OA17153A - Uracyl spirooxetane nucleosides - Google Patents

Abstract

The present invention relates to compounds of the formula I:

Description

Uracyl Splrooxetane Nucleosides

Background of the Invention

This invention relates to spirooxetanc nucleosides and nuclcotides that arc inhibitors of the hepatitis C virus (HCV).

HCV is a single stranded, positive-sense RNA virus belonging to the Flaviviridae family of viruses in the hepacivirus genus. The NS5B région of the RNA polygone encodes a RNA dépendent RNA polymerase (RdRp), which is esscntial to viral réplication. Following the initial acute infection, a majority of infected individuals develop chronic hepatitis because HCV replicates prcfercntially in hépatocytes but is not directly cytopathie. In particular, the lack of a vigorous T-lymphoeytc response and the high propensity of the virus to mutate appcar to promotc a high rate of chronic infection. Chronic hepatitis can progress to liver fibrosis, leading to cirrhosis, end-stage liver disease, and HCC (hepatocellular carcinoma), making it the leading causeof liver transplantations. There arc six major HCV génotypes and more than 50 subtypes, which arc dîfferently distributed gcographically. HCV génotype 1 is the prédominant génotype in Europe and in the US. The extensive gcnetic hctcrogcncity of HCV has important diagnostic and clinîcal implications, perhaps explaining difficultics in vaccine development and the lack of response to current therapy.

Transmission of HCV can occur through contact with contaminatcd blood or blood products, for exemple following blood transfusion or intravenous drug use. The introduction of diagnostic tests used in blood screening has lcd to a downward trend in post-transfusion HCV incidence. However, given the slow progression to the end-stage liver disease, the existing infections will continue to présent a scrious medical and économie burden for décades.

Current HCV therapy is based on (pegylated) interferon-alpha (IFN-α) in combination wîth ribavirin. This combination therapy yields a sustained virologie response tn more than 40% of patients infected by génotype 1 HCV and about 80% of those infected by génotypes 2 and 3. Beside the limited efficacy against HCV génotype 1, this combination therapy has significant side effects and is poorly tolcrated in many patients. Major side effects include influenza-like symptoms, hématologie abnormalities, and neuropsychiatrie symptoms. Hcncc there is a need for more effective, convenient and better-tolcratcd treatments.

-2Rcccntly, therapy possibilit ies hâve extended towards the combination of a HCV protease inhibitor (e.g. Tclaprcvir or boccprcvir) and (pegylated) interferon-alpha (IFNa) ! ribavirin.

Expérience with HIV drugs, in particular with HIV protease inhibitors, has taught that sub-optimal pharmacok inc tics and complex dosing régimes quickly rcsult in inadvertent compliance failurcs. This in tum means that the 24 hour trough concentration (minimum plasma concentration) for the respective drugs in an HIV régime frcqucntly falls below the IC90 or ED90 threshold for large parts of the day. It is considcrcd that a 24 hour trough level of at least the IC50, and more rcalistically, the IC90 or ED90, is essential to slow down the development of drug escape mutants. Achicving the necessary pharmacokinctics and drug metabolism to allow such trough levels provides a stringent challenge to drug design.

The NS5B RdRp is essentiel for réplication of the single-strandcd, positive sense, HCV RNA genome. This enzyme has elicited significant interest among médicinal chcmists. Both nucleoside and non-nuclcoside inhibitors of NS5B are known. Nucleoside inhibitors can act as a chain terminator or as a compétitive inhibitor, or as both. In order to be active, nucleoside inhibitors hâve to be taken up by the cell and converted in vivo to a triphosphate. Thîs conversion to the triphosphate is commonly mediated by cellular kinases, which imparts additional structural rcquircmcnts on a potential nucleoside polymerase inhibitor. In addition this limits the direct évaluation of nucleosides as inhibitors of HCV réplication to cell-bascd assays capable of in situ phosphorylation.

Severai attempts hâve been made to develop nucleosides as inhibitors of HCV RdRp, but while a handful of compounds hâve progressed into clinical development, none hâve proceedcd to registration. Amongst the problems which HCV-targetcd nucleosides hâve encountered to date are toxicity, mutagenicity, lack of sclectivity, poor efficacy, poor bioavailability, sub-optimal dosage régimes and ensuing high pill burden and cost of goods.

Spirooxetane nucleosides, in particular l-(8-hydroxy-7-(hydroxy- methyl)-l,6dioxaspiro[3.4]octan-5-yl)pyrimidinc-2,4-dionc dérivatives and their use as HCV inhibitors arc known from WO2010/130726, and WO2012/062869, including CAS-1375074-52-4.

-3Thcrc is a need for HCV inhibitors that may ovcrcome at least onc of the disadvantages of current HCV therapy such as side effects, limited eflicacy, the cmcrgîng of résistance, and compliance failures, or improve the sustained viral response.

The présent invention conccms a group of HCV-inhibiting uracyl spirooxctanc dérivatives with useful properties regardîng onc or more of the following parameters: antiviral eflicacy towards at least onc of the following génotypes ta, lb, 2a, 2b, 3,4 and 6, favorable profile of résistance development, lack of toxicity and gcnotoxicity, favorable pharmacokinctics and pharmacodynamies and case of formulation and administration.

Description ofthe Invention

In onc aspect the présent invention provides compounds that can be represented by the

including any possible stereoiso mer thereof, wherein:

R⁹ is Cj-Côalkyl, phenyl, Cj-Cîcycloalkyl or Cj-Cîalkyl substituted with 1,2 or 3 substituents each independently selected from phenyl, naphtyl, Cî-Cecycloatkyt, hydroxy, or Ci-Ceaikoxy;

or a pharmaceutically acceptable sait or solvaté thereof.

Of particular interest are compounds of formula 1 or subgroups thereof as defined herein, that hâve a structure according to formula la:

(la)

-4In one embodiment of the présent invention, R⁹ is Ci-Côalkyl, phenyl, Cj-C7cycloalkyl or Ci-Cjalkyl substituted with 1 substituent selected from phenyl, Ci-Cecycloalkyl, hydroxy, or Cj-Ccalkoxy. In another embodiment of the present invention, R⁹ in Formula I or la is Ci-Cealkyl or CrC2alkyl substituted with phenyl Ci-C2alkoxy or Ci-Cécycloalkyl. In a more preferred embodiment, R⁹ is C2-C₄alky! and in a most preferred embodiment, R⁹ is Apropyl.

A preferred embodiment according to the invention is a compound according to

including any pharmaceutically acceptable sait or solvaté thereof and the use of compound (V) in the synthesis of a compound according to Formula I, la or Ib.

The invention further relates to a compound of formula V:

including any pharmaceutically acceptable sait or solvaté thereof and the use of compound (V) in the synthesis of a compound according to Formula I, la or lb.

In addition, the invention relates to a compound of formula VI:

including any stereochcmical form and/or pharmaceutically acceptable sait or soivate thereof.

Additionally, the invention relates to a pharmaceutical composition comprising a compound according to Formula I, la or Ib, and a pharmaceutically acceptable carrier. The invention also relates to a product containing (a) a compound of formula I, la or Ib a, and (b) another HCV inhibitor, as a combined préparation for simultaneous, separatc or sequential use in the treatment of HCV infections

Yet another aspect of the invention relates to a compound according to Formula I, la or Ib or a pharmaceutical composition according to the présent invention for use as a médicament, preferably for use in the prévention or treatment of an HCV infection in a mammal.

In a further aspect, the invention provides a compound of formula 1 la or Ib or a pharmaceutically acceptable sait, hydrate, or soivate thereof, for use in the treatment or prophylaxie (or the manufacture of a médicament for the treatment or prophylaxie) of HCV infection. Représentative HCV génotypes in the context of treatment or prophylaxie in accordance with the invention include génotype lb (prévalent in Europe) or la (prévalent inNorth Amerîca). The invention also provides a method for the treatment or prophylaxie of HCV infection, in particular of the génotype la or 1 b.

Of particular intereet is compound 8a mentioned in the section “Exemples” as well as the pharmaceutically acceptable acid addition salts of this compound.

The compounds of formula I hâve several ccntcrs of chirality, in particular at the carbon atoms Γ, 2’, 3', and 4'. Although the stcrcochemistry at these carbon atoms is fîxed, the compounds may display at Ieast 75%, preferably at Ieast 90%, such as in excess of 95%, or of 98%, cnantiomeric purity at each of the chiral centers.

-6The phosphores ccntcr can bc présent as Rp or Sp, or a mixture of such stcrcoisomcrs, including racemates. Diastercoisomcrs resulting from the chiral phosphores ccntcr and a chiral carbon atom may exist as well.

The compounds of formula I are represented as a defined stercoisomcr, cxccpt for the stcrcoisomerism at the phosphorous atom. The absolutc configuration of such compounds can be determined using art-known methods such as, for example, X-ray diffraction or NMR and/or implication from starting materiais of known stcrcochemistry. Pharmaceutical compositions in accordance with the invention will preferably comprise stcreoisomcrically pure forms of the indicated stercoisomcr of the particular compound of formula I.

Pure stereoisomcric forms of the compounds and intermediates as mentioned herein arc defined as isomers substantially free of other cnantiomcric or diastereomcric forms of the same basic molccular structure of said compounds or intermediates. In particular, the term “stcrcoisomerically pure” conccms compounds or intermediates having a stereoisomcric excess of at least 80% (i.c. minimum 90% of one isomer and maximum 10% of the other possible isomers) up to a stereoisomcric excess of 100% (i.c. 100% of one isomer and none of the other), more in particular, compounds or intermediates having a stereoisomcric excess of 90% up to 100%, even more in particular having a stereoisomcric excess of 94% up to 100% and most in particular having a stereoisomcric excess of 97% up to 100%, or of 98% up to 100%. The terms “cnantiomcrically pure” and “diastcrcomerically pure” should bc understood in a similar way, but then having regard to the cnantiomcric excess, and the diastcreomeric excess, respectively, of the mixture in question.

Pure stereoisomcric forms of the compounds and intermediates of this invention may be obtained by the application of art-known procedures. For instance, enantiomers may bc separated from each other by the sélective crystallization of their diastcreomeric salts with optically active acids or bases. Examples thereof are tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid and camphorsulfonic acid. Altemativcly, enantiomers may bc separated by chromatographie techniques using chiral stationary layers. Said pure stcreochemically isomcric forms may also be derived from the corresponding pure stcreochemically isomcric forms of the appropriate starting materiais, provided that the réaction occurs stcrcospecifïcally. Preferably, if a spécifie stercoisomcr is desired, said compound is synthesized by stereospecifïc methods of préparation. These methods will advantagcously cmploy enantiomerically pure starting materiais.

-7The diastereomcric raccmatcs of the compounds of formula I can bc obtained separately by conventional methods, Appropriate physical séparation methods that may advantageously bc employed are, for cxamplc, sélective crystallization and chromatography, e.g. column chromatography.

The pharmaceutically acceptable addition salts comprise the therapeutically active nontoxic acid and base addition sait forms of the compounds of formula I. Of interest arc the free, i.e. non-salt forms of the compounds of formula I, or of any subgroup of compounds of formula I specified herein.

The pharmaceutically acceptable acid addition salts can conveniently bc obtained by treating the base form with such appropriate acid. Appropriate acids comprise, for cxamplc, inorganic acids such as hydrohalic acids, e.g. hydrochforic or hydrobromic 15 acid, sulfuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propionic, hydroxyacctic, lactic, pyruvic, oxalic (i.e. ethanedioie), malonic, succinic (i.e. butanedioie acid), malcic, fumaric, malic (i.e. hydroxylbutanedioic acid), tartaric, citric, mcthancsulfonîc, cthanesulfonic, bcnzcncsulfonic, p-toluencsulfonic, cyclamîc, salicylic, p-aminosalicylic, palmoic and the like acids.

Conversely said sait forms can bc converted by treatment with an appropriate base into the free base form.

The compounds of formula I containing an acidic proton may also be converted into their non-toxic métal or amine addition sait forms by treatment with appropriate 25 organic and inorganic bases. Appropriate base sait forms comprise, for cxamplc, the ammonium salts, the alkali and earth alkalinc métal salts, e.g. the lithium, sodium, potassium, magnésium, calcium salts and the like, salts with organic bases, e.g. the bcnzathinc, AT-mcthyl-D-glucaminc, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like.

The term solvatés covcrs any pharmaceutically acceptable solvatés that the compounds of formula I as well as the salts thereof, arc able to form. Such solvatés are for example hydrates, alcoholatcs, e.g. cthanolatcs, propanolatcs, and the like.

Some of the compounds of formula I may also exist in their tautomeric form. For example, tautomeric forms of amide (-C(=O)-NH-) groups arc îminoalcohols (-C(OH)=N-), which can bccomc stabilized in rings with aromatic charactcr. The uridine base is an cxamplc of such a form. Such forms, although not explîcitly

-8indicatcd in the structural formulae represented herein, arc intended to be includcd within the scope of the présent invention.

Short description of the Figure

Figure 1: In vivo efficacy of compound 8a and CAS-1375074-52-4 as determined in a humanized hépatocyte mouse model.

Définitions

As used herein C|-C_nalkyl as a group or part of a group defines saturated straight or branchcd chain hydrocarbon radicale having from 1 to n carbon atoms. Accordingly, C|-C₄alkyr as a group or part of a group defines saturated straight or branchcd chain hydrocarbon radicals having from 1 to 4 carbon atoms such as for example methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, 2-mcthyl-l-propyl, 2-mcthyl-2-propyl. Ci-Ccalkyl cncompasses Ci-C₄alkyl radicals and the higher homologues thereof having 5 or 6 carbon atoms such as, for exemple, 1-pcntyl, 2-pcntyl, 3-pcntyl, 1-hcxyl, 2-hcxyl, 2-mcthyl-l-butyl, 2-methyl-l-pcntyl, 2-cthyl-l-butyl, 3-mcthyl-2-pentyl, and the like. Of interest amongst Ci-Qjalkyl is Ci-C₄alkyl.

*Ci-C_Balkoxy’ means a radical -O-C]-C_aalkyl wherein Ci-C_nalkyl is as defined above. Accordingly, ‘Ci-Cealkoxy’ means a radical -O-Ci-Ccalkyl wherein Ci-Cealkyl is as defined above. Examples of Ci-Cealkoxy arc methoxy, cthoxy, n-propoxy, or isopropoxy. Of interest is ‘Ci-C2alkoxy’, cncompassing methoxy and cthoxy.

“Cî-Cecycloalkyl” includes cydopropyl, cyclobutyl, cydopentyl, and cyclohexyl.

In one embodiment, the term “phenyl-Ci-Cealkyl” is benzyl.

As used herein, the term ‘(=0)’ or ‘oxo’ forms a carbonyl moiety when attached to a carbon atom. It should be noted that an atom can only be substituted with an oxo group when the valency of that atom so permits.

The term monophosphatc, diphosphatc or triphosphate ester refers to groups:

o o o o o o —o—j³—OH . —o—y—O—|—OH · —o—|—o—|—o—|—OH

OH OH OH OH OH OH

Where the position of a radical on a molecular moiety is not specified (for example a substituent on phenyl) or is represented by a floating bond, such radical may be

-9positioned on any atom of such a moiety, as long as the resulting structure is chemically stable. When any variable is présent more than once in the molécule, each définition is independent.

Whenever used herein, the term ‘compounds of formula Γ, or ‘the présent compounds’ or similar terms, it is meant to include the compounds of Formula I, la and Ib, including the possible stcrcochcmically isomeric forms, and their pharmaceutically acceptable salts and solvatés.

IO The présent invention also inciudes isotopc-labclcd compounds of formula I or any subgroup of formula I, wherein one or more of the atoms is replaced by an isotope that difFers from the one(s) typically found in nature. Examples of such isotopes include isotopes of hydrogen, such as ²H and ³H; carbon, such as ⁿC, ¹³C and ^HC; nitrogcn, such as ^l3N and ^l3N; oxygen, such as ^ISO, ^l7O and ^l8O; phosphorus, such as ^3lP and ³²P, 15 sulphur, such as ³⁵S; fluorine, such as ¹⁸F; chlorine, such as ³⁶C1; bromine such as ⁷⁵Br, ⁷⁶Br, ⁷⁷Br and ⁸²Br; and iodine, such as ^l23I, ^l241,¹²⁵I and ^,3,I. Isotope- labeied compounds ofthe invention can be prepared by processes analogous to those described herein by using the appropriatc isotopc-labclcd rcagents or starting materials, or by artknown techniques. The choicc ofthe isotope included in an isotope-labcled compound 20 dépends on the spécifie application of that compound. For exemple, for tissue distribution assays, a radioactive isotope such as ³H or ^HC is incorporated. For radioimaging applications, a positron emitting isotope such as ⁿC, ^l8F, ^l3N or ^lsO will be useful. The incorporation of deuterium may providc greater mctabolic stability, resulting in, e.g. an increased in vivo half life ofthe compound or reduced dosage 25 requirements.

General Synthetic Procedures

The following schemes are just meant to bc illustrative and are by no means limiting the scope.

The startingmaterial I-[(4R,5R,7R,8R)-8-hydroxy-7-(hydroxymethyl)-l,6-dioxaspiro[3.4]octan-5-yl]pyrimidinc-2,4(l//,3//)-dionc (1) can be prepared as exemplified in WO2010/130726. Compound (1) is converted into compounds ofthe présent invention via a p-mcthoxybenzyl protectcd derivative (4) as exemplified in the following Scheme 1.

CHjCN-HjOqOTO),

Scheme 1

In Scheme 1, R⁹ can bc Ci-Cealkyl, phenyl, naphtyl, C3-C?cyc!oalkyl or Ci-Cîalkyl substituted with 1,2 or 3 substituents each independently selected from phenyl, Ca-Côcycloalkyl, hydroxy, or Cj-Cealkoxy, preferably R⁹ is Ci-Cealkyl or Ci-Cîalkyl substituted with phenyl, Ci-Cîalkoxy or Cj-Cecycloalkyl, even more preferably R⁹ is Cî-C4alkyl and most preferably R⁹ is i-propyl.

In a further aspect, the présent invention concems a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I as specified herein, and a pharmaceutically acceptable carrier. Said composition may contain from 1% to 50%, or from 10% to 40% of a compound of formula I and the remainder of the composition is the said carrier. A therapeutically effective amount in this context is an amount sufïîcient to act in a prophylactic way against HCV infection, to inhibit HCV, to stabilize or to rcduce HCV infection, in infected subjects or subjects being at risk of becoming infected. In still a further aspect, this invention relates to a process of preparing a pharmaceutical composition as specified herein, which comprises intimatcly mixing a pharmaceutically acceptable carrier with a therapeutically effective amount of a compound of formula I, as specified herein.

-11Thc compounds of formula I or of any subgroup thereof may bc formulated into various pharmaceutical forms for administration purposes. As appropriate compositions there may bc cited ail compositions usually employed for systcmically administering drugs. To prépare the pharmaceutical compositions of this invention, an effective amount of the particular compound, optionally in addition sait form or métal compiex, as the active ingrédient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a widc variety of forms depending on the form of préparation desired for administration. These pharmaceutical compositions arc désirable in unitary dosage form suitable, particularly, for administration orally, rcctally, percutancously, or by parentéral injection. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may bc employed such as, for exemple, water, glycols, oils, alcohols and the like in the case of oral liquid préparations such as suspensions, syrups, élixirs, émulsions and solutions; or solid carriers such as starchcs, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powdcrs, pii 1s, capsules, and tablets. Because of their easc in administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid pharmaceutical carriers arc obviously employed. For parentéral compositions, the carrier will usually comprise stérile water, at least in large part, though other ingrédients, for exemple, to aid solubility, may be included. Injectable solutions, for exemple, mey bc prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also bc prepared in which case appropriate liquid carriers, suspending agents and the like may bc employed. Also included are solid form préparations intendcd to bc converted, shortly before use, to liquid form préparations. In the compositions suitable for pcrcutancous administration, the carrier optionally comprises a pénétration cnhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introducc a sîgnificant de Ict crions effect on the skin. The compounds of the présent invention may also bc administered via oral inhalation or insufflation in the form of a solution, a suspension or a dry powder using any art-known delivery System.

It is cspecially advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for easc of administration and uniformity of dosage.

Unit dosage form as used herein refers to physically discrète units suitable as unitary dosages, each unit containing a predetermined quantity of active ingrédient calculated to producc the desired therapeutic effect in association with the rcquired pharmaceutical carrier. Examples of such unit dosage forms arc tablets (including

-12scorcd or coated tablets), capsules, pi Ils, supposi tories, powder packets, wafers, injectable solutions or suspensions and the like, and segregated multiples thereof.

The compounds of formula I show activity against HCV and can bc used in the treatment and/or prophylaxie of HCV infection or discascs associated with HCV. The latter include progressive liver fibrosis, inflammation and necrosis leading to cirrhosis, end-stage liver disease, and HCC. The compounds of this invention morcovcr are believed to bc active against mutated strains of HCV and show a favorable pharmacokinetic profile and hâve attractive properties in terms of bioavailability, including an acceptable half-life, AUC (area under the curve) and peak values and lacking unfavorable phenomena such as insufficient quick onset and tissue rétention.

The in vitro antiviral activity against HCV of the compounds of formula I can bc tested in a cellular HCV replicon system based on Lohmann et al. (1999) Science 285:110-113, with the further modifications described by Krieger et al. (2001) Journal of Virology 75:4614-4624 (incorporated herein by reference), which is further exemplified in the exemples section. This model, while not a complété infection model for HCV, is widely acecpted as the most robust and efficient model of autonomous HCV RNA réplication currently availabie. It will bc appreciated that it is important to distinguish between compounds that specificaliy interfère with HCV functions from those that exert cytotoxic or cytostatic effects in the HCV replicon model, and as a conséquence cause a decrease in HCV RNA or linked reporter enzyme concentration. Assays are known in the field for the évaluation of cellular cytotoxicity based for example on the activity of mitochondrial enzymes using fluorogenic redox dycs such as rcsazurin. Furthermorc, cellular countcr scrccns exist for the évaluation of non-sclcctive inhibition of linked reporter gene activity, such as fîrcfly luciferase. Appropriate cell types can be equipped by stable transfection with a luciferase reporter gene whose expression is dépendent on a constituévely active gene promoter, and such cells can be used as a counter-scrcen to eliminate non-selcctive inhibitors.

Due to their anti-HCV properties, the compounds of formula I, including any possible stereoisomers, the pharmaceutically acceptable addition salts or solvatés thereof, arc useful in the treatment of warm-blooded animais, in particular humans, infected with HCV, and in the prophylaxis of HCV infections. The compounds of the présent invention may therefore bc used as a mcdicine, in particular as an anti-HCV or a HCVinhibiting mcdicine. The présent invention also relates to the use of the présent compounds in the manufacture of a médicament for the treatment or the prévention of HCV infection. In a further aspect, the présent invention relates to a method of treating

-13a warm-bloodcd animal, in particular human, infected by HCV, or being at risk of bccoming infected by HCV, said method comprising the administration of an anti-HCV effective amount of a compound of formula I, as specified herein. Said use as a mcdicinc or method of treatment comprises the systemic administration to HCVinfcctcd subjects or to subjects susceptible to HCV infection of an amount effective to combat the conditions associated with HCV infection.

In general it is contemplated that an antiviral effective daily amount would be from about 1 to about 30 mg/kg, or about 2 to about 25 mg/kg, or about 5 to about 15 mg/kg, or about 8 to about 12 mg/kg body weight. Average daily doses can be obtained by multiplying thèse daily amounts by about 70. It may be appropriate to administer the required dose as two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing about 1 to about 2000 mg, or about 50 to about 1500 mg, or about 100 to about 1000 mg, or about 150 to about 600 mg, or about 100 to about 400 mg of active ingrédient per unit dosage form.

As used herein the term about” has the meaning known to the person skilled in the art. In certain embodiments the term “about” may be left out and the exact amount is meant. In other embodiments the term “about” means that the numerical following the term “about” is in the range of ± 15%, or of ± 10%, or of ± 5%, or of ± 1%, of said numerical value.

-I4Examolcs

Scheme 2

Synthesis of compound (8a)

POCh (5)

OH

A.

Et₃N, -5°C,1 h

(6a)

Et₃N. NMI, -20°C, 5h

CAN

CH₃CN-H₂O(3œ7). 10°C, 15 h

Synthesis of compound (2)

Compound (2) canbc prepared by dissolvîng compound (1) in pyridine and adding l,3-dichloro-l,l,3,3-tetraisopropyldisiloxanc. The reaction is stirred at room température until complété. The solvent îs removed and the product rcdissolved in 10 CH2CI2 and washed with saturated NaHCOj solution. Drying on MgSO₄ and removal ofthe solvent gives compound (2).

Synthesis of compound (3)

Compound (3) is prepared by reacting compound (2) with p-mcthoxybenzylchloride în the présence of DBU as the base in CHjCN.

Synthesis of compound (4)

Compound (4) is prepared by cleavage of the bis-sily 1 protccting group in compound (3) using TBAF as the fluoridc source.

Synthesis of compound (6a)

A solution of isopropyl alcohol (3.86 mL,0.05mol) and triethylamine (6.983 mL, 0.05mol) în dichloromethane (50 mL) was added to a stirred solution of POCb (5)

-15(5.0 mL, 0.0551mol) in DCM (50 mL) dropwise over a period of 25 min at -5°C. After the mixture stirred for lh, the solvent was evaporated, and the residue was suspended in ether (100 mL). The triethylamine hydrochloride sait was filtered and washed with ether (20 mL). The filtrate was concentrated, and the residue was distilled to give the (6) as a colorless liquid (6.1g, 69 %yield).

Synthesis of compound (7a)

To a stirred suspension of (4) (2.0 g, 5.13 mmol) in dichloromethane (50 mL) was added triethylamine (2,07 g, 20.46 mmol) at room température. The reaction mixture was cooled to -20°C, and then (6a) (1.2 g, 6.78mmol) was added dropwise over a period of 10min. The mixture was stirred at this température for 15min and then NM1 was added (0.84 g, 10.23 mmol), dropwise over a period of 15 min. The mixture was stirred at -15°C for 1 h and then slowly warmed to room température in 20 h. The solvent was evaporated, the mixture was concentrated and purified by column chromatography using petroleum cther/EtOAc (10:1 to 5:1 as a gradient) to give (7a) as white solid (0.8 g, 32 % yield).

Synthesis of compound (8a)

To a solution of (7a) in CHjCN (30 mL) and H₂O (7 mL) was add CAN portion wise below 20°C. The mixture was stirred at 15-20°C for 5h under N₂. Na₂SOj (370 mL) was added dropwise into the reaction mixture below 15°C, and then Na₂COj (370 mL) was added. The mixture was filtered and the filtrate was extracted with CH₂CI₂ (100 mL*3). The organic layer was dried and concentrated to give the residue. The residue was purified by column chromatography to give the target compound (8a) as white solid. (Yield: 55%) ‘H NMR (400 MHz, CHLOROFORM-7) δ ppm 1.45 (dd, 7=7.53, 6.27 Hz, 6 H), 2.65 2.84 (m, 2 H), 3.98 (td, J=10.29, 4.77 Hz, 1 H), 4.27 (t, 7=9.66 Hz, 1 H), 4.43 (ddd, 7=8.91,5.77,5.65 Hz, 1 H), 4.49 - 4.61 (m, 1 H), 4.65 (td, 7=7.78, 5.77 Hz, 1 H), 4.73 (d, 7=7.78 Hz, 1 H), 4.87 (dq, 7=12.74,6.30 Hz, 1 H), 5.55 (br. s„ 1 H), 5.82 (d, 7=8.03 Hz, 1 H), 7.20 (d, 7=8.03 Hz, 1 H), 8.78 (br. s., 1 H); ^JIP NMR (CHLOROFORM-7) 6 ppm-7.13; LC-MS: 375 (M+1 )+

-16Scheme 3

Synthesis of compound (VI)

Step I: Synthesis of compound (9)

Compound (1), CAS 1255860-33-3 (1200 mg, 4.33 mmol) and l,8-bis(dimcthylamino)naphthalene (3707 mg, 17.3 mmol) were dissolved in 24.3 mL of trimethylphosphate. The solution was cooled to 0°C. Compound (5) (1.21 mL, 12.98 mmol) was added, and the mixture was stirred well maintaining the température at 0°C for 5 hours. The reaction was quenched by addition of 120 mL of tctracthylammonium bromide solution (IM) and extracted with CH2CI2 (2x80 mL). Purification was done by préparative HPLC (Stationary phase: RP XBridgc Prcp C18 OBD-10gm, 30x150mm, mobile phase: 0.25% NH4HCO3 solution in water, CHjCN), yiclding two fractions. The purest fraction was dissolved in water (15 mL) and passed through a manually packcd Dowcx (H*) column by clution with water. The end of the clution was determined by checking UV absorbance of eluting fractions. Combined fractions were frozen at -78°C and lyophilized. Compound (9) was obtained as a white flufïy solid (303 mg, (0.86 mmol, 20% yield), which was used immediatcly in the following réaction.

-17Step 2: Préparation of compound (Vl)

Compound (9) (303 mg, 0.86 mmol) was dissolved in 8 mL water and to this solution was added N.N’-DicyclohexyM-morpholine carboxamidine (253.8 mg, 0.86 mmol) dissolved in pyridine (8.4 mL). The mixture was kept for 5 minutes and then evaporated to dryness, dried ovemight in vacuo ovemight at 37°C. The résidu was dissolved in pyridine (80 mL). This solution was added dropwise to vigorously stirred DCC (892.6 mg, 4.326 mmol) in pyridine (80 mL) at reflux température. The solution was kept refluxing for 1.5h during which some turbïdity was observed in the solution. The réaction mixture was cooled and evaporated to dryness. Diethylether (50 mL) and water (50 mL) were added to the solid résidu. N'N-dicyclohcxylurea was filtered off, and the aqueous fraction was purified by préparative HPLC (Stationary phase: RP XBridge Prep Cl8 OBD-ΙΟμτη, 30x 150mm, mobile phase: 0.25% NH4HCOJ solution in water, CHjCN), yielding a white solid which was dried ovemight in vacuo at 38°C. (185 mg, 0.56 mmol, 65% yield). LC-MS: (M+H)⁺: 333.

'H NMR (400 MHz, DMSO-dô) d ppm 2.44 - 2.59 (m, 2 H) signal faits under DMSO signal, 3.51 (td, >9.90,5.50 Hz, 1 H), 3.95 - 4.11 (m, 2 H), 4,16 (d, >10.34 Hz, 1 H), 4.25 - 4.40 (m, 2 H), 5.65 (d, >8.14 Hz, 1 H), 5.93 (br. s., 1 H), 7.46 (d, >7.92 Hz, 1 H), 2H's not observed

Biological exemples

Renllcon Assays

The compounds of formula I were examîncd for activity in the inhibition of HCV-RNA réplication in a cellular assay. The assay was used to demonstratc that the compounds of formula I inhibited a HCV functional cellular rcplicating cell fine, also known as HCV rcplicons. The cellular assay was based on a bicistronic expression construct, as described by Lohmann et al. (1999) Science vol. 285 pp. 110-113 with modifications described by Krieger et al. (2001) Journal of Virology 75:4614-4624, in a multi-targct screening strategy.

RepHeon assay (A)

In essence, the method was as follows. The assay utilized the stably transfccted cell line

Huh-7 luc/nco (hercafter referred to as Huh-Luc). This cell line harbors an RNA encoding a bicistronic expression construct comprising the wild type NS3-NS5B régions of HCV type Ib translatcd froman internai ribosome entry site (IRES) from enccphalomyocarditis virus (EMCV), prcccded by a reporter portion (FfL-luciferasc), and a sclectable marker portion (nco^R, néomycine phosphotransfcrasc). The construct is bordered by 5’ and 3’ NTRs (non-translatcd régions) from HCV génotype lb.

-18Continucd culture ofthe rcplicon cells in the présence ofG418 (neo^R) is dépendent on the réplication ofthe HCV-RNA. The stably transfected rcplicon cells that express

HCV-RNA, which replicates autonomously and to high levels, encoding inter alia luciferase, were used for screening the antiviral compounds.

The replicon cells wcrc platcd in 384-well plates in the presence of the test and control compounds which were added in various concentrations. Following an incubation of three days, HCV réplication was measured by assaying luciferase activity (using standard luciferase assay substrates and rcagents and a Pcrkin Elmcr VicwLux™ ultraHTS microplate imager). Replicon cells in the control cultures hâve high luciferase expression in the absence ofany inhibitor. The inhibitory activity of the compound on luciferase activity was monitored on the Huh-Luc cells, enabling a dose-response curve for each test compound. EC50 values wcrc then calculated, which value represents the amount ofthe compound required to decrease the level of detected luciferase activity by 50%, or more specifically, the ability of the genetically linked HCV rcplicon RNA to rcplicatc.

Results (A)

Table 1 shows the replicon results (ECso, rcplicon) and cytotoxicity results (CCso (μΜ) (Huh-7)) obtained for the compound of the exemples given above.

Table 1.

Compound number	ECso (μΜ) (HCV)	CCso (μΜ) (Huh-7)
8a	0.13 (n = 4)	> 100

Replicon assays (B)

Further rcplicon assays were performed with compound 8a of which the protocols and results arc disclosed below.

Assay 1

The anti-HCV activity of compound 8a was tested in cell culture with rcplicon cells generated using rcagents from the Bartcnschlagcr laboratory (the HCV 1b bicistronic subgenomic luciferase reporter rcplicon clone ET). The protocol induded a 3-day incubation of 2500 rcplicon colis in a 384-wcll format in a ninc-point 1:4 dilution sériés ofthe compound. Dose response curves were generated based on the firefly luciferase

-19rcad-out. In a variation of this assay, a 3 day incubation of3000 cells in a 96-wcll format in a ninc-poînt dilution séries was followed by qRT-PCR Taqman détection of

HCV genome, and normalized to the cellular transcript, RPL13 (of the ribosomal subunit RPL13 gene) as a control for compound inhibition of cellular transcription.

Assay 2

The anti-HCV activity of compound 8a was tested in cell culture with replicon cells generated using rcagcnts from the Bartcnschlager laboratory (the HCV 1 b bicîstronic subgcnomic lucifcrasc reporter replicon clone ET or Huh-Luc-Nco). The protocol included a 3-day incubation of 2 x 10⁴ replicon cells in a 96-well format in a six-point 1:5 dilution séries of the compound. Dose response curves were generated based on the luciferase read-out.

Assay 3

The anti-HCV activity of compound 8a was tested in cell culture with replicon cells generated using reagents from the Bartcnschlager laboratory (the HCV 1 b bicîstronic subgcnomic luciferase reporter replicon clone ET or Huh-Luc-Nco). The protocol included either a 3-day incubation of 8 x 1 θ’ cells or 2 x 10⁴ cells in a 96-well format in an cight-point 1:5 dilution séries of the compound. Dose response curves were generated based on the luciferase read-out.

Results

Table 2 shows the average replicon results (ECso, replicon) obtained for compound 8a following assays as given above.

Table 2:

Assay	Average ECso value (8a):
1	57 μΜ (n = 8)
2	17.5 μΜ(η=4)
3	> 100μΜ(η=1)

Primary human hépatocyte lit vitro assay

The anti-HCV activity of compound 8a was determined in an in vitro primary human hépatocyte assay. Protocols and results are disclosed below.

-20Protocol

Hcpatocyte isolation and culture

Primary human hépatocytes (PHH) were prepared from patients undergoing partial hcpatcctomy for métastases or benign tumors. Fresh human hépatocytes were isolated 5 from encapsulated liver fragments using a modification of the two-step collagénase digestion method. Briefly, encapsulated liver tissue was placed in a custom-made perfusion apparatus and hcpatic vcsscls were cannulated with tubing attached multichannel manifold. The liver fragment was mit ially perfused for 20 min with a prewarmed (37°C) calcium-frcc buffer supplemcnted with ethylene glycol tetraacetic 10 acid (EGTA) followed by perfusion with a prewarmed (37°C) buffer containing calcium (CaCI₂, H₂O₂) and collagénase 0.05% for 10 min. Then, liver fragment was gcntly shaken to free liver cells in Hépatocyte Wash Medium. Cellular suspension was filtered through a gauze-lincd funncl. Cells were centrifuged at low speed centrifugation. The supematant, containing damaged or dead hépatocytes, non parenchymal cells and débris was removed and pcllcted hépatocytes were rc-suspcndcd in Hépatocyte Wash Medium. Viability and cell concentration were determined by trypan btue exclusion test.

Cells were resuspended in complété hcpatocyte medium consisting of William’s medium (invitrogcn) supplemcnted with 100IU/L insulin (Novo Nordisk, France), and 10% heat inactivated fêtai calf sérum (Biowest, France), and seeded at a density 1,8x106 viable cells onto 6 well plates that had been prccoatcd with a type I collagen from calfskin (Sigma-Aldrich, France) The medium was rcplaced 16-20 hours later with fresh complété hépatocyte medium supplemented with hydrocortisone hcmisuccinate (SERB, Paris, France), and cells were left in this medium until HCV inoculation. The cultures were maintaincd at 37°C in a humidified 5% CO2 atmosphère.

The PHHs were inoculated 3 days after seeding. JFHl-HCVcc stocks were used to 30 inoculate PHHs for 12 hours, at a multiplicity of infection (MOI) of 0.1 ffu per cell.

After a 12-hours incubation at 37°C, the inoculum was removed, and monolayers were washed 3 times with phosphatc-buïïcrcd saline and incubated in complète hcpatocyte medium containing 0.1% dimethylsufoxidc as carrier contrat, 100 lU/ml of IFNalpha as négative control or elsc incrcasing concentrations of compound 8a. The cultures then 35 were maintained during 3 days.

-21Ouantitation of HCV RNA

Total RNA was prepared from cultured cells or from filtered culture supematants using the RNeasy or Qiamp viral RNA minikit respectively (Qiagen SA, Courtaboeuf,

France) according to the manu facturer’s recommendations. HCV RNA was quantified in cells and culture supematants using a strand-spccifïc reverse rcal-timc PCR technique described previously (Carrière M and al 2007):

Reverse transcription was performed using primers described previously located in the 50 NCR région of HCV genome, tag-RCl (5’-GGCCGTCATGGTGGCGAATAAGTCTAGCCATGGCGTTAGTA-3 ’) and RC21 (5’-CTCCCGGGGCACTCGCAAGC-3*) for the négative and positive strands, respectively. After a dénaturation step performed at 70^QC for 8 min, the RNA template was incubatcd at 4^QC for 5 min in the présence of200 ng oftag-RCl primer and 1.25 mM of each dcoxynucleosidc triphosphate (dNTP) (Promega, Charbonnières, France) in a total volume of 12 μΙ.

Reverse transcription was carried out for 60 min at 60°C in the présence of 20 U RNaseOutTM (Invitrogen,Cergy Pontoise, France) and 7.5 U ThermoscriptTM reverse transcriptase (Invitrogen), in the buffer rccommcndcd by the manufacturer. An additional treatment was applied by adding 1 μΐ (2U) RNascH (Invitrogen) for 20 min at 37°C.

The first round of nested PCR was performed with 2 μΐ of the cDNA obtained in a total volume of 50 μΐ, containing 3 U Taq polymerase (Promega), 0.5 mM dNTP, and 0.5 μΜ RC1 (5*-GTCTAGCCATGGCGTTAGTA-3’) and RC2I primers for positivcstrand amplification, or Tag (5’-GGCCGTCATGGTGGCGAATAA-3*) and RC21 primers for négative strand amplification. The PCR protocol consistcdof 18 cycles of dénaturation (94°C for 1 min), annealing (55°C for 45 sec), and extension (72°C for 2 min). The cDNA obtained was purified using the kit from Qiagen, according to the manu facturer *s instructions.

The purified product was then subjcctcd to rcal-time PCR. The réaction was carried out using the LightCyclcr 480 SYBR Green I Master (2x con) Kit (Roche, Grenoble,

France), with LC480 instruments and technology (Roche Diagnostics). PCR amplifications were performed in a total volume of 10 μΙ, containing 5 μΐ of Sybrgreen

I Master Mix (2x), and 25 ng of the 197R (5’-CTTrCGCGACCCAACACTAC-3’) and

104 (5’-AGAGCCATAGTGGTCTGCGG-3*) primers. The PCR protocol consistcd of one step of initial dénaturation for 10 min at 94°C, followed by40 cycles of

-22dcnaturation (95°C for 15 sec), annealing (57°C for 5 sec), and extension (72°C for 8 sec).

The quantitation of 28Sr RNA by spécifie RT-PCR was used as an internai standard to express the results of HCV positive or négative strands per pg of total hépatocyte RNA. Spécifie primers for 28 S rRNA were designed using the Oligo6 software 5’-TTGAAAATCCGGGGGAGAG-3’(nt2717-2735) and 50-ACATTGTTCCAACATGCCAG-30 (nt 2816-2797). Reverse transcription was performed using AMV reverse transcriptase (Promcga), and the PCR protocol consisted ofonc step of initial dénaturation for 8 min at 95^qC, followed by 40 cycles of dénaturation (95°C for 15 sec), annealing (54°C for 5 sec), and extension (72°C for 5 sec).

Results

Table 3 shows the anti-HCV activity of compound 8a as determined in the in vitro primary human hépatocyte assay described above. The numbers are expressed as 10⁶ HCV RNA copîes/pg of total RNA. Results of two independent experiments (Exp 1 and Exp 2) arc given. The data per experiment is the average of two measurements.

Table 3: Effect of compound 8a on positive strand HCV-RNA levels în primary human hépatocytes (expressed as 10⁶ HCV RNA copics/pg of total RNA).

Table 3.

	Exp. 1	Exp. 2
No HCV	0	0
HCV control	3.56	5.53
ÏFNa (lOOIU/mL)	1.48	1.59
8a (0.195 pM)	2.18	1.12
8a (0.78 pM)	2.25	1.3
8a (3.12 pM)	1.09	0.94
8a (12.5 pM)	2.17	1.3
8a(50 pM)	0.94	1.33

-23In vivo effîcacy assay

The in vivo effîcacy of compound 8a and CAS-1375074-52-4 was determined in a humanized hépatocyte mouse mode! (PBX-mouse) as previously described in Inoue et. al (Hcpatology. 2007 Apr;45(4):92t-8) and Tenato et. al. (Am J Pathol 2004;!65-901912) with the following spécification: Test animais: HCV G ta -infected PXB-mice, male or fematc, >70 % replacement index of human hépatocytes. Dosing was performed p.o for 7 days at doses indicated below wherein QD represents a single dose per day, B1D represents two doses per day.

Effîcacy of compound 8a was compared to CAS-1375074-52-4. Results arc indicated in Figure t. The Figure shows the log drop HCV viral RNA after dosing for a period of 7 days.

Figure 1 clearly shows that a dosing of 1 OOmg/kg QD for CAS 1375074-52-4 (indicated as *, n=4) does not rcsult in a significant log drop in HCV viral RNA. This in strong contrast to each of the indicated dose regimens for compound 8a, were a ciear log drop is observed for 100 mg/kg QD (indicated as ♦, n=3), 200mg/kg QD (indicated as ·, n=4), 50mg/kg BlD(indicatcd as , n=4). The most pronounced log drop effect in viral RNA is observed after a 7 day dosing of compound 8a at ! 00 mg/kg B1D (indicated as n=4).

Claims (11)

1. Claims

   1. A compound of formula I:

   5 including any possible stcrcoisomcr thereof, wherein:

   R’ is Cj-Cealkyl, phenyl, Cî-Cîcycloalkyl or Ci-Cîalkyl substituted with 1, 2 or 3 substituents each independently selected from phenyl, naphtyl, Cj-Cecycloalkyl, hydroxy, or Ci-Cealkoxy;

   10 or a pharmaceutically acceptable sait or solvaté thereof.
2. 2. A compound according to claim 1 which is of formula la:
3. 3. A compound according to claim 1 or 2, wherein R⁹ is Ci-Cealkyl or Ci-C2alkyl substituted with phenyl Ci-Cjalkoxy or Cî-Cecycloalkyl.
4. 4. A compound according to any one of claims 1 to 3, wherein R⁹ is C2-C₄alkyl. 20
5. 5. A compound according to any one of claims 1 to 4, wherein R⁹ is Apropyl.
6. 6. A compound according to any one of claims 1 to 5, which is of formula Ib:

   5 including pharmaceutically acceptable sait or solvaté thereof.
7. 8. The use of compound (V) in the synthesis of a compound according to any onc of claims 1-6.
8. 10 9. A compound of formula VI:

   including any stercochcmical form and/or pharmaceutically acceptable sait or solvaté thereof.

   15 10. A pharmaceutical composition comprising a compound according to any of claims

   1 to 6, and a pharmaceutically acceptable carrier.

   -26·
9. 11. A compound according to any of claims 1 to 6 or claim 9 or a pharmaceutical composition according to claim 10, for use as a mcdicament.
10. 12. A compound according to any of claims 1 to 6 or claim 9, or a pharmaceutical

    5 composition according to claim 9, for use in the prévention or treatment of an HCV infection in a mammal.
11. 13. A product containing (a) a compound of formula I as defined in any one ofclaims 1 to 6 or claim 9, and (b) another HCV inhibitor, as a combined préparation for

    10 simultancous, separate or scquential use in the treatment of HCV infections.