1 λ⁶benzo[1,2,4]thiadiazin-3-yl)-3-(3-methyl-butyl)-1-1-oxa-3-aza-tricyclo[6.2.1. 0²’⁷undec-5-en-4-one

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<img file="BRPI0809685A2_D0198.tif" />

The acid methyl ester (rac-di-exo)-3-[[2-(7-iodo-1,1-dioxol,4-dihydro-lÀ⁶Crude -benzo[l,2,4]thiadiazin-3-yl)-acetyl]-(3-methylbutyl)-amino]7-oxa-bicyclo[2.2.l]heptane-2-carboxylic acid was dissolved in ethanol (5 mL), and a 21% by weight solution of sodium ethoxide in ethanol (1 mL, 2.73 mmol) was added to the above solution. The mixture was stirred at 60°C for 6 hours and allowed to cool to 25°C. The mixture was poured into 0.5 M aqueous hydrochloric acid solution (100 mL). The product began to precipitate and was collected by vacuum filtration. The precipitate was purified by flash column chromatography (Teledyne Isco RediSep column; 100% ethyl acetate) to provide the desired product, (rac-di-exo)-6-hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro-1-⁶-benzo[1,2,4]thiadiazin-3-yl)-3-(3-methylbutyl)-1-oxa-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-4-one (0.066 g, 0.118 mmol, 21.7% in two steps), as a white solid.^XH NMR (400

MHz, DMSO-d₆) δ: 0.92 (6H, d, J = 6.2 Hz), 1.41 - 1.73 (7H, m), 2.52 2.52 (1H, m), 2.94 - 3.07 (1H, m), 3.81 - 3.92 (2H, m), 4.75 (2H, d, J = 2.2 Hz), 7.37 (1H, d, J = 8.5 Hz), 8.00 (1H, dd, Ji = 8.6 Hz, J₂ = 2.2 Hz), 8.08 (1H, s). LC-MS (ESI) calcd for C21H24IN3O5S 557.05, found 558.2 [M+H⁺Anal, calcd para C2iH₂4lN₃THE₅S*0.5 II₂O: C, 44.53; H,

4.45; N, 7.45; found C, 44.69; H, 4.15; N, 7.45.

Example 34 (1R,2S,7R,8S)-6-Hydroxy-5-(7-iodo-1, 1-dioxo-1,4-dihydro-lÀ⁶benzo[1,2,4Jthiadiazin-3-yl)-3-(3-methyl-butyl)-3-azatricyclo[6.2.1.0²-⁷undec-5-en-4-one

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<img file="BRPI0809685A2_D0199.tif" />

a) Ethyl ester of (lS,2R,3S,47?)-3-[[2-(7-Iodo-l,ldioxo-l,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-acetyl]-(3-methyl-butyl)-amino]-bicyclo[2.2. l]heptane-2-carboxylic acid

<img file="BRPI0809685A2_D0200.tif" />

Acid (7-Iodine-1, 1-dioxo-1,4-dihydro- lA⁶-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in U.S. Patent Application 2008/0031852, 0.2 g, 0.546 mmol) was dissolved in anhydrous N,N-dimethylformamide (5 mL). Ethyl ester of (1S,2R,3S,4R)-3(3-Methylbutylamino)-bicyclo[2.2.1]heptane-2-carboxylic acid (prepared as described in Example 22a, 0.138 g, 0.546 mmol) was added followed by l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.11 g, 0.573 mmol). Then W-methylmorpholine (0.116 g, 1.15 mmol) was added to the reaction mixture above. The mixture was stirred at 25 °C for 16 h. The solution was poured into 1.0 M aqueous hydrochloric acid solution (100 mL). The aqueous layer was extracted with ethyl acetate (2 x 100 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under vacuum to give the crude product, ethyl ester of (1S,2J?,3S,4J?)-3-[[2-(7-iodo-1,1-dioxo-1,4-dihydro-1A⁶-benzo[1,2,4]thiadiazin-3-yl)-acetyl]-(3-methyl-butyl)-amino]-bicyclo[2.2.1]heptane-2

216/317 carboxylic acid, as a light yellow oil, which was used in the next step without any further purification. LC-MS (ESI) calcd for C24H32IN3O5S 601.11, found 602.6 [M+H⁺],

b) (1 R,2 S,7R, 8 S)-6-Hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydrolÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-(3-methyl-butyl)-3-aza-tricyclo[6.2.1.0²·⁷] undec-5-em-4-ona

<img file="BRPI0809685A2_D0201.tif" />

<img file="BRPI0809685A2_D0202.tif" />

The ethyl ester of (1S,2R,3S,4R)-3-[[2-(7-iodo-1,1dioxo-1,4-dihydro-1A⁶Crude -benzo[1,2,4]thiadiazin-3-yl)-acetyl]-(3-methylbutyl)amino]-bicyclo[2.2.1]heptane-2-carboxylic acid (0.546 mmol) was dissolved in ethanol (5 mL), and a 21% by weight solution of sodium ethoxide in ethanol (1 mL, 2.73 mmol) was added to the above solution. The mixture was stirred at 60°C for 6 hours and allowed to cool to 25°C. The mixture was poured into 0.5 M aqueous hydrochloric acid solution (100 mL). The product began to precipitate and was collected by vacuum filtration. The precipitate was purified by flash column chromatography (Teledyne Isco RediSep column; 40% ethyl acetate in hexanes) to provide the desired product, (lR,2S,7R,8S)-6-hydroxy-5-(7-iodo-l,l-dioxo-l,4-dihydro-l⁶-benzo[1,2,4]thiadiazin-3-yl)-3-(3-methyl-butyl)-3-aza-tricycle [6. 2.1.0²>⁷]undec-5-en-4-one (0.122 g, 0.2 mmol, 40.3% in two steps), as a light yellow solid. NMR (400 MHz, DMSO-dg) δ: 0.92 (6H, d, J =5.4 Hz), 1.16 - 1.63 (9H, m), 2.99 - 3.12 (2H, m), 3.62 - 3.71 (2H, m), 7.37 (1H, d, J = 8.3 Hz), 7.99 (1H, dd, Ji = 8.6 Hz, J₂ = 1.6 Hz), 8.08 (1H, s). hC-MS (ESI) calcd for C22H26IN3O4S 555.07, found 556.3 [M+H⁺].

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Example 35

Cyclopropanesulfonic acid /3-[(177,28,777,88)-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²-⁷]undec-5-en-5-yl]-l,1dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-amide

<img file="BRPI0809685A2_D0203.tif" />

(177.2S,77?,8S)-3-(4-fluoro-benzyl)-6-hydroxy-5-(7-iodo-1,ldioxo-1,4-dihydro-lÀ^the-benzo[1,2,4]thiadiazin-3-yl)-3-aza-tricyclo[6.2.1.0²>⁷undec-5-en-4-one (prepared as described in Example 19, 81 mg, 0.14 mmol), cyclopropanesulfonic acid amide (132 mg, 1.09 mmol), sarcosine (7V-methylglycine) (18 mg, 0.20 mmol), copper(I) iodide (26 mg, 0.14 mmol), and potassium phosphate (173 mg, 0.82 mmol) were combined and dissolved in 7V,7V-dimethylformamide (4 mL). The flask was degassed and fed back with nitrogen (3x). The reaction was stirred at 100°C for 4 h. The mixture was allowed to cool to 25°C, diluted with ethyl acetate (20 mL), and extracted with a saturated aqueous sodium bicarbonate solution (2 x 20 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated under vacuum. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 8% methanol in dichloromethane) yielded the desired product, cyclopropanesulfonic acid /3-[(177.2S,777.8S)-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-l,l-dioxo-l,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-amide (76 mg, 0.13 mmol, 96%), as a white powder. Ή NMR (400 MHz, DMSO-de) B: 0.85 - 1.68 (8H, m), 2.50 - 2.62 (2H, m), 2.83 - 2.97 (3H, m), 3.45 - 3.47 (1H,

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m), 4.56 (1H, bs), 5.17 - 5.20 (1H, m), 7.03 -7.07 (2H, m), 7.20 - 7.23 (2H, m), 7.63 - 7.69 (2H, m), 8.01 (1H, s), LC-MS (ESI) calculated for C27H27FN4O6S2 586.14, found 587.4

Example 36 (rgc-di-exo)-A-{3-[3-(3,3-Dimethyl-butyl)-6-hydroxy-4-oxo-3-aza tricyclo|6.2.1.0²-⁷]undeca-5,9-dien-5-yl]-1, 1-dioxo-1,4-dihydro- 1À⁶benzo) 1,4]thiazin-7-yl)-methanesulfonamide

<img file="BRPI0809685A2_D0204.tif" />

a) Acid methyl ester (rgc-di-exo)-3-{(3,3-Dimethyl-butyl)10 [2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- 1À⁶benzo[1,4]thiazin-3-yl)-acetyl]-amino}-bicyclo[2.2. l]hept-5-ene-2carboxylic acid

<img file="BRPI0809685A2_D0205.tif" />

To a stirred solution of methyl ester of (rgc-di15 exo)-3-(3,3-dimethylbutylamino)-bicyclo[2.2.l]hept-5-ene-2-carboxylic acid (prepared as described in Example 32a, 103 mg, 0.410 mmol) in anhydrous Α,Α-dimethylformamide (3 mL) under a nitrogen atmosphere

219/317 nio, acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1A⁶-benzo[l,4]thiazin-3-yl)-acetic acid (prepared as described in Example 29a, 136 mg, 0.410 mmol), IV-methylmorpholine (87.1 mg, 0.861 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (82.3 mg, 0.431 mmol) were added sequentially. After stirring at 25°C for 90 min, 1.0 M aqueous hydrochloric acid solution (10 mL) and saturated aqueous brine solution were added to the reaction mixture. The mixture was extracted with ethyl acetate (3 x 10 mL) and the combined organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The crude product, methyl ester of (racdi-exo)-3-{(3,3-dimethylbutyl)-[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1⁶-benzo[1,4]thiazin-3-yl)-acetyl]-amino}-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid was used directly in the next step without further purification. LC-MS calculation for C26H35N3O7S2 showed 565.19, found 566.5 [M+H⁺],

b) (rac-di-exo)-7V-{3-[3-(3,3-Dimethyl-butyl)-6-hydroxy-4-oxo-3aza-tricyclo[6.2.1.0²>⁷]undeca-5,9-dien-5-yl]-1,1 -dioxo-1,4-dihydro- 1À⁶benzo[I,4]thiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0206.tif" />

The methyl ester of (rac-di-exo)-3-{(3,3-dimethylbutyl)[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- lÀ⁶Crude -benzo[1,4]thiazin-3-yl)-acetyl]-amino}-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid was dissolved in ethanol (10 mL). To this solution was added a 21% by weight solution of sodium ethoxide in ethanol (0.399 g, 1.23 mmol) and the mixture

The reaction mixture of 220/317 was stirred at 25°C for 2.5 h. A 1.0 M aqueous hydrochloric acid solution (10 mL) was added and, after stirring for another 30 min, an additional 1.0 M aqueous hydrochloric acid solution (5 mL) was added, under which the product precipitated. The solid was collected by vacuum filtration, washed with a 1.0 M aqueous hydrochloric acid solution (5 mL), and then dried under vacuum to provide the desired product, (rac-diexo)-A-{3-[3-(3,3-dimethylbutyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²-⁷] undeca-5,9-dien-5-yl]-1,1 -dioxo-1,4-dihydro- lÀ⁶-benzo[1,4]thiazin-7-yl}methanesulfonamide (59 mg, 0.111 mmol, 27.1% in two steps), as a yellowish cinnamon-colored solid.^LH NMR (400 MHz, CDCh) δ: 1.00 - 1.01 (9H, m), 1.26 - 1.29 (2H, m), 1.51 - 1.70 (4H, m), 2.57 - 2.63 (1H, m), 3.07 (3H, s), 3.10 - 3.17 (2H, m), 3.24 - 3.26 (1H, m), 3.35 3.39 (III, m), 3.39 - 3.99 (III, m), 4.90 (0.7H, d, J = 16.7 Hz), 5.20 (0.3H, d, J = 16.2 Hz), 5.27 (0.7H, d, J = 16.9 Hz), 5.73 (0.3H, d, J =

16.1 Hz), 6.17 - 6.22 (1H, m), 6.36 - 6.39 (1H, m), 7.17 (1H, s), 7.22 -

7.25 (2H, m), 7.56 - 7.61 (1H, m), 7.67 - 7.69 (1H, m). LC-MS calcd for C25H31N3O6S2 533.17, found 534.4 [M+H⁺].

Example 37 (rac-di-eA:o)-A-í3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza20 tricycle [6.2,1.0²-⁷1undeca-5,9-dien-5-yl]-l, 1-dioxo-1,4-dihydro- 1À⁶ benzo[1,41thiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0207.tif" />

a) Methyl ester of (rac-di-exo)-3-{(4-fluoro-benzyl)-[2221/317 (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,4]thiazin-3yl)-acetyl]-amino}-bicyclo[2,2.1]hept-5-ene-2-carboxylic

<img file="BRPI0809685A2_D0208.tif" />

To a stirred solution of methyl ester of (rac-diexo)-3-(4-fluoro-benzylamino)-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (prepared as described in Example 9b, 112.7 mg, 0.410 mmol) in anhydrous α,α-dimethylformamide (3 mL) under a nitrogen atmosphere, (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1α-⁶-benzo[1,4] thiazin-3-yl)-acetic acid (prepared as described in Example 29a, 136 mg, 0.410 mmol), α-methylmorpholine (87.1 mg, 0.861 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (82.3 mg, 0.431 mmol) were added sequentially. After stirring at 25°C for 90 min, 1.0 M aqueous hydrochloric acid solution (10 mL) and saturated aqueous brine solution (10 mL) were added to the reaction mixture. The mixture was extracted with ethyl acetate (3 x 10 mL) and the combined organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The crude product, methyl ester of (rac-diexo)-3-{(4-fluorobenzyl)-[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1-⁶-benzo[l,4]thiazin-3-yl)-acetyl]-amino}-bicyclo[2.2.1]hept-5-ene2-carboxylic acid was used in the next step without any further purification. LC-MS calculation for C27H28FN3O7S2 showed 589.14, found 590.5 [M+H⁺],

c) (rac-di-exo)-7V-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undeca-5,9-dien-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,4]thiazin-7-yl}-methanesulfonamide

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<img file="BRPI0809685A2_D0209.tif" />

The acid methyl ester (rac-di-exo)-3-{(4-fluoro-benzyl)-[2(7-methanesulfonylamino-1,1-dioxo 1,4-dihydro-lÀ⁶Crude -benzo[1,4]thiazin-3yl)-acetyl]-amino}-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid was dissolved in ethanol. To this solution was added a 21% by weight solution of sodium ethoxide in ethanol (0.399 g, 1.23 mmol) and the reaction mixture was stirred at 25°C for 2.5 h. A 1.0 M aqueous hydrochloric acid solution (10 mL) was added and, after stirring for another 30 min, an additional 1.0 M aqueous hydrochloric acid solution (5 mL) was added, under which the product precipitated. The solid was collected by vacuum filtration, washed with 1.0 M aqueous hydrochloric acid solution (5 mL), and then dried under vacuum to provide the desired product, (rac-di-exo)-N-{3[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1,0²’⁷]undeca-5,9dien-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,4]thiazin-7-yl}-methanesulfonamide (44 mg, 0.079 mmol, 19.3% in two steps), as a cinnamon-colored solid, bi-NMR (400 MHz, CDC1₃) δ: 0.83 - 0.93 (1H, m), 1.26 1.29 (2H, m), 1.51 (1H, d, J = 8.4 Hz), 1.69 (1H, d,J = 9.5 Hz), 2.59 (1H, d, J = 8.3 Hz), 3.05 (3H, s), 3.09 (1H,s) (2H, m), 7.19 - 7.26 (3H, m), 7.56 (1H, d, J = 6.8 Hz), 7.68 (1H, s). LC-MS calcd for C26H24FN3O6S2 557.11, found 558.1 [M+H⁺],

Example 38 (rac-di-exo]-N- {3-[6-Hydroxy-3-(3-methylbutyl)-4-oxo-3-aza

223/317 tricycle [6.2.1.O²’^y]undeca-5,9-dien-5-yl]-l, 1-dioxo-1,4-dihydro- 1À⁶benzof 1,4]thiazin-7-yl} -methanesulfonamide

<img file="BRPI0809685A2_D0210.tif" />

a) (rac-di-exo)-3-[[2-(7-methanesulphonylamino-1,1-dioxo-1,4-dihydro-lÀ) acid methyl ester⁶-benzo[1,4]thiazin-3-yl)-acetyl](3-methylbutyl)-amino]-bicyclo[2,2.1]hept-5-ene-2-carboxylic acid

<img file="BRPI0809685A2_D0211.tif" />

To a stirred solution of methyl ester of (rac-diexo)-3-(3-methylbutylamino)-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (prepared as described in Example 31b, 97.2 mg, 0.410 mmol) in JV, anhydrous dimethylformamide (3 mL) under a nitrogen atmosphere, (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1-⁶-benzo[1,4]thiazin-3-yl)-acetic acid (prepared as described in Example 29a, 136 mg, 0.410 mmol), TV-methylmorpholine (87.1 mg, 0.861 mmol), and l-(315-dimethylaminopropyl)-3-ethylcarbodiimide chloride (82.3 mg, 0.431 mmol) were added sequentially. After stirring at 25°C for 90 min, 1.0 M aqueous hydrochloric acid solution (10 mL) and saturated aqueous brine solution (10 mL) were added to the reaction mixture. The mixture was extracted with ethyl acetate (3 x 10 mL) and the layers

224/317 combined organic compounds were dried over sodium sulfate, filtered, and concentrated under vacuum. The crude product, methyl ester of (rac-diexo)-3-[[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1]⁶benzo[1,4]thiazin-3-yl)-acetyl]-(3-methylbutyl)-amino]-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid was used in the next step without further purification. LC-MS calculation for C25H33N3O7S2 showed 551.18, found to be 552.3 [M+H⁺],

b) (rac-di-exo)-/V-{3-[6-Hydroxy-3-(3-methylbutyl)-4-oxo-3-azatricyclo [6.2.1,0²’⁷]undeca-5,9-dien-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,4]thiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0212.tif" />

The methyl ester of (rac-di-exo)-3-[[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- lÀ⁶Crude -benzo[1,4]thiazin-3-yl)-acetyl]-(3-methylbutyl)-amino]-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid was dissolved in ethanol (10 mL). To this solution was added a 21% by weight solution of sodium ethoxide in ethanol (0.399 g, 1.23 mmol) and the reaction mixture was stirred at 25°C for 2.5 h. A 1.0 M aqueous hydrochloric acid solution (10 mL) was added and, after stirring for another 30 min, an additional 1.0 M aqueous hydrochloric acid solution (5 mL) was added, under which the product precipitated. The solid was collected by vacuum filtration, washed with 1.0 M aqueous hydrochloric acid solution (5 mL), and then dried under vacuum to provide the desired product, (rac-di-exo)-N-{3[6-hydroxy-3-(3-methylbutyl)-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undeca-5,9dien-5-yl]-1,1-dioxo- 1,4-dihydro- lÀ⁶-benzo[I,4]thiazin-7-yl}-methanesulfonamide (48.6 mg, 0.094 mmol, 22.8% in two steps), as a solid

225/317 cinnamon color. Ui NMR (400 MHz, CDCI3) δ: 0.96 - 1.01 (611, m), 1.26 1.29 (2H, m), 1.48 - 1.73 (5H, m), 2.58 - 2.63 (1H, m), 3.07 (3H, s), 3.08 - 3.17 (1H, m), 3.25 (1H, s), 3.37 - 3.40 (1H, m), 3.89 - 3.97 (1H, m), 4.90 (0.7H, d, J = 17 CDCI3 Hz), 5.21 (0.3H, d, J- 17.4 Hz), 5.28 (0.7H, 5 d, J = 16.7 Hz), 5.72 (0.3H, d, J = 17.1 Hz), 6.17 - 6.22 (1H, m), 6.35 6.39 (HI, m), 7.23 (1H, d, J = 8.5 Hz), 7.38 (HI, s), 7.56 - 7.62 (HI, m), 7.68 -7.69 (1H, m). LC-MS calculation for C24II29N3O6S2 519.15, found 520.2 [M+H⁺].

Example 39 irac-di-exo)-TV-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²-⁷]undeca-5,9-dien-5-yl]-1,1 -dioxo-1,4-dihydro- 1λ⁶benzoph 1,2,4]thiadiazin-7-yl)-7V-methyl-methanesulfonamide

<img file="BRPI0809685A2_D0213.tif" />

a) (rac-di-exo)-3-(4-fluoro-benzyl)-6-hydroxy-5-(7-iodo-1,1-di15 oxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-aza-tricyclo[6.2.1.0²·⁷_undeca-5,9-dien-4-one

<img file="BRPI0809685A2_D0214.tif" />

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To a stirred solution of methyl ester of (rac-diexo)-3-(4-fluoro-benzylamino)bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (prepared as described in Example 9b, 400 mg, 1.454 mmol) in anhydrous N,N-dimethylformamide (14 mL) under a nitrogen atmosphere, acid (7-iodo-1,1-dioxo-1,4-dihydro-1⁶-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in US Patent Application 2008/0031852, 532.1 mg, 1.454 mmol), IV-methylmorpholine (309 mg, 3.053 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (292 mg, 1.527 mmol) were added sequentially. After stirring at 25°C for 4 h, triethylamine (441 mg, 4.362 mmol) was added, and the mixture was stirred at 50°C for 2 h. The reaction mixture was allowed to cool to 25°C and continued to be stirred for 16 h. Upon addition of a 1.0 M aqueous hydrochloric acid solution (10 mL), a white precipitate formed which was collected by vacuum filtration, washed with a 1.0 M aqueous hydrochloric acid solution (5 mL), and dried under vacuum to provide the desired product, (rac-di-exo)-3-(4-fluoro-benzyl)-6-hydroxy-5(7-iodo-1,1-dioxo-1,4-dihydro-1).⁶-benzo[1,2,4]thiadiazin-3-yl)-3-aza-tricyclo[6.2.1,0²,⁷undeca-5,9-dien-4-one (280.2 mg, 0.474 mmol, 32.6%), as a white solid.^XH NMR (400 MHz, DMSO-d₆) δ: 1.16 - 1.20 (2H, m), 1.38 (1H, d, J = 9.4 Hz), 1.63 (1H, d, J = 9.3 Hz), 2.86 (1H, bs), 3.21 (1H, bs), 3.41 (1H, d, J = 9.2 Hz), 4.53 (1H, d, J = 15.6 Hz), 5.05 (1H, d, J= 15.6 Hz), 6.13 - 6.16 (1H, m), 6.35 - 6.37 (1H, m), 7.15 (2H, t, J= 8.4 Hz), 7.33 - 7.38 (3H, m), 7.99 - 8.01 (1H, m), 8.09 (1H, s). LCMS calcd for C24H19FIN3O4S 591.01, found 592.4 [M+H⁺],

b) (rac-di-exo)-Af-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²>⁷]undeca-5,9-dien-5-yl]~1,1-dioxo-1,4-dihydro- lÀ⁶-benzo [1,2,4] thiadiazin-7-yl} - A-methyl-methanesulfonamide

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<img file="BRPI0809685A2_D0215.tif" />

(rac-di-exo)-3-(4-fluoro-benzyl)-6-hydroxy-5-(7-iodo-1,1-dioxo1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-aza-tricycle [6.2.1.0²-⁷Undeca-5,9-dien-4-one (0.100 g, 0.1692 mmol), potassium triphosphate (0.1075 g, 0.5076 mmol), sarcosine (0.0094 g, 0.1015 mmol), and copper(I) iodide (0.00128 g, 0.0677 mmol) were combined. Anhydrous N,N-dimethylformamide (3 mL) was added followed by N-methylmethanesulfonamide (0.09223 g, 0.8460 mmol). The solution was degassed and fed back with nitrogen. The mixture was stirred at 100°C for 16 h. Upon cooling, the mixture was diluted with ethyl acetate (200 mL), washed with 1.0 M aqueous hydrochloric acid solution (2 x 100 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to a solid. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 100% ethyl acetate in hexanes) yielded the desired product, (rac-di-exo)-7V-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²,⁷]undeca-5,9-dien-5-yl]-l, 1-dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazine-T-ilj-A-methyl-methanesulfonamide (0.00105 g, 0.0184 mmol, 10.8%), as a pale yellow solid.^XH NMR (400 MHz, CDC13) δ: 1.27 (3H, s), 1.56 (1H, d, J = 9.5 Hz), 1.66 (1H, d, J = 9.2 Hz), 2.19 (lH, s), 2.64 (1H, s), 2.75 (1H, d, J = 9.3 Hz), 3.19 (1H, bs), 3.39 (III, d, J = 9.4 Hz), 3.44 (1H, bs), 3.75 (III, s), 4.32 (1H, d, J = 15.4 Hz), 5.22 (1H, d, J = 14.8 Hz), 6.11 - 6.13 (1H, m), 6.36 -6.38 (III, m), 6.97 (1H, d, J = 8.6 Hz), 7.04 (2H, t, J = 8.6 Hz), 7.20 - 7.24 (2H, m), 7.85 (1H, dd, Ji = 8.6 Hz, J₂ = 2.2 Hz), 8.22 (1H, s). LC-MS calcd forC26H25FN4OõS2 572.12, found 573.3 [M+H⁺].

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Example 40 /3-(lR,2S,7R,8S)-[6-hydroxy--3-(3-methyl-butyl)-4-oxo-3-azathicyclo[6.2.1.0²&lt;⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- 1A⁶benzo[l,2₁4]thiadiazin-7-yl) -amide of cyclopropanesulfonic acid

<img file="BRPI0809685A2_D0216.tif" />

A reaction flask was loaded with copper(I) iodide (20 mg, 0.11 mmol), sarcosine (7V-methyl-ylglycine) (14.7 mg, 0.17 mmol), cyclopropanesulfonamide (125 mg, 1.04 mmol), (17α,2S,7R,8S)-6-hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro-1A⁶-benzo[1, 2,4]thiadiazin-3-yl)-3-(3-methyl-butyl)-3-aza-tricyclo[6.2.1.0²>⁷undec-5em-4-one (prepared as described in Example 34, 115 mg, 0.21 mmol) and potassium phosphate (176 mg, 0.83 mmol). The flask was degassed and back-filled with nitrogen, and then anhydrous N,N-dimethylformamide (5 mL) was added. The resulting suspension was vigorously shaken at 100°C for 3 h and then allowed to cool to 25°C. The mixture was passed through a Celite plug and washed with 10% methanol/dichloromethane. The filtrate was concentrated under vacuum, and the residue was purified by pre-HPLC [Luna 5gm C18 column (2) 100A AXIA 150 x 21.2 mm, 5 microns, 30%-95% in 7 min @ 30 mL/min flow rate, 0.05% trifluoroacetic acid in acetonitrile / 0.05% trifluoroacetic acid in water] to provide the desired product, /3-(17?,2S,7R,8S)-[6-hydroxy-3-(3-methylbutyl)-4-oxo-3-aza-tricyclo[6.2.1.0²⁷]undec-5-en-5-yl]-l, l-dioxo-1,4 -dihydro-lA⁶-benzo[1,2,4]thiadiazin-7-yl}-cyclopropanesulf229/317phonic acid-amide (25 mg, 0.046 mmol, 22%), as a white solid. HI NMR (400 MHz, DMSO-cZg) δ: 0.92 - 0.98 (10H, m), 1.20 - 1.64 (9H, m), 2.52 (1H, m), 2.62 (1H, s), 2.68 (1H, m), 3.00 (1H, d, J = 9.6 Hz), 3.07 (1H, m), 3.61 (1H, d, J= 10.4 Hz), 3.66 (1H, m), 7.50 - 7.59 (3H, m), 10.17 (1H, 5 s). LC-MS (ESI) calcd for C25H32N4O6S2 548.18, found 549.4

[M+H⁺].

Example 41

7V-(3-[(IR, 2S,7R, 8Sf3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azat.riciclo[6,2.1.0²-⁷]undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro- 1Ã⁶10 benzo) 1,2,4]thiadiazin-7-yl}-benzenesulfonamide

<img file="BRPI0809685A2_D0217.tif" />

(lR,2S,7R,8S)-3-(4-fluoro-benzyl)-6-hydroxy-5-(7-iodo-1,ldioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-aza-tricyclo[6.2.1.0²,⁷undec-5-en-4-one (prepared as described in Example 19, 0.10 g, 0.17 mmol), cyclopropanesulfonic acid amide (0.21 mg, 1.36 mmol), sarcosine (7V-methylglycine) (0.02 g, 0.26 mmol), copper(I) iodide (0.03 g, 0.17 mmol), and potassium phosphate (0.22 g, 1.02 mmol) were combined and dissolved in 7V,7V-dimethylformamide (10 mL). The flask was degassed and fed back with nitrogen (3x). The reaction was stirred at 100 °C for 4 h. The mixture was allowed to cool to 25 °C, diluted with ethyl acetate (20 mL), and extracted with a saturated aqueous sodium bicarbonate solution (2 x 20 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated at

230/317 vacuum. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 8% methanol in dichloromethane) yielded the desired product, 7V-{3-[(177,23,777,8S)-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0².⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÃ⁶-benzo[l,2,4]thiadiazin-7-yl}-benzenesulfonamide (0.10 g, 0.16 mmol, 92%), as a white powder.^]H NMR (400 MHz, DMSO-dg) □: 1.16 -1.19 (3H, m), 1.38 -1.57 (5H, m), 2.98 (1H, d, J = 8.4 Hz), 3.51 (1H, d, J = 9.2 Hz), 4.40 (1H, d, J = 15.6 Hz), 4.94 (1H, d, J- 15.6 Hz), 7.11 - 7.15 (1H, m), 7.29 -7.32 (2H, m), 7.39 -7.47 (2H, m), 7.54 -7.64 (4H, m), 7.74 - 7.82 (3H, m), 10.74 (1H, s). LC-MS (ESI) calcd for C30H27FN4O6S2 622.69, found 623.3 [M+H⁺],

Example 42 {3-[6-hydroxy-3-(3-methylbutyl)-4-oxo-1-oxa-3-aza-tricyclobenzyl 1,2,4]thiadiazin-7-yl}-amide of (rac-di-exo)cyclopropanesulfonic acid

<img file="BRPI0809685A2_D0218.tif" />

A reaction flask was loaded with copper(I) iodide (20 mg, 0.11 mmol), sarcosine (7V-methylglycine) (14.7 mg, 0.17 mmol), cyclopropanesulfonamide (125 mg, 1.04 mmol), (rac-di-exo)-6-hydroxy-5(7-iodo-1,1-dioxo-1,4-dihydro-lÀ⁵-benzo[1,2,4]thiadiazin-3-yl)-3-(3-methylbutyl)-1-oxa-3-aza-tricyclo[6.2.1.0²’⁷undec-5-en-4-one (prepared as described in Example 33, 115 mg, 0.21 mmol) and potassium phosphate

231/317 (175 mg, 0.82 mmol). The flask was degassed and back-filled with nitrogen, and then anhydrous Α,Α-dimethylformamide (5 mL) was added. The resulting suspension was vigorously shaken at 100°C for 5 h and then allowed to cool to 25°C. The mixture was passed through a Celite plug and washed with 10% methanol/dichloromethane. The filtrate was concentrated under vacuum, and the residue was purified by prep-HPLC [Luna 5Dm Cl8 column (2) 100A AXIA 150 x 21.2 mm, 5 microns, 30%95% in 7 min @ 30 mL/min flow rate, 0.05% trifluoroacetic acid in acetonitrile/ 0.05% trifluoroacetic acid in water] to provide the desired product, {3-[6-hydroxy-3-(3-methylbutyl)-4-oxo-1-oxa-3-azatricyclo[6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ^the(rac-di-exo)-cyclopropanesulfonic acid-benzo[1,2,4]thiadiazin-7-yl}-amide (17 mg, 0.031 mmol, 15%), as a white solid. Ή NMR (400 MHz, DMSO-dô) δ: 0.91 - 0.96 (10H, m), 1.40 - 1.76 (7H, m), 2.69 (1H, m), 3.00 (1H, bs), 3.29 (1H, m), 3.84 (2H, m), 4.74 (2H, m), 7.51 - 7.59 (3H, m), 10.17 (1H, s). LC-MS (ESI) calculated for C24H3oN40?S2 550.16, found 551.4 [M+H⁺]

Example 43 {3-(3-(3,3-dimethyl-butyl)-6-hydroxy-4-oxo-3-aza-tricyclo [6.2.1.0²’⁷]undeca5.9-dien-5-yl|-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-amide of (rac-di-exo)-cyclopropanesulfonic acid

<img file="BRPI0809685A2_D0219.tif" />

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a) Methyl ester of (rac-di-exo)-3-{(3,3-Dimethylbutyl)[2-(7-iodo-1,1-dioxo-1,4-1 λ⁶-[1,2,4]thiadiazin-3-yl)-acetyl]-amino}-bicyclo [2.2. l]hept-5-ene-2-carboxylic acid

<img file="BRPI0809685A2_D0220.tif" />

To a solution of methyl ester of (rac-di-exo)-3-(3,3-dimethylbutylamino)-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (prepared as described in Example 32a, 227.8 mg, 0.907 mmol) in N,N-dimethylformamide (3 mL) was added (7-iodo-1,1-dioxo-1,4-dihydro-1^the-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in US Patent Application 2008/0031852, 398.2 mg, 1.088 mmol) and the mixture was vortexed until all the material was completely dissolved. A 1.0 M solution of Aj/V-dicyclohexylcarbodiimide in dichloromethane (1.179 mL, 1.179 mmol) was added to the above solution and the mixture was stirred at 25°C for 18 h under a nitrogen atmosphere. The 7V,7V-dicyclohexylurea precipitate was removed by filtration over Celite, the filter cake was washed with dichloromethane (3 x 5 mL), and the solvent was removed under vacuum. The residue was dissolved in a 1:1 mixture of ethyl acetate and hexanes (20 mL), washed with saturated aqueous brine solution, saturated aqueous sodium bicarbonate solution, and water. The layers were separated, and the aqueous layers were extracted back with the 1:1 mixture of ethyl acetate and hexanes (20 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum to provide the crude product, methyl ester of (rac-di-exo)-3-{(3,3-dimethylbutyl)[2-(7-iodo-1,1-dioxo-1,4-dihydro-11-lÀ⁶-benzo[1,2,4]thiadiazine-3-yl)-acetyl]-amino} -bicyclo[2,2,1]hept-5-ene-2-carboxylic acid, as a yellow oil233/317

I, which was used in the next step without any further purification. LC-MS calculation for C24H30IN3O5S 599.10, found 600.3 [M+H⁺],

b) (rac-di-exo)-3-(3,3-Dimethyl-butyl)-6-hydroxy-5-(7-iodo-1,1dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-aza-tricyclo[6.2.1.

0²-⁷undeca-5,9-dien-4-one

<img file="BRPI0809685A2_D0221.tif" />

The acid methyl ester (rac-di-exo)-3-{(3,3-dimethyl-butyl)[2-(7-iodo-1,1-dioxo-1,4-dihydro-lÀ⁶Crude -benzo[1,2,4]thiadiazin-3-yl)-acetyl]amino}-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (877.2 mg, 0.907 mmol) was dissolved in ethanol (20 mL). To this solution was added a 21% by weight solution of sodium ethoxide in ethanol (1.176 g, 3.628 mmol) and the reaction mixture was stirred at 25°C for 2 h. A 1.0 M aqueous hydrochloric acid solution (20 mL) was added, at which point the product began to precipitate. The reaction mixture was stirred for another 30 min before the precipitate was collected by vacuum filtration. The solid was washed with 1.0 M aqueous hydrochloric acid solution followed by water and then vacuum dried. The resulting solid was then ground with methanol, filtered, and vacuum dried. Further purification by flash column chromatography (Teledyne Isco column) was performed.

RediSep; 0 to 50% ethyl acetate in hexanes) provided the desired product, (rac-di-exo)-3-(3,3-dimethyl-butyl)-6-hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-aza-tricyclo[6.2.1.0²·⁷] undeca-5,9-dien-4-one (154.1 mg, 0.272 mmol, 30%) as a white solid. Ή NMR (400 MHz, CDCI3) δ: 1.01 (9H, s), 1.32 - 2.00 (4H, m),

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2.19 (III, d, J = 2.2 Hz), 2.75 (1H, d, J = 9.5 Hz), 3.10 - 3.52 (4H, m), 3.78 - 3.88 (HI, m), 6.17 - 6.20 (1H, m), 6.40 - 6.46 (III, m), 7.01 (III, d, J= 8.6 Hz), 7.84 (HI, dd, Ji = 8.5 Hz, J₂ = 1.5 Hz), 8.22 (III, s). LCMS calcd for C23H26IN3O4S 567.07, found 568.3 [M+H⁺],

c) ({3-(3-(3,3-Dimethyl-butyl)-6-hydroxy-4-oxo-3-aza-tricyclo [6.

2. 1.0²’⁷] undeca-5,9-dien-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-amide of (rac-di-exo)-cyclopropanesulfonic acid

<img file="BRPI0809685A2_D0222.tif" />

(rac-di-exo)-3-(3,3-Dimethyl-butyl)-6-hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-aza-tricyclo[6.2.1.0²·⁷Undeca-5,9-dien-4-one (0.155 g, 0.255 mmol), potassium triphosphate (0.271 g, 1.275 mmol), sarcosine (N-methyl glycine) (0.027 g, 0.306 mmol), and copper(I) iodide (0.024 g, 0.128 mmol) were combined. Anhydrous N,N-dimethylformamide (7 mL) was added followed by cyclopropanesulfonic acid amide (0.155 g, 1.275 mmol). The solution was degassed and fed back with nitrogen. The mixture was stirred at 100 °C for 16 h. Under cooling, the mixture was diluted with ethyl acetate (200 mL), washed with 1.0 M aqueous hydrochloric acid solution (2 x 100 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to a solid. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 100% ethyl acetate in hexanes) yielded the desired product, {3-[3-(3,3-dimethylbutyl)-6-hydroxy-4-oxo-3aza-tricyclo[6.2.1.0²-⁷]undeca-5,9-dien-5-yl]-1,1-dioxo-1,4-dihydro- 1λ⁶benzo[l,2,4]thiadiazin-7-yl}-amide of (rac-di-exo)-cyclopropane235/317 sulfonic acid (0.089 g, 0.159 mmol, 62.5%), as a pale yellow solid. 1H NMR (400 MHz, CDC1₃) δ: 1.00 (9H, s), 1.14 -1.21 (2H, m), 1.24 - 1.37 (2H, m), 1.51 - 1.63 (2H, m), 2.49 - 2.61 (1H, m), 2.73 -2.76 (1H, m), 3.09 - 3.16 (1H, m), 3.19 - 3.25 (1H, m), 3.38 - 3.46 (2H, m), 5 3.79 -3.88 (1H, m), 6.17 - 6.19 (1H, m), 6.38 - 6.40 (1H, m), 7.18 -7.25 (1H, m), 7.57 (1H, s), 7.63 -7.66 (1H, m), 7.73 -7.79 (1H, m). LC-MS (ESI) calculated for C26H32N4O6S2 560.18, found 561.6 [M+H⁺].

Example 44

7V-[3-(1R,2S,7R,8S)-3-Cyclopentyl-6-hydroxy-4-oxo-3-aza-¹⁰ tricycle[6.2.1.0²7]undec-5-en-5-yl)-1,1-dioxo-1,4-dihydro- 1λ⁶- benzo[1,2,4] thiadiazin-7-yl]-methanesulfonamide

<img file="BRPI0809685A2_D0223.tif" />

a) Ethyl ester of (1S,2R,3S,4R)-3-Cyclopentylaminobicyclo[2.2.1]heptane-2-carboxylic acid

<img file="BRPI0809685A2_D0224.tif" />

<img file="BRPI0809685A2_D0225.tif" />

Cyclopentanone (0.12 mL, 1.38 mmol) was added to a solution of ethyl ester of (1S,2R,3S,4R)-3-amino-bicyclo[2.2.1]heptane-2-carboxylic acid (prepared as described in Example 6k, 230 mg, 1.26 mmol) in anhydrous methanol (10 mL) at 25°C under a

236/317 nitrogen atmosphere. After stirring for 10 min, glacial acetic acid (0.5 mL) and sodium cyanoborohydride (260 mg, 3.15 mmol) were added sequentially, and the resulting mixture was stirred at 50°C for 30 min. The reaction mixture was poured into saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous brine solution, dried over sodium sulfate, and filtered. The filtrate was concentrated under vacuum to provide the desired product, ethyl ester of (1S,2R,3S,4R)-3-cyclopentylamino-bicyclo[2.2.1]heptane-2-carboxylic acid (237 mg, 0.94 mmol, 75%), as a yellow oil. LC-MS (ESI) calcd for C15H25NO2 251.19, found 252.0 [M+H⁺],

b) 7V-[3-(17?,2S,71?,8S)-3-Cyclopentyl-6-hydroxy-4-oxo-3-azatricycle [6.2.1.0²’⁷]undec-5-en-5-yl)-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl]-methanesulfonamide

<img file="BRPI0809685A2_D0226.tif" />

To a stirred solution of methyl ester of (1S,21?,3S,41?)-3-cyclopentylamino-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid (150 mg, 0.60 mmol) and (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1A⁶-benzo[1,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 181 mg, 0.54 mmol) in anhydrous α,α-dimethylformamide (5 mL) under a nitrogen atmosphere, α-methylmorpholine (0.12 mL, 1.08 mmol) and λ-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (104 mg, 0.54 mmol) were added sequentially. The mixture was stirred at 25°C for 45 min, triethylamine (0.25 mL, 1.76 mmol) was added, and the resulting mixture was stirred at 50°C for 60 h.

The 237/317 reaction mixture was allowed to cool to 25°C, diluted with ethyl acetate, washed with 1.0 M aqueous hydrochloric acid solution and saturated aqueous brine solution, dried over magnesium sulfate, and filtered. The filtrate was concentrated under vacuum and the residue was purified by prep HPLC [Luna 5Dm C18 column (2) 100A AXIA 150 x 21.2 mm, 5 microns,

30%-95% in 7 min @ 30 mL/min flow rate, 0.05% trifluoroacetic acid in acetonitrile / 0.05% trifluoroacetic acid in water] to provide the desired product, 7V-[3-(lR,2S,7R,8S)-3-cyclopentyl-6hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²>⁷]un-dec-5-en-5-yl)-1,1-dioxo-1,410 dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl]-methanesulfonamide (80 mg, 0.15 mmol, 26%), as a yellow solid. Ή NMR (400 MHz, DMSOd₆)S: 1.20 - 1.65 (8H, m), 1.75 - 1.95 (6H, m), 2.42 (1H, s), 2.60 (1H, s), 2.99 (III, d, J = 9.2 Hz), 3.05 (3H, s), 3.60 (1H, d, J= 9.2 Hz), 3.93 (III, m), 7.48 -7.58 (3H, m), 10.17 (1H, s), LC-MS (ESI) calcd for

C23H28N4O6S2 520.15, found 521.4 [M+H⁺].

Example 45 {3-[6-hydroxy-3-(3-methyl-butyl-4-oxo-3-aza-tricyclo[6.2.1,0²>⁷] undeca-5,9dien-5-yl]-1,1 -dioxo-1,4-dihydro- lÀ⁶-benzo| 1,2,41 Thiadiazin-7-yl}-(rac-di-exo)-cyclopropanesulfonic acid amide

<img file="BRPI0809685A2_D0227.tif" />

a) (rac-di-exo)-3-[[2-(7-Iodo-1,1-dioxo-1,4-dihydro-lÀ) acid methyl ester⁶-benzo[1,2,4]thiadiazin-3-yl)-acetyl]-(3-methyl-butyl)amino]-bicyclo[2.2. l]hept-5-ene-2-carboxylic acid

238/317

<img file="BRPI0809685A2_D0228.tif" />

To a solution of methyl ester of (rac-di-exo)-3-(3-methylbutylamino)-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (prepared as described in Example 31b, 214.8 mg, 0.906 mmol) in N,N-dimethylformamide (3 mL) was added (7-iodo-1,1-dioxo-1,4-dihydro-1⁶-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in US Patent Application 2008/0031852, 397.7 mg, 1.087 mmol) and the mixture was vortexed until all the material had been completely dissolved. A 1.0 M solution of A,A-dicyclohexylcarbodiimide in dichloromethane (1.178 mL, 1.178 mmol) was added to the above solution and the mixture was stirred at 25°C for 18 h under a nitrogen atmosphere. The Α,Α-dicyclohexylurea precipitate was removed by filtration over Celite, the filter cake was washed with dichloromethane (3 x 5 mL), and the solvent was removed under vacuum. The residue was dissolved in a 1:1 mixture of ethyl acetate and hexanes (20 mL), washed with saturated aqueous brine solution, saturated aqueous sodium bicarbonate solution, and water. The layers were separated, and the aqueous layers were extracted back with the 1:1 mixture of ethyl acetate and hexanes (20 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum to provide the crude product, methyl ester of (rac-di-exo)-3-[[2-(7-iodo-1,1dioxo-1,4-dihydro-1]^the-benzo[1,2,4]thiadiazin-3-yl)-acetyl]-(3-methylbutyl)amino]-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid, as a yellow oil, which was used in the next step without any further purification. LC-MS calcd for C23H28IN3O5S 585.08, found 586.3 [M+H⁺].

b) (rac-di-exo)-6-Hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro- 1λ⁶

239/317 benzo [1,2,4]thiadiazin-3-yl)-3-(3-methyl-butyl)-3-aza-tricyclo[6.2.1 Ό²>⁷umdeca-5,9-dien-4-one

<img file="BRPI0809685A2_D0229.tif" />

The acid methyl ester (rac-di-exo)-3-[[2-(7-iodo-1,1-dioxol,4-dihydro-lÀ⁶Crude -benzo[l,2,4]thiadiazin-3-yl)-acetyl]-(3-methyl-butyl)-amino]bicyclo[2.2.l]hept-5-ene-2-carboxylic acid was dissolved in ethanol (20 mL). To this solution was added a 21% by weight solution of sodium ethoxide in ethanol (1.174 g, 3.624 mmol) and the reaction mixture was stirred at 25°C for 2 h. A 1.0 M aqueous hydrochloric acid solution (20 mL) was added, at which point the product began to precipitate. The reaction mixture was stirred for another 30 min before the precipitate was collected by vacuum filtration. The solid was washed with 1.0 M aqueous hydrochloric acid solution followed by water and then vacuum dried. The resulting solid was then ground with methanol, filtered, and vacuum dried. Further purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 50% ethyl acetate in hexanes) yielded the desired product, (rac-di-exo)-6-hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro-IgA⁶-benzo[1,2,4]thiadiazin-3-yl)-3-(3-methyl-butyl)-3aza-tricyclo[6.2.1.0²’⁷undeca-5,9-dien-4-one (28.7 mg, 0.0519 mmol, 5.7% in two steps), as a dirty white solid.^XH NMR (400 MHz, CDC1₃) δ: 0.98 (3H, d, J = 3.3 Hz), 0.99 (3H, d, J = 2.9 Hz), 1.47 - 1.68 (6H, m), 2.75 (1H, d, J = 9.3 Hz), 3.08 - 3.32 (2H, m), 3.39 - 3.44 (2H, m), 3.82 - 3.89 (1H, m), 6.18 - 6.20 (1H, m), 6.39 - 6.41 (1H, m), 6.99 (1H, d, J= 8.6 Hz), 7.84 (1H, d, J = 10.8 Hz), 8.21 (1H, s). LC-MS calculation for C22H24IN3O4S 553.05, found 554.1 [M+H⁺].

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c) (3-16-IIhydroxy-3-(3-methyl-butyl)-4-oxo-3-aza-tricyclo[6.2.1. 0²7]undeca-5,9-dien-5-iri-1,1 -dioxo-1,4-dihydro- lÀ⁶(rac-di-exo)-cyclopropanesulfonic acid-benzo[1,2,41thia-diazin-7-yl}-amide

<img file="BRPI0809685A2_D0230.tif" />

(rac-di-exo)-6-Hydroxy-5-(7-iodo-1,1-dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-3-yl)-3-(3-methyl-butyl)-3-aza-tricyclo[6.2.1.0²·⁷Undeca-5,9-dien-4-one (0.029 g, 0.052 mmol), potassium triphosphate (0.055 g, 0.259 mmol), sarcosine (α-mctyl glycine) (0.006 g, 0.062 mmol), and copper(I) iodide (0.005 g, 0.026 mmol) were combined. Anhydrous N,N-dimethylformamide (7 mL) was added followed by cyclopropanesulfonic acid amide (0.031 g, 0.259 mmol). The solution was degassed and fed back with nitrogen. The mixture was stirred at 100°C for 16 h. Upon cooling, the mixture was diluted with ethyl acetate (200 mL), washed with 1.0 M aqueous hydrochloric acid solution (2 x 100 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to a solid. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 100% ethyl acetate in hexanes) yielded the desired product, {3-[6-hydroxy-3-(3-methylbutyl)-4-oxo-3-azatricyclo[6.2.1.0²’⁷]undeca-5,9-dien-5-yl]-l, 1-dioxo-1,4-dihydro-lÀ⁶benzo[l,2,4]thiadiazin-7-yl}-amide of (rac-di-exo)cyclopropanesulfonic acid (0.009 g, 0.016 mmol, 31.6%), as a pale yellow solid. 1H NMR (400 MHz, CDC1₃) δ: 0.98 - 1.08 (6H, m), 1.17 - 1.26 (311, m), 1.52 - 1.73 (4H, m), 2.48 - 2.62 (2H, m), 2.75 - 2.77 (1H, m), 3.08 - 3.15 (1H, m), 3.39 - 3.44 (2H, m), 3.83 - 3.91 (1H, m), 6.19 (1H, dd, Ji = 5.5 Hz, J₂ = 3.1 Hz), 6.40 (1H, dd, Ji = 5.4 Hz, J₂ = 4.0 Hz), 7.01 (1H, s),

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7.24 (1H, t, J = 7.1 Hz), 7.63 - 7.65 (1H, m), 7.70 - 7.73 (III, m). LC-MS (ESI) calculated for C25H30N4O6S2 546.16, found 547.4 [M+H⁺],

Example 46 {3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²-⁷]undeca5,9-dien-5-yl]-l, 1-dioxo-1,4-dihydro-lÃ⁶-benzo[l,2,4]thiadiazin-7-yljamide of (mc-di-exo)cyclopropanesulfonic acid

<img file="BRPI0809685A2_D0231.tif" />

(rac-di-exo)-3-(4-fluoro-benzyl)-6-hydroxy-5-(7-iodo-1,1-dioxol,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-aza-tricyclo[6.2.1.0²’⁷unde10 ca-5,9-dien-4-one (prepared as described in Example 39a, 0.169 g, 0.285 mmol), potassium triphosphate (0.303 g, 1.426 mmol), sarcosine (N-methylglycine) (0.031 g, 0.342 mmol), and copper(I) iodide (0.027 g, 0.146 mmol) were combined. Anhydrous α,α-dimethylformamide (7 mL) was added followed by cyclopropanesulfonic acid amide (0.173 g,

1,426 mmol). The solution was degassed and fed back with nitrogen. The mixture was stirred at 100°C for 16 h. Upon cooling, the mixture was diluted with ethyl acetate (200 mL), washed with 1.0 M aqueous hydrochloric acid solution (2 x 100 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to a solid. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 100% ethyl acetate in hexanes) yielded the desired product, {3-[3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²-⁷]undeca-5,9-dien-5-yl]-l, 1 -dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-amide acid (rac-di

242/317 exo)-cyclopropanessulfonic acid (0.093 g, 0.159 mmol, 55.8%), as a pale yellow solid. Hl NMR (400 MHz, CDCI3) δ: 0.97 - 1.04 (2H, m), 1.14 - 1.20 (2H, m), 1.53 (1H, d, J = 9.4 Hz), 1.66 (1H, d, J = 9.5 Hz), 2.48 - 2.61 (2H, m), 2.74 (1H, d, J = 9.4 Hz), 3.37 - 3.41 (2H, m), 4.32 (1H, d, J= 15.3 Hz), 5.19 (1H, d, J = 14.8 Hz), 6.09 - 6.11 (1H, m), 6.33 - 6.36 (1H, m), 7.02 (2H, t, J=8.6 Hz), 7.16 -7.23 (3H, m), 7.63 (III, dd, Ji = 9.3 Hz, J₂ = 2.3 Hz), 7.77 (1H, d, J = 2.3 Hz), 8.04 (1H, s). LC-MS (ESI) calculated for C27H25FN4O6S2 584.12, found 585.2 [M+H⁺],

Example 47

7V-[3-(1R,2S,7R,8SL(3-Cyclooentyl-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²,⁷]undec-5-en-5-yl)-1,1-dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-7-yl]-methanesulfonamide-/V-isopropyl carbamate

IV-[3-(1R,2S,7R,8S)-3-cyclopentyl-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl)-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl]-methanesulfonamide (prepared as described in Example 44, 48.5 mg, 0.093 mmol) was dissolved in anhydrous pyridine (2 mL) under a nitrogen atmosphere. A 1.0 M solution of isopropyl chloroformate in toluene (0.46 mL, 0.46 mmol) was added slowly. The resulting mixture was stirred at 25°C for 15 min. LC-MS showed the end of the reaction. The reaction was stopped with methanol (1 mL) and the resulting mixture was concentrated under vacuum. The residue was purified.

243/317 by prep-HPLC [Luna column 5μη C18 (2) 100Ω ACHIA 150 χ 21.2 mm, 5 microns, 30%-95% in 7 min @ 30 mL/min flow rate, 0.05% trifluoroacetic acid in acetonitrile/ 0.05% trifluoroacetic acid in water] to provide the desired product, 7V-[3-(lR,2S,7R,8S)-(3-cyclopentyl-65hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl)-1,1 -dioxo-1,4dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl]-methanesulfonamide-7V-isopropyl carbamate (15 mg, 0.025 mmol, 27%), as a yellow solid, HI NMR (400 MHz, DMSO-d₆) δ: 1.19 (6H, d, J = 6.0 Hz), 1.30 - 1.65 (8H, m), 1.75 - 1.95 (6H, m), 2.43 (1H, m), 2.61 (1H, m), 3.00 (1H, m), 3.55 (1H, m), 3.62 (3H, s), 3.95 (1H, m), 4.94 (1H, m), 7.57 (1H, d, J = 9.2

Hz), 7.64 (1H, dd, J = 8.8, 1.6 Hz), 7.97 (1H, s). LC-MS (ESI) calculated for C27H34N4O8S2 606.16, found 607.3 [M+H⁺].

Example 48 tricycle [6.2.1.0²’⁷you_ndec-5-en-5-yl)-1,1-dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4] thiadiazin-7-yl]-methanesulfonamide

<img file="BRPI0809685A2_D0232.tif" />

<img file="BRPI0809685A2_D0233.tif" />

a) Methyl ester of (rac-di-exo)-3-Cyclopentylamino-7oxa-bicyclo[2.2.1]heptane-2-carboxylic acid

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<img file="BRPI0809685A2_D0234.tif" />

Cyclopentanone (0.56 mL, 6.37 mmol) was added to a solution of (rac-di-exo)-3-amino-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid methyl ester (prepared as described in Example 8a, 1.10 g, 6.37 mmol) in anhydrous methanol (15 mL) at 25°C under a nitrogen atmosphere. After stirring for 20 min, glacial acetic acid (0.75 mL) and sodium cyanoborohydride (1.0 g, 15.92 mmol) were added sequentially, and the resulting mixture was stirred at 50°C for 1 h. The reaction mixture was poured into a saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate. The combined organic layers were washed with a saturated aqueous brine solution, dried over sodium sulfate, and filtered. The filtrate was concentrated under vacuum to give the desired product, methyl ester of (rac-diexo)-3-cyclopentylamino-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid (1.06 g, 4.43 mmol, 70%), as a yellow oil. LC-MS (ESI) calculation for C13H21NO3 239.15, found 240.2 [M+H⁺],

b) (rac-di-exo)-lV-[3-(3-Cyclopentyl-6-hydroxy-4-oxo-1 l-oxa-3aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl)-l, 1-dioxo-l,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl]-methanesulfonamide

<img file="BRPI0809685A2_D0235.tif" />

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To a stirred solution of methyl ester of (rac-diexo)-3-cyclopentylamino-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid (170 mg, 0.71 mmol) and (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro)⁶-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 197 mg, 0.60 mmol) in anhydrous A,A-dimethylformamide (5 mL) under a nitrogen atmosphere, IV-methylmorpholine (0.132 mL, 1.20 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (115 mg, 0.60 mmol) were added sequentially. The mixture was stirred at 25°C for 1 h, triethylamine (0.34 mL, 2.4 mmol) was added and the resulting mixture was stirred at 50°C for 1 h. The reaction mixture was allowed to cool to 25°C, diluted with ethyl acetate, washed with 1.0 M aqueous hydrochloric acid solution and saturated aqueous brine solution, dried over magnesium sulfate, and filtered. The filtrate was concentrated under vacuum and the residue was purified by prep-HPLC [Luna 5qm C18 column (2) 100 Â AXIA 150 x 21.2 mm, 5 microns, 30%-95% in 7 min @ 30 mL/min flow rate, 0.05% trifluoroacetic acid in acetonitrile / 0.05% trifluoroacetic acid in water] to provide the desired product, (rac-di-exo)-A-[3-(3-cyclopentyl-6-hydroxy-4-oxo-1-oxa-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl)-1,1 -dioxo-1,4-dihydro- 1À⁶benzo[l,2,4]thiadiazin-7-yl]-methanesulfonamide (120 mg, 0.23 mmol, 38%), as a dirty white solid.^XH NMR (400 MHz, DMSO-úe/ δ: 1.48 - 1.96 (12H, m), 3.05 (3H, s), 3.30 (1H, d, J = 8.0 Hz), 3.89 (III, d, J = 9.2 Hz), 4.13 (1H, m), 4.57 (III, s),4.74 (1H, s), 7.49 -7.58 (3H, m), 10.17 (1H, s) LC-MS (ESI) calculated for C22H26N4O7S2 522.12, found 523.4 [M+H).⁺],

Example 49

N-{3-[(28.7RL3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²>⁷]dodec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- 1λ⁶benzo[1,2,4]thiadiazin-7-yl)-methanesulfonamide

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<img file="BRPI0809685A2_D0236.tif" />

a) 4-Oxa-tricycle [5.2.2.0²’⁶undecane-3,5-dione

<img file="BRPI0809685A2_D0237.tif" />

4-Oxa-tricycle[5.2.2.0²-⁶]undec-8-ene-3,5-dione (4.00 g,

A concentration of 22.45 mmol) was dissolved in ethyl acetate (100 mL). Palladium in 10% carbon (400 mg) was added. The flask was degassed and fed back with hydrogen gas via a flask. The mixture was stirred at 25 ° for 16 h. The mixture was passed through a Celite plug and the filtrate was concentrated under vacuum to provide a thick, clear oil.

Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 30% ethyl acetate in hexanes) yielded the desired product, 4-oxa-tricyclo[5.2.2.0²’⁶]undecane-3,5-dione (2.92 g, 16.20 mmol, 72%), as a white powder. Ή NMR (400 MHz, DMSO-dõj δ: 1.55 - 1.64 (6H, m), 1.76 (2H, d, J = 9.2 Hz), 2.25 (2H, s), 3.11 (2H, s). LC-MS (ESI) calcd for C1OH12O3 180.20, found 181.0 [M+H⁺].

b) Methyl monoester of (2S,3R)-Bicyclo[2,2,2]octane-2,3-dicarboxylic acid

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<img file="BRPI0809685A2_D0238.tif" />

4-Oxa-tricyclo[5.2.2.0] was added.²’⁶[Undec-3,5-dione (0.90 g, 4.99 mmol)] was dissolved in toluene (50 mL) and carbon tetrachloride (50 mL). Quinine (1.78 g, 5.49 mmol) was added, and the mixture was cooled to -55 °C. Methanol (0.61 mL, 14.97 mmol) was added dropwise to the above mixture. The reaction was stirred at -55 °C for 18 h. The reaction was heated to 25 °C and concentrated under vacuum. The crude material was dissolved in ethyl acetate (50 mL) and washed with 1.0 M aqueous hydrochloric acid solution (2 x 40 mL). The organic layer was further washed with saturated aqueous brine solution (20 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a clear oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 50% ethyl acetate in hexanes) yielded the desired product, methyl monoester of (2S,3R)-bicyclo[2,2,2]octane-2,3dicarboxylic acid (1.10 g, 5.18 mmol, 92%), as a clear oil, hl NMR (400 MHz, DMSO-dõ/δ: 1.31 (2H, dd, Ji = 20.0 Hz, J₂ = 12.4 Hz), 1.52 1.54 (4H, m), 1.63 (1H, t, J = 10.4 Hz), 1.75 (1H, t, J = 9.6 Hz), 1.87 (2H, bs), 2.84 (2H, dd, Ji = 29.6 Hz, J₂ = 10.8 Hz), 3.52 (3H, s), 12.01 (1H, s). LC-MS (ESI) calculated for CnHieCH 212.24, found 213.1 [M+H⁺].

c) (2R,3S)-3-Benzyloxycarbonylamino-bicyclo[2,2,2]octane-2-carboxylic acid methyl ester

<img file="BRPI0809685A2_D0239.tif" />

OMe ; NHCbz

H

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Methyl monoester of (2S,3R)-Bicyclo[2,2,2]octane-2,3-dicarboxylic acid (1.01 g, 4.76 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL). The flask was degassed and back-filled with nitrogen, and the mixture was cooled to 0 °C. Triethylamine (1.99 mL, 14.28 mmol) was added, followed by the dropwise addition of ethyl chloroformate (0.91 mL, 9.52 mmol) with vigorous stirring. The mixture was stirred at 0 °C for 1 h. Sodium azide (0.93 g, 14.28 mmol) was dissolved in water (5 mL) and added to the reaction mixture at 0°C. The mixture was stirred at 0°C for 5 min. The ice bath was removed. The mixture was heated to 25°C and stirred for 2 h. The mixture was poured into water (50 mL) and the product extracted in ethyl acetate (50 mL). The organic layer was further washed with a half-saturated aqueous solution of sodium bicarbonate (2 x 20 mL), a saturated aqueous brine solution (20 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to yield a clear oil. The oil was dissolved in anhydrous benzene (10 mL) and refluxed with stirring under nitrogen for 2 h. Upon cooling to 25°C, the solution was concentrated under vacuum to yield a slightly yellow oil. The oil was dissolved in dichloromethane (10 mL) and benzyl alcohol (0.54 mL, 5.24 mmol) was added followed by triethylamine (1.33 mL, 9.52 mmol). The mixture was refluxed under nitrogen for 16 h. Upon cooling to 25°C, the solution was concentrated under vacuum to yield a golden oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 20% ethyl acetate in hexanes) yielded the desired product, methyl ester of (2R,3S/-3-benzyloxycarbonyl-aminobicyclo[2,2,2]octane-2-carboxylic acid (0.58 g, 1.83 mmol, 38%), as a clear oil, Ή NMR (400 MHz, CDC1₃) δ: 1.18 - 1.28 (2H, m), 1.42 - 1.50 (5H, m), 1.73 - 1.96 (3H, m), 2.88 (1H, d, Ji = 5.6 Hz), 3.27 (1H, s), 3.42 (3H, s), 4.00 - 4.04 (1H, m), 4.97 (2H, dd, Ji = 46.4 Hz, J₂ = 12.8 Hz), 7.06 (1H, d, J = 9.6 Hz), 7.24 -7.34 (4H, m). LC-MS (ESI) calcd for C18H23NO4 317.38, found 317.9 [M+H⁺],

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d) Methyl ester chloride of (2R,3S)-3-Aminobicyclo[2,2,2]octane-2-carboxylic acid

THE

<img file="BRPI0809685A2_D0240.tif" />

Methyl ester of (2R,3S/-3-Benzyloxycarbonylaminobicyclo[2,2,2]octane-2-carboxylic acid) (0.57 g, 1.79 mmol) was dissolved in ethyl acetate (20 mL). Palladium in 10% carbon (60 mg) was added. The flask was degassed and fed back with hydrogen gas via a balloon. The mixture was stirred at 25°C for 16 h. The mixture was passed through a Celite plug and the filtrate was concentrated under vacuum to provide a thick, clear oil. The oil was dissolved in diethyl ether (6 mL) and added dropwise with vigorous stirring to a mixture of 4.0 M hydrochloric acid solution in 1,4-dioxane (1.02 mL) and diethyl ether (10 mL). The desired product began to precipitate as a white solid. The mixture was stirred for 20 min. The precipitate was collected by vacuum filtration and washed with additional diethyl ether (5 mL). The solid was further dried under vacuum for 1 h to give the desired product, methyl ester chloride of /2R,3S)-3-amino-bicyclo[2,2,2]octane-2-carboxylic acid (0.33 g, 1.50 mmol, 84%), as a white powder.^XH NMR (400 MHz, DMSO-dgj δ: 1.38 (2H, dd, Ji = 21.2 Hz, J₂ = 13.6 Hz), 1.55 - 1.63 (5H, m), 1.76 - 1.89 (3H, m), 3.02 (1H, dd, Ji = 10.0 Hz, J₂ = 2.4 Hz), 3.47 (1H, bs), 3.65 (3H, s), 7.97 (3H, s). LC-MS (ESI) calculated for C10II17NO2 (free amine) 183.25, found 184.2 [M+H⁺].

e) (2R,3SL3-(4-fluoro-benzylamino)bicyclo[2,2,2] octane-2-carboxylic acid methyl ester

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<img file="BRPI0809685A2_D0241.tif" />

Methyl ester chloride of (2R,3S)-3-Amino-bicyclo[2,2,2]octane-2-carboxylic acid (0.34 g, 1.54 mmol) was dissolved in methanol (10 mL). Sodium acetate (0.25 g, 3.08 mmol) was added followed by powdered molecular sieves of 4A (0.34 g) and 4-fluorobenzaldehyde (0.16 mL, 1.54 mmol). Sodium cyanoborohydride (0.19 g, 3.08 mmol) was added and the mixture was stirred at 25°C for 16 h. The mixture was poured into a mixture of saturated aqueous solution of sodium bicarbonate (20 mL) and ethyl acetate (30 mL). After stirring, both layers were passed through a Celite plug. The organic layer was further washed with a saturated aqueous solution of sodium bicarbonate (10 mL), a saturated aqueous solution of brine (10 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the crude product, methyl ester of (2R,3S)-3-(4-fluorobenzylamino)-bicyclo[2,2,2]octane-2-carboxylic acid (0.32 g, 1.11 mmol, 72%), as a clear oil. LC-MS (ESI) calcd for C17H22FNO2 291.36, found 292.2 [M+H⁺].

f) A-{3-[(2S,7RL3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.2.0²>⁷]dodec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4] 20 thiadiazin-7-yl}-methanesulfonamide

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<img file="BRPI0809685A2_D0242.tif" />

Methyl ester of /2R,3S/-3-(4-fluorobenzylamino)-bicyclo[2,2,2]octane-2-carboxylic acid (93 mg, 0.32 mmol) was dissolved in anhydrous N,N-dimethylformamide (4 mL). Acid (7-methanesulfonylamino-1,1dioxo-1,4-dihydro-1⁶-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 107 mg, 0.32 mmol) was added followed by α-methylmorpholine (74 pL, 0.67 mmol). The mixture was stirred until everything was dissolved, approximately 5 min. l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide chloride (65 mg, 0.34 mmol) was added and the mixture was stirred at 25°C for 16 h. The reaction was stopped by adding a saturated aqueous solution of sodium bicarbonate (20 mL). The mixture was extracted with ethyl acetate (3 x 30 mL). The combined organic layers were washed with saturated aqueous brine solution (20 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to give a golden oil. The oil was dissolved in ethanol (5 mL). A 21% by weight solution of sodium ethoxide in ethanol (0.36 mL, 0.96 mmol) was added. The reaction was refluxed for 16 h. The reaction was stopped by adding a 1.0 M aqueous hydrochloric acid solution (10 mL). The mixture was extracted with ethyl acetate (3 x 20 mL). The organic layer was then washed with a saturated sodium bicarbonate solution (2 x 20 mL), a saturated aqueous brine solution (20 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a clear oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 20% ethyl acetate in dichloromethane) yielded the desired product, IV-{3-[(2S,7R)-3-(4-fluoro252/317benzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.2.0²>⁷]dodec-5-en-5-yl]-l,l-dioxo-1,4-dihydro-lA⁶-benzo[l,2,4]thiadiazin-7-yl}-methanesulfonamide (0.11 g, 0.19 mmol, 59%), as a white powder.^XH NMR (400 MHz, DMSO-de/ô: 1.39 (2H, d, J = 8.0 Hz), 1.54 - 1.59 (8H, m), 1.91 (1H, s), 5 2.14 (1H, s), 3.06 (3H, s), 3.75 (1H, d, J = 11.6 Hz), 4.28 (1H, d, J = 15.2

Hz), 5.03 (1H, d, J = 15.6 Hz), 7.13 -7.17 (2H, m), 7.34 - 7.37 (2H, m), 7.50 - 7.60 (3H, m), 10.18 (1H, s). LC-MS (ESI) calcd for C26H27FN4O6S2 574.64, found 575.1 [M+H⁺], mp: 203.8 - 205.7 °C. ee = 94.4% [HPLC analysis: Chiralpak AS-RH 4.6 x 250 mm, 5 microns, 0.8 10 mL/min, 310 nm).

Example 50 czs-IV-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.2.0²’⁷]dodec-5-en-5-yl]-1,1-dioxo-1.4-dihydro- 1A⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0243.tif" />

a) Cis-Bicyclo[2,2,2]octane-2,3dicarboxylic acid methyl monoester

<img file="BRPI0809685A2_D0244.tif" />

4-Oxa-tricycle[5.2.2.0²’⁶]undec-8-ene-3,5-dione (1.00 g, 5.61

A concentration of 253/317 mmol) was dissolved in methanol (20 mL). Palladium in 10% carbon (100 mg) was added. The flask was degassed and fed back with hydrogen gas via a balloon. The mixture was stirred at 25°C for 16 h. The mixture was passed through a Celite plug and the filtrate was left to stand at 25°C for 72 h. The solution was then concentrated under vacuum to provide a clear oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 60% ethyl acetate in hexanes) yielded the desired product, cisbicyclo[2,2,2]octane-2,3-dicarboxylic acid methyl monoester (1.10 g, 5.18 mmol, 92%), as a clear oil. Ή NMR (400 MHz, DMSO-d₆/B: 1.31 (2H, dd, JI = 20.0 Hz, J₂ = 12.4 Hz), 1.52 - 1.54 (4H, m), 1.63 (1H, t, J = 10.4 Hz), 1.75 (1H, t, J = 9.6 Hz), 1.87 (2H, bs), 2.84 (2H, dd, JI = 29.6 Hz, J2 = 10.8 Hz), 3.52 (3H, s), 12.01 (1H, s). LC-MS (ESI) calcd for CuHiôCA 212.24, found 213.2 [M+H⁺].

b) cisSBenzyloxycarbonylamino bicyclo[2,2,2]octane-2-carboxylic acid methyl ester

THE

OMe ^^NHCbz

Methyl monoester of cis-bicyclo[2,2,2]octane-2,3-dicarboxylic acid (1.09 g, 5.14 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL). The flask was degassed and back-filled with nitrogen, and the mixture was cooled to 0°C. Triethylamine (2.15 mL, 15.42 mmol) was added, followed by the dropwise addition of ethyl chloroformate (0.98 mL, 10.28 mmol) with vigorous stirring. The mixture was stirred at 0°C for 1 h. Sodium azide (1.00 g, 15.42 mmol) was dissolved in water (7 mL) and added to the reaction mixture at 0°C. The mixture was stirred to

0°C for 5 min. The ice bath was removed. The mixture was heated to 25°C and stirred for 2 h. The mixture was poured into water (70 mL) and the

254/317 The product was extracted in ethyl acetate (70 mL). The organic layer was further washed with a half-saturated aqueous sodium bicarbonate solution (2 x 30 mL), a saturated aqueous brine solution (30 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to give a clear oil. The oil was dissolved in anhydrous benzene (10 mL) and refluxed with stirring under nitrogen for 2 h. Upon cooling to 25°C, the solution was concentrated under vacuum to give a slightly yellow oil. The oil was dissolved in dichloromethane (7 mL) and benzyl alcohol (0.58 mL, 5.65 mmol) was added followed by triethylamine (1.43 mL, 10.28 mmol). The mixture was refluxed under nitrogen for 16 h. Upon cooling to 25°C, the solution was concentrated under vacuum to yield a golden oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 20% ethyl acetate in hexanes) yielded the desired product, cis-3-benzyloxycarbonylaminobicyclo[2,2,2]octane-2-carboxylic acid methyl ester (1.32 g, 4.16 mmol, 81%), as a clear oil. Ή NMR (400 MHz, CDC1₃) □: 1.18 -1.28 (2H, m),l.42 - 1.50 (5H, m), 1.73 - 1.96 (3H, m), 2.88 (1H, d, Ji = 5.6 Hz), 3.27 (1H, s), 3.42 (3H, s), 4.00 - 4.04 (1H, m), 4.97 (2H, dd, Ji = 46.4 Hz, J₂ = 12.8 Hz), 7.06 (1H, d, J = 9.6 Hz), 7.24 -7.34 (4H, m). LC-MS (ESI) calcd for C18H23NO4 317.38, found 317.92 [M+H⁺].

c) Methyl ester chloride of cis-3-aminobicyclo[2,2,2]octane-2-carboxylic acid

THE

HCI

Methyl ester of CZS-3-Benzyloxycarbonylamino-bicyclo[2,2,2]octane-2-carboxylic acid (0.67 g, 2.11 mmol) was dissolved in ethyl acetate (10 mL). Palladium carbonate 5% (97 mg) was added. The flask was degassed and refilled with hydrogen gas via a balloon.

The 255/317 mixture was stirred at 25°C for 16 h. The mixture was passed through a Celite plug and the filtrate was concentrated under vacuum to provide a thick, clear oil. The oil was dissolved in diethyl ether (6.7 mL) and added dropwise, with vigorous stirring, to a mixture of 4.0 M hydrochloric acid solution in 1,4-dioxane (1.2 mL) in diethyl ether (12 mL). The desired product began to precipitate as a white solid. The mixture was stirred for 20 min. The precipitate was collected by vacuum filtration and washed with additional diethyl ether (5 mL). The solid was then dried under vacuum for 1 h to give the desired product, cis-3-amino-bicyclo[2,2,2]octane-2-carboxylic acid methyl ester chloride (0.35 g, 1.59 mmol, 75%), as a white powder. Ή NMR (400 MHz, DMSO-dgj δ: 1.38 (2H, dd, Ji = 21.2 Hz, J₂ = 13.6 Hz), 1.55 - 1.63 (5H, m), 1.76 - 1.89 (3H, m), 3.02 (1H, dd, Ji = 10.0 Hz, J₂ = 2.4 Hz), 3.47 (1H, bs), 3.65 (3H, s), 7.97 (3H, s). LC-MS (ESI) calculated for C10H17NO2 (free amine) 183.25, found 184.05 [M+H⁺].

d) cis-3-(4-fluoro-benzylamino)-bicyclo[2,2,2]octane-2-carboxylic acid methyl ester

<img file="BRPI0809685A2_D0245.tif" />

Methyl ester chloride of cis-3-amino-bicyclo[2,2,2]octan-2-carboxylic acid (0.34 g, 1.55 mmol) was dissolved in methanol (10 mL). Sodium acetate (0.25 g, 3.10 mmol) was added followed by powdered molecular sieves of 4A (0.34 g) and 4-fluorobenzaldehyde (0.17 mL, 1.55 mmol). Sodium cyanoborohydride (0.20 g, 3.10 mmol) was added and the mixture was stirred at 25°C for 16 h. The mixture was poured into a saturated aqueous sodium bicarbonate solution.

256/317 (20 mL) and ethyl acetate (30 mL). After stirring, both layers were passed through a Celite plug. The organic layer was further washed with saturated aqueous sodium bicarbonate solution (10 mL), saturated aqueous brine solution (10 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the crude product, cis-3-β4-fluorobenzylamino)-bicyclo[2,2,2]octan-2-carboxylic acid methyl ester (0.34 g, 1.17 mmol, 75%), as a clear oil. LCMS (ESI) calcd for C17H22FNO2 291.36, found 292.18 [M+H⁺].

d) cys-A-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricycle [6.2.2.0²·⁷] dodec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0246.tif" />

Methyl ester of cis-3-(4-fluorobenzylamino)-bicyclo[2,2,2]octane-2-carboxylic acid (0.20 g, 0.69 mmol) was dissolved in anhydrous N,N-dimethylformamide (8 mL). Acid (7-methanesulfonylamino-1,1dioxo-1,4-dihydro-1⁶-benzo[1,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 0.23 g, 0.69 mmol) was added followed by 77-methylmorpholine (0.16 mL, 1.45 mmol). The mixture was stirred until everything was dissolved, approximately 5 min. l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.14 g, 0.72 mmol) was added and the mixture was stirred at 25°C for 4 h. Triethylamine (0.29 mL, 2.07 mmol) was added and the mixture was stirred at 50°C for 16 h. Upon cooling to 25°C, the solution was diluted with ethyl acetate (50 mL) and washed with 1.0 M aqueous hydrochloric acid solution (2 x 50 mL).

257/317 saturated aqueous brine solution (20 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to give a golden oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 20% ethyl acetate in dichloromethane) yielded the desired product, cis-A-{3-[3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[ 6.2.2.0²>⁷]dodec-5-en-5-yl]-l,l-dioxo-l,4-dihydro-lÀ⁶-benzo[l,2,4] thiadiazin-7-yl}-methanesulfonamide (0.30 g, 0.52 mmol, 76%), as a white powder. Ή NMR (400 MHz, DMSO-d6j δ: 1.39 (2H, d, J = 8.0 Hz), 1.54 - 1.59 (8H, m), 1.91 (1H, s), 2.14 (1H, s), 3.06 (3H, s), 3.75 (1H, d, J = 11.6 Hz), 4.28 (1H, d, J = 15.2 Hz), 5.03 (1H, d, J = 15.6 Hz), 7.13 7.17 (2H, m), 7.34 -7.37 (2H, m), 7.50 -7.60 (3H, m), 10.18 (1H, s) calcd for. C26H27FN4O6S2 574.64, found 575.4 [M+H⁺], mp: 203.8 - 205.7°C. Anal, calcd for C26H27FN4O₆S2«0.4 PhMe: C, 56.57; H, 4.98; N, 9.16; found C, 57.09; H, 5.08; N, 9.38.

Example 51 /2jR,7ShA-[3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatetracyclo[6.3.2.0²>⁷.0^g-^u]tridec-5-en-5-yl]-l, 1-dioxo-l^-dihydro-lÀ⁶benzol 1,2,4]thiadiazin-7-yl)-methanesulfonamide

<img file="BRPI0809685A2_D0247.tif" />

a)exo-4,4a,5,5a,6,6a-Hexahydro-4,6-ethene-1H-cycloprop[/] isobenzofuran-1,3(3aH)-dione and endo-4,4a,5.5a,6,6ahexahydro-4,6-ethene-1H-cycloprop[/]isobenzofuran-1,3(3aH)-dione

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<img file="BRPI0809685A2_D0248.tif" />

Furan-2,5-dione (5.32 g, 54.26 mmol) was partially dissolved in xylene (100 mL). Cyclohepta-1,3,5-triene (5 g, 54.26 mmol) was added. The reaction was stirred at 144°C for 5 h. The mixture was allowed to cool to 25°C and concentrated under vacuum. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 20% ethyl acetate in hexanes) yielded the desired products, exo-4,4a,5,5a,6,6a-hexahydro-4,6-etheno-1H-cycloprop[/]isobenzofuran-1,3(3aH)-dione (7.46 g, 39.22 mmol, 72%) and endo-4,4a,5,5a,6,6a-hexahydro-4,6-etheno-1H-cycloprop[/]isobenzofuran-1,3(3aH)-dione (0.88 g, 4.63 mmol, 8%), as white solids. Ή NMR (400 MHz, CDCh) δ: exo: 0.27 - 0.42 (2H, m), 1.11 -1.15 (2H, m), 3.25 (2H, t, J = 1.6 Hz), 3.46 - 3.50 (2H, m), 5.89 (2H, dd, Ji = 4.8 Hz, J₃ = 3.2 Hz);

endo: 0.14 - 0.26 (2H, m), 1.12 - 1.16 (2H, m), 3.09 (2H, s), 3.40 - 3.44 (2H, m), 5.94 (2H, dd, Ji = 4.8 Hz, J₂ = 3.2 Hz). LC-MS (ESI) calcd for C11H10O3 190.20, found exo 191.0; endo 191.3 [M+H⁺],

b) (6R,7S)-7-(methoxycarbonyl)tricyclo[3.2.2.0]²-⁴non-8-ene-6-carboxylic acid

<img file="BRPI0809685A2_D0249.tif" />

exoA,4a,5,5a,6,6a-Hexahydro-4,6-etheno-1H-cycloprop[/] isobenzofuran-1,3(3aH)-dione (1.00 g, 5.26 mmol) was dissolved in methanol (20 mL). The reaction was stirred at 25 °C for 72 h. The mixture was

259/317 concentrated under vacuum to provide a clear oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 60% ethyl acetate in hexanes) yielded the desired product, (6R,7S)-7-(methoxycarbonyl)tricyclo[3.2.2.0] acid.²>⁴]non-8-ene-6-carboxylic acid (1.11 g, 5.00 mmol, 95%), as a white solid.^XH NMR (400 MHz, DMSO-dg) δ: 0.01 - 0.10 (2H, m), 0.93 - 1.02 (2H, m), 2.98 - 3.05 (4H, m), 3.45 (3H, s), 5.68 - 5.77 (2H, m), 11.94 (1H, s). LC-MS (ESI) calcd for C12H14O4 222.24, found 223.2 [M+H⁺],

b) Methyl (6S,7R)-7-{[(benzyloxy)carbonyl] amino}tricycle [3.2. 2.0²>⁴non-8-ene-6-carboxylate

<img file="BRPI0809685A2_D0250.tif" />

(6R,7S/-7-(methoxycarbonyl)tricyclo[3.2.2.0²’⁴Non-8-ene-6-carboxylic acid (0.78 g, 3.53 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL). The flask was degassed and back-filled with nitrogen, and the mixture was cooled to 0 °C. Triethylamine (1.48 mL, 10.59 mmol) was added, followed by the dropwise addition of ethyl chloroformate (0.67 mL, 7.06 mmol) with vigorous stirring. The mixture was stirred at 0 °C for 1 h. Sodium azide (0.69 g, 10.59 mmol) was dissolved in water (5 mL) and added to the reaction mixture at 0 °C. The mixture was stirred at 0°C for 5 min. The ice bath was removed. The mixture was heated to 25°C and stirred for 2 h. The mixture was poured into water (70 mL) and the product extracted in ethyl acetate (70 mL). The organic layer was further washed with half-saturated aqueous sodium bicarbonate solution (2 x 30 mL), saturated aqueous brine solution (30 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a clear oil. The oil was dissolved in anhydrous benzene (10 mL) and refluxed with stirring under nitrogen for 2 h. Upon cooling...

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At 25°C the solution was concentrated under vacuum to yield a slightly yellow oil. The oil was dissolved in dichloromethane (10 mL) and benzyl alcohol (0.40 mL, 3.88 mmol) was added followed by triethylamine (0.98 mL, 7.06 mmol). The mixture was refluxed under nitrogen for 16 h. Upon cooling to 25°C the solution was concentrated under vacuum to yield a golden oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 20% ethyl acetate in hexanes) yielded the desired product, methyl (6S, 7Rj-7-{[(benzyloxy)carbonyl]amino}tricyclo[3.2.2.0²’⁴non-8-ene-6-carboxylate (0.62 g, 1.89 mmol, 54%) as a clear oil. Ή NMR (400 MHz, CDC1₃) δ: 0.17 - 0.22 (2H, m), 0.90 1.05 (2H, m), 2.98 - 3.14 (3H, m), 3.48 (3H, s), 4.38 - 4.43 (1H, m), 5.00 - 5.12 (2H, m), 5.77 (1H, t, J = 7.2 Hz), 6.05 (1H, t, J = 7.2 Hz), 7.29 7.37 (511, m). LC-MS (ESI) calcd for C19H21NO4 327.37, found 328.3 [M+H⁺],

c) Methyl hydrochlorate (6S, 7R/-7-aminotricycle [3.

2.2.0²’⁴nonane-6-carboxylate

<img file="BRPI0809685A2_D0251.tif" />

Methyl (6S, 7R)-7-{[(benzyloxy)carbonyl]amino }tricyclo[3.2.2. 0²-⁴Non-8-ene-6-carboxylate (0.61 g, 1.85 mmol) was dissolved in ethyl acetate (10 mL). Palladium in carbon 5% (0.10 g) was added. The flask was degassed and fed back with hydrogen gas via a flask. The mixture was stirred at 25°C for 16 h. The mixture was passed through a Celite plug and the filtrate was concentrated under vacuum to provide a thick, clear oil. The oil was dissolved in diethyl ether (6 mL) and added dropwise, with vigorous stirring, to a mixture of a 4.0 M hydrochloric acid solution in 1,4-dioxane (0.93 mL) in diethyl ether (11 mL). The mixture was concentrated and dried under vacuum to provide the

261/317 desired product, methyl chloride (6S, 7R)-7-aminotricyclo[3,2,2,0²[4]nonane-6-carboxylate (0.43 g, 1.85 mmol, 100%), as a sticky gum. LC-MS (ESI) calcd for C11H17NO2 (free amine) 195.26, found 196.5 [M+H⁺],

d) Methyl (6S,7R)-7-[(4-fluoro-benzyl)amino]tricycle [3.2.2.

0²’⁴nonane-6-carboxylate

<img file="BRPI0809685A2_D0252.tif" />

Methyl chloride (6S,7R)-7-aminotricyclo[3.2.2.0²’⁴]nonane-6-carboxylate (0.43 g, 1.85 mmol) was dissolved in methanol (10 mL). Sodium acetate (0.30 g, 3.70 mmol) was added followed by powdered molecular sieves of 4α (0.40 g) and 4-fluorobenzaldehyde (0.20 mL, 1.85 mmol). Sodium cyanoborohydride (0.23 g, 3.70 mmol) was added and the mixture was stirred at 25°C for 16 h. The mixture was poured into a mixture of saturated aqueous sodium bicarbonate solution (20 mL) and ethyl acetate (30 mL). After agitation, both layers were passed through a Celite plug. The organic layer was further washed with saturated aqueous sodium bicarbonate solution (10 mL), saturated aqueous brine solution (10 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the crude product, methyl (6S,7R/-7-[(4-fluoro-benzyl)amino]tricyclo[3.2.2.0²-⁴[nonane-6-carboxylate as a clear oil. LC-MS (ESI) calcd for C17H22FNO2 291.36, found 292.2 [M+H⁺].

d) (2R,7 Sj-A-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tetracyclo[6.3.2.0²-⁷.0⁹¹ ^tridcc-S-en-S-il] -1,1 -dioxo-1,4-dihydro- lÀ⁶-benzo

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[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0253.tif" />

Methyl (6S,7R)-7-[(4-fluoro-benzyl)amino]tricyclo[3.2.2.0²’⁴]nonane-6-carboxylate (0.15 g, 0.49 mmol) was dissolved in anhydrous N,N-dimethylformamide (8 mL). Acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1⁶-benzo[1,2,4]thiadiazin3-yl)-acetic acid (prepared as described in Example 1g, 0.16 g, 0.49 mmol) was added followed by IV-methylmorpholine (0.11 mL, 1.03 mmol). The mixture was stirred until everything was dissolved, approximately 5 min. l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.10 g, 0.51 mmol) was added and the mixture was stirred at 25°C for 4 h. Triethylamine (0.20 mL, 1.47 mmol) was added and the mixture was stirred at 50°C for 16 h. Upon cooling to 25°C, the solution was diluted with ethyl acetate (50 mL) and washed with 1.0 M aqueous hydrochloric acid solution (2 x 50 mL), saturated aqueous brine solution (20 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a golden oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 20% ethyl acetate in dichloromethane) yielded the desired product, (2R,7S)-7V-{3-[3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-azatetracyclo[ 6.3.2.0²>⁷.0⁹-¹¹]tridec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (0.23 g, 0.40 mmol, 82%), as a white powder. Ή NMR (400 MHz, DMSO-d₆) δ: 0.44 - 1.28 (10H, m), 2.29 (1H, bs), 3.06 (3H, s), 3.82 (1H, d, J = 11.6 Hz), 4.40 (III, d, J = 14.0 Hz), 4.98 (1H, d, J = 15.6 Hz), 7.12 -7.60 (7H, m), 10.19 (111, s). LC-MS (ESI) calcd for C27H27FN4O6S2 586.65, found 587.5

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[M+H⁺],

Example 52 (2S,7R)-7V-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3azatetracyclo[6.3.2.07707¹ qtridec-S-en-S-yl] -1,1-dioxo-l ,4-dihydro-1λ⁶ benzol 1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0254.tif" />

a) (6S,7R)-7-(methoxycarbonyl)tricyclo[3.2.2. 0]²>⁴non-8-ene-6-carboxylic

<img file="BRPI0809685A2_D0255.tif" />

endo-4,4a,5,5a,6,6a-Hexahydro-4,6-etheno-1H-cycloprop[isobenzofuran-1,3(3aH)-dione] (prepared as described in Example 51a, 0.86 g, 4.50 mmol) was dissolved in methanol (20 mL). The reaction was stirred at 25°C for 72 h. The mixture was concentrated to provide a clear oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 60% ethyl acetate in hexanes) yielded the desired product, (6S,7R)-7-(methoxycarbonyl)tricyclo[3.2.2.0]²’⁴]non-8-ene-6-carboxylic acid (0.94 g, 4.23 mmol, 94%) as a white solid.^XH NMR (400 MHz, CDC1₃) δ: 0.06 - 0.17 (2H, m), 1.26 1.38 (2H, m), 2.74 - 2.85 (2H, m), 3.15 - 3.19 (211, m), 3.67 (3H, s), 5.85

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- 5.86 (2H, m). LC-MS (ESI) calcd for C12II14O4 222.24, found 223.5 [M+H⁺].

b) Methyl (6R,7S/-7-{[(benzyloxy)carbonyl]amino}tricycle [3.2.2. 0²,4]non-8-ene-6-carboxylate

<img file="BRPI0809685A2_D0256.tif" />

NHCBZ

(6S,7R)-7-(methoxycarbonyl) tricyclo[3.2.2.0] acid²’⁴Non-8-ene-6-carboxylic acid (0.68 g, 3.07 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL). The flask was degassed and back-filled with nitrogen, and the mixture was cooled to 0°C. Triethylamine (1.28 mL, 9.21 mmol) was added, followed by the dropwise addition of ethyl chloroformate (0.58 mL, 6.14 mmol) with vigorous stirring. The mixture was stirred at 0°C for 1 h. Sodium azide (0.60 g, 9.21 mmol) was dissolved in water (5 mL) and added to the reaction mixture at 0°C. The mixture was stirred at 0°C for 5 min. The ice bath was removed. The mixture was heated to 25°C and stirred for 2 h. The mixture was poured into water (70 mL) and the product extracted in ethyl acetate (70 mL). The organic layer was further washed with half-saturated aqueous sodium bicarbonate solution (2 x 30 mL), saturated aqueous brine solution (30 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a clear oil. The oil was dissolved in anhydrous benzene (10 mL) and refluxed with stirring under nitrogen for 2 h. Upon cooling to 25°C, the solution was concentrated under vacuum to yield a slightly yellow oil. The oil was dissolved in dichloromethane (10 mL) and benzyl alcohol (0.35 mL, 3.38 mmol) was added followed by triethylamine (0.86 mL, 6.14 mmol). The mixture was refluxed under nitrogen for 16 h. Upon cooling to 25°C, the solution was concentrated under vacuum to yield a golden oil. Irrigation by flash column chromatography (Column

265/317 'Teledyne Isco R'édiSep; 0 to 20% ethyl acetate in hexanes) provided the desired product, methyl (6R,7S)-7-{[(benzyloxy)carbonyl]amino}tricyclo [3.2.2. 0²H]non-8-ene-6-carboxylate (0.36 g, 1.10 mmol, 36%) as a clear oil. NMR (400 MHz, CDC1₃) δ: 0.11 - 0.19 (2H, m), 0.13 - 1.34 (2H, m), 2.77 - 3.03 (3H, m), 3.62 (3H, s), 4.01 - 4.07 (1H, m), 5.07 5.14 (2H, m), 5.79 - 5.85 (2H, m), 7.30 - 7.38 (5H, m). LC-MS (ESI) calcd for C19H21NO4 327.37, found 328.3 [M+H⁺],

c) Methyl (6R,7S)-7-aminotricyclo[3.2.2.0²>⁴nonane-6-carboxylate

<img file="BRPI0809685A2_D0257.tif" />

Methyl (6R,7S/-7-{[(benzyloxy)carbonyl]amino}tricyclo[3.2.2. 0²>⁴non-8-ene-6-carboxylate (0.35 g, 1.07 mmol) was dissolved in ethyl acetate (10 mL). Palladium in carbon 5% (0.04 g) was added. The flask was degassed and back-filled with hydrogen gas via a balloon. The mixture was stirred at 25°C for 16 h. The mixture was passed through a Celite plug and the filtrate was concentrated under vacuum to give the crude product, methyl (6R,7S)-7-aminotricyclo[3.2.2.0²-⁴]nonane-6-carboxylate, as a thick, clear oil, which was used in the next step without further purification. LC-MS (ESI) calcd for C11H17NO2 (free amine) 195.26, found 196.3 [M+H⁺],

d) Methyl (6R,7S/-7-[(4-fluoro-benzyl)amino]tricyclo [3.2.2.

0²’⁴nonane-6-carboxylate

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<img file="BRPI0809685A2_D0258.tif" />

Methyl (6R,7S)-7-aminotricyclo[3.2.2.0²-⁴Crude nonane-6-carboxylate was dissolved in methanol (10 mL). Acetic acid (0.12 mL, 2.14 mmol) was added followed by 4-fluorobenzaldehyde (0.12 mL, 1.075 mmol). Sodium cyanoborohydride (0.14 g, 2.14 mmol) was added and the mixture was stirred at 60°C for 16 h. The mixture was cooled to 25°C and poured into a mixture of saturated aqueous sodium bicarbonate solution (20 mL) and ethyl acetate (30 mL). After stirring, both layers were passed through a Celite plug. The organic layer 10 was further washed with saturated aqueous sodium bicarbonate solution (10 mL), saturated aqueous brine solution (10 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide the crude product, methyl (SR, 7S)-7-[(4-fluoro-benzyl)amino]tricyclo[3.2.2.0²’⁴]nonane-6-carboxylate, as a clear oil, which was used in the following step 15 without further purification. LC-MS (ESI) calcd for C17H22FNO2 291.36, found 292.0 [M+H⁺].

e) (2S,77?Á^^-{3'[3*(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatetracyclo[6.3 .2.0²’⁷.0⁹’¹ ^tridec-S-en-õ-il]-1,1 -dioxo-1,4-dihydro- lÀ⁶-benzo [1, 2,4]thiadiazin-7-yl}-ethanesulfonamide

<img file="BRPI0809685A2_D0259.tif" />

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Methyl (6R, 7S/-7-[(4-fluoro-benzyl)amino]tricycle [3.2.2.0²·⁴Crude nonane-6-carboxylate was dissolved in anhydrous N,A-dimethylformamide (8 mL). Acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydrol⁶-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 0.22 g, 0.66 mmol) was added followed by N-methylmorpholine (0.15 mL, 1.39 mmol). The mixture was stirred until everything was dissolved, approximately 5 min. l-(3-l)imethylaminopropyl)-3-ethylcarbodiimide chloride (0.13 g, 0.69 mmol) was added and the mixture was stirred at 25°C for 4 h. Triethylamine (0.28 mL, 1.98 mmol) was added and the mixture was stirred at 50°C for 16 h. Upon cooling to 25°C, the solution was diluted with ethyl acetate (50 mL) and washed with 1.0 M aqueous hydrochloric acid solution (2 x 50 mL), saturated aqueous brine solution (20 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to provide a golden oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 20% ethyl acetate in dichloromethane) yielded the desired product, (2S,7R/-7V-{3-[3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-aza-tetracyclo[6.3. 2.0²’⁷.0⁹>ⁿ]tridec-5-en-5-yl]-l, l-dioxo-l,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (0.50 g, 0.86 mmol, 80% after three steps), as a white powder. Ή NMR (400 MHz, DMSO-d) δ: 0.33 1.34 (10H, m), 2.30 (1H, bs), 3.06 (3H, s), 3.81 (1H, d, J = 11.2 Hz), 4.48 (1H, d, J = 15.6 Hz), 4.98 (1H, d, J= 14.8 Hz), 7.13 -7.60 (7H, m), 10.19 (1H, s). LC-MS (ESI) calcd for C27H27FN4O6S2 586.65, found 587.3 [M+H⁺],

Example 53 (1 R,2 S,7R,88)-5-( 1,1 -Dioxo-7-pyrrolidin-1 -yl-1,4-dihydro- 1À⁶benzo[1,2,4]thiadiazin-3-yl)-6-hydroxy-3-(3-methyl-butyl)-3-azatricyclo[6.2.1.0²’⁷Jundec-5-en-4-one

268/317

<img file="BRPI0809685A2_D0260.tif" />

Pyrrolidine (15 pL, 0.180 mmol), sarcosine (JV-methyl glycine) (3.2 mg, 0.018 mmol), copper(I) iodide (3.4 mg, 0.018 mmol), and potassium phosphate (57.3 mg, 0.270 mmol) were placed in a flask under a nitrogen atmosphere. (lR,2S,7R,8S)-6-Hydroxy-5-(7iodo-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-3-(3-methyl-butyl)-3-aza-tricyclo[6.2.1.0²’⁷undec-5-en-4-one (prepared as described in Example 34, 50 mg, 0.090 mmol) was dissolved in 7V,1V-dimethylformamide (1 mL) and added to the above mixture. The mixture was stirred at 80 °C for 26 h. Additional pyrrolidine (50 pL, 0.60 mmol), sarcosine (7V-methylglycine) (10 mg, 0.056 mmol), and copper(I) iodide (15 mg, 0.079 mmol) were added and the mixture was stirred at 80 °C for another 16 h until LC-MS analysis indicated the end of the reaction. The reaction was repeated on the same scale as described above and after stirring at 80 °C for 24 h, additional pyrrolidine (50 pL, 0.60 mmol), sarcosine (TV-methyl glycine) (10 mg, 0.056 mmol), and copper(I) iodide (15 mg, 0.079 mmol) were added and the mixture was stirred at 80 °C for another 18 h until LC-MS analysis indicated the end of the reaction. Both batches were combined and filtered through a Celite plug. The filter cake was washed with 10% methanol in dichloromethane (2 x 2 mL) and α,7V-dimethylformamide (2 mL). The filtrate was concentrated under vacuum and the residue was purified by prep-HPLC [Luna 5pm C18 column (2) 100 Ω AXIA 150 x 21.2 mm, 5 microns, 30%-95% in 7 min @ 30 mL/min flow rate, 0.05% trifluoroacetic acid in acetonitrile / 0.05% trifluoroacetic acid in water] to provide the

269/317 desired product, (IR,2 S,7R,88)-5-(1,1-dioxo-7-pyrrolidin-1-yl-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-6-hydroxy-3-(3-methyl-butyl)-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-4-one (36.7 mg, 0.074 mmol, 40.9%), as a yellow solid. - 2.98 (1H, m), 3.03 - 3.10 (III, m), 3.27 - 3.30 (411, m), 3.60 - 3.71 (2H, m), 6.71 (1H, d, J = 2.3 Hz), 6.90 (III, dd, Ji = 9.2 Hz, J2 = 2.7 Hz), 7.41 (1H, d, J = 9.5 Hz). LC-MS (ESI) calcd for C26H34N4O4S 498.23, found 499.4 [M+H⁺[Analysis, calculated for C26H34N4O4S*0.5 H2O: C, 61.51; H, 6.95; N, 11.04; found: C, 61.45, H, 6.74, N, 10.91.]

Example 54 {3-[(1R,2S,7R,88)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- 1λ⁶ benzo[l,2,4]thiadiazin-7-1I}-amide of pyridine-3-sulfonic acid

<img file="BRPI0809685A2_D0261.tif" />

(1R,2S,7R,8S)-3-(4-fluoro-benzyl)-6-hydroxy-5-(7-iodo-1,1dioxo-1,4-dihydro-lÀ⁶-benzo [1,2,4]thiadiazin-3-yl)-3-aza-tricycle [6.2.1.0²· ⁷Jundec-5-en-4-one (prepared as described in Example 19, 100 mg, 0.17 mmol), pyridine-3-sulfonic acid amide (81 mg, 0.51 mmol), sarcosine (N-methyl glycine) (23 mg, 0.26 mmol), copper(I) iodide (33 mg, 0.17 mmol), and potassium phosphate (216 mg, 1.02 mmol) were combined and dissolved in JV,IV-dimethylformamide (10 mL). The flask

270/317 was degassed and fed back with nitrogen (3 x). The reaction was stirred at 100°C for 4 h. The mixture was cooled to 25°C, diluted with ethyl acetate (20 mL), and extracted with saturated aqueous sodium bicarbonate solution (2 x 20 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated under vacuum. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 8% methanol in dichloromethane) yielded the desired product, pyridine-3-sulfonic acid {3-[(1R,2S,7R,8S)-3-(4-fluorobenzyl)6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1 -dioxo-1,4dihydro- lA⁶-benzo[l,2,4]thiadiazin-7-yl}-amide (56 mg, 0.09 mmol, 54%), as a white powder. NMR (400 MHz, DMSO-dó) δ: 1.38 - 1.58 (6H, m), 2.61 (1H, s), 3.00 (1H, d, J = 7.6 Hz), 3.52 (1H, d, J = 8.4 Hz), 4.40 (1H, d, J = 15.6 Hz), 4.94 (1H, d, J = 15.2 Hz), 7.13 (2H, t, J = 8.4 Hz), 7.29 -7.32 (2H, m), 7.42 - 7.50 (3H, m), 7.60 -7.63 (1H, m), 8.11 (1H, d, J = 7.6 Hz), 8.79 (1H, d, J = 4.4Hz), 8.87 (1H, s), 10.94 (1H, s), 13.99 (1H, s). LC-MS (ESI) calcd for C29H26FN5O6S2 623.68, found 624.2 [M+H⁺],

Example 55 (1R, 28.71?, 88)-7V-{3-[3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²-⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- 1A⁶benzo[1,2,41 thiadiazin-7-iI}-sulfamide

<img file="BRPI0809685A2_D0262.tif" />

a) Benzyl [!V-{3-[(1R,2S,7R,8S)-3-(4-fluoro-benzyl)-6-hydroxy4-oxo-3-aza-tricyclo[6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro271/317

THE⁶-benzo[1,2,4]thiadiazin-7-yl}-sulfamoyl]carbamate

<img file="BRPI0809685A2_D0263.tif" />

Benzyl alcohol (35 g/L, 0.338 mmol) was dissolved in dichloromethane (3 mL) and cooled to 0°C. Chlorosulfonyl isocyanate (29.4 g/L, 0.338 mmol) was added and the mixture was stirred at 0°C for 2.5 h. Triethylamine (47 g/L, 0.34 mmol) was then added, followed by a solution of (1R,2S,7R,8S)5-)-5-(-amino-1,1-dioxo-1,4-dihydro-⁶-benzo[1,2,4]thiadiazin-3-yl)-3-(4fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²’⁷[undec-5-en-4-one (prepared as described in Example 30, 81.4 mg, 0.169 mmol) in dichloromethane (2 mL) were added at 0°C and the mixture was stirred at 25°C for 17 h. The mixture was extracted with water (2 x 2 mL) and saturated aqueous brine solution (2 mL). The organic layer was concentrated under vacuum and further dried for 16 h under high vacuum to give the crude product, benzyl [ÍV-{3-[(177,2S,71?,8S)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl]-sulfamoyl]carbamate, which was used in the next step without any further purification. LC-MS (ESI) calcd for C32H30FN5O8S2 695.15, found 696.6 [M+H⁺],

b) (177.2 S, 7R.8 S)-N-{3-]3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3aza tricyclo[6.2.1.0²-⁷]undec-5-en-5-il]-1,1-dioxo-1,4-dihi-dro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-sulfamide

272/317

<img file="BRPI0809685A2_D0264.tif" />

Benzyl [7V-{3-[(1R,2S,7R,8S)-3-(4-fluoro-benzyl)-6-hydroxy-4oxo-3-aza-tricyclo[6.2.1.0²-⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- 1Ã⁶Benzo[l,2,4]thiadiazin-7-yl}-sulfamoyl]carbamate was dissolved in meta-nol (5 mL) and the mixture was degassed and fed back with nitrogen (3%). Palladium in carbon (10% dry, 180 mg) was added and the mixture was degassed and fed back with hydrogen gas via flask. The mixture was stirred at 25°C for 16 h. The mixture was passed through a Celite plug and the filtrate was concentrated under vacuum to provide the crude product, which was further purified by pre-HPLC [Luna 5μ C18 column (2) 100A AXIA 150 x 21.2 mm, 5 microns, 30%-95% in 7 min @ 30 mL/min flow rate, 0.05% trifluoroacetic acid in acetonitrile/ 0.05% trifluoroacetic acid in water] to provide the desired product, (lR,2S,7R,8S)-7V-{3-[3-(4-fluoro-benzyl)15 6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-l, 1-dioxo-1,4dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-sulfamide (55.7 mg, 0.099 mmol, 58.7% in two steps), as a white solid. UI NMR (400 MHz, DMSO-de) δ: 1.19 - 1.24 (2H, m), 1.40 - 1.60 (4H, m), 2.51 - 2.54 (1H, m), 2.64 - 2.65 (1H, m), 3.04 (1H, d, J = 9.3 Hz), 3.53 (1H, d, J = 9.3

Hz), 4.42 (1H, d, J = 15.7 Hz), 4.96 (1H, d, J = 15.6 Hz), 7.15 (2H, m), 7.31 - 7.34 (H, m), 7.43 (1H, dd, Ji = 9.4 Hz, J₂ = 2.3 Hz), 7.51 - 7.55 (2H, m), 9.96 (1H, s). LC-MS (ESI) calcd for C24H24FN5O6S 561.12, found 562.5 [M+H⁺Anal, calcd for C24H24FN5OõSo0,5 II2O: C, 50.51; H, 4.42; N, 12.27; found: C, 50.42, H, 4.35, N, 11.90.

Example 56

273/317 (1/7.2 S, 777.8 8)-A-[3-(3-benzyl-6-hydroxy-4-oxo-3-αζαtricyclo[6.2.1 0²-'^z]undec-5-en-5-yl)-l, 1-dioxo-1,4-dihydro- lA⁶-benzo[1,2,4] thiadiazin-7-yl]-methanesulfonamide

<img file="BRPI0809685A2_D0265.tif" />

a) Ethyl ester of acid (1S,2/7,3S,4/7)-3-benzylamino-bicyclo[2.2. l]carboxylic heptane-2-

<img file="BRPI0809685A2_D0266.tif" />

Benzaldehyde (0.454 mL, 4.47 mmol) and 10 drops of glacial acetic acid were added sequentially to a solution of ethyl ester of (1S,2/7,3S,4/7)-3-amino-bicyclo[2.2.1]heptane-2-carboxylic acid (prepared as described in Example 6k, 0.82 g, 4.47 mmol) in methanol (15 mL) at 25°C. Sodium cyanoborohydride (0.703 g, 11.2 mmol) was added, and the reaction mixture was stirred at 25°C for 2 h, and then partitioned between half-saturated aqueous sodium bicarbonate solution (150 mL) and ethyl acetate (2 x 150 mL). The organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by flash column chromatography (Teledyne Isco RediSep column; 0 to 35% ethyl acetate in hexanes) to provide the desired product, ethyl ester of (1S,2/7,3S,4R)-3-benzylamino-bicyclo[2.2.1]hep acid.

274/317 ethane-2-carboxylic acid (0.875 g, 3.22 mmol, 72%), as a clear oil, U-I NMR (400 MHz, CDC1₃) δ: 1.06 - 1.16 (2H, m), 1.20 - 1.23 (2H, m), 1.28 (3H, t, J- 7.0 Hz), 1.46 - 1.61 (2H, m), 1.93 - 1.97 (1H, m), 2.33 - 2.34 (1H, m), 2.43 - 2.44 (1H, m), 2.59 - 2.62 (1H, m), 2.98 3.00 (1H, m), 3.71 (1H, d, J = 14.0 Hz), 3.85 (1H, d, J = 13.3 Hz), 4.14 (2H, q, J= 7.3 Hz), 7.20 -7.24 (1H, m), 7.27 -7.33 (4H, m).

b) (11?, 2 S, 7R,8 S)-/V-[3-(3-Benzyl-6-hydroxy-4-oxo-3-aza-tricycle [6.2.1.0²’⁷]undec-5-en-5-yl)-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4] thiadiazin-7-yl]

-methanesulfonamide

<img file="BRPI0809685A2_D0267.tif" />

Acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- 1λ⁶benzo[l,2,4] thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g 0.200 g, 0.600 mmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.121 g, 0.631 mmol) and IV-methylmorpholine (0.139 mL, 1.26 mmol) were sequentially added to a solution of ethyl ester of (1S,21?,3S,4R)-3-benzylamino-bicyclo[2.2.l]heptane-2-carboxylic acid (0.164 g, 0.600 mmol) in 7V,7V-dimethylformamide (5 mL) at 25°C. The reaction mixture was stirred at 25°C for 2 h, and then partitioned between 1.0 M aqueous hydrochloric acid solution (100 mL) and ethyl acetate (2 x 100 mL). The organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was dissolved in ethanol (15 mL) at 25°C. A 21% by weight solution of sodium ethoxide in ethanol (0.972 mL, 3.00 mmol) was added and the mixture of

275/317 The reaction was heated to 60°C for 1 h. After cooling to 25°C, the reaction mixture was partitioned between 1.0 M aqueous hydrochloric acid solution (100 mL) and ethyl acetate (2 x 100 mL). The organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by flash column chromatography (Teledyne Isco RediSep column; 40 to 100% ethyl acetate in hexanes) to provide the desired product, (lR,2S,7R,8S)-A-[3-(3-benzyl-6-hydroxy-4oxo-3-aza-tricyclo[6.2.1.0²,⁷]undec-5-en-5-yl)-1,1-dioxo-1,4-dihydro- 1λ⁶benzo[l,2,4]thiadiazin-7-yl]-methanesulfonamide (0.120 g, 0.222 mmol, 37%), as a white solid. Ή NMR (400 MHz, DMSO-dgj δ: 1.15 1.21 (2H, m), 1.39 - 1.61 (4H, m), 2.52 (1H, bs), 2.64 (1H, bs), 3.05 (3H, s), 3.30 (2H, bs), 3.54 (1H, d, J= 9.5 Hz), 4.43 (1H, d, J = 16.4 Hz), 4.99 (1H, d, J = 15.5 Hz), 7.23 -7.28 (2H, m), 7.33 (2H, m), 7.48 -7.51 (2H, m), 7.55 -7.57 (2H, m), 10.17 (1H, s). calculated for C25H26N4O6S2 542.13, found 543.2 [M+H⁺].

Example 57

HI?,2S,7R,8S)-A-[3-(6-Hydroxy-3-isobutyl-4-oxo-3-azatricyclo[6.2.1.0²’⁷lundec-5-en-5-yl)-1,1-dioxo-1,4-dihydro-lÃ⁶-benzol l,2,4]thiadiazin-7-yl]-methanesulfonamide

<img file="BRPI0809685A2_D0268.tif" />

a) Ethyl ester of (1S,2R,3S,4R)-3-isobutylaminobicyclo[2.2.1]heptane-2-carboxylic acid

276/317

<img file="BRPI0809685A2_D0269.tif" />

Isovaleraldehyde (0.374 mL, 4.10 mmol) and 10 drops of glacial acetic acid were added sequentially to a solution of ethyl ester of (1S,27?,3S,4R)-3-amino-bicyclo[2.2.1]heptane-2-carboxylic acid (prepared as described in Example 6k, 0.750 g, 4.09 mmol) in methanol (12 mL) at 25°C. Sodium cyanoborohydride (0.643 g, 10.2 mmol) was added, and the reaction mixture was stirred at 25°C for 2 h, and then partitioned between half-saturated aqueous sodium bicarbonate solution (150 mL) and ethyl acetate (2 x 150 mL). The organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum to provide the desired product, ethyl ester of (18,2R,3S,4R)-3-isobutylamino-bicyclo[2.2.1]heptane-2-carboxylic acid, as a clear oil, which was used in the next step without any further purification.

b) (1R,2S,7R,8>S)-7V-[3-(6-hydroxy-3-isobutyl-4-oxo-3-aza-tricycle [6.2.1,0²’⁷]undec-5-en-5-yl)-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4] thiadiazin - 7 - yl] - methanesulfonamide

<img file="BRPI0809685A2_D0270.tif" />

Acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- 1λ⁶benzo[l,2,4] thiadiazin-3-yl)-acetic acid (prepared as described in Example

277/317 lg, 0.317 g, 0.951 mmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.192 g, 1.00 mmol) and IV-methylmorpholine (0.220 mL, 2.00 mmol) were sequentially added to a solution of crude (18,2R,3S,4R)-3-isobutylamino-bicyclo[2.2.1]heptane-2-carboxylic acid ethyl ester (0.228 g, 0.952 mmol) in R,R-dimethylformamide (4 mL) at 25°C. The reaction mixture was stirred at 25°C for 2 h, and then partitioned between 1.0 M aqueous hydrochloric acid solution (100 mL) and ethyl acetate (2 x 100 mL). The organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was dissolved in ethanol (15 mL) at 25°C. A 21 wt% sodium ethoxide solution in ethanol (1.23 mL, 3.80 mmol) was added, and the reaction mixture was heated at 60°C for 1.5 h. After cooling to 25°C, the reaction mixture was partitioned between 1.0 M aqueous hydrochloric acid solution (150 mL) and ethyl acetate (2 x 150 mL). The organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by flash column chromatography (Teledyne Isco RediSep column; 40 to 100% ethyl acetate in hexanes) to provide the desired product, (lR,2S,7R,8S)-7V-[3-(6-hydroxy-3-isobutyl-4-oxo-3-aza-tricyclo[6.2.1,0²’⁷]undec-5-en-5-yl)-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl]-methanesulfonamide (0.096 g, 0.190 mmol, 20%), as a white solid. HI NMR (400 MHz, DMSOd₆) δ: 0.83 (3H, d, J = 7.2 Hz), 0.93 (3H, d, J = 7.1 Hz), 1.19 - 1.21 (1H, m), 1.28 - 1.32 (1H, m), 1.43 - 1.63 (3H, m), 2.09 - 2.15 (1H, m), 2.54 (1H, bs), 2.61 - 2.61 (1H, m), 2.74 - 2.79 (1H, m), 3.05 (3H, s), 3.30 (1H, bs), 3.60 (1H, d, J = 9.2 Hz), 3.73 - 3.79 (1H, m), 7.49 - 7.52 (1H, m), 7.56 -7.58 (2H, m), 10.17 (1H, s). LC-MS (ESI) calculated for C22H28N4O6S2 508.15, found 509.4 [M+H⁺].

Example 58 (1R,2S,7R,8S)-7V-{3-[3-(3-chloro-4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²’⁷1undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo

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[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0271.tif" />

a) Ethyl ester of (1S,2R,3S,4R)-3-(3-Chloro-4-fluorobenzylamino)-bicyclo[2.2. l]heptane- 2-carboxylic

<img file="BRPI0809685A2_D0272.tif" />

3-Chloro-4-fluoro-benzaldehyde (0.710 mL, 4.48 mmol) and 10 drops of glacial acetic acid were added sequentially to a solution of ethyl ester of (1S,2R,3S,4R)-3-amino-bicyclo[2.2.1]heptane 2-carboxylic acid (prepared as described in Example 6k, 0.82 g, 4.47 mmol) in methanol (15 mL) at 25°C. Sodium cyanoborohydride (0.709 g, 11.3 mmol) was added, and the reaction mixture was stirred at 25°C for 2 h, and then partitioned between half-saturated aqueous sodium bicarbonate solution (150 mL) and ethyl acetate (2 x 150 mL). The organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by flash column chromatography (Teledyne Isco RediSep column; 0 to 35% ethyl acetate in hexanes) to provide the desired product, ethyl ester of (1S,2R,3S,4R)-3-(3-chloro-4-fluorobenzylamino)-bicyclo[2.2.1]heptane-2

279/317 carboxylic acid (1.12 g, 3.44 mmol, 77%), as a clear oil, HI NMR (400 MHz, CDC1₃) δ: 1.04 - 1.16 (2H, m), 1.18 - 1.24 (2H, m), 1.29 (3H, t, J = 7.4 Hz), 1.44 - 1.61 (2H, m), 1.91 - 1.94 (1H, m), 2.27 - 2.28 (1H, m), 2.42 - 2.43 (1H, m), 2.60 (1H, d, J = 7.7 Hz), 2.91 (1H, d, J = 8.8 Hz), 3.64 (1H, d, J = 13.9 Hz), 3.79 (1H, d, J = 14.2 Hz), 4.14 (2H, q, J = 7.0 Hz), 7.02 - 7.06 (1H, m), 7.13 -7.17 (1H, m), 7.36 -7.38 (1H, m).

b) (1 R,2S,7R,8S)-/V-{3-[3-(3-chloro-4-fluoro-benzyl)-6-hydroxy-4oxo-3-aza-tricyclo[6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶benzoph 1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0273.tif" />

Acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- 1λ⁶benzo[l,2,4] thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 0.258 g, 0.774 mmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (0.156 g, 0.814 mmol) and TV-methylmorpholine (0.170 mL, 1.55 mmol) were sequentially added to a solution of ethyl ester of (lS,2R,3S,4R)-3-(3-chloro-4-fluorobenzylamino)-bicyclo[2.2.l]heptane-2-carboxylic acid (0.252 g, 0.773 mmol) in A,A-dimethylformamide (4 mL) at 25°C. The reaction mixture was stirred at 25°C for 2 h, and then partitioned between 1.0 M aqueous hydrochloric acid solution (100 mL) and ethyl acetate (2 x 100 mL). The organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was dissolved in ethanol (20 mL) at 25°C. A 21% solution in

280/317 wt of sodium ethoxide in ethanol (1.00 mL, 3.09 mmol) was added and the reaction mixture was heated to 60°C for 1 h. After cooling to 25°C, the reaction mixture was partitioned between 1.0 M aqueous hydrochloric acid solution (100 mL) and ethyl acetate (2 x 100 mL). The 5 organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by flash column chromatography (Teledyne Isco RediSep column; 40 to 100% ethyl acetate in hexanes) to provide the desired product, (lR,2S,7R,8S)-N-{3-[3-(3-chloro-4-fluorobenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en10 5-il] -1, 1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (0.196 g, 0.332 mmol, 43%), as a white solid. Ή NMR (400 MHz, DMSO-d₆? δ: 1.04 - 1.21 (4H, m), 1.40 - 1.60 (3H, m), 2.54 2.63 (1H, m), 3.05 (3H, s), 3.31 - 3.40 (2H, m), 3.55 - 3.58 (1H, m), 4.45 (1H, d, J = 16.1 Hz), 4.91 (1H, d, J = 14.7 Hz), 7.31 -7.35 (2H, m), 7.51 15 7.57 (4H, m), 10.17 (1H, s), 13.96 (1H, s). LC-MS (ESI) calcd for

C25H24CIFN4O6S2 594.08, found 595.3 [M+H⁺],

Example 59 (rac-di-exo)-/V-f3-[6-Hydroxy-3-(3-methyl-butyl)-4-oxo-1 l-oxa-3-azatricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- 1À620 benzo[1,2,4] thiadiazin-7-ίΠ -AT-methyl-methanesulfonamide

<img file="BRPI0809685A2_D0274.tif" />

(rac-di-exo)-N-{3-[6-hydroxy-3-(3-methyl-butyl)-4-oxo-1 l-oxa-3281/317 aza-tricycle

[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lA⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (prepared as described in Example 25, 67 mg, 0.128 mmol) was dissolved in TV,TV-dimethylformamide (2 mL). Potassium carbonate (35 mg, 0.25 mmol) and iodomethane (0.008 mL, 0.128 mmol) were added sequentially. The reaction was stirred at 25°C for 2 h. The reaction was stopped by adding 1.0 M aqueous hydrochloric acid solution (20 mL). The mixture was extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with saturated aqueous brine solution (50 mL), dried over sodium sulfate, filtered, and concentrated under vacuum to give an oil. Purification by flash column chromatography (Teledyne Isco RediSep column; 0 to 100% ethyl acetate in hexanes) yielded the desired product, (rac-di-exo)-TV-{3-[6-hydroxy-3-(3-methylL-butyl)-4-oxo-1-oxa-3-aza-tricyclo[6.2.1.0²·⁷] undec-5-en-5-yl]-1, l-dioxo-1,4-dihydrolA⁶-benzo[1,2,4]thiadiazin-7-yl}-TV-methyl-methanesulfonamide (36 mg, 0.067 mmol, 52%), as a white solid. UI NMR (400 MHz, DMSOd)₆) δ: 0.92 (6H, d, J = 6.2 Hz), 1.16 - 1.65 (7H, m), 2.79 - 2.93 (2H, m), 2.96 (3H, s), 3.26 (3H, s), 3.62 - 3.69 (1H, m), 3.76 - 3.83 (1H, m), 4.60 (2H, d, J = 21.8 Hz), 7.27 (1H, d, J = 7.3 Hz), 7.55 (1H, d, J = 8.5 Hz), 7.65 (1H, s). LC-MS (ESI) calculated for C23H30N4O7S2 538.16, found 539.4 [M+H⁺],

Example 60

N-{3-\{lR,2S,7 R,8S)-3 -[3-fluoro-4-methyl-benzyl)-6-hydroxy-4oxo-3-aza-tricyclo[6.2.1.0²7]undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro- 1A⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

282/317

<img file="BRPI0809685A2_D0275.tif" />

a) Ethyl ester of (1S,2R',3S,4Rj-3-(3-fluoro-4-methylbenzylammo)-bicyclo[2.2.1]heptane-2-carboxylic acid

THE

<img file="BRPI0809685A2_D0276.tif" />

<img file="BRPI0809685A2_D0277.tif" />

F

Ethyl ester of (1S,2R,3S,4R)-3-amino-bicyclo[2.2.1]heptane-2-carboxylic acid (prepared as described in Example 6k, 300 mg, 1.637 mmol) was suspended in ethanol (5 mL). 3-fluoro-4-methylbenzaldehyde (0.2 mL, 1.637 mmol) was added followed by glacial acetic acid (0.1 mL, 3.724 mmol) and powdered molecular sieves of

4. Sodium cyanoborohydride (0.593 g, 9.406 mmol) was added and the mixture was stirred under nitrogen at 50°C for 18 h. Upon cooling, the mixture was filtered through Celite. The filtrate was diluted with half-saturated aqueous sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (2 x 75 mL). The layers were separated and the organic layer was washed with saturated aqueous brine solution, dried over sodium sulfate, filtered, and concentrated under vacuum. Further purification by flash column chromatography (Teledyne Isco RediSep column; I^the Column: 0 to 25% ethyl acetate in hexanes; 2^the Column: 0 to 20% ethyl acetate in hexanes) provided the product

283/317 desired, ethyl ester of (1S,2R,3S,4R)-3-(3-fluoro-4-methylbenzylamino)-bicyclo[2.2.1]heptane-2-carboxylic acid (221 mg, 0.724 mmol, 44%), as a thick, clear oil, HI NMR (400 MHz, CDCl3) δ: 1.04 - 1.15 (2H, m), 1.21 (1H, d, J = 10.1 Hz), 1.29 (3H, t, J = 7.1 Hz), 1.42 - 1.63 (4H, m), 1.94 (1H, dt, J = 10.2 Hz, J₂ = 2.0 Hz), 2.25 (3H, s), 2.43 (1H, d, J = 3.5 Hz), 2.59 (1H, dd, Ji = 8.6 Hz, J₂ = 1.6 Hz), 2.93 (1H, dd, J1 = 8.6 Hz, J₂ = 1.6 Hz), 3.65 (1H, d, J = 13.4 Hz), 3.79 (1H, à, J = 14.2 Hz), 4.15 (2H, q, J = 7.1 Hz), 6.94 (1H, d, J = 7.9 Hz), 6.98 (III, d, J = 10.9 Hz), 7.08 (1H, t, J = 7.8 Hz). LC-MS (ESI) calcd for C18H24FNO2 305.18, found 305.9 [M+H⁺].

b) Ethyl ester of (1S,2R,3S,4R)-3-{(3-fluoro-4-methylbenzyl)-[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- 1A⁶-benzo[1,2,4]thiadiazin-3-yl)-acetyl]-amino}-bicyclo[2.2. l]heptane-2-carboxylic acid

<img file="BRPI0809685A2_D0278.tif" />

A solution of ethyl ester of (1S/2R,3S,4R)-3-(3-fluoro-4-methylbenzylamino)-bicyclo[2.2.1]heptane-2-carboxylic acid (92 mg, 0.30 mmol), (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1⁶-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 100 mg, 0.30 mmol) and l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide chloride (60 mg, 0.315 mmol) in 7V,7V-dimethylformamide (4.0 mL) was added .ZV-methylmorpholine (0.07 mL, 0.63 mmol). After stirring at 25°C for 3 h, the mixture was poured into 1.0 M aqueous hydrochloric acid solution and extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with a saturated aqueous brine solution, dried over sodium sulfate, filtered, and concentrated.

284/317 vacuum-packed to provide the crude product, ethyl ester of (1S,2R,3S,4R)-3-{(3-fluoro-4-methylbenzyl)-[2-(7-methanesulfonylamino-1,Γ-dioxo-1,4-dihydro-1A⁶-benzo[1,2,4]thiadiazine-3-yl)-acetyl]-amino}-bicyclo[2.2.1]heptane-2-carboxylic acid, as an orange oil. The crude product was used in the next step without further purification. LC-MS (ESI) calcd for C28H33FN4O7S2 620.18, found 621.4 [M+H⁺].

c) A-{3-[(1R,2S,7R,8S)-3-(3-fluoro-4-methyl-benzyl)-6-hydroxy-4oxo-3-aza-tricyclo[6.2.1.0²>⁷]undec-5-en-5-íl] 1, 1-dioxo-1,4-dihydro-lÀ⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0279.tif" />

The ethyl ester of (1S,2R,3S,4R)-3-{(3-fluoro-4-methylbenzyl)-[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- 1A⁶Crude -benzo[1,2,4]thiadiazin-3-yl)-acetyl]-amino}-bicyclo[2.2.1]heptane-2-carboxylic acid was dissolved in ethanol (5 mL) at 25 °C. A 21% by weight solution of sodium ethoxide in ethanol (0.224 mL, 0.60 mmol) was added and the reaction mixture was heated at 60 °C for 90 min. After cooling to 25 °C, the reaction mixture was stirred for 18 h at 25 °C. The mixture was partitioned between 1.0 M aqueous hydrochloric acid solution (100 mL) and ethyl acetate (2 x 100 mL). The organic layers were washed with a saturated aqueous brine solution, dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by flash column chromatography (Teledyne Isco RediSep column; I^the Column: 0 to 10% ethyl acetate in hexanes; 2^the Column: 30 to 80% ethyl acetate in hexanes) to provide the desired product, A-{3-[(lR,2S,7R,8S)-3-(3

285/317 /Líor-4-methi7-benzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1,0²’⁷]undec-5-en5-yl]-1,1-dioxo-1,4-dihydro-1A⁶-benzo[1,2,4]thiadiazine-7-yl}-methanesulfonamide (53 mg, 0.092 mmol, 31% in two steps), as a white solid. III NMR (400 MHz, DMSO-d₆), 4.42 (1H, d, J = 15.5 Hz), 4.92 (III, d, J = 15.5 Hz), 7.01 -7.07 (2H, m), 7.23 (1H, t, J = 8.1 Hz), 7.49 -7.58 (3H, m), 10.17 (1H, s). LC-MS (ESI) calculated for C26H27FN4O6S2 574.14, found 575.4 [M+H⁺],

Example 61 (1R,S,7R,8S)-5-(7-Bromo-1,1-dioxo-1.4-dihydro-1A⁶-pyrido[2.3e][1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricycle [6.2.1.0²-⁷undec-5-en-4-one

<img file="BRPI0809685A2_D0280.tif" />

F

a) 2-amino-5-bromo-pyridine-3-sulfonic acid amide

THE,_ZThe IVF^sVV^Bri Ü H₂N^N^

5-Bromopyridine-2-ylamine (10 g, 57.8 mmol) was added to chlorosulfonic acid (40 mL, 602 mmol) with stirring at 25°C. The mixture was heated to 160°C with stirring for 3 h. Upon cooling to 25°C, the brown solution was carefully poured over ice (~500

286/317

(g) The resulting precipitate was collected by vacuum filtration and washed with water to give the intermediate, 2-amino-5-bromopyridine-3-sulfonyl chloride, as a beige solid. The solid was suspended in a 15% aqueous ammonium hydroxide solution. The reaction mixture was stirred for 45 min. Everything was completely dissolved at this point. The mixture was cooled to 0°C and the pH was adjusted to ~8 by the careful addition of 12.0 M aqueous hydrochloric acid solution. A solid precipitated and was collected by vacuum filtration, washed with water (2 x 50 mL) and dried under vacuum to give the desired product, 2-amino-5-bromo-pyridine-3-sulfonic acid amide (7.48 g, 29.7 mmol, 51%), as a light beige solid. Ή NMR (400 MHz, DMSO-d₆) δ: 6.69 (2H, bs), 7.54 (2H, bs), 7.91 (1H, d, J = 2.5 Hz), 8.22 (1H, d, J = 2.3 Hz).

b) Ethyl ester of 7V-(5-Bromo-3-sulfamoyl-pyridin-2-yl)-malonamic acid

<img file="BRPI0809685A2_D0281.tif" />

2-amino-5-bromo-pyridine-3-sulfonic acid amide (2.5 g, 9.9 mmol) was dissolved in 1,4-dioxane (50 mL). Ethyl ester of chlorocarbonyl acetic acid (1.9 mL, 14.85 mmol) was added and the mixture was heated to 90°C with stirring for 2 h. Upon cooling to 25°C, the mixture was poured into a half-saturated aqueous sodium bicarbonate solution (250 mL). A solid precipitated and was collected by vacuum filtration, washed with water (50 mL) and dried under vacuum to give the desired product, ethyl ester of A-(5-bromo-3-sulfamoylpyridin-2-yl)-malonamic acid (2.5 g, 6.82 mmol, 69%), as a light beige solid. Ή NMR (400 MHz, DMSO-d₆/ô: 1.19 (3H, t, J = 7.0 Hz), 3.67 (2H, s), 4.10 (2H, q, J = 7.3 Hz), 7.78 (2H, bs), 8.33 (1H, d, J = 2.3 Hz), 8.70

287/317 (1H, d, J = 2.4 Hz), 9.85 (1H, bs).

c) Acid ethyl ester (7-bromo-l, 1-dioxo-1,4-dihydro-ΙΑ⁶

-pyrido[2,3-e][l,2,4]thiadiazin-3-yl)-acetic

<img file="BRPI0809685A2_D0282.tif" />

H

In a sealed tube, ethyl ester of 7V-(5-bromo-3-sulfamoyl-pyridin-2-yl)-malonamic acid (2.2 g, 6.0 mmol) was suspended in toluene (44 mL), and triethylamine (11 mL, 78.9 mmol) was added. The mixture was heated to 110°C with stirring for 30 min. The solid was completely dissolved, although an immiscible oily residue was observed at the bottom of the flask. Upon cooling to 25°C, ethyl acetate (~50 mL) was added. Everything became miscible. The solution was concentrated under vacuum to give a golden oil. The oil was dissolved in methanol (~50 mL) and concentrated under vacuum to give the crude product, ethyl ester of (7-bromo-1,1-dioxo-1,4-dihydro)15lA⁶-pyrido[2,3-e][l,2,4]thiadiazin-3-yl)-acetic acid (2.18 g, >6.0 mmol, 100%, still containing some solvent), as a golden oil. LC-MS (ESI) calculated for CiOHiOBrN3O4S 346.96, found 348.1 (100%), 349.2 (10%), 350.2 (99%) [M+H⁺].

d) Acetate (7-bromo-1, 1-dioxo-1,4-dihydro-1A⁶-pirido[2,3-e]

[1,2,4]thiadiazin-3-yl) sodium

<img file="BRPI0809685A2_D0283.tif" />

The ethyl ester of (7-bromo-1,1-dioxo-1,4-dihydro-1A) acid⁶pyrido[2,3-e][1,2,4]thiadiazin-3-yl)-acetic acid (1.14 g, 3.27 mmol) was dissolved

288/317 of methanol (20 mL). Solid sodium hydroxide (0.392 g, 9.8 mmol) was added followed by water (10 mL). Everything was completely dissolved within ~5 minutes. After ~20 minutes, a solid began to precipitate. The mixture continued to be stirred for 10 min.

The product was collected by vacuum filtration, washed with methanol (~5 mL) and dried under vacuum to provide the desired product, acetate (7-bromo-1,1-dioxo-1,4-dihydro-1).⁶-pyrido[2,3e][1,2,4]thiadiazin3-yl) sodium (0.48 g, 1.5 mmol, 46%), as a white solid. !HNMR (400 MHz, D₂O) δ: 3.30 (2H, s), 8.31 (1H, d, J = 2.2 Hz), 8.57 (1H, d, J = 1.6 Hz). Note: Successive NMR acquisitions of the product in DMSO-dō indicated decarboxylation over a period of ~30 min to yield 1,1-dioxide of 7-bromo-3-methyl-4H-pyrido[2,3-e][1,2,4]thiadiazine. However, the product appears to be stable as the solid sodium salt form.

e) (IR, 2S,7R, 8S)-5-(7-bromo-1,1-dioxo-1,4-dihydro-lÀ^the-pyrido[2,3-e] [1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricycle [6.2.1.0²’⁷undec-5-en-4-one

<img file="BRPI0809685A2_D0284.tif" />

Ethyl ester of (1S,2R,3S,4R)-3-(4-fluorobenzylamino)bicyclo[2.2.1]heptane-2-carboxylic acid (prepared as described in Example 61, 0.213 g, 0.73 mmol), acetate (7-bromo-1,1-dioxo-1,4dihydro-1A⁶-pyrido[2,3-e][1,2,4]thiadiazin-3-yl) sodium (0.25 g, 0.73 mmol) and O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (0.305 g, 0.803 mmol) were combined and dissolved in A,A-dimethylformamide (1.5 mL). The mixture was vigorously stirred for

289/317 for about 1 min until everything was dissolved. The solution continued to be stirred for 1 h. Triethylamine (0.5 mL, 3.65 mmol) was added and the mixture was stirred at 50°C for 16 h. The mixture was concentrated under vacuum to a volume of ~5 mL. Water (~20 mL) was added and the product precipitated. The solid was collected by vacuum filtration and purified by flash column chromatography (Merck silica gel 60, 40-63 pm; 35% ethyl acetate in hexanes) to give the desired product (0.24 g, 0.44 mmol, 60%) as a brittle, white foam. A portion of the product (0.07 g, 0.128 mmol) was recrystallized from n-propanol (0.5 mL) to give the desired pure product, 5-(7-bromo l,l-dioxo l,4-dihydro-l-⁶-pyrido[2,3-e][1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²>⁷undec-5-en-4-one (0.044 g, 0.081 mmol, 63%), as a yellow powder. 1H4 NMR (400 MHz, DMSO-d₆) δ: 1.13 - 1.23 (2H, m), 1.38 -1.60 (4H, m), 2.51 (1H, s), 2.63 (1H, s), 3.00 (1H, d, J =

8.4 Hz), 3.55 (1H, d, J = 9.4 Hz), 4.41 (1H, d, J = 14.7 Hz), 4.95 (1H, d, J = 15.4 Hz), 7.14 (2H, t, J= 9.0 Hz), 7.33 (2H, dd, J1 = 8.6 Hz, J₂ = 5.5 Hz), 8.66 (1H, s), 8.82 (1H, s). LC-MS (ESI) calcd for C₂3H₂oBrFN404S 546.04, found 547.2 [M+H⁺].

Example 62 /V-{3-[(1R,2S,7R,8S)-3-/4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-pyrido[2,3e][1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0285.tif" />

Methanesulfonamide (0.348 g, 3.66 mmol), potassium phosphate

290/317 sio (0.078 g, 0.366 mmol), L-proline (0.021 g, 0.183 mmol) and copper iodide (0.035 g, 0.183 mmol) were combined and suspended in dimethyl sulfoxide (0.5 mL). The flask was degassed and backfilled with argon. The mixture was heated, with stirring, at 110°C for 5 min. 5-(7Bromo-1,1-dioxo-1,4-dihydro-lA⁶-pyrido [2,3-e][1,2,4] thiadiazin-3-yl)-3-(4fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²’⁷The compound undec-5-en-4-one (prepared as described in Example 62, 0.1 g, 0.183 mmol) was dissolved in dimethyl sulfoxide (0.5 mL) and transferred to a reaction mixture. The mixture was stirred at 110°C for 16 h. Upon cooling, the mixture was poured into ethyl acetate (150 mL) and saturated aqueous ammonium solution (100 mL). The mixture was stirred and the mixture was passed through a Celite plug. The organic layer was separated from the filtrate, washed with saturated aqueous ammonium chloride solution (50 mL), saturated aqueous brine solution (50 mL), dried over magnesium sulfate, filtered, and concentrated under vacuum to a brown wax solid. Flash column chromatography (Merck silica gel 60, 40-63 pm; 0 to 50% ethyl acetate in dichloromethane) yielded the desired product, 7V-{3[(1R,2S,7R,8S)-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-azatricyclo[

6.2.1,0²>⁷]undec-5-en-5-yl]-1,1-dioxo- 1,4-dihydro- lA⁶-pyrido[2,3-e][1,2,4]thiadiazin-7-yl}-methanesulfonamide (0.018 g,0.032 mmol, 17.5%), as a beige solid.^THEH NMR (400 MHz, DMSO-d₆) δ: 1.04 - 1.52 (6H, m), 2.37 - 2.41 (1H, m), 2.53 - 2.56 (1H, m), 3.05 (3H, s), 3.25 - 3.34 (2H, m), 4.25 (1H, d, J = 15.1 Hz), 4.93 (1H, d, J = 15.0 Hz), 7.11 (2H, t, J = 9.1 Hz), 7.27 (2H, dd, JI = 8.7 Hz, J₂ = 5.4 Hz), 7.81 (1H, s), 8.36 (1H, s), 10.01 (1H, bs). LC-MS (ESI) calcd for C24H24FNSO6S2 561.12, found 562.4 [M+H⁺].

Example 63 (IR,2 8.7R, 88)-5-( 1,1-Dioxo- 1,4-dihydro- lA⁶-pirido[2. δε] [1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricycle [6.2.1.0²·⁷] undec-5-en-4-ona

291/317

<img file="BRPI0809685A2_D0286.tif" />

F

5-(7-13romo-1,1-dioxo-1,4-dihydro-lÀ⁶-pyrido[2,3-e][1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²’⁷The compound undec-5en-4-one (prepared as described in Example 62, 0.07 g, 0.128 mmol) was dissolved in methanol (8 mL). Ammonium formate (0.2 g, 3.17 mmol) followed by palladium in 10% carbon (wet, 0.1 g) was added. The mixture was stirred at 50°C for 3 h. The mixture was filtered through Celite and the filtrate was concentrated under vacuum. The residue was dissolved in water (5 mL) and ethyl acetate (50 mL). The mixture was stirred and the layers were separated. The organic layer was dried over magnesium sulfate, filtered, and concentrated under vacuum to a clear oil. Flash column chromatography (Merck silica gel 60, 40-63 pm; 20-40% ethyl acetate in hexanes) yielded the desired product, (12?,2S,7R,85)-5-/1,1-dioxo-1,4-dihydro-1⁶-pyrido[2,3-e][1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy3-aza-tricycle [6.2.1.0²’⁷]undec-5-en-4-one (0.0441 g, 0.094 mmol, 74%), as a brittle, white foam.^XH NMR (400MHz, DMSO-cZe) δ: 1.11-1.61 (6H, m), 2.51 (1H, s), 2.63 (1H, s), 2.99 (1H, d, J = 9.4 Hz), 3.54 (1H, d, J = 9.4 Hz), 4.41 (1H, d, J = 15.7 Hz), 4.95 (1H, d, J = 15.4 Hz), 7.14 (2H, t, J= 8.6 Hz), 7.33 (2H, dd, Ji = 8.6 Hz, J₂ = 5.4 Hz), 7.51 (1H, dd, Ji = 7.8 Hz, J₂ = 4.5 Hz), 8.34 (1H, d, J = 7.6 Hz), 8.68 (1H, dd, Ji = 4.6 Hz, J₂ = 1.7 Hz). LC-MS (ESI) calcd for C23H_2]FN₄O4S 468.13, found 469.4 [M+H⁺].

Example 64 (12?,2S,72?,8S)-5-(l,l-dioxo-1,4-dihydro-lÀ⁶-pyrido[4.3-e][l,2,4]thiadiazin292/317

3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricycle [6.2.1.0²·⁷] undec-5-en-4-ona

<img file="BRPI0809685A2_D0287.tif" />

a) Amide of 4-azidopyridine-3-sulfonic acid

<img file="BRPI0809685A2_D0288.tif" />

Amide of 4-Chloropyridine-3-sulfonic acid (4 g, 20.77 mmol) and sodium azide (13.7 g, 210 mmol) were combined. Anhydrous N,N-dimethylformamide (80 mL) and water (30 mL) were added. The mixture was stirred at 90°C for 2 h. Upon cooling, the mixture was diluted with saturated aqueous ammonium chloride solution (200 mL). The product was extracted in ethyl acetate (6 x 200 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated under vacuum to give the desired product, 4-azido-pyridine-3-sulfonic acid amide (3.75 g, 18.83 mmol, 91%), as a pale yellow solid. HI NMR (400 MHz, DMSO-d) δ: 7.54 (211, bs), 7.57 (1H, d, J = 5.4 Hz), 8.68 (1H, d, J = 5.5 Hz), 8.81 (1H, s).

b) Amide of 4-aminopyridine 3-sulfonic acid

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<img file="BRPI0809685A2_D0289.tif" />

Amide of 4-Azidopyridine-3-sulfonic acid (3.75 g, 18.83 mmol) was dissolved in methanol (80 mL). Sodium borohydride (0.712 g, 18.83 mmol) was carefully added in portions. Vigorous effervescence was observed. The mixture continued to be stirred at 25°C for 25 min. The mixture was concentrated under vacuum to a thick yellow sludge. The residue was dissolved in a mixture of ethyl acetate (200 mL) and saturated aqueous ammonium chloride solution (200 mL). The aqueous layer was extracted back with ethyl acetate (6 x 200 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated under vacuum to give the desired product, 4-aminopyridine-3-sulfonic acid amide (1.8 g, 10.4 mmol, 55%), as a pale yellow solid.^XH NMR (400 MHz, DMSO-dg) δ: 6.63 (2H, bs), 6.68 (1H, d, J = 5.4 Hz), 7.40 (2H, bs), 8.06 (1H, d, J = 5.5 Hz), 8.43 (1H, s).

c) Acid methyl ester (1,1-dioxo-1,4-dihydro-lÀ⁶-pyrido [4,3 -e] [1,2,4] thiadiazin-3-yl) -acetic

<img file="BRPI0809685A2_D0290.tif" />

H

Amide of 4-Aminopyridine-3-sulfonic acid (0.25 g, 1.44 mmol) was suspended in malonic acid methyl diester (5 mL, 43.75 mmol). The flask was purged with nitrogen and the mixture was stirred at 180°C for 60 min. Upon cooling, the mixture was diluted with ethyl acetate (5 mL), causing immediate precipitation of a byproduct.

294/317 unwanted. The solid was removed by vacuum filtration and washed with ethyl acetate (2 mL). The filtrate was passed through a silica gel plug, eluted with ethyl acetate followed by methanol in 5% ethyl acetate. The fractions containing the methanol in 5% ethyl acetate were combined and concentrated under vacuum to provide the desired product, methyl ester of (1,1-dioxo-1,4-dihydro-1) acid.⁶-pyrido[4,3e][l,2,4]thiadiazin-3-yl)-acetic acid (0.135 g, 0.529 mmol, 37%) as a yellow oil. LC-MS (ESI) calculated for C9H9N3O4S 255.03, found 256.0 [M+H⁺],

d) Acetate (1,1-dioxo-1,4-dihydro-lÀ⁶-pyrido[4,3-e][1,2,4]thiadiazin-3-yl) sodium

<img file="BRPI0809685A2_D0291.tif" />

Acid methyl ester (1,1-dioxo-1,4-dihydro-lÀ⁶-pyrido[4,3-e][1,2,4]thiadiazin-3-yl)-acetic acid (0.13 g, 0.51 mmol) was dissolved in methanol (3 mL). Sodium hydroxide (0.08 g, 2.0 mmol) was dissolved in water (1.5 mL). The solutions were combined and stirred at 25°C for 3 h. The mixture was concentrated under vacuum to give the desired product, acetate (1,1-dioxo-1,4-dihydro-l-⁶-pyrido[4,3-e][1,2,4]thiadiazin-3-yl) sodium, as a yellow oil. LC-MS (ESI) calculated for C9H7N3O4S (free acid) 241.02, found 242.2 [M+H⁺],

e) (IR,28,7R,88)-5-( 1,1 -dioxo-1,4-dihydro- lÀ⁶-_pirido[4,3-e] [1,2,4] thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricycle [6.2.1. 0²·⁷] undec-5-en-4-ona

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<img file="BRPI0809685A2_D0292.tif" />

F

Acetate (l,l-dioxo-1,4-dihydro-lÀ⁶-pyrido[4,3-e][l,2,4]thiadiazin-3-yl) sodium, ethyl ester of (1S,277,3S,477)-3-(4-fluorobenzylamino)-bicyclo[2.2.1]heptane-2-carboxylic acid (prepared as described in Example 61, 0.149 g, 0.51 mmol) and O-(7azabenzotriazol-1-11)-77,77,77/17-tetramethyluronium hexafluorophosphate (0.194 g, 0.51 mmol) were combined and dissolved in anhydrous 77,77-dimethylformamide (1 mL). The mixture was stirred at 25°C for 1 h. Triethylamine (0.35 mL, 2.6 mmol) was added and the mixture was stirred at 50°C for 16 h. Upon cooling, the mixture was diluted with ethyl acetate (100 mL), washed with saturated aqueous ammonium chloride solution (2 x 25 mL) and saturated aqueous brine solution (25 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated under vacuum. Purification by flash column chromatography (Merck silica gel 60, 40-63 pm, 75% to 100% ethyl acetate in hexanes) followed by trituration of the resulting oil with a 1:1 mixture of hexanes and diethyl ether (2 mL) yielded the desired product, (177,2S,777,8S)-5-(l,1-dioxo-1,4-dihydro-l).⁶-pyrido[4,3-e) [1,2,4]thiadiazin-3-yl)-3 -(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.

2.1.0²-⁷]undec-5-en-4-one (0.0184 g, 0.039 mmol, 7.7% in two steps), as a pale yellow powder. LC-MS (ESI) calculated for C23H21FN4O4S 468.13, found 469.2 [M+H⁺], ^XH NMR (400 MHz, DMSO-d₆/δ: 1.01 - 1.54 (6H, m), 2.35 (1H, d, 3.7 Hz), 2.46 (1H, s), 2.53 (1H, s), 3.26 (1H, d, J = 9.5 Hz), 4.23 (1H, d, J = 15.8 Hz), 4.94 (1H, d, J = 15.0 Hz), 7.08 -7.13 (3H, m), 7.24 -7.28 (2H, m), 8.44 (III, d, J = 4.8 Hz), 8.67 (1H, s).

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Example 65 (177.2S,777.88)-5-(1,1-dioxo-1,4-dihydro-lÃ⁶-pyrido[3,2e][1,2,4]thiadiazin-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-azatricyclo[6.2.1.0²·⁷] undec-5-en-4-ona

<img file="BRPI0809685A2_D0293.tif" />

a) 2-Benzylsulfanyl-3-nitro-pyridine

<img file="BRPI0809685A2_D0294.tif" />

Ethanol (150 mL) was added to a mixture of 2-chloro-3-nitropyridine (5 g, 31.54 mmol) and potassium carbonate (4.8 g, 34.7 mmol). Benzyl mercaptan (4.09 mL, 34.7 mmol) was added followed by water (30 mL). The mixture was stirred at 25°C for 4 h. Water (350 mL) was added and the product precipitated. The solid was collected by vacuum filtration, washed with water (100 mL) and dried under vacuum for 4 h to give the desired product, 2-benzylsulfaniI-3-nitropyridine (6.6 g, 26.8 mmol, 15 85%), as a yellow powder.^XH NMR (400 MHz, DMSO-de) δ: 4.47 (2H,

s), 7.21 -7.31 (3H, m), 7.40 - 7.45 (3H, m), 8.58 (1H, dd, Ji = 8.6 Hz, J₂= 1.7 Hz), 8.83 (1H, dd, J₂ = 4.7 Hz, J₂ = 1.7 Hz).

b) 3-Nitropyridine-2-sulfonyl chloride

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<img file="BRPI0809685A2_D0295.tif" />

2-Benzylsulfanyl-3-nitropyridine (6 g, 24.39 mmol) was dissolved in dichloromethane (84 mL). Acetic acid (12 mL) and water (24 mL) were added. The mixture was cooled to 0°C with vigorous stirring. 1,3-dichloro-5,5-dimethylimidazolidine-2,4-dione (14.4 g, 73.17 mmol) was added in portions as a suspension in dichloromethane (48 mL). The mixture was allowed to slowly warm to 25°C and continued to be stirred for 16 h. The mixture was poured into a 5% aqueous sodium metabisulfite solution (100 mL) and stirred well. Dichloromethane (200 mL) and a 20% aqueous dibasic potassium phosphate solution (100 mL) were added and the mixture was stirred well. The layers were separated. The desired crude product, 3-nitropyridine-2-sulfonyl chloride, dissolved in the organic layer, was used directly in the next step without any further isolation or characterization.

c) 3-nitropyridine-2-sulfonic acid amide

OO

<img file="BRPI0809685A2_D0296.tif" />

The organic layer was cooled to 0 °C and concentrated aqueous ammonium hydroxide solution (25 mL) was added. The mixture was stirred for 10 min. The mixture was washed with 10% aqueous citric acid solution (added until pH &lt; 7, -200 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated under vacuum. The resulting solid was ground with a 1:1 mixture of hexanes and ethyl acetate (-15 mL), collected by vacuum filtration, and vacuum dried for 16 h to give the desired product, 3-nitropyridine-2-sulfonic acid amide (3.1 g, 15.26 mmol, 63%), as a beige solid. Ή NMR (400

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MHz, DMSO-d₆j δ: 7.89 (III, d, J = 4.7 Hz), 7.95 (2H, s), 8.48 (1H, dd, JI = 8.6 Hz, J₂ = 1.6 Hz), 8.92 (HI, d, J = 4.7 Hz).

d) Amide of 3-aminopyridine-2-sulfonic acid

<img file="BRPI0809685A2_D0297.tif" />

3-Nitropyridine-2-sulfonic acid amide (3 g, 14.77 mmol) and iron powder (5 g, &lt;10 microns) were suspended in ethanol (100 mL). Saturated aqueous ammonium chloride solution (60 mL) was added and the mixture stirred at 105 °C for 1.5 h. Upon cooling to 25 °C, ethyl acetate (200 mL) was added and the mixture was vigorously stirred. The entire mixture was filtered through a Celite plug. The resulting filtrate was diluted with ethyl acetate (100 mL) and the layers were separated. The aqueous layer was extracted back with ethyl acetate (3 x 200 mL). The organic layers were combined, dried over magnesium sulfate, filtered, and concentrated under vacuum until dry. The resulting solid was triturated with a 3:1 mixture of hexanes and ethyl acetate (~10 mL). The solid was collected by vacuum filtration and vacuum dried for 16 h to give the desired product, 3-aminopyridine-2-sulfonic acid amide (1.95 g, 11.27 mmol, 76%), as a dirty white powder. HI NMR (400 MHz, DMSO-dõ? δ: 5.98 (2H, bs), 7.19 20 - 7.21 (1H, m), 7.32 (2H, bs), 7.79 -7.80 (1H, m), 7.90 - 7.91 (1H, m).

e) Ethyl ester of (l,l-dioxo-l,4-dihydro-l)⁶-aunt-

2,4,8-triazanaphthalen-3-yl)-acetic

<img file="BRPI0809685A2_D0298.tif" />

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3-aminopyridine-2-sulfonic acid amide (1.8 g, 10.4 mmol) was dissolved in 1,4-dioxane (45 mL). Ethyl ester of chlorocarbonyl acetic acid (1.57 mL, 12.48 mmol) was added. The mixture was stirred at 90°C for 1.5 h. Upon cooling, the mixture was diluted with ethyl acetate (300 mL) and washed with half-saturated aqueous sodium bicarbonate solution (100 mL) followed by saturated aqueous brine solution (100 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated under vacuum. The resulting oily residue was treated with toluene (36 mL) and triethylamine (9 mL). The mixture was stirred at 110°C in a sealed tube for 1.5 h. The residue never completely dissolved. Upon cooling, the mixture was concentrated under vacuum to yield a thick oil. Trituration with a minimal amount of ethyl acetate (~5 mL) solidified the product. The solids were collected by vacuum filtration and dried under vacuum for 16 h to provide the desired product, ethyl ester of (1,1-dioxo-1,4-dihydro-1-⁶-thia-2,4,8-triazenaphthalen-3-yl)-acetic acid (0.3 g, 1.11 mmol, 11%), as a white solid. NMR (400 MHz, DMSO)₆) δ: 1.21 (3H, t, J = 7.0 Hz), 3.71 (2H, s), 4.16 (2H, quartet, J = 7.1 Hz), 7.70 -7.76 (2H, m), 8.62 (1H, dd, Ji = 4.0 Hz, J₂ = 1.5 Hz), 12.29 (1H, bs). LC-MS (ESI) calcd for C10H11N3O4S 269.05, found

270.1 [M+H⁺],

f) Sodium salt of (1,1-dioxo-1,4-dihydro-1) acid⁶-tia[2,4,8]- triazanaphthalen- 3 - il) - acetic

<img file="BRPI0809685A2_D0299.tif" />

Acid ethyl ester (1,1-dioxo-1,4-dihydro-lÀ⁶-thia-2,4,8triazenaphthalen-3-1I)-acetic acid (0.25 g, 0.928 mmol) was dissolved in methanol (5 mL) at approximately 60°C. Sodium hydroxide (0.111 g,

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A solution of 2.79 mmol) was dissolved in water (2.5 mL). Upon cooling to 25 °C, the solutions were combined. The mixture was stirred at 25 °C for 3 h. The mixture was stored at -40 °C for 16 h. The mixture was concentrated under vacuum to provide the crude product, sodium salt of (1,1-dioxo-1,4-dihydro-l) acid.⁶-thia-2,4,8-triazenaphthalen-3-yl)-acetic acid (~ 0.928 mmol), as a yellow film, which was used in the next step without further purification.

g) (lR,2S,7R,8S)-5-(l, 1-dioxo-1,4-dihydro-lÀ⁶-thia-2,4,8triazanaphthalen-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0²·⁷] undec-5-en-4-ona

<img file="BRPI0809685A2_D0300.tif" />

F

The sodium salt of the acid (1,1-dioxo-1,4-dihydro-1)⁶Crude thia-2,4,8-triazenaphthalen-3-yl)-acetic acid (-0.928 mmol), ethyl ester of (1S,2R,38,417)-3-/4-fluoro-benzylamino)-bicyclo[2.2.l]heptane-2-carboxylic acid (0.27 g, 0.928 mmol) and O-(7-azabenzotriazol-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate (0.494 g, 1.3 mmol) were combined and dissolved in anhydrous A,A-dimethylformamide (2 mL). The mixture was stirred at 25°C for 1.5 h. Triethylamine (0.697 mL, 5 mmol) was added and the mixture was stirred at 50°C for 16 h. Upon cooling, the mixture was diluted with ethyl acetate (100 mL), washed with saturated aqueous ammonium chloride solution (2 x 25 mL) and saturated aqueous brine solution (25 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated under vacuum. The resulting solid was triturated with ethyl acetate (-15 mL) and the product solidified. The solid was collected.

301/317 by vacuum filtration and vacuum dried for 16 h to provide the desired product, (1R,2S,7R,85)-5-(1,1-dioxo-1,4-dihydro-lÀ⁶-thia-2,4,8-triazanaphthalen-3-yl)-3-(4-fluoro-benzyl)-6-hydroxy-3-aza-tricyclo[6.2.1.0² 7]undec-5en-4-one (0.03 g, 0.064 mmol, 6.9%), as a white powder. Ή NMR(400

MHz, DMSO-dejô: 0.92 -1.05 (2H, m), 1.18 - 1.46 (4H, m), 2.26 (1H, d, J = 3.1 Hz), 2.36 (1H, d, J = 9.5 Hz), 2.45 (1H, d, J = 3.0 Hz), 3.15 (1H, d, J = 9.4 Hz), 4.13 (1H, d, J = 15.6 Hz), 4.86 (1H, d, J = 15.6 Hz), 7.01 7.05 (2H, m), 7.16-7.20 (2H, m), 7.39 - 7.42 (1H, m), 7.50 (1H, dd, JI = 8.6 Hz, J₂ = 1.6 Hz), 8.26 (1H, dd, J1 = 4.6 Hz, J₂ = 1.5 Hz). LC-MS (ESI) calcd for C23H21FN4O4S 468.13, found 469.2 [M+H¹].

Example 66

7V->3-[(1R,25,7R,8S)-3-(2-cycloproyl-ethyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²-⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- 1À⁶benzoph 1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0301.tif" />

a) Ethyl ester of (1S,2R,3S,4R)-3-(2-cyclopropylethylamino)-bicyclo[2.2.1]heptane-2-carboxylic acid

<img file="BRPI0809685A2_D0302.tif" />

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A solution of cyclopropylacetaldehyde in dichloromethane

1.4 M (prepared as described in Example 23a, 3.4 mL, 4.76 mmol) was added to a solution of ethyl ester of (18,212,3S,412)-3amino-bicyclo[2.2.1]heptane-2-carboxylic acid (prepared as described in Example 6k, 580 mg, 3.17 mmol) in anhydrous methanol (15 mL) at 25 °C under a nitrogen atmosphere. After stirring for 20 min, glacial acetic acid (0.6 mL) was added. The solution was cooled to 0 °C, sodium triacetoxyborohydride (1.7 g, 7.93 mmol) was added, and the resulting mixture was stirred at 25 °C for 20 h. The reaction mixture was stopped with saturated aqueous sodium bicarbonate solution (25 mL) and extracted with ethyl acetate (3 x 60 mL). The combined organic layers were washed with saturated aqueous brine solution, dried over sodium sulfate, and filtered. The filtrate was concentrated under vacuum to provide the desired product, ethyl ester of (1S,212.3S,412)-3-(2-cyclopropylethylamino)-bicyclo[2.2.1]heptane-2-carboxylic acid (676.4 mg, 2.69 mmol, 84.9%), as a yellow oil. LC-MS (ESI) calcd for C15H25NO2 251.19, found 252.0 [M+H⁺],

b) Ethyl ester of (1S,2R,3S,4R)-3-{(2-cyclopropylethyl)-[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- 1A⁶-benzo[1,2,4]thiadiazin-3-yl)-acetyl]-aminoj-bicyclo[2.2.1]heptane-2-carboxylic acid

To a stirred solution of ethyl ester (1S,2R,3S,412)-3-(2-cyclopropylethylamino)-bicyclo[2.2.1]heptane-2-carboxylic acid (338.2 mg, 1.35 mmol) in anhydrous Α,Α-dimethylformamide (10 mL) under a nitrogen atmosphere, (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro

303/317 lÀ⁶-benzo[1,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 493 mg, 1.48 mmol), α-methylmorpholine (0.33 mL, 2.96 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (284 mg, 1.48 mmol) were added sequentially. After stirring at 25°C for 21 h, additional acid (7-methanesulfonylamino-1,1-dioxo-1,4-dihydro-1α) was added.⁶-benzo[l,2,4]thiadiazin-3-yl)-acetic acid (prepared as described in Example 1g, 150 mg, 0.45 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide chloride (85 mg, 0.44 mmol) were added. After another 26 h, 1.0 M aqueous hydrochloric acid solution (12 mL) was added and the mixture was extracted with ethyl acetate (3 x 60 mL). The combined organic layers were washed with saturated aqueous brine solution (15 mL), dried over sodium sulfate, filtered, and concentrated under vacuum to provide the crude product, ethyl ester of (1S',2R,3S,4R)-3-{(2-cyclopropylethyl)-[2-(7methanesulfonylamino-1,1-dioxo-1,4-dihydro-1A⁶-benzo[1,2,4]thiadiazin-3-yl)-acetyl]-amino)-bicyclo[2.2.1]heptane-2-carboxylic acid, as a red oil, which was used in the next step without any further purification. LC-MS (ESI) calcd for C25H34N4O7S2 566.19, found 567.4 [M+H⁺],

c) 7V-{3-[(1R,2S,7R,8S)-3-(2-cyclopropyl-ethyl)-6-hydroxy-4oxo-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-l, 1-dioxo- 1,4-dihydrolÀ⁶-benzo[1,2,4]thiadiazin-7-yl)-methanesulfonamide

A solution of ethyl ester of the acid (lS,2R,3S,4R)-3-{(2

304/317 cyclopropyl-ethyl)-[2-(7-methanesulfonylamino-1,1-dioxo-1,4-dihydro- 1λ⁶A 21% by weight solution of sodium ethoxide in ethanol (2.4 mL, 7.41 mmol) was added to benzo[1,2,4]thiadiazine-3-yl)-acetyl]-amino}-bicyclo[2.2.1]heptane-2-carboxylic acid in absolute ethanol (11 mL). After stirring at 25°C for 23 h, an additional 21% by weight solution of sodium ethoxide in ethanol (1.0 mL, 3.09 mmol) was added. After stirring at 25°C for 6 h, an additional 21% by weight solution of sodium ethoxide in ethanol was added and the mixture was stirred for another 24 h. The mixture was then acidified with an aqueous solution of hydrochloric acid.

1.0 M (22 mL) was extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The crude mixture was purified by pre-HPLC [Luna 5pm C18 column (2) 100A AXIA 150 x 21.2 mm, 5 microns, 30%-95% in 7 min @ 30 mL/min flow rate, 0.05% trifluoroacetic acid in acetonitrile/ 0.05% trifluoroacetic acid in water] to provide the desired product, 7V43-[(1R,2S,7R,8S)-3-(2-cyclopropylethyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷] undec-5-en-5-yl]-l,l-dioxol,4-dihydro-lÀ⁶-benzo[l,2,4]thiadiazin-7-yl}-methanesulfonamide (160.7 mg, 0.309 mmol, 22.9% in two steps), as a solid.^]H NMR (400

MHz, CDC1₃) δ: 0.09 -0.15 (2H, m), 0.49 - 0.54 (2H, m), 0.64 - 0.73 (1H, m), 1.27 - 1.31 (2H, m), 1.48 - 1.78 (6H, m), 2.55 - 2.56 (1H, m), 2.74 2.80 (1H, m) m), 7.62 -7.68 (2H, m). LC-MS (ESI) calcd for C23H28N4O6S2

520.15, found 521.4 [M+H⁺].

Example 67

IV-{3-[(1R,2S,7R,8S)-3-(4-fluoro-benzyl)-6,9-dihydroxy-4-oxo-3-azatricyclo[6,2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo [1.2.4]thiadiazin-7-ill-methanesulfonamide

305/317 _F

A-{3-[(1R,2S,7R,8S)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricyclo[6.2.1.0²-⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazine-7-yl}-methanesulfonamide (prepared as described in Example 6, 168 mg, 0.299 mmol) was dissolved in dimethyl sulfoxide (15 mL). A buffer reaction was prepared by combining anhydrous D-glucose (3.3 g), GDH-102 (375 mg), and NADP⁺ (570 mg) in a 100 mM aqueous potassium phosphate solution (pH 8.0) (700 mL). Lyophilized MCYP-P1 Cll (1800 mg, 1500 nmol, 0.84 nmol P450/mg, Codexis, Inc.) was dissolved in a 50 mM aqueous potassium phosphate solution (pH 8.0) (29 mL). The following components were added in order to a 2.8 L Fernbach flow regulator flask: the reaction buffer, followed by the MCYP-PICII stock solution, then the A-{3-[(1R,2S,7R,8S)-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-azatricyclo[6. 2.1.0 solution]}²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lA⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide. The vessel used to make the stock solution of A-{3-[(1R,2S,7R,8S)-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²-⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lA⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide was washed with 0.1 M aqueous potassium phosphate solution (pH 8.0) (6 mL) and this solution was then added to the flask, bringing the total reaction volume to 750 mL. The mixture was then incubated for 24 h at 30°C with gentle shaking, followed by freezing at -80°C. Methanol (1.5 L) was added to the thawed reaction mixture resulting in the formation of a precipitate. The solids were removed as pellets by centrifugation for 45 min at 10,000 rpm. The supernatant was concentrated under vacuum and the residue was...

306/317 purified by prep-HPLC to provide the desired product, 7V-{3[(l/?_;2S,77?,8S)-3-f4-fluoro-benzyl)-6,9-dihydroxy-4-oxo-3-aza-tricyclo[6.2.

1.0²’⁷]undec-5-en-5-yl]-1,1- dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (49.5 mg, 0.086 mmol, 29%), as a white solid.^XH NMR (500 MHz, DMSO-d₆) δ: 1.21 (1H, dd, Ji = 14.7 Hz, J₂ = 4.8 Hz), 1.46 - 1.59 (3H, m), 2.44 (III, s), 2.53 (1H, s), 2.87 (1H, d, J = 8.7 Hz), 3.07 (3H, s), 3.39 (1H, d, J = 8.5 Hz), 3.80 (III, d, J = 6.0 Hz), 4.42 (1H, d, J = 15.5 Hz), 4.96 (1H, d, J = 15.7 Hz), 7.16 (2H, t, J = 8.7 Hz), 7.32 -7.34 (2H, m), 7.52 (1H, dd, Ji = 9.0 Hz, J₂ = 2.3 Hz), 7.59 7.60 (2H, m), 10.23 (HI, s), 14.08 (1H, s), 15.03 (Ui, bs), LC-MS (ESI) calcd for C25H25FN4O7S2 576.11, found 577.5 [M+H⁺].

Example 68

7V-{3-[(17?.2S,7F?,8SF3-(4-fluoro-benzyl)-6-hydroxy-4oxo-3-aza-tricyclo[6.2.1.0] L-arginine salt²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- 1À⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0303.tif" />

NH₂

<img file="BRPI0809685A2_D0304.tif" />

NH

N-{3-[(1R,2S,7R,8S) -3- (4-fluoro-benzyl) -6-hydroxy-4-oxo-3aza-tricycle

[6.2.1.0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ^the-benzo[1,2,4]thiadiazine-7-yl}-methanesulfonamide (prepared as described in Example 6, 0.280 g, 0.499 mmol) was dissolved in acetonitrile (5.0 mL). A 0.1 M aqueous L-arginine solution (3.0 mL, 0.3 mmol) was added, followed by the addition of an L-arginine solution.

307/317 in 0.1 M 1-propanol (2.0 mL, 0.2 mmol). After stirring for 6 h at 23°C, the flask was opened to the atmosphere and the suspension was stirred for 16 h. The solid was collected by filtration and further dried under vacuum at 23°C to provide the desired product, L-arginine salt, N-{3[(17?, 2S,7R,8S)-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²>⁷undec

-5-en-5-yl]-1,1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (0.257 g, 0.341 mmol, 68%), as a crystalline solid.^XH NMR (300 MHz, DMSO-d₆) δ: 0.96 - 1.17 (2H, m), 1.28 (1H, app t, J = 10.0 Hz), 1.35 - 1.82 (7H, m), 2.33 (1H, app d, J = 3.0 Hz), 2.43 (1H, d, J = 9.3 Hz), 2.97 (3H, s), 3.00 - 3.17 (2H, m), 3.23 (1H, d, J = 9.3 Hz), 4.21 (1H, d, J = 15.3 Hz), 4.94 (1H, d, J = 15.3 Hz), 7.04 7.15 (3H, m), 7.27 (2H, dd, J = 5.7,8.7 Hz), 7.35 (1H, dd, J = 2.5,8.9 Hz), 7.35 - 7.51 (4H, m), 8.82 (1H, br s), 15.29 (1H, br s). Anal, calcd for C3iH₃9FN₈O8S₂«H20: C, 49.46; H, 5.49; N, 14.88; 0, 19.13; S, 8.52; F, 2.52; found: C, 49.49; H, 5.23; N, 14.96; 0, 18.69; S, 8.82; F, 2.81. mp = 216°C (DSC).

Example 69

L-lysine salt of /V'-{3-[(1R,2S,7R,8S')-3-(4-fluoro-benzyl)-6-hydroxy-4oxo-3-aza-tricyclo[6.2.1.0²-⁷]undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro- 1À⁶benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide

<img file="BRPI0809685A2_D0305.tif" />

NH₂

CO₂H

7V-{3-[(lR,2S,7/?,8S)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza308/317 tricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (prepared as described in Example 6, 0.090 g, 0.160 mmol) was dissolved in acetonitrile (2.5 mL). An aqueous solution of L-lysine (0.469 mL of a 50 mg/mL solution in water, 0.160 mmol) was added. The solvent was allowed to evaporate under a stream of nitrogen and ethanol (0.5 mL) was added. The mixture was stirred at 35°C for 2 days and then immersed in an ultrasonic bath. Water (0.5 mL) was added and the mixture was stirred at 23°C for 3 days. The solid was collected by filtration and further dried under vacuum at 23°C to provide the desired product, lysine salt, N-{3[(177.2S,77?,8S)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²>⁷] undec-5-en-5-yl]-l,l-dioxo-l,4-dihydro-lÀ⁶ (0.070 g, 0.096 mmol, 60%), as a crystalline solid.^XH NMR (300 MHz, DMSO-d₆) δ: 0.96 - 1.15 (2H, m), 1.22 - 1.76 (10H, m), 2.34 (1H, app d, J=2.7 Hz), 2.43 (III, d, J = 9.3 Hz), 2.74 - 2.78 (2H, m), 2.97 (3H, s), 3.18 - 3.29 (1H, m), 4.21 (1H, d, J = 15.3 Hz), 4.95 (1H, d, J = 15.6 Hz), 7.07 -7.18 (3H, m), 7.27 (2H, dd, J - 5.7,8.7 Hz), 7.36 (1H, dd, J= 2.4,8.7 Hz), 7.44 (1H, d, J = 2.4 Hz), 15.31 (1H, br s). Anal, calcd for C3iH39FNõO8S2*H20: C, 51.37; H, 5.70; N, 11.59; O, 19.87; S, 8.85; F, 2.62; found: C, 51.13; H, 5.52; N, 11.63; O, 20.07; S, 9.20; F, 2.71. pf = 200°C (DSC).

Example 70

7)1-13-((177,28,777,88)-3-(4-fluorobenzyl)-6-hydroxy-,4-oxo-3-azatricyclo[6.2.1.0] hemimagnesium salt²'⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydrolÃ⁶-benzo(1,2,41thiadiazin-7-yl)-methanesulfonamide

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<img file="BRPI0809685A2_D0306.tif" />

A-{3-[(l R,2S,7R,8S)-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricycle [6.2.1.0²-⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lA⁶-benzo[1,2,4]thiadiazine-7-yl}-methanesulfonamide (prepared as described in Example 6, 0.465 g, 0.829 mmol) was dissolved in acetone (9.0 mL).

A 7-8% by weight solution of magnesium methoxide in methanol (0.593 mL, 0.414 mmol) was added. The solvent was evaporated and the residue was then diluted with water (0.9 mL) and acetone (1.8 mL). The resulting mixture was stirred at 23°C for 16 h. The solid was collected by filtration and further dried under vacuum at 23°C to provide the desired product, 7V-{3-[(lR,28,77?,88)-3-(4-fluorobenzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0] hemimagnesium salt, trihydrate²’⁷]undec-5-en-5-yl]-1, 1-dioxo-1,4-dihydro-lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (0.377 g, 0.602 mmol, 73%), as a crystalline solid. Π4 NMR (300 15 MHz, DMSO-d₆) δ: 0.96 - 1.17 (2H, m), 1.22 - 1.58 (4H, m), 2.33 (1H, br s), 2.44 (1H, d, J= 9.6 Hz), 2.98 (3H, s), 3.23 (114, d, J = 9.3 Hz), 4.21 (1H, d, J= 14.7 Hz), 4.94 (1H, d, J = 15.3 Hz), 7.03 -7.19 (3H, m), 7.21 7.48 (4H, m), 9.81 (114, br s), 15.35 (114, br s). Anal, calcd for C25H₂4N4O₆FS₂*0.5 Mg*3 H₂O: C, 47.98; H, 4.83; N, 8.95; O, 23.01; S, 20 10.25; F, 3.04; Mg, 1.94; found: C, 47.66; H, 4.89; N, 8.98; O,

23.00; S, 11.36; F, 3.09; Mg, 1.82. mp = 184°C (DSC).

Example 71

N-{3-[(1R.2S,7R,8S)-3 -(4-fluoro-benzyl)-6-hydroxy-4-oxo3-aza-tricyclo[6.2.1.0] sodium salt²-⁷]undec-5-en-5-yl]-1,1 -dioxo-1,4-dihydro- IA⁶310/317 benzofl,2,4] thiadiazin-7-yl)-methanesulfonamide

<img file="BRPI0809685A2_D0307.tif" />

7V-{3-[(lR.2S,7R,8S/-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricycle [6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lA⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (prepared as described in Example 6, 0.407 g, 0.726 mmol) was suspended in ethanol (11.0 mL). A 1.0 M aqueous sodium hydroxide solution (0.726 mL, 0.726 mmol) and water (1.0 mL) were added. The mixture was seeded with a crystal of the sodium salt of 7V-{3-[(lR.2S,7R,8S/-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1.0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (produced from a separate batch) and the mixture was then stirred at 23°C for 1 day. The solid was collected by filtration and further dried under vacuum at 23°C to give the desired product, sodium salt, hydrate of 7V-{3[(lR.2S,7R,8S/-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1. 0²’⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lÀ⁶-benzo[1,2,4]thiadiazin-7yl}-methanesulfonamide (2.25 molar equiv. water) (0.235 g, 0.377 mmol, 52%), as a crystalline solid. Ή NMR (300 MHz, DMSO-d₆) δ: 0.99 1.11 (2H, m), 1.28 (1H, app t, J = 10.2 Hz), 1.36 - 1.53 (3H, m), 2.33 (1H, app d, J= 2.7 Hz), 2.42 (1H, d, J = 9.3 Hz), 2.97 (3H, s), 3.22 (1H, d, J = 9.3 Hz), 4.20 (1H, d, J = 15.3 Hz), 4.95 (1H, d, J = 15.3 Hz), 7.09 7.16 (3H, m), 7.25 -7.36 (3H, m), 7.42 (1H, d, J = 2.4 Hz), 9.79 (1H, s), 15.32 (1H, s). Anal, calcd para C₂5H₂4FN₄No₆S₂*2.25 H₂O: C, 48.19; H, 4.61; N, 8.99; O, 21.18; S, 10.29; F, 3.05; Na, 3.69; found: C, 48.14; H, 4.67; N, 8.97; O, 21.07; S, 10.25; F, 3.13; Na, 3.87. pf =

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182-188°C (DSC).

Example 72

Potassium salt of 7V-{3-[(1R.2S,7R,8S)-3-(4-fluoro-benzyl)-6-hydroxy-4oxo-3-aza-tricyclo[6.2.1.0²>⁷]undec-5-en-5-ill-1,1-dioxo-1,4-dihydro- 1A⁶benzo[1,2,4]thiadiazin-7-yl]-methanesulfonamide

<img file="BRPI0809685A2_D0308.tif" />

7V-{3-[(IR.2S,7R,8Sj-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-azatricycle [6.2.1.0²>⁷]undec-5-en-5-yl]-l, 1-dioxo- 1,4-dihydro-lA⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide (prepared as described in Example 6, 0.281 g, 0.501 mmol) was dissolved in methyl ethyl ketone (8.0 mL). A 0.5 M aqueous potassium hydroxide solution (1.0 mb, 0.500 mmol) was added. The solution was seeded with potassium salt of 2V-{3-[(JR.2S,7R,8Sj-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6. 2.1,0²>⁷]undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lA⁶-benzo[1,2,4]thiadiazin-7-yl}-methanesulfonamide crystalline, (produced from a separate batch) and the resulting mixture was then stirred at 23°C for 3 h. The solid was collected by filtration and further dried under vacuum at 23°C to give the desired product, potassium salt, hydrate of 7V-{3[(lR.2S,7R,8Sj-3-(4-fluoro-benzyl)-6-hydroxy-4-oxo-3-aza-tricyclo[6.2.1,0²-⁷] undec-5-en-5-yl]-1,1-dioxo-1,4-dihydro- lA⁶-benzo[1,2,4]thiadiazin-7-yl}methanesulfonamide (0.75 molar water equiv.) (0.127 g, 0.207 mmol, 41 %), as a crystalline solid. Ή NMR (300 MHz, DMSO-de) δ: 0.99 1.11 (2H, m), 1.27 (1H, app t, J = 10.3 Hz), 1.36 -1.54 (3H, m), 2.33 (1H,

312/317 br s), 2.42 (1H, d, J = 9.0 Hz), 2.95 (3H, s), 3.22 (1H, d, J = 9.3 Hz), 4.20 (1H, d, J = 15.3 Hz), 4.96 (1H, d, J = 15.6 Hz), 7.09 -7.15 (3H, m), 7.25 7.34 (3H, m), 7.41 (1H, d, J = 2.7 Hz), 9.84 (1H, br s), 15.30 (1H, s). Anal, calcd for C₂₅H₂₄FKN4O₆S2*0.75 H₂O: C, 49.05; H, 4.20; N, 9.15; 0.17.64; S, 10.48; F, 3.10; K, 6.39; found: C, 48.82; H, 4.11; N, 9.06; 0.17.35; S, 10.37; F, 3.18; K, 6.75. mp - 278°C (DSC).

Biological Tests

The ability of the compounds in Formula 1 to inhibit HCV replication can be demonstrated in the following in vitro tests.

The compounds were tested for HCV polymerase inhibition. The tests were performed on a 96-well streptavidin-coated flash plate using 20 nM enzyme, 0.5 qCi of [a³³P]GTP, 0.6 μΜ GTP and 250 nM 5'biotinylated oligo (rG13)/poly rC in 20 mM Tris-HCI, pH 7.5, 5 mM MgCl₂The reaction consisted of 5 mM dithiothreitol, 0.1 g/L bovine serum alumina, and 100 U/mL RNase inhibitor. The reaction was stopped by aspiration after 75 min at 28°C, and the plate was washed several times. After washing and drying the plate, the incorporated radioactivity was counted using a Microbeta scintillation counter. ICso values ​​were calculated relative to the uninhibited control, and the inhibition data were fitted to a 4-parameter ICso equation. For very potent inhibitors, the data were fitted to a strongly dependent quadratic equation to obtain IC50 values.

The test results (IC50 values) for the compounds of Formula I are summarized in Table 1, where in this document ++++ means inhibition of NS513 polymerase with IC50 values ​​less than 0.02 μM, +++ means IC50 values ​​between 0.02 μM and 0.1 μM, ++ means IC50 values ​​between 0.1 μM and 1 μM, and + means IC50 values ​​between 1 μM and 100 μM. The test results, for example, numbers 64 and 65 in Table 1 are EC50 values, where in this document

313/317 document **** means inhibition of HCV replication EC50 values ​​less than 0.02 μM, *** means EC50 values ​​between 0.02 μM and 0.1 μM, ** means EC50 values ​​between 0.1 μM and 1 μM, and * means EC50 values ​​between 1 μM and 100 μM.

Table 1

<td>Example No.</td><td>ic₅₀</td>

<td> 1</td><td> ++++</td>

<td> 2</td><td> ++++</td>

<td> 3</td><td> ++</td>

<td> 4</td><td> ++++</td>

<td> 5</td><td> 4-4-</td>

<td> 6</td><td> ++++</td>

<td> 7</td><td> 4-4-</td>

<td> 8</td><td> ++++</td>

<td> 9</td><td> +++</td>

<td> 10</td><td> +++</td>

<td> 11</td><td> ++++</td>

<td> 12</td><td> ++++</td>

<td> 13</td><td> 4-4-4-</td>

<td> 14</td><td> ++++</td>

<td> 15</td><td> ++++</td>

<td> 16</td><td> ++++</td>

<td> 17</td><td> +++</td>

<td> 18</td><td> +++</td>

<td> 19</td><td> +</td>

<td> 20</td><td> ++</td>

<td> 21</td><td> +++</td>

<td> 22</td><td> 4-4-4-</td>

<td> 23</td><td> ++++</td>

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<td> 24</td><td> ++</td>

<td> 25</td><td> ++++</td>

<td> 26</td><td> ++</td>

<td> 27</td><td> +</td>

<td> 28</td><td> ++</td>

<td> 29</td><td> ++++</td>

<td> 30</td><td> ++</td>

<td> 31</td><td> +++</td>

<td> 32</td><td> +++</td>

<td> 33</td><td> +</td>

<td> 34</td><td> +</td>

<td> 35</td><td> +++</td>

<td> 36</td><td> +++</td>

<td> 37</td><td> ++++</td>

<td> 38</td><td> +++</td>

<td> 39</td><td> +</td>

<td> 40</td><td> +++</td>

<td> 41</td><td> +++</td>

<td> 42</td><td> +++</td>

<td> 43</td><td> +++</td>

<td> 44</td><td> 4-4-</td>

<td> 45</td><td> +++</td>

<td> 46</td><td> ++++</td>

<td> 47</td><td> +</td>

<td> 48</td><td> 4-4-4-</td>

<td> 49</td><td> ++++</td>

<td> 50</td><td> ++++</td>

<td> 51</td><td> +++</td>

<td> 52</td><td> +++</td>

<td> 53</td><td> +</td>

<td> 54</td><td> ++</td>

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<td> 55</td><td> ++++</td>

<td> 56</td><td> ++++</td>

<td> 57</td><td> +++</td>

<td> 58</td><td> ++++·</td>

<td> 59</td><td> ++</td>

<td> 60</td><td> +++</td>

<td> 61</td><td> +</td>

<td> 62</td><td> +++</td>

<td> 63</td><td> ++</td>

<td> 64</td><td> **</td>

<td> 65</td><td> **</td>

<td> 66</td><td> +++</td>

<td> 67</td><td> ++++</td>

Replicon tests with HCV (Replicon EC50 (pM))

The cell culture component of the test is carried out essentially as described by Bartenschlager et al., Hepatology 2002, 35, 694-703, where exponential growth of HCV Huh-7/C24 replicon cells are seeded at 4.5 x 103 cells/well in 96-well plates and 24 hours later are treated with a six-point half-log concentration of the compound. After 72 hours of exposure, the medium is discarded from the compound test plate and the 10-layer cells are lysed by adding the 150 L lysis mixture (Genospectra) with incubation at 53°C for 45 minutes. Following incubation, each lysate is thoroughly mixed and 5 L (NS3 probe) or 10 L (GAPOH probe) of each lysate is then transferred to the capture plate and analyzed by bDNA assay.

Branched DNA (bDNA) Tests

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Based on sequences provided by NS3 [AJ242652], Genospectra (Fremont, CA, USA) designed and synthesized probes for these analytes (along with GAPDH). Cellular bDNA analysis is performed essentially as described in the Genospectra protocol (details in Shyamala, V, et al., Anal. Biochem. 1999, 266, 140-7), where specific target capture extenders, label extenders, and blocking probes are added to the capture plate after the addition of 5 or 10 pL of cell lysate. After overnight annealing, during which the target RNA is captured onto the plate via interaction with the capture extenders, the plate is washed and then amplified (which binds via label extenders) and label probes are sequentially added.

After subsequent addition of chemiluminescent substrate (dioxetane), each plate is read by a luminometer (Wallac 1420 Multilabel HTS Counter Victor 2). The luminescence signal is proportional to the amount of mRNA present in each lysate. In addition to the samples, background controls (without probe) containing only cell lysate are also included in each bDNA test plate, and the average signal from these control wells is subtracted from the sample reading before analysis. The percentage of the drug-free control is determined for both NS3 and GAPDH signals, also for each compound. The percentage of inhibition is determined for each concentration of the compound relative to the drug-free control to calculate the EC50.

Replicon HEV Testing Protocol Based on Luciferase

Huh-Iuc/neo-ET HCV replicon cells were seeded exponentially at 6χ¹⁰³ Cells/well in 96-well test plates. 24 hours later, the cells were treated with various concentrations of the compound in triplicate. After 72 hours of exposure to the compound, luciferase activity in the wells was determined using the

317/317 Bright-Gio reagent (Promega, Madison, Wisconsin) with a luminometer (Wallac 1420 Multilabel UTS Counter Victor 2). The background control was treated with replicon cells with 100 nM BILN-2061, an HCV protease inhibitor. The % inhibition was determined for each concentration of the compound relative to the negative control (no compound) to calculate the EC50.

It should be understood that the preceding description is illustrative and explanatory in nature and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, the technician will recognize obvious modifications and variations that can be made without departing from the spirit of the invention.