HK1160472A - Uracyl cyclopropyl nucleotides - Google Patents

Description

Uracil cyclopropyl nucleotide

Technical Field

The present invention relates to novel nucleotides which are inhibitors of the polymerase of the Hepatitis C Virus (HCV) and their use in the treatment or prevention of HCV.

Background

HCV is a single-stranded, positive-sense RNA virus belonging to the Flaviviridae family (Flaviviridae) in the hepacivirus genus. The NS5B region of the RNA polygene encodes an RNA-dependent RNA polymerase (RdRp), which is essential for viral replication. Following initial acute infection, most infected individuals develop chronic hepatitis because HCV replicates preferentially in hepatocytes rather than directly cytopathic. In particular, the lack of a robust T lymphocyte response and a high propensity for viral mutation appears to promote a high proportion of chronic infections. Chronic hepatitis can progress to liver fibrosis, leading to cirrhosis, end-stage liver disease, and HCC (hepatocellular carcinoma), making it the leading cause of liver transplantation.

There are six major HCV genotypes and more than 50 subtypes, which have different geographical distributions. HCV genotype 1 is the predominant genotype in europe and the united states. The extensive genetic heterogeneity of HCV has important diagnostic and clinical implications, perhaps accounting for difficulties in vaccine development and lack of response to current therapies.

Transmission of HCV can occur by contact with contaminated blood or blood products, for example after use in blood transfusion or intravenous drug injection. Diagnostic tests introduced for blood screening have resulted in a reduced incidence of HCV post-transfusion. However, due to the slow progression to advanced liver disease, existing infections will persist as a serious medical and economic burden for decades.

Current HCV therapy is based on (pegylated) interferon-alpha (IFN- α) in combination with ribavirin (ribavirin). This combination therapy produces a sustained viral response in more than 40% of patients infected with genotype 1HCV and about 80% of patients infected with genotypes 2 and 3. In addition to the limited efficacy for HCV genotype 1, this combination therapy has significant side effects and poor tolerability in many patients. Major side effects include flu-like symptoms, hematologic abnormalities and neuropsychiatric symptoms. Thus, there is a need for more effective, convenient and better tolerated treatments.

The dosing experience of HIV drugs, particularly HIV protease inhibitors, has shown suboptimal pharmacokinetics and complex dosing regimens, rapidly leading to unnoticed compliance failures (inadvertant compliance failures). This in turn means that in an HIV treatment regimen, the 24 hour trough concentration (lowest plasma concentration) of each drug often drops below IC during most of the day₉₀Or ED₉₀The threshold value of (2). It is believed that at least IC₅₀24 hours trough level, and more practically, IC₉₀Or ED₉₀Is necessary to slow down the development of drug escape mutations (drug escape mutants). Achieving the desired pharmacokinetics and drug metabolism to allow such trough levels provides a serious challenge for drug design.

NS5B RdRp is essential for the replication of a single-stranded, positive-sense HCV RNA genome. Such enzymes are of great interest to pharmaceutical chemists. Nucleoside and non-nucleoside inhibitors of NS5B are known. Nucleoside inhibitors can act both as chain terminators and as competitive inhibitors, which interfere with the binding of nucleotides to the polymerase. When acting as a chain terminator, the nucleoside homologue must be taken up by the cell and converted to a triphosphate in vivo. This conversion to triphosphates is typically mediated by cellular kinases, which makes additional structural requirements for potential nucleoside polymerase inhibitors. Furthermore, this limits the direct assessment of nucleosides as inhibitors of HCV replication to cell-based assays (cell-based assays) that can be phosphorylated in situ.

Various attempts have been made to develop nucleosides as inhibitors of HCV RdRp, and while few compounds are in clinical development, none go through registration. HCV-targeted nucleosides have heretofore encountered problems of toxicity, mutagenicity, lack of selectivity, poor therapeutic efficacy, poor bioavailability, suboptimal dose therapy and subsequent high drug burden, and item cost.

Several patents and patent applications and scientific publications disclose nucleoside analogs having HCV inhibitory activity. WO 2004/002999 discloses modified 2 'and 3' -nucleoside prodrugs for the treatment of flaviviridae infections. WO 2008/043704 discloses 4-amino-1- ((2R, 3S, 4S, 5R) -5-azido-4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydrofuran-2-yl) -1H-pyrimidin-2-one and ester derivatives as HCV polymerase inhibitors.

There is a need for HCV inhibitors that can overcome the disadvantages of current HCV therapies such as side effects, limited efficacy, emerging resistance, and compliance failures, as well as improve sustained viral responses.

The present invention relates to a group of HCV inhibiting 1- (7-hydroxy-6-hydroxymethyl-5-oxa-spiro [2.4] hept-4-yl) -1H, 3H-pyrimidine-2, 4-dione derivatives having useful properties with respect to one or more of the following parameters: antiviral efficacy, favorable profile of resistance development, absence of toxicity and genotoxicity, favorable pharmacokinetics and pharmacodynamics, and ease of formulation and administration. The compound 1- ((4R, 6R, 7S) -7-hydroxy-6-hydroxymethyl-5-oxa-spiro [2.4] hept-4-yl) -1H, 3H-pyrimidine-2, 4-dione, also known as 2 '-deoxy-2' -spiro-cyclopropyluridine, has been disclosed in J.am, chem.Soc., 1992, 114, 4007-.

The compounds of the present invention are also attractive due to the fact that they lack activity against other viruses, particularly against HIV. Patients infected with HIV often also suffer from concurrent infections such as HCV. Treatment of such patients with HCV inhibitors, which also inhibit HIV, can lead to the emergence of drug-resistant HIV strains.

Description of the invention

In one aspect, the present invention provides compounds that may be represented by formula I:

including any possible stereoisomers thereof, wherein:

R¹is hydrogen or halo;

R⁴is a monophosphate ester group, a diphosphate ester group or a triphosphate ester group; or R⁴Is a radical of the formula

R⁷Is phenyl, optionally selected from 1, 2 or 3 each independently from halo, C₁-C₆Alkyl radical, C₃-C₆Alkenyl radical, C₁-C₆Alkoxy radical, C₁-C₆Substituent substitutions of alkoxycarbonyl, hydroxyl and amino; or R⁷Is naphthyl; or R⁷Is indolyl or N-C₁-C₆An alkoxycarbonyl indolyl group;

R⁸is hydrogen, C₁-C₆Alkyl, benzyl;

R^8′is hydrogen, C₁-C₆Alkyl, benzyl; or

R⁸And R^8′Together with the carbon atom to which they are attached form C₃-C₇A cycloalkyl group;

R⁹is C₁-C₁₀Alkyl radical, C₃-C₇Cycloalkyl radical, C₃-C₆Alkenyl, benzyl or phenyl, the phenyl being optionally selected from 1, 2 or 3 each independently from the group consisting of hydroxy, C₁-C₆Alkoxy, amino, mono-and di-C₁-C₆Substituted with alkyl amino;

or a pharmaceutically acceptable salt or solvate thereof.

In another aspect, the present invention relates to the use of a compound of formula I as specified herein for the inhibition of HCV. Or is thatFor use in the manufacture of a medicament for the preparation of a compound of formula I as specified herein. The invention also relates to a composition of matter having the structure described above, but wherein R⁴Is a hydrogen intermediate to begin with the process for preparing the compound of formula I.

The group-NH-C (R)⁸)(R^8′) -C (═ O) -forming amino acid residues, which include natural and non-natural amino acid residues. These amino acid residues (wherein R⁸Is hydrogen) is of particular importance. In the examples that follow, R^8’Instead of hydrogen, the configuration possessed at the asymmetric carbon atom may be an L-amino acid configuration. Examples are alanine (Ala), valine (Val), isoleucine (Ile) and phenylalanine (Phe) residues, in particular L-Ala, L-Val, L-Ile and L-Phe. Amino acid residue (wherein R⁸And R^8’Together with the carbon atom to which they are attached form C₃-C₇Cycloalkyl) is 1, 1-cyclopropylamino acid or 1, 1-cyclobutylamino acid.

A subgroup of compounds of formula I are those compounds of formula I as defined herein, or a subgroup of compounds of formula I, wherein R¹Is hydrogen; or wherein R is¹Is iodo.

A subgroup of compounds of formula I are those compounds of formula I as defined herein, or a subgroup of compounds of formula I, wherein R⁴Is a radical of the formula

A subgroup of compounds of formula I are those compounds of formula I as defined herein, or a subgroup of compounds of formula I, wherein:

(a)R⁷is phenyl, optionally selected from 1, 2 or 3 each independently from halo, C₁-C₆Alkyl radical, C₃-C₆Alkenyl radical, C₁-C₆Alkoxy, hydroxy and amino; or R⁷Is naphthyl; or R⁷Is indolyl; or R⁷Is N-tert-butoxycarbonylindoleA group;

(b)R⁷is phenyl, optionally selected from 1, 2 or 3 each independently from halo, C₁-C₆Alkyl radical, C₃-C₆Alkenyl and C₁-C₆Substituent substitution of alkoxy; or R⁷Is naphthyl;

(c)R⁷is phenyl, optionally substituted by halo or C₁-C₆Alkyl substituted, or R⁷Is naphthyl;

(d)R⁷is phenyl, consisting of C₁-C₄Alkoxycarbonyl substitution;

(e)R⁷is phenyl, consisting of C₁-C₂Alkoxycarbonyl substitution;

(f)R⁷is phenyl, optionally substituted by chloro or C₁-C₆Alkyl substitution; or R⁷Is naphthyl;

(g)R⁷is phenyl, optionally selected from 1, 2 or 3 each independently from halo and C₁-C₆Alkyl substituent substitution;

(h)R⁷is phenyl, optionally substituted by 1 or 2 substituents each independently selected from halo, C₁-C₆Alkyl radical, C₃-C₆Alkenyl radical, C₁-C₆Alkoxy, hydroxy and amino; or R⁷Is naphthyl; or R⁷Is indolyl; or R⁷Is N-tert-butoxycarbonylindolyl;

(i)R⁷is phenyl, optionally substituted by 1, selected from halo, C₁-C₆Alkyl radical, C₃-C₆Alkenyl radical, C₁-C₆Alkoxy, hydroxy and amino; or R⁷Is naphthyl; or R⁷Is indolyl; or R⁷Is N-tert-butoxycarbonylindolyl;

(j)R⁷is phenyl, optionally substituted by 1, selected from halo, C₁-C₆Alkyl radical, C₃-C₆Alkenyl and C₁-C₆Substituent substitution of alkoxy;

(k)R⁷is naphthyl;

(l)R⁷is 5-indolyl or N-tert-butoxycarbonyl-5-indolyl.

In one embodiment, R, which in a compound of formula I or any subgroup thereof is indolyl⁷The radical being 5-indolyl or N-C₁-C₆R of alkoxycarbonyl indolyl⁷The radical is N-tert-butoxycarbonyl-5-indolyl, in particular N-tert-butoxycarbonyl-5-indolyl. The indolyl group, when attached at its 5-position, can be represented as follows:

wherein R is^7aIs hydrogen or C₁-C₆Alkoxycarbonyl, or R^7aIn particular hydrogen or tert-butyloxycarbonyl.

A subgroup of compounds of the formula I are those compounds of the formula I or a subgroup of compounds of the formula I as defined herein, wherein R⁸Is hydrogen and R^8’Is methyl or C₁-C₆Alkyl groups such as isopropyl or isobutyl. A subgroup of compounds of the formula I is those compounds of the formula I or a subgroup of compounds of the formula I as defined herein, whereinThe moiety is glycyl, alanyl or valyl (Gly, Ala or Val; especially Gly, L-Ala or L-Val).

A subgroup of compounds of the formula I is those compounds of the formula I or a subgroup of compounds of the formula I as defined herein, wherein

Part has a structure

Wherein R is⁸Is hydrogen and R^8’Is hydrogen, C₁-C₆Alkyl, benzyl; or

R⁸Is hydrogen and R^8’Is hydrogen or C₁-C₆An alkyl group;

R⁸is hydrogen and R^8’Is C₁-C₂An alkyl group;

R⁸is hydrogen and R^8’Is methyl.

In one embodiment, R⁸And R^8’Together with the carbon atom to which they are attached form C₃-C₇A cycloalkyl group; or especially form C₃-C₄A cycloalkyl group; or especially to form cyclopropyl.

A subgroup of compounds of the formula I is those compounds of the formula I or a subgroup of compounds of the formula I as defined herein, wherein

(a)R⁹Is C₁-C₁₀Alkyl radical, C₃-C₇Cycloalkyl radical, C₃-C₆Alkenyl or benzyl;

(b)R⁹is C₁-C₈Alkyl or benzyl;

(c)R⁹is C₁-C₆Alkyl or benzyl;

(d)R⁹is C₁-C₆An alkyl group;

(e)R⁹is C₁-C₄An alkyl group; or

(f)R⁹Is methyl, ethyl, isopropyl, 1-methyl-propyl, isobutyl, butyl or tert-butyl;

(g)R⁹is benzyl;

(h)R⁹is cyclopentyl, 5-hexenyl, 2,2-dimethyl-butyl, octyl, 2-propyl-pentyl.

Of interest are the compounds mentioned in the experimental section and their pharmaceutically acceptable salts or solvates. Of particular interest are the compounds listed in the experimental section under compound numbers 1, 3, 5, 9, 10, 11, 12, 13, 14, 15.

The compounds of the formula I have several chiral centers, in particular at the carbon atoms 1 ', 3 ' and 4 '. Although the stereochemistry at these carbon atoms is fixed, the compounds may exhibit an enantiomeric purity (enantiomeric purity) at each chiral center of at least 75%, preferably at least 90%, e.g., greater than 95%.

Chirality may also be present in substituents, e.g. at R⁴Is thatWhen it is in R carrying carbon⁸(where R is⁸And R^8’Are different) and possess chirality at the phosphorus atom. The phosphorus center may exist as Rp or Sp, or a mixture of such stereoisomers, including racemates. Diastereomers arising from chiral phosphorus centers and chiral carbon atoms may also be present.

In another aspect, the present invention provides a compound of formula I, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, for use in (or in the manufacture of a medicament for) treating or preventing HCV infection. In the context of treatment or prevention according to the present invention, representative HCV genotypes include genotype 1b (prevalent in europe) or 1a (prevalent in north america). The present invention also provides a method for treating or preventing HCV infection, particularly genotype 1a or 1 b.

The compounds of formula I are represented by the defined stereoisomers. The absolute configuration of such compounds can be determined using methods known in the art, such as X-ray diffraction or NMR and/or known from starting materials of known stereochemistry. The pharmaceutical compositions according to the invention should preferably comprise preparations which are substantially stereoisomerically pure with the indicated stereoisomer.

Pure stereoisomeric forms of the compounds and intermediates referred to herein are defined as isomers substantially free of other enantiomeric or diastereomeric (diastereomeric) forms of the same basic molecular structure of the compounds or intermediates. In particular, the term "stereoisomerically pure" relates to a compound or intermediate having a stereoisomeric excess of at least 80% (i.e. at least 90% of one isomer and at most 10% of the other possible isomers) up to a stereoisomeric excess of 100% (i.e. 100% of one isomer and no other isomers), more particularly a compound or intermediate having a stereoisomeric excess of 90% up to 100%, even more particularly a compound or intermediate having a stereoisomeric excess of 94% up to 100%, and most particularly a compound or intermediate having a stereoisomeric excess of 97% up to 100%. The terms "enantiomerically pure" and "diastereomerically pure" are to be understood in the same way, but refer to the enantiomeric and diastereomeric excess, respectively, in the mentioned mixtures.

Pure stereoisomeric forms of the compounds and intermediates of the present invention may be obtained by carrying out procedures known in the art. For example, enantiomers may be separated from one another by selective crystallization of their diastereomeric salts with optically active acids or bases. Examples thereof are tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid and camphorsulfonic acid. Alternatively, enantiomers can be separated by chromatographic techniques using chiral solid phases. The pure stereochemically isomeric forms may also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction proceeds stereospecifically. If a particular stereoisomer is desired, it is preferred that the compound be synthesized by stereospecific methods of preparation. These processes will advantageously use enantiomerically pure starting materials.

The diastereomeric racemates of the compounds of formula I can be obtained separately by conventional methods. Suitable physical separation methods which can advantageously be used are, for example, selective crystallization and chromatography, for example column chromatography.

Pharmaceutically acceptable addition salts include the therapeutically active non-toxic acid and base addition salt forms of the compounds of formula I. Of interest are the free forms, i.e. non-salt forms, of the compounds of formula I or any subgroup of compounds of formula I as specified herein.

Pharmaceutically acceptable acid addition salts can be conventionally obtained by treating the base form with such a suitable acid. Suitable acids include, for example, inorganic acids such as hydrohalic acids (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, phosphoric acid, and the like; or organic acids such as acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid (i.e., oxalic acid), malonic acid, succinic acid (i.e., succinic acid), maleic acid, fumaric acid, malic acid (i.e., malic acid), tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, salicylic acid, p-aminosalicylic acid, pamoic acid (pamoic acid), and the like. Conversely, the salt form may be converted to the free base form by treatment with a suitable base.

The compounds of formula (I) containing an acidic proton may also be converted into their non-toxic metal or amine addition salt forms by treatment with suitable organic and inorganic bases. Suitable base salt forms include, for example, ammonium salts, alkali and alkaline earth metal salts, such as lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, such as benzathine (benzathine), N-methyl-D-glucamine, hydrabamine (hydrabamine) salts, and salts with amino acids, such as arginine, lysine and the like.

The term "solvate" includes any pharmaceutically acceptable solvate that the compound of formula I and salts thereof may form. Such solvates are, for example, hydrates, alkoxides such as ethoxides, propoxides and the like.

Certain compounds of formula (I) may also exist in their tautomeric form. For example, the tautomeric form of an amide (-C (═ O) -NH-) group is an iminoalcohol (-C (oh) ═ N-), which can become stable in a ring with aromatic character. Uridine bases are examples of such forms. Such forms, although not explicitly indicated in the structural formulae represented herein, are also intended to be encompassed within the scope of the present invention.

"C" as used herein₁-C₄Alkyl "when taken as a group or part of a group defines a saturated straight or branched chain hydrocarbon radical having from 1 to 4 carbon atoms, such as methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-methyl-2-propyl. "C₁-C₆Alkyl "includes C₁-C₄Alkyl and its higher homologues having 5 or 6 carbon atoms, such as 1-pentyl, 2-pentyl, 3-pentyl, 2-dimethylpropyl, 1-hexyl, 2-methyl-1-butyl, 2-methyl-1-pentyl, 2-ethyl-1-butyl, 3-methyl-2-pentyl, and the like. At C₁-C₆Of importance in the alkyl radical is C₁-C₄An alkyl group. "C₁-C₁₀Alkyl "includes C₁-C₆Alkyl groups and their higher homologues having 7, 8, 9 or 10 carbon atoms, such as heptyl, 2-heptyl, 3-heptyl, 2-methylhexyl, octyl, 2-octyl, 3-octyl, nonyl, 2-nonyl, 3-nonyl, 2-butylpentyl, decyl, 2-decyl, and the like. At C₁-C₁₀Of importance in the alkyl radical is C₁-C₆An alkyl group.

“C₁-C₆Alkoxy "means-O-C₁-C₆Alkyl radical, wherein C₁-C₆Alkyl is as defined above. C₁-C₆Examples of alkoxy groups are methoxy, ethoxy, n-propoxy or isopropoxy.

“C₃-C₇Cycloalkyl "includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. Of importance are cyclopropyl, cyclopentyl and cyclohexyl.

The term "C_3-6Alkenyl "as a group or part of a group defines straight and branched chain hydrocarbon radicals having from 3 to 6 carbon atoms and having saturated carbon-carbon bonds and at least one double bond, e.g. 1-propenyl, 2-propenyl (or allyl), 1-butenyl, 2-buteneA phenyl group, a 3-butenyl group, a 2-methyl-2-propenyl group, a 2-pentenyl group, a 3-pentenyl group, a 2-hexenyl group, a 3-hexenyl group, a 4-hexenyl group, a 2-methyl-2-butenyl group, a 2-methyl-2-pentenyl group, etc. In one embodiment, linkage C_3-6The carbon atoms from the alkenyl group to the rest of the molecule are saturated. At C_3-6Of the alkenyl groups, important is C_3-4An alkenyl group. At C_3-6Alkenyl or C_3-4Of the alkenyl groups are those which are important and have one double bond.

The term "halo" broadly refers to fluoro, chloro, bromo, and iodo.

The term "(═ O)" or "oxo" as used herein, when attached to a carbon atom, forms a carbonyl group. It must be noted that an atom can only be substituted by an oxo group when the valency of the atom permits.

The term "monophosphate, diphosphate or triphosphate" refers to the following groups:

as used herein, the position of a group on any molecular moiety used in the definition may be anywhere on such moiety, so long as it is chemically stable. The definitions are independent when any variable can occur more than once in any part.

Whenever used herein, the term "compound of formula I" or "compound of the invention" or similar terms are intended to encompass compounds of formula I, including possible stereochemically isomeric forms and pharmaceutically acceptable salts and solvates thereof.

The invention also includes isotopically-labeled compounds of formula I, or any subgroup of formula I, in which one or more atoms are replaced by an isotope other than those typically found in nature. Examples of such isotopes include isotopes of hydrogen, for example²H and³h; carbon, e.g.¹¹C、¹³C and¹⁴c; nitrogen, e.g.¹³N and¹⁵n; oxygen such as¹⁵O、¹⁷O and¹⁸o; phosphorus such as³¹P and³²p; sulfur, e.g.³⁵S; fluorine such as¹⁸F; chlorine such as³⁶Cl; bromine as⁷⁵Br、⁷⁶Br、⁷⁷Br and⁸²br; and iodine such as¹²³I、¹²⁴I、¹²⁵I and¹³¹I. isotopically-labeled compounds of the present invention can be prepared by processes analogous to those described herein or by techniques known in the art using appropriate isotopically-labeled reagents or starting materials. The choice of isotope included in the isotopically labeled compound depends on the particular application of the compound. For example, for tissue distribution testing, incorporation of radioactive isotopes, e.g.³H or¹⁴C. For imaging applications, positron emitting isotopes, for example¹¹C、¹⁸F、¹³N or¹⁵O would be useful. Incorporation of deuterium can provide greater metabolic stability, resulting in, for example, increased in vivo half-life of the compound or decreased required dosage.

General synthetic method

The starting material, 2 '-deoxy-2' -spirocyclopropyl uridine, may be prepared as described in j.am, chem.soc., 1992, 114, 4007-. A compound of formula I wherein R⁴Is a groupCan be prepared by reacting the starting material with phosphoroamidate (phosphoroamiditic acid ester)1 d. The latter may be prepared by reacting alcohol 1a with POCl₃In the presence of a base, thus obtaining phosphoryl dichloride 1b, which is then reacted with an amino acid 1 c.

A compound of formula I wherein R¹Is halo and can be prepared by first converting intermediate 1e to its hydroxy-protected form If, which is then halogenated, for example with N-halosuccinimide, to Ig, for example with N-iodosuccinimide to Ih. Suitable hydroxy protecting groups are alkylated silyl groups, especially hindered alkylated silyl groups, such as tert-butyldimethylsilyl, triisopropylsilyl or 1, 1, 3, 3-tetraisopropyl-disiloxane-1, 3-diyl (TIPDS). These groups are introduced by reaction of the starting alcohol with the appropriate silyl chloride derivative and can thereafter be removed with a fluoride compound such as tetrabutylammonium fluoride (TBAF) to give compound Ih. These reactions are shown in the following figures, where Pg is a hydroxyl protecting group such as the silyl groups described above.

In another aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I as specified herein and a pharmaceutically acceptable carrier. A therapeutically effective amount herein refers to an amount sufficient to act prophylactically against, stabilize or reduce HCV infection in an infected patient or a patient at risk of infection. In another aspect, the present invention relates to a process for preparing a pharmaceutical composition as specified herein, which comprises intimately mixing a pharmaceutically acceptable carrier with a therapeutically effective amount of a compound of formula I as specified herein.

Thus, for administration purposes, the compounds of the present invention, or any subgroup thereof, may be formulated in a variety of pharmaceutical forms. Suitable compositions which may be cited are all compositions of drugs which are usually intended for systemic administration. To prepare the pharmaceutical compositions of this invention, an effective amount of the particular compound, optionally in addition salt form or metal complex, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are suitably in single dosage form, particularly suitable for administration orally, rectally, subcutaneously or by parenteral injection. For example, in preparing the compositions in oral dosage form, in the case of oral liquid preparations such as suspensions, syrups, elixirs, emulsions and solutions, any of the usual pharmaceutical media may be employed, such as water, glycols, oils, alcohols and the like; or in the case of powders, pills, capsules and tablets, solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like may be employed. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will typically comprise sterile water, at least in large part, although other ingredients, for example, to increase solubility, may be included. For example, injectable solutions may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations. In compositions suitable for subcutaneous administration, the carrier optionally contains a penetration enhancer and/or a suitable wetting agent, optionally in admixture with any natural, suitable additives in minor proportions, which additives do not cause a significant injurious effect on the skin. The compounds of the present invention may also be administered in the form of a solution, suspension or dry powder by oral inhalation or insufflation using any delivery system known in the art.

It is particularly advantageous to formulate the above pharmaceutical compositions in unit dosage form for convenient administration and consistent dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, suppositories, powder packets, wafers, injectable solutions or suspensions and the like, and segregated multiples thereof.

The compounds of formula I exhibit activity against HCV and can be used in the treatment and prevention of HCV infection or HCV-related diseases. The latter includes progressive liver fibrosis, inflammation and necrosis leading to cirrhosis, end-stage liver disease and HCC. Furthermore, it is believed that various compounds of the present invention are active against mutant strains of HCV. In addition, many of the compounds of the present invention exhibit good pharmacokinetic profiles and have attractive properties in terms of bioavailability, including acceptable half-life, AUC (area under the curve) and peak values, and lack of undesirable phenomena such as insufficiently rapid onset and tissue retention.

The in vitro antiviral activity of the compounds of formula I against HCV can be determined according to Lohmann et al (1999) Science 285: 110-: 4614-4624 (incorporated herein by reference) was tested in the cellular HCV replicon system, which is further exemplified in the examples section. This model, although not a complete infection model for HCV, is widely accepted as the most reliable (robust) and efficient model of autonomous HCV RNA replication currently available. In the HCV replicon model, it is understood that it is important to distinguish compounds that specifically interfere with HCV function, and from these interferences, the compounds exert toxic effects or cytostatic effects and thus cause a decrease in HCV RNA or linked reporter enzyme concentrations. Methods for assessing cytotoxicity based on, for example, granuloglandular enzyme activity using redox fluorescent dyes such as resazurin are known in the art. Furthermore, there are cytometric screens for assessing non-selective inhibition of the activity of linked reporter genes, such as firefly luciferase. Suitable cell types can be equipped with luciferase reporter genes by stable transfection, the expression of which is dependent on a constitutively active gene promoter, and such cells can be used as a counter-screen to exclude non-selective inhibitors.

Due to their antiviral properties, in particular their anti-HCV properties, the compounds of formula I, including any of the possible stereoisomers, pharmaceutically acceptable addition salts or solvates thereof, are useful in the treatment of warm-blooded animals, especially humans, infected with HCV, and in the prevention of HCV infection. The present invention also relates to a method of treating a warm-blooded animal, especially a human, infected with, or at risk of infection with, HCV, which comprises administering an anti-HCV effective amount of a compound of formula I as specified herein.

The compounds of the invention can therefore be used as medicaments, in particular as anti-HCV medicaments. The use as a medicament or method of treatment comprising systemically administering to a patient infected with HCV or susceptible to HCV an effective amount to combat a condition associated with HCV infection.

The invention also relates to the use of a compound of the invention in the manufacture of a medicament for the treatment or prevention of HCV infection.

It is generally contemplated that an antivirally effective daily amount is from about 0.01 to about 700mg/kg, or about 0.5 to about 400mg/kg, or about 1 to about 250mg/kg, or about 2 to about 200mg/kg, or about 10 to about 150mg/kg of body weight. It may be appropriate to administer the required dose in two, three, four or more sub-doses at appropriate intervals throughout the day. The sub-doses may be formulated in unit dosage forms, for example containing from about 1 to about 6000mg, or from about 50 to about 5000mg, or from about 100 to about 2000mg, or from about 200 to about 1000mg, or from about 100 to about 600mg, or from about 200 to about 500mg of active ingredient per unit dosage form.

The present invention also relates to a combination of a compound of formula I, a pharmaceutically acceptable salt or solvate thereof, and another antiviral compound, particularly another anti-HCV compound. The term "combination" may refer to a product containing (a) a compound of formula I as specified above and (b) optionally another anti-HCV compound, as a combined preparation for simultaneous, separate or sequential use in the treatment of HCV infection.

anti-HCV compounds that can be used in such combinations include HCV polymerase inhibitors, HCV protease inhibitors, inhibitors of other targets in the HCV life cycle, and immunomodulators and combinations thereof. HCV polymerase inhibitors include NM283 (valopicitabine), R803, JTK 109, JTK 003, HCV-371, HCV-086, HCV-796 and R-1479, R-7128, MK-0608, VCH-759, PF-868554, GS9190, XTL-2125, NM-107, GSK625433, R-1626, BILB-1941, ANA-598, IDX-184, IDX-375, MK-3281, MK-1220, ABT-333, PSI-7851, PSI-6130, VCH-916. HCV protease inhibitors (NS2-NS3 inhibitors and NS3-NS4A inhibitors) include BILN-2061, VX-950 (telaprevir), GS-9132(ACH-806), SCH-503034(boceprevir), TMC435350 (also known as TMC435), TMC493706, ITMN-191, MK-7009, BI-12202, BILN-2065, BI-201335, BMS-605339, R-7227, VX-500, BMS650032, VBY-376, VX-813, SCH-6, PHX-1766, ACH-1625, IDX-136, IDX-316. Examples of HCV NS5A inhibitors are BMS790052, A-831, A-689, NIM-811 and DEBIO-025 are examples of NS5B cyclophilin inhibitors.

Other inhibitors targeted in the HCV life cycle include NS3 helicase; (ii) a metalloprotease inhibitor; antisense oligonucleotide inhibitors such as ISIS-14803 and AVI-4065; siRNA's such as SIRPLEX-140-N; vector-encoded short hairpin rna (shrna); DNAzymes; HCV-specific ribozymes such as hepazyme (heptazyme), rpi.13919; entry inhibitors such as HepeX-C, HuMax-HepC; alpha glucosidase inhibitors such as cigavir, UT-231B, etc.; KPE-02003002; and BIVN 401.

Immunomodulators include natural and recombinant interferon isoforms (isofroms) including alpha-interferon, beta-interferon, gamma-interferon and omega-interferon, such as Intron ARoferon-ACanferon-A300AdvaferonInfergenHumoferonSumiferon MPAlfaferoneIFN-betaAnd ferronPolyethylene glycol derivatized (pegylated) interferon compounds, e.g., PEG interferon-alpha-2 a (Pegasys)) PEG Interferon-alpha-2 b (PEG-Intron)) And pegylated IFN- α -con 1; long acting formulations and derivatizations of interferon compounds such as albumin-fused interferon albuferon α (albumin-interferon α); compounds that stimulate interferon synthesis in cells such as resiquimod; an interleukin; compounds that enhance the development of a type 1 helper T cell response such as SCV-07; TOLL-like receptor agonists such as CpG-10101 (activin) and etoricide; thymosin alpha 1; ANA-245; ANA-246; histamine dihydrochloride; propafenone; tetrachlorodecaoxide; apremizin (ampligen); IMP-321; KRN-7000; antibodies such as sivarsai (civacir) and XTL-6865; and prophylactic and therapeutic vaccines such as Innovac C and HCV E1E2/MF 59.

Other antiviral agents include ribavirin, amantadine (amantadine), virilizine (viramidine), nitrozole, and galantamine; telbivudine; NOV-205; talibaclin (taribavirin); inhibitors of internal ribosome entry; broad spectrum viral inhibitors such as IMPDH inhibitors and mycophenolic acid and derivatives thereof and include, but are not limited to VX-497 (merimepodib), VX-148 and/or VX-944); or a combination of any of the above.

Specific drugs for use in the combination include interferon-alpha (IFN-alpha), pegylated interferon-alpha or ribavirin, as well as therapeutic agents based on antibodies targeting anti-HCV epitopes, small interfering RNA (si RNA), ribozymes, DNAzymes, antisense RNA, small molecule antagonists such as NS3 protease, NS3 helicase, and NS5B polymerase.

In another aspect, the present invention provides a combination of a compound of formula I and an anti-HIV compound as specified herein. The latter are preferably HIV inhibitors with positive effects on drug metabolism and/or pharmacokinetics, which improve bioavailability. An example of such an HIV inhibitor is ritonavir (ritonavir). Accordingly, the present invention also provides pharmaceutical compositions comprising (a) a compound of formula I or a pharmaceutically acceptable salt or solvate thereof; and (b) ritonavir or a pharmaceutically acceptable salt thereof. The compound ritonavir, its pharmaceutically acceptable salts and processes for its preparation are disclosed in WO 94/14436.

The present invention also relates to a process for the preparation of a combination as described herein, comprising the step of combining a compound of formula I as specified above with another drug, e.g. an antiviral agent, including anti-HCV or anti-HIV drugs, in particular those mentioned above.

The combinations may find use in the manufacture of medicaments for the treatment of HCV infection in a mammal infected with HCV, in particular a combination comprising a compound of formula I as specified above and interferon-alpha (IFN-alpha), pegylated interferon-alpha or ribavirin. Alternatively, the present invention provides a method of treating a mammal, particularly a human, infected with HCV, which comprises administering to said mammal an effective amount of a combination as specified above. In particular, the treatment includes systemic administration of the combination, and an effective amount refers to an amount effective to treat a clinical condition associated with HCV infection.

In one embodiment, the above combination is formulated in the form of a pharmaceutical composition as described above, containing the active ingredients described above and a carrier. Each active ingredient may be formulated separately and each formulation may be administered simultaneously, or one formulation containing both may be provided, and other active ingredients may be provided if desired. In the foregoing examples, the combination may also be formulated as a combined preparation for simultaneous, separate or sequential use in HCV treatment. The combination may take any of the forms described above. In one embodiment, the two components are formulated in one dosage form, e.g., a fixed dose combination. In a particular embodiment, the present invention provides a composition comprising (a) a therapeutically effective amount of a compound of formula I, including its possible stereoisomeric forms, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, and (b) a therapeutically effective amount of ritonavir or a pharmaceutically acceptable salt thereof, and (c) a carrier.

The individual components of the combination of the invention may be administered separately at different times during the course of therapy or simultaneously in separate or single combinations. The invention is intended to include all such treatment regimens, either simultaneous or alternating treatment and the term "administering" is to be construed accordingly. In a preferred embodiment, the separate dosage forms are administered simultaneously.

In one embodiment, the combination of the invention contains ritonavir or a pharmaceutically acceptable salt thereof in an amount sufficient to clinically improve the bioavailability of the compound of formula I relative to the bioavailability when said compound of formula I is administered alone. Alternatively, the combination of the invention contains ritonavir or a pharmaceutically acceptable salt thereof in an amount sufficient to increase the amount of the compound of formula I selected from t relative to the at least one pharmacokinetic variable when said compound of formula I is administered alone_1/2、C_min、C_max、C_ssAt least one pharmacokinetic variable of AUC at 12 hours or AUC at 24 hours.

The combinations of the invention may be administered to humans at dosage levels of the individual components contained in the combination within the specified dosage ranges, for example the compound of formula I as described above, and ritonavir or a pharmaceutically acceptable salt may be present at dosage levels ranging from 0.02 to 10.0 g/day.

The weight ratio of the compound of formula I to ritonavir ranges from about 30: 1 to about 1: 15, or about 15: 1 to about 1: 10, or about 15: 1 to about 1: 1, or about 10: 1 to about 1: 1, or about 8: 1 to about 1: 1, or about 5: 1 to about 1: 1, or about 3: 1 to about 1: 1, or about 2: 1 to about 1: 1, of antigen. The compound of formula I and ritonavir may be administered, preferably orally, simultaneously, once or twice daily, wherein the amount of the compound of formula I per dose is as described above; and the amount of ritonavir per dose is from 1 to about 2500mg, or about 50 to about 1500mg, or about 100 to about 800mg, or about 100 to about 400mg, or 40 to about 100mg of ritonavir.

Examples

The following examples are intended to illustrate the invention and should not be construed as limiting its scope.

In each case, retention times (Rt (min)) and the observed m/z are given. Two retention times are indicated in particular when the separation of the two diastereomers is observed in LC-MS. When the compound does not give a stereochemical designation for the phosphorus atom, the compound is a 1: 1 mixture of the two phosphorus-diastereomers. In some cases, such mixtures are separated, but the exact stereochemical configuration is not known. Such compounds are denoted a and B and can be characterized by their physicochemical properties.

Example 1: synthesis of Compound (1)

To 1-naphthalenol (1.0 eq, 69.4mmol, 10.0g) in diethyl ether (250ml) was added phosphorus oxychloride (1.0 eq, 69.4mmol, 6.5ml) and the solution was cooled to-78 ℃. Anhydrous N, N-diisopropylethylamine (DIPEA; 1.0 equiv., 69.4mmol, 12.1ml) was added and the resulting solution was left to warm to room temperature overnight. The white slurry was filtered under an inert atmosphere and all volatiles were removed to give a as a colorless liquid, which was used in the next step without further purification.

A (1.0 eq, 4.6mmol, 1.0g) and benzyl 2-amino-propionate hydrochloride (1.0 eq, 4.6mmol, 1.2g) in CH₂Cl₂The solution in (40ml) was cooled to-80 ℃. Anhydrous DIPEA (2.0 equiv., 9.3mmol, 1.6ml) was added dropwise. After 1 hour the reaction was allowed to warm to room temperature. Stirring was continued for a further 1 hour and the solvent was removed under reduced pressure. Anhydrous ether was added and the precipitate was filtered and washed twice with anhydrous ether under argon atmosphere. The filtrate was evaporated to dryness to give B, which was stored as a 0.97M solution in Tetrahydrofuran (THF) at-18 ℃.

To a solution of C (1.0 equiv., 0.59mmol, 150mg) in anhydrous THF (6ml) was added tert-butyl MgCl (1.5 equiv., 0.89mmol, 521. mu.l, 1.7M solution in THF) at room temperature. B (1.4 equiv., 0.83mmol, 852. mu.l of a 0.97M solution in THF) was added dropwise and the mixture was stirred at room temperature for 2.5 h. 30 drops of saturated NH were added₄Aqueous Cl solution and the reaction mixture was evaporated on silica and then purified by column chromatography (on CH)₂Cl₂0-5% methanol) to give 1(74mg, 19% yield, 96% purity) as a mixture of diastereomers.¹H NMR(400MHz，DMSO-d₆)δppm 0.39-0.61(m，3H)1.01-1.12(m，1H)1.18-1.33(m，3H)3.88-4.09(m，3H)4.16-4.31(m，1H)4.31-4.42(m，1H)4.96-5.16(m，2H)5.35-5.49(m，2H)5.95(s，1H)6.25-6.37(m，1H)7.26-7.35(m，5H)7.36-7.62(m，5H)7.74(d，J＝8.02Hz，1H)7.95(d，J＝7.82Hz，1H)8.11(t，J＝7.92Hz，1H)11.31(br.s.，1H)。LC-MS：R_t2.21 min, 620(M-H)^-。

The following compounds were prepared using procedures analogous to example 1. The compounds are separated as a mixture of diastereomers. For compound (7), the diastereomers were separated and tested separately.

Compound (2)

¹H NMR(400MHz，DMSO-d₆)δppm 0.47-0.62(m，3H)1.04-1.17(m，7H)1.24-1.34(m，3H)2.20(s，3H)3.12-3.25(m，1H)3.86-3.98(m，2H)3.99-4.07(m，1H)4.09-4.23(m，1H)4.24-4.35(m，1H)5.02-5.17(m，2H)5.36-5.46(m，1H)5.48-5.57(m，1H)5.92-5.99(m，1H)6.16-6.30(m，1H)7.23-7.39(m，7H)7.51-7.60(m，1H)11.31(br.s.，1H)。LC-MS：R_t2.64 min, 660M/z (M-H)^-。

Compound (3)

¹H NMR(400MHz，DMSO-d₆)δppm 0.47-0.62(m，3H)1.01-1.09(m，1H)1.14(d，6H)1.17-1.24(m，3H)3.67-3.84(m，1H)3.85-3.96(m，1H)3.99-4.07(m，1H)4.09-4.23(m，1H)4.23-4.35(m，1H)4.78-4.89(m，1H)5.34-5.44(m，1H)5.51-5.59(m，1H)5.90-5.96(m，1H)5.96-6.07(m，1H)7.11-7.25(m，3H)7.31-7.40(m，2H)7.52-7.63(m，1H)11.31(br.s.，1H)。LC-MS：R_t2.24 min&2.36 min, M/z 522(M-H)^-。

Compound (4)

¹H NMR(400MHz，DMSO-d₆)δppm 0.43-0.63(m，3H)1.00-1.11(m，1H)1.31-1.46(m，6H)3.84-3.92(m，1H)3.96-4.06(m，1H)4.09-4.20(m，1H)4.22-4.32(m，1H)5.06(s，2H)5.33-5.43(m，1H)5.47-5.56(m，1H)5.88-6.01(m，2H)7.10-7.23(m，3H)7.24-7.40(m，7H)7.46-7.60(m，1H)11.30(br.s.，1H)。LC-MS：R_t2.07 min, 584(M-H)^-。

Compound (5)

¹H NMR(400MHz，DMSO-d₆)δppm 0.44-0.67(m，3H)0.99-1.33(m，7H)3.74-3.97(m，2H)3.97-4.10(m，3H)4.10-4.24(m，1H)4.24-4.39(m，1H)5.32-5.45(m，1H)5.51-5.62(m，1H)5.88-5.98(m，1H)5.98-6.12(m，1H)7.11-7.27(m，3H)7.30-7.44(m，2H)7.52-7.66(m，1H)11.31(br.s.，1H)。LC-MS：R_t2.10 min&2.23 min, M/z 508(M-H)^-。

Compound (6)

¹H NMR(400MHz，DMSO-d₆)δppm 0.38-0.61(m，3H)1.01-1.12(m，1H)3.73-3.86(m，2H)3.90-3.98(m，1H)4.00-4.11(m，1H)4.23-4.33(m，1H)4.33-4.43(m，1H)5.09(s，2H)5.35-5.49(m，2H)5.96(s，1H)6.08-6.27(m，1H)7.22-7.61(m，9H)7.74(d，J＝7.69Hz，1H)7.95(d，J＝7.10Hz，1H)8.12(d，J＝7.72Hz，1H)11.30(br.s.，1H)。LC-MS：R_t2.12 min, M/z 606(M-H)^-。

Compound (7)

¹H NMR(400MHz，DMSO-d₆)δppm 0.46-0.63(m，3H)1.01-1.13(m，1H)1.20-1.31(m，3H)3.84-3.98(m，2H)3.99-4.06(m，1H)4.10-4.23(m，1H)4.24-4.34(m，1H)5.02-5.14(m，2H)5.35-5.44(m，1H)5.53-5.61(m，1H)5.90-5.98(m，1H)6.11-6.24(m，1H)7.14-7.24(m，2H)7.28-7.42(m，7H)7.52-7.61(m，1H)11.31(br.s.，1H)。LC-MS：R_t2.98 min&3.07 min, M/z 604(M-H)^-。

Compound (7a) (isomer A)

¹H NMR(400MHz，DMSO-d₆)δppm 0.45-0.61(m，3H)；1.01-1.15(m，1H)；1.24(d，J＝6.46Hz，3H)；3.82-3.95(m，2H)；3.96-4.07(m，1H)；4.13-4.24(m，1H)；4.24-4.34(m，1H)；5.09(s，2H)；5.36-5.48(m，1H)；5.58(d，J＝7.63Hz，1H)；5.95(s，1H)；6.20(t，J＝11.35Hz，1H)；7.17(d，J＝7.82Hz，2H)；7.29-7.43(m，7H)；7.55(d，J＝7.63Hz，1H)；11.33(br.s.，1H)。LC-MS：R_t4.02 min, 604(M-H) M/z^-。

Compound (7B) (isomer B)

¹H NMR(400MHz，DMSO-d₆)δppm¹H NMR(400MHz，DMSO-d₆)δppm 0.48-0.61(m，3H)；1.02-1.13(m，1H)；1.26(d，J＝7.04Hz，3H)；3.86-3.98(m，2H)；3.99-4.05(m，1H)；4.09-4.20(m，1H)；4.24-4.32(m，1H)；5.03-5.13(m，2H)；5.34-5.44(m，1H)；5.57(d，J＝8.02Hz，1H)；5.94(s，1H)；6.18(dd，J＝12.91，10.17Hz，1H)；7.21(d，J＝8.61Hz，2H)；7.30-7.41(m，7H)；7.57(d，J＝8.22Hz，1H)；11.32(brS., 1H). LC-MS: rt 4.07 min, M/z 604(M-H)^-。

Compound (8)

¹H NMR(400MHz，DMSO-d₆)δppm 0.46-0.65(m，3H)1.02-1.11(m，1H)1.14(t，J＝7.05Hz，3H)1.18-1.32(m，3H)3.73-3.962(m，2H)3.97-4.09(m，3H)4.11-4.24(m，1H)4.24-4.36(m，1H)5.34-5.46(m，1H)5.53-5.62(m，1H)5.90-5.98(m，1H)6.05-6.18(m，1H)7.17-7.28(m，2H)7.43(d，J＝8.80Hz，2H)7.54-7.62(m，1H)11.33(br.s.，1H)。LC-MS：R_t2.41 min&2.51 min, M/z 542(M-H)^-。

Compound (9)

¹H NMR(400MHz，DMSO-d₆)δppm 0.41-0.64(m，3H)0.99-1.14(m，1H)1.16-1.33(m，3H)3.82-3.97(m，2H)4.02(d，J＝5.28Hz，1H)4.08-4.23(m，1H)4.23-4.34(m，1H)5.01-5.15(m，2H)5.34-5.45(m，1H)5.56(d，J＝8.02Hz，1H)5.95(s，1H)6.04-6.17(m，1H)7.19(d，J＝7.43Hz，3H)7.26-7.41(m，7H)7.51-7.62(m，1H)11.31(br.s.，1H)。LC-MS：R_t1.98 min, M/z 570(M-H)^-。

Compound (10)

¹H NMR(400MHz，DMSO-d₆)δppm 0.43-0.66(m，3H)0.83(d，J＝5.09Hz，6H)1.03-1.12(m，1H)1.12-1.34(m，11H)1.53-1.66(m，1H)3.76-4.09(m，5H)4.10-4.24(m，1H)4.24-4.36(m，1H)5.34-5.50(m，1H)5.56(d，J＝7.63Hz，1H)5.92-5.98(m，1H)5.99-6.12(m，1H)7.14-7.24(m，3H)7.36(t，J＝7.53Hz，2H)7.52-7.63(m，1H)11.32(br.s.，1H)。LC-MS：R_t2.53 min, M/z 594(M + H)⁺。

Compound (11)

¹H NMR(400MHz，DMSO-d₆)δppm 0.43-0.63(m，3H)0.86(s，9H)0.99-1.12(m，1H)1.19-1.32(m，3H)3.62-3.71(m，1H)3.72-3.80(m，1H)3.80-3.97(m，2H)4.02(br.s.，1H)4.08-4.24(m，1H)4.24-4.37(m，1H)5.29-5.46(m，1H)5.55(d，J＝7.43Hz，1H)5.94(d，J＝7.24Hz，1H)6.00-6.13(m，1H)7.07-7.25(m，3H)7.35(t，J＝7.73Hz，2H)7.49-7.66(m，1H)11.31(br.s.，1H)。LC-MS：R_t2.08 min, M/z 552(M + H)⁺。

Compound (12)

¹H NMR(400MHz，DMSO-d₆)δppm 0.46-0.63(m，3H)0.81-0.90(m，3H)1.04-1.12(m，1H)1.17-1.36(m，5H)1.45-1.56(m，2H)3.73-4.09(m，5H)4.10-4.24(m，J＝11.32，11.32，5.66，5.46Hz，1H)4.24-4.36(m，1H)5.33-5.46(m，1H)5.52-5.60(m，1H)5.91-5.98(m，1H)5.98-6.09(m，1H)7.10-7.28(m，3H)7.29-7.43(m，2H)7.51-7.65(m，1H)11.29(br.s.，1H)。LC-MS：R_t2.63 min&2.74 min, M/z 538(M + H)⁺。

Compound (13)

¹H NMR (400MHz, chloroform-d) δ ppm 0.54-0.78(m, 3H)0.85-0.98(m, 6H)1.20-1.33(m, 1H)1.33-1.48(m, 3H)1.84-2.01(m, J ═ 10.05, 6.68, 3.34, 3.34Hz, 1H)3.64-4.52(m, 9H)5.52-5.78(m, 1H)5.99-6.10(m, 1H)7.14-7.59(m, 6H)8.64(br.s., 1H). LC-MS: r_t2.57 min&2.68 min, M/z 536(M-H)^-。

Compound (14)

¹H NMR(400MHz，DMSO-d₆)δppm 11.25-11.38(1H，m)7.54-7.63(1H，m)7.32-7.42(2H，m)7.14-7.25(3H，m)5.97-6.06(1H，m)5.92-5.97(1H，m)5.53-5.60(1H，m)5.36-5.44(1H，m)4.98-5.07(1H，m)4.25-4.37(1H，m)4.09-4.25(1H，m)4.00-4.09(1H，m)3.87-3.98(1H，m)3.67-3.84(1H，m)1.70-1.88(2H，m)1.44-1.69(6H，m)1.16-1.27(3H，m)1.02-1.15(1H，m)0.46-0.64(3H，m)。LC-MS：R_t2.65 min&2.76 min, M/z 548(M-H)^-。

Compound (15)

¹H NMR(400MHz，DMSO-d₆)δppm 0.42-0.62(m，3H)0.74-0.86(m，3H)1.02-1.10(m，1H)1.11-1.32(m，5H)1.34-1.52(m，2H)3.80-4.01(m，4H)4.06(t，J＝6.36Hz，1H)4.16-4.32(m，1H)4.32-4.42(m，1H)5.38(d，J＝5.48Hz，1H)5.40-5.51(m，1H)5.95(s，1H)6.14-6.35(m，1H)7.40-7.63(m，5H)7.74(d，J＝5.67Hz，1H)7.95(d，J＝6.06Hz，1H)8.05-8.18(m，1H)11.30(br.s.，1H)。LC-MS：R_t2.19 min, 588(M + H)⁺。

Compound (16)

¹H NMR(400MHz，DMSO-d₆)δppm 0.43-0.64(m，3H)1.02-1.11(m，1H)1.19-1.30(m，3H)1.62(s，9H)3.86-3.99(m，2H)4.03(t，J＝6.05Hz，1H)4.11-4.24(m，1H)4.25-4.35(m，1H)5.04-5.13(m，2H)5.33-5.42(m，1H)5.48-5.56(m，1H)5.93-5.99(m，1H)6.00-6.13(m，1H)6.66(d，J＝2.93Hz，1H)7.09-7.21(m，1H)7.23-7.36(m，5H)7.40-7.47(m，1H)7.47-7.58(m，1H)7.69(d，J＝2.93Hz，1H)，7.97(d，J＝8.20Hz，1H)11.28(br.s.，1H)。LC-MS：R_t3.54 min ═&3.60 min, M/z 711(M + H)⁺。

Compound (17)

¹H NMR(400MHz，DMSO-d₆)δppm 0.42-0.66(m，3H)0.95-1.13(m，4H)2.76-2.87(m，1H)2.87-3.01(m，1H)3.76-4.14(m，7H)5.32-5.43(m，1H)5.50-5.57(m，1H)5.91-6.01(m，1H)6.09-6.27(m，1H)7.00-7.08(m，2H)7.11-7.35(m，8H)7.46-7.56(m，1H)11.30(br.s.，1H)。LC-MS：R_t2.69 min&2.78 min, M/z 586(M + H)⁺。

Compound (18)

¹H NMR(400MHz，DMSO-d₆)δppm 0.46-0.63(m，3H)1.02-1.13(m，1H)1.17-1.28(m，3H)1.30-1.43(m，2H)1.47-1.60(m，2H)1.94-2.07(m，2H)3.75(s，3H)3.79-4.09(m，5H)4.09-4.24(m，1H)4.25-4.37(m，1H)4.88-5.14(m，4H)5.33-5.44(m，1H)5.55(d，J＝8.00Hz，1H)5.67-5.80(m，1H)5.80-5.90(m，1H)5.89-6.03(m，2H)6.64-6.74(m，1H)6.84-6.94(m，1H)7.13-7.23(m，1H)7.54-7.65(m，1H)11.31(br.s.，1H)。LC-MS：R_t3.44 min-&3.51 min, M/z 634(M + H)⁺。

Compound (19)

¹H NMR(400MHz，DMSO-d₆)δppm 0.41-0.62(m，3H)1.00-1.11(m，1H)1.23(s，12H)3.81-3.96(m，2H)3.96-4.05(m，1H)4.06-4.20(m，1H)4.20-4.31(m，1H)5.00-5.12(m，2H)5.31-5.42(m，1H)5.52(d，J＝8.02Hz，1H)5.93(s，1H)5.98-6.11(m，1H)7.00-7.12(m，2H)7.25-7.38(m，7H)7.46-7.61(m，1H)11.29(br.s.，1H)。LC-MS：R_t2.44 min, M/z 628(M + H)⁺。

Compound (20)

¹H NMR(400MHz，DMSO-d₆)δppm 0.40-0.62(m，3H)1.01-1.10(m，1H)1.19-1.30(m，3H)3.84-3.96(m，2H)3.99-4.06(m，1H)4.08-4.22(m，1H)4.22-4.33(m，1H)5.00-5.12(m，2H)5.39(br.s.，1H)5.45-5.52(m，1H)5.91-6.02(m，2H)6.37(s，1H)6.92(t，J＝10.37Hz，1H)7.27-7.41(m，8H)7.44-7.61(m，1H)11.12(br.s.，1H)11.29(br.s.，1H)。LC-MS：R_t1.89 min, 611(M + H) with M/z⁺。

Compound (21)

¹H NMR(400MHz，DMSO-d₆)δppm 0.43-0.63(m，3H)0.71-0.85(m，3H)1.01-1.14(m，1H)1.48-1.73(m，2H)3.64-3.80(m，1H)3.85-3.97(m，1H)3.97-4.07(m，1H)4.07-4.23(m，1H)4.24-4.34(m，1H)5.01-5.16(m，2H)5.32-5.46(m，1H)5.50-5.60(m，1H)5.93-5.98(m，1H)6.00-6.11(m，1H)7.08-7.24(m，3H)7.26-7.43(m，7H)7.50-7.62(m，1H)11.30(br.s.，1H).LC-MS：R_t2.83 min ═&2.94 min, M/z 586(M + H)⁺。

Compound (22)

¹H NMR(400MHz，DMSO-d₆)δppm 0.45-0.63(m，3H)1.02-1.18(m，7H)1.29(d，J＝6.06Hz，3H)2.20(s，3H)3.12-3.28(m，1H)3.85-3.98(m，2H)3.99-4.09(m，1H)4.09-4.23(m，1H)4.23-4.34(m，1H)5.02-5.16(m，2H)5.35-5.45(m，1H)5.45-5.55(m，1H)5.92-6.02(m，1H)6.08-6.24(m，1H)6.93(d，J＝7.63Hz，1H)7.10-7.21(m，2H)7.34(br.s.，5H)7.50-7.63(m，1H)11.32(br.s.，1H)。LC-MS：R_t2.40 min, M/z 628(M + H)⁺。

Compound (23)

¹H NMR(400MHz，DMSO-d₆)δppm 0.46-0.63(m，3H)0.85(t，J＝6.65Hz，3H)1.04-1.12(m，1H)1.15-1.32(m，13H)1.44-1.56(m，2H)3.73-4.08(m，5H)4.10-4.24(m，1H)4.24-4.36(m，1H)5.35-5.45(m，1H)5.55-5.63(m，1H)5.92-5.98(m，1H)6.05-6.20(m，1H)7.17-7.27(m，2H)7.43(d，J＝8.80Hz，2H)7.55-7.62(m，1H)11.32(br.s.，1H)。LC-MS：R_t3.72 min, 626(M-H)^+-。

Compound (24)

¹H NMR(400MHz，DMSO-d₆)δppm 0.46-0.65(m，3H)0.81(t，J＝7.40Hz，3H)1.02-1.17(m，4H)1.17-1.31(m，3H)1.49(dq，J＝7.28，7.11Hz，2H)3.67-3.87(m，1H)3.87-3.99(m，1H)3.99-4.10(m，1H)4.10-4.24(m，1H)4.24-4.37(m，1H)4.63-4.81(m，1H)5.32-5.44(m，1H)5.50-5.62(m，1H)5.90-5.97(m，1H)5.97-6.07(m，1H)7.12-7.25(m，3H)7.32-7.41(m，2H)7.54-7.62(m，1H)11.30(s，1H).LC-MS：R_t2.58 min&2.69 min, M/z 536(M-H)^-。

Compound (25)

¹H NMR(400MHz，DMSO-d₆)δppm 0.45-0.63(m，3H)1.04-1.09(m，1H)1.10-1.17(m，6H)1.22-1.33(m，3H)2.09-2.21(m，3H)2.78(spt，J＝6.91Hz，1H)3.84-3.97(m，2H)3.98-4.07(m，1H)4.09-4.24(m，1H)4.24-4.34(m，1H)5.03-5.16(m，2H)5.34-5.45(m，1H)5.48-5.57(m，1H)5.92-6.00(m，1H)6.07-6.17(m，1H)6.93(d，J＝7.82Hz，1H)7.12(d，J＝7.82Hz，1H)7.15(s，1H)7.27-7.39(m，5H)7.58(d，J＝8.02Hz，1H)11.30(br.s.，1H)。LC-MS：R_t2.44 min, M/z 628(M + H)⁺。

Compound (26)

¹H NMR(400MHz，DMSO-d₆)δppm 11.27-11.35(1H，m)7.51-7.64(1H，m)7.39-7.48(2H，m)7.17-7.28(1H，m)6.27-6.38(1H，m)5.90-5.97(1H，m)5.53-5.60(1H，m)5.34-5.42(1H，m)4.26-4.38(1H，m)4.14-4.26(1H，m)3.97-4.12(3H，m)3.85-3.96(1H，m)2.34-2.44(2H，m)2.17-2.28(1H，m)2.05-2.17(1H，m)1.67-1.88(2H，m)1.11-1.19(3H，m)1.04-1.11(1H，m)0.46-0.64(3H，m)。LC-MS：R_t2.57 min, 568(M-H)^-。

Compound (27)

¹H NMR(400MHz，DMSO-d₆)δppm 11.27-11.36(1H，m)7.50-7.65(1H，m)7.30-7.43(2H，m)7.12-7.25(3H，m)5.86-5.98(2H，m)5.50-5.59(1H，m)5.36-5.45(1H，m)4.24-4.36(1H，m)4.14-4.24(1H，m)3.98-4.11(1H，m)3.87-3.97(1H，m)3.52-3.60(3H，m)1.28-1.42(6H，m)1.02-1.13(1H，m)0.45-0.64(3H，m)。LC-MS：R_t1.57 min, M/z 510(M + H)⁺。

Compound (28)

¹H NMR(400MHz，DMSO-d₆)δppm 11.17-11.41(1H，m)7.48-7.64(1H，m)7.30-7.41(2H，m)7.12-7.26(3H，m)5.91-5.96(1H，m)5.74-5.86(1H，m)5.49-5.57(1H，m)5.32-5.41(1H，m)4.77-4.90(1H，m)4.26-4.36(1H，m)4.14-4.25(1H，m)3.99-4.10(1H，m)3.87-3.95(1H，m)1.28-1.41(6H，m)1.11-1.20(6H，m)1.04-1.10(1H，m)0.45-0.63(3H，m)。LC-MS：R_t2.41 min, 536(M-H) M/z^-。

Compound (29)

¹H NMR(400MHz，DMSO-d₆)δppm 11.24-11.38(1H，m)7.49-7.65(1H，m)7.29-7.41(2H，m)7.11-7.23(3H，m)6.41-6.56(1H，m)5.90-5.97(1H，m)5.51-5.60(1H，m)5.34-5.44(1H，m)4.26-4.38(1H，m)4.15-4.26(1H，m)3.95-4.08(3H，m)3.87-3.95(1H，m)1.19-1.33(2H，m)1.02-1.17(5H，m)0.92-1.02(1H，m)0.45-0.63(3H，m)。LC-MS：R_t1.59 min, M/z 522(M + H)⁺。

Compound (30)

¹H NMR(400MHz，DMSO-d₆)δppm 11.23-11.38(1H，m)7.53-7.64(1H，m)7.29-7.43(2H，m)7.11-7.26(3H，m)5.85-6.01(2H，m)5.50-5.62(1H，m)5.34-5.46(1H，m)4.25-4.38(1H，m)4.09-4.25(1H，m)3.99-4.09(1H，m)3.86-3.98(1H，m)3.59-3.77(1H，m)1.30-1.43(9H，m)1.14-1.28(3H，m)1.00-1.13(1H，m)0.44-0.64(3H，m)。LC-MS：R_t1.90 min, 538(M + H) M/z⁺。

Compound (31)

¹H NMR(400MHz，DMSO-d₆)δppm 11.19-11.38(1H，m)7.52-7.63(1H，m)7.30-7.41(2H，m)7.12-7.24(3H，m)5.90-6.01(2H，m)5.52-5.60(1H，m)5.33-5.43(1H，m)4.79-4.92(1H，m)4.24-4.35(1H，m)4.09-4.24(1H，m)3.99-4.08(1H，m)3.85-3.97(1H，m)3.51-3.67(1H，m)1.45-1.71(2H，m)1.10-1.19(6H，m)1.03-1.10(1H，m)0.75-0.84(3H，m)0.47-0.62(3H，m)。LC-MS：R_t2.36 min&2.46 min, M/z 536(M-H)^-。

Compound (32)

¹H NMR(400MHz，DMSO-d₆)δppm11.21-11.40(1H，m)7.49-7.66(1H，m)7.30-7.43(2H，m)7.12-7.25(3H，m)6.21-6.31(1H，m)5.90-5.98(1H，m)5.50-5.59(1H，m)5.36-5.44(1H，m)4.26-4.37(1H，m)4.15-4.26(1H，m)3.98-4.11(3H，m)3.88-3.97(1H，m)2.32-2.45(2H，m)2.17-2.29(1H，m)2.04-2.18(1H，m)1.67-1.88(2H，m)1.11-1.21(3H，m)1.01-1.11(1H，m)0.45-0.65(3H，m)。LC-MS：R_t1.76 min, 536(M + H) M/z⁺。

Compound (33)

¹H NMR(400MHz，DMSO-d₆)δppm 0.45-0.65(m，3H)0.93-1.04(m，1H)1.04-1.17(m，5H)1.21-1.35(m，2H)3.86-3.95(m，1H)3.95-4.09(m，3H)4.16-4.26(m，1H)4.27-4.38(m，1H)5.35-5.43(m，1H)5.54-5.62(m，1H)5.94(s，1H)6.50-6.64(m，1H)7.17-7.26(m，2H)7.38-7.47(m，2H)7.50-7.63(m，1H)11.31(s，1H)。LC-MS：R_t2.29 min, 554(M-H)^-。

Compound (34)

¹H NMR(400MHz，DMSO-d₆)δppm 0.44-0.65(m，3H)1.01-1.13(m，1H)1.18-1.33(m，6H)3.84-4.08(m，3H)4.12-4.37(m，4H)4.99-5.16(m，2H)5.31-5.43(m，1H)5.50-5.61(m，1H)5.91-6.00(m，1H)6.00-6.17(m，1H)7.21-7.40(m，6H)7.40-7.50(m，1H)7.50-7.63(m，2H)7.69-7.83(m，1H)11.29(br.s.，1H)。LC-MS：R_t2.96 min, 644(M + H) M/z⁺。

Example 2:

(C) compound (F) of the iodinated analog of (a) was prepared using the following procedure.

To a solution of (D) (1.44g, 2.9mmol) in anhydrous DMF (30ml) was added N-iodosuccinimide (1.63g, 7.25mmol, 2.5 equiv.) at room temperature. The reaction mixture was heated to 110 ℃ and stirred at this temperature overnight. After cooling to room temperature, by adding NaHSO₃In saturated NaHCO₃The reaction was quenched as a 7.5% w/v solution in the solution. The mixture was again saturated NaHCO₃The solution (250ml) was diluted and extracted with ethyl acetate (3 × 200 ml). The combined organic layers were dried (Na)₂SO₄) Filtered and concentrated to give a yellow oil. After purification by column chromatography (heptane/ethyl acetate 10 to 30% gradient) white solid (E) was obtained (1.44g, 80%).¹HNMR(400MHz，DMSO-d₆)δppm 0.54(dt，J＝10.05，5.12Hz，1H)0.62-0.70(m，1H)0.76(dt，J＝10.05，5.12Hz，1H)0.87-1.14(m，29H)3.74-3.82(m，1H)3.96(dd，J＝12.88，2.34Hz，1H)4.09(dd，J＝12.88，3.90Hz，1H)4.53(d，J＝7.80Hz，1H)5.73(s，1H)7.89(s，1H)11.73(s，1H)。LC-MS：R_t8.94 min, 645(M + Na)⁺。

To a suspension of (E) (1.24g, 1.99mmol) in methanol (20ml) was added ammonium fluoride (369mg, 5 equiv.) at room temperature. The reaction mixture was warmed to 50 ℃ under argon and stirred for 7 hours. After concentrating the mixture, the residue obtained is purified by column chromatography (dichloromethane/methanol ester 2.5 to 10% gradient). Thus was obtained (F) as a white solid (711mg, 92%).¹H NMR(400MHz，DMSO-d₆)δppm 0.50-0.65(m，3H)1.05(t，J＝5.95Hz，1H)3.56-3.67(m，1H)3.70-3.80(m，2H)4.05-4.15(m，1H)5.13-5.27(m，2H)5.85(s，1H)8.44(s，1H)11.65(br.s.，1H)。LC-MS：R_t1.32 min, M/z 403(M + Na)⁺。

Example 3: synthesis of phosphoramidate (35-38)

(F) (120mg, 0.316mmol), pre-dried by co-evaporation with pyridine, was dissolved in N-methylimidazole (0.3ml, 3.79mmol, 12 equivalents) and anhydrous dichloromethane (3.2ml) and an approximately 1M solution of the appropriate phosphoroamidate chloride (1.2 equivalents) was added under argon at room temperature. The reaction was stirred for 3 hours. Additional reagents are added if necessary. After complete consumption of the starting materials, the reaction mixture was diluted with dichloromethane and washed with 0.5M aqueous HCl. The aqueous layer was extracted with dichloromethane and the combined organic layers were dried (Na)₂SO₄) Filtered and concentrated. The residue was purified by column chromatography (dichloromethane/methanol gradient 1 to 10%) to yield the product 36-39 as a white solid (yield 68-77%).

Compound (35)

¹H NMR(400MHz，DMSO-d₆)δppm 0.50-0.62(m，3H)1.01-1.11(m，1H)1.19-1.31(m，3H)3.84-4.00(m，2H)4.01-4.09(m，1H)4.11-4.33(m，2H)5.02-5.14(m，2H)5.29-5.41(m，1H)5.83-5.95(m，1H)6.00-6.14(m，1H)7.11-7.23(m，3H)7.26-7.40(m，7H)7.93(s，1H)11.69(br.s.，1H)。LC-MS：R_t5.27 min, M/z 698(M + H)⁺。

Compound (36)

¹H NMR(400MHz，DMSO-d₆)δppm 0.44-0.64(m，3H)0.70-0.86(m，3H)1.00-1.12(m，1H)1.46-1.73(m，2H)3.67-3.82(m，1H)3.84-3.98(m，1H)4.00-4.10(m，1H)4.10-4.36(m，2H)4.98-5.15(m，2H)5.27-5.40(m，1H)5.83-5.94(m，1H)5.94-6.07(m，1H)7.09-7.24(m，3H)7.26-7.43(m，7H)7.87-7.99(m，1H)11.69(br.s.，1H)。LC-MS：R_t5.58 min, 712(M + H) M/z⁺。

Compound (37)

¹H NMR(400MHz，DMSO-d₆)δppm 0.48-0.64(m，3H)1.02-1.11(m，1H)1.14(t，J＝6.93Hz，3H)1.18-1.30(m，3H)3.74-3.86(m，1H)3.86-3.97(m，1H)3.98-4.10(m，3H)4.11-4.37(m，2H)5.27-5.42(m，1H)5.84-5.92(m，1H)5.93-6.06(m，1H)7.11-7.27(m，3H)7.31-7.42(m，2H)7.93(s，1H)11.70(br.s.，1H)。LC-MS：R_t4.24 min, 653(M + NH)₄)⁺。

Compound (38)

¹H NMR(400MHz，DMSO-d₆)δppm 0.49-0.63(m，3H)1.02-1.10(m，1H)1.15(d，J＝5.07Hz，6H)1.18-1.25(m，3H)3.70-3.84(m，1H)3.86-3.97(m，1H)4.02-4.10(m，1H)4.12-4.34(m，2H)4.78-4.90(m，1H)5.29-5.40(m，1H)5.85-5.91(m，1H)5.91-5.99(m，1H)7.12-7.24(m，3H)7.31-7.40(m，2H)7.93(s，1H)11.70(br.s.，1H)。LC-MS：R_t4.74 min, M/z 667(M + NH)₄)⁺。

Biological examples

Replicon assay

The activity of the compounds of formula I to inhibit HCV RNA replication was examined in cellular assays directed to the identification of compounds that inhibit HCV functional cell replication lines (also referred to as HCV replicons). Cellular assays are described in Lohmann et al (1999), Science vol.285pp.110-113 and by Krieger et a1 (2001), journal of Virology 75: 4614-4624, based on a bicistronic (bicistronic) expression construct using a multi-target screening strategy.

The assay utilizes the stably transfected cell line Huh-7Luc/neo (hereinafter referred to as Huh-Luc). This cell line harbors a bicistronic expression construct encoded by RNA (harbors) via a reporter moiety (FfL-luciferase) and a selectable marker moiety (neo)^RNeomycin phosphotransferase) containing the wild-type NS3-NS5B region of HCV type 1b translated from an Internal Ribosome Entry Site (IRES) from encephalomyocarditis virus (EMCV). The building blocks are bordered by the 5 'and 3' NTRs (untranslated regions) of HCV type 1 b. Replicon cells in G418 (neo)^R) Continuous culture in the presence depends on the replication of HCV RNA. Stably transfected replicon cells expressing hcv rna, which replicate autonomously and encode high amounts of inter alia luciferase, are used for screening antiviral compounds.

Replicon cells were placed in 384-well plates in the presence of added test and control compounds at different concentrations. After three days of culture, luciferase activity was measured by assaying for luciferase activity (using standard luciferase assay substrates and reagents with Perkin Elmer ViewLux^TMultraHTS microtiter plate imager) measures HCV replication. Replicon cells in the control culture had high luciferase expression in the absence of any inhibitor. The inhibitory activity of the compounds on luciferase activity was monitored on Huh-Luc cells to generate dose-response curves for each test compound. Then calculate EC₅₀A value representing the amount of compound required to reduce the level of luciferase activity detected by 50%Or more specifically, the ability to replicate a genetically related HCV replicon RNA.

Cytotoxicity

Cytotoxicity was determined in the Huh7-CMV-Luc replicon assay. Replicon cells (2500 cells/well) stably transformed with a luciferase reporter gene under the control of a Cytomegalovirus (CMV) constitutive promoter were cultured in the presence or absence of a concentration of the test compound. At 37 ℃ in moist 5% CO₂After three days of culture in the atmosphere, cell proliferation was quantified by measuring Luc activity and measured in CC₅₀Values are expressed (cytotoxicity, 50% inhibitory concentration of cell growth). The assay was performed on 384-well plates.

HIV test method

The compounds of the invention were tested for efficacy against wild-type Human Immunodeficiency Virus (HIV). The antiviral activity was evaluated using a cellular assay according to the following procedure. The human T-cell line MT4 was genetically engineered with the Green Fluorescent Protein (GFP) and HIV-specific promoter, HIV-1 Long Terminal Repeat (LTR). This cell line, termed MT4LTR-EGFP, can be used in vitro to evaluate the anti-HIV activity of the compounds studied. In HIV-1 infected cells, a Tat protein is produced which up-regulates the LTR promoter and ultimately leads to stimulation of GFP reporter protein production, so that ongoing HIV-infection can be measured with a fluorometer. Effective concentration values such as 50% Effective Concentration (EC) can be measured₅₀) And is usually expressed in μ M concentration. EC (EC)₅₀The value is defined as the concentration of test compound that reduces fluorescence of HIV-infected cells by 50%. HIV-1 infection was monitored using a scanning microscope. Image analysis allows very sensitive detection of viral infections. Measurements are taken prior to cell necrosis, which typically occurs about five days after infection, specifically three days after infection. EC against wild-type IIIB strains are listed in column IIIB of the table₅₀The value is obtained.

The results in Table 1 demonstrate that the compounds of the present invention exhibit activity against HCV while lacking anti-HIVAnd (4) activity. They show advantageous results in terms of toxicity and have an acceptable selectivity index (EC)₅₀And CC₅₀Ratio of).

Results

Table 1 shows the replicon results (EC) obtained for the example compounds given above₅₀Replicon) and cytotoxic outcome (CC)₅₀(. mu.m) (Huh-7)). HIV activity (EC) is also given₅₀HIV (μ M)) and cytotoxicity (CC) in HIV cell lines₅₀(μM)(MT-4))。

TABLE 1

"-" means that no test results were obtained

Claims (15)

1. A compound of formula I:

including any possible stereoisomers thereof, wherein:

R¹is hydrogen or halo;

R⁴is a monophosphate ester group, a diphosphate ester group or a triphosphate ester group; or R⁴Is a radical of the formula

R⁷Is phenyl, optionally selected from 1, 2 or 3 each independently from halo, C₁-C₆Alkyl radical, C₃-C₆Alkenyl radical, C₁-C₆Alkoxy radical, C₁-C₆Substituent substitutions of alkoxycarbonyl, hydroxyl and amino; or R⁷Is naphthyl; or R⁷Is indolyl or N-C₁-C₆An alkoxycarbonyl indolyl group;

R⁸is hydrogen, C₁-C₆Alkyl, benzyl;

R^8′is hydrogen, C₁-C₆Alkyl, benzyl; or

R⁸And R^8′Together with the carbon atom to which they are attached form C₃-C₇A cycloalkyl group;

R⁹is C₁-C₁₀Alkyl radical, C₃-C₇Cycloalkyl, benzyl or phenyl, the phenyl group being optionally selected from 1, 2 or 3 each independently of the others from hydroxy, C₁-C₆Alkoxy, amino, mono-and di-C₁-C₆Substituted with alkyl amino;

or a pharmaceutically acceptable salt or solvate thereof.

2. A compound according to claim 1, wherein R⁴Is a radical of the formula

3. A compound according to any one of claims 1-2, wherein R⁷Is phenyl, optionally selected from 1, 2 or 3 each independently from halo, C₁-C₆Alkyl radical, C₃-C₆Alkenyl and C₁-C₆Substituent substitution of alkoxy; or R⁷Is naphthyl.

4. A compound according to any one of claims 1-2, wherein R⁷Is phenyl, optionally substituted by 1, 2 or 3 substituents each independently selected from halo and C₁-C₆Alkyl substituents.

5. A compound according to any one of claims 1-2, wherein R⁷Is phenyl, optionally substituted by halo or C₁-C₆Alkyl substitution; or R⁷Is naphthyl.

6. A compound according to any one of claims 1 to 5, wherein R⁸Is hydrogen and R^8’Is hydrogen or C₁-C₆An alkyl group.

7. A compound according to any one of claims 1 to 5, wherein

The radical having the structure

Wherein R is⁸Is hydrogen and R^8’Is hydrogen, C₁-C₆Alkyl, benzyl.

8. A compound according to claim 7, wherein R⁸Is hydrogen and R^8’Is C₁-C₂An alkyl group.

9. A compound according to claim 7, wherein R⁸Is hydrogen and R^8’Is methyl.

10. The compound according to any one of claims 1-9, wherein R⁹Is C₁-C₁₀Alkyl radical, C₃-C₇Cycloalkyl radical, C₃-C₆Alkenyl or benzyl.

11. A compound according to claim 10, wherein R⁹Is C₁-C₈Alkyl or benzyl.

12. A compound according to claim 10, wherein R⁹Is methyl, ethyl, isopropyl, 1-methyl-propyl, isobutyl, butyl, tert-butyl, benzyl, cyclopentyl, 5-hexenyl, 2-dimethyl-butyl, octyl, 2-propyl-pentyl.

13. A compound according to claim 10, wherein R⁹Is ethyl, isopropyl, butyl, benzyl, cyclopentyl, 5-hexenyl, 2-dimethyl-butyl or 2-propyl-pentyl.

14. A pharmaceutical composition comprising an antiviral effective amount of a compound of formula I as defined in any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

15. The use of a compound according to any one of claims 1-13 as an HCV inhibitor.