Hcv ns3 protease inhibitors

[0001] The present invention relates to macrocyclic compounds that are useful as NS3 hepatitis C virus (HCV) protease inhibitors, their synthesis, and their use for treating or preventing HCV infection.

BACKGROUND OF THE INVENTION [0002] Hepatitis C (HCV) is a serious health problem that leads to chronic liver diseases, such as cirrhosis and liver cancer, in a significant number of infected people, estimated at 2-15% of the world population. About 3.9 million infected people live in the United States alone, according to the American Center for Disease Control (US) Center for Disease Control), approximately five times as many people as those infected with human immunodeficiency virus (HIV). According to the World Health Organization, more than 170 million people are infected worldwide, with at least 3 to 4 million people infected each year. After infection, about 20% of people get rid of the virus, but the remaining HCV persists for the rest of their lives. Ten to twenty percent of chronically infected people develop cirrhosis or cancer. The viral disease is transmitted parenterally through infected blood and blood products, contaminated needles or sexually and directly from infected mothers or pregnant mothers to their offspring.

[0003] Current treatments for HCV infections, which are limited to immunotherapy with recombinant interferon α alone or in combination with the ribavirin nucleoside analogue, provide limited clinical benefit. In addition, there are no HCV vaccines. Therefore, there is an urgent need to develop improved therapeutic agents that will successfully combat chronic HCV infection. The current state of the art in the treatment of HCV infection is discussed in the following documents: B. Dymock et al., "Novel approaches to the treatment of hepatitis C virus infection," Antiviral Chemistry & Chemotherapy, 11: 79-96 (2000); H. Rosen et al., "Hepatitis C virus: current understanding and prospects for future therapies," Molecular Medicine Today, 5: 393-399 (1999); D. Moradpour et al., "Current and evolving therapies for hepatitis C," European J. Gastroenterol. Hepatol., 11: 1189-1202 (1999), R. Bartenschlager, "Candidate Targets for Hepatitis C Virus-Specific Antiviral Therapy," Intervirology, 40: 378-393 (1997); GM Lauer and BD Walker, "Hepatitis

C Virus Infection, "N. Engl. J. Med., 345: 41-52 (2001); BW Dymock," Emerging therapies for hepatitis C virus infection, "Emerging Drugs, 6: 13-42 (2001); and C Crabb, "Hard-Won Advances Spark Excitement about Hepatitis C," Science: 506-507 (2001).

[0004] Some virus-encoded enzymes are putative targets for therapeutic interventions, including metalloprotease (NS2-3), serine protease (NS3), helicase (NS3) and RNA-dependent RNA polymerase (NSSB). The NS3 protease is located in the N-terminal domain of the NS3 protein and is considered the main target of drugs because it is responsible for intramolecular cleavage at the NS3 / 4A site and further intermolecular processing in the NS4A / 4B, NS4B / 5A and NS5A / 5B junctions. In previous studies, specific groups of peptides, such as hexapeptides, as well as tripeptides, were discussed in U.S. Patent Nos. US2005 / 0020503, US2004 / 0229818 and US2004 / 00229776, showing degrees of activity in inhibiting NS3 proteases. International patent application WO 03/064455 describes macrocyclic peptides that are useful as HCV NS3 protease inhibitors. It is an object of the present invention to provide further compounds that have HCV NS3 protease activity.

Summary of the invention [0005] The present invention relates to novel macrocyclic compounds of formula (I) and / or pharmaceutically acceptable salts or hydrates thereof. These compounds are useful in inhibiting HCV NS3 (non-structural 3) protease (hepatitis C virus), preventing or treating one or more symptoms of HCV infection, either as compounds or their pharmaceutically acceptable salts or hydrates (if appropriate), or as components of the composition pharmaceutical, in combination or not with other HCV anti-viral agents, anti-inflammatory agents, immunomodulatory agents, antibiotics or vaccines. More specifically, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt or hydrate thereof: In particular, the invention relates to a compound of formula (I) or a pharmaceutically acceptable salt or hydrate thereof:

<img file="PL1924593T3_D0001.tif" />

wherein:

piq are both 1;

R¹ means CONR¹⁰SO 2 R⁶;

R² is C1-C6 alkyl or C2-C6 alkenyl; wherein said alkyl or alkenyl is optionally substituted with 1 to 3 halo;

R³ is C1-C8 alkyl or C3-C8 cycloalkyl;

R⁵ is H;

R⁶ is C3-C6 cycloalkyl;

Y is C (= O);

Z is O;

M is C1-C12 alkylene or C1-C12 alkenylene; and each R¹⁰ is independently H or C1-C6 alkyl.

[0006] The present invention also includes pharmaceutical compositions containing a compound of the present invention and methods for preparing such pharmaceutical compositions. The present invention further relates to methods of treating or preventing one or more symptoms of HCV infection.

[0007] Other embodiments, aspects and features of the present invention are either more specifically described or will be apparent from the following description, examples and appended claims.

Detailed description of the invention [0008] The present invention includes compounds of formula I above, and pharmaceutically acceptable salts and / or hydrates thereof. These compounds and their pharmaceutically acceptable salts and / or hydrates are HCV protease inhibitors (e.g., HCV NS3 protease inhibitors). The present invention also includes compounds of formulas II-a and III-a in which all variables are as defined for formula I.

<img file="PL1924593T3_D0002.tif" />

[0009] A first embodiment of the present invention is a compound of formula I, II-a or

III-a, or a pharmaceutically acceptable salt or hydrate thereof, in which R¹ stands for CONHSO2R^6; and all other variables are as originally defined (i.e. as defined in the summary of the invention). In the first aspect of the first practical implementation of R.¹ stands for CONHSO2R^6, in which R⁶ is C3-C5 cycloalkyl; and all other variables are defined as defined in the first practical implementation. In the first aspect of the first practical implementation of R.¹ stands for CONHSO2R⁶in which R⁶ is cyclopropyl; and all other variables are as defined in the first practical implementation.

[0010] A second embodiment of the invention is a compound of formula I, II-a or III-a, or a pharmaceutically acceptable salt or hydrate thereof, wherein R² is C 1 -C 6 -alkyl or C 2 -C 6 alkenyl and all other variables are as originally defined or as defined in any one of the previous embodiments. In the first aspect of the second practical implementation of R.² is C1-C4-alkyl or C2-C4 alkenyl; and all other variables are as originally specified or as defined in one of the previous practical implementations. In the second aspect of the second practical implementation of R.² is C2-C4 alkenyl; and all other variables are as originally specified or as defined in one of the previous practical implementations. In the description of the second aspect of the second practical implementation_R2 means vinyl; and all other variables are as defined in the third practical implementation or as defined in any of the previous practical implementation. In the third aspect of the second practical implementation of R.² is C1-C4 alkyl; and all other variables are as originally specified or as defined in any previous practical implementation. In the description of the third aspect of the third practical implementation of R.² means ethyl and all other variables are as defined in the third embodiment or as defined in any of the previous embodiments.

[0011] A third embodiment of the present invention is a compound of formula I, II-a or III-a, or a pharmaceutically acceptable salt or hydrate thereof, wherein R³ is C3-C8 cycloalkyl or C1-C8 alkyl and all other variables are as originally defined or as defined in any one of the previous embodiments. In the first aspect of the third practical implementation of R.³ is C5-C7 cycloalkyl or C1-C8 alkyl; and all other variables are as defined in the third practical implementation or as defined in any of the previous practical implementation. In the second aspect of the third practical implementation of R.³ is C5-C6 cycloalkyl or C1-C8 alkyl; and all other variables are as defined in the third practical implementation or as defined in any of the previous practical implementation. In the third aspect of the third practical implementation of R.³ is propyl or butyl; and all other variables are as defined in the third practical implementation or as defined in any of the previous practical implementation. In the description of the third aspect of the third practical implementation of R.³ means i-propyl, n-butyl or t-butyl and all other variables are as defined in the third embodiment or as defined in any of the previous embodiments. In the fourth aspect of the third practical implementation of R.³ is cyclopentyl or cyclohexyl; and all other variables are as defined in the third practical implementation or as defined in any of the previous practical implementation.

[0012] A fourth embodiment of the present invention is a compound of formula I, II-a or

III-a, or a pharmaceutically acceptable salt or hydrate thereof, in which M is C1-C10 alkylene or C2-C10 alkenylene (including linear and branched alkylene or alkenylene); and all other variables are as originally specified or as defined in any previous practical implementation. In a first aspect of the fourth embodiment, M is C1-C8 alkylene or C2-C8 alkenylene (including linear and branched alkylene or alkenylene) and all other variables are as originally defined or as defined in any of the previous embodiments. In a second aspect of the fourth embodiment, M is C4 alkylene or C4 alkenylene (including linear and branched alkylene or alkenylene) and all other variables are as defined in the fourth embodiment or as defined in any of the previous embodiments. In a third aspect of the fourth embodiment, M is C5 alkylene or C5 alkenylene (including linear and branched alkylene or alkenylene); and all other variables are as defined in the fourth practical implementation or as defined in any of the previous practical implementation. In a fourth aspect of the fourth embodiment, M is C6 alkylene or C6 alkenylene (including linear and branched alkylene or alkenylene); and all other variables are as defined in the fourth practical implementation or as defined in any of the previous practical implementation. In a fifth aspect of the fourth embodiment, M is C7 alkylene or C7 alkenylene (including linear and branched alkylene or alkenylene); and all other variables are as defined in the fourth practical implementation or as defined in any of the previous practical implementation. In a sixth aspect of the fourth embodiment, M is C8 alkylene or C8 alkenylene (including linear and branched alkylene or alkenylene); and all other variables are as defined in the fourth practical implementation or as defined in any of the previous practical implementation. In a seventh aspect of the fourth embodiment, M is C9 alkylene or C9 alkenylene (including linear and branched alkylene or alkenylene); and all other variables are as defined in the fourth practical implementation or as defined in any of the previous practical implementation. In an eighth aspect of the fourth embodiment, M is C10 alkylene or C10 alkenylene (including linear and branched alkylene or alkenylene); and all other variables are as defined in the fourth practical implementation or as defined in any of the previous practical implementation. In a ninth aspect of the fourth embodiment, M is selected from the following; and all other variables are as defined in the fourth practical implementation or as defined in any of the previous practical implementation.

<img file="PL1924593T3_D0003.tif" />

[0013] A fifth embodiment of the present invention is a compound, or a pharmaceutically acceptable salt or hydrate thereof, selected from the group consisting of the following compounds.

<img file="PL1924593T3_D0004.tif" />

τπ-ίΐ.ι ι ⁷···

ΠΙ- $ Τ ο

1Τ 1-ίΟ

Jll ί-Ί

I.TI-U ιιι-; · 2

LI1-71 small

1Π-Μ

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<img file="PL1924593T3_D0007.tif" />

[0014] Other embodiments of the invention relate to the following:

(a) a pharmaceutical composition comprising an effective amount of a compound of formula I, II-a or III-a and a pharmaceutically acceptable carrier.

(b) a pharmaceutical composition (a), further comprising a second therapeutic agent selected from the group consisting of HCV antiviral agent, immunomodulating agent and anti-infective agent.

(c) a pharmaceutical composition (b), wherein the HCV antiviral agent is an antiviral agent selected from the group consisting of an HCV protease inhibitor and an HCV NS5B polymerase inhibitor.

(d) a pharmaceutical combination which is (i) a compound of formula I, II-a or III-a, and (ii) a second therapeutic agent selected from the group consisting of an HCV antiviral agent, an immunomodulatory agent and an anti-infective agent, wherein the compound of formula I, II-a or III-a and the second therapeutic agent are each provided in an amount that causes the combination to be effective in inhibiting HCV NS3 protease, or in treating or preventing HCV infection.

(e) combination (d), wherein the HCV antiviral agent is an antiviral agent selected from the group consisting of an HCV protease inhibitor and an HCV NS5B polymerase inhibitor.

(f) a method of inhibiting HCV NS3 protease in a subject in need thereof which comprises administering to the subject an effective amount of a compound of formula I, II-a or III-a.

(g) a method of preventing or treating HCV infection in a subject in need thereof which comprises administering to the subject an effective amount of a compound of formula I, II-a or III-a.

(h) a method (g) wherein the compound of formula I, II-a or III-a is administered in combination with an effective amount of at least one second therapeutic agent of the selected group consisting of an HCV antiviral agent, immunomodulatory agent and an anti-viral agent infective.

(i) the method (h), wherein the HCV antiviral agent is an antiviral agent selected from the group consisting of an HCV protease inhibitor and an HCV NS5B polymerase inhibitor.

(j) a method of inhibiting HCV NS3 protease in a subject in need thereof which comprises administering to the subject a pharmaceutical composition (a), (b), or (c) or a combination of (d) or (e).

(k) a method for preventing or treating HCV infection in a subject in need thereof which comprises administering to the subject a pharmaceutical composition (a), (b), or (c) or a combination of (d) or (e).

[0015] The present invention also includes a compound of the present invention (i) for use in, (ii) for use as a medicament for, or (iii) for use in the preparation of a medicament for: (a) inhibiting HCV NS3 protease, or (b) for the prevention or treatment of HCV infection. In these uses, the compounds of the present invention may optionally be used in combination with one or more second therapeutic agents selected from HCV antiviral agents, anti-infective agents, and immunomodulatory agents.

[0016] Additional embodiments of the invention relate to the pharmaceutical compositions, combinations and methods outlined in (a) - (k) above and the uses mentioned in the previous paragraph, wherein the compound of the present invention applied therein is a compound of one of the practical embodiments, aspects, classes, subclasses or descriptions of the compounds described above. In all these embodiments, the compound may optionally be used in the form of a pharmaceutically acceptable salt or hydrate, if appropriate.

[0017] As used herein, the term "alkyl" refers to any linear or branched chain alkyl group having a number of carbon atoms in the specified range. Thus, for example, "C 1-6 alkyl" (or "C 1 -C 6 alkyl") refers to all hexyl and pentyl alkyl isomers as well as n-, iso-, s and t-butyl, n and isopropyl, ethyl and methyl. As another example, "C 1-4 alkyl" refers to n-, iso-, si-t-butyl, n and isopropyl, ethyl and methyl.

[0018] The term "haloalkyl" means an alkyl group in which hydrogen has been replaced by a halogen. The term "alkoxy" refers to the group "alkyl-O-".

[0019] The term "alkylene" refers to any linear or branched chain alkylene group having a number of carbon atoms in the specified range. Thus, for example, "C1-6 alkylene-" refers to any of linear or branched C1 to C6 alkylene. The class of alkylene with particular importance in relation to the invention is - (CH2) 1-6-, and the subclasses with special significance include :-( CH2) 1-4 -, - (CH2) 1-3 -, - (CH2) 1- 2-i-CH2-. Alkylene-CH (CH 3) - is also of interest.

[0020] The term "alkenylene" refers to any divalent alkenylene group having a linear or branched chain number of carbon atoms in the specified range.

[0021] The term "cycloalkyl" refers to any cyclic alkane or alkene ring having a number of carbon atoms in the specified range. Thus, for example, "C3-8 cycloalkyl" (or "C3-C8 cycloalkyl") means a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl group. The term "cycloalkoxy" means the group "cycloalkyl-O-".

[0022] The term "halogen" (or "halo") means fluoro, chloro, bromo and iodo (optionally further fluoro, chloro, bromo and iodo).

[0023] Unless expressly stated otherwise, all ranges cited herein are included. For example, a heteroaryl ring described as containing "1 to 3 heteroatoms" means that the ring may contain 1, 2 or 3 heteroatoms. It is also to be understood that any scope cited herein includes within its scope all subranges in that range. Oxidized forms of N and S heteroatoms are within the scope of this invention.

[0024] If any variable (e.g. R¹⁰⁾ occurs more than once in any element or in Formula I, II-a or III-a, or in any other formula representing and describing the compounds of the present invention, its definition at each occurrence is independent of its definition at every other occurrence. Combinations of substituents and / or variables are also permissible only if stable compounds form as a result of the combination.

[0025] Unless specifically stated otherwise, substitution by said substituent is allowed on any ring atom (e.g., aryl, heteroaromatic or saturated heterocyclic ring), provided that such ring substitution is chemically allowed and results in a stable compound . A "stable" compound is a compound that can be obtained and isolated, and its structure and properties remain or can be caused to remain substantially unchanged for a period of time sufficient to apply the compound for the purposes described herein (e.g., therapeutic or prophylactic administration to a subject) .

[0026] As a result of the selection of substituents and substitution patterns, some compounds of the present invention may have asymmetric centers and may exist as mixtures of stereoisomers or as individual diastereomers or enantiomers.

All isomeric forms of these compounds, whether separated or in mixtures, are within the scope of the present invention.

[0027] One skilled in the art will readily recognize that some of the compounds of the present invention may exist as tautomers. For the purposes of this invention, reference to a compound of formula I, II-a or III-a, is a reference to a compound per se, or to any of its tautomers per se, or to a mixture of two or more tautomers.

[0028] The compounds of the present invention are useful in inhibiting HCV protease (e.g. HCV NS3 protease) and preventing or treating HCV infection. For example, the compounds of the present invention are useful in the treatment of HCV infection after suspected past exposure to HCV, through such routes as blood transfusion, exchange of body fluids, bites, accidental needle pricks or exposure to patient's blood during surgery.

[0029] The compounds of the present invention are useful in the preparation and implementation of screening tests for antiviral compounds. For example, the compounds of the present invention are useful for isolating enzyme mutants, which are excellent screening tools for more powerful antiviral compounds. In addition, the compounds of the present invention are useful in determining or determining the binding site of other antiviral agents to HCV protease, e.g. by competitive braking. Thus, the compounds of the present invention are commercial products sold for these purposes.

[0030] The compounds of the present invention may be administered in the form of pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to a salt that has the efficacy of the parent compound and which is not biologically or otherwise undesirable (e.g., neither toxic nor otherwise harmful to the recipient). Suitable salts include acid addition salts, which can, for example, be formed by mixing a solution of the compound of the present invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, acetic acid, trifluoroacetic acid or benzoic acid. Many compounds of the invention contain an acidic moiety, in which case their corresponding pharmaceutically acceptable salts may contain alkali metal salts (e.g. sodium or potassium salts), alkaline earth metal salts (e.g. calcium or magnesium salts) and salts formed with suitable organic ligands such as quaternary ammonium salts. Also in the presence of an acid (-COOH) or alcohol group, pharmaceutically acceptable esters can be used to change the solubility or hydrolytic properties of the compound.

[0031] The term "administration" and variants thereof (e.g., "administering" a compound) in relation to a compound of the invention means providing a compound or a prodrug of the compound to a subject in need of treatment. If a compound of the invention or a prodrug thereof is provided in combination with one or more active agents (e.g. antiviral agents useful for treating HCV infection), it is contemplated that "administration" and variations thereof, each also includes simultaneous and sequential delivery of a compound or salt (or hydrate) and other agents.

[0032] As used herein, the term "composition" is intended to include a product containing the specified ingredients as well as any product that results, directly or indirectly, from a combination of the specified ingredients.

[0033] "Pharmaceutically acceptable" means that the ingredients of the pharmaceutical composition must be compatible with each other (compatible) and not harmful to the recipient.

[0034] The term "subject" (alternatively, herein referred to as "patient") as used herein refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.

[0035] The term "effective amount" as used herein means that the amount of active ingredient or pharmaceutical agent that elicits the biological or therapeutic response of a tissue, system, animal or human that is sought by a scientist, veterinarian, physician or other clinician . In one embodiment, the effective amount is a "therapeutically effective amount" to alleviate the symptoms of the disease or condition being treated. In another embodiment, the effective amount is a "therapeutically effective amount" for preventing symptoms of the disease or condition being prevented. The term also includes the amount of active substance sufficient to inhibit HCV NS3 protease and thereby elicit the response sought (i.e., "effective inhibition amount"). If the amount of active ingredient (i.e. the active ingredient) is administered in the form of a salt, references to the amount of active ingredient refer to the free acid or free base of the compound.

[0036] To inhibit HCV NS3 protease and prevent or treat HCV infection, the compounds of the present invention, optionally in salt or hydrate form, can be administered by any means that provides contact of the active agent with the site of action of the active agent. They can be administered by any conventional means available for use in combination with pharmaceuticals, either as single therapeutic agents or in combination with therapeutic agents. They can be administered alone, but are usually administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice. The compounds of the invention may, for example, be administered orally, parenterally (including subcutaneous, intravenous, intramuscular, intra-sternal or infusion injection), by inhalation of an aerosol or rectally, in a dosage unit form of a pharmaceutical composition containing an effective amount of the compound and conventional non-pharmaceutically toxic acceptable carriers, adjuvants and substrates. Liquid preparations suitable for oral administration (e.g. suspensions, syrups, elixirs and the like) may be prepared according to methods known in the art and may use any conventional medium such as water, glycols, oils, alcohols and the like. Solid preparations suitable for oral administration (e.g. powders, tablets, capsules and tablets) may be prepared according to methods known in the art and may use such solid excipients as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like. Parenteral compositions can be prepared according to methods known in the art and usually use sterile water as a carrier and optionally other ingredients such as solubilizing agents. Injectable solutions may be prepared according to methods known in the art in which the carrier comprises saline solution, glucose solution or solution containing a mixture of saline and glucose. A further description of methods suitable for use in preparing the pharmaceutical compositions of the present invention and ingredients suitable for use in said compositions is found in Remington's Pharmaceutical Sciences, 18th edition, issued by AR Gennaro, Mack Publishing Co., 1990.

[0037] The compounds of the present invention may be administered orally in a dose range of 0.001 to 1000 mg / kg body weight of a mammal (e.g. human) per day in a single dose or in divided doses. One preferred dose range is 0.01 to 500 mg / kg body weight per day orally in single or divided doses. Another preferred dose range is 0.1 to 100 mg / kg body weight per day orally in one or two divided doses. For oral administration, the compositions may be provided in the form of tablets or capsules containing from 1.0 to 500 milligrams of active ingredient, in particular: 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250 , 300, 400 and 500 mg of active substance for symptomatic dosage adjustment for the patient being treated. The specific dose level and frequency of dosing for a particular patient can be varied and depends on many factors, including the effect of the specific compound used, metabolic stability and duration of action of this compound, age, weight, general health, sex, diet, method and time of administration, the rate of excretion, the combination of drugs, the stage of the particular disease state and the host being treated.

[0038] As mentioned above, the present invention also relates to a method of inhibiting HCV NS3 protease, inhibiting HCV replication or preventing or treating HCV infection with a compound of the present invention in combination with one or more therapeutic agents and a pharmaceutical composition comprising a compound of the present invention and one or more therapeutic agents selected from the group consisting of HCV antiviral agent, an immunomodulating agent and an anti-infective agent. Such anti-HCV active therapeutic agents include, but are not limited to, ribavirin, levovirin, viramidine, thymosine alpha-1, R7025 (enhanced interferon (Roche)), interferon-β, interferon-α, pegylated interferon-α (peginterferon-α), combination of interferon-α and ribavirin, combination of peginterferon-α and ribavirin, combination of interferon-α and levovirin, and combination of peginterferon-α and levovirin. Interferon-α includes, without limitation, recombinant interferon-uta (such as interferon Roferon available from Hoffmann-LaRoche, Nutley, NJ) pegylated interferon-α2a (Pegasys ™) interferon-a2b (such as Intron A-interferon available from Schering Corp. , Kenilworth, NJ) pegylated interferon-a2b (PegIntron ™) 'recombinant interferon consensus (e.g., alpha-1 interferon), albuferon (interferon-α bound to human serum albumin (Human Genome Sciences)), and purified interferon-α product. Amgen's recombinant interferon consensus bears the trade name Infergen®. Levovirin is the L-enantiomer of ribavirin, which showed immunomodulatory activity similar to ribavirin. Viramidine is a ribavirin analog disclosed in WO 01/60379 (provided to ICN Pharmaceuticals). According to the method of the present invention, the individual components of the combination may be administered separately at different times during the application of the treatment or simultaneously in divided or single combination forms.

[0039] For the treatment of HCV infection, the compounds of the present invention may also be administered in combination with an agent that is an HCV NS3 serine protease inhibitor. HCV NS3 Serine Protease is an important viral enzyme and has been described as an excellent target for inhibiting HCV replication. Both substrate and non-substrate based inhibitors, HCV NS3 protease inhibitors are disclosed in WO 98/22496, WO 98/46630, WO 99/07733, WO 99/07734, WO 99/38888, WO 99/50230, WO 99 / 64442, WO 00/09543, WO 00/59929, GB-2337262, WO 02/48116, WO 02/48172, and U.S. Patent No. 6,323,180.

[0040] Ribavirin, levovirin and viramidine may exert anti-HCV effects by modulating intracellular guanine nucleotide sets by inhibiting intracellular inosine monophosphate dehydrogenase (IMPDH) enzyme. IMPDH is a speed limiting enzyme in the biosynthetic pathway in de novo biosynthesis of guanine nucleotides. Ribavirin is easily intracellularly phosphorylated and the monophosphate derivative is an IMPDH inhibitor. Thus, inhibition of IMPDH is another useful target for the discovery of inhibitors of HCV replication. Therefore, the compounds of the present invention can also be administered in combination with an IMPDH inhibitor such as VX-497, which is disclosed in WO 97/41211 and WO 01/00622 (granted to Vertex); another IMPDH inhibitor such as that disclosed in WO 00/25780 (to BristolMyers Squibb); or mycophenolate mofetil [see AC Allison and EM Eugui, Agents Action, 44 (Suppl.): 165 (1993)].]].

[0041] For the treatment of HCV infection, the compounds of the present invention may also be administered in combination with the antiviral agent amantadine (1aminoadamantane) [for a full description of this agent, see J. Kirschbaum, Anal. Profiles Drug Subs. 12: 1-36 (1983)].)].

[0042] For the treatment of HCV infection, the compounds of the present invention may also be administered in combination with an antiviral agent polymerase R7128 (Roche).

[0043] The compounds of the present invention may also be combined for the treatment of HCV infection with the 2'-C-branched ribonucleoside antiviral disclosed in RE Harry-O'kuru, et al., J. Org. Chem., 62: 1754-1759 (1997); MS Wolfe, et al., Tetrahedron Lett., 36: 7611-7614 (1995); U.S. Patent No. 3,480, 613 (November 25, 1969); International Patent Application No. WO 01/90121 (November 29, 2001); International Patent Application No. WO 01/92282 (December 6, 2001); and International Patent Application No. WO 02/32920 (April 25, 2002); and International Patent Application No. WO 04/002999 (January 8, 2004); and International Patent Application No. WO 04/003000 (January 8, 2004); and International Patent Application No. WO 04/002422 (January 8, 2004). Such 2'-branched ribonucleosides include, without limitation, 2'-C-methyl-cytidine, 2'-C-methyl-uridine, 2'-C-methyl-adenosine, 2'-C-methyl-guanosine and 9- (2- C-methyl-e-Dribofuranosyl) -2,6-diaminopurine and the corresponding C-2 ', C-3', and C-5 'hydroxy amino acid ester of hydroxyl and the corresponding optionally substituted cyclic 1,3-propandiol esters of 5'- derivatives phosphate.

[0044] The compounds of the present invention may also be combined for the treatment of HCV infection with other nucleosides having anti-HCV properties such as disclosed in WO 02/51425 (July 4, 2002), provided to Mitsubishi Pharma Corp .; WO

01/79246, WO 02/32920, WO 02/48165 (June 20, 2002), and WO2005003147 (January 13

2005) (including R1656, (2'R) -2'-deoxy-2'-fluoro-2'-C-methylcytidine, shown as compounds 3-6 On page 77) provided by Pharmasset, Ltd .; WO 01/68663 (September 20, 2001), issued to ICN Pharmaceuticals; WO 99/43691 (September 2, 1999); WO 02/18404 (March 7, 2002), US2005 / 0038240 (February 17, 2005) and WO2006021341 (March 2, 2006), including 4'-azido nucleosides such as R1626, 4'-azidocytidine, given to HoffmannLaRoche; US 2002/0019363 (February 14, 2002); WO 02/100415 (December 19, 2002); WO 03/026589 (April 3, 2003); WO 03/026675 (April 3, 2003); WO 03/093290 (November 13, 2003); US 2003/0236216 (December 25, 2003); US 2004/0006007 (8 January 2004); WO 04/011478 (February 5, 2004); WO 04/013300 (February 12, 2004); US 2004/0063658 (1 April 2004); and WO 04/028481 (April 8, 2004).

[0045] For the treatment of HCV infection, the compounds of the present invention may also be administered in combination with an agent that is an HCV NS5B polymerase inhibitor. Such HCV NS5B polymerase inhibitors that can be used as combination therapy include, but are not limited to, those disclosed in WO 02/057287, US 6777395, WO

02/057425, US 2004/0067901, WO 03/068244, WO 2004/000858, WO 04/003138 and WO

2004/007512. Other such HCV polymerase inhibitors include, but are not limited to, valopicitabine (NM-283; Idenix) and 2'-F-2'-beta-methylcytidine (see also WO 2005/003147, issued to Pharmasset, Ltd.).

[0046] In one embodiment, the HCV NS5B NS5B polymerase inhibitors that are used in combination with the present HCV NS3 protease inhibitors are selected from the following compounds: 4-amino-7- (2-C-methyl-eD-arabinofuranosyl) -7H pyrrolo [2,3-d] pyrimidine; 4-amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4-methylamino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4-dimethylamino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4cyklopropylamino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 418 amino-7- (2-C-vinyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4-amino-7- (2-Chydroksymetylo-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4-amino-7- (2-Cfluorometylo-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4-amino-5-methyl-7- (2-Cmetylo-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4-amino-7- (2-C-methyl-e-Dribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine-5-carboxylic acid; 4-amino-5-bromo-7- (2-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4-amino-5-chloro-7- (2-Cmetylo-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4-amino-5-fluoro-7- (2-C-methyl-D-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 2,4-diamino-7- (2-C-methyl-e-D-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 2-amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 2-amino-4-cyclopropylamino-7- (2-C-methyl-e-D-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 2-amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidin-4 (3H) -one; 4-amino-7- (2-C-ethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4-amino-7- (2-C, 2-O-dimethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidin-4 (3H) -one; 2 amino-5-metylo-7- (2-C, 2-O-dimethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidin-4 (3H) -one; 4-amino-7- (3-deoxy-2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4-amino-7- (3-deoxy-2-C-methyl-ED-arabinofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4-amino-2-fluoro-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4-amino-7- (3-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4 amino-7- (3-C-methyl-eD-xylofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4-amino-7- (2,4-di-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; 4-amino-7- (3-deoxy-3-fluoro-2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine; and the corresponding 5'-triphosphates; or a suitable pharmaceutically acceptable salt thereof.

[0047] The compounds of the present invention may also be combined for the treatment of HCV infection with non-nucleoside HCV polymerase inhibitors such as disclosed in WO 01/77091 (October 18, 2001), issued to Tularik, Inc .; WO 01/47883 (July 5, 2001), issued to Japan Tobacco, Inc .; WO 02/04425 (17 January 2002), issued to Boehringer Ingelheim; WO 02/06246 (January 24, 2002), granted to Istituto di Ricerche di Biologia Moleculare P. Angeletti SPA; WO 02/20497 (March 3, 2002); WO 2005/016927 (in particular JTK003), issued to Japan Tobacco, Inc .; and HCV-796 (Viropharma Inc.).

[0048] In one embodiment, the non-nucleoside HCV NS5B polymerase inhibitors that are used in combination with the present HCV NS3 protease inhibitors are selected from the following compounds: 14-cyclohexyl-6- [2- (dimethylamino) ethyl] -7- oxo-5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocine-11-carboxylic acid; 14-cyclohexyl-6- (2-morpholin-4-ylethyl) -5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocine-11-carboxylic acid; 14-cyclohexyl-6- [2- (dimethylamino) ethyl] -3-methoxy-5,6,7,8-tetrahydroindolo [2,1a] [2,5] benzodiazocine-11-carboxylic acid; 14-cyclohexyl-3-methoxy-6-methyl5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazoeine-11-carboxylic acid; methyl ({[(14-cyclohexyl-3-methoxy-6-methyl-5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocin-11ylo) carbonyl] amino} sulfonyl) acetate; ({[(14-cyclohexyl-3-methoxy-6-methyl-5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocin-11-yl) carbonyl] amino} sulfonyl) acetic acid; 14-cyclohexyl-N - [(dimethylamino) sulfonyl] -3-methoxy-6-methyl-5,6,7,819 tetrahydroindolo [2,1-a] [2,5] benzodiazocine-11-carboxamide; 3-chloro-14-cyclohexyl-6- [2- (dimethylamino) ethyl] -7-oxo-5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocine-11-carboxylic acid; N '- (11-carboxy-14-cyclohexyl-7,8-dihydro-6H-indolo [1,2e] [1,5] benzoxazocin-7-yl) -N, N-dimethylethane-1,2-diamino bis (trifluoroacetate); 14-cyclohexyl-7,8-dihydro-6H-indolo [1,2-e] [1,5] benzoxazocine-11-carboxylic acid; 14-cyclohexyl-6-methyl-7-oxo-5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocine-11-carboxylic acid; 14-cyclohexyl-3-methoxy-6-methyl-7-oxo-5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocine-11-carboxylic acid; 14-cyclohexyl-6- [2- (dimethylamino) ethyl] -3-methoxy-7-oxo-5,6,7,8-tetrahydroindolo [2,1a] [2,5] benzodiazocine-11-carboxylic acid; 14-cyclohexyl-6- [3- (dimethylamino) propyl] -7-oxo-5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocine-11-carboxylic acid; 14-cyclohexyl-7-oxo-6- (2-piperidin-1-ylethyl) -5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocine-11-carboxylic acid; 14-cyclohexyl-6- (2-morpholin-4-ylyl) -7-oxo-5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocine-11-carboxylic acid; 14-cyclohexyl-6- [2- (diethylamino) ethyl] -7-oxo-5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocine-11-carboxylic acid; 14-cyclohexyl-6- (1-methylpiperidin-4-yl) -7-oxo-5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocine-11-carboxylic acid; 14-cyclohexyl-N - [(dimethylamino) sulfonyl] -7-oxo-6- (2-piperidin-1-ylethyl) -5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocine 11-carboxamide; 14-cyclohexyl-6- [2- (dimethylamino) ethyl] -N - [(dimethylamino) sulfonyl] -7-oxo-5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocine-11-carboxamide ; 14-cyclopentyl-6- [2- (dimethylamino) ethyl] -7-oxo-5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocine-11-carboxylic acid; 14-cyclohexyl-5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocine 11-carboxylic acid; 6-allyl-14-cyclohexyl-3-methoxy-5,6,7,8-tetrahydroindolo [2,1a] [2,5] benzodiazocine-11-carboxylic acid; 14-cyclopentyl-6- [2- (dimethylamino) ethyl] 5,6,7,8-tetrahydroindolo [2,1-a] [2,5] benzodiazocine-11-carboxylic acid; 14-cyclohexyl-6- [2- (dimethylamino) ethyl] -5,6,7,8-tetrahydroindolo [2,1a] [2,5] benzodiazocine-11-carboxylic acid; 13-cyclohexyl-5-methyl-4,5,6,7-tetrahydrofuro [3 ', 2': 6.7] [1,4] diazocino [1,8-a] indole-10-carboxylic acid; Cyclohexyl-6- [2- (dimethylamino) ethyl] -7-oxo-6,7,8,9-tetrahydro-5H-indolo [2,1a] [2,6] benzodiazonine-12-carboxylic acid; 15-cyclohexyl-8-oxo-6,7,8,9-tetrahydro5H-indolo [2,1-a] [2,5] benzodiazonine-12-carboxylic acid; 13-cyclohexyl-6-oxo-6,7-dihydro-5H-indolo [1,2-d] [1,4] benzodiazepine-10-carboxylic acid; and their pharmaceutically acceptable salts.

[0049] The above indole-based tetracyclic HCV NS5B polymerase inhibitors can be obtained according to the following AE methods, as shown below, in which various variables can be selected for the specific tetracyclic indole compound to be obtained:

Method A [0050]

<img file="PL1924593T3_D0008.tif" />

The intermediate 2-bromoindole (prepared as described in published international patent application WO2004087714) was functionalized on indole nitrogen by introducing precursor functionality of W '/ X' into one or both elements of the W / X chain. The Pd-mediated coupling methodology (e.g. Suzuki, Stille, etc.) then introduced C2 aromatic, containing precursor Z '/ Y' functionality into one or both of the Z / Y connection elements. A functional group treatment followed by ring closure provided the tetracyclic system. Deprotection of the ester then provided target indole carboxylic acids, with a C2 aromatic compound attached to the indole nitrogen.

Method B [0051]

<img file="PL1924593T3_D0009.tif" />

After attachment of the chain to form the corresponding 2-haloaromatic system, Pd-mediated ring closure provided a fused tetracyclic system. Deprotection of the ester then provided target indole carboxylic acids, with a C2 aromatic chain attached to indole nitrogen.

Method C [0052]

<img file="PL1924593T3_D0010.tif" />

The aromatic C2 system was first introduced using Pd-mediated coupling methodology (Suzuki, Stille, etc.). Then the chain was added, with cyclization to indole nitrogen, finally closing the ring. Deprotection of the ester then provided target indole carboxylic acids, with a C2 aromatic chain attached to indole nitrogen.

Method D [0053]

<img file="PL1924593T3_D0011.tif" />

Condensed tetracyclic intermediate products obtained by AC methods undergo treatment on functional groups in the chain before deprotection of the ester to provide target C2-chain indole carboxylic acids.

Method E

<img file="PL1924593T3_D0012.tif" />

The indole C2-chain carboxylic acids obtained by AD methods were then derivatized by treatment with carboxylate functionality to obtain compounds from the carboxylate or carboxamide substitution. When carrying out the above synthetic sequences, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules concerned. This can be achieved by using methods of commonly used group protection such as those described in

Protective Groups in Organic Chemistry, ed. JFW McOmie, Plenum Press, 1973; and TW Greene &amp; PGM Wuts, Protective Groups in Organic Synthesis, John Wiley &amp; Sons, 3rd Edition, 1999. Protecting groups can be removed in a convenient next step using methods known in the art.

[0055] The inhibitory activity of HCV NS3 protease of the present compounds can be tested using assays known in the art. One such test is the "time-resolved fluorescence" (TRF) HCV NS3 protease fluorescence test, as described in Example 56. Other examples of such tests are described in, e.g., international patent application

WO2005 / 046712. Compounds useful as HCV NS3 protease inhibitors have Ki less than 50 μΜ, more preferably less than 10 μΜ, and even more preferably less than 100 nM.

[0056] The present invention also includes a process for preparing compounds of formula I, II-a, or III-a. Compounds of the present invention can be readily prepared according to the following reaction schemes and examples, or modifications thereof, using readily available starting materials, reagents and common synthetic procedures. In these reactions, it is also possible to use variants that are known to those skilled in the art, but are not mentioned in detail. In addition, other methods for preparing compounds of the invention will be apparent to those skilled in the art in light of the following reaction schemes and examples. Unless otherwise indicated, all variables are as defined above. The following reaction schemes and examples are only intended to illustrate the invention and practice. Examples should not be construed as limiting the scope or spirit of the invention.

General description of the synthesis:

[0057] The compounds of the present invention can be synthesized as outlined in General Schemes 1 and 2.

Diagram 1

<img file="PL1924593T3_D0013.tif" />

[0058] Scheme 1 (n = 0-9) shows the synthesis of a representative molecule. An appropriately protected 4-hydroxyproline derivative (e.g., carbamated protected nitrogen and ester protected acid can be reacted with carbonyl diimidazole or an equivalent reagent and then reacted with the correspondingly substituted isoindoline or tetrahydroisoquinoline. Alkenyl functionality can be introduced at this or a later stage by palladium catalysed reaction of a halogen substituent such as chloride, bromide and iodide, or other functionality such as tryflate, with an organometallic reagent such as vinyl- or allyl-trialkyltin. Alternatively, alkenyl functionality can be introduced prior to reaction with protected prolinol.

[0059] Scheme 2 shows the synthesis of olefin containing the amino acid portion. An amino acid (either commercially available or readily obtained using known methods in the art) in which the acid functionality is protected in the form of an ester (for example, R = methyl) can be converted to amides A by olefinic carboxylic acid coupling using a wide range of agents coupling agents used in peptides known to those skilled in the art, such as DCC, EDC, BOP, TBTU, etc. The preparation of sulfonamides B can be accomplished by reaction with the appropriate sulfonyl chloride in an organic solvent (e.g. THF) with an amine base as a scavenger. Urea derivatives C can be obtained by reacting an aminoester with a reagent such as carbonyl diimidazole to form the isocyanate intermediate (Catalano et al., WO 03/062192) followed by the addition of a second amine containing olefin. Alternatively, phosgene, diphosgene or triphosgene may be used instead of carbonyl diimidazole. Cyanoguanidine D derivatives can be obtained by reacting an amino acid ester with diphenyl C-cyanocarbonimidate in an organic solvent followed by the addition of a second olefin-containing amine. Carbamate E derivatives can be obtained by reacting an olefin containing alcohol with carbonyldiimidazole (or phosgene, triphosgene or diphosgene) in an organic solvent followed by addition of an aminoester.

Diagram 2

<img file="PL1924593T3_D0014.tif" />

[0060] After functionalizing the amine, the ester can be hydrolyzed under various basic conditions known to those skilled in the art (Theodora W. Greene, Protective Groups in Organic Synthesis, 3rd edition, John Wiley and Sons, 1999).

[0061] Deprotection of the carbamate protecting group in the proline portion can be accomplished using a variety of methods known to those skilled in the art (Theodora W. Greene, Protective Groups in Organic Synthesis, Third Edition, John Wiley and Sons, 1999).

[0062] To complete the synthesis of compounds of the present invention, the amino acid derivative may be conjugated to a proline derivative using a wide range of coupling agents used in peptides known to those skilled in the art, such as DCC, EDC, BOP, TBTU, etc. ( see Diagram 1). The macrocyclization is then carried out by olefin metathesis using a series of catalysts, described in the literature for this purpose. At this stage, the olefinic bond formed in the ring-closing metathesis can optionally be hydrogenated to give a saturated or functionalized bond, such as cyclopropanation. The proline ester is then hydrolyzed under basic conditions and coupled with a cyclopropylaminoacid ester (the corresponding alkenyl or alkylcyclopropane portion of the molecule can be obtained as previously described (LlinasBrunet et al., US 6,323,180) and subjected to an additional basic hydrolysis step, providing final compounds. The proline ester can also be hydrolyzed and directly coupled to the appropriately functionalized acyl cyclopropylamino acid sulfonamide (which can be prepared according to Wang XA et al., WO2003 / 099274) to provide final compounds.

[0063] Olefin metathesis catalysts include the following ruthenium based compounds: F: Miller et al., J. Am. Chem. Soc 1996, 118, 9606; G: Kingsbury et al. J. Am. Chem. Soc 1999, 121, 791; H: Scholl et al., Org. Lett. 1999, 1, 953; Hoveyda et al. US2002 / 0107138; K: Furstner and colleagues,

J. Org. Chem 1999, 64, 8275. The usefulness of these catalysts in ring-closing metathesis is well known in the literature (e.g. Trnka and Grubbs, Acc. Chem. Res. 2001, 34, 18).

<img file="PL1924593T3_D0015.tif" />

List of abbreviations [0064]

BOP Benzotriazol-1-yl-oxy-tris- (dimethylamino) phosphonic DCC hexafluorophosphate Dicyclohexylcarbodiimide CH3CN Acetonitrile

DBU 1,8-Diazabicyclo [5.4.0] undec-7-ene

<td>DCE</td><td>dichloroethane</td><td>DCM</td><td>dichloromethane</td>

<td>DMAP</td><td>4-Dimethylaminopyridine</td><td>DIPEA</td><td>diisopropylethylamine</td>

<td>DMF</td><td>dimethylformamide</td><td>DMSO</td><td>dimethyl sulfoxide</td>

<td>EDC</td><td colspan="3">N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide</td>

<td>Et 3 N</td><td>triethylamine</td><td>Et 2 O</td><td>Diethyl ether</td>

<td>EtOC</td><td>Ethyl acetate</td><td>EtOH</td><td>Ethanol</td>

<td>HATU</td><td colspan="3">O- (7-Azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate</td>

<td>HBr</td><td>Hydrobromic acid</td><td></td><td></td>

<td>HCI</td><td>Hydrochloric acid</td><td>HOAc</td><td>Acetic acid</td>

<td>HOAt</td><td>1-Hydroxy-7-azabenzotriazole</td><td>LiOH</td><td>Lithium hydroxide</td>

<td>MeOH</td><td>methanol</td><td>MgSO4</td><td>Magnesium sulfate</td>

<td>NaHCO 3</td><td>Hydrogen sodium carbonate</td><td>Na2SO4</td><td>Sodium sulfate</td>

<td>NaOH</td><td>Sodium hydroxide</td><td>NH 4 Cl</td><td>Ammonium chloride</td>

<td>NH4OH</td><td>Ammonium hydroxide</td><td>Pd / C</td><td>Palladium on coal</td>

Pd (PPh3) 4 tetrakis (triphenylphosphine) palladium (0)

<td>PhMe</td><td>Toluene</td><td>PPh3</td><td>triphenylphosphine</td>

<td>RT</td><td>room temperature</td><td>THF</td><td>Tetrahydofuran</td>

TBTU O-Benzotriazol-1-yl-N, N, N ', N'-tetramethyluronium tetrafluoroborate

EXAMPLE 1 (5R, 7S, 10S) -10-Butyl-N - ((1R, 2S) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropyl) -3,9,12-trioxo-1,6, 7,9,10,11,12,14,15,16-decahydro-5H-2.22: 5.8-dimethane-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide (III-1) [0065 ]

<img file="PL1924593T3_D0016.tif" />

Step 1: 4-Chloroisisoindoline [0066] α α

[0067] A mixture of 3-chlorophthalic acid anhydride (9 g, 49.2 mmol) and formamide (100 ml) was heated to 125 ° C and stirred for 3 hours. Then water (300 ml) was added and the mixture was cooled to room temperature. The mixture was filtered and the resulting white solid washed with water and dried to give 4-chloro-1H-isoindole-1,3 (2H) -dione (7.7 g, 86% yield).

[0068] To the solid 4-chloro-1H-isoindole-1,3 (2H) -dione (4.0 g, 22.0 mmol), borane-THF complex (1M / THF, 88.1 mL, 88 , 1 mmol). After the addition was complete, the reaction mixture was heated to reflux (80 ° C) and stirred for 6 hours. The reaction mixture was then cooled to 0 ° C, methanol (2.8 mL, 88.1mmol) was carefully added dropwise and the reaction mixture was warmed to room temperature. HCl (6N) was added until the mixture acidified and then the mixture was concentrated. The crude product was dissolved in 1M HCl and extracted twice with ethyl ether and twice with dichloromethane. The aqueous layer was adjusted to pH = 11 using solid NaOH and extracted three times with ethyl acetate. The combined ethyl acetate extracts were dried over Na2SO4, filtered and concentrated to give 4-chloroisisoindoline (1.8 g, 53% yield). LRMS (ESI) m / z 154 [(M + H)⁺; calculated for C8H9ClN: 154].

Step 2: 1-tert-Butyl 2-methyl (2S, 4R) -4 - {[(4-chloro-1,3-dihydro-2H-isoindol-2-yl) carbonyl] oxy} pyrrolidine-1,2-dicarboxylate [ 0069]

<img file="PL1924593T3_D0017.tif" />

[0070] To a solution of N-Boc proline methyl ester (2.87 g, 11.7 mmol) in DMF (15 mL) at 0 ° C was added carbonyldiimidazole (1.9 g, 11.7 mmol). The reaction was warmed to room temperature and stirred for 30 minutes. Then a solution of 4-chloroisisoindoline (1.8 g, 11.7 mmol) in DMF (10 ml) was added and the reaction mixture was heated to 50 ° C and stirred for 2 hours. The reaction mixture was poured into ethyl ether and 0.5 M HCl and the layers were separated. The organic layer was washed with water, dried over Na2SO4, filtered and concentrated. The crude product was purified on silica gel (elution gradient 10% to 90% ethyl acetate in hexanes) to give 1-tert-butyl 2-methyl (2S, 4R) -4 - {[(4-chloro-1,3-dihydro-2H-isoindole -2-yl) carbonyl] oxy} pyrrolidine-1,2-dicarboxylate (3.3 g, 66% yield). LRMS (ESI) m / z 325 [(M + H-Boc)⁺; calculated for C15H18ClN2O4: 325].

Step 3: 1-tert-Butyl 2-methyl (2S, 4R) -4 - {[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl) carbonyl] oxy} pyrrolidine-1,2-dicarboxylate [ 0071]

<img file="PL1924593T3_D0018.tif" />

[0072] 1-tert-butyl 2-methyl (2S, 4R) -4 - {[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl) carbonyl] oxy} pyrrolidine-1,2-dicarboxylate solution (40 mg, 0.09 mmol), vinyl tributyltinate (36 mg, 0.11 mmol) and cesium fluoride (31 mg, 0.21 mmol) in dioxane (0.5 mL) was degassed with N2 for 15 minutes. Then bis (tributylphosphine) palladium (0) (2 mg, 0.005 mmol) was added and the reaction vessel was sealed and heated to 100 ° C for 18 hours. After cooling, the reaction mixture was concentrated and purified by silica gel chromatography (10% to 90% ethyl acetate in hexanes) to give 1-tert-butyl 2-methyl- (2S, 4R) -4 - {[(4-vinyl-1,3 dihydro-2H-isoindol-2-yl) carbonyl] oxy} pyrrolidine-1,2-dicarboxylate (10 mg, 25% yield). LRMS (ESI) m / z 317 [(M + H-Boc)⁺; calculated for C17H21N2O4: 317].

Step 4: Methyl N - [(pent-4-enyloxy) carbonyl] -L-norleucyl- (4R) - {[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl) carbonyl] oxy} -L -prolinate [0073]

<img file="PL1924593T3_D0019.tif" />

[0074] For a flask containing 1-tert-butyl 2-methyl- (2S, 4R) -4 - {[(4-vinyl-1,3-dihydro2H-isoindol-2-yl) carbonyl] oxy} pyrrolidine-1, 2-dicarboxylate (60 mg, 0.14 mmol) was added a 4M solution of HCl in dioxane (2 mL). After 1 hour, LC-MS analysis showed complete consumption of starting material and formation of the desired Boc product. Volatiles were then removed in vacuo, and the crude product was taken into DMF (2 mL).

[0075] To this mixture was added N - [(pent-4-en-1-yloxy) carbonyl] -L-norleucine (41 mg, 0.17 mmol) (obtained according to the procedure below), DIPEA (0.076 ml, 0, 43 mmol), EDC (54 mg, 0.28 mmol) and HOAt (44 mg, 0.28 mmol). Stirred at room temperature for 30 minutes, total amine consumption was demonstrated using LC-MS. The reaction mixture was then worked up with 0.5N HCl and EtOAc. The organic layer was washed with brine and dried over MgSO4. The solvent was then removed in vacuo and the crude product was purified on silica gel (10-90% EtOAc / hexanes) to provide 60 mg (79% yield) of methyl N - [(pent-4-enyloxy) carbonyl] -L-norleucyl- (4R) -4 - {[(4-winylo1,3-dihydro-2H-isoindol-2-yl) carbonyl] oxy} -L-prolinate. LRMS (ESI) m / z 542 [(M + H)⁺; calculated for C29H40N3O7: 542].

Step 5: Methyl (5R, 7S, 10S) -10-Butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16.16 decahydro-5H-2.22: 5,8-dimethane-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate [0076]

<img file="PL1924593T3_D0020.tif" />

[0077] Methyl N - [(pent-4-enyloxy) carbonyl] -L-norleucyl- (4R) -4 - {[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl) carbonyl] oxy solution } -L-proline (60 mg, 0.11 mmol) in DCE (20 ml) was degassed with N2 for 15 minutes. Zhan ruthenium RC-301 metathesis catalyst (Zhan Catalyst I (described as J on page 43), RC-301, Zannan Pharma Ltd.) (7 mg, 0.01mmol) was then added. The solution was then heated to 100 ° C for 1 hour. After this time, LC-MS and TLC analysis showed complete consumption of starting material and the formation of almost a single product that had the desired mass. The solvent was then removed in vacuo, and the crude product was purified on silica gel (5-70% EtOAc / hexane) to provide 45 mg (79% yield) of methyl (5R, 7S, 10S) -10-butyl-3,9,12-trioxo1 , 6,7,9,10,11,12,14,15,16-decahydro-5H-2,22: 5,8-dimethano-4,13,2,8,11benzodioksatriazacykloicosine-7-carboxylate. LRMS (ESI) m / z 514 [(M + H)⁺; calculated for C27H36N3O7: 514].

Stage 6: (5R, 7S, 10S) -10-Butyl-N - ((1R, 2S) -1 - {[(cyklopropylsulfonylo) amino] carbonyl} -2winylocyklopropylo) -3,9,12-trioxo-1,6,7, 9,10,11,12,14,15,16-decahydro-5H-2.22: 5.8-dimethane-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide [0078] To the methyl solution (5R, 7S, 10S) -10-butyl-3,9,12-triokso1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22: 5,8-dimethano-4 , 13,2,8,11 benzodioxatriazacycloicosine-7-carboxylate (45 mg, 0.09 mmol) in THF (2 mL), MeOH (0.5 mL) and water (1 mL) LiOH (21 mg, 0 mg) , 87 mmol). The reaction mixture was heated to 40 ° C and stirred for 1 hour, during which time the total consumption of the starting methyl ester was observed using LC-MS. The mixture was then worked up with 0.5N HCl and EtOAc. The organic layer was then dried over K2CO3, and the solvent was removed in vacuo. The crude product was taken in DMF (1 mL).

[0079] To the above solution was added (1R, 2S) -1 {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropananoate chloride (Llinas-Brunet et al. US03 / 15755 and Wang et al., WO 03/099274) (32 mg , 0.12 mmol), TBTU (51 mg, 0.16 mmol) and DIPEA (0.071 mL, 0.40 mmol) and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was directly purified by reverse phase HPLC to give (5R, 7S, 10S) 10-butyl-N - ((1R, 2S) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropyl) -3,9, 12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22: 5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7 carboxamide (27 mg, 47% yield). ¹H NMR (500 MHz, ppm, CDCl3) δ 10.01 (s, 1H), 7.27 (m, 2H), 7.12 (d, 1H), 7.04 (s, 1H) , 6.40 (d, J = 16.1 Hz, 1H), 6.08 (m, 1H), 5.76 (m, 1H), 5.44 (s, 1H), 5, 36 (d, 1H), 5.25 (d, 1H), 5.14 (d, 1H), 4.80-4.68 (m, 3H), 4.59 (d, 1H) ), 4.44 (m, 2H), 4.38 (m, 1H), 4.28 (m, 1H), 3.95 (m, 1H), 3.77 (dd, 1H ), 2.94 (m, 1H), 2.43 (m, 2H), 2.29 (d, 2H), 2.06 (m, 2H), 1.94 (m, 1H) ), 1.78 (m, 4H), 1.45 (m, 1H), 1.38-1.06 (m, 5H), 1.04 (d, 2H), 0.92 ( t, 3H) ppm. LRMS (ESI) m / z 712 [(M + H)⁺; calculated for C35H46N5O9S: 712].

Example 2 (5R, 7S10S) -10-tert-Butyl-N - ((1R, 2S) -1 - {[(cyclopropylsulfonyl) aminolcarbonyl} -2-vinylcyclopropyl) -3,9,12-trioxo-1,6,7, 9,10,11,12,14,15,16-decahydro-5H-2.22: 5-Dimethane-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide (II-2) [0080]

<img file="PL1924593T3_D0021.tif" />

[0081] Example 2 was prepared according to the procedure used for Example 1 except that 3-methyl-N - [(pent-4-enyloxy) carbonyl] -L-valine (obtained according to the procedure below) was used instead of N - [(pent -4-en-1-yloxy) carbonyl] -L-norleucine in step 4. ¹H NMR (500 MHz, ppm, CDCl3) δ 9.90 (s, 1H), 7.28 (m, 2H), 7.13 (m, 2H), 6.31 (d, J = 15 , 9 Hz, 1H), 6.04 (m, 1H), 5.74 (m, 1H), 5.45 (m, 2H), 5.27 (d, 1H), 5, 16 (d, 1H), 4.774.66 (m, 3H), 4.55 (d, 1H), 4.48 (t, 1H), 4.41-4.35 (m, 2H ), 4.27 (m, 1H), 3.93 (m, 1H), 3.74 (dd, 1H), 2.93 (m, 1H), 2.45 (d, 2H ), 2.32 (m, 2H), 2.10-1.95 (m, 2H), 1.74 (m, 1H), 1.47 (m, 1H), 1.37 ( m, 2H), 1.07 (s, 9H) ppm. LRMS (ESI) m / z 712 [(M + H)⁺; calculated for C35H46N5O9S: 712].

Example 3 (5R, 7S, 10S) -10-tert-Butyl-N - ((1R, 2S) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropyl) -15,15-dimethyl-3,9, 12-trioxo-1,6,7,9,10,11,12,14,15,16-dekahydro5H-2,22: 5,8-dimethano-4,13,2,8,11-benzodioxatriazacycloicosine-7 carboxamide (III-8) [0082]

<img file="PL1924593T3_D0022.tif" />

Step 1: 1-Bromo-2 3-bis (bromomethyl) benzene [0083]

<img file="PL1924593T3_D0023.tif" />

[0084] A suspension of 3-bromo-o-xylene (196 g, 1.06 mol), N-bromosuccinimide (377 g, 2.15 mol) and benzoyl peroxide (0.26 g, 1.0 mmol) in carbon tetrachloride (1800 ml) was heated to reflux under nitrogen for 15 hours. The contents of the reaction flask were cooled, filtered and the filtrate evaporated. The crude material was distilled in a high vacuum. The main fractions were distilled between 88 ° C and 152 ° C. 108 g of pure material was recovered. 182 g of slightly crude material were recovered which could be used in the next reaction. ¹H NMR (CDCl3) δ (ppm) 7.56 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.26 (s, 1 H), 7.16 (t, J = 8.0 Hz, 1H), 4.84 (s, 2H), 4.64 (s, 2H).

Step 2: 2-Benzyl-4-bromoisoindoline [0085]

<img file="PL1924593T3_D0024.tif" />

[0086] Potassium bicarbonate (204 g, 2.04 mol) was suspended in acetonitrile (12 L) and the mixture was heated to 80 ° C. Solutions of 1-bromo-2,3-bis (bromomethyl) benzene (280 g, 0.82 mol in 500 ml acetonitrile) and benzylamine (87.5 g, 0.82 mol in 500 ml acetonitrile) were added simultaneously via addition funnel over time. 1 hour. The reaction mixture was stirred at 77 ° C for 16 hours. The contents of the reaction flask were cooled, filtered and the solvent removed by evaporation. The reaction was partitioned between 1M K2CO3 and EtOAc. The organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated. Flash column chromatography (elution gradient: heptane to 10% EtOAc in heptane) provided after evaporation the title compound as a light oil,¹H NMR (CDCl3) δ (ppm) 7.41-7.39 (m, 2H), 7.37-7.34 (m, 2H), 7.32-7.27 (m, 2H) , 7.10-7.03 (m, 2H), 4.02 (s, 2H), 3.97 (s, 2H), 3.91 (s, 2H). LRMS (ESI) m / z 289 [(M + H)⁺; calculated for C15H15BrN: 289].

[0087] Converted into the HCl salt with HCl / MeOH. MTBE was added and the solid filtered off to give 118 g of product as the HCl salt.

Step 3: 2-Benzyl-4-vinylisoindoline [0088]

<img file="PL1924593T3_D0025.tif" />

[0089] A solution of 2-benzyl-4-bromoisoindoline (16.7 g, 58.0 mmol) and tributyl (vinyl) tin (20.3 mL, 69.6 mmol) in toluene (400 mL) was degassed by bubbling nitrogen through solution for 0.25 hours. Tetrakis (triphenylphosphine) palladium (0) (1.30 g, 1.16 mmol) was added and the resulting solution was heated at 100 ° C in an oil bath under nitrogen for 24 hours. The contents of the reaction flask were cooled, evaporated and subjected to flash column chromatography eluting with 95/5 hexane / ethyl acetate to give, after evaporation, the title compound as a light oil which turns pink on standing. LRMS (ESI) m / z 236 [(M + H)⁺; calculated for C17H18N: 236].

Step 4: 4-Vinylisoindoline [0090]

NH [0091] A solution of 2-benzyl-4-vinylisoindoline (58 mmol) in 1,2-dichloroethane (150 mL) was placed in a 1 L round bottom flask under nitrogen. An addition funnel containing a solution of 1-chloroethyl chloroformate (7.5.1 mL, 69.6 mmol) in 1,2-dichloroethane was attached. The reaction flask was cooled in an ice bath and the contents of the dropping funnel were added dropwise over 20 minutes, maintaining an internal reaction temperature &lt;5 ° C. After the addition, the reaction flask was allowed to warm to room temperature, then heated to reflux for 45 minutes. The contents of the reaction flask were cooled to room temperature, then the solvent was removed by evaporation. Methanol (200 ml) was added and the contents of the reaction flask were heated to reflux for 30 minutes. The reaction flask was cooled and the solvent removed by evaporation. Water (200 ml) was added and the resulting mixture was washed with ethyl acetate (2 x 250 ml). The aqueous layer was basified with 2N sodium hydroxide, then extracted with methylene chloride (4 x 250 mL). The combined organic extracts were dried over anhydrous sodium sulfate, filtered and the filtrate evaporated. The residue was subjected to flash column chromatography eluting with methylene chloride / methanol / ammonium hydroxide 97/3 / 0.3 to 95/5 / 0.5. Evaporation of the fractions gave the title compound as a brown oil, 6.00g (41.4 mmol, 71% yield for two steps). LRMS (ESI) m / z 146 [(M + H)⁺; calculated for C10H12N: 146].

Step 5: 1-tert-Butyl 2-methyl (2S, 4R) -4 - {[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl) carbonyl] oxy} pyrrolidine-1,2-dicarboxylate [ 0092]

<img file="PL1924593T3_D0026.tif" />

[0093] A solution of 1-tert-butyl 2-methyl (2S, 4R) -4-hydroxypyrrolidine-1,2-dicarboxylate (10.1 g, 41.4 mmol) in DMF (90 ml) under nitrogen was cooled to 0 ° C. Solid 1,1'-carbonyldiimidazole (6.70 g, 41.4 mmol) was added to the reaction. The contents of the reaction flask were warmed to room temperature and after 2 hours a solution of 4-vinylisoindoline (6.00 g, 41.4 mmol) in DMF (10 mL) was added. The reaction was heated at 60 ° C in an oil bath for 2 hours, then cooled and poured into water and 5% potassium bisulfate. The resulting mixture was extracted with ethyl acetate (4 x 250 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and evaporated. Flash column chromatography eluting with 70/30 hexane / ethyl acetate gave the title compound as a white foam, 13.9 g (33.4 mmol, 81% yield). LRMS (ESI) m / z 417 [(M + H)⁺; calculated for C22H29N2O6: 417].

Step 6: (3R, 5S) -5- (methoxycarbonyl) pyrrolidin-3-yl-4-vinyl-1,3-dihydro-2-Hindoindole-2 H-carboxylate hydrochloride [0094]

<img file="PL1924593T3_D0027.tif" />

[0095] 1-tert-butyl 2-methyl (2S, 4R) -4 - {[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl) carbonyl] oxy} pyrrolidine-1,2-dicarboxylate solution (13.9 g, 33.4 mmol) in ethyl acetate (700 mL) was cooled in an ice bath, saturated with hydrogen chloride gas. The reaction flask was sealed and allowed to warm to room temperature. After 3.5 hours, the solvent was removed by evaporation to give the title compound as a gray solid, 11.2 g, 95% yield).¹H NMR (500 MHz, ppm, CD3OD) δ 7.47-7.45 (m, 1H), 7.32-7.31 (m, 1H), 7.26-7.21 (m, 1 H), 6.79-5.73 (m, 1H), 5.79-5.73 (m, 1H), 5.46 (s, 1H), 5.41-5.38 (m , 1H), 4.80-4.72 (m, 4H), 3.91 (s, 3H), 3.74-3.63 (m, 2H), 2.77-2.71 (m, 1H), 2.51-2.46 (m, 1H). LRMS (ESI) m / z 317 [(M + H)⁺; calculated for C17H21N2O4: 317].

Step 7: Methyl N - {[(2,2-dimethylpent-4-enyl) oxy] carbonyl} -3-methyl-L-valyl- (4R) -4 {[(4-vinyl-1,3-dihydro- 2H-isoindol-2-yl) carbonyl] oxy} -L-prolinate [0096]

<img file="PL1924593T3_D0028.tif" />

[0097] To (3R, 5S) -5- (methoxycarbonyl) pyrrolidin-3-yl-4-vinyl-1,3-dihydro-2H-isoindole-2-carboxylate hydrochloride solution (2.00 g, 5.67 mmol) and N - {[(2,2-dimethylpent-4-yl) oxy] carbonyl} -3-methyl-L-valine (1.54 g, 5.67 mmol) in DMF (100 ml) EDC (1, 41 g, 7.37 mmol), HOBt (1.00 g, 7.37 mmol) and DIPEA (3.16 mL, 22.8 mmol). The reaction mixture was stirred at room temperature for 18 hours and then diluted with ethyl acetate and aqueous NaHCO3. The layers were separated and the organic layer was washed with water and brine, dried over Na2SO4, filtered and concentrated. The crude residue was purified on silica gel (elution gradient 5% to 50% ethyl acetate in hexanes) to give methyl N - {[(2,2-dimethylpent-4-enyl) oxy] carbonyl} -3-methyl-L-valyl- (4R) -4 - {[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl) carbonyl] oxy} -Lprolinate (2.75 g, 85% yield) as a white foam. LRMS (ESI) m / z 570 [(M + H)⁺; calculated for C31H44N3O7: 570].

Step 8: Methyl (5R, 7S, 10S) -10-tert-butyl-15,15-dimethyl-3,9,12-trioxo1,6,7,9,10,11,12,14,15,16- decahydro-5H-2.22: 5,8-dimethane-4,13,2,8,11 benzodioxatriazacycloicosine-7-carboxylate [0098]

<img file="PL1924593T3_D0029.tif" />

[0099] Methyl N - {[(2,2-dimethylpent-4-enyl) oxy] carbonyl} -3-methyl-Lvalyl- (4R) -4 - {[(4-vinyl-1,3-dihydro- 2H-isoindol-2-yl) carbonyl] oxy} -L-prolate (2.46 g, 4.32 mmol) in anhydrous dichloromethane (450 ml) was flushed with nitrogen for 15 minutes. Then a solution of bis (tricyclohexylphosphine) -3-phenyl-1H-inden-1-ylidenorutene dichloride (Neolyst M1 catalyst purchased from Strem) (0.40 g, 0.43 mmol) in degassed anhydrous dichloromethane (50 ml) was added dropwise over 30 minutes. . The reaction mixture was stirred at room temperature during which time 0.2 g catalysts were added approximately every 8-12 hours. The progress of the reaction was monitored by HPLC until the reaction was completed after 48 hours. The residue was purified by flash chromatography on silica gel, eluting with 10-70% EtOAc / hexane to give methyl (5R, 7S, 10S) -10-tert-butyl-15,15-dimethyl-3,9,12-trioxo- 1,6,7,9,10,11,12,14,15,16 decahydro-5H-2.22: 5,8-dimethane-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate (1, 85 g, 76% yield). LRMS (ESI) m / z 542 [(M + H)⁺; calculated for C29H40N3O7: 542].

Step 9: (5R, 7S, 10S) -10-tert-Butyl-15,15-dimethyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16 decahydro -5H-2.22: 5,8-dimethane-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylic acid [0100]

<img file="PL1924593T3_D0030.tif" />

[0101] For methyl solution (5R, 7S, 10S) -10-tert-butyl-15,15-dimethyl-3,9,12-trioxo 1.6.7.9.10.11.12.14.15.16-Decahydro-5H-2, 22: 5,8-dimethane-4,13,2,8,11 benzodioxatriazacycloicosine-7-carboxylate (0.9 g, 1.67 mmol) in THF: H2O (2: 1, 45 mL) LiOH (0 , 40, 16.7 mmol). The reaction mixture was heated to 40 ° C and stirred for 1 hour. The reaction mixture was diluted with aqueous HCl, and extracted with EtOAc. The combined EtOAc layers were washed with water, brine, dried over Na2SO4, filtered and concentrated. The product was used without further purification. LRMS (ESI) m / z 528 [(M + H)⁺; calculated for C28H38N3O7: 528].

Stage 10: (5R, 7S, 10S) -10-tert-Butyl-N- (1R, 2S) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropyl) 15,15-dimethyl-3,9,12 -trioxo 1.6.7.9.10.11.12.14.15.16- decahydro-5H-2.22: 5.8-dimethane-4,3,3,2,8,11 benzodioxatriazacycloicosine-7-carboxamide Acid solution (5R, 7S, 10S) -10-tert-butyl-15,15-dimethyl-3,9,12-trioxo 1.6.7.9.10.11.12.14.15.16- decahydro-5H-2.22: 5.8-dimethane-4,13,2 , 8,11-benzodioxatriazacycloicosine-7-carboxylic acid (100 mg, 0.19 mmol), (1R, 2S) -1 {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropanoate chloride (Llinas-Brunet et al. US03 / 15755 and Wang et al. WO 03/099274) (76 mg, 0.28 mmol), O - (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium phosphorhexafluoride (HATU, 108 mg, 0.28 mmol), DIPEA (0.073 mL, 0.42 mmol) and 4-dimethylaminopyridine (2 mg ) in dichloromethane (5 mL) was stirred at 40 ° C for 1 hour. The reaction solution was diluted with aqueous saturated NaHCO3, and extracted with EtOAc. The combined EtOAc layers were washed with water, brine, dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography eluting with 3% MeOH / CH2Cl2 to give (5R, 7S, 10S) -10-tert-butyl-N - ((1R, 2S) -1 {[(cyclopropylsulfonyl) amino] carbonyl} -2 -winylocyklopropylo) 15,15-dimethyl-3,9,12triokso-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22: 5,8-dimetano- 4,13,2,8,11 benzodioxatriazacycloicosine-7-carboxamide (80 mg, 57% yield).¹H NMR (400 MHz, ppm, CDCl3) δ 7.48 (s, 1H), 7.23 (s, 1H), 7.12 (d, 1H), 6.23 (d, J = 15 , 9 Hz, 1H), 5.94 (m, 1H), 5.76 (m, 1H), 5.50 (m, 2H), 5.43 (s, 1H), 5, 24 (d, J = 16.6 Hz, 1H), 5.11 (d, 1H), 4.70 (s, 2H), 4.61 (d, 1H), 4.48 (m , 3H), 4.35 (d, 1H), 4.14 (d, 1H), 3.74 (d, 1H), 3.34 (d, 1H), 2.89 (m , 1H), 2.43 (dd, 2H), 2.06 (m, 1H), 1.93 (m, 1H), 1.89 (dd, 1H), 1.43 (d , 1H), 1.25 (m, 3H), 1.09 (s, 3H), 1.06 (s, 9H), 0.86 (s, 3H). LRMS (ESI) m / z 740 [(M + H)⁺; calculated for C37H50N5O9S: 740].

Example 4 (5R, 7S, 10S) -10-tert-Butyl-N - ((1R, 2S) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropyl) -3,9,12-trioxo-6 , 7,9,10,11,12,14,15,16,17-decahydro-1H, 5H-2.23: 5.8-dimethane-4,3,3,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide (III -12) [0103]

<img file="PL1924593T3_D0031.tif" />

[0104] The title compound was prepared according to the procedure used for Example 3, except that 3-methyl-N - [(hex-5-enyloxy) carbonyl] -L-valine (obtained according to the procedure below) was used instead of N - {[ (2,2-dimethylpent-4-enyl) oxy] carbonyl} -3-methyl-L-valine in step 7. ¹H NMR (500 MHz, ppm, CD3OD) δ 9.13 (s, 1H), 7.26 (t, 1H),

7.23 (d, 1H), 7.16 (d, 1H), 6.39 (d, J = 16.4 Hz, 1H), 6.08 (m, 1H), 5.76 (m, 1H), 5.38 (s, 1H), 5.29 (d, 1H), 5.12 (d, 1H), 4.79 (d, 1H), 4.73-4, 63 (m, 4H), 4.41 (s, 1H), 4.37 (q, 1H),

4.24 (d, 1H), 3.96 (dd, 1H), 3.77 (quin. 1H), 2.94 (m, 1H), 2.51 (q, 1H), 2, 29-2.13 (m, 4H), 1.87 (dd, 1H), 1.68 (m, 2H), 1.53 (quin. 2H), 1.44 (dd, 1H ), 1.25 (m, 2 H), 1.05 (s, 9 H), LRMS (ESI) m / z 726 [(M + H)⁺; calcd for C36H48NSO9S: 726].

Example 5 (5R, 7S, 10S) -10-Butyl-N - ((1R, 2S) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropyl) -3,9,1,2-trioxo-6, 7,9,10,11,12,14,15,16,17-decahydro-1H, 5H-2.23: 5.8-dimethane-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide (III- 133) [0105]

<img file="PL1924593T3_D0032.tif" />

[0106] The title compound was prepared according to the procedure used for Example 3, except that 3-methyl-N - [(hex-5-enyloxy) carbonyl] -L-norleucine (obtained according to the procedure below) was used instead of N - {[ (2,2-dimethylpent-4-yl) oxy] carbonyl} -3-methyl-L-valine in step 7. ¹H NMR (500 MHz, ppm, CD3OD) δ

7.24 (t, 1H), 7.23 (d, 1H), 7.15 (d, 1H), 6.91 (d, 1H), 6.37 (d, J = 16, 1 Hz, 1H), 6.07 (m, 1H), 5.75 (m, 1H), 5.39 (s, 1H), 5.29 (d, 1H), 5.12 ( d, 1H), 4.77 (d, 1H), 4.66 (m, 3H), 4.57 (m, 1H), 4.47 (q, 1H), 4.39 ( q, 1H), 4.27 (d, 1H), 3.90 (dd, 1H), 3.77 (quin, 1H), 2.96 (m, 1H), 2.49 ( q, 1H), 2.29 (m, 1H), 2.22 (m, 3H), 1.88 (dd, 1H), 1.75 (m, 2H), 1.64 ( m, 2H), 1.52 (m, 2H), 1.39 (m, 5H), 1.27 (m, 1H), 1.18 (m, 1H), 1.09 ( m, 2H), 0.94 (t, 3H). LRMS (ESI) m / z 726 [(M + H)⁺; calcd for C36H48N5O9S: 726].

Example 6 (5R, 7S, 10S) -10-Butyl-N - ((1R, 2S) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropyl) -3,9,12-trioxo-1,6, 7,9,10,11,12,14,15,16,17,18-dodecahydro-5H-2,24:

5,8-dimethane-4,13,2,8,11-benzodioxatriazacyclodocosine-7-carboxamide (III-198) [0107]

<img file="PL1924593T3_D0033.tif" />

[0108] The title compound was prepared according to the procedure used for Example 3 except that N - [(hept-6-en-1-yloxy) carbonyl] -L-norleucine (obtained according to the procedure below) was used instead of N - {[( 2,2-dimethylpent-4-enyl) oxy] carbonyl} -3-methyl-L-valine in step 7. ¹H NMR (500 MHz, ppm, CD3OD) δ 9.26 (s, 1H), 7.39 (d, 1H),

7.24 (t, 1H), 7.15 (d, 1H), 6.30 (d, J = 15.9 Hz, 1H), 6.20 (m, 1H), 5.75 ( m, 1H), 5.53 (s, 1H), 5.31 (d, 1H), 5.12 (d, 1H), 4.70 (m, 4H), 4.43 ( dd, 1H), 4.34 (m, 2H), 4.27 (q, 1H), 3.91 (dd, 1H), 3.79 (quin, 1H), 3.31 ( m, 1H), 2.97 (m, 1H), 2.31 (m, 1H), 2.22 (m, 3H), 1.89 (dd, 1H), 1.74 ( m, 2H), 1.66 (m, 1H), 1.56 (m, 3H), 1.38 (m, 8H), 1.19 (m, 1H), 1.09 ( m, 2H), 0.94 (t, 3H). LRMS (ESI) m / z 740 [(M + H)⁺; calculated for C37H50N5O9S: 740]. Example 7 (5R, 7S, 10S) -10-tert-Butyl-N - ((1R, 2S) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropyl) -15,15-dimethyl-3,9, 12-thioxo-6,7,9,10,11,12,14,15,16,17-decahydro38 1H, 5H-2.23: 5,8-dimethane-4,13,2,8,11-benzodioxatriazacyclohenicosine -7-carboxamide (III199) [0109]

<img file="PL1924593T3_D0034.tif" />

[0110] The title compound was prepared according to the procedure used for Example 3 except that N - {[(2,2-dimethylhex-5-enyl) oxy] carbonyl} -3-methyl-L-valine (prepared according to the procedure below) used instead of N - {[(2,2-dimethylpent-4-yl) oxy] carbonyl} -3-methyl-L-valine in step 7. ¹H NMR (500 MHz, ppm, CD3OD) δ 9.17 (s, 1H), 7.27 (t, J = 7.5 Hz, 1H), 7.21 (t, J = 7.5 Hz , 2H), 7.16 (d, J = 7.5 Hz, 1H), 6.38 (d, J = 16 Hz, 1H), 6.03 (m, 1H), 5.79 (m, 1H), 5.32 (m, 2H), 5.13 (m, 1H), 4.82-4.77 (m, 1H), 4.73-4.61 (m , 4H), 4.48 (s, 1H), 4.39 (m, 1H), 4.19 (d, J = 12 Hz, 1H), 3.96 (m, 1H), 2.96 (m, 1H), 2.59-2.55 (m, 1H), 2.35-2.12 (m, 4H), 1.89 (m, 1H), 1, 49-1.23 (m, 6H), 1.51-0.98 (m, 14H), 0.95-0.85 (m, 4H). LRMS (ESI) m / z 754 [(M + H)⁺; calculated for C38H52N5O9S: 754].

Example 8

5R, 75,10S) -10-tert-Butyl-N - ((1R, 2R) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2etylocyklopropylo) -3,9,12-trioxo-1,6,7 , 9,10,11,12,14,15,16,17,18-dodecahydro-5H-2.22: 5.8-dimethane-4,3,3,2,8,11-benzodioxatriazacycloicosine-7-carboxamide (III-200 ) [0111]

<img file="PL1924593T3_D0035.tif" />

[0112] A solution of Example 2 (0.32 mg, 0.45 mmol) and palladium on carbon (10 wt%, 0.03 g) in EtOAc (10 mL) was vigorously stirred under a hydrogen balloon for 1 hour. The reaction mixture was filtered and concentrated. The residue was purified by reverse phase HPLC (DeltaPak C18 column), using 40-65% CH3CN in water (with NH4OAc 1 g / L). Fractions were concentrated, diluted with aqueous saturated NaHCO3 (20 mL) and extracted with CH2Cl2 (3 x 70 mL). The combined CH2Cl2 layers were washed with water (50 mL), dried over Na2SO4, filtered and concentrated to give (5R, 7S, 10S) -10-tert-butyl-N - ((1R, 2R) -1 {[(cyclopropylsulfonyl) amino] carbonyl } -2-ethylcyclopropyl) -3,9,12-triokso1,6,7,9,10,11,12,14,15,16,17,18-dodecahydro-5H-2,22: 5,8-dimethano -4,13,2,8,1139 benzodioxatriazacycloicosine-7-carboxamide (0.31 g, 97% yield). ¹H NMR (CD3OD ppm) δ 7.23 (t, 1H), 7.14 (d, 1H), 7.10 (d, 1H), 7.02 (d, 1H), 5.52 (s, 1H), 4.74-4.60 (m, 4

H), 4.48-4.30 (m, 4H), 3.88 (d, 1H), 3.75 (s, 1H), 2.99 (m, 1H), 2.62 ( m, 1H), 2.41 (m, 2

H), 2.14 (m, 1H), 1.79 (m, 1H), 1.65-1.51 (m, 6H), 1.47-1.19 (m, 5H), 1.07 (s, 9H), 0.99 (t,

H). LRMS (ESI) m / z 716 [(M + H)⁺; calculated for C35H50N5O9 S: 716].

Example 9 (5R, 7S, 10S) -10-tert-Butyl-N - ((1R, 2R) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropyl) -3,9,12-trioxo-6, 7,9,10,11,12,14,15,16,17,18,19-dodecahydro-1 H, 5H2,23: 5,8-dimethano-4,13,2,8,11-benzodioxatriazacyclohenicosine-7 carboxamide (III-201) [0113]

<img file="PL1924593T3_D0036.tif" />

[0114] The title compound was obtained in Example 4 using the procedure described for Example 8. ¹H NMR (500 MHz, ppm, CD3OD) δ 7.23 (t, 1H), 7.14 (d, 1H), 7.10 (d, 1H), 7.02 (d, 1H) , 5.36 (s, 1H), 4.71 (m, 3H), 4.64 (t, 1H), 4.56 (m, 1H), 4.40 (m, 2H) , 4.24 (d, 1H), 3.96 (dd, 1H), 3.72 (quin, 1H), 2.98 (m, 1H), 2.58 (m, 1H) , 2.49 (m, 2H), 2.15 (t, 1H), 1.691,19 (m, 15H), 1.09 (m, 1H), 1.06 (s, 9H) , 0.98 (t, 3H). LRMS (ESI) m / z 730 [(M + H)⁺; calculated for C36H52N5O9S: 730].

Example 10 (5R, 7S, 10S) -10-Butyl-N - ((1R, 2R) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropyl) -3,9,12-trioxo-6,7, 9,10,11,12,14,15,16,17,18,19-dodecahydro-1H, 5H2,23: 5,8-dimethane-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide ( III-202) [0115]

<img file="PL1924593T3_D0037.tif" />

[0116] The title compound was obtained in Example 5 using the procedure described for Example 8. ¹H NMR (500 MHz, ppm, CD3OD δ 7.23 (t, 1H), 7.14 (d, 1H), 7.09 (d, 1H), 6.99 (d, 1H),

5.39 (s, 1H), 4.76-4.61 (m, 4H), 4.43 (m, 3H), 4.29 (d, 1H), 3.92 (dd, 1H), 3.69 (quin, 1H), 2.99 (m, 1H), 2.57 (m, 1H), 2.51 (m, 2H), 2.19 (tt, 1H), 1.77 (m, 1H), 1.70-1.30 (m, 20

H), 1.17 (m, 2H), 1.10 (m, 2H), 0.99 (t, 3H), 0.95 (t, 3H). LRMS (ESI) m / z 730 [(M + H)⁺; calculated for C36H5zN5O9S: 730].

Example 11 (5R, 7S, 10S) -10-Butyl-N - ((1R, 2R) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropyl) -3,9,12-trioxo-1,6, 7,9,10,11,12,14,15,16,17,18,19,20-tetradecihydro-5H2,24: 5,8-dimethano-4,13,2,8,11-benzodioksatriazacyklodocosine-7- carboxamide (III-203) [0117]

<img file="PL1924593T3_D0038.tif" />

[0118] The title compound was obtained in Example 6 using the procedure described for Example 8. ¹H NMR (500 MHz, ppm, CD3OD) δ 7.2 (t, 1H), 7.15 (d, 1H), 7.11 (d, 1H), 5.55 (s, 1H) , 4.70 (m, 4H), 4.49 (m, 1H), 4.38 (t, 1H), 4.29 (m, 2H), 3.94 (dd, 1H) , 3.73 (quin, 1H), 3.00 (m, 1H), 2.63 (quin, 1H), 2.51 (m, 1H), 2.38 (m, 1H) , 2.20 (tt, 1H), 1.76 (quin, 1H), 1.68-1.07 (m, 24H), 1.00 (t, 3H), 0.95 (t , 3H). LRMS (ESI) m / z 744 [(M + H)⁺; calculated for C37H54N5O9S: 744].

Example 12 (5R, 7S, 10S) -10-tert-Butyl-N - ((1R, 2R) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropyl) -15.15-dimethyl-3.9, 12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18dodekahydro-5H-2,22: 5,8-dimethano-4,13,2,8,11- benzodioxatriazacycloicosine-7-carboxamide (III-204) [0119]

<img file="PL1924593T3_D0039.tif" />

[0120] The title compound was obtained in Example 3 using the procedure described for Example 8. ¹H NMR (400 MHz, ppm, CD3OD) δ 9.06 (s, 1H), 7.22 (dd, 1H), 7.13 (d, 1H), 7.07 (d, 1H) , 5.51 (s, 1H), 4.72 (d, 2H), 4.68 (d, 2H), 4.44 (d, 2H), 4.28 (m, 2H) , 3.87 (dd, 1H),

3.28 (m, 1H), 2.98 (d, 1H), 2.85 (m, 3H), 2.52 (m, 1H), 2.43 (m, 2H), 2.15 (m, 1H), 1.151.17 (m, 3H), 1.41 (m, 2H), 1.30 (m, 1H), 1.21 (m, 4H), 1.08 (m, 1H), 1.06 (s, 3H), 1.05 (s, 9H), 0.98 (t, 3H), 0.81 (s, 3H). LRMS (ESI) m / z 744 [(M + H)⁺; calculated for

C37H54N5O9S: 744].

Example 13 (5R, 7S, 10S) -10-tert-Butyl-N - ((1R, 2R) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropyl) -15.15-dimethyl-3.9, 12-trioxo-6,7,9,10,11,12,14,15,16,17,18,19dodekahydro-1H, 5H-2,23: 5,8-dimethano-4,13,2,8, 11-benzodioxatriazacyclohenicosine-7-carboxamide (III-205) [0121]

<img file="PL1924593T3_D0040.tif" />

[0122] The title compound was obtained in Example 7 using the procedure described for Example 8. ¹H NMR (500 MHz, ppm, CD3OD δ 9.09 (s, 1H), 7.24 (t, J = 7.5 Hz, 1H), 7.15 (d, J = 7.5 Hz, 1H), 7.10 (d, J = 7.5 Hz, 1H), 5.53 (s, 1H), 4.75-4.59 (m, 4H), 4.44-4 , 37 (m, 3H), 4.20 (d, J = 12 Hz, 1H), 3.95-3.91 (m, 1H), 3.31 (m, 2H), 2, 99-2.96 (m, 1H), 2.62-2.46 (m, 3H),

2.17-2.13 (m, 1H), 1.67-1.50 (m, 6H), 1.37-1.18 (m, 7H), 1.15-0.96 ( m, 16H), 0.80 (s, 3H). LRMS (ESI) m / z 758 [(M + H)⁺; calculated for C38H56N5O9S: 758].

Alternative receiving:

Step 1: 1-Bromo-2,3-bis (bromomethyl) benzene [0123]

<img file="PL1924593T3_D0041.tif" />

[0124] To a suspension of 3-bromo-o-xylene (999 g, 5.40 mol) in chlorobenzene (9 L) at room temperature was added N-bromosuccinimide (1620 g, 9.1 Mol) and benzoyl peroxide (2.6 g , 10.8 mmol). The reaction mixture was heated to 80 ° C and stirred under nitrogen for 18 hours. The reaction mixture was cooled to 70 ° C and an additional portion of NBS (302 g, 1.7 mol) was added. The reaction mixture was heated to 80 ° C and stirred under nitrogen for 22 hours. The reaction mixture was cooled to room temperature, diluted with heptane (6 L) and filtered. The precipitate was washed with heptane (4 L) and the combined filtrates were evaporated. The crude product was dissolved in heptane (2 L) and chloroform (200 mL) and filtered through basic alumina (500 g). The alumina layer was washed with heptane (4 L) and the combined filtrates were evaporated to give 1-bromo-2,3-bis (bromomethyl) benzene (1760 g, crude mass) which was used without further purification.¹H NMR (CDCl 3) δ (ppm) 7.56 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.16 (t, J = 8.0 Hz, 1H), 4.84 (s, 2H), 4.64 (s, 2H).

Step 2: 2-Benzyl-4-bromoisoindoline hydrochloride [0125]

<img file="PL1924593T3_D0042.tif" />

[0126] Potassium bicarbonate (657 g, 6.56 mol) was suspended in MeCN (17 L) and the mixture was heated to 80 ° C. Solutions of crude 1-bromo-2,3-bis (bromomethyl) benzene (900 g, 2.63 mol in 1 L MeCN) and benzylamine (281 g, 2.63 mol in 1 L MeCN) were added simultaneously from the dropping funnels over time hours. The reaction mixture was stirred at 77 ° C for 2 hours and then cooled to room temperature and stirred for 16 hours. The contents of the reaction flask were cooled, filtered and the solvent removed by evaporation. The reaction was partitioned between water (6 L) and EtOAc (2 L). The pH was adjusted to&gt; 9 by addition of 1M K2CO3, the layers were separated and the aqueous layer was extracted with an additional portion of EtOAc (2 L). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and evaporated. The crude oil was diluted with EtOH (300 mL) and cooled to 0 ° C. Methanolic HCl was added to acidify the mixture, then MTBE (700 mL) and the mixture was sonicated, followed by stirring for 15 hours. MTBE (1 L) was added and the mixture was filtered and washed with 20% EtOH in MTBE, then MTBE. The solid was air dried to give 2-benzyl-4-bromoisoindoline hydrochloride (211 g). An additional portion of the product (86 g) was isolated by concentrating the mother liquors. LRMS (ESI) m / z 289 [(M + H)⁺; calculated for C15H15BrN: 289].

Step 3: 4-Bromoisoindoline [0127]

<img file="PL1924593T3_D0043.tif" />

[0128] To a solution of 2-benzyl-4-bromoisoindoline hydrochloride (11 g, 30.96 mmol) in 200 mL EtOAc was added 1M NaOH (100 mL) and the mixture was stirred for 30 minutes. The organic layer was separated, washed with brine, dried over anhydrous Na2SO4 and the solvent was evaporated into an oil which was azeotroped with toluene (50 mL). The oil was dissolved in chlorobenzene (50 ml) and added to a stirred solution of 4A molecular sieves (5 g). After 10 minutes, 1-chloroethylchloroformate (5.6 mL, 51mmol) was added dropwise over 5 minutes. The reaction mixture was then heated to 90 ° C for 2 hours, cooled to room temperature and filtered. The solids were washed with chlorobenzene (5 ml) and methanol (40 ml). The filtrate was heated to 70 ° C for 1 hour, allowed to cool and stirred at room temperature overnight. The solids were filtered off, washed with chlorobenzene (2 ml) and hexane and dried to give 6.84 g of the title compound. LRMS (ESI) m / z 198.1 [(M + H)⁺; calculated for C8H9BrN: 198.0].

Step 4: 1-t-Butyl 2-methyl (2S, 4R) -4 - {[(4-bromo-1,3-dihydro-2H-isoindol-2-yl) carbonyl] oxy} pyrrolidine-1,2-dicarboxylate [ 0129]

<img file="PL1924593T3_D0044.tif" />

[0130] To a solution of proline methyl (2S, 4R) -BOC-4-hydroxyester (126.3 g, 515 mmol) in DMF (960 mL) at 0 ° C was added N, N'-carbonyldiimidazole (83.51 g, 515 mmol). The reaction mixture was stirred at room temperature for 3 hours. 4-Bromoisoindoline hydrochloride (120 g, 515 mmol) and diisopropylethylamine (96.3 mL, 540 mmol) were added and the reaction mixture was heated to 50 ° C for 6 hours, then allowed to cool to room temperature and stirred overnight. The reaction mixture was partitioned between EtOAc (3 L) and 10% aqueous KHSO4 (6 L), the aqueous layer re-extracted with EtOAc (2 L) and the combined organic layers were washed with 10% aqueous NaHCO3, brine, dried over Na2SO4 and the solvent evaporated to foam (239 g). LRMS (ESI) m / z 471.0 [(M + H)⁺; calculated for C20H26BrN2O6: 471.1].

Step 5: 1-t-Butyl 2-methyl (2S, 4R) -4 - {[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl) carbonyl] oxy} pyrrolidine-1,2-dicarboxylate

<img file="PL1924593T3_D0045.tif" />

[0131] [0132] For 2-methyl (2S, 4R) -4 - 1-t-butyl solution [{4-bromo-1,3-dihydro-2H-isoindol2-yl) carbonyl] oxy} pyrrolidine-1 , 2-dicarboxylate (10.0 g, 21.3 mmol) in ethanol (200 mL) potassium vinyl trifluoroborate (4.28 g, 32 mmol) and triethylamine (4.5 mL, 32 mmol) were added, followed by the dichloro chloride adduct [ 1,1-bis (diphenylphosphine) ferrocene] palladium (II) dichloromethane (175 mg, 0.21mmol). The reaction mixture was heated to reflux for 6 hours, cooled to room temperature, diluted with 10% aqueous KHSO4 and ethanol was removed by evaporation in vacuo. The aqueous residue was extracted with EtOAc and the organic layer was washed with brine, dried over Na2SO4, the solvent was evaporated and the crude product was purified by silica gel chromatography eluting with 4060% EtOAc / hexane to give, after evaporation, the title compound (8.18 g). LRMS (ESI) m / z 417.2 [(M + H)⁺; calculated for C22H29N2O6: 417.2].

Step 6: (3R, 5S) -5- (methoxycarbonyl) pyrrolidine-3-14-vinyl-1,3-dihydro-2 hydrochloride

H-isoindole-2-carboxylate [0133]

<img file="PL1924593T3_D0046.tif" />

[0134] A mixture of 1-t-butyl 2-methyl (2S, 4R) -4 - {[(4-vinyl-1,3-dihydro-2H-isoindol-2-yl) carbonyl] oxy} pyrrolidine-1,2-dicarboxylate (18.0 g, 43.2 mmol) and HCl / dioxane (4 M) (43.2 mL, 173 mmol) was stirred at room temperature for 2 hours. The reaction mixture was concentrated to remove dioxane, then concentrated with Et 2 O to give (3R, 5S) -5- (methoxycarbonyl) pyrrolidin-3-yl-4-vinyl-1,3-dihydro-2-Hisoindole-2-carboxylate hydrochloride as an off-white solid ( 15 g), which was used without further purification. LRMS (ESI) m / z 317 [(M + H)⁺; calculated for C17H21N2O4: 317].

Step 7: Methyl N - {[(2,2-dimethylhex-5-en-1-yl) oxy] carbonyl} -3-methyl-L-valyl (4R) -4 - {[(4-vinyl-1, 3-dihydro-2H-isoindol-2-yl) carbonyl] oxy} -L-proline [0135]

<img file="PL1924593T3_D0047.tif" />

[0136] To (3R, 5S) -5- (methoxycarbonyl) pyrrolidin-3-yl-4-vinyl-1,3-dihydro-2H-isoindole-2-carboxylate hydrochloride solution (5.0 g, 14.2 mmol) and N - {[(2,2-dimethylhex-5enyl) oxy] carbonyl} -3-methyl-L-valine (4.0 g, 14.2 mmol) in DMF (20 ml) at room temperature DIPEA ( 2.5 mL, 14.2 mmol), EDC (5.5 g, 28.4 mmol), and HOAt (1.9 g, 14.2 mmol). After 18 hours, the reaction mixture was poured into Et2O and extracted with 1 N HCl. The aqueous layer was extracted with EtOAc, and the combined organic layers were washed with 1 N HCl, water, NaHCO 3 and brine. The organic layer was dried over MgSO 4 and the solvent removed in vacuo. The crude product was purified on silica gel (30% EtOAc in hexanes) to provide 4.2 g of the title compound as a thick oil. LRMS (ESI) m / z 584.4 [(M + H)⁺; calculated for C32H46N3O7: 584.3].

Step 8: Methyl (5R, 7S, 10S, 18E) -10-tert-butyl-15,15-dimethyl-3,9,12-trioxo6,7,9,10,11,12,14,15,16, 17-decahydro-1H, 5H-2,23: 5,8-dimethano-4,13,2,8,11benzodioksatriazacyklohenicosine-7-carboxylate

<img file="PL1924593T3_D0048.tif" />

[0138] For methyl solution N - {[(2,2-dimethylhex-5-en-1-yl) oxy] carbonyl} -3-methyl-L-valyl- (4R) -4 - {[(4-vinyl-1 , 3-dihydro-2H-isoindol-2-yl) carbonyl] oxy} -Lprolinate (4.7 g, 8.05 mmol) in degassed (bubbling nitrogen for 30 minutes) DCM (1410 ml) Zhan 1B catalyst (Zhan catalyst 1B, RC-303, Zannan Pharma Ltd.) (0.591 g, 0.805 mmol). The mixture was then stirred at room temperature under N atmosphere₂. After 19 hours, the reaction was complete and DMSO (57 μΐ, 0.805 mmol) was added. The mixture was stirred for 2 hours and the mixture concentrated in vacuo to ~ 70 mL. The crude product was then directly purified on silica gel (elution gradient, 0-50% EtOAc in hexanes) to provide 4.4 g of the title compound as an oil. LRMS (ESI) m / z 556.3 [(M + H)⁺; calculated for C30H42N3O7: 556.3].

Step 9: Methyl (5R, 7S, 10S) -10-tert-butyl-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17, 18.19-dodecahydro-1H, 5H-2.23: 5,8-dimethane-4,13,2,8,11 benzodioxatriazacyclohenicosine-7-carboxylate [0139]

<img file="PL1924593T3_D0049.tif" />

[0140] For (5R, 7S, 10S, 18E) -10-tert-butyl-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15, methyl solution 16,17-decahydro-1H, 5H-2.23: 5,8-dimethane-4,13,2,8,11 benzodioxatriazacyclohenicosine-7-carboxylate (4.4 g, 7.92 mmol) in EtOAc (79 mL) Pd / C (0.421 g, 0.396 mmol) was added. Then a H2 balloon was placed on the reaction flask. The flask was quickly evacuated and filled with H2. After 17 hours, the reaction was complete as determined by LC-MS. The Pd / C was filtered through glass wool and the crude product was purified on silica gel (elution gradient, 0-60% EtOAc in hexanes) to provide 4.01 g of the title compound as a white powder. LRMS (ESI) m / z 558.4 [(M + H)⁺; calculated for C30H44N3O7: 558.3].

Step 10: (5R, 7S, 10S) -10-tert-Butyl-15,15-dimethyl-3,9,12-trioxo6,7,9,10,11,12,14,15,16,17,18 , 19-dodecahydro-1H, 5H-2.23: 5,8-dimethane-4,13,2,8,11 benzodioxatriazacyclohenicosine-7-carboxylic acid [0141]

<img file="PL1924593T3_D0050.tif" />

[0142] For (5R, 7S, 10S) -10-tert-butyl-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16 methyl solution, 17,18,19-dodecahydro-1H, 5H-2.23: 5,8-dimethane-4,13,2,8,11 benzodioxatriazacyclohenicosine-7-carboxylate (5.76 g, 10.33 mmol) in THF (41.3 mL), MeOH (41.3 mL), and water (20.7 mL) LiOH (4.33 g, 103 mmol) was added at room temperature. After complete conversion (45 minutes), as assessed by LC-MS, the reaction was worked up, partitioning between Et2O and 1N HCl. The aqueous layer was then extracted with EtOAc. The combined organic layers were dried over MgSO 4 and the solvent removed in vacuo to afford 5.53 g of the title compound which was used without further purification. LRMS (ESI) m / z 544.4 [(M + H)⁺; calculated for C29H42N3O7: 544.3].

Stage 11: (5R, 7S, 10S) -10-tert-Butyl-N - ((1R, 2R) -1 {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropyl) 15,15-dimethyl-3,9,12triokso -6,7,9,10,11,12,14,15,16,17,18,19-dodecahydro-1H, 5H-2,23: 5,8-dimethano-4,13,2,8,11benzodioksatriazacyklohenicosine -7-carboxamide (III-205) [0143] For (5R, 7S, 10S) hydrochloride solution -10-tert-butyl-15,15-dimethyl-3,9,12 trioxo-6,7,9,10,11 , 12,14,15,16,17,18,19-dodecahydro-1H, 5H-2.23: 5,8-dimethane-4,13,2,8,11 benzodioxatriazacyclohenicosine-7-carboxylic acid (5.53 g, 10.17 mmol) and (1R, 2R) 1-amino-N- (cyclopropylsulfonyl) -2-ethylcyclopropanecarboxamide (3.28 g, 12.21mmol) in DMF (50.9 mL) DIPEA (7 , 11 ml, 40.7 mmol) and HATU (5.03 g, 13.22 mmol). After complete conversion (1 hour), the reaction mixture was partitioned between EtOAc and 1N HCl. The organic layer was washed with brine three times, dried over MgSO 4, and the solvent was removed in vacuo. The crude product was then purified on silica gel (elution gradient, 20-80% EtOAc in hexanes) to provide 5.8 g of the title compound as a white powder.

Example 14 (5R, 7S, 10S) -10-tert-Butyl-N - ((1R, 2S) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropyl) -3,9,12-trioxo-1, 6,7,9,10,11,12,14,15,16,17,18-dodecahydro-5H2,22: 5,8-dimethane-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide ( III-5) [0144]

<img file="PL1924593T3_D0051.tif" />

Step 1: Methyl (5R, 7S, 10S) -10-tert-butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,165,16 decahydro-5H-2, 22: 5,8-dimethane-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate [0145]

<img file="PL1924593T3_D0052.tif" />

[0146] Methyl (5R, 7S, 10S) -10-tert-butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2, 22: 5,8-dimethane-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate was prepared according to the procedure used for methyl (5R, 7S, 10S) -10-tert-butyl -15.15-dimethyl-3.9 12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22: 5,8-dimetano4,13,2,8,11-benzodioxatriazacycloicosine-7 -carboxylate (example 3, step 8) except that that 3-methyl-N - [(pent-4-enyloxy) carbonyl] -L-valine (obtained according to the procedure below) was used instead of N - {[(2,2-dimethylpent-4-enyl) oxy] carbonyl} -3 -methyl-Lvaline in step 7. LRMS (ESI) m / z 514 [(M + H)⁺; calculated for C27H36N3O7: 514].

Step 2: Methyl (5R, 75.10S) -10-tert-butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18dodecahydro- 5H-2.22: 5,8-dimethane-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate [0147]

<img file="PL1924593T3_D0053.tif" />

[0148] For (5R, 7S, 10S) methyl solution -10-tert-butyl-3,9,12-trioxo1,6,7,9,10,11,12,14,15,16,17,18- dodecahydro-5H-2.22: 5,8-dimethane-4,13,2,8,11 benzodioxatriazacycloicosine-7-carboxylate (0.10 g, 0.20 mmol) in ethyl acetate (7 ml) added 10% palladium on carbon (0.01 g). The reaction mixture was stirred under a hydrogen balloon for 5 hours at room temperature. The contents of the reaction flask were filtered through celite and the filtrate was evaporated. The crude product was used without further purification (0.09 g, 90% yield). LRMS (ESI) m / z 516 [(M + H)⁺; calculated for C27H38N3O7: 516].

Stage 3: (5R, 7S, 10S) -10-tert-Butyl-N - ((1R, 2S) -1 {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropyl) -3,9,12-triokso1,6,7 , 9,10,11,12,14,15,16,17,18-dodecahydro-5H-2.22: 5,8-dimethane-4,3,3,2,8,11 benzodioxatriazapycycloicosine-7-carboxamide [0149] To (5R, 7S, 10S) methyl solution -10-tert-butyl-3,9,12-trioxo1,6,7,9,10,11,12,14,15,16,17,18-dodecahydro-5H- 2.22: 5,8-dimethane-4,13,2,8,11 benzodioxatriazacycloicosine-7-carboxylate (90 mg, 0.18 mmol) in THF (2 mL) and MeOH (0.5 mL), LiOH (1N 1.75 mL, 1.75 mmol) was added. The reaction mixture was heated to 40 ° C and stirred for 1 hour, during which time the total consumption of the starting methyl ester was observed using LC-MS. The mixture was then worked up with 0.5N HCl and EtOAc. The organic layer was then dried over K2CO3, and the solvent was removed in vacuo. The crude product was taken in DMF (1 mL).

[0150] To the above solution was added (1R, 2S) -1 {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropananoate chloride (51 mg, 0.19 mmol), TBTU (77 mg, 0.24 mmol) and DIPEA (0.07 mL, 0.40 mmol) and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was directly purified by reverse phase HPLC to afford (5R, 7S, 10S) -10-tert-butylN - ((1R, 2S) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropyl) -3 , 9,12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18-dodecahydro-5H-2.22: 5, 8-dimethane-4,13,2,8 , 11-benzodioxatriazacycloicosine-7-carboxamide (34 mg, 28% yield). ¹H NMR (500 MHz, ppm, CD3OD) δ 9.14 (s, 1H), 7.23 (t, 1H), 7.13 (d, 1H), 7.10 (d, 1H) , 5.75 (quin, 1H), 5.53 (s, 1H), 5.29 (d, 1H), 5.12 (d, 1H), 4.75-4.59 (m, 5 H), 4.42 (m, 2H), 4.34 (s, 1H), 4.30 (d, 1H), 3.88 (dd, 1H), 3.75 (m, 1 H), 3.60 (q, 2H), 2.95 (m, 1H), 2.63 (m, 1H), 2.41 (m, 2H), 2.26-2.12 (m, 2H), 1.88 (dd, 1H), 1.79 (m, 1H), 1.56 (m, 3H), 1.41 (m, 3H),

1.25 (m, 2H), 1.17 (t, 2H), 1.06 (s, 9H). LRMS (ESI) m / z 714 [(M + H)⁺; calculated for C35H48N5O9S: 714].

Example 15 (5R, 7S, 10S) -10-tert-Butyl-N - ((1R, 2S) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropyl) -15,15-dimethyl-3,9, 12-trioxo-1,6,7,9,10,11,12,14,15,16,17,18dodekahydro-5H-2,22: 5,8-dimethano-4,13,2,8,11- benzodioxatriazacycloicosine-7-carboxamide (III-206) [0151]

<img file="PL1924593T3_D0054.tif" />

[0152] The title compound was prepared according to the procedure used for Example 14 (using steps 2 and 3) except that methyl (5R, 7S, 10S) -10-tert-butyl-15,15-dimethyl-3,9,12-trioxo -1,6,7,9,10,11,12,14,15,16-decahydro-5H-2.22: 5,8-dimethane4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate (example 3 step 1) was used instead of methyl (5R, 7S, 10S) -10-tert-butyl-3,9,12-trioxo-1,6,7,9,10,11,12,14,15, 16-decahydro-5H- 2.22: 5,8-dimethane-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate in step 2. ¹H NMR (400 MHz, ppm, CDCl 3) δ 9.91 (s, 1H), 7.22 (t, 1H), 7.09 (d, 2H), 7.05 (d, 1H) ), 5.77 (m, 1H), 5.60 (s, 1H), 5.45 (d, 1H), 5.29 (s, 1H), 5.15 (d, 1H) ), 4.72 (q, 2H), 4.40-4.55 (m, 4H), 4.30 (d, 1H), 4.25 (d, 1H), 3.78 ( dd, 1H), 3.26 (d, 1H), 2.91 (m, 1H), 2.50 (m, 3H), 2.39 (m, 3H), 2.11 ( m, 1H), 1.98 (m, 2H), 1.51 (m, 2H), 1.38 (m, 4H), 1.18 (m, 1H), 1.04 ( s, 9H), 1.01 (t, 3H), 0.79 (s, 3H). LRMS (ESI) m / z 742 [(M + H)⁺; calcd for C37H52N5O9S: 742].

Example 16 (5R, 7S, 10S) -10-tert-Butyl-N - ((1R, 2R) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropyl) -3,9,12-trioxo-1, 6,7,9,10,11,12,14,15,16-Decahydro-5H-2.22: 5.8-dimethane-4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxamide (III-16) [0153]

<img file="PL1924593T3_D0055.tif" />

[0154] For (5R, 7S, 10S) methyl solution -10-tert-butyl-3,9,12-trioxo1,6,7,9,10,11,12,14,15,16-decahydro-5H- 2.22: 5,8-dimethane-4,13,2,8,11 benzodioxatriazacycloicosine-7-carboxylate (Example 14, step 1) (60 mg, 0.12 mmol) in THF (1 mL) and MeOH ( 0.5 ml) LiOH (1N 1.17 ml, 1.17 mmol) was added. The reaction mixture was heated to 40 ° C and stirred for 1 hour, at which time total consumption of the starting methyl ester was observed using LC-MS. The mixture was then worked up with 0.5N HCl and EtOAc. The organic layer was then dried over K2CO3, and the solvent was removed in vacuo. The crude product was taken in DMF (1 mL).

[0155] To the above solution, (1R, 2R) -1 {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropanamininium chloride (32 mg, 0.12 mmol), TBTU (48 mg, 0.15 mmol) and DIPEA were added (0.044 mL, 0.25 mmol) and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was directly purified by reverse phase HPLC to give (5R, 7S, 10S) 10-tert-butyl-N - ((1R, 2R) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropyl) -3, 9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22: 5,8-dimethano-4,13,2,8,11-benzodioksatriazacykloicosine- 7-carboxamide (55 mg, 67% yield). ¹H NMR (500 MHz, ppm, CD3OD δ 7.33 (d, 1H), 7.26 (t, 1H), 7.16 (d, 1H),

6.39 (d, J = 15.7 Hz, 1H), 6.13 (m, 1H), 5.37 (s, 1H), 4.69 (m, 4H), 4.47- 4.28 (m, 4H), 3.89 (m, 1H), 3.83 (d, 1H), 2.98 (m, 1H), 2.40 (m, 2H), 2.31 (m, 1H), 2.11 (t, 1H), 1.99 (s, 1H), 1.73 (s, 1H), 1.60 (m, 2H), 1.52 (m, 1H), 1.29-1.15 (m, 3H), 1.08 (s, 9H), 0.98 (t, 3H). LRMS (ESI) m / z 714 [(M + H)⁺; calculated for C35H48N5O9S: 714].

Example 17 (SR, 7S, 10S) -10-tert-Butyl-N - ((1R, 2R) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropyl) -3,9,12-trioxo-6, 7,9,10,11,12,14,15,16,17-decahydro-1H, 5H-2.23: 5.8-dimethane-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxamide (III- 207) [0156]

<img file="PL1924593T3_D0056.tif" />

Step 1: Methyl (5R, 7S, 10S) -10-tert-butyl-3,9,12-trioxo-6,7,9,10,11,1 2,14,15,16,17 decahydro-1H, 5H -2.23: 5,8-dimethane-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxylate [0157]

<img file="PL1924593T3_D0057.tif" />

[0158] Methyl (5R, 7S, 10S) -10-tert-butyl-3,9,12-trioxo-6,7,9,10,11,1 2,14,15,16,17-decahydro-1H, 5H -2.23: 5,8-dimethane-4,13,2,8,11-benzodioxatriazacyclohenicosine-7-carboxylate was prepared according to the procedure used for methyl (5R, 7S, 10S) -10-tert-butyl-15,15-dimethyl-3, 9,12-trioxo-1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22: 5,8-dimethano-4,13,2,8,11-benzodioksatriazacykloicosine- 7-carboxylate (example 3, step 8) except that that 3-methyl-N - [(hex-5-enyloxy) carbonyl] -L-valine (obtained according to the procedure below) was used instead of N - {[(2,2-dimethylpent-4-enyl) oxy] carbonyl} -3-methyl -L-valines in step 7. LRMS (ESI) m / z 528 [(M + H)⁺; calculated for C28H38N3O7: 528].

Stage 2: (5R, 7S, 10S) -10-tert-Butyl-N - ((1R, 2R) -1 {[(cyclopropylsulfonyl) amino] carbonyl} -2- ethylcyclopropyl) -3,9,12-triokso6,7,9 , 10,11,12,14,15,16,17-decahydro-1H, 5H-2.23: 5,8-dimethane-4,3,3,2,8,11 benzodioxatriazacyclohenicosine-7- carboxamide [0159] Example 17 was obtained according to the procedure used for example 16 except for methyl use (5R, 7S, 10S) -10-tert-butyl-3,9,12-triokso6,7,9,10,11,12,14,15,16,17-decahydro-1H, 5H-2,23: 5,8-dimethane-4,13,2,8,11 benzodioxatriazacyclohenicosine-7-carboxylate instead of methyl- (5R, 7S, 10S) -10-tert-butyl-3,9,12-trioxo-1,6,7,9, 10,11,12,14,15,16-decahydro-5H-2.22: 5,8-dimethane4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate (Example 14, step 1). ¹H NMR (500 MHz, ppm, CD3OD) δ 9.06 (s, 1H), 7.27 (t, 1H), 7.24 (d, 1H), 7.18 (d, 1H) , 6.40 (d, J = 16.4 Hz, 1H), 6.11 (m, 1H), 5.39 (t, 1H), 4.80 (d, 1H), 4.69 (m, 4H), 4.42 (s, 1H), 4.25 (d, 1H), 3.97 (dd, 1H), 3.79 (quin, 1H), 2.98 (m, 1H), 2.50 (q, 1H), 2.78 (m, 2H), 2.15 (m, 1H), 1.77-1.54 (m, 8H) , 1.32-1.19 (m, 4H), 1.11 (m, 1H), 1.07 (s, 9H), 0.98 (t, 3H). LRMS (ESI) m / z 728 [(M + H)⁺; calcd for C36H50N5O9S: 728].

Example 18 (5R, 7S, 10S) -10-tert-Butyl-N - ((1R, 2R) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropyl) -15.15-dimethyl-3.9, 12-trioxo-6,7,9,10,11,12,14,15,16,17-dekahydro1H, 5H-2,23: 5,8-dimethano-4,13,2,8,11-benzodioksatriazacyklohenicosine- 7-carboxamide (III208) [0160]

<img file="PL1924593T3_D0058.tif" />

Step 1: Methyl (5R, 7S, 10S) -10-tert-butyl-15,15-dimethyl-3,9,12-trioxo6,7,9,10,11,12,14,15,16,17- decahydro-1H, 5H-2.23: 5,8-dimethane-4,13,2,8,11 benzodioxatriazacyclohenicosine-7-carboxylate [0161]

<img file="PL1924593T3_D0059.tif" />

[0162] Methyl (5R, 7S, 10S) -10-tert-butyl-15,15-dimethyl-3,9,12-trioxo-6,7,9,10,11,12,14,15,16,17- decahydro-1H, 5H-2.23: 5,8-dimethane-4,13,2,8,11 benzodioxatriazacyclohenicosine-7-carboxylate was prepared according to the procedure used for methyl (5R, 7S, 10S) -10-tert-butyl-15 15-dimethyl-3,9,12-triokso1,6,7,9,10,11,12,14,15,16-decahydro-5H-2,22: 5,8-dimethano-4,13,2 , 8,11-benzodioxatriazacycloicosine-7-carboxylate (example 3, step 8) except that that N {[(2,2-dimethylhex-5-enyl) oxy] carbonyl} -3-methyl-L-valine (obtained according to the procedure below) was used instead of N - {[(2,2-dimethylpent-4-enyl) oxy] carbonyl} -3-methyl-L-valine in step 7. LRMS (ESI) m / z 556 [(M + H)⁺; calculated for C30H42N3O7: 556]. Stage 2: (5R, 7S, 10S) -10-tert-Butyl-N - ((1R, 2R) -1 {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropyl) 15,15-dimethyl-3,9,12triokso -6,7,9,10,11,12,14,15,16,17-decahydro-1H, 5H-2,23: 5,8-dimethano-4,13,2,8,11benzodioksatriazacyklohenicosine-7-carboxamide [0163] Example 18 was prepared according to the procedure used for Example 16 except for methyl use (5R, 7S, 10S) -10-tert-butyl-15,15-dimethyl-3,9,12-triokso6,7,9,10,11,12,14,15,16,17-decahydro-1H, 5H-2.23: 5,8-dimethane-4,13,2,8,11 benzodioxatriazacyclohenicosine-7-carboxylate instead of methyl- (5R, 75.10S) -10-tert-butyl-3,9,12-trioxo-1, 6,7,9,10,11,12,14,15,16-decahydro-5H-2.22: 5,8-dimethane4,13,2,8,11-benzodioxatriazacycloicosine-7-carboxylate (Example 14, step 1). ¹H NMR (500 MHz, ppm, CD3OD) δ 10.05 (s, 1H), 7.24 (m, 2H), 7.17 (d, 1H), 7.11 (d, 1H) , 6.61 (s, 1H), 6.28 (d, J = 16.4 Hz, 1H), 5.95 (m, 1H), 5.58 (m, 1H), 5, 31 (s, 1H), 4.71 (m, 2H), 4.55 (m, 2H), 4.46 (d, 2H), 4.29 (dd, 1H), 4, 17 (d, 1H), 3.89 (d, 1H), 3.32 (d, 1H), 2.92 (m, 1H), 2.59 (m, 1H), 2, 21-2.30 (m, 2H), 2.08 (m, 1H), 1.60-1.78 (m, 6H), 1.22-1.31 (m, 5H), 1.06 (s, 9H), 1.04 (t, 3H), 0.093 (t, 3H), 0.87 (s, 3H). LRMS (ESI) m / z 756 [(M + H)⁺; calculated for C38H54N5O9S: 756].

Preparation of N - [(pent-4-eN-yloxy) carbonyl] -L-norleucine:

[0164]

<img file="PL1924593T3_D0060.tif" />

[0165] To a solution of 1-penten-4-ol (0.95 g, 11.0 mmol) in DMF (15 mL) at 0 ° C was added carbonyldiimidazole (1.79 g, 11.0 mmol). The reaction mixture was warmed to room temperature and stirred for 30 minutes. Then Lnorleucine methyl ester hydrochloride (2.0 g, 11.0 mmol) was added, the reaction mixture was heated to 50 ° C and stirred for 15 minutes. After cooling, the reaction mixture was diluted with ethyl ether and washed twice with water. The organic layer was dried over sodium sulfate, filtered and concentrated. The crude product was purified by silica gel chromatography (elution gradient 10 to 90% ethyl acetate in hexanes) to provide 2.1 g (74% yield) of methyl N - [(pent-4-en-1-yloxy) carbonyl] -L -norleucinate in the form of clear oil.

[0166] To a stirred solution of N - [(pent-4-enyloxy) carbonyl] -Norleucinate methyl solution (8.50g, 33.03 mmol) in THF (20 mL) was added 1N NaOH (20 mL). This reaction solution was stirred at room temperature for 3 hours, then acidified to pH 3 with 1N HCl and extracted (3 x 250 mL) with EtOAc. The combined EtOAc layers were washed with 50 mL water, 50 mL brine, dried over sodium sulfate, filtered and concentrated to give 7.09 g (88% yield) of the title product as a clear oil. LRMS (ESI) m / z 244 [(M + H)⁺; calculated for C12H22NO4: 244].

Preparation of 3-methyl-N - [(pent-4-enyloxy) carbonyl] -L-valine:

[0167]

<img file="PL1924593T3_D0061.tif" />

[0168] A solution of 4-pentenol (7.22 g, 83.8 mmol) and triphosgene (11.3 g, 38.1 mmol) in dioxane (160 mL) was cooled to 0 ° C, then DIPEA (9.85 g) was added dropwise , 76.2 ml). The white suspension was stirred vigorously for 1 hour at 25 ° C, then cooled to 0 ° C. A 1N NaOH solution (76.2 mL) and t-butylglycine (10.0 g, 76.2 mmol) were added. The resulting suspension was heated to 25 ° C and stirred for 18 hours. About half of the dioxane was removed in vacuo, the solution was poured into 1N NaOH (100 mL) and washed with dichloromethane (3 x 150 mL). The aqueous layer was acidified with 6N HCl and the desired product was extracted with dichloromethane (3 x 150 mL). The combined organic layers were dried over MgSO4 and concentrated to give 13.7 g (73.9% yield) of 3-methyl-N - [(pent-4-enyloxy) carbonyl] -L-valine as a colorless oil. LRMS (ESI) m / z 244 [(M + M⁺; calculated for C12H22NO4: 244].

Preparation of N - [(hex-5-en-1-yloxy) carbonyl] -L-norleucine:

[0169]

<img file="PL1924593T3_D0062.tif" />

[0170] N - [(Hex-5-en-1-yloxy) carbonyl] -L-norleucine was prepared according to the procedure for N - [(pent-4-en-1-yloxy) carbonyl] -L-norleucine using 5- hexenol instead of 4-pentenol. LRMS (ESI) m / z 258 [(M + H)⁺; calculated for C13H24NO4: 258].

Preparation of 3-Methyl-N - [(hex-5-enyloxy) carbonyl] -L-valine:

[0171]

<img file="PL1924593T3_D0063.tif" />

[0172] 3-Methyl-N - [(hex-5-enyloxy) carbonyl] -L-valine was prepared according to the procedure for

3-methyl-N - [(pent-4-enyloxy) carbonyl] -L-valine using 5-hexenol instead of 4-pentenol. LRMS (ESI) m / z 258 [(M + H)⁺; calculated for C13H24NO4: 258].

Preparation of N- [hept-6-en-1-yloxy) carbonyl] -L-norleucine:

[0173]

<img file="PL1924593T3_D0064.tif" />

[0174] N - [(Hept-6-en-1-yloxy) carbonyl] -L-norleucine was prepared according to the procedure for N [(pent-4-en-1-yloxy) carbonyl] -L-norleucine using 6-heptenol instead of 4-pentenol. LRMS (ESI) m / z 272 [(M + H)⁺; calculated for C14H26NO4: 272].

Preparation of N - {[(2,2-Dimethylpent-4-enyl) oxy] carbonyl} -3-methyl-L-valine:

[0175]

<img file="PL1924593T3_D0065.tif" />

Step 1: 2,2-Dimethylpent-4-en-1-ol [0176]

<img file="PL1924593T3_D0066.tif" />

[0177] A solution of 2,2-dimethyl-4-pentenoic acid (6.0 g, 46.8 mmol) in anhydrous THF was cooled in an ice bath to 0 ° C. Lithium aluminum hydride in THF (56.2 mL, 56.2 mmol) was added in a free stream and the reaction allowed to warm to 25 ° C. The reaction mixture was stirred for 1 hour before pouring into 1N HCl and diethyl ether. The organic layer was separated, dried over MgSO4 and concentrated to provide 2,2-dimethylpent-4-en-1-ol as a clear oil (4.7 g, 87.9% yield).

Step 2: N - {[(2,2-Dimethylpent-4-enyl) oxy] carbonyl} -3-methyl-L-valine [0178] DIPEA (2.48 g, 19.2 mmol) was added dropwise at 0 ° C to a solution of 2,2-dimethylpent-4-en-1ol (2.24 g, 19.6 mmol) and triphosgene (2.56 g, 8.64 mmol) in 60 mL of dioxane. The resulting white suspension was stirred for 5 minutes at 0 ° C, then allowed to warm to 25 ° C over 1 hour. The suspension was cooled to 0 ° C in an ice bath, then 1N NaOH (19.2 mL) and L-tert-butylglycine (2.52 g, 19.2 mmol) were added. The reaction mixture was heated to 25 ° C and stirred for 72 hours. Dioxane was removed in vacuo and the reaction mixture was basified to pH 12 with 1N NaOH. The aqueous layer was extracted with dichloromethane (3 x 150 mL), then acidified to pH ~ 1 with 6N HCl. The aqueous layer was extracted with dichloromethane (3 x 150 mL). The combined organic layers were dried over MgSO4 and concentrated to give N - {[(2,2-dimethylpent-4-enyl) oxy] carbonyl} -3-methyl-L-valine as a white powder (4.26 g, 82.7% yield). LRMS (ESI) m / z 272 [(M + H)⁺; calculated for C14H26NO4: 272].

Preparation of N - {[(2,2-Dimethylhex-5-enyl) oxy] carbonyl} -3-methyl-L-valine:

[0179]

<img file="PL1924593T3_D0067.tif" />

Step 1: Ethyl 2,2-dimethylhex-5-enate [0180]

<img file="PL1924593T3_D0068.tif" />

[0181] To a stirred solution of diisopropylamine (13.38 mL, 94.70 mmol) in anhydrous THF (50 mL), at -70 ° C and under nitrogen, 2.5 M n-BuLi in ether (36) was slowly added. , 50 ml,

91.25 mmol). After stirring for 15 minutes, ethyl isobutyrate (11.51 mL, 86.09 mmol) in THF (50 mL) was then added dropwise to this reaction solution, stirred for 20 minutes before dropping 4-bromo-1-butene (9.79 ml, 96.42 mmol) in HMPA (20 ml). The reaction solution was then stirred to -50 ° C for 5 hours, quenched by the addition of 1M HCl (10 mL) and water (100 mL), followed by extraction (3 x 125 mL) with ether. The combined ether layers were washed with water (4 x 70 mL), aqueous saturated NaHCO3 (2 x 70 mL), dried over Na2SO4, filtered and concentrated. The crude product was flash chromatographed on 120 g silica gel 60, eluting with 1-20% EtOAc / hexane to afford the title product as a clear oil (11.01g, 75% yield). LRMS (ESI) m / z 171 [(M + H)⁺; calculated for C10H19O2: 171].

Step 2: 2,2-Dimethylhex-5-en-1-ol [0182]

<img file="PL1924593T3_D0069.tif" />

[0183] To a stirred solution of 1M LAH in ether (142.14 mL, 142.14 mmol), at 0 ° C and under nitrogen, ethyl 2,2-dimethylhex-5-enoate (11.00 g, 64.61mmol) was added dropwise. ) dissolved in

100 ml anhydrous ether over 1 hour. This reaction solution was stirred at 22 ° C for hours, then quenched by the addition of water (3 mL), 1M NaOH (11 mL) and water (9 mL), dried over Na 2 SO 4, filtered and concentrated to give the title product (7.22 g,

87,09 %). ¹1 H NMR (500 MHz, CDCl 3) δ 5.85-5.77 (m, 1H); 5.01 (d, 1H); 4.93 (d, 1H); 3.33 (d, 2H); 2.03 (m, 2H); 1.34 (m, 2H); 0.89 (m, 6H) ppm.

Step 3: N - {[(2,2-Dimethylhex-5-enyl) oxy] carbonyl} -3-methyl-L-valine [0184] To a mixed solution of 2,2-dimethylhex-5-en-1-ol ( 10.75 g, 83.85 mmol) in anhydrous 1,4-dioxane (100 mL), at 0 ° C and under nitrogen, triphosgene (13.69 g, 46.12 mmol) was carefully added followed by DIPEA (14, 61 ml, 83.85 mmol). This reaction solution was stirred at 22 ° C for 1 hour, cooled to 0 ° C and 1N NaOH (83.85 mL, 83.85 mmol) and L-tert-leucine (11.00 g, 83.85 mmol) were slowly added. , then stirred at 22 ° C for 20 hours. The reaction solution was basified to pH 10 with 1N NaOH, washed with CH2Cl2 (3 x 100 mL), acidified to pH 5 with 1N HCl and extracted with CH2Cl2 (3 x 150 mL). The combined CH2Cl2 layers were washed with water (100 mL), dried over Na2SO4, filtered and concentrated to give the title product (20.26 g, 84.66%).¹1 H NMR (500 MHz, CDCl 3) δ 5.85-5.77 (m, 1H); 5.24 (d, 1H); 5.01 (d, 1H); 4.93 (d, 1H); 4.20 (d, 1H); 3.86 (d, 1H); 3.79 (d, 1H); 2.01 (m, 2H); 1.36 (m, 2H); 1.04 (s, 9H); 0.92 (m, 6H) ppm. LRMS (ESI) m / z 286 [(M + H)⁺; calculated for C15H28NO4: 286].

Preparation of ethylcyclopropanamininium chloride:

(1R, 2R-1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-

<img file="PL1924593T3_D0070.tif" />

[0186] Mixture of (1R, 2S) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropanedinium chloride (Llinas-Brunet et al. US03 / 15755 and Wang et al. WO 03/099274) (0.05 g, 0.187 mmol ) and palladium on carbon (10 wt.%, 0.01g) in EtOAc (5 mL) was vigorously stirred under a hydrogen atmosphere provided by a hydrogen balloon for 1 hour. The reaction mixture was filtered and concentrated to give (1R, 2R) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropananoate chloride (0.045 g, 89% yield).

Example 19 (5R, 7S, 10S) -10-tert-Butyl-N - ((1R, 2S) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropyl) -15.15-dimethyl-3.9, 12-trioxo-6,7,9,10,11,12,14,15,16,17,18,19dodekahydro-1H, 5H-2,23: 5.8-dimethano-4,13,2,8,11- benzodioxatriazacyclohenicosine-7-carboxamide (III-210) [0187]

<img file="PL1924593T3_D0071.tif" />

Example 19 (5R, 7S, 10S) -10-tert-butyl-15,15-dimethyl-3,9,12 trioxo-6,7,9,10,11,12,14,15.16 , 17,18,19-dodecahydro-1H, 5H-2.23: 5,8-dimethane-4,13,2,8,11 benzodioxatriazacyclohenicosine-7-carboxylic acid (Example 13, alternative preparation, step 4) using the procedure for Example 3, step 10. ¹H NMR (500 MHz, CD3OD, ppm) δ 7.25-7.09 (m, 3H), 5.82-5.74 (m, 1H), 5.35-5.29 (m, 2 H), 5.15-5.12 (m, 1H), 4.75-4.59 (m, 3H), 4.45-4.38 (m, 2H), 4.21-4 , 12 (m, 1H), 4.13-4.09 (m, 1H), 3.95-3.92 (m, 1H), 2.98-2.94 (m, 1H) , 2.62-2.54 (m, 1H), 2.49-2.46 (m, 2H), 2.25-2.21 (m, 1H),

2.19-2.13 (m, 1H), 1.90-1.88 (m, 1H), 1.52 (m, 2H), 1.48-1.45 (m, 1H ), 1.40-1.18 (m, 6H), 1.15-1.00 (m, 14H), and 0.81 (m, 4H). LRMS (ESI) m / z 756.4 [(M + H)⁺; calculated for C38H53N5O9S: 755.9].

Example 20 (5R, 7S, 10S, 18E) -10-Cyclohexyl-N - ((1R, 2S) -1 {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropyl) -15.15-dimethyl-3.9 , 12triokso-6,7,9,10,11,12,14,15,16,17-decahydro-1H, 5H-2,23: 5,8-dimethano-7-4,13,2,8,11benzodioksatriazacyklohenicosine -carboxamide (III-225) [0189]

<img file="PL1924593T3_D0072.tif" />

[0190] Example 20 was prepared using the procedures of Example 13, alternative preparation steps 1, 2, 4 and 5 using (2 S) -cyclohexyl acid ({[(2,2-dimethylhex-5-en1-yl) oxy] carbonyl } amino) acetic in step 1 and (1R, 2S) -1 {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropanamininium chloride in step 5. ¹H NMR (500 MHz, CD3OD, ppm) δ 7.26 (m, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.15 (d, J = 9.5 Hz , 1H), 6.38 (d, J = 9.5 Hz, 1H), 5.99-6.02 (m, 1H), 5.74-5.80 (m, 1H), 5.29-5.34 (m, 2H), 5.11-5.14 (m, 1H), 4.79-4.81 (m, 2H), 4.64-4.72 ( m, 3H), 4.56 (d, J = 11.5 Hz, 1H), 4.364.40 (m, 2H), 4.18 (d, J = 11.5 Hz, 1H), 4.10 (d, J = 5.5 Hz, 0.5 H), 3.91-3.94 (dd, J = 11.5,

3.5 Hz, 1H), 3.34 (d, J = 11.0 Hz, 1H), 2.95-2.97 (m, 1H), 2.52-2.56 (m, 1H), 2.16-2.35 (m,

5 H), 1.65-1.82 (m, 8H), and 0.85-1.43 (m, 17H). LRMS (ESI) m / z 780.4 [(M + H)⁺; calculated for C40H53N5O9S: 780.9].

Example 21 (5R, 7S, 10S) -10-Cyclohexyl-N - ((1R, 2R) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropyl) -15,15-dimethyl-3,9,12- 6,7,9,10,11,12,14,15,16,17,18,19dodekahydro-trioxo-1H, 5H-2,23: 5,8-dimethano-4,13,2,8,11- benzodioxatriazacyclohenicosine-7-carboxamide (III-226) [0191]

<img file="PL1924593T3_D0073.tif" />

[0192] Example 21 was prepared in Example 20 using the procedure described for Example 8. ¹H NMR (500 MHz, CDCl3, ppm) δ 10.13 (s, 1H), 7.22 (t, J = 7.5 Hz, 1H), 7.10 (d, J = 7.5 Hz , 1H), 7.05 (d, J = 7.5 Hz, 1H), 6.73 (s, 1H), 5.40 (d, J = 9.5 Hz, 1H), 5 , 36 (m, 1H), 4.674.76 (m, 2H), 4.55 (d, J = 15.5 Hz, 1H), 4.44 (d, J = 14.5 Hz, 1 H), 4.41 (d, J = 11.0 Hz, 1H), 4.29-4.39 (m, 2H), 4.16 (d, J = 11.0 Hz, 1H) , 3.82-3.85 (dd, J = 11.5, 3.5 Hz, 1H), 3.25 (d, J = 11.0 Hz, 1H), 2.95 (m, 1 H), 2.51-2.59 (m, 2H), 2.36-2.44 (m, 2H), 1.73-1.76 (m, 5H), and 0.79 ( br s, 2H). LRMS (ESI) m / z 784.4 [(M + H)⁺; calcd for C40H57N5O9S: 784.4].

Alternative preparation of (1R, 2R) -1-amino-N- (cyclopropylsulfonyl) -2-ethylcyclopropanecarboxamide hydrochloride:

[0193]

<img file="PL1924593T3_D0074.tif" />

Step 1: tert-Butyl ((1R, 2R) -1 {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropyl) carbamate:

[0195] The hydrogenation reactor was charged with methanol (1000 ml) suspension of tert-butyl ((1R, 2S) -1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-vinylcyclopropyl) carbamate (164 g, 0.50 mol) (Wang et al., US 6,995

174) and 5% Ru / C (dry, 7.5 wt%, 12.4 g) and mixing was established. The reactor was placed under nitrogen (20 psig) and brought to atmospheric pressure three times to remove residual oxygen. Then hydrogen was pressurized (50 psig) in the reactor. After 20 hours, the reactor was brought to atmospheric pressure. The reaction suspension was then transferred to the reaction and filtered through solka flok (34 grams, wetted w / 100 mL with methanol) to provide a clear, light brown solution. The flock was washed with methanol (200 ml x 2). The combined methanol solutions were concentrated under reduced pressure to provide the crude product as a white solid (153 g). The crude product was suspended in ethyl acetate (800 ml), heated to 40 ° C and held for 30 minutes. The solution was then inoculated, held for 30 minutes, and heptane (500 mL) was added through an addition funnel over 30 minutes. The partially crystallized solid was cooled to room temperature and held overnight, after which additional heptane (500 mL) was added. After one hour, additional heptane (250 mL) was added via a dropping funnel, and the white suspension was held for one hour. The solution was filtered and the solid washed with heptane / EtOAc (500 mL, 4: 1) and dried under reduced pressure to give tert-butyl ((1R, 2R) 1 - {[(cyclopropylsulfonyl) amino] carbonyl} -2-ethylcyclopropyl) carbamate ( 125.9 g).

Step 2: (1R, 2R) -1-amino-N- (cyclopropylsulfonyl) -2-ethylcyclopropanecarboxamide hydrochloride:

A solution of the product from step 1 above (92 g, 0.28 mol) in DCM (1200 ml) was cooled to 0 ° C and HCl was bubbled through the solution for 10 minutes, the cooling bath was removed and the reaction mixture was stirred for 2 hours. Nitrogen was bubbled through the reaction mixture for 5 minutes and the volatiles were evaporated. The residue was azeotroped with DCM (x3) to give an off-white powder (75 g). LRMS (M + H)⁺ calculated = 233; found 233.

Preparation of (2S) -cyclohexyl ({[2,2-dimethylhex-5-en-1-yl) oxy] carbonyl} amino) acetic acid:

[0197]

<img file="PL1924593T3_D0075.tif" />

[0198] (2S) -Cyclohexyl ({[(2,2-dimethylhex-5-en-1-yl) oxy] carbonyl} amino) acetic acid was prepared according to the procedure for 3-methyl-N - [(pent-4enyloxy) carbonyl] -L-valine using (2S) -amino (cyclohexyl) acetic acid and 2,2-dimethylhex-5-en-1-ol. LRMS (ESI) m / z 312.3 [(M + H)⁺; calculated for C17H30NO4: 312.2]. Example 22

HCV NS3 protease fluorescence test "time-resolved fluorescence" (TRF) [0199] The HCV NS3 protease fluorescence test was carried out in a final volume of 100μ1 solution in assay buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 15% glycerin, 0.15% Triton X-100, 10 mM DTT, and 0.1% PEG 8000. NS3 protease was preincubated with various concentrations of inhibitors for 10-30 minutes. The peptide substrate in the assay was Ac-C (Eu) -DDMEE-Abu- [COO] -XSAK (QSY7) -NH2, in which Eu is a europium-labeled group, Abu is a 1-aminobutanoic acid which contains an ester bond with 2- hydroxypropane (X). Hydrolysis of the peptide under the action of the NS3 protease causes the separation of the fluorophore from the quencher, giving rise to fluorescence. Protease activity was induced by the addition of TRF peptide substrate (final concentration 50-100 nM). The reaction was terminated by adding after 1 hour at room temperature with 100 μΐ 500 mM MES, pH 5.5. The fluorescence product was detected using either a Victor V2 or pulsed fluorimeter (Perkin Elmer Life and Analytical Sciences) with excitation at 340 nm and emission at 615 nm with a 50-400 μs delay. Test concentrations of various forms of enzymes with a signal to background ratio of 10-30 were selected. Inhibition constants were obtained using a four-parameter fit.

[0200] The compounds in Examples 1-21 were found to have a Ki value of less than 100 nM (e.g., less than 1 nM) in the NSF TRF protease assay as described above.

Adam Kruszewski

Patent Attorney