Inhibitors of flaviviridae viruses

BACKGROUND OF THE INVENTION

Viruses belonging to the Flaviviridae family include at least three distinguishable species, including pestiviruses, flaviviruses and hepaciviruses (Calisher et al., J. Gen. Virol., 1993, 70, 37-43). Although pestiviruses such as bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV), and the disease of borderline disease (BDV) sheep), they cause many economically important animal diseases, their significance in relation to morbidity in humans is much less characterized (Moennig, V. et al., Adv. Vir. Res. 1992, 48, 53-98). Flaviviruses are responsible for serious human diseases, which include dengue fever and yellow fever, and hepaciviruses cause infection in people with the hepatitis C virus.

Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide (Boyer, N. et al. J Hepatol. 32: 98-112, 2000) and therefore a large part of current Virus research aims to develop better treatments for chronic HCV infection in humans (Di Besceglie, AM and Bacon, BR, Scientific American, Oct .: 80-85, (1999); Gordon, CP et al., J. Med. Chem. 2005, 48, 1-20, Maradpour, D. et al., Nat. Rev. Micro. 2007, 5 (6), 453-463). A review of many methods of HCV treatment was made by Bymock et al. In Antiviral Chemistry & Chemotherapy, 11: 2; 79-95 (2000).

Currently, two antiviral compound, ribavirin, a nucleoside analogue, and interferon-alpha (α) (IFN) are used for the treatment of chronic HCV infection in humans. Ribavirin alone does not effectively reduce viral RNA levels, is highly toxic, and is known to cause anemia. It is noted that the combination of IFN and ribavirin is effective in the eradication of chronic hepatitis C (Scott, LJ et al. Drugs 2002, 62, 507-556), but a sustained benefit is obtained in less than half of the patients in whom treatment.

Infections with the Flaviviridae family of viruses cause significant mortality, morbidity and significant economic losses worldwide. Alkinyl-substituted thiophenes having Flaviviridae virus activity have been disclosed by Chana et al., WO 2008058393; Wunberga et al., WO 2006072347; and Chana et al., WO 2002100851 (e.g. WO 2008058393 discloses a compound of 5- (3,3-dimethyl-1-butyl-1-yl) -3 - [(trans-4-methoxycyclohexyl) [trans-4-methylcyclohexylcarbonyl] amino] ] -2-thiophenecarboxylic acid); but currently none of them is a clinically approved antiviral curative. There is still a need to develop effective treatments for Flaviviridae infections.

- EP 2523950

SUMMARY OF THE INVENTION

Compounds of formula I are provided:

<img file="PL2523950T3_D0001.tif" />

or a pharmaceutically acceptable salt thereof, wherein: R² is selected from the group consisting of

<img file="PL2523950T3_D0002.tif" />

Het is an optionally substituted 3-12-membered heterocyclyl or an optionally substituted 3-14-membered heteroaryl;

wherein each substituted Het substituted is one or more Q⁴; every Q⁴, independently, is selected from the group consisting of halogen, oxo, oxide, -NO2, -N (= O), -SR⁴⁰, -S (O) R⁴⁰, -S (O) 2R⁴⁰, -S (O) 2 NR⁴⁰R⁴¹, -NR⁴⁰C (O) R⁴¹, -NR⁴⁰C (O) NR⁴¹R⁴², -NR⁴⁰S (O) 2 R⁴¹, -OP (O) R⁴¹R⁴², -P (O) R⁴¹R⁴²

NO⁴⁰S (O) R⁴

-P (O) OR⁴¹R⁴

-P (O) (OR⁴¹) OR⁴

-C (O) NR⁴¹R⁴²an optionally substituted C 1-6 alkyl, an optionally substituted C 2-6 alkenyl, an optionally substituted C 2-6 alkynyl, an optionally substituted C 3-6 cycloalkyl, an optionally substituted C 6-12 aralkyl, an optionally substituted C 6-12 aryl, an optionally substituted 3-14 membered heteroaryl, an optionally substituted C 1-6 alkyloxy, an optionally substituted C 2-6 alkenyloxy, an optionally substituted C 2-6 alkynyloxy, an optionally substituted C 3-6 cycloalkyloxy, an optionally substituted C 6-12 aryloxy, an optionally substituted 3-14-membered heteroaryloxy, an optionally substituted 4- 4 A 12-membered heterocyclyloxy, an optionally substituted -C (O) C 1-6 alkyl, an optionally substituted -C (O) C 2-6 alkenyl, an optionally substituted -C (O) C 2-6 alkynyl, an optionally substituted -C (O) C 3 - 6 cycloalkyl,optionally substituted-C (O) C6-12 aryl, an optionally substituted -C (O) - 3-14-membered heteroaryl, optionally substituted

-C (O) C6-12 aralkyl, an optionally substituted 3-10-membered heterocyclyl, -OH, -NR⁴¹R⁴², -C (O) OR⁴⁰, -CN, -N₃, -C (= NR⁴³) NR⁴¹R⁴², -C (= NR⁴³) OR⁴⁰, -NR⁴⁰C (= NR⁴³) NR⁴¹R⁴², -NR⁴¹C (O) OR⁴⁰ and -OC (O) NR⁴¹R⁴²;

every R⁴⁰, R⁴¹ and R⁴²is independently selected from the group consisting of H, an optionally substituted C1-12 alkyl, an optionally substituted C2-12 alkenyl, an optionally substituted C2-12 alkynyl, an optionally substituted C3-12 cycloalkyl, an optionally substituted C6-14 aryl, optionally substituted 3 -14-membered heteroaryl, an optionally substituted 3-12-membered heterocyclyl, an optionally substituted 3-18-membered heteroarylalkyl and an optionally substituted C6-18 aralkyl;

- EP 2523950 or R⁴¹ and R⁴² taken together with the atoms to which they are attached, form a 3- to 10-membered heterocyclyl;

every R⁴³is independently selected from the group consisting of H, an optionally substituted C1-12 alkyl, an optionally substituted C2-12 alkenyl, an optionally substituted C2-12 alkynyl, an optionally substituted C3-12 cycloalkyl, an optionally substituted C6-14 aryl, optionally substituted 3 -14-membered heteroaryl, an optionally substituted 3-12-membered heterocyclyl, an optionally substituted 3-18-membered heteroaralkyl, an optionally substituted C6-18 aralkyl, -CN, -C (O) R⁴⁴, -CHO and -S (O) 2R⁴⁴;

every R⁴⁴, separately, is an optionally substituted C 1-12 alkyl;

wherein each substituted Q⁴, substituted with R⁴⁰, substituted with R⁴¹, substituted with R⁴², substituted with R⁴³ or substituted R⁴⁴ is independently substituted with one or more Q's⁵; every Q⁵, separately, is selected from the group consisting of halogen, oxo, oxide, -NO2,

-N (= O), -SR⁵⁰, -S (O) R⁵⁰, -S (O) 2R⁵⁰, -S (O) 2 NR⁵⁰R⁵¹, -NR⁵⁰C (O) R⁵¹, -NR⁵⁰C (O) NR⁵¹R⁵², -NR⁵⁰S (O) R⁵¹, -NR⁵⁰S (O)₂R⁵¹, -OP (O) R⁵¹R⁵², -P (O) R⁵¹R⁵², -P (O) OR⁵¹R⁵², -P (O) (OR⁵¹) OR⁵², -C (O) NR⁵¹R⁵²an optionally substituted C1-6 alkyl, an optionally substituted C2-6 alkenyl, an optionally substituted C2-6 alkynyl, an optionally substituted C3-6 cycloalkyl, optionally substituted ^C6-12 aralkyl, an optionally substituted C6-12 aryl, an optionally substituted 3-14 membered heteroaryl, an optionally substituted C1-6 alkyloxy, an optionally substituted C2-6 alkenyloxy, an optionally substituted C2-6 alkynyloxy, an optionally substituted C3-6 cycloalkyloxy, optionally a substituted C 6-12 aryloxy, an optionally substituted 3-14-membered heteroaryloxy, an optionally substituted 4-12-membered heterocycloxy, an optionally substituted -C (O) C 1-6 alkyl, an optionally substituted -C (O) C 2-6 alkenyl optionally substituted -C (O) C2-6 alkynyl, an optionally substituted -C (O) C3-6 cycloalkyl, an optionally substituted -C (O) C6-12 aryl, an optionally substituted -C (O) - 3-14-membered heteroaryl, optionally substituted-C (O) C6-12 aralkyl, an optionally substituted 3-10-membered heterocyclyl, -OH, -NR⁵¹R⁵², -C (O) OR⁵⁰, -CN, -N3, -C (= NR⁵³) NR⁵¹R⁵², -C (= NR⁵³) OR⁵⁰, -NR⁵⁰C (= NR⁵³) NR⁵¹R⁵², -NR⁵¹C (O) OR⁵⁰ and -OC (O) NR⁵¹R⁵²;

every R⁵⁰, R⁵¹ and R⁵²is independently selected from the group consisting of H, an optionally substituted C1-12 alkyl, an optionally substituted C2-12 alkenyl, an optionally substituted C2-12 alkynyl, an optionally substituted C3-12 cycloalkyl, an optionally substituted C6-14 aryl, optionally substituted 3 -14-membered heteroaryl, an optionally substituted 3-12-membered heterocyclyl, an optionally substituted 3-18-membered heteroarylalkyl and an optionally substituted C6-18 aralkyl;

or R⁵¹ and R⁵² taken together with the atoms to which they are attached, form a 3- to 10-membered heterocyclyl;

every R⁵³is independently selected from the group consisting of H, an optionally substituted C1-12 alkyl, an optionally substituted C2-12 alkenyl, an optionally substituted C2-12 alkynyl, an optionally substituted C3-12 cycloalkyl, an optionally substituted C6-14 aryl, optionally substituted 3 -14-membered heteroaryl, an optionally substituted 3-12-membered heterocyclyl, an optionally substituted 3-18-membered heteroaralkyl, an optionally substituted C6-18 aralkyl, -CN, -C (O) R⁵⁴, -CHO and -S (O) 2R⁵⁴;

every R⁵⁴, independently, is an optionally substituted C 1-12 alkyl;

wherein each substituted Q⁵, substituted with R⁵⁰, substituted with R⁵¹, substituted with R⁵², substituted with R⁵³ or substituted R⁵⁴ is independently substituted with one or more Q's⁶; every Q⁶, independently, is selected from the group consisting of halogen, oxo, oxide,

-WELL₂, -N (= O), -SR⁶⁰, -S (O) R⁶⁰, -S (O) 2R⁶⁰, -S (O) 2 NR⁶⁰R⁶¹, -NR⁶⁰C (O) R⁶¹, -NR⁶⁰C (O) NR⁶¹R⁶², -NR⁶⁰S (O) R⁶¹, -NR⁶⁰S (O)₂R⁶¹, -OP (O) R⁶¹R⁶², -P (O) R⁶¹R⁶², -P (O) OR⁶¹R⁶², -P (O) (OR⁶¹) OR⁶², -C (O) NR⁶¹R⁶², C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, C6-12 aralkyl, C6-12 aryl,

EP-2323950 3-14-membered heteroaryl, C 1-6 alkyloxy, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 3-6 cycloalkyloxy, C 6-12 aryloxy, 3-14-membered heteroaryloxy, 4-12-membered heterocycloxy , -C (O) C1-6 alkyl, - C (O) C2-6 alkenyl, -C (O) C2-6 alkynyl, -C (O) C3-6 cycloalkyl, -C (O) C1-6 haloalkyl , -C (O) C6-12 aryl, -C (O) - 3-14-membered heteroaryl, -C (O) C6-12 aralkyl, 3-10-membered heterocyclyl, -OH, -NR⁶¹R⁶², -C (O) OR⁶⁰, -CN, -N3, -C (= NR⁶³) NR⁶¹R⁶², -C (= NR⁶³) OR⁶⁰, -NR⁶⁰C (= NR⁶³) NR⁶¹R⁶², -NR⁶¹C (O) OR⁶⁰ and -OC (O) NR⁶¹R⁶²;

every R⁶⁰, R⁶¹ and R⁶², independently, is selected from the group consisting of H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C1-12 haloalkyl, C6-14 aryl, 3-14-membered heteroaryl, 3 -12-membered heterocyclyl, 3-18-membered heteroarylalkyl and C6-18 aralkyl; or R⁶¹ and R⁶² taken together with the atoms to which they are attached, form a 3- to 10-membered heterocyclyl;

every R⁶³, independently, is selected from the group consisting of H, C1-12 alkyl, ^C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C6-14 aryl, 3-14-membered heteroaryl, 3-12-membered heterocyclyl, 3-18-membered heteroarylalkyl, C6-18 aralkyl, -CN, C (O) R⁶⁴, -CHO and -S (O)₂R⁶⁴; and every R⁶⁴, independently, is C1-12 alkyl.

The description discloses a compound for use in a method of treating Flaviviridae infection by administering a compound of formula I in an effective amount to a patient in need thereof. The compound of formula I is administered to a human in need thereof, such as a person who is infected with a virus of the Flaviviridae family, for example a person infected with HCV. In one example, the treatment effect is a reduction in one or more viral loads or an RNA clearance in a patient.

In another example, the disclosure discloses a compound for use in a method of treating and / or preventing a disease caused by a viral infection, wherein the viral infection is caused by a virus selected from the group consisting of dengue virus, yellow fever virus, West Nile virus, Japanese virus encephalitis, tick-borne encephalitis virus, Kunjin virus, murray brain virus Murray, St Louis encephalitis virus, OM hemorrhagic fever virus, bovine viral diarrhea virus, Zika virus and hepatitis C virus; by administering to a patient in need thereof a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

In another embodiment, a compound of formula I is provided for use in the treatment of Flaviviridae virus infection. In another aspect of this embodiment, infection with the Flaviviridae virus is an acute or chronic infection with the HCV virus. In another aspect of this embodiment, the treatment effect is a reduction in one or more viral loads or an RNA clearance in a patient. In another aspect of this embodiment, the treatment effect is a reduction in HCV viral load or clearance of HCV RNA in a subject.

In another embodiment, there is provided a pharmaceutical composition comprising a compound of formula I and one or more pharmaceutically acceptable carriers or one or more pharmaceutically acceptable excipients. The pharmaceutical composition of formula I may additionally contain one or more additional therapeutic agents. One or more additional therapeutic agents may be, without limitation, selected from: interferons, ribavirin or its analogues, inhibitors of HCV NS3 proteases, alpha-glucosidase 1 inhibitors, hepatoprotectants, nucleoside or nucleotide inhibitors of HCV NS5B polymerase, HCV NS5B non-nucleoside inhibitors, NS5A inhibitors, NS5A inhibitors HCV, TLR-7 agonists, cyclophilin inhibitors, HCV IRES inhibitors, pharmacokinetic enhancers and other drugs for HCV therapy,

In another example, the disclosure discloses a compound for use in a method for treating the symptoms or effects of HCV infection or for preventing them in an infected animal,

EP 2523950 which method comprises administering to an animal (i.e. treatment of said animal) a combined pharmaceutical composition or formulation comprising an effective amount of a compound of formula I and a second compound having anti-HCV activity.

In another embodiment, compounds of formula I and their pharmaceutically acceptable salts and all racemates, enantiomers, diastereomers, tautomers, polymorphs, pseudopolymorphs and amorphous forms thereof are provided.

The description discloses processes and novel intermediates that are useful in the preparation of compounds of formula I.

The description discloses novel methods for the synthesis, analysis, separation, isolation, purification, characterization and testing of compounds of formula I.

The invention includes combinations of aspects and embodiments as well as preferences as outlined throughout the specification.

DETAILED DESCRIPTION

A detailed reference will now be made to specific embodiments of the invention, examples of which are illustrated by the attached structures and formulas. Although the invention will be described in connection with the aforementioned embodiments, it should be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents that may be included within the scope of the invention as defined herein. In a preferred embodiment R² means

<img file="PL2523950T3_D0003.tif" />

In another preferred embodiment of R² means

<img file="PL2523950T3_D0004.tif" />

In another preferred embodiment of R² means

<img file="PL2523950T3_D0005.tif" />

In another embodiment, Het is an optionally substituted 3-12-membered heterocyclyl or an optionally substituted 3-14-membered heteroaryl, wherein the optionally substituted 3-12-membered heterocyclyl or the optionally substituted 3-14-membered heteroaryl contain from one to four heteroatoms selected from one another. from O, S or N. In another embodiment, Het is an optionally substituted 3-12-membered heterocyclyl containing one or two heteroatoms selected from O, S or N. In another embodiment, Het is an optionally substituted 5-10-membered heteroaryl containing from one to four heteroatoms selected from O, S or N. In another embodiment, Het is an optionally substituted tetrahydrofuranyl. In another embodiment, Het is optionally substituted tetrahydrofuran-3-yl. In another embodiment, Het is optionally substituted pyridinyl. In another embodiment, Het is an optionally substituted pyridazinyl. In another embodiment, Het is

- optionally substituted tetrahydro-2H-pyranyl. In another embodiment, Het is an optionally substituted piperidinyl. In another embodiment, Het is optionally substituted pyrrolidinyl. In another embodiment, Het is optionally substituted tetrahydrothiophenyl. In another embodiment, Het is optionally substituted pyrazinyl. In another embodiment, Het is optionally substituted 1H-tetrazolyl. In another embodiment, Het is optionally substituted azetidinyl. In another embodiment, Het is optionally substituted tetrahydrofuranyl. In another embodiment, Het is optionally substituted tetrahydrofuran-3 (S) -yl. In another embodiment, Het is optionally substituted tetrahydrofuran-3 (R) -yl. In another embodiment, Het is optionally substituted tetrahydro-2H-furo [2,3-b] furanyl. In another embodiment, Het is an optionally substituted thiazolyl. In another embodiment, Het is optionally substituted 1H-imidazolyl. In another embodiment, Het is an optionally substituted 4H-1,2,4-triazolyl. In another embodiment, Het is optionally substituted 1H-pyrazolyl. In another embodiment, Het is optionally substituted with 1,3,4-thiadiazolyl. In another embodiment, Het is optionally substituted quinolinyl. In another embodiment, Het is optionally substituted [1,2,4] triazolo [4,3-a] pyridinyl. In another embodiment, Het is an optionally substituted thiophenyl. In another embodiment, Het is optionally substituted 1,2,4-thiadiazolyl. In another embodiment, Het is an optionally substituted pyrimidinyl. In another embodiment, Het is an optionally substituted 1H-1,2,3-triazolyl. In another embodiment, Het is optionally substituted with 1,3,4-oxadiazolyl. In another embodiment, Het is optionally substituted imidazo [1,2-b] pyridazinyl.

In another embodiment, the compounds of formula I are represented by the formula

<img file="PL2523950T3_D0006.tif" />

where R² is selected from the group consisting of

<img file="PL2523950T3_D0007.tif" />

<img file="PL2523950T3_D0008.tif" />

<img file="PL2523950T3_D0009.tif" />

or a pharmaceutically acceptable salt thereof, the remaining variables being defined as for formula I.

In another embodiment of Formula III, Het is an optionally substituted 3-12-membered heterocyclyl or an optionally substituted 3-14-membered heteroaryl, wherein the optionally substituted 3-12-membered heterocyclyl or the optionally substituted 3-14-membered heteroaryl contain from one to the other. four heteroatoms selected from O, S or N. In another aspect of this embodiment, Het is optionally substituted

In another aspect of this embodiment, Het is optionally substituted pyridinyl. In another aspect of this embodiment, Het is an optionally substituted pyridazinyl. In another aspect of this embodiment, Het is optionally substituted tetrahydro-2H-pyranyl. In another aspect of this embodiment, Het is an optionally substituted piperidinyl. In another aspect of this embodiment, Het is optionally substituted pyrrolidinyl. In another aspect of this embodiment, Het is optionally substituted tetrahydrothiophenyl. In another aspect of this embodiment, Het is optionally substituted pyrazinyl. In another aspect of this embodiment, Het is optionally substituted 1H-tetrazolyl. In another aspect of this embodiment, Het is optionally substituted azetidinyl. In another aspect of this embodiment, Het is optionally substituted tetrahydrofuranyl. In another aspect of this embodiment, Het is optionally substituted tetrahydrofuran-3-yl. In another aspect of this embodiment, Het is optionally substituted tetrahydrofuran-3 (S) -yl. In another aspect of this embodiment, Het is optionally substituted tetrahydrofuran-3 (R) -yl. In another aspect of this embodiment, Het is optionally substituted tetrahydro-2H-furo [2,3-b] furanyl. In another aspect of this embodiment, Het is an optionally substituted thiazolyl. In another aspect of this embodiment, Het is optionally substituted 1H-imidazolyl. In another aspect of this embodiment, Het is an optionally substituted 4H-1,2,4-triazolyl. In another aspect of this embodiment, Het is optionally substituted 1H-pyrazolyl. In another aspect of this embodiment, Het is optionally substituted with 1,3,4-thiadiazolyl. In another aspect of this embodiment, Het is optionally substituted quinolinyl. In another aspect of this embodiment, Het is optionally substituted [1,2,4] triazolo [4,3-a] pyridinyl. In another aspect of this embodiment, Het is an optionally substituted thiophenyl. In another aspect of this embodiment, Het is optionally substituted 1,2,4-thiadiazolyl. In another aspect of this embodiment, Het is an optionally substituted pyrimidinyl. In another aspect of this embodiment, Het is optionally substituted 1H-1,2,3-triazolyl. In another aspect of this embodiment, Het is optionally substituted with 1,3, 4-oxadiazolyl. In another aspect of this embodiment, Het is an optionally substituted imidazo [1,2-b] pyridazinyl.

In another embodiment according to formula III² means

<img file="PL2523950T3_D0010.tif" />

In another aspect of this embodiment, Het is an optionally substituted 3-12-membered heterocyclyl or an optionally substituted 3-14-membered heteroaryl, wherein the optionally substituted 3-12-membered heterocyclyl or the optionally substituted 3-14-membered heteroaryl contain from one to four. heteroatoms selected from O, S or N. In another aspect of this embodiment, Het is an optionally substituted 3-12-membered heterocyclyl or an optionally substituted 3-14-membered heteroaryl, wherein the optionally substituted 3-12-membered heterocyclyl or optionally substituted 3- 14-membered

The heteroaryl has from one to four heteroatoms selected from O or N. In another aspect of this embodiment, Het is an optionally substituted 3-12-membered heterocyclyl containing one or two heteroatoms selected from 0, S or N. In another aspect, of this embodiment Het is an optionally substituted 3-12-membered heterocyclyl containing one or two heteroatoms selected from O or N. In another aspect of this embodiment, Het is an optionally substituted 5-10-membered heteroaryl having one to four heteroatoms selected from 0, S or N. In another aspect of this embodiment, Het is optionally substituted pyridinyl. In another aspect of this embodiment, Het is an optionally substituted pyridazinyl. In another aspect of this embodiment, Het is optionally substituted tetrahydro-2H-pyranyl. In another aspect of this embodiment, Het is an optionally substituted piperidinyl. In another aspect of this embodiment, Het is optionally substituted pyrrolidinyl. In another aspect of this embodiment, Het is optionally substituted tetrahydrothiophenyl. In another aspect of this embodiment, Het is optionally substituted pyrazinyl. In another aspect of this embodiment, Het is optionally substituted 1H-tetrazolyl. In another aspect of this embodiment, Het is optionally substituted azetidinyl. In another aspect of this embodiment, Het is optionally substituted tetrahydrofuranyl. In another aspect of this embodiment, Het is optionally substituted tetrahydrofuran-3-yl. In another aspect of this embodiment, Het is optionally substituted tetrahydrofuran-3 (S) -yl. In another aspect of this embodiment, Het is optionally substituted tetrahydrofuran-3 (R) -yl. In another aspect of this embodiment, Het is optionally substituted tetrahydro-2H-furo [2,3-b] furanyl. In another aspect of this embodiment, Het is an optionally substituted thiazolyl. In another aspect of this embodiment, Het is optionally substituted 1H-imidazolyl. In another aspect of this embodiment, Het is an optionally substituted 4H-1,2,4-triazolyl. In another aspect of this embodiment, Het is optionally substituted 1H-pyrazolyl. In another aspect of this embodiment, Het is optionally substituted with 1,3,4-thiadiazolyl. In another aspect of this embodiment, Het is optionally substituted quinolinyl. In another aspect of this embodiment, Het is optionally substituted [1,2,4] triazolo [4,3-a] pyridinyl. In another aspect of this embodiment, Het is an optionally substituted thiophenyl. In another aspect of this embodiment, Het is optionally substituted 1,2,4-thiadiazolyl. In another aspect of this embodiment, Het is an optionally substituted pyrimidinyl. In another aspect of this embodiment, Het is optionally substituted 1H-1,2,3-triazolyl. In another aspect of this embodiment, Het is an optionally substituted 1,3,4-oxadiazolyl. In another aspect of this embodiment, Het is an optionally substituted imidazo [1,2-b] pyridazinyl. In another aspect of this embodiment, Het is optionally substituted [1,2,4] triazolo [4,3-a] pyridinyl. In another aspect of this embodiment, Het is an optionally substituted thiophenyl. In another aspect of this embodiment, Het is optionally substituted 1,2,4-thiadiazolyl. In another aspect of this embodiment, Het is an optionally substituted pyrimidinyl. In another aspect of this embodiment, Het is optionally substituted 1H-1,2,3-triazolyl. In another aspect of this embodiment, Het is an optionally substituted 1,3,4-oxadiazolyl. In another aspect of this embodiment, Het is an optionally substituted imidazo [1,2-b] pyridazinyl. In another aspect of this embodiment, Het is optionally substituted [1,2,4] triazolo [4,3-a] pyridinyl. In another aspect of this embodiment, Het is an optionally substituted thiophenyl. In another aspect of this embodiment, Het is optionally substituted 1,2,4-thiadiazolyl. In another aspect of this embodiment, Het is an optionally substituted pyrimidinyl. In another aspect of this embodiment, Het is optionally substituted 1H-1,2,3-triazolyl. In another aspect of this embodiment, Het is an optionally substituted 1,3,4-oxadiazolyl. In another aspect of this embodiment, Het is an optionally substituted imidazo [1,2-b] pyridazinyl. In another aspect of this embodiment, Het is optionally substituted 1,2,4-thiadiazolyl. In another aspect of this embodiment, Het is an optionally substituted pyrimidinyl. In another aspect of this embodiment, Het is optionally substituted 1H-1,2,3-triazolyl. In another aspect of this embodiment, Het is an optionally substituted 1,3,4-oxadiazolyl. In another aspect of this embodiment, Het is an optionally substituted imidazo [1,2-b] pyridazinyl. In another aspect of this embodiment, Het is optionally substituted 1,2,4-thiadiazolyl. In another aspect of this embodiment, Het is an optionally substituted pyrimidinyl. In another aspect of this embodiment, Het is optionally substituted 1H-1,2,3-triazolyl. In another aspect of this embodiment, Het is an optionally substituted 1,3,4-oxadiazolyl. In another aspect of this embodiment, Het is an optionally substituted imidazo [1,2-b] pyridazinyl.

In another embodiment, the compound of formula I or III is

<img file="PL2523950T3_D0011.tif" />

<img file="PL2523950T3_D0012.tif" />

- 9 - EP 2523950

<img file="PL2523950T3_D0013.tif" />

definitions

Unless otherwise stated, the following terms and phrases, as used in the description, are intended to have the following meanings. The fact that a specific term or phrase is not clearly defined should not be associated with indeterminacy or lack of precision, but rather it should be recognized that the terms used in the description were used in their typical meaning. In the case of the use of the trade names in the description, the intention of the applicants is independent

Refers to a product bearing the trade name and active pharmaceutical ingredient (s) of the product bearing this trade name.

The term "treatment", and its grammatical equivalents, when used in the context of treating a disease, means slowing or halting the progression of the disease or alleviating at least one symptom of the disease, and more preferably ameliorating more than one symptom of the disease. For example, treatment of hepatitis C virus infection may include a reduction in HCV viral load in a person infected with HCV and / or a reduction in the severity of icterus occurring in a subject infected with HCV.

"Alkyl" means a hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms. For example, an alkyl group may have from 1 to 20 carbon atoms (i.e., C1-C20 alkyl), from 1 to 10 carbon atoms (i.e., C1-C10 alkyl) or from 1 to 6 carbon atoms (i.e., C1-C6 alkyl). . Examples of suitable alkyl groups include (but are not limited to) methyl (Me, -CH 3), ethyl (Et, -CH 2 CH 3), 1-propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3), 2-propyl ( i-Pr, i-propyl, -CH (CH3) 2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH (CH3) 2), 2-butyl (s-Bu, s-butyl, -CH (CH3) CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C (CH3) 3),

1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 2 CH 3), 2-pentyl (-CH (CH 3) CH 2 CH 2 CH 3), 3-pentyl (-CH (CH 2 CH 3) 2),

2-methyl-2-butyl (-C (CH3) 2CH2CH3), 3-methyl-2-butyl (-CH (CH3) CH (CH3) 2), 3-methyl-1-butyl (-CH2CH2CH (CH3) 2 ), 2-methyl-1-butyl (-CH 2 CH (CH 3) CH 2 CH 3), 1-hexyl (-CH 2 CH 2 CH 2 CH 2 CH 2 CH 3), 2-hexyl (-CH (CH 3) CH 2 CH 2 CH 2 CH 3), 3-hexyl (-CH (CH 2 CH 3) (CH 2 CH 2 CH 3) ), 2-methyl-2-pentyl (-C (CH 3) 2 CH 2 CH 2 CH 3), 3-methyl-2-pentyl (-CH (CH 3) CH (CH 3) CH 2 CH 3), 4-methyl-2-pentyl (-CH (CH 3) ) CH 2 CH (CH 3) 2), 3-methyl-3-pentyl (-C (CH 3) (CH 2 CH 3) 2), 2-methyl-3-pentyl (-CH (CH 2 CH 3) CH (CH 3) 2), 2.3 -dimethyl-2-butyl (-C (CH3) 2 CH (CH3) 2), 3,3-dimethyl-2-butyl (-CH (CH3) C (CH3) 3 and octyl (- (CH2) 7CH3).

"Alkoxy" means a group having the formula -O-alkyl in which the alkyl group, as defined above, is attached to the parent molecule via an oxygen atom. The alkyl part of the alkoxy group may have from 1 to 20 carbon atoms (i.e., C1-C20 alkoxy), from 1 to 12 carbon atoms (i.e., C1-C12 alkoxy) or from 1 to 6 carbon atoms (i.e., C1-C6 alkoxy) . Examples of suitable alkoxy groups include (but are not limited to) methoxy (-O-CH3 or -OMe), ethoxy (-OCH2CH3 or -OEt), t-butoxy (-OC (CH3) 3 or -OtBu) and the like similar.

"Haloalkyl" means an alkyl group, as defined above, in which one or more of the hydrogen atoms of the alkyl group is replaced with a halogen atom. The alkyl part of the haloalkyl group may have from 1 to 20 carbon atoms (i.e., C1-C20 haloalkyl), from 1 to 12 carbon atoms (i.e., C1-C12 haloalkyl) or from 1 to 6 carbon atoms (i.e., C1-C6 alkyl). . Examples of suitable haloalkyl groups include (but are not limited to) -CF3, -CHF2, -CFH2, -CH2CF3 and the like.

"Alkenyl" means a hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, i.e. a sp 2 carbon-carbon double bond. For example, an alkenyl group may have from 2 to 20 carbon atoms (i.e., C2-C20 alkenyl), from 2 to 12 carbon atoms (i.e., C2-C12 alkenyl) or from 2 to 6 carbon atoms (i.e., C2-C6 alkenyl) . Examples of suitable alkenyl groups include (but are not limited to) vinyl (-CH = CH 2), allyl (-CH 2 CH = CH 2), cyclopentenyl (-C 5 H 7) and 5-hexenyl (-CH 2 CH 2 CH 2 CH 2 CH = CH 2).

"Alkynyl" means a hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, i.e. a carbon-carbon triple bond sp. For example an alkynyl group may have from 2 to 20 carbon atoms (i.e., C2-C20 alkynyl; ), from 2 to 12 carbon atoms (i.e., C2-C12 alkynyl) or from 2 to 6 carbon atoms (i.e., C2-C6 alkynyl). Examples of suitable alkynyl groups include (but are not limited to) acetylene (-CeCH), propargyl (-CH)₂CeCH) and the like.

"Alkylene" refers to a saturated, branched or straight chain radical or cyclic hydrocarbon radical having two monovalent radical centers derived from the removal of two hydrogen atoms from the same or from two different atoms

EP 2523950 of the parent carbon of the alkane. For example, an alkylene group may have from 1 to 20 carbon atoms, from 1 to 10 carbon atoms or from 1 to 6 carbon atoms. Typical alkylene radicals include (but are not limited to) methylene (-CH2-), 1,1-ethylene (-CH (CH3) -), 1,2-ethylene (-CH2CH2-), 1,1-propylene (- CH (CH 2 CH 3) -), 1,2-propylene (-CH 2 CH (CH 3) -), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -) and the like.

"Alkenylene" refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical having two monovalent radical centers derived from the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene. For example, an alkenylene group may have from 1 to 20 carbon atoms, from 1 to 10 carbon atoms or from 1 to 6 carbon atoms. Typical alkenylene radicals include (but are not limited to) 1,2-ethylene (-CH = CH-).

"Alkynyl" refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical having two monovalent radical centers derived from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkyne. For example, an alkynylene group may have from 1 to 20 carbon atoms, from 1 to 10 carbon atoms or from 1 to 6 carbon atoms. Typical alkynyl radicals include (but are not limited to) acetylene (-CeC-), propargyl (-CH)₂CeC-) and 4-pentynyl (-CH₂CH₂CH₂CeC-).

"Alkylyn" refers to a saturated, branched or straight-chain radical having two radical centers derived from the removal of three hydrogen atoms from two carbon atoms of a parent alkane. For example, an alkyl group may have from 2 to 20 carbon atoms, from 2 to 10 carbon atoms, or from 2 to 6 carbon atoms. Typical alkylin radicals include (but are not limited to) 1,2-ethyl (-CH2CH =), 1,2-propyl (-CH2C (CH3) =), 1,3-propyl (-CH2CH2CH =), 1 , 4-butylyn (-CH2CH2CH2CH =) and the like.

"Aryl" means a monovalent aromatic hydrocarbon radical obtained by removing one hydrogen atom from a single carbon atom of a parent aromatic ring system. For example, an aryl group may have from 6 to 20 carbon atoms, from 6 to 14 carbon atoms, or from 6 to 12 carbon atoms. Typical aryl groups include (but are not limited to) radicals derived from benzene (e.g., phenyl), substituted benzene, naphthalene, anthracene, biphenyl and the like.

"Arylene" refers to an aryl which is defined above having two monovalent radical centers derived from the removal of two hydrogen atoms from the same or from two different carbon atoms of a parent aryl. Typical arylene radicals include (but are not limited to) phenylene.

"Arylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal carbon atom or a sp3 carbon atom, is replaced with an aryl radical. Typical arylalkyl groups include (but are not limited to) benzyl, 2-phenylethan-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, naphthobenzyl, 2-naphthophenyletan-1-yl and the like. The aralkyl group may contain from 6 to 20 carbon atoms, e.g. the alkyl residue has from 1 to 6 carbon atoms and the aryl portion has from 6 to 14 carbon atoms.

"Cycloalkyl" refers to a saturated or partially unsaturated ring having from 3 to 7 carbon atoms in the case of a unicycle, from 7 to 12 carbon atoms in the case of a bicycle and up to about 20 carbon atoms in the case of a polycyclic. Monocyclic cycloalkyl groups have from 3 to 6 ring atoms, and usually 5 or 6 ring atoms. Bicyclic cycloalkyl groups have from 7 to 12 ring atoms, e.g. arranged in a bicyclic system (4,5), (5.5), (5,6) or (6,6), or 9 or 10 ring atoms arranged in a bicyclic system (5.6) or (6.6). The cycloalkyl groups include mono-, bi- and polycyclic hydrocarbon rings, whether coupled, bridged or spiro. Non-limiting examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, and

- 1-Cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, bicyclo [3.1.0] hex-6-yl and the like.

"Cycloalkylene" refers to a cycloalkyl group, as defined above, having two monovalent radical centers derived from the removal of two hydrogen atoms from the same or two different carbon atoms of a cycloalkyl parent. Typical cycloalkylene radicals include (but are not limited to) cyclopropylene, cyclobutylene, cyclopentylene and cyclohexylene.

"Cycloalkylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal carbon atom or a sp 3 carbon atom, is replaced with a cycloalkyl radical as defined above. Typical cycloalkylalkyl groups include (but are not limited to) cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexenylmethyl, 2-cyclohexylethan-1-yl, 2-cyclohexenyl ethan-1-yl, 2-cyclopropylethan-1-yl, 2-cyclopentyl ethane. -1-yl and the like. The cycloalkylalkyl group may contain from 4 to 26 carbon atoms, e.g. the alkyl residue has from 1 to 6 carbon atoms and the cycloalkyl moiety is as defined above.

"Cycloalkylalkylene" refers to a cycloalkylalkyl group, as defined above, having two monovalent radical centers derived from the removal of one hydrogen atom from a cycloalkylalkyl alkyl portion and from removal of a second hydrogen atom from a cycloalkyl portion of a cycloalkylalkyl. Non-limiting examples of cycloalkylalkylene radicals include:

<img file="PL2523950T3_D0014.tif" />

<img file="PL2523950T3_D0015.tif" />

"Halogen" refers to F, Cl, Br or I.

The term "haloalkyl" as used herein refers to an alkyl group as defined herein that is substituted with at least one halogen. Examples used in the description of branched or straight chain "haloalkyl" groups include (but are not limited to) methyl, ethyl, propyl, isopropyl, n-butyl and t-butyl independently substituted by one or more halogens, e.g. fluoro, chloro, bromo and iodo. The term "haloalkyl" should be interpreted to include substituents such as perfluoroalkyl groups such as -CF3.

The term "haloalkoxy" as used herein refers to the group -OR^andwhere R^and is a haloalkyl group, as defined herein. Non-limiting examples of haloalkoxy groups include -O (CH2) F, -O (CH) F2 and -OCF3.

The terms "heterocycle" or "heterocyclyl" refer to a saturated or partially saturated cyclic group having from 1 to 14 carbon atoms and from 1 to 6 heteroatoms selected from N, S, P or O and include single-ring and polycyclic systems, including conjugate ring systems. , bridged or spiro-type. The terms "heterocycle" or "heterocyclyl" as used herein include, without limitation, for example, the heterocycles described in Paquette, Leo A .; Principles of Modern Heterocyclic Chemistry (WA Benjamin, New York, 1968), especially in Chapters 1, 3, 4, 6, 7 and 9; The Chemistry of Heterocyclic Compounds, A Series of Monographs (John Wiley &amp; Sons, New York, 1950 to present), especially in volumes 13, 14, 16, 19 and 28; and J. Am. Chem. Soc. (1960) 82: 5566. In one embodiment, the atom (s)

The carbon, nitrogen, phosphorus or sulfur of the heterocyclic group may be oxidized to give C (= O), N-oxide, phosphine, sulfinyl or sulfonyl residues.

Substituted heterocyclyl include, as one example, heterocyclic rings substituted with any of the substituents disclosed herein, including oxo groups. A non-limiting example of a carbonyl substituted heterocyclyl is:

<img file="PL2523950T3_D0016.tif" />

Examples of heterocycles include, for example, but are not intended to be limiting, dihydropyridyl, tetrahydropyridyl (piperidyl), tetrahydrothiophenyl, sulfur-oxidised tetrahydrothiophenyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, azetidinyl, 2-pyrrolidonyl, tetrahydrofuranyl, decahydoquinolinyl, octahydroisoquinolinyl, pyranyl, morpholinyl and bis -tetrahydrofuranyl:

<img file="PL2523950T3_D0017.tif" />

"Heterocyclyl" or "heterocyclylene" refers to "heterocycle" or "heterocyclyl", as defined above, having two monovalent radical centers derived from the removal of two hydrogen atoms from the same or from two different carbon atoms of a parent heterocycle, from the removal of two hydrogen atoms from two the nitrogen atoms of the parent heterocycle or removing hydrogen from nitrogen and removing the hydrogen from the carbon of the parent heterocycle. Non-limiting examples of heterocyclic or heterocyclylene include:

<img file="PL2523950T3_D0018.tif" />

"Heteroaryl" refers to monovalent aromatic heterocyclyl having at least one ring heteroatom. Thus, "heteroaryl" refers to an aromatic group having from 1 to 14 carbon atoms and from 1 to 6 heteroatoms selected from oxygen, nitrogen, sulfur or phosphorus. In the case of polycyclic systems, for example, the term "heteroaryl" includes ring-linked, bridged or spiro-conjugated systems having aromatic and non-aromatic rings. In one embodiment, the ring (s) of carbon, nitrogen, or sulfur of the heteroaryl group may be oxidized to give C (= O), N-oxide, sulfinyl or sulfonyl residues.

Examples of heteroaryls include, but are not limited to, pyridyl, thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, tianaphenyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, 6H-1, 2,5-thiadiazinyl, 2H, 6H-1,5,2-dithiazinyl, thienyl, tiantrenyl, isobenzofuranyl, chromenyl, xantenyl, phenoxatinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl, naphthyridinyl,

- quinazolyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl, phenanthyrinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazine, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl , piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl and isatinoil. "Heterocyclylene" refers to a heterocyclyl, as defined herein, obtained by replacing a hydrogen atom from a carbon atom or heterocyclyl heteroatom with an open valency. Likewise, "heteroarylene" refers to aromatic heterocyclylene.

"Heterocyclylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal carbon atom or a sp3 carbon atom, is replaced with a heterocyclyl radical (i.e., a heterocyclyl-alkylene-residue). Typical heterocyclyl alkyl groups include (but are not limited to) heterocyclyl-CH2-,

2-heterocyclyl) ethan-1-yl and the like, wherein the "heterocyclyl" portion includes any one of the heterocyclyl groups described above, including those described in Principles of Modern Heterocyclic Chemistry. The skilled person will also understand that the heterocyclyl group may be attached to the alkyl part of the heterocyclyl alkyl via a carbon bond or a carbon-heteroatom bond, provided that it will be attached in a chemically stable manner. The thick heterocyclylalkyl contains from 2 to 20 carbon atoms and

1-6 heteroatoms, e.g. the alkyl part of the heterocyclylalkyl group contains from 1 to 6 carbon atoms and the heterocyclyl radical contains from 1 to 14 carbon atoms. Examples of heterocyclylalkyl include, for example, but are not intended to be limiting, 5-membered heterocycles containing sulfur, oxygen, phosphorus and / or nitrogen, such as pyrrolidinylmethyl,

2-tetrahydrofuranylethan-1-yl and the like, 6-membered sulfur, oxygen and / or nitrogen containing heterocycles such as piperidinylmethyl, morpholinylmethyl, piperidinylethyl, teterahydropyranyl and the like.

"Heteroarylalkyl" refers to an alkyl group, as defined herein, in which a hydrogen atom has been replaced with a heteroaryl group, as defined herein. Non-limiting examples of heteroarylalkyl include -CH2-pyridinyl, -CH2-pyrrolyl, -CH2-oxazolyl, -CH2-indolyl, -CH2-isoindolyl, -CH2-purinyl, -CH2-furanyl, -CH2-thienyl, -CH2-benzofuranyl, -CH2-benzothiophenyl, -CH2-carbazolyl, -CH2-imidazolyl, -CH2-thiazolyl, -CH2-isoxazolyl, -CH2-pyrazolyl, -CH2-isothiazolyl, -CH2-quinolyl, -CH2-isoquinolyl, - CH2-pyridazole, -CH2-pyrimidyl, -CH2-pyrazole, -CH (CH3) -pyridinyl, -CH (CH3) -pyrrolyl, - CH (CH3) -oxazolyl, -CH (CH3) -indolyl, -CH (CH3) -isoindolyl, -CH (CH3) -nynyl, -CH (CH3) -furanyl, - CH (CH3) -tinyl, -CH (CH3) -benzofuranyl, -CH (CH3) -benzothiophenyl, -CH (CH3) -carbazolyl, -CH (CH3) -imidazolyl, -CH (CH3) -thiazolyl, -CH (CH3) -isoxazolyl, -CH (CH3) -pyrazolyl,

The term "heterocyclyloxy" refers to a heterocyclyl group attached to an adjacent atom via oxygen.

If a sulfur atom is present, it may have different levels of oxidation, i.e. S, SO, SO2 or SO3. All of these oxidation levels are included within the scope of the invention.

If there is a phosphorus atom, it may have different levels of oxidation, i.e. POR^andR^bR^c, PO2R^andR^b or PO3R^andR^bwhere every R^and, R^b and R^c are independently selected from H, C1-12 alkyl, ^C2-12 alkenyl, C2-12 alkynyl, C6-14 aryl, 3-12-membered heterocycle, 3-18-membered heteroarylalkyl, C6-18 aralkyl; or two taken together (with or without oxygen atoms) form a 5 to 10-membered heterocycle. All of these oxidation levels are included within the scope of the invention.

The term "optionally substituted" with respect to a particular residue of a compound of the formula of the invention, for example an "optionally substituted aryl" refers to a residue having no or one or more substituents.

The term "substituted" with respect to alkyl, alkylene, aryl, aralkyl, alkoxy, heterocyclyl, heteroaryl, carbocyclyl, etc., e.g. "substituted alkyl", "substituted alkylene", "substituted aryl", "substituted aralkyl", "substituted heterocyclyl "I

- "substituted carbocyclyl" mean respectively alkyl, alkylene, aryl, aralkyl, heterocyclyl, carbocyclyl, in which one or more hydrogen atoms have been independently substituted with a non-hydrogen substituent. Divalent groups may also be substituted in a similar manner. Unless otherwise indicated, typical substituents include (but are not limited to) -X, -R^b, -ABOUT^-, = O, -OR^b, -SR^b, -S^-, -NR^b2

N⁺R^b3, = NR^b, -CX₃, -CN,

NHC (= O) R^b, -OC (= O) R^b, -NHC (= O) NR^b2, -S (= O) 2-, S (= O) R^b, -OP (= O) (OR^b) 2, -P (= O) (OR^b) 2,

-P (= O) (OH) 2, -P (O) (OR^b)(ABOUT^-), -C (= O) R^b, -C (= O) X, -C (S) R^b, -C (O) OR^b, -C (O) O^-, -C (S) OR^b .

-OCN, -SCN, -N = C = O, -NCS, -NO, -NO2, = N2, -N3,

-S (= O) 2OH, -S (= O) 2 R^b, - OS (= O) 2OR^b, -S (= O) 2 NR^b2

-P (= O) (O^-) 2,

-C (O) SR^b, -C (S) SR^b, -C (O) NR^b2, -C (S) NR^b2, -C (= NR^b) NR^b2, wherein each X is independently halogen: F, Cl, Br or I; and every R^b is independently H, alkyl, aryl, aralkyl, a heterocycle or a protecting group or a prodrug moiety. Alkylene, alkenylene and alkynylene groups may also be substituted in a similar manner. Unless otherwise indicated, when the term "substituted" is used in conjunction with groups such as aralkyl, which have two or more residues that may be substituted, the substituents may be attached to an aryl moiety, an alkyl moiety, or both.

Those skilled in the art will recognize that when residues such as "alkyl", "aryl", "heterocyclyl", etc. are substituted with one or more substituents, they can alternatively be referred to as "alkylene", "arylene", " heterocyclyl "etc. (i.e., indicating that at least one of the hydrogen atoms of the parent" alkyl "," aryl "," heterocyclyl "residues has been replaced with the indicated substituent (s)). When residues such as "alkyl", "aryl", "heterocyclyl", etc. are referred to herein as "substituted" or are shown schematically as substituted (or optionally substituted, e.g. when the number of substituents is from zero to a specified positive integer ), the terms "alkyl", "aryl", "heterocyclyl" etc. are to be understood as interchangeable with the terms "alkylene", "arylene", "heterocyclylene", etc.

Those skilled in the art will recognize that the compounds of the invention may exist in solvated or hydrated form. The scope of the invention includes such forms. Those skilled in the art will also know that the compounds of the invention can be esterified. Esters and other physiologically functional derivatives and prodrug forms of a compound of the invention are described herein.

"Ester" means any ester of the compound wherein any of the -COOH functionalities of the molecule is replaced by a -C (O) OR functional group or wherein any of the -OH functional groups are replaced by a -OC (O) R functional group in which the R ester residue is any carbon containing group that forms a stable ester residue, including (but not limited to) alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl and their substituted derivatives.

The term "prodrug" as used herein refers to any compound which upon administration into the biological system generates a therapeutic substance, i.e. an active ingredient, as a result of a spontaneous chemical reaction (s), a catalyzed chemical reaction (s). ) by enzyme, photolysis and / or metabolic chemical reaction (s). The prodrug is thus a covalently modified analog or latent form of the therapeutically active compound. Examples of prodrugs include ester moieties, quaternary ammonium moieties, glycol moieties, and the like.

One of skill in the art will recognize that substituents and other moieties of compounds of Formula I or III should be selected such that a compound will be sufficient and stable enough to provide a pharmaceutically useful compound from which a pharmaceutical composition with acceptable stability can be obtained. . Compounds of formula I that exhibit such stability are considered within the scope of the invention.

Those skilled in the art will recognize that the compounds of the invention may contain one or more chiral centers. The scope of the invention includes such forms.

- EP 2523950

The compound of formula I or III and its pharmaceutically acceptable salts may exist in the form of various polymorphs or pseudopolymorphs. The term "crystalline polymorphism" as used herein means the ability of a crystalline compound to exist in the form of various crystalline structures. Polymorphism can generally occur in response to changes in temperature, pressure or both. Polymorphism may also be the effect of changes in the crystallization process. Polymorphs can be distinguished based on various physical properties known in the art, such as X-ray diffraction patterns, solubility, and melting point. The crystalline polymorphism may be the result of differences in crystal packing (packing polymorphism) or differences in packing between different conformers of the same molecule (conformational polymorphism). The term "crystalline pseudopolymorphism" as used herein means the ability of a hydrate or solvate of a compound to exist in the form of various crystalline structures. The pseudopolymorphs of the invention may occur due to differences in crystal packing (pseudopolymorphism of packing) or due to differences in packing between different conformers of the same molecule (conformational pseudopolimorphism). The invention includes all polymorphs and pseudopolymorphs of compounds of Formulas I-II and their pharmaceutically acceptable salts. The pseudopolymorphs of the invention may occur due to differences in crystal packing (pseudopolymorphism of packing) or due to differences in packing between different conformers of the same molecule (conformational pseudopolimorphism). The invention includes all polymorphs and pseudopolymorphs of compounds of Formulas I-II and their pharmaceutically acceptable salts. The pseudopolymorphs of the invention may occur due to differences in crystal packing (pseudopolymorphism of packing) or due to differences in packing between different conformers of the same molecule (conformational pseudopolimorphism). The invention includes all polymorphs and pseudopolymorphs of compounds of Formulas I-II and their pharmaceutically acceptable salts.

The compound of formula I or III and its pharmaceutically acceptable salts may also exist in the form of an amorphous solid. The term amorphous solid as used in the description means a solid body in which there is no long range order in relation to the position of atoms in the solid. This definition also applies if the crystal size is two nanometers or less. To obtain the amorphous forms of the invention, additives, including solvents, may be used. The invention includes all amorphous forms of the compounds of formula I and their pharmaceutically acceptable salts.

Some of the compounds described herein contain one or more chiral centers or may otherwise be present in the form of multiple stereoisomers. The scope of the invention includes mixtures of stereoisomers as well as purified enantiomers or mixtures enriched enantiomerically / diastereomerically. The scope of the invention also includes the individual isomers of the compounds represented by the formula of the invention as well as their fully or partially balanced mixtures. The present invention also includes the individual isomers of the compounds represented by the above formulas as mixtures with their isomers wherein one or more chiral centers are inverted.

The term "chiral" refers to molecules that have the property of not being able to apply them to their own mirror image, while the term "achiral" refers to molecules that can be superimposed on their own mirror image.

The term "stereoisomers" refers to compounds that have identical chemical composition, but differ in the distribution of atoms or groups in space.

"Diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of each other. Diastereomers exhibit different physical properties, e.g. melting points, boiling points, spectral properties and reactivity. Diastereomer mixtures can be separated by high resolution analytical procedures, such as electrophoresis and chromatography.

The term "enantiomers" refers to the stereoisomers of a compound that are their intact non-charge mirror images.

The term "atropisomers" refers to the stereoisomers of a compound that arise from the inhibition of rotation around individual bonds in which stereoisomers of spatial latency for rotation are so large that it is possible to isolate individual conformers. Atropisomers exhibit axial chirality. Atropisomers can be thermally balanced, and the interconversion barrier can be measured kinetically. Atropisomycosis may occur irrespective of the presence of other forms of chiral isomerism.

- EP 2523950

The stereochemical definitions and rules used herein are generally in agreement with SP Parker, ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley &amp; Sons, Inc., New York.

Many organic compounds exist in optically active forms, i.e. they exhibit the ability to twist the plane of polarized light in the plane. When describing the optically active compound, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule around its chiral center (s). To indicate the sign of twisting of polarized light in a plane by a given compound, the prefixes di I or (+) and (-) are used, where (-) or I means that the compound is left-handed. The relationship to the prefixes (+) or d is dextrorotary.

The given stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate and may arise when stereoselection or stereospecificity are not present in a chemical reaction or in a chemical process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric forms devoid of optical activity.

The invention includes salts or solvates of the compounds described herein, including combinations thereof, such as a salt solvate. The compounds of the invention may exist in solvated forms, e.g. hydrated forms, as well as in unsolvated forms, and the invention encompasses all these forms.

Typically, but not necessarily, the salts of the invention are pharmaceutically acceptable salts. Salts encompassed by the term "pharmaceutically acceptable salts" mean nontoxic salts of the compounds of the invention.

Examples of suitable pharmaceutically acceptable salts include salts formed by the addition of an inorganic acid, such as chloride, bromide, sulfate, phosphate and nitrate; salts formed by the addition of an organic acid, such as acetate, galactarate, propionate, succinate, lactate, glycolate, malate, tartrate, citrate, maleate, fumarate, methanesulfonate, p-toluenesulfonate and ascorbate; salts with acidic amino acids, such as aspartate and glutamate; alkali metal salts, such as sodium salt and potassium salt; alkaline earth metal salts, such as magnesium salt and calcium salt; ammonium salt; organic basic salts, such as a trimethylamine salt, a triethylamine salt, a pyridine salt, a picolin salt, a dicyclohexylamine salt and a N, N'-dibenzylethylenediamine salt; and salts with basic amino acids, such as lysine salt and arginine salt. The salts may in some cases be hydrates or ethanol solvates.

The "approximately" modifier used in connection with the quantity takes into account the given value and has the meaning dictated by the context (eg it takes into account the error rate associated with the measurement of a given quantity).

Whenever the compound described herein is substituted with more than one same group, it should be understood that these groups may be the same or different, i.e. each group is selected independently. Wavy lines,

<img file="PL2523950T3_D0019.tif" />

indicate bonding sites in the form of covalent bonds with adjacent substructures, groups, residues or atoms.

In some cases, the compounds of the invention may also exist in the form of tautomeric isomers. Although only one delocalized resonance structure can be represented, all such forms are contemplated as being within the scope of the invention. Enamine tautomers can be for example for purine, pyrimidine,

Immunol, imidazole, guanidine, amidine and tetrazolium systems and all their possible tautomeric forms are included within the scope of the invention.

Selected substituents including compounds of Formula I or III may exist in a recurrent degree. In this context, a "recursive substituent" means that a substituent may specify a different occurrence of itself. Multiple specifications may be direct or indirect via a sequence of other substituents. Due to the recursive nature of such substituents, theoretically, in any embodiment, a large number of compounds may be present. A typical expert in the field of medicinal chemistry understands that the total number of such substituents is reasonably limited by the desired properties that one wants to obtain for a given compound. Such properties include, but are not limited to, physical properties such as molecular weight, solubility or log P, application properties, such as activity against the intended purpose, and practical properties such as ease of synthesis. Recursive substituents may be the intended aspect of the invention. For a typical expert in the field of medical chemistry, the universality of these substituents will be obvious. To the extent that the recursive substituents are in an embodiment of the invention, they may specify another occurrence of themselves 0, 1, 2, 3 or 4 times.

The compounds of formula I or III also include molecules that have inbuilt isotopes of atoms specified in specific molecules. Non-limiting examples of these isotopes include D, T,¹⁴C ¹³C ¹⁸O and ¹⁵N.

Protecting groups

In the context of the invention, protecting groups include prodrug and chemical moieties of protecting groups.

Protecting groups are available, commonly known and used, and are optionally used to prevent side reactions with the shielded group during synthetic procedures, i.e. during the pathways or methods of preparing the compounds of the invention. The decision about which groups should be screened, if at all, and the nature of the chemical group protecting the PG will depend on the reaction chemistry that should be protected (eg acidic, alkaline conditions, oxidizing, reducing or other) and the intended direction of synthesis. The PG groups need not be, and generally are not, the same if the compound is substituted with multiple PG groups. In general, the PG group will be used to shield functional groups, such as carboxyl, hydroxyl, thio or amino groups, and thus to prevent side reactions or otherwise to increase the efficiency of the synthesis process. The order of uncovering to obtain free, unobstructed groups depends on the intended direction of synthesis and on the reaction conditions that will be used, and the uncovering may occur in any order determined by the expert in the field.

Different functional groups of the compounds of the invention may be protected. For example, protecting groups for -OH groups (whether hydroxy, carboxylic acid, phosphonic acid or other functional groups) include "ether- or ester-forming groups". Ether or ester forming groups have the ability to function as chemical protecting groups in the synthetic schemes described herein. However, some hydroxyl-protecting groups and the thio group are neither ether forming groups nor ester-forming groups, of which those skilled in the art realize that these include amides as shown below.

A very large number of hydroxyl-protecting groups and amide-forming groups and the corresponding chemical cleavage reactions are described in Protective Groups in Organic Synthesis,

Theodora W. Greene and Peter GM Wuts (John Wiley &amp; Sons, Inc., New York, 1999, ISBN 0-47116019-9) ("Greene"). See also Kocienski, Philip J .; Protecting Groups (Georg Thieme Verlag Stuttgart, New York, 1994), which is incorporated herein by reference in its entirety. Particularly, Chapter 1, Protecting Groups: An Overview, pages 1-20, Chapter 2, Hydroxyl Protecting Groups,

EP 2523950 pages 21-94, section 3, Diol Protecting Groups, pp. 95-117, section 4, Carboxyl Protecting Groups, pp. 118-154, section 5, Carbonyl Protecting Groups, pp. 155-184. For protecting groups for carboxylic acid, phosphonic acid, phosphonate, sulphonic acid and other protecting groups for acids, see Greene, as shown below. These groups include, for example, but are not intended to be limiting, esters, amides, hydrazides and the like.

Protecting groups forming ether and ester

The ester-forming groups include: (1) phosphonate ester-forming groups such as phosphonamidate esters, phosphorothioate esters, phosphonate esters and phosphono-bis-amidates;

(2) carboxyl ester forming groups; and (3) sulfur ester forming groups such as sulfonate and sulfinate.

Metabolites of the compounds of the invention

The metabolic products of the compounds described herein may be formed in vivo, for example, by oxidation, reduction, hydrolysis, amidation, esterification and the like reactions to which a compound is administered primarily due to enzymatic processes. These products are usually identified by the preparation of an isotope labeled (e.g., C¹⁴ or H³a compound of the invention, parenterally administering it in a detectable dose (e.g., greater than about 0.5 mg / kg) to an animal, such as a rat, mouse, guinea pig, monkey, or human, waiting for a sufficiently long time to allow metabolic processes to occur ( usually from about 30 seconds to 30 hours), and isolation of its conversion products from urine, blood or other biological samples. These products are easily isolated because they are labeled (others are isolated by using antibodies that have the ability to bind epitopes that have survived in the metabolite). The metabolite structures are determined by conventional methods, e.g. by MS or NMR analysis. In general, metabolite analysis is performed in the same way as conventional drug metabolism studies that are well known to those skilled in the art.

Described and illustrated are definitions and substituents of the compounds of the invention of various genera and subgenuses. The skilled person should be aware that by using any combination of the definitions and substituents set forth above, no unrealistic forms or relationships should be obtained. The term "unreal forms or compounds" means structures of compounds that violate appropriate scientific rules (such as, for example, compounds in which the carbon atom forms more than four covalent bonds), or compounds that are too unstable to be isolated and obtained from these are pharmaceutically acceptable dosage forms.

Pharmaceutical formulations

Formulations with conventional carriers and excipients are formulated for the compounds of the invention, which are selected in accordance with standard practice. The tablets will contain excipients, lubricants, fillers, binders and the like. The aqueous formulations are prepared in a sterile form, and if they are intended to be administered via a non-oral route, they will generally be isotonic. All formulations will optionally include excipients, such as those set out in the Handbook of Pharmaceutical Excipients (1986), the manual incorporated herein by reference in its entirety. Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkyl cellulose, hydroxyalkyl methyl cellulose, acid

- stearin and the like. The pH of the formulation is in the range of from about 3 to about 11, but is usually from about 7 to about 10.

Although it is possible to administer the active ingredients themselves, it may be advantageous to prepare them in the form of pharmaceutical formulations. The formulations of the invention, for both veterinary and human applications, contain at least one active ingredient together with one or more acceptable carriers and optionally together with other therapeutic components. The carrier (s) must (must) be &amp; quot; acceptable &amp; quot; in the sense that it must (must) be compatible with the other ingredients of the formulation and that it must (must) be physiologically harmless to the recipient .

The formulations include those carriers that are suitable for the aforementioned routes of administration. The formulations may for convenience be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Techniques and formulations can generally be found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.), A publication incorporated herein by reference in its entirety. The methods include the step of combining the active ingredient with the carrier, which consists of one or more additional ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or disintegrated solid carriers or both, and then, if necessary, by shaping the product.

Formulations of the invention suitable for oral administration may be in the form of discrete units, such as capsules, cachets or tablets, each containing a predetermined quantity of active ingredient; in the form of a powder or granules; in the form of a solution or a suspension in an aqueous or non-aqueous liquid; or in the form of an oil-in-water liquid emulsion or in the form of a water-in-oil liquid emulsion. The active ingredient can also be administered in the form of a bolus, electuary or paste.

The tablet is made by compression or molding, optionally with one or more additional ingredients. Compressed tablets may be made by compressing in a suitable machine the active ingredient in a free-floating form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active agent or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. Tablets may optionally be coated or scored and optionally formulated so as to provide a sustained or controlled release of the active ingredient.

When administered to the eye or other external tissues, e.g. to the mouth and skin, the formulations are preferably applied topically in the form of an ointment or cream containing the active ingredient (s) in an amount of, for example, from 0.075 to 20 % w / w (including content of active ingredient (s) in the range between 0.1% and 20% in increments of 0.1% w / w, for example 0.6% w / w, 0.7% w / w, etc.) , preferably from 0.2 to 15% w / w, and most preferably from 0.5 to 10% w / w. In the case of ointment formulations, the active ingredients may be used with either a paraffinic or water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base.

If desired, the aqueous phase of the cream base may contain, for example, at least 30% w / w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups, such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof. The topical formulations may desirably include a compound that enhances the absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such penetration enhancers include dimethylsulfoxide and related analogs.

The oily phase of the emulsions according to the invention can be obtained from known components in a known manner. Although this phase may contain only an emulsifier (otherwise known as an emulsifier),

It preferably contains a mixture of at least one emulsifier with a fat or oil or with both fat and oil. Preferably, the hydrophilic emulsifier is present together with a lipophilic emulsifier that acts as a stabilizer. It is also beneficial to include both oil and fat. The emulsifier (s) with or without the stabilizer (s) together form a so-called emulsifying wax, and the wax together with the oil and fat form a so-called emulsifying base for the ointment that forms the oil dispersed phase of the cream formulation.

Examples of emulsifiers and emulsion stabilizers suitable for use in the formulation of the invention include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

The choice of suitable oils or fats for the formulation depends on the desired cosmetic properties that you want to achieve. The cream should preferably be a non-greasy, non-staining and washable product with a suitable consistency to ensure that it will not leak from tubes or other containers. Straight or branched, mono- or di-basic alkyl esters such as diisodipate, isocetyl stearate, coconut fatty acid diethyl glycol diester, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a branched chain ester mixture known as Crodamol CAP, with the most preferred esters being the last three mentioned. They can be used alone or in combination, depending on the properties required.

The pharmaceutical formulations of the invention contain one or more compounds of the invention together with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents. Pharmaceutical formulations containing the active ingredient may be in any form suitable for the intended method of administration. For example, for oral use, tablets, pastilles, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compositions for oral administration may be prepared according to any method known in the art for the preparation of pharmaceutical compositions, and such compositions may contain one or more agents, including sweetening agents, flavoring agents, coloring agents and preservatives that ensure a pleasant taste preparation. Tablets containing the active ingredient mixed with a non-toxic pharmaceutically acceptable excipient that is suitable for the preparation of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binding agents, such as cellulose, microcrystalline cellulose, starch, gelatin or acacia; and slime agents, such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques, including by microencapsulation, to delay disintegration and adsorption in the gastrointestinal tract and thereby to obtain long-term action over a longer period of time. For example, a time delay material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax may be used.

Formulations for oral use may also be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert diluent in the form of a solid, for example with calcium phosphate or kaolin, or in the form of soft gelatin capsules for which the active ingredient is mixed. with water or with an oil medium, such as peanut oil, liquid paraffin or olive oil.

- 22 - EP 2523950

The aqueous suspensions according to the invention contain active materials mixed with excipients suitable for the preparation of aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents, such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of alkylene oxide with acid fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long-chain aliphatic alcohol (e.g., heptadecetylene-oxycetanol), a condensation product of ethylene oxide with a partial ester derived from fatty acid and hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate).

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those described herein, and flavoring agents may be added to provide a pleasant oral preparation. These compositions can be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules of the invention suitable for preparing an aqueous suspension by the addition of water comprise an active ingredient mixed with a dispersing or wetting agent, a suspending agent and one or more preservatives. Examples of suitable dispersing or wetting agents and suspending agents are disclosed above. Additional excipients, e.g. sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture thereof. Suitable emulsifying agents include naturally occurring gums, such as acacia gum and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters from ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose.

The pharmaceutical compositions of the invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to methods known in the art using suitable dispersing or wetting agents and suspending agents which are mentioned herein. The sterile injectable preparation may also be a sterile solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol, or it may be prepared as a lyophilized powder. Acceptable vehicles and solvents that may be used include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile solidified oils can conventionally be used as a solvent or suspending medium. For this purpose, any inert solid oil may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may also be used in the preparation of injectables.

- EP 2523950

The amount of active ingredient that can be combined with the carrier material to obtain a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a time-release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of active material associated with a suitable and practical in use amount of carrier material that may range from about 5 to about 95% of the total composition (weight: weight). The pharmaceutical composition can be prepared so that readily measurable amounts for administration are obtained. For example, an aqueous solution for intravenous infusion may contain from about 3 to 500 μg of active ingredient per milliliter of the solution so that an infusion can be made at the rate of about 30 ml / hour.

Formulations suitable for ocular administration include eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient. The active ingredient is preferably present in such formulations at a concentration of from 0.5 to 20%, more preferably from 0.5 to 10%, in particular at a concentration of about 1.5% w / w.

Formulations suitable for topical administration to the mouth include lozenges comprising the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles containing the active ingredient in an inert basis such as gelatin and glycerol or sucrose and acacia; and mouthwashes containing the active ingredient in a suitable liquid carrier.

Formulations for rectal administration may be in the form of a suppository with a suitable base containing, for example, cocoa butter or a salicylate.

Formulations suitable for pulmonary administration or for nasal administration have a particle size of, for example, in the range of from 0.1 to 500 μm (including particle sizes in the range of between 0.1 and 500 μm in increments of, e.g. 0.5 μm, 1 μm, 30 μm, 35 μm). μm, etc.) and are administered by means of rapid nasal inhalation or inhalation through the mouth to reach the alveoli. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration can be prepared according to conventional methods and can be provided with other therapeutic agents, such as compounds used so far in the treatment or prophylaxis of infections as described herein.

Formulations suitable for vaginal administration may be in the form of pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers that are considered suitable in the art.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions that may contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the recipient for which they are intended; and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents.

The formulations are in unit dose or multi-dose containers, e.g. in sealed ampoules and vials, and can be stored lyophilized, requiring only the addition of a sterile liquid carrier, e.g. water for injection, immediately before use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the previously described types. Preferred unit dose formulations are those comprising a daily dose of the active ingredient or a unit daily sub-dose thereof, as specified above in the description, or a suitable fraction thereof.

It is to be understood that in addition to the ingredients in a particular manner, the formulations of the invention mentioned above may contain other agents conventionally used in

In view of the type of formulation in question, for example those suitable for oral administration may contain flavorings.

The compounds of the invention may also be formulated so as to provide controlled release of the active ingredient, allowing dosage at longer intervals or improving the pharmacokinetic profile or toxicity profile of the active ingredient. Accordingly, the invention also provides compositions comprising one or more compounds of the invention from which the formulations with sustained or controlled release are obtained.

The effective dose of the active ingredient depends at least on the nature of the condition being treated, the toxicity, whether the compound is used prophylactically (lower doses) or in the case of active viral infection, delivery method and pharmaceutical formulation and will be determined by the physician using conventional dose escalation studies. It can be expected that the effective dose will be from about 0.0001 to about 100 mg / kg body weight per day; typically from about 0.01 to about 10 mg / kg body weight per day; even more often from about .01 to about 5 mg / kg body weight per day; most often from about .05 to about 0.5 mg / kg body weight per day. For example, a candidate daily dose for an adult with a body weight of approximately 70 kg will range from 1 mg to 1000 mg, preferably between 5 mg and 500 mg,

In yet another embodiment, the application discloses pharmaceutical compositions comprising a compound of formula I and / or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient.

Routes of administration

One or more compounds of the invention (referred to herein as active ingredients) are administered by any route appropriate to the condition to be treated. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) and the like. It should be recognized that the preferred route of administration may vary depending on, for example, the condition receiving the treatment. An advantage of the compounds of the invention is that they are bioavailable when administered orally and can be administered in this way.

Combination treatment, including combined HCV

In another embodiment, the compounds of the invention may be combined with one or more active agents. Non-limiting examples of suitable combinations include combinations of one or more compounds of the invention with one or more interferons, ribavirin or analogues thereof, HCV NS3 protease inhibitors, NS5a inhibitors, alpha-glucosidase inhibitors 1, hepatoprotectants, mevalonate decarboxylase antagonists, renin-angiotensin antagonists, other anti-fibrotic agents, nucleoside or nucleotide inhibitors of HCV NS5B polymerase, HCV NS5B polymerase inhibitors, HCV NS5A inhibitors, TLR-7 agonists, cyclophilin inhibitors, HCV IRES inhibitors, pharmacokinetic enhancers, and other HCV therapeutics.

More specifically, one or more compounds of the invention may be combined with one or more compounds selected from the group consisting of the following compounds:

1) interferons, e.g. pegylated rIFN-alpha 2b (PEG-Intron), pegylated rIFN-alpha 2a (Pegasys), rIFN-alpha 2b (Intron A), rIFN-alpha 2a (Roferon-A), interferon alpha (MOR- 22, OPC-18, Alfaferone, Alfanative, Multiferon, subalin), interferon alfacon-1 (Infergen), interferon alpha-n1 (Wellferon), interferon alpha-n3 (Alferon), interferon-beta (Avonex, DL-8234), interferon-omega

- EP 2523950 (omega DUROS, Biomed 510), albinterferon alfa-2b (Albuferon), IFN alpha XL, BLX-883 (Locteron), DA-3021, glycosylated interferon alfa-2b (AVI-005), PEG-Infergen, PEGylated interferon lambda (PEGylated IL-29) and a belerophone;

2) ribavirin and its analogues, e.g. ribavirin (Rebetol, Copegus) and tarenavirine (Viramidine);

3) HCV NS3 protease inhibitors, e.g. boceprevir (SCH-503034, SCH-7), telaprevir (VX-950), VX-813, TMC-435 (TMC435350), ABT-450, BI-201335, BI-1230, MK-7009, SCH-900518, VBY-376, VX-500, GS-9256, GS-9451, BMS-790052, BMS-605339, PHX-1766, AS-101, YH-5258, YH5530, YH5531 and ITMN- 191 (R-7227);

4) alpha glucosidase 1 inhibitors, e.g. celgosivir (MX-3253), Miglitol and UT-231 B;

5) hepatoprotectors, e.g. emericasan (IDN-6556), ME-3738, GS-9450 (LB-84451), silibilin and MitoQ;

6) nucleoside or nucleotide inhibitors of HCV NS5B polymerase, e.g. R1626, R7128 (R4048), IDX184, IDX-102, PSI-7851, BCX-4678, valopicitabine (NM-283) and MK-0608;

7) HCV NS5B non-nucleoside inhibitors, e.g. filibruv (PF-868554), ABT-333, ABT-072, BI-207127, VCH-759, VCH-916, JTK-652, MK-3281, VBY-708, VCH -222, A848837, ANA-598, GL60667, GL59728, A-63890, A-48773, A-48547, BC-2329, VCH-796 (nesbuvir), GSK625433, BILN-1941, XTL-2125 and GS-9190;

8) HCV NS5A inhibitors, e.g. AZD-2836 (A-831), AZD-7295 (A-689) and BMS-790052;

9) TLR-7 agonists, e.g. imiquimod, 852A, GS-9524, ANA-773, ANA-975, AZD-8848 (DSP-3025), PF-04878691 and SM-360320;

10) cyclophilin inhibitors, e.g. DEBIO-025, SCY-635 and NIM811;

11) HCV IRES inhibitors, e.g. MCI-067;

12) substances that improve pharmacokinetic properties, e.g. BAS-100, SPI-452, PF-4194477, TMC-41629, GS-9350, GS-9585 and roxithromycin;

13) other drugs for the treatment of HCV, e.g. thymosin alpha 1 (Zadaxin), nitazoxanide (Alinea, NTZ), BIVN-401 (virostat), PYN-17 (altirex), KPE02003002, actilon (CPG-10101), GS-9525 , KRN-7000, civacir, GI-5005, XTL-6865, BIT225, PTX-111, ITX2865, TT-033i, ANA 971, NOV-205, tarvacin, EHC-18, VGX-410C, EMZ-702, AVI 4065 , BMS-650032, BMS-791325, Bawituximab, MDX-1106 (ONO-4538), Oglufanid, FK-788 and VX-497 (merimepodib);

14) mevalonate decarboxylase antagonists, e.g. statins, HMGCoA synthase inhibitors (e.g., hymeglusin), squalene synthesis inhibitors (e.g., scrapylic acid);

15) angiotensin II receptor antagonists, e.g. losartan, irbesartan, olmesartan, candesartan, valsartan, telmisartan, eprosartan;

16) angiotensin converting enzyme inhibitors, e.g. captopril, zofenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, fosinopril;

17) other anti-fibrotic agents, e.g. amiloride and

18) endothelin antagonists, e.g. bosentan and ambrisentan.

In yet another embodiment, the application discloses pharmaceutical compositions comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, in combination with at least one additional active agent and a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient. In yet another embodiment, the application provides a combination of a pharmaceutical agent with two or more therapeutic agents in a unit dosage form. Thus, it is also possible to combine any compound of the invention with one or more active agents in a unit dosage form.

- EP 2523950

Combination therapy can be administered in simultaneous or sequential mode. In the case of sequential administration, the combination may be administered in two or more administrations.

Co-administration of a compound of the invention with one or more other active agents generally refers to the simultaneous or sequential administration of a compound of the invention and one or more other active agents, such that a therapeutically effective amount of a compound of the invention and one or more other active agents are present together in the patient's body.

Co-administration includes administration of unit dosages of the compounds of the invention prior to or after a single dose of one or more other active agents, e.g., administering the compounds of the invention with a difference of a few seconds, minutes or hours to / from the administration of one or more other agents active. For example, a unit dose of a compound of the invention may be administered in the first instance, and a unit dose of one or more other active agents will be administered within a few seconds or minutes. Alternatively, a unit dose of one or more other active agents may be administered first, followed by a unit dose of a compound of the invention in a few minutes or seconds. In some cases, it may be desirable to administer a unit dose of a compound of the invention in the first place, after which, after a period of several hours (e.g., 1-12 hours), a unit dose of one or more other active agents will be administered. In other cases, it may be desirable to administer a unit dose of one or more other active agents first and then, after a period of several hours (e.g., 1-12 hours), a unit dose of a compound of the invention will be administered.

Combination therapy may provide "synergy" and "synergistic effect", i.e. a situation where the active ingredients used together give a better effect than the sum of the effects that are obtained when using these compounds separately. A synergistic effect can be achieved when the active ingredients are: (1) combined in one formulation and administered or delivered simultaneously in a combined formulation; (2) delivered alternately or in parallel in separate formulations; or (3) in some other scheme. For the alternate treatment delivery, a synergistic effect can be achieved when the compounds are administered or delivered sequentially, e.g. in separate tablets, pills or capsules, or by separate injections in different syringes.

The application discloses a compound for use in methods of treating HCV in a patient, which methods include: administering to the subject a therapeutically effective amount of a compound of Formula I or III or a pharmaceutically acceptable salt, pharmaceutically acceptable solvate and / or ester thereof. In a preferred aspect, the compound of Formula I or III is, at least, 70% single diastereomer, 80% single diastereomer, 90% single diastereomer, or most preferably 95% single diastereomer.

The application also discloses a compound for use in methods of treating HCV in a patient, which methods include: administering to the subject a therapeutically effective amount of a compound of formula I or III, or a pharmaceutically acceptable salt, pharmaceutically acceptable solvate and / or ester thereof, and at least one additional active healing factor. In a preferred aspect, the compound of Formula I or III is, at least, 70% single diastereomer, 80% single diastereomer, 90% single diastereomer, or most preferably 95% single diastereomer.

The application also discloses a compound for use in methods of treating HCV in a patient, which methods include: administering to the subject a therapeutically effective amount of a compound of formula I or III, or a pharmaceutically acceptable salt thereof, pharmaceutically acceptable

- a solvate and / or ester, and at least one additional active therapeutic agent selected from the group consisting of one or more compounds of the invention with one or more interferons, ribavirin or its analogues, HCV NS3 protease inhibitors, inhibitors NS5a, alphaglucosidase 1 inhibitors, hepatoprotectants, mevalonate decarboxylase antagonists, renin-angiotensin system antagonists, other anti-fibrotic agents, nucleoside or nucleotide inhibitors of HCV NS5B polymerase, HCV NS5B polymerase inhibitors, HCV NS5A inhibitors, TLR-7 agonists, cyclophilin inhibitors, IRES inhibitors HCV, substances that improve the pharmacokinetics and other drugs for the treatment of HCV. In a preferred aspect, the compound of Formula I or III is,

In yet another embodiment, the application provides the use of a compound of Formula I or III, or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate and / or ester, for the preparation of a medicament for the treatment of an HCV infection in a patient. In a preferred aspect of this embodiment, the compound of formula I or III is, at least, 70% single diastereomer, 80% single diastereomer, 90% single diastereomer or most preferably 95% single diastereomer.

In yet another embodiment, the application provides the use of a compound of Formula I or III, or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate and / or ester, in the treatment of HCV infection. In a preferred aspect of this embodiment, the compound of formula I or III is, at least, 70% single diastereomer, 80% single diastereomer, 90% single diastereomer or most preferably 95% single diastereomer.

Those skilled in the art will recognize that treatment of a viral infection such as HCV can be characterized in many ways and measured using a variety of endpoints. The scope of the invention is intended to cover all such characteristics.

Examples of synthesis

In the detailed description of experiments, specific abbreviations and acronyms were used. Table 1 lists many of the abbreviations and acronyms used, although most of them will be understood by those skilled in the art.

Table 1. List of abbreviations and acronyms.

<td>Abbreviation</td><td>Importance</td>

<td>ac</td><td>acetyl</td>

<td>ACN</td><td>acetonitrile</td>

<td>AIBN</td><td>2,2'-azobis (2-methylpropionitrile)</td>

<td>BINAP</td><td>2,2'-bis (diphenylphosphino) -1,1'-binaphthyl</td>

<td>Bn</td><td>benzyl</td>

<td>BnBr</td><td>benzyl bromide</td>

<td>BSA</td><td>bis (trimethylsilyl) acetamide</td>

<td>BzCI</td><td>benzoyl chloride</td>

<td>CDI</td><td>carbonyldiimidazole</td>

<td>DABCO</td><td>1,4-diazabicyclo [2.2.2] octane</td>

- EP 2523950

<td>cares</td><td>dibenzylideneacetone</td>

<td>DBN</td><td>1,5-diazabicyclo [4.3.0] non-5-ene</td>

<td>DDQ</td><td>2,3-dichloro-5,6-dicyano-1,4-benzoquinone</td>

<td>DBU</td><td>1,5-diazabicyclo [5.4.0] undec-5-ene</td>

<td>DCA</td><td>dichloroacetamide</td>

<td>DCC</td><td>dicyclohexylcarbodiimide</td>

<td>DCE</td><td>1,2-dichloroethane</td>

<td>DCM</td><td>dichloromethane</td>

<td>Stop.</td><td>degrees</td>

<td>DIAD</td><td>diisopropylazodicarboxylate</td>

<td>DIEA</td><td>N, N- diisopropylethylamine</td>

<td>DMAP</td><td>4-dimethylaminopyridine</td>

<td>DME</td><td>1,2-dimethoxyethane</td>

<td>DMTCl</td><td>dimethoxytrityl chloride</td>

<td>DMSO</td><td>dimethyl sulfoxide</td>

<td>DMTr</td><td>4,4'-dimetoksytrytyl</td>

<td>DMF</td><td>dimethylformamide</td>

<td>EtOAc</td><td>ethyl acetate</td>

<td>ES, ESI</td><td>electrospray ionization</td>

<td>HMDS</td><td>hexamethyldisilazane</td>

<td>HPLC</td><td>high-pressure liquid chromatography</td>

<td>LC</td><td>liquid chromatography</td>

<td>LDA</td><td>lithium diisopropylamide</td>

<td>LRMS</td><td>low-resolution mass spectrum</td>

<td>MCPBA</td><td>meta-chloroperbenzoic acid</td>

<td>MeCN</td><td>acetonitrile</td>

<td>MeOH</td><td>methanol</td>

<td>MMTC</td><td>monomethoxythritol chloride</td>

<td>m / z or m / e</td><td>mass-to-load ratio</td>

<td>MH +</td><td>weight plus 1</td>

<td>MH^-</td><td>mass minus 1</td>

<td>MsOH</td><td>methanesulfonic acid</td>

<td>MS or ms</td><td>mass spectrum</td>

<td>NBS</td><td>N-bromosuccinimide</td>

<td>Virgin Mary</td><td>N-methylpyrrolidone</td>

<td>ph</td><td>phenyl</td>

- EP 2523950

<td>room temp.</td><td>room temperature</td>

<td>TBAF</td><td>tetrabutylammonium fluoride</td>

<td>TES</td><td>triethylsilyl</td>

<td>THF</td><td>tetrahydrofuran</td>

<td>THP</td><td>tetrahydropyran</td>

<td>TMSCl</td><td>chlorotrimethylsilane</td>

<td>TMSBr</td><td>bromotrimethylsilane</td>

<td>TMSI</td><td>iodotrimethylsilane</td>

<td>TMSOTf</td><td>(Trimethylsilyl) trifluoromethanesulfonate</td>

<td>TEA</td><td>triethylamine</td>

<td>TBA</td><td>tributylamine</td>

<td>TBAP</td><td>tributylammonium pyrophosphate</td>

<td>TBSCl</td><td>t-butyldimethylsilyl chloride</td>

<td>TEAB</td><td>Triethylammonium bicarbonate</td>

<td>TFA</td><td>trifluoroacetic acid</td>

<td>TLC or tlc</td><td>thin layer chromatography</td>

<td>tr</td><td>triphenylmethyl</td>

<td>tol</td><td>4-methylbenzoyl</td>

<td>Turbo Grignard</td><td>a 1: 1 mixture of isopropylmagnesium chloride and lithium chloride</td>

<td>xantphos</td><td>4,5-bis (diphenylphosphino) -9,9-dimethylxanthene</td>

<td>δ</td><td>parts per million downfield from tetramethylsilane</td>

General schemes

The compounds of the invention can be synthesized by means of several pathways comprising the steps of forming key bonds as shown in AC schemes wherein the carboxylate substituent R is either a protecting group, such as an alkyl ester (where necessary), or the free acid itself. The alkyl ester protecting group can easily be removed by saponification with an alkali metal hydroxide in a protic solvent, such as water or alcohol, and this process can be further facilitated by the use of ethereal solvent and / or heating mixtures. Alternatively, they can be removed by dealkylation by heating with an alkali metal halide in an aprotic solvent. As can be seen, the substituents on Het may be modified following the other linking steps through, e.g.

EP 2523950

- 30 Scheme A

<img file="PL2523950T3_D0020.tif" />

The bond between L and Het can be formed by displacing X at Het, where X is a leaving group such as a halide, a sulfinate, a sulfonate or a phosphate residue. The reaction is readily carried out by deprotonation of LH with a base such as sodium hydride or potassium hexamethyldisilazide, or further facilitated by the presence of a quaternary amine; the reaction can be carried out in a variety of solvents such as THF, dioxane, dichloromethane, NMP, DMF or DMSO, and can be accelerated by heating.

Scheme B

<img file="PL2523950T3_D0021.tif" />

Binding between R³ and L may be formed by nucleophilic displacement of the leaving group X at R³. The leaving groups can be very different and include (but are not limited to) halide, carboxylate, sulfinate, sulfonate or phosphate residues, and can be formed from the corresponding alcohol in situ by treatment with reagents such as dialkyl azodicarboxylates. The reaction may also be facilitated by the deprotonation of Het-LH with a base such as sodium hydride or potassium hexamethyldisilazide, or it is facilitated by the presence of a quaternary amine; the reaction can be carried out in a variety of solvents such as THF, dioxane, dichloromethane, NMP, DMF or DMSO, and can be accelerated by heating.

Diagram C

<img file="PL2523950T3_D0022.tif" />

<img file="PL2523950T3_D0023.tif" />

The starting material in Scheme A can be synthesized as shown in Scheme C. Substituted 3-aminothiophenes II can be formed by reductive amination of the YR compound³-LR (where Y is an aldehyde or a ketone, and R and R^and are optional protecting groups) or by direct alkylation (where Y is a leaving group such as a halide, sulfinate, sulfonate or phosphate residue) of 3-aminothiophene I (see application

- WO2008 / 58393). In the latter case, the alkylation can be facilitated by the deprotonation of the amine with a base such as sodium hydride or potassium hexamethyldisilazide, and can be carried out in a variety of solvents such as THF, dioxane, dichloromethane, NMP, DMF or DMSO, and can be further accelerated. through heating. In cases when R³ is the aromatic reaction can be catalyzed by Pd (J. Org. Chem., 2000, 65, 1158-1174). Alternatively, compound II can be formed by coupling the amine to 3-iodothiophene IV catalyzed by Pd (J. Org. Chem., 2000, 65, 1158-1174). Amine II is converted to amide III by acylation with a carboxylic acid derivative, such as an acyl chloride or anhydride, in the presence of a base such as pyridine, or a quaternary amine in an inert solvent such as dichloromethane. Alternatively, compound IV can be converted to compound III directly via Cu-catalysed amidation (J. Am. Chem. Soc., 2002, 124, 7421-7428).

The starting material for Scheme B can be formed in an analogous manner, wherein the leaving group X is formed in the final step by standard methods from the precursor alcohol.

The synthesis of iodothiophene IV is illustrated below for the case where R¹ = tBu, and other variants can be synthesized in the same way:

Diagram D

<img file="PL2523950T3_D0024.tif" />

nBuLi (2.2 eq.) THF, -78 ° C, 1 h followed by I₂, THF

<img file="PL2523950T3_D0025.tif" />

65%

To a solution of 5- (3,3-dimethyl-but-1-ynyl) -thiophene-2-carboxylic acid (6.2 g, 30 mmol, see US Patent 5,851,421) in THF (100 mL) was added a dropwise solution of nBuLi (at 2.0 M in pentane, 33 ml, 66 mmol) at -78 ° C. After the addition, the reaction mixture was stirred at -78 ° C for 1 h. A solution of I 2 (7.7 g, 30 mmol) in THF (100 mL) was slowly added to the flask (over about 15 min). After a further 10 minutes, the reaction was quenched with a 1 N HCl solution (50 mL) and the reaction was warmed to room temperature. The volatiles were removed in vacuo and the residue was dissolved in ether (500 mL). The organic solution was washed with a 1 M Na2S2O3 solution (100 mL x 2), brine (100 mL), and then dried over Na2SO4.

Diagram E

<img file="PL2523950T3_D0026.tif" />

To a solution of 5- (3,3-dimethyl-but-1-ynyl) -3-iodo-thiophene-2-carboxylic acid (1.0 g, 3.0 mmol) and DMF (20 μθ in dry dichloromethane (10 ml) was added oxalyl chloride (508 μL, 6.0 mmol) at room temperature. After stirring at room temperature for 90 min the reaction mixture was concentrated in vacuo to remove volatiles The residue was dissolved in pyridine (5 mL) and methanol (5 mL) and stirred for 2 h. The volatiles were removed in vacuo and the residue was partitioned between ether (150 mL) and saturated NH 4 Cl solution

- EP 2523950 (50 ml). The organic layer was washed with saturated NH 4 Cl solution (50 mL) and dried over Na 2 SO 4. After concentration in vacuo, the residue was purified by silica gel chromatography (EtOAc / hexanes) to give the desired product (835 mg, 80%).

5- (3,3-dimethyl-but-1-ynyl) -3-iodo-thiophene-2-cynthesis of carboxylic acid ester

<img file="PL2523950T3_D0027.tif" />

A mixture of 5-bromo-thiophene-2-carboxylic acid ethyl ester (7 g, 30 mmol), copper iodide (1.2 g, 6 mmol), triethylamine (20 mL) in DMF (100 mL) was degassed in a 350 mL pressure bottle. . Then tris (dibenzylideneacetone) dipalladium (0) (2.1 g, 3 mmol) and 3,3-dimethyl-but-1-yn (18.3 ml, 150 mmol) were added and heated at the temperature of steps for 3 hours. The reaction mixture was filtered through celite and washed with ethyl acetate. The solution was diluted with water and extracted twice with ethyl acetate. The organic phases were combined and washed with water. After drying and concentration, the impure residue was purified by flash chromatography to give 6.9 g (95%) of 5- (3,3-dimethyl-but-1-ynyl) -thiophene-2-carboxylic acid ethyl ester as a yellow oil. .

To a solution of 5- (3,3-dimethyl-but-1-ynyl) -thiophene-2-carboxylic acid ethyl ester (6.9 g) in THF (100 mL) was added LiOH (1.5 N, 100 mL). The mixture was stirred at room temperature for 4 hours. The reaction mixture was acidified with HCl to pH = 2, and the volatiles were removed in vacuo. The obtained beige solid was collected by filtration, washed with water, and then dried overnight to give 6.2 g of the product, which was used without further purification.

To a solution of 5- (3,3-dimethyl-but-1-ynyl) -thiophene-2-carboxylic acid (6.2 g, 30 mmol, see US Patent 5,851,421) in THF (100 mL) was added a dropwise solution of nBuLi (at 2.0 M in pentane, 33 ml, 66 mmol) at -78 ° C. After the addition, the reaction mixture was stirred at -78 ° C for 1 h. A solution of I 2 (7.7 g, 30 mmol) in THF (100 mL) was slowly added to the flask (over about 15 min). After a further 10 minutes, the reaction was quenched with a 1 N HCl solution (50 mL) and the reaction was warmed to room temperature. The volatiles were removed in vacuo and the residue was dissolved in ether (500 mL). The organic solution was washed with a 1 M Na2S2O3 solution (100 mL x 2), brine (100 mL), and then dried over Na2SO4.

To a solution of 5- (3,3-dimethyl-but-1-ynyl) -3-iodo-thiophene-2-carboxylic acid (1.0 g, 3.0 mmol) and DMF (20 μΐ) in dry dichloromethane (10 ml) was added chloride oxalyl (508 μΐ, 6.0 mmol) at room temperature. After stirring at room temperature for 90 min, the reaction mixture was concentrated in vacuo to remove volatiles. The residue was dissolved in pyridine (5 mL) and methanol (5 mL) and stirred for 2 h. The volatiles were removed in vacuo and the residue was partitioned between ether (150 mL) and saturated NH 4 Cl solution (50 mL). The organic layer was washed with saturated NH 4 Cl solution (50 mL) and dried over MgSO 4 (50 mL)

- 33 - EP 2523950

Na2SO4. After concentration in vacuo, the residue was purified by silica gel chromatography (EtOAc / hexanes) to give the desired product (835 mg, 80%).

<img file="PL2523950T3_D0028.tif" />

NaH (36 mg of a 60% oil dispersion, 0.90 mmol) was added portionwise to a solution of trimethylsulfoxonium chloride (116 mg, 0.90 mmol) in DMSO (2.0 mL) at room temperature. After 15 minutes a solution of 5- (3,3-dimethyl-butyl-1-ynyl) -3 - [(4-methyl-cyclohexanecarbonyl) - (4-oxo-cyclohexyl) -amino] -thiophene-2-carboxylic acid methyl ester (343) was added dropwise. mg, 0.75 mmol) (WO 2008/058393) in THF (2.0 ml). After 3 hours, brine was added and the reaction mixture was extracted with ethyl acetate. The combined organic layers were washed with water, brine, and then dried over Na 2 SO 4 and concentrated to give a pale foam. Purification by flash column chromatography on silica gel with methanol in dichloromethane at 5% gave the product (216 mg, 61%) as a white off-white solid.

NaH (20 mg of a 60% oil dispersion, 0.50 mmol) was added to the solution

3-Hydroxypyridine (48 mg, 0.50 mmol) in DMF (2.0 ml) at room temperature. After stirring for 10 min, a solution of the epoxide of the previous step (216 mg, 0.46 mmol) in DMF (2.0 mL) was added. The reaction mixture was heated at 100 ° C for 6 h, cooled and partitioned between ethyl acetate and sat. NH 4 Cl solution. The organic layer was separated, washed with a 5% aqueous LiCl solution, brine, and then dried over Na 2 SO 4 and concentrated to give a dark orange residue. Purification by flash column chromatography on silica gel with methanol in dichloromethane at 5% yielded the desired product (87 mg), which was contaminated with unidentified impurity. This material was used in the next step without any additional purification.

A 1.0 N NaOH (0.50 mL) solution was added to a solution of the methyl ester from the previous step (87 mg, 0.15 mmol) in THF (0.75 mL) and methanol (0.75 mL) at 0 ° C. After 1 hour, the reaction mixture was concentrated to dryness, yielding an orange gum which was purified by preparative HPLC to give the desired product (4 mg, 1.3% for two steps) as the trifluoroacetic acid salt. MS (m / z): 553.0 [M + H]; HPLC retention time: 3.36 min (a mixture of 2-98% acetonitrile: water with trifluoroacetic acid at a concentration of 0.05%).

- 34 - EP 2523950

Example 15 - Compound 2: 5- (3,3-Dimethyl-but-1-ynyl) -3 - [[4-hydroxy-4- (tetrahydrofuran3 -ff-yloxymethyl) -cyclohexyl] - (4-methyl-cyclohexanecarbonyl) acid - amino] thiophene-2-carboxylic

Synthesis of 5- (3,3-dimethyl-but-1-ynyl) -3- (1,4-dioxa-spiro [4.5] dec-8-aminoamino) -thiophene-2-carboxylic acid methyl ester

<img file="PL2523950T3_D0029.tif" />

A mixture of 5- (3,3-dimethyl-but-1-ynyl) -3-iodo-thiophene-2-carboxylic acid methyl ester (0.5 g, 1.5 mmol), palladium acetate (0.033 g, 0.15 mmol), BINAP (0.093). g.

0.15 mmol), cesium carbonate (0.733 g, 2.25 mmol) and 1,4-dioxa-spiro [4.5] dec-8-ylamine (0.706 g,

4.5 mmol) in toluene (8 ml) was degassed with N 2 and then heated at 110 ° C for 8 h. The reaction mixture was diluted with ethyl acetate, filtered through a pad of Celite and purified by silica gel chromatography (hexane / ethylacetate). 0-40% concentration) to give the title compound with a yield of

50%. MS (m / z): 378.1 [M + H]; HPLC retention time: 5.12 min (a mixture of 2-98% acetonitrile: water with trifluoroacetic acid at 0.05% concentration).

Synthesis of 5- (3,3-dimethyl-but-1-ynyl) - (4-oxo-cyclohexylamino) -thiophene-2-carboxylic acid ethyl ester

<img file="PL2523950T3_D0030.tif" />

A mixture of 5- (3,3-dimethyl-but-1-ynyl) -3- (4-oxocyclohexylamino) thiophene-2-carboxylic acid methyl ester (0.5 g, 1.5 mmol) and HCl (24 mmol, HCl at 4 M) in THF / H2O (6 mL) was heated to 45 ° C for 2 h. The reaction mixture was diluted with ethyl acetate (20 mL) and the organic phase was washed with H 2 O (3 x 100 mL), sat. NaHCO3 solution (1 x 100 mL), and then dried over Na2SO4. The solution was filtered and concentrated to give the title compound in a yield of 96%. MS (m / z): 334.1 [M + H]; HPLC retention time: 4.530 min (a mixture of 2-98% acetonitrile: water with trifluoroacetic acid at 0.05% concentration).

EP 2523950

- 35 Scheme Ί

<img file="PL2523950T3_D0031.tif" />

NaH (140 mg of a 60% oil dispersion, 3.50 mmol) was added portionwise to a solution of trimethylsulfoxonium chloride (452 ​​mg, 3.51 mmol) in DMSO (8.0 mL) at room temperature. After 15 minutes, a solution of acid methyl ester was added dropwise

5- (3,3-dimethyl-but-1-ynyl) -3 - [(4-methyl-cyclohexanecarbonyl) - (4-oxo-cyclohexyl) amino] -thiophene-2-carboxylic acid (1.34 g, 2.93 mmol ) in THF (8.0 ml). After 3 hours, brine was added and the reaction mixture was extracted with ethyl acetate. The combined organic layers were washed with water, brine, and then dried over Na 2 SO 4 and concentrated to give a viscous thick oil. Trituration with ethyl ether and hexanes gave the methyl ester of the acid

5- (3,3-dimethyl-but-1-ynyl) -3 - [(4-methyl-cyclohexanecarbonyl) - (1-oxa-spiro [2.5] oct-6-yl) amino] thiophene-2-carboxylic acid (509 mg, 37%) as a colorless solid.

NaH (55 mg of a 60% oil dispersion, 1.37 mmol) was added to a solution of (R) - (-) - 3-hydroxytetrahydrofuran (120 mg, 1.36 mmol) in dry THF (4.0 mL) at 0 ° C. After 5 minutes, 5- (3,3-dimethyl-but-1-ynyl) -3 - [(4-methyl-cyclohexanecarbonyl) - (1-oxa-spiro [2.5] oct-6-yl) -amino methyl ester was added. ] thiophene-2-carboxylic acid in the form of a solid (125 mg, 0.265 mmol) and the reaction mixture was heated in a 75 ° C oil bath for 2.5 days. The reaction mixture was cooled and evaporated to dryness to give a brown residue which was purified by C18 reverse phase column chromatography (100% water to 10% acetonitrile / water). The product containing fractions were combined and evaporated to give 5- (3) acid, 3-dimethyl-but-1-ynyl) -3 - [[4-hydroxy-4- (tetrahydrofuran-3-yloxymethyl) -cyclohexyl] - (4-methyl-cyclohexanecarbonyl) -amino] -thiophene-2-carboxylic acid (30 mg, 20%) as a colorless solid. MS (m / z): 546.0 [M + H]⁺; HPLC retention time 4.32 min (a mixture of 2-98% acetonitrile: water with trifluoroacetic acid at

0.05%).

Example 16 - Compound 3: 5- (3,3-Dimethyl-but-1-ynyl) -3 - [[4-hydroxy-4- (tetrahydrofuran-35-yloxymethyl) -cyclohexyl] - (4-methyl-cyclohexanecarbonyl) acid - amino] thiophene-2-carboxylic

- EP 2523950

<img file="PL2523950T3_D0032.tif" />

Compound 3 (32 mg, 21%) was synthesized in an analogous manner to Example 15 but using (S) - (+) - 3-hydroxytetrahydrofuran instead of (R) - (-) - 3-hydroxytetrahydrofuran: MS (m / z) C): 546.0 [M + H] +; HPLC retention time 4.32 min (a mixture of 2-98% acetonitrile: water with trifluoroacetic acid at 0.05% concentration).

Example 20 - Compound 4: Synthesis of 5- (3,3-dimethyl-but-1-ynyl) -3 - {(4-methylcyclohexanecarbonyl) - [4-methyl-cyclohexanecarbonyloxy) -4-tetrahydro-furan-3-yloxymethyl) - cyclohexyl] -amino} -thiophene-2-carboxylic acid

Scheme 11

<img file="PL2523950T3_D0033.tif" />

Compound 4

NaH (370 mg of a 60% oil dispersion, 9.25 mmol) was added in portions to a solution of trimethylsulfonium iodide (1.88 g, 9.25 mmol) in DMSO (16.0 mL) at room temperature. After 15 minutes a solution of 5- (3,3-dimethyl-butyl-1-ynyl) -3 - [(4-oxo-cyclohexylamino) -thiophene-2-carboxylic acid methyl ester (1.10 g, 3.30 mmol) in THF was added dropwise (16.0). ml). After 3 hours, brine was added and the reaction mixture was extracted with ethyl acetate. The combined organic layers were washed with water, brine, and then dried over Na 2 SO 4 and concentrated to give a viscous thick oil. Purification by silica gel column chromatography (Teledyne Isco Redisep Rf Gold ™) with dichloromethane gave the two epoxide isomers from which the first one that eluted was the desired ester

5- (3,3-dimethyl-but-1-ynyl) -3- (1-oxa-spiro [2.5] oct-6-ylamino] -thiophene-2-carboxylic acid methyl ester (400 mg, 35%) ) in the form of a colorless solid.

A solution of (S) - (+) - 3-hydroxytetrahydrofuran (259 mg, 2.94 mmol) in dry NMP (1 mL) was added to a solution of KOtBu (271 mg, 2.42 mmol) in dry NMP (1 mL). The reaction mixture was stirred at room temperature for 15 min, then a solution of 5- (3,3-dimethyl-but-1-ynyl) -3- (1-oxa-spiro [2.5] oct-6-ylamino] - methyl ester was added. Thiophene-2-carboxylic acid (200 mg, 0.576 mmol) in dry NMP (4.0 mL) and the reaction mixture was heated in a 40 C oil bath for 24 h. The reaction mixture was poured into ice water, cooled to 0 C, neutralized with an acid solution. 5% citric acid to pH = 5-6 and extracted with ethyl acetate The organic layer was washed with 5% LiCl, brine and then dried and concentrated to give a brown oil which was collected with dichloromethane / MeOH (5.0 ml). 1. 0 ml) and treated dropwise with a solution of TMSCH2N2 (0.35 ml, 0.692 mmol, concentration 2 M in hexane). After 20 minutes, the volatiles were removed in vacuo and the impure residue was purified by silica gel column chromatography (Teledyne Isco Redisep Rf Gold ™), eluting first with 100% dichloromethane, and finally with 20% ethyl acetate in dichloromethane, affording the ester 5- (3,3-dimethyl-but-1-ynyl) -3- [4-hydroxy-4- (tetrahydrofuran-3-yloxymeth) -cyclohexylamino] -thiophene-2-carboxylic acid methyl ester (120 mg, 48%) in the form of a colorless solid.

Triethylamine (1.02 mL, 7.34 mmol) was added to the acid methyl ester solution

5- (3,3-dimethyl-but-1-ynyl) -3- [4-hydroxy-4- (tetrahydrofuran-3-yloxymethyl) cyclohexylamino] -thiophene-2-carboxylic acid (200 mg, 0.459 mmol) in dichloromethane (12.0 ml). After 10 minutes, the solution was cooled to -78 ° C and TESOTf (0.87 mL, 3.85 mmol) was added dropwise. The reaction mixture was stirred for 30 min at -78 ° C, quenched by addition of ice, diluted with saturated NaHCO 3 solution and extracted with dichloromethane. The organic layer was separated, dried over Na2SO4, concentrated and purified by column chromatography with ethyl acetate in hexane from 0 to 20% yielding 5- (3,3-dimethyl-but-1-yl) -3- [4] methyl ester. -hydroxy-4- (tetrahydro-furan-3-yloxymethyl) -4-triethylsilanyloxy-cyclohexylamino] -thiophene-2-carboxylic acid (200 mg, 79%) as a yellow oil.

5- (3,3-Dimethyl-but-1-ynyl) -3- [4-hydroxy-4- (tetrahydrofuran-3-yloxymethyl) -4-triethylsilanyloxy-cyclohexylamino] -thiophene-2-carboxylic acid methyl ester (200 mg, 0.36 mmol) was dissolved in pyridine (2 ml). After 10 minutes, pure 4-methyl-cyclohexanecarbonyl chloride (233 mg, 1.44 mmol) was added dropwise. The reaction solution was heated at 110 ° C overnight then concentrated to obtain a residue which was purified by column chromatography with ethyl acetate in hexane (from 0 to 20%) to give 5- (3,3- dimethyl-but-1-ynyl) -3 - {(4-methyl-cyclohexanecarbonyl) - [4- (4-methyl-cykloheksanokarbonyloksy) -4- (tetrahydro-furan-3-yloxymethyl) -cyclohexyl] -amino} -thiophene-2-carboxylic acid (80 mg, 32%) as a colorless solid.

NaOH (1.17 ml of a 1.0 N aqueous solution) was added dropwise to a solution of 5- (3,3-dimethyl-but-1-ynyl) -3 - {(4-methyl-cyclohexanecarbonyl) - [4- (4-methyl) methyl ester. -cyclohexanecarbonyloxy) -4- (tetrahydrofuran-3-yloxymethyl) -cyclohexyl] -amino} thiophene-2-carboxylic acid (80 mg, 0.117 mmol) in methanol (2 ml) / THF (2 ml) / H2O (2 ml) ) in room temperature. The solution was heated at 70 ° C overnight and then cooled to room temperature. The volatiles were removed in vacuo and the obtained residue was purified by HPLC with CH3CN (TFA 0.1%) / H2O (TFA 0.1%) to give compound 4 (23.3 mg, 36%) as a colorless solid. MS (m / z): 546.0 [M + H] &lt;+&gt;; HPLC retention time 4.169 min (a mixture of 2-98% acetonitrile: water with trifluoroacetic acid at 0.05% concentration).

- EP 2523950

Synthesis of (15,6) -4,6-dimethyl-cyclohex-3-enecarboxylic acid and chloride

Scheme 2

<img file="PL2523950T3_D0034.tif" />

H₂Oh, then Na₂SO₃ (Aq)

H₂ABOUT₂ 30% (aq), LiOH ^ H₂O, THF,

<img file="PL2523950T3_D0035.tif" />

ABOUT

OH (COCI)₂

DMF

<img file="PL2523950T3_D0036.tif" />

O.Cl

4S-benzyl-3- (4,6S-dimethyl-cyclohex-3-ene-1S-carbonyl) -oxazolidin-2-one, prepared according to a method similar to that described in J. Am. Chem. Soc. 110 (4), 1988,

1238-1256, dissolved in THF (1000 ml) and H 2 O (350 ml). The solution was cooled in an ice bath and 30% H 2 O 2 (36 ml, 354 mmol) was added slowly, and LiOH * H 2 O (S) (9.90 g, 263 mmol) was added in one portion. The reaction mixture was allowed to slowly warm to RT overnight. and stirred for 16 h. Then the reaction mixture was cooled in an ice bath. Na2SO3 (60 g, 472 mmol) was dissolved in H2O (400 mL) and very slowly added to the cooled reaction mixture. The solution was stirred for 1 h, then separated into layers. The organic compounds were removed under reduced pressure. The aqueous layer was added back to the concentrated organic layer and washed with CH 2 Cl 2 (2 x 500 mL). The pH of the aqueous layer was adjusted to 2 by the slow addition of concentrated HCl. The aqueous layer was extracted with EtOAc (4 x 300 mL) and dried over Na2SO4.

4,6-S-dimethyl-cyclohex-3-ene-1S-carboxylic acid (944 mg, 6.17 mmol) was dissolved in CH₂cl₂ (10 ml) and DMF (20 μl) was added. The solution was cooled to 0 ° C and then slowly added to it (COCl).₂ (700 μ, 7.38 mmol). The reaction mixture was stirred in an ice bath for 1 hour and then concentrated. The residue was taken up with hexanes and concentrated; Co-evaporation with hexanes was carried out once more. The acid chloride was used without further purification.

Example 23 - Compound 5: 5- (3,3-Dimethyl-but-1-ynyl-3- {4,6ff-dimethyl-cyclohex-3-enisobonyl) - [4-hydroxy-4- (tetrahydrofuran-3- acid) oxymethyl) -cyclohexyl] -amino} -thiophene-2-carboxylic

Diagram 12

- EP 2523950

<img file="PL2523950T3_D0037.tif" />

Compound 5

5- (3,3-Dimethyl-but-1-ynyl) -3- (1-oxa-spiro [2.5] oct-6-ylamino) -thiophene-2-carboxylic acid methyl ester was prepared similar to the process in scheme 11 above using trimethylsulfoxonium chloride instead of trimethylsulfonium iodide. A solution of 5- (3,3-dimethyl-but-1-ynyl) -3- (1-oxa-spiro [2.5] oct-6-ylamino) -thiophene-2-carboxylic acid methyl ester (362 mg, 0.575 mmol) in pyridine (11 mL) was treated with 4,6R-dimethyl-cyclohex-3-enocarbonyl-1R-chloride (325 mg, 2.11 mmol, prepared analogously to scheme 2) and heated to 85 ° C for 22 h. After cooling, the reaction mixture was concentrated , diluted with ethyl acetate, washed with a 1 M HCl solution (100 ml) and dried over NaSO4. The solution was filtered and purified on silica gel, obtaining 3 - [[4-chloromethyl-4- (4) methyl ester, 6-dimethyl-cyclohex-3-enokarbonyloksy) -cyclohexyl] - (4,6-dimethyl-cyclohex-3-enecarbonyl) -amino] -5- (3,3-dimethyl-but-1-ynyl) thiophene-2-carboxylic acid (34 mg, 0.05 mmol). The thick yellow oil was collected with NMP (2 mL), treated with (5) - (+) - 3-hydroxytetrahydrofuran (36 μL, 0.5 mmol) and K-OtBu (45 mg, 0.4 mmol) and then heated to 75 ° C. ° C for 1 h. After cooling, the reaction mixture was neutralized with a 1 M HCl solution, diluted with ethyl acetate (100 mL), and the organic layer was washed with brine (100 mL) and dried over NaSO4. The solution was filtered and the volatiles removed in vacuo. The obtained residue was purified by HPLC with CH 3 CN (TFA 0.1%) / H 2 O (TFA 0.1%) to give the desired products as a colorless solid. Isomer A (Compound 5A): MS (m / z): 558.0 [M + H] &lt;+&gt;; HPLC retention time 7. 95 min (mixture with a concentration of 2-98% acetonitrile: water with trifluoroacetic acid at a concentration of 0.05%), run time 30 min. Isomer B (Compound 5B); MS (m / z): 558.2 [M + H] &lt;+&gt;; HPLC retention time 8.02 min (a mixture of 2-98% acetonitrile: water with trifluoroacetic acid at a concentration of 0.05%), run time 30 min.

Example 24 - Compound 6: 5- (3,3-Dimethyl-but-1 - {(4,65-dimethyl-cyclohex-3-enocarbonyl [4-hydroxy-4-tetrahydro-furan-3 (S) -yloxymethyl] acid ) -cyclohexyl] -amino} -thiophene-2-carboxylic

EP 2523950

<img file="PL2523950T3_D0038.tif" />

<img file="PL2523950T3_D0039.tif" />

Compound 6 was synthesized in an analogous manner to Example 23, wherein the acylation was carried out with 4,6S-dimethyl-cyclohex-3-enocarbonyl chloride. MS (m / z): 558.3 ​​[M + H].

Example 27 - Compound 7: 5- (3,3-Dimethyl-but-1-ynyl) - [[4-hydroxy-4- (tetrahydrofuran3 (ff) -yloxymethyl) -cyclohexyl] - (15) -4-methyl acid cyclohex-3-enecarbonyl) -amino] -thiophene-2-carboxylic

<img file="PL2523950T3_D0040.tif" />

Compound 7 Scheme 15

<img file="PL2523950T3_D0041.tif" />

4,4-Dimethyl-2-oxo-tetrahydrofuran-3-yl ester of acrylic acid (R) (2.92 g, 15.9 mmol) in dichloromethane (20 ml) and hexanes (3 ml) was cooled to -10 ° C and treated with titanium tetrachloride (2.4 ml, concentration 2.4 M in dichloromethane, 2.4 mmol). The red solution was stirred for 15 min and isoprene (2.4 ml, 23.8 mmol) was added dropwise over 5 min. After stirring for 1.5 h, an additional portion of isoprene (2.4 mL, 23.8 mmol) was added and the reaction mixture was stirred at -10 to 0 ° C for 2.5 h. After cooling to -10 ° C, the reaction was quenched with ammonium chloride (sat. Aq. ). Water and ethyl acetate: hexanes (1: 1) were added. The organic layer was separated and the aqueous layer was extracted again with the ethyl acetate: hexanes (1: 1) mixture. The combined organic layers were dried over sodium sulfate, filtered and concentrated.

4,4-Dimethyl-2-oxo-tetrahydrofuran-3-yl ester of 4-methyl-cyclohex-3- (S) -encarboxylic acid (3.34 g, 13.2 mmol) in THF (25 mL), water (2.5 mL) and methanol (2.5 ml) was treated with lithium hydroxide monohydrate (2.8 g, 66.2 mmol) and heated to 50 ° C with stirring. After 1 hour, the reaction mixture was treated with a 1 M HCl solution (about 25 mL). The mixture was extracted with hexanes: ethyl acetate (200 mL: 15 mL), dried over sodium sulfate, filtered and concentrated to 2.4 g of a white semi-solid. The residue was redissolved in hexanes: dichloromethane (100 mL, 95: 5), washed with water, dried over sodium sulfate, filtered and concentrated to 1.68 g (91% yield) of (1S) -4-methyl-cyclohex-3-enecarboxylic acid. in the form of a white powder.

EP 2523950

- 41 Scheme 16

<img file="PL2523950T3_D0042.tif" />

Compound 7

(1S) -4-Methyl-cyclohex-3-enecarboxylic acid (209 mg, 1.5 mmol), dried azeotropically by evaporation from toluene, treated with trisodium potassium phosphate (383 mg, 1.8 mmol), suspended in dichloromethane (4 ml) and treated dimethylformamide (2 drops). The reaction mixture was cooled to 0 ° C and oxalyl chloride (0.3 mL, 3.2 mmol) was added dropwise. The reaction mixture was allowed to warm to ambient temperature with stirring for 2 h. After filtration of the solids, the solution was concentrated, treated with hexanes and concentrated again to give 4-methyl-cyclohex-3-enocarbonyl chloride (S) as a light yellow oil which was immediately used in the reaction. next stage.

(1S) -4-Methyl-cyclohex-3-enecarboxylic acid chloride (1.5 mmol), 5- (3,3-dimethyl-but-1-ynyl) -3- (1,4-dioxa-spiro) methyl ester 4.5] dec-8-ylamino) -thiophene-2-carboxylic acid (159 mg, 0.42 mmol) and trisodium potassium phosphate (266 mg, 1.25 mmol) are suspended in dichloroethane (1 ml), sealed with a cap and heated to temperature

90 ° C. After 16 hours, the reaction mixture was cooled and partitioned between ethyl acetate and water. The organic layer was separated and the aqueous layer was extracted again with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered and concentrated. Flash chromatography (15: 60% EtOAc: hexanes) afforded 128 mg (61% yield) of the desired 5- (3,3-dimethyl-but-1-ynyl) -3 - [methyl ester] [( 1,4-dioxa-spiro [4.5] dec-8-yl) - ((1S) -4-methyl-cyclohex-3-enocarbonyl) -amino] thiophene-2-carboxylic acid in the form of a white foam.

5- (3,3-Dimethyl-but-1-ynyl) -3 - [(1,4-dioxa-spiro [4.5] dec-8-yl) ((1S) -4-methyl-cyclohex-5-methyl ester 3-enocarbonyl) -amino] -thiophene-2-carboxylic acid (116 mg,

0.23 mmol) was dissolved in THF (1.8 ml) and treated with a 4 M HCl solution (0.9 ml).

The reaction mixture was heated to 45 ° C and stirred for 4.5 h. It was added

Additional 25 ml HCl solution (0.2 ml) was added and the solution was stirred for 2 h at 45 ° C. Ethyl acetate was added and the organic layer was separated, and then washed with brine, sodium bicarbonate (sat.aq) and brine. The organic layer was dried over sodium sulfate, filtered and concentrated to 98 mg of the desired 5-3,3-dimethyl-but-1-ynyl) -3 - [(1S) -4-methyl-cyclohex-3-enocarbonyl methyl ester) - (4-oxo-cyclohexyl) amino] -thiophene-2-carboxylic acid in the form of a white foam.

Trimethylsulfoxonium chloride (39 mg, 0.3 mmol) in DMSO (1.5 ml) was treated with sodium hydride (10 mg, 60% oil dispersion, 0.25 mmol) and stirred at ambient temperature for 10 min. 5- (3,3-Dimethylbut-1-ynyl) -3 - [(4-methyl-cyclohex-3-enocarbonyl) - (4-oxo-cyclohexyl) -amino] -thiophene-2-carboxylic acid methyl ester (S) was added dropwise. ) in THF (1 mL + 0.5 mL) and the reaction mixture was stirred for 1 h. The orange solution was treated with 5% citric acid until pH ~ 4 and partitioned between water and ethyl acetate. The organic layer was separated and the aqueous layer was extracted again with ethyl acetate. The combined organic layers were washed with water and brine and dried over sodium sulfate. After filtration and concentration, the residue was purified by flash chromatography (25:75 EtOAc: hexanes),

(R) -Tetrahydro-furan-3-ol (89 mg, 1.01 mmol) in 1-methyl-pyrrolidin-2-one (1 ml) was treated with potassium tert-butoxide (90.5 mg, 0.81 mmol) and stirred at ambient temperature for 15 minutes. This slightly turbid solution was added to 5- (3,3-dimethyl-but-1-ynyl) -3 - [(4-methyl-cyclohex-3-enocarbonyl) -l-oxa-spiro [2.5] octane methyl ester. -yl) amino] -thiophene-2-carboxylic acid (S) (34 mg, 0.073 mmol). The reaction mixture was sealed and heated to 40 ° C for 16 h. After cooling, the mixture was treated with a 2 M HCl solution until pH ~ 3, partitioned between ethyl acetate and water and separated. The aqueous layer was extracted again with ethyl acetate and the combined organic layers were washed with water, brine and dried over sodium sulfate. After filtration and concentration, the residue was purified by HPLC with CH 3 CN (TFA 0. 1%) / H 2 O (TFA 0.1%), yielding 22 mg (55% yield) of compound 7 as a white powder: MS (m / z): 544.0 [M + H] +; HPLC retention time 4.20 min (a mixture of 2-98% acetonitrile: water with trifluoroacetic acid at a concentration of 0.05%).

Example 28 - Compound 8: 5- (3,3-Dimethyl-but-1-ynyl-3 - [[4-hydroxy-4- (tetrahydrofuran-3 (5) -yloxymethyl) -cyclohexyl] - (15) -4- acid methyl-cyclohex-3-enecarbonyl) -amino] -thiophene-2-carboxylic

<img file="PL2523950T3_D0043.tif" />

Connection 8

Compound 8 was synthesized in a similar manner to Example 27 but using (S) -tetrahydro-furan-3-ol instead of (R) -tetrahydro-furan-3-ol: MS (m / z): 544.1 [M + H ] +; HPLC retention time 4.20 min (a mixture of 2-98% acetonitrile: water with trifluoroacetic acid at a concentration of 0.05%).

- EP 2523950

Example 29 - Compound 9: 5- (3,3-Dimethyl-but-1-ynyl) -3 - [[4-hydroxy-4- (tetrahydrofuran3 (5) -yloxymethyl) -cyclohexyl] - (1ff) -4 methyl-cyclohex-3-enecarbonyl) -amino] -thiophene-2-carboxylic

<img file="PL2523950T3_D0044.tif" />

Compound 9

The 4,4-dimethyl-2-oxo-tetrahydrofuran-3-yl ester of acrylic acid (R) was prepared as follows: 3- (S) -hydroxy-4,4-dimethyl-dihydro-furan-2-one ( 2.60 g, 20 mmol) and diisopropylethylamine (5.2 mL, 30 mmol) in dichloromethane (25 mL) was cooled to -10 ° C, acryloyl chloride (2.03 mL, 25 mmol) was added dropwise and stirred for 2 h. Concentration of HCl was added. 1 M (20 ml) and the organic layer was washed with sodium bicarbonate and water. The organic layer was dried over sodium sulfate, filtered and concentrated. Flash chromatography (10:40 EtOAc: hexanes mixture) gave 2.09 g (57% yield) of the desired 4,4-dimethyl-2-oxo-tetrahydrofuran-3-yl acrylic acid ester (R) in in the form of a clear oil.

Compound 9 was prepared in a similar manner to Example 27 but using (1R) -4-methyl-cyclohex-3-enoic acid acid (1R) -chlorid acid chloride (1S) -4-methyl-cyclohex-3-enecarboxylic acid and (S ) -tetrahydro-furan-3-ol instead of (R) -tetrahydro-furan-3-ol: MS (m / z): 544.0 [M + H] +; HPLC retention time 4.22 min (a mixture of 2-98% acetonitrile: water with trifluoroacetic acid at a concentration of 0.05%).

Example 30 - Compound 10: 5- (3,3-Dimethyl-but-1-ynyl) -3 - [[4-hydroxy-4-tetrahydrofuran3 (ff) -yloxymethyl) -cyclohexyl] - (1ff) - 4-methylcyclohex-3-enecarbonyl) -amino] -thiophene-2-carboxylic

<img file="PL2523950T3_D0045.tif" />

Connection 10

Compound 10 was prepared in a similar manner to Example 29, but using (R) -tetrahydro-furan-3-ol instead of (S) tetrahydrofuran-3-ol: MS (m / z): 544.1 [M + H] +; HPLC retention time 4.20min (a mixture of 2-98% acetonitrile: water with trifluoroacetic acid at 0.05% concentration).

Example 31: Compounds 16, 17 (reference) and 18

<img file="PL2523950T3_D0046.tif" />

- EP 2523950

2,4-Dibromothiophene (6 g, 24.8 mmol), PdCl2 (PPh3) 2 (522 mg, 0.74 mmol) and Cul (283 mg, 1.49 mmol) were placed in a 250 mL round bottom flask which was then sealed with a rubber pad. septum. The flask was evacuated and refilled with argon three times, followed by addition of DMF (150 ml) and TEA (30 ml). 3,3-Dimethylbut-1-yn (2.87 mL, 23.56 mmol) was added. The reaction mixture was heated at 45 ° C for two hours, during which time 2,4-dibromothiophene was completely consumed. The insoluble material was removed by filtration and the filtrate was concentrated in vacuo. The residue was partitioned between EtOAc (150 mL) and aqueous ammonium hydroxide (2 mL of a solution of 28-30% by weight diluted in 100 mL of water). The organic phase was separated, washed with 5% aqueous LiCl solution and brine, dried over Na 2 SO 4 and filtered. The filtrate was concentrated. The residue was purified by chromatography on silica gel eluting with hexane to give compound 11 (5.1 g) as a pale yellow liquid.

HPLC retention time: 5.169 min (a mixture of 5-95% acetonitrile with 0.05% TFA: water with 0.05% TFA).

<img file="PL2523950T3_D0047.tif" />

A mixture of compound 11 (0.773 g, 3.18 mmol), Cul (30 mg, 0.160 mmol), cesium carbonate (2.072 g, 6.36 mmol), 2-acetylcyclohexanone (90 mg, 0.636 mmol) and 1,4-dioxa-spiro [4.5 ] dec8-ylamine (1.0 g, 6.36 mmol) in DMF (1.6 mL) was degassed with N 2, then heated to 80 ° C for 16 h in a sealed tube. The reaction mixture was diluted with EtOAc, filtered through a pad of diatomaceous earth, washed with 5% aqueous LiCl solution, dried over sodium sulfate, filtered and concentrated. Flash compound (EtOAc: hexanes) gave compound 12.

MS (m / z): 320.20 [M + H].

<img file="PL2523950T3_D0048.tif" />

A solution of compound 12 (4.16 g, 13.0 mmol) in 1,2-dichloroethane (40 ml) was cooled to 0 ° C and treated with a solution of (1S, 6S) -4,6-dimethylcyclohex-3-n-carbonyl chloride (3.88 g, 24 mmol, prepared in the same way as in scheme 2) in 20 ml of 1,2-dichloroethane. The reaction mixture was allowed to slowly warm to room temperature and stirred for 17 hours, after which it was diluted with DCM, washed twice with saturated NH4Cl solution (aq), dried over MgSO4, filtered and concentrated. The residue was purified on silica gel, yielding a mixture of (1S, 6S) -N- (5-dimethylbut-1-ynyl) thiophen-3-yl) -4,6-dimethyl-N- (1). , 4-dioxaspiro [4.5] decan-8-yl) cyclohex-3-enecarboxamide and the ketal hydrolysis product (5.60 g, 12.3 mmol). The mixture was collected with THF (70 ml), treated with a 4 N HCl solution (aq) and stirred at 45 ° C for 90 minutes. THF was removed under reduced pressure and the resulting aqueous layer was extracted three times with EtOAc. The combined aqueous layers were washed with a saturated NaHCO3 solution (aq), water and brine, dried over MgSO4, filtered and concentrated to 5.05 g of ketone product 13.

<img file="PL2523950T3_D0049.tif" />

A solution of trimethylsulfoxonium chloride (0.47 g, 3.64 mmol) in THF (8 mL) / DMSO (8 mL) was treated with NaH (0.126 g, 3.16 mmol) at room temperature for 20 minutes. A solution of compound 13 (1 g, 2.43 mmol) in THF (8 mL) was added dropwise over 7 minutes and stirring was continued for 0.5 h. The reaction mixture was cooled to 0 ° C and quenched with 10% citric acid (200 mL). . The aqueous layer was extracted with ethyl acetate (300 mL) and the combined organic phases were washed with brine (500 mL), dried over Na 2 SO 4 and concentrated. The contaminated material was purified by silica gel chromatography to obtain (1S, 6S) -N- (5- (3,3-dimethylbut-1-yl) thiophen-3-yl) -4,6-dimethyl-N - (3 R, 6S) -1-oxaspiro [2.5] octan-6-yl) cyclohex-3-ene carboxamide (14) (0.25 g, 0.58 mmol).

A solution of compound 14 (0.25 g, 0.58 mmol) in THF (3 mL) was cooled to -78 ° C and treated with a solution of LDA (2 M in THF, 2.35 mmol). After 2 hours, the reaction mixture was bubbled with CO2 (g) for 15 min. The reaction mixture was allowed to warm to room temperature and quenched with NH3Cl solution (sat.). The aqueous layer was extracted with ethyl acetate (50 mL) and the combined organic phases were washed with brine (100 mL), dried over Na 2 SO 4 and concentrated to give compound 15. The crude was dissolved in NMP (1 mL) and treated with potassium tert-butoxide (2.5 mmol) and (S) -tetrahydrofuran-3-ol (2.5 mmol). The reaction mixture was heated to 40 ° C for 16 h. The reaction mixture was then cooled and neutralized with aqueous HCl (1 M). The product was extracted with ethyl acetate,

- EP 2523950

CH3CN (TFA 0.1%) / H2O (TFA 0.1%) to give compound 16. MS (m / z): 558.1 [MH] -; HPLC retention time 4.47 min (a mixture of 2-98% acetonitrile: water with trifluoroacetic acid at a concentration of 0.1%), run time 6 min.

Compound 17 (reference)

<img file="PL2523950T3_D0050.tif" />

.0

Compound 17

A solution of compound 16 (0.025 g, 0.044 mmol) in DCM / MeOH (5 ml / 1 ml) was treated with trimethylsilyldiazomethane (2 M in hexanes, 0.22 ml) for 30 min. The reaction mixture was concentrated and purified by HPLC with CH 3 CN (TFA 0.1%) / H 2 O (TFA 0.1%) to give the title compound as a solid. MS (m / z): 572.1 [MH] -; HPLC retention time

5.08 min (mixture with a concentration of 2-98% acetonitrile: water with trifluoroacetic acid at a concentration of 0.1%), run time 6 min.

Compound 18

<img file="PL2523950T3_D0051.tif" />

Ο,

Ό °

Compound 18 was synthesized in a manner analogous to compound 18, using (R) -tetrahydrofuran-3-ol. MS (m / z): 558.1 [M + H] &lt;+&gt;; HPLC retention time 4.48 min (a mixture of 2-98% acetonitrile: water with trifluoroacetic acid at a concentration of 0.1%), run time 6 min.

Example 32 +

ΗΝ-Ο / \

HATU.DIEA, DCM, mer .0

HATU (32.5 g, 85.5 mmol, 1.1 eq.) And N, O-dimethylhydroxylamine hydrochloride (8.3 g, 85.5 mmol, 1.1 eq.) Were placed in a round bottom flask containing 300 mL of dichloromethane. DIEA (40 mL, 233 mmol, 3.0 eq) was added, followed by a solution of d9-pivalic acid (8.6 g, 77.7 mmol, 1.0 eq) in DCM (25 mL). The reaction mixture was stirred at room temperature until complete consumption of the carboxylic acid. The solution was concentrated in vacuo, removing volatiles, and the residue was partitioned between DCM (200 mL) and saturated NH4Cl solution (100 mL). The aqueous phase was extracted with DCM (200 mL) and the combined organic phases were dried over Na 2 SO 4 and then concentrated. The residue was purified by silica gel chromatography to give compound 19 (5.1 g, 33 mmol, 42% yield) as a colorless oil.

- EP 2523950

<img file="PL2523950T3_D0052.tif" />

Methyl 3-amino-5-iodothiophene-2-carboxylate (6.24 g, 22 mmol, 1.0 eq.) And ketal (5.16 g, 33 mmol, 1.5 eq.) Were dissolved in 27 mL of acetic acid in a round bottom flask. Sodium triacetoxyborohydride (7.0 g, 33 mmol, 1.5 eq) was added in portions at room temperature. After completion of the reaction, water (30 ml) was added and the mixture was poured into EtOAc (200 ml). The phases were separated and the organic layer was washed with H 2 O and brine, and then dried over Na 2 SO 4. The volatiles were removed in vacuo and the residue was purified by chromatography (3.0 equiv.) With silica to give compound 20 (7.1 g, 17 mmol, 76% yield) as a yellow solid.

(COCI)₂ (3.0 equiv). ^ 3 ^ 04 (1.25 eq. DCM, DMF (k_andt).

0 ° C to room temperature, 2 hours_r

<img file="PL2523950T3_D0053.tif" />

<img file="PL2523950T3_D0054.tif" />

<img file="PL2523950T3_D0055.tif" />

Compound 20 (7.1 g, 17 mmol) and freshly ground K3PO4 (7.12 g, 33 mmol) were suspended in DCE (40 mL) in a 250 mL round bottom flask. The solution was cooled to 0 ° C in an ice-water bath. The syringe (R) -4-methylcyclohex-3-enocarbonyl chloride (7.9 g, 50 mmol) in DCE (25 mL) was added dropwise via syringe. After the addition, the reaction mixture was stirred overnight under reflux. The reaction mixture was diluted with DCM (200 mL) and the organic phase was washed with a saturated NH4Cl solution (2 x 100 mL). After drying over Na 2 SO 4, the organic layer was concentrated to give a yellow foamy solid. The residue was purified by silica gel chromatography to obtain compound 21 (5.5 g, 10 mmol, 60% yield) as a white off-white solid.

<img file="PL2523950T3_D0056.tif" />

A solution of compound 21 (1.0 g, 1.9 mmol, 1.0 eq.) In THF (12.0 mL) in a round bottom flask was cooled to 0 ° C. Isopropylmagnesium chloride (2.0 M in THF, 2.1 mmol, 1.1 eq.) Was added dropwise and the mixture was stirred for 30 min.

A solution of Weinreb 19 amide (322 mg, 2.1 mmol, 1.1 eq.) In THF (1.0 mL) was slowly added and the resulting solution was gradually warmed to room temperature and stirred overnight. The reaction mixture was poured into a saturated solution of NH4Cl (50 mL) and extracted with ice

DCM (2 x 100 mL). The combined organic phases were dried over Na₂SO₄ and then concentrated. The contaminated compound 22 was used in the next step without purification.

<img file="PL2523950T3_D0057.tif" />

To a solution of compound 22 (97 mg, 0.19 mmol, 1.0 eq.) And

Dimethyl 1-diazo-2-oxopropylphosphonate (110 mg, 0.57 mmol, 3.0 eq) in MeOH (3.0 mL) was added K 2 CO 3 (105 mg, 0.758 mmol, 4.0 eq). The resulting solution was stirred overnight at room temperature. The reaction was then partitioned between EtOAc (50 mL) and 1 N HCl solution (50 mL). The organic phase was dried over Na 2 SO 4 and then concentrated. The impure material was purified by silica gel column chromatography to obtain compound 23 (70 mg, 0.138 mmol, 73% yield) as a white solid.

<img file="PL2523950T3_D0058.tif" />

Subsequent synthetic transformations necessary to obtain compound 24 were carried out in a similar manner as described in example 27. MS (m / z): 553.1 [M + H] +; HPLC retention time 4.46 min (a mixture of 2-98% acetonitrile: water with 0.05% trifluoroacetic acid in 6 min).

Example 33: Compound 29

- EP 2523950

<img file="PL2523950T3_D0059.tif" />

<img file="PL2523950T3_D0060.tif" />

Step_1: Synthesis of (R) -methyl-5-iodo-3- (4-methyl-N- (4-oxocyclohexyl) cyclohex-3-ene carboxamido) thiophene-2-carboxylate (25)

A mixture of compound 21 (2.0 g, 3.6 mmol) and HCl (40 mmol, HCl 1 N) in THF (20 mL) was heated at 45 ° C for 20 min. The reaction mixture was diluted with ethyl acetate and the organic layer was separated and then washed with sodium bicarbonate (sat aq) and brine. The organic layer was dried over sodium sulfate, filtered and concentrated to give compound 25. The contaminated material was used in the next step without further purification.

MS (m / z): 502.2 [M + H]; HPLC retention time: 2.67 min (a mixture of 2-98% acetonitrile: water with 0.05% formic acid over 3.5 min).

Step 2: Synthesis of methyl 5-iodo-3 - ((R-4-methyl-N - ((3S, 6S) -1-oxaspiro [2.5] octan-6-yl) cyclohex-3-enoicarboxamido) thiophene-2-carboxylate ( 26)

Trimethysulfoxonium chloride (592 mg, 4.6 mmol) in DMSO (10 mL) was treated with sodium hydride (162 mg, 60% oil dispersion, 4.03 mmol) and stirred at ambient temperature for 30 min. Compound 25 (the residue from the previous step) in THF (10 ml) was added dropwise and the reaction mixture was stirred for 30 min. The orange solution was treated with 10% citric acid until pH ~ 4 and partitioned between water and ethyl acetate. The organic layer was separated and the aqueous layer was extracted again with ethyl acetate. The combined organic layers were washed with water and brine and dried over sodium sulfate. After filtration and concentration, the residue was purified by flash chromatography (EtOAc: hexanes) to give compound 26 (1.3 g, 2.52 mmol, 69%) as a white solid.

MS (m / z): 515.9 [M + H]; HPLC retention time: 2.76 min (a mixture of 2-98% acetonitrile: water with 0.05% formic acid over 3.5 min).

- EP 2523950

Step 3: Synthesis of 5- (4-hydroxy-3,3-dimethylbut-1-ynyl) -3 - ((R) -4-methyl-N - ((3S, 6S) -1oxaspiro [2.5] octane-6 methyl) cyclohex-3-enecarboxamido) methyl thiophene-2-carboxylate (27)

Compound 26 (260 mg, 0.5 mmol), 2,2-dimethylbut-3-yn-1-ol (150 mg, 1.5 mmol), bis (triphenylphosphine) palladium (II) dichloride (18 mg, 0.025 mmol), CuI ( 9.5 mg, 0.05 mmol) and triethylamine (1 mL) were dissolved in DMF (5 mL) in a sealed tube. The mixture was heated at 80 ° C for 5 h. Then the reaction mixture was poured into EtOAc (200 mL) and washed with NH₄Cl (2 x 50 ml) and 5% LiCl (2 x 50 ml). The organic layer was separated and the aqueous layer was extracted again with ethyl acetate. The combined organic layers were washed with water and brine and dried over sodium sulfate. After filtration and concentration, the residue was purified by flash chromatography (EtOAc: hexanes) to give compound 27 (203 mg, 0.42 mmol, 84%) as a white solid.

MS (m / z): 486.1 [M + H]; HPLC retention time: 2.60 min (a mixture of 2-98% acetonitrile: water with 0.05% formic acid over 3.5 min).

Step 4: Synthesis of 5- (4-Hydroxy-3,3-dimethyl-1-in-1-ynyl) -3 - ((R-4-methyl-N- (3S, 6S) -1oxaspiro [2.5] octane-6-yl) ) cyclohex-3-enecarboxamido) thiophene-2-carboxylic acid (28)

Compound 27 (203 mg, 0.42 mmol) was dissolved in THF (5 mL) and water (3 mL). LiOHH ​​was added₂O (176 mg). The reaction mixture was stirred at ambient temperature for one day and then quenched with aqueous 10% citric acid (5 mL). The reaction mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried and concentrated to give compound 28 (186 mg).

Step 5: Synthesis of 5- (4-Hydroxy-3,3-dimethyl-1-in-1-ynyl) -3 - ((R) -N - ((1R, 4S) -4-hydroxy-4 (((S) - tetrahydrofuran-3-yloxy) methyl) cyclohexyl) -4-methylcyclohex-3-encarboxamido) thiophene-2-carboxylic acid (29) (S) -Tetrahydrofuran-3-ol (187 mg, 2.1 mmol) in 1-methyl-pyrrolidine 2-one (4.0 ml) was treated with potassium tert-butoxide (95 mg, 0.848 mmol) and stirred at ambient temperature for 20 minutes. Compound 28 (100 mg, 0.21 mmol) was added to this mixture. The reaction mixture was heated at 35 ° C for 16 hrs under an inert atmosphere. After cooling, the mixture was treated with aqueous 10% citric acid until pH ~ 3, partitioned between ethyl acetate and water and separated. The aqueous layer was extracted again with ethyl acetate and the combined organic layers were washed with water, brine and dried over sodium sulfate.

MS (m / z): 560.1 [M + H] &lt;+&gt;

Biological examples

Antiviral activity

Another aspect of the invention relates to a compound for use in methods of inhibiting viral infections, which methods include the step of treating a sample or object suspected of requiring such inhibition with a composition of the invention.

In the context of the invention, samples that are believed to contain a virus include natural or man-made materials, such as living organisms; tissue or cell cultures; biological samples, such as samples of biological materials (blood, serum, urine, cerebrospinal fluid, tears, sputum, saliva, tissue samples and the like); laboratory samples; food, water or air samples; samples

- bio-products, such as cell extracts, especially recombinant cells synthesizing a desired glycoprotein; and the like. Typically, the sample will be suspected of containing the organism that causes the viral infection, some of which is pathogenic, such as the oncogenic virus. The sample may be located in any medium, including water and mixtures of organic solvent with water. Samples include living organisms, such as humans, and man-made materials such as cell cultures.

If desired, the antiviral activity of a compound of the invention after application of the composition can be observed by any method, including by direct and indirect methods to detect such activity. As methods for determining such activity, both quantitative and qualitative methods as well as semi-quantitative methods are considered. Typically, one of the screening methods described above is used, but any other method, such as observing the physiological characteristics of the living organism, is also applicable.

The antiviral activity of a compound of the invention can be measured using standard screening protocols that are known. For example, the antiviral activity of a compound can be measured using the following general protocols.

Cellular immunodetection of flaviviruses

BHK21 or A549 cells are treated with trypsin, counted and diluted to 2 × 10⁵ cells / ml in Hama F-12 media (A549 cells) or in RPMI-1640 medium (BHK21 cells), which are supplemented with fetal bovine serum (FBS) at 2% and penicillin / streptomycin at a concentration of 1 %. The cells are plated in clean 2x -10 well 2x10 tissue culture plates⁴ per well and placed at 37 ° C with a CO2 concentration of 5% overnight. The next day, the cells are infected with the virus at a multiplicity of infection (MOI) index of 0.3 in the presence of test compounds at various concentrations for 1 hour at 37 ° C and a CO2 concentration of 5% for a further 48 hours. Cells are washed once with PBS buffer and fixed with cold methanol for 10 min. After two washes with PBS buffer, the fixed cells are blocked with PBS containing FBS at 1% and Tween-20 at 0.05% for 1 hour at room temperature. A solution of the primary antibody (4G2) is then added at a concentration of 1:20 to 1: 100 in PBS containing FBS at 1% and Tween-20 at a concentration of 0.05% and incubated for 3 hours. The cells are then washed three times with PBS, and incubated for one hour with horseradish peroxidase (HRP) -conjugated anti-mouse IgG (Sigma, dilution 1: 2000). After washing three times with PBS buffer, 50 microliters of a 3,3 ', 5,5'-tetramethylbenzidine (TMB) substrate solution (Sigma) is added to each well for two minutes. The reaction is stopped with the addition of 0.5 M sulfuric acid. The plates are read at 450 nm absorbance for the quantitative determination of the viral load. After measurement, the cells are washed three times with PBS buffer and then incubated with propidium iodide for 5 min. The plate is read in a Tecan Safire ™ reader (excitation 537 nm, emission 617 nm) for quantification of the number of cells. The dose-response curves are determined by comparing the average absorbance and logarithm of the concentrations of the test compounds. The EC50 value is calculated by non-linear regression analysis. A positive control such as N-nonyl-deoxynojirimycin may be used.

Cellular analysis of the cytopathic effect caused by flaviviruses

For testing for West Nile virus or Japanese encephalitis virus, BHK21 cells are trypsinized and diluted to a concentration of 4 x 10⁵ cells / ml in RPMI-1640 medium supplemented with FBS at a concentration of 2% and

- Penicillin / streptomycin at a concentration of 1%. For testing for dengue virus, Huh7 cells are trypsinized and diluted to a concentration of 4 x 10⁵ cells / ml in DMEM medium supplemented with FBS at 5% and penicillin / streptomycin at a concentration of 1%. Place 50 microliters of cell suspension (2 x 10⁴ cells) per well in 96-well plates with an optical bottom made of PIT polymer (Nunc). The cells are cultured overnight in culture medium at 37 ° C with a CO 2 concentration of 5% and then infected with West Nile virus (e.g. strain B956) or Japanese encephalitis virus (e.g., Nakayama strain) at MOI = 0.3 or dengue virus (e.g., DEN-2 NGC strain) at MOI = 1, in the presence of test compounds at various concentrations. Plates containing the virus and test compounds are further incubated at 37 ° C with a CO2 concentration of 5% for 72 hours. After the incubation is complete, 100 microliters of CellTiter-Glo ™ reagent are added to each well. The contents are mixed for 2 minutes on an orbital shaker to induce cell lysis. The plates are incubated at room temperature for 10 minutes to stabilize the luminescence signal. Luminescence is read using a plate reader. A positive control such as N-nonyl-deoxynojirimycin may be used.

Antiviral activity on the mouse dengue infection model

The compounds are tested in vivo on a mouse model of dengue virus infection (Schul et al. J. Infectious Dis. 2007; 195: 665-74). AG129 mice from six to ten weeks of age (B &amp; K Universal Ltd, HII, UK) are housed in independently ventilated cages. The mice are injected intraperitoneally with 0.4 ml of the dengue 2 TSV01 virus suspension. Blood samples are collected by puncturing the back of the eye socket under general anesthesia with isoflurane. Blood samples are taken in tubes containing sodium citrate to a final concentration of 0.4% and immediately centrifuged for 3 minutes at 6000 g to obtain plasma. Plasma (20 microliters) is diluted in 780 microliters of RPMI-1640 medium and frozen rapidly in liquid nitrogen to perform plaque analysis. The remaining plasma behaves to determine the level of the cytokine and NS1 protein.

To test the antiviral activity, the compound of the invention dissolves in a carrier fluid, e.g. in 10% ethanol, 30% PEG 300, and 60% D5W (dextrose in 5% water); or in composition: 6 N HCl (1.5 equiv.): 1 N NaOH (pH set at 3.5): 100 mM citrate buffer, pH 3.5 (0.9% v / v: 2.5% v / v: 96.6 % v / v). Thirty-six AG129 mice aged 6-10 weeks are divided into six groups of six mice in each. All mice are infected with the dengue virus as described above (day 0). Group 1 is administered by oral route by gavage of 0.2 mg / kg of the compound of the invention in an amount of 200 ml / mouse twice daily (first time in the morning and second time in the late afternoon) for three consecutive days, starting on day 0 (the first dose immediately prior to infection) dengue). Groups 2, 3 and 4 are administered in the same manner 1 mg / kg, 5 mg / kg and 25 mg / kg, respectively. A positive control such as (2R, 3R, 4R, 5R) -2- (2-amino-6-hydroxy-purin-9-yl) -5-hydroxymethyl-3-methyltetrahydrofuran-3,4-diol may be used, giving by oral gavage in the amount of 200 microliters / mouse in the same way as in the previous groups. Still another carrier fluid is administered.

On the third day after the infection, blood samples are taken from the mouse in an amount of approximately 100 microliters (anticoagulated with sodium citrate) by puncturing the back of the eye after general anesthesia with isoflurane. Plasma from each blood sample is obtained by centrifugation and quickly frozen in liquid nitrogen to perform plaque analysis. The collected plasma samples are examined using plaque analysis as described by Schul et al. Cytokines are also investigated as described by Schul. The NS1 protein peptide is tested using the kit

- 53 - EP 2523950

Platelia ™ (BioRad Laboratories). An indicator of the presence of an antiviral effect is a reduction in the level of cytokines and / or NS1 protein.

Typically, about 5-100-fold viral load reduction, more typically 10-60-fold, most often 20-30-fold, is achieved at doses of the compounds of the invention of 5-50 mg / kg.

HCV determination protocol

The anti-HCV activity of the compounds of the invention was tested on a human Huh-7 hepatoma cell line bearing the HCV replicon. The designation included the following steps:

Stage 1: preparation of the compound and serial dilutions

Serial dilutions were prepared in 100% DMSO in a 384-well plate.

The solution containing the compound at 225 times the starting concentration of the final serial dilution was prepared in 100% DMSO and 15 μΐ was added to the pre-selected wells in the 384-well column of the 384-well plate. The remaining wells of the 384-well plate were filled with 10 μΐ of 100% DMSO except columns 23 and 24, to which 10 μl of an HCV protease inhibitor (ITMN-191) at 500 μΜ in 100% DMSO was added. The HCV protease inhibitor was used as a control providing 100% inhibition of HCV replication. Then the plate was placed on the Biomek FX station workstation to start the serial dilution. Serial dilutions were made in ten cycles of 3-fold dilutions from columns 3 to 12 or from columns 13 to 22.

Step 2: preparation of the plate with cell culture and addition of the compound

To each well of a black 384-well propylene plate, 90 μl cell medium containing 1600 suspended Huh7 cells bearing the HCV replicon was added via the Biotek uFlow workstation. A solution of the compound in a 0.4 μl volume was transferred from a serial dilution plate to a cell culture plate using a Biomek FX workstation. The DMSO concentration at the final assay conditions was 0.44%. The plates were incubated for 3 days at 37 ° C with a CO2 concentration of 5% and 85% humidity.

Step 3: detection of cytotoxicity and suppression of virus replication

a) Cytotoxicity assay: The medium in a 384-well cell culture plate was aspirated using a Biotek EL405 plate washer. 50 Pl of a solution containing Calcein AM at a concentration of 400 nM in PBS at 100% concentration was added to each well of the plate using the Biotek uFlow workstation. The plate was incubated for 30 minutes at room temperature before measuring the fluorescence signal (emission 490 nm, excitation 520 nm) with a Perkin Elmer Envision plate reader.

b) Determination of viral replication inhibition: A solution of calcein-PMS in a 384-well cell culture plate was aspirated using a Biotek EL405 plate washer. 20 μl of Dual-Glo luciferase buffer (Promega, Dual-Glo Luciferase Assay Reagent, Cat. No. E298B) was added to each well plate using the Biotek uFlow workstation. The plate was incubated for 10 minutes at room temperature. Then 20 μl of a solution containing a 1: 100 mixture of the Dual-Glo Stop &amp; Glo substrate (Promega, Dual-Glo Luciferase Assay Reagent, Cat. No. E313B) and a Dual-Glo Stop &amp; Glo buffer (Promega, Dual-Glo) were added to each well of the plate. Luciferase Assay Reagent, cat. No. E314B) using the Biotek uFlow workstation. The plate was incubated at room temperature for 10 minutes before measuring the luminescence signal with a Perkin Elmer Envision plate reader.

Stage 4: calculations

- EP 2523950

Cytotoxicity in percent was determined by measuring the effect of converting Calcein AM to a fluorescent product. The average fluorescence signal of control wells containing DMSO was defined to be 100% non-toxic. Individual fluorescence signals for wells treated with the test compound were divided by the average signal for control wells containing DMSO, and then multiplied by 100%, resulting in a viability in percent. Anti-replicative activity against HCV was determined based on the luminescence signal of the test well as compared to control wells containing DMSO. The background signal was determined on the basis of the mean luminescence signal for wells treated with the HCV protease inhibitor and subtracted from the signal for the test wells, as well as from the signal for control wells containing DMSO.

% iinhibition = 100% / [(EC₅₀/ [L])^b + 1], where b is the Hill coefficient. See, for reference, Hill, AV, The Possible Effects of Aggregation of the Molecules of Hemoglobin on its Dissociation Curves, J. Physiol. 40: iv-vii. (1910).

Inhibition values ​​in% at a particular concentration, e.g. 2 μΜ, can also be determined from the above formula.

Based on the studies, it was found that certain compounds of the invention inhibit viral replication as shown in Table 1:

Table 1

<td>Relationship</td><td>Inhibition in% at 2 μΜ</td>

<td>1</td><td>99.9</td>

<td>2</td><td>99.98</td>

<td>3</td><td>99.97</td>

<td>4</td><td>99.64</td>

<td>5A</td><td>99.97</td>

<td>5B</td><td>99.95</td>

<td>6</td><td>99.88</td>

<td>7</td><td>99.98</td>

<td>8</td><td>99.94</td>

<td>9</td><td>100</td>

<td>10</td><td>100</td>

<td>16</td><td>99.99</td>

<td>17 (reference)</td><td>78.85</td>

<td>18</td><td>100</td>

<td>24</td><td>100</td>

<td>29</td><td>100</td>

The individual pharmacological responses that are observed can vary according to and depending on the particular compound chosen or whether pharmaceutical carriers are present, as well as the type of formulation and method of administration used, and such

The expected variability or expected differences in results are considered in accordance with the practice of the invention.

Although particular embodiments of the invention have been illustrated and described in detail throughout the description, the invention is not limited thereto. The above detailed descriptions are given as an embodiment of the invention and should not be construed as limiting the invention in any way.

Proxy:

PPAWSO "ATENTOWA 'BELLEPAT"

Izabela Szychulskc-Howranek ul. Słowackiego 44, 37-700 Przeniwśl tel. (016) 7J2-37-77 fax: (016) 675-02-87 phone (0608) 503-081 e-mati <a href="mailto:bellepat@cp.pl">bellepat@cp.pl</a> NIP: 795-207-16-72 REGON: 1803505 (6

RIGHTS mgr liabelś Sjychuhka-hamfiri entry number 31S2

EP 2523950