JP2016525114A - Combinations comprising tricyclohexadecahexaene derivatives for use in the treatment of hepatitis C virus - Google Patents

Abstract

The present disclosure relates generally to antiviral compounds, and more specifically, combinations of compounds capable of inhibiting the function of NS5A protein encoded by hepatitis C virus (HCV), compositions comprising such combinations, and functions of NS5A protein Relates to a method for inhibiting.

Description

(Cross-reference of related applications)
This application enjoys the benefit of US Provisional Application No. 61 / 847,388, filed July 17, 2013, all of which is incorporated herein by reference.

  The present invention relates generally to antiviral compounds, and more specifically, combinations of compounds capable of inhibiting the function of NS5A protein encoded by hepatitis C virus (HCV), compositions comprising the combinations, And to a method of inhibiting the function of NS5A protein.

  HCV is a major human pathogen, with an estimated 170 million people infected worldwide-about five times the number of human immunodeficiency virus type 1 infections. A significant proportion of these HCV-infected individuals develop severe progressive liver disease, including cirrhosis and hepatocellular carcinoma.

  Over the past decade, the standard treatment for treating chronic HCV has used a combination of pegylated interferon and ribavirin. The treatment is not the highest success rate in achieving a sustained viral response (SVR) for the six major HCV genotypes (especially less successful for genotype 1), and Cause many side effects. When recently approved drugs targeting HCV NS3 / 4A proteases (PIs) (Victorellis® and Insibec®) are administered with pegylated interferon and ribavirin, the proportion of patients experiencing SVR and SVR There is a significant improvement in the duration of treatment necessary to achieve. However, it is clear and urgent to develop additional therapies to combat protease inhibitor resistance, improve efficacy across all HCV genotypes, and advance antiviral therapy towards the ultimate goal of interferon-free therapy. Is needed.

  HCV is a plus-strand RNA virus about 9500 nucleotides long, with a single open reading frame (ORF) encoding a single large polyprotein that is about 3000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases, yielding structural and nonstructural (NS) proteins. In the case of HCV, the production of mature nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. The first is a metalloprotease that is thought to cleave the NS2-NS3 junction; the second is a serine protease (also referred to herein as NS3 protease) contained within the N-terminal region of NS3. Mediates all subsequent cleavages both NS in downstream, ie in cis at the NS3-NS4A cleavage site and in trans for the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites . The NS4A protein is a cofactor for NS3 protease. The formation of NS3-NS4A complex is necessary for proper protease activity. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5A is a multifunctional protein required for viral RNA replication and virion assembly. NS5B (also referred to herein as HCV polymerase) is an RNA-dependent RNA polymerase involved in RNA virus synthesis.

  Throughout the HCV genome, considerable diversity is found due to the high error rate of the coding RNA-dependent RNA polymerase that lacks proofreading ability within the nucleotide and encoded amino acid sequence. The clinical significance of HCV genetic diversity is the tendency of mutations to occur during treatment with monotherapy, thus using HCV inhibitors that are pan-genotype subjects and act through independent mechanisms Combination therapy is desired.

  Compounds that selectively inhibit HCV viral replication and are useful in the treatment of HCV-infected patients are desirable. In particular, a compound that effectively inhibits the function of NS5A protein is desirable. The functions and important roles of NS5A protein in HCV replication include, for example, the following documents: Non-patent document 1; Non-patent document 2; Non-patent document 3; Non-patent document 4; Non-patent document 5; Patent Document 7; described in Patent Document 1 of C. Rice et al.

  A method for identifying compounds that exhibit synergistic inhibition of HCV replicon activity when combined with an HCV NS5A inhibitor (eg, BMS-790052 (Patent Document 2 filed on July 13, 2011)) is described. ing. Briefly, each compound is essentially inactive or has little activity when tested individually against several NS5A resistant mutants and tested in combination with NS5A targeted compounds. In some cases it only has a synergistic inhibitory activity. The synergistic compound was identified using titration of the test compound in the presence of a fixed concentration of HCV NS5A inhibitor (eg BMS-790052).

WO2006093867 PCT / US2011 / 043785

S. L. Tan, et al., Virology, 284: 1-12 (2001) K.-J. Park, et al., J. Biol. Chem., 30711-30718 (2003) T. L. Tellinghuisen, et al., Nature, 435, 374 (2005) R. A. Love, et al., J. Virol, 83, 4395 (2009) N. Appel, et al., J. Biol. Chem., 281, 9833 (2006) L. Huang, J. Biol. Chem., 280, 36417 (2005) M. Gao, et al, Nature (2010)

［式中、
R¹'およびR¹'は、アルコキシアルキル、アルキル、シクロアルキルおよびピラニルから独立して選択され、該シクロアルキルおよび該ピラニルは、所望により、アルキル、ハロ、ハロアルキル、ヒドロキシおよびヒドロキシアルキルから独立して選択される1、2または3つの置換基で置換されていてもよく；
R²およびR²'は、同一または異なるアルキル基である］
の化合物またはその医薬的に許容される塩を提供する。 In a first aspect, the present invention provides a combination comprising an NS5A targeting compound and an NS5A synergist, the combination being resistant to only the NS5A targeting compound when administered. Resulting in synergistic anti-HCV activity against a mutant comprising the given mutation, wherein the NS5A synergist is of formula (I):

[Where:
R ¹ ′ and R ¹ ′ are independently selected from alkoxyalkyl, alkyl, cycloalkyl and pyranyl, wherein the cycloalkyl and pyranyl are optionally independently of alkyl, halo, haloalkyl, hydroxy and hydroxyalkyl. Optionally substituted with 1, 2 or 3 selected substituents;
R ² and R ² ′ are the same or different alkyl groups]
Or a pharmaceutically acceptable salt thereof.

  In a first embodiment of the first aspect, the present invention provides a composition comprising said combination and one or more pharmaceutically acceptable carriers. In a second embodiment, the composition comprises one or two other compounds having anti-HCV activity. In a third embodiment, the at least one other compound is interferon or ribavirin. In a fourth embodiment, the interferon is selected from interferon α 2B, pegylated interferon α, pegylated interferon λ, consensus interferon, interferon α 2A, and lymphoblastoid interferon τ. In a fifth embodiment, the composition comprises one or two other compounds having anti-HCV activity, wherein at least one other compound is HCV protease, HCV polymerase for the treatment of HCV infection. It is effective to inhibit the function of a target selected from HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein and IMPDH.

  In a second aspect, the present invention provides a method for treating HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of said combination, or a pharmaceutically acceptable salt combination thereof. provide. In a first embodiment of the second aspect, the method comprises administering one or two other compounds having anti-HCV activity before, after or simultaneously with the combination or pharmaceutically acceptable salt thereof. A method is provided that is further characterized by: In a second embodiment, the at least one other compound is interferon or ribavirin. In a third embodiment, the interferon is selected from interferon α 2B, pegylated interferon α, pegylated interferon λ, consensus interferon, interferon α 2A and lymphoblastoid interferon τ. In a fourth embodiment, at least one other compound is used for the treatment of HCV infection, HCV protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein and It is effective to inhibit the function of a target selected from IMPDH.

[式中、
R¹'およびR¹'は、アルキルおよびシクロアルキルから独立して選択され、該シクロアルキルは、所望により、アルキル、ハロ、ハロアルキル、ヒドロキシおよびヒドロキシアルキルから独立して選択される1、2または3つの置換基で置換されていてもよい]
の化合物またはその医薬的に許容される塩を提供する。 In another embodiment, the invention provides a combination comprising an NS5A targeting compound and an NS5A synergist, comprising a mutation that, when administered, confers resistance only to said NS5A targeting compound Provides synergistic anti-HCV activity to the body, wherein the NS5A synergist is of formula (II):

[Where
R ¹ ′ and R ¹ ′ are independently selected from alkyl and cycloalkyl, which is optionally selected from 1, 2 or 3 independently selected from alkyl, halo, haloalkyl, hydroxy and hydroxyalkyl May be substituted with two substituents]
Or a pharmaceutically acceptable salt thereof.

  Other aspects of the invention may include suitable combinations of the embodiments described herein.

  Still other aspects and embodiments may be found in the description herein.

  The description of the invention herein should be construed in accordance with the laws and principles of chemical bonding. In some cases, it may be necessary to remove a hydrogen atom to place a substituent at any given position.

In some cases, the number of carbon atoms in any particular group is described before the group designation. For example, the term “C _2-6 alkenyl” refers to an alkenyl group containing 2 to 6 carbon atoms. Where these designations are made, they take precedence over all other definitions contained herein.

  It should be understood that the compounds encompassed by the present invention are suitably stable for use as pharmaceuticals.

  The definition of any substituent or variable at a particular position in the molecule is intended to be independent of its definition in other parts of the molecule.

  All patents, patent applications, and references mentioned herein are hereby incorporated by reference in their entirety. In the case of inconsistencies, the disclosure (including definitions) of this application shall prevail.

As used herein, the following terms have the meanings specified.
As used herein, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

  Unless otherwise specified, all aryl, cycloalkyl, and heterocyclyl groups of the invention can be substituted as described in their respective definitions. For example, the aryl portion of an arylalkyl group may be substituted as described in the definition of the term “aryl”.

As used herein, the term “NS5A synergist” alone exhibits weaker activity against HCV wild type than NS5A targeted compound, but when combined with NS5A targeted compound, NS5A targeted compound alone A molecule that exhibits an EC ₅₀ potency that is at least three times greater than the potency.

As used herein, the term “synergistic anti-HCV activity” refers to an EC ₅₀ potency that is three or more times greater than that of the NS5A targeted compound alone.

  As used herein, the term “NS5A targeted compound” refers to an NS5A protein, most commonly within (but not limited to) at least one resistant substitution within the first 100 residues of NS5A. Refers to a molecule that inhibits HCV replication.

  The term “alkoxy” as used herein refers to an alkyl group attached to the parent molecular group through an oxygen atom.

  The term “alkoxyalkyl” as used herein refers to an alkyl group substituted with one, two, or three alkoxy groups.

  The term “alkyl” as used herein refers to a group derived from a straight or branched chain saturated hydrocarbon having from 1 to 7 carbon atoms.

  The term “cycloalkyl” as used herein refers to a 3 to 7 membered monocyclic saturated carbocyclic ring.

  The term “halo” as used herein refers to Cl, Br, F, or I.

  The term “haloalkyl” as used herein refers to an alkyl group substituted with 1, 2, 3, or 4 halogen atoms.

  The term “hydroxy” as used herein refers to —OH.

  The term “hydroxyalkyl” as used herein refers to an alkyl group substituted with one, two, or three hydroxy groups.

  There are asymmetric centers in the compounds of the present invention. These centers are indicated by the symbol “R” or “S” depending on the configuration of substituents around the chiral carbon atom. It should be understood that the present invention encompasses all stereochemical isomers or mixtures thereof that have the ability to inhibit NS5A. The individual stereoisomers of the compounds can be prepared synthetically from commercially available starting materials with chiral centers, or can be used to produce a mixture of enantiomer products and then convert to a mixture of diastereomers. It can be prepared by performing separations such as subsequent separation or recrystallization, chromatographic techniques, or direct separation of enantiomers on chiral chromatography columns. The specific stereochemical starting compounds are either commercially available or can be prepared and resolved by methods known to those skilled in the art.

  Certain compounds of the present invention may also exist in different stable conformational forms, which may be separable. Torsional asymmetry (eg, due to steric hindrance or ring distortion) due to restricted rotation about an asymmetric single bond may allow separation of different conformers. The present invention includes each conformational isomer of these compounds and mixtures thereof.

  The compounds of the present invention also exist as tautomers; therefore, the present invention also includes all tautomers.

  The term “compounds of the invention” and equivalent expressions are meant to include the compounds making up the combinations of the invention and pharmaceutically acceptable enantiomers, diastereomers and salts thereof. Similarly, references to intermediates are meant to include their salts where allowed by the content.

The present invention is meant to include all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. As a general example, without limitation, isotopes of hydrogen include deuterium and tritium. Carbon isotopes include ¹³ C and ¹⁴ C. Isotopically labeled compounds of the invention are generally suitable in place of unlabeled reagents used elsewhere, by conventional techniques known to those skilled in the art or by methods analogous to those described herein. Can be produced using a simple isotope-labeling reagent. Such compounds can have a variety of potential uses, for example, as standards and reagents in the determination of biological activity. In the case of stable isotopes, such compounds may have the ability to conveniently modify biological, pharmacological, or pharmacokinetic properties.

  The compounds of the present invention can exist as pharmaceutically acceptable salts. As used herein, the term “pharmaceutically acceptable salt” refers to a salt or zwitterionic form of a compound of the present invention, which is water or oil-soluble or dispersible and of the appropriate medical judgment. In range, suitable for use in contact with the patient's tissue without undue toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit / risk ratio Effective for intended use. The salt can be prepared during final isolation and purification of the compound or can be prepared separately by reacting a suitable nitrogen atom with a suitable acid. Typical acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, hydrogen sulfate, butyrate, camphorate, Camphorsulfonate; digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethane sulfonate, lactate, maleate, mesitylene sulfonate, methane sulfonate, naphthylene sulfonate, nicotinate, 2-naphthalene sulfonate, oxalate, pamoate ( palmoate), pectate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, succinate, tartrate, tricate B acetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para - toluenesulfonate, and undecanoate. Examples of acids that can be used to form pharmaceutically acceptable addition salts include inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid) and organic acids (e.g., oxalic acid, maleic acid, Succinic acid and citric acid).

  Base addition salts are prepared by reacting a carboxy group with a suitable base (e.g., a hydroxide, carbonate or bicarbonate of a metal cation) or with ammonia, or an organic primary, secondary, or tertiary amine. By reacting, it can be produced during the final isolation and purification of the compound. Pharmaceutically acceptable salt cations include lithium, sodium, potassium, calcium, magnesium, and aluminum, and non-toxic amine cations (e.g., ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine). , Triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N, N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N, N-dibenzylphenethylamine, and N, N '-Dibenzylethylenediamine). Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, and piperazine.

  When a therapeutically effective amount of each compound of the combination, as well as its pharmaceutically acceptable salt, can be administered as a raw chemical for use in therapy, the active ingredient is a pharmaceutical composition. Can exist as Accordingly, the present invention provides a pharmaceutical comprising a therapeutically effective amount of a compound comprising a combination, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients. Further provided is a composition. As used herein, the term “therapeutically effective amount” refers to the total amount of each active ingredient that is sufficient to show a meaningful patient benefit (eg, sustained reduction in viral load). When applied to an individual active ingredient administered alone, the term refers to the amount of the ingredient alone. When applied to a combination, the term refers to the combined amount of active ingredients that provide a therapeutic effect, whether combined, sequentially or simultaneously. The compounds relating to the combination and pharmaceutically acceptable salts thereof are as described above. The carrier, diluent or excipient must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the recipient thereof. According to another aspect of the invention, a medicament comprising mixing a combination compound or pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable carriers, diluents or excipients. A method of manufacturing the formulation is also provided. As used herein, the term “pharmaceutically acceptable” is within the scope of appropriate medical judgment, commensurate with a reasonable benefit / risk ratio, excessive toxicity, irritation, allergic reaction, or other problem or A compound, substance, composition, and / or dosage form that is suitable for use in contact with a patient's tissue without complications and that is effective for its intended use.

  The pharmaceutical preparation may be in unit dosage form containing a predetermined amount of active ingredient per unit dose. The dosage concentration at which the compound of the present invention is about 0.01 to about 250 milligrams per kilogram ("mg / kg") body weight per day, preferably about 0.05 to about 100 mg / kg body weight per day, is It is typical in monotherapy for prevention and treatment. Usually, the pharmaceutical composition of the present invention is administered from about 1 to about 5 times per day or continuously. Such administration can be used as long-term treatment or emergency treatment. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form depends on the condition being treated, the severity of the condition, the frequency of administration, the route of administration, the rate of elimination of the compound used, the duration of the treatment, and the age of the patient, Can vary by gender, weight, and condition. Preferred unit dosage formulations are those containing a daily dose or less of the active ingredient as described herein above, or an appropriate fraction thereof. Usually, treatment is initiated at a dosage that is significantly less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the conditions is reached. In general, it is most desirable to administer the compound at a concentration level that would normally achieve an antiviral effect without causing any harmful or toxic side effects.

  When a composition of the invention includes a compound of the invention in combination with one or more other therapeutic or prophylactic agents, both the compound and the other drug are typically administered normally in a monotherapy regimen. Present at a dose concentration of about 10 to 150%, more preferably about 10 to 80% of the total dose.

  The pharmaceutical formulation can be any suitable route, e.g. oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral ( Subcutaneous, intradermal, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, intravenous, or intradermal injection or infusion may be indicated. Such formulations can be prepared by any method known in the pharmaceutical art (eg, by associating the active ingredient with a carrier or excipient). Oral administration or administration by injection is preferred.

  Pharmaceutical formulations adapted for oral administration include capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whipping agents; or oil-in-water liquid emulsions or water-in-oil It may be a separate unit, such as an emulsion.

  For example, for oral administration, in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Powders are prepared by finely pulverizing the compound to a suitable fine particle size and mixing it with a pharmaceutical carrier such as an edible carbohydrate (eg starch or mannitol). Flavoring agents, preservatives, dispersing agents, and coloring agents may also be present.

  Capsules are manufactured by preparing a powder mixture as described above and filling a molded gelatin sheath. Prior to the filling step, a fluidizing agent and a lubricant (eg, colloidal silica, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol) can be added to the powder mixture. Disintegrants or solubilizers (eg, agar, calcium carbonate or sodium carbonate) can be added to increase the availability of the drug when the capsule is ingested.

  In addition, if desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can be added to the mixture. Suitable binders include starch, gelatin, natural sugars (eg glucose or β-lactose), corn syrup, natural and synthetic gums (eg acacia, tragacanth or sodium alginate), carboxymethylcellulose, polyethylene glycol and the like. Examples of lubricants used in these dosage forms include sodium oleate and sodium chloride. Examples of lubricants used in these dosage forms include sodium oleate and sodium chloride. Examples of the disintegrant include, but are not limited to, starch, methyl cellulose, agar, bentonite, and xanthan gum. Tablets are formulated, for example, by preparing a powder mixture, granulating or filling, adding a lubricant and disintegrant, and pressing into tablets. The powder mixture comprises a suitably powdered compound and the above diluent or base, and optionally a binder (e.g., carboxymethylcellulose, alginate, gelatin, or polyvinylpyrrolidone), a dissolution retardant ( For example, paraffin), absorption enhancers (eg quaternary salts) and / or absorbents (eg bentonite, kaolin or calcium hydrogen phosphate). The powder mixture can be granulated by wetting with a binder (eg syrup, starch paste, acadia mucilage, or a solution of cellulosic or polymeric material) and pressing through a sieve. As an alternative to granulation, the powder mixture can be passed through a tablet machine to obtain an incompletely shaped slag and then ground into granules. The granules can be smoothed by the addition of stearic acid, stearate, talc, or mineral oil to prevent adhesion to the tablet mold. The smoothed mixture is then compressed into tablets. Also, the compounds of the present invention can be combined with a free flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a shellac sealing coat, a sugar or polymer coating, and a wax polish coating may be applied. Dyestuffs can be added to these coatings to distinguish different unit doses.

  Oral solutions (eg, solutions, syrups, and elixirs) can be prepared in dosage unit form so that a given quantity contains a predetermined amount of the compound. Syrups can be made by dissolving the compound in a suitably flavored aqueous solution, while elixirs can be made by using a non-toxic vehicle. Solubilizers and emulsifiers (e.g. ethoxylated isostearyl alcohol and polyoxyethylene sorbitol ether), preservatives, flavoring additives (e.g. peppermint oil or natural sweeteners, or saccharin or other artificial sweeteners) can also be added .

  If desired, unit dosage formulations for oral administration can be microencapsulated. The formulation can also be made to delay or sustain release, for example, by coating or incorporating particulate material into polymers, waxes, and the like.

  The compounds of formula (I), and pharmaceutically acceptable salts thereof, can also be administered in the form of liposome delivery systems (e.g., small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles). . Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.

  The combination of compounds and pharmaceutically acceptable salts thereof can also be delivered by using monoclonal antibodies as individual carriers to which the compound molecules are bound. The compounds may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinyl pyrrolidone, pyran copolymers, polyhydroxypropyl methacrylamide phenol, polyhydroxyethyl aspartamide phenol, or polyethylene oxide polylysine substituted with a palitoyl residue. In addition, the compounds are a class of biodegradable polymers useful for achieving controlled release of drugs, such as polylactic acid, polepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans. , Polycyanoacrylates, and hydrogel cross-linked or amphiphilic block copolymers.

  Pharmaceutical formulations suitable for transdermal administration may be individual patches intended to maintain intimate contact with the recipient's epidermis for an extended period of time. For example, the active ingredient can be delivered from the patch by iontophoresis, as generally described in Pharmaceutical Research 1986, 3 (6), 318.

  Pharmaceutical formulations suitable for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

  In the treatment of the eye or other external tissue, for example mouth and skin, the formulations are preferably applied as topical ointment or cream. When formulated into an ointment, the active ingredient may be used with either a paraffinic or water-miscible ointment base. Alternatively, the active ingredient can be formulated in a cream with an oil-in-water cream base or a water-in-oil base.

  Pharmaceutical formulations suitable for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier (especially an aqueous solvent).

  Pharmaceutical formulations suitable for topical administration in the mouth include lozenges, pastilles and mouth washes.

  Pharmaceutical formulations suitable for rectal administration may be suppositories or enemas.

  Pharmaceutical formulations suitable for nasal administration wherein the carrier is a solid include, for example, course powders having a particle size of 20-500 microns, which is a method of ingesting olfactory drugs, i.e. in the nose. Administered by rapid inhalation through a nasal cavity from a container of powder close together. Suitable formulations wherein the carrier is a liquid for administration as a nasal spray or nasal spray include aqueous or oily solutions of the active ingredient.

  Pharmaceutical formulations suitable for administration by inhalation include particulate powder or particulate mist, which can be generated using various types of metered pressure aerosols, nebulizers, or inhalers.

  Pharmaceutical formulations suitable for intravaginal administration may be pessaries, tampons, creams, gels, pastes, foams or spray formulations.

  Pharmaceutical formulations suitable for parenteral administration include aqueous and non-aqueous, which may include antioxidants, buffers, bacteriostats, and salts that render the formulation isotonic with the blood of the intended recipient. As well as aqueous and non-aqueous sterile suspensions that may contain suspending and thickening agents. The formulation may be in single or multi-dose containers, such as sealed ampoules and vials, with the addition of a sterile liquid carrier (e.g. water for injection) just prior to use. It may be stored in a lyophilized state as required. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

  In addition to the ingredients specifically described above, the formulation may include other agents conventional in the art in view of the type of formulation (e.g., formulations suitable for oral administration may include flavoring agents). It should be understood.

  The term “patient” includes both humans and other mammals.

  The term “treatment” includes: (i) preventing the onset of a disease, disorder, or condition in a patient susceptible to the disease, disorder, and / or condition but not yet diagnosed; ii) suppression of the disease, disorder, or symptom, ie, inhibiting its progression; and (iii) reducing the disease, disorder, or symptom, ie, causing regression of the disease, disorder, and / or symptom. Say.

  The compounds of the present invention can also be administered with a cyclosporine, such as cyclosporin A. Cyclosporine A has been shown to be active against HCV in clinical trials (Hepatology 2003, 38, 1282; Biochem. Biophys. Res. Commun. 2004, 313, 42; J. Gastroenterol. 2003, 38, 567).

Table A below lists some illustrative examples of compounds that can be administered with the compounds of the present invention. The compounds of the present invention can be administered with other anti-HCV active compounds in combination therapy, either together or separately, or by mixing the compounds into a composition.
Table A

  The compounds of the present invention may be used as experimental reagents. The compounds may be useful in the design of viral replication assays, validation of animal assay systems and providing research tools for structural biology studies to further enhance knowledge of HCV disease mechanisms. Furthermore, the compounds of the invention are useful for establishing or determining the binding site of other antiviral compounds, for example, by competitive inhibition.

  The compounds of the present invention may also be used to treat or prevent viral contamination of substances, such that such substances (e.g. blood, tissue, surgical instruments and surgical gowns, laboratory instruments and gowns, and blood collection) Or may reduce the risk of viral infection in laboratory or medical personnel or patients in contact with blood transfusion equipment and materials.

  The present invention is intended to encompass compounds of formula (I) made by synthetic or metabolic processes, including those occurring in the human or animal body (in vivo) or processes occurring in vitro. .

  Abbreviations used in this application, particularly including those in the following illustrative schemes and examples, are well known to those skilled in the art. Some of the abbreviations used are: RT or rt is room temperature or retention time (determined by context); rett is retention time; min or mins is minutes; TFA is trifluoroacetic acid; min or mins is minutes; ACN or MeCN is acetonitrile; DCM is dichloromethane; DIEA or DiPEA or DIPEA is diisopropylethylamine; HATU is O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetra Methyluronium hexafluorophosphate; h or hr or hrs is time; MeOH is methanol; dppf is diphenylphosphinoferrocene; EtOAc is ethyl acetate; OAc is acetate; DMSO is dimethyl sulfoxide; TBTU is 2- (1H-benzo Triazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate; Me is methyl; and DMF is N, N-dimethylformamide.

  While this disclosure is described in connection with specific embodiments, it is not intended to limit the scope of the invention. On the other hand, the present disclosure includes all alternatives, modifications, and equivalents as can be included within the scope of the claims. Thus, the following examples, including specific embodiments, illustrate one illustration of the present invention, which examples are for the purpose of illustrating specific embodiments and are the most useful and useful in its methods and conceptual aspects and It is understood that this is presented to provide what is believed to be an easily understood description.

  Starting materials can be obtained from commercial sources or prepared by well-established literature methods known to those skilled in the art. The acid precursor for the final process can be prepared according to the method described in US Provisional Application No. 13/933495, filed July 2, 2013.

LC / MS condition 1
Column = Ascentis Express C18, 2.1 X 50 mm, 2.7 um
Solvent A = CH ₃ CN (2%) + 10 mM NH ₄ COOH / H ₂ O (98%)
Solvent B = CH ₃ CN (98%) + 10 mM NH ₄ COOH / H ₂ O (2%)
Start% B = 0; End% B = 100
Gradient time = 1.4 minutes; Stop time = 4 minutes Stop time = 4 minutes Flow rate = 1 mL / min; Wavelength = 220 nm

LC / MS condition 2
Column = Waters BEH C18, 2.0 x 50 mm, 1.7 μm
Solvent A = ACN (5%) + H ₂ O (95%) (with 10 mM NH ₄ OAc)
Solvent B = ACN (95%) + H ₂ O (5%) (with 10 mM NH ₄ OAc)
Start% B = 0; End% B = 100
Gradient time = 3 minutes Flow rate = 1 mL / min Wavelength = 220 nm
Temperature = 50 ° C

LC / MS condition 3
Column: Waters Phenomenex C18, 2.0 x 30 mm, 3 μm particles Mobile phase A: 10% MeOH: 90% Water: 0.1% TFA
Mobile phase B: 90% MeOH: 10% Water: 0.1% TFA
Gradient: 0% B, 0-100% B over 3 minutes, then hold at 100% B for 1 minute Flow rate: 0.8 mL / min Detection: 220 nm
Temperature: 40 ° C

LC / MS condition 4
Column: Waters BEH C18, 2.0 x 50 mm, 1.7 μm particles
Mobile phase A: 5:95 acetonitrile: water (containing 10 mM ammonium acetate)
Mobile phase B: 95: 5 acetonitrile: water (containing 10 mM ammonium acetate)
Gradient: 0% B, 0-100% B over 3 minutes, then 0.5% hold at 100% B Flow rate: 1 mL / min
Detection: UV at 220 nm
Temperature: 50 ° C

実施例B-1 工程a

4-ブロモベンゼン-1,2-ジアミン(2.5 g, 13.37 mmol)/DCM(30 mL)の溶液に、(S)-2-((tert-ブトキシカルボニル)アミノ)-3,3-ジメチルブタン酸(3.09 g, 13.37 mmol)、DIPEA(2.334 mL，13.37 mmol)およびHATU(5.08 g, 13.37 mmol)を加えた。該反応混合溶液を、室温にて18時間撹拌した。該反応混合溶液を、水で希釈して、DCMで抽出した。有機相を、塩水で洗浄して、Na₂SO₄で乾燥し、濾過し、濃縮した。粗製物質を、40 g Redisep Silica column, 溶離液としてCHCl₃/MeOHを用いるISCOにより精製して、(S)-tert−ブチル(1-((2-アミノ-4-ブロモフェニル)アミノ)-3,3-ジメチル-1-オキソブタン-2−イル)カルバメート(1.82 g)を黄色の固体として得た。LC (条件1)：R_t = 2.13分. LC/MS：[M+H₂O]⁺ C₁₇H₂₇BrN₂O₄として計算：計算値；402.12；実測値402.2. ¹H NMR (DMSO-d₆, δ = 2.50 ppm, 400 MHz)：δ 9.35 - 9.21 (m, 1 H), 7.07 (d, J = 8.5 Hz, 1 H), 6.91 (d, J = 2.0 Hz, 1 H), 6.80 - 6.60 (m, 1 H), 5.25 - 5.01 (m, 2 H), 4.07 - 3.89 (m, 1 H), 1.52 - 1.34 (m, 9 H), 1.02 - 0.86 (m, 9 H). Example B-1

Example B-1 Step a

To a solution of 4-bromobenzene-1,2-diamine (2.5 g, 13.37 mmol) / DCM (30 mL), add (S) -2-((tert-butoxycarbonyl) amino) -3,3-dimethylbutanoic acid. (3.09 g, 13.37 mmol), DIPEA (2.334 mL, 13.37 mmol) and HATU (5.08 g, 13.37 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with water and extracted with DCM. The organic phase was washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated. The crude material was purified by ISCO using 40 g Redisep Silica column, CHCl ₃ / MeOH as eluent to give (S) -tert-butyl (1-((2-amino-4-bromophenyl) amino) -3 , 3-Dimethyl-1-oxobutan-2-yl) carbamate (1.82 g) was obtained as a yellow solid. LC (Condition 1): R _t = 2.13 min. LC / MS: Calculated as [M + H ₂ O] ⁺ C ₁₇ H ₂₇ BrN ₂ O ₄ : Calculated; 402.12; Found 402.2. ¹ H NMR (DMSO- d ₆ , δ = 2.50 ppm, 400 MHz): δ 9.35-9.21 (m, 1 H), 7.07 (d, J = 8.5 Hz, 1 H), 6.91 (d, J = 2.0 Hz, 1 H), 6.80 -6.60 (m, 1 H), 5.25-5.01 (m, 2 H), 4.07-3.89 (m, 1 H), 1.52-1.34 (m, 9 H), 1.02-0.86 (m, 9 H).

酢酸(15 mL)を、(S)-tert−ブチル(1-((2-アミノ-4-ブロモフェニル)アミノ)-3,3-ジメチル-1-オキソブタン-2−イル)カルバメート(1.8 g, 4.50 mmol)に加えて、該反応混合液を、終夜65℃に加熱した。揮発性成分を、真空除去して、残留物を、無水CH_２Cl_２(2 x 15 mL)と共に共蒸留した。有機相を、飽和NaHCO_３溶液、塩水で洗い、Na₂SO₄で乾燥し、濃縮して、(S)-tert−ブチル(1-(6-ブロモ-1H-ベンゾ[d]イミダゾール−2−イル)-2,2-ジメチル プロピル)カルバメート(1.68 g)を、黄色の固体として得た。LC(条件1)：R_t = 2.19分. LC/MS：[M+H]⁺ C₁₇H₂₅BrN₃O₂として計算：計算値；381.11；実測値382.2. ¹H NMR (DMSO-d₆, δ = 2.50 ppm, 300 MHz)：δ 12.46 - 12.27 (m, 1 H), 7.82 - 7.65 (m, 1 H), 7.59 - 7.41 (m, 1 H), 7.29 (dt, J = 1.9, 8.5 Hz, 1 H), 7.12 - 6.90 (m, 1 H), 4.64 (d, J = 9.8 Hz, 1 H), 1.44 - 1.27 (m, 9 H), 0.88 (br. s., 9 H). Example B-1, step b

Acetic acid (15 mL) was added to (S) -tert-butyl (1-((2-amino-4-bromophenyl) amino) -3,3-dimethyl-1-oxobutan-2-yl) carbamate (1.8 g, 4.50 mmol) and the reaction mixture was heated to 65 ° C. overnight. The volatile components were removed in vacuo and the residue was co-distilled with anhydrous CH ₂ Cl ₂ (2 × 15 mL). The organic phase is washed with saturated NaHCO ₃ solution, brine, dried over Na ₂ SO ₄ , concentrated, and (S) -tert-butyl (1- (6-bromo-1H-benzo [d] imidazole-2- Yl) -2,2-dimethylpropyl) carbamate (1.68 g) was obtained as a yellow solid. LC (condition 1): R _t = 2.19 min. LC / MS: Calculated as [M + H] ⁺ C ₁₇ H ₂₅ BrN ₃ O ₂ : calculated; 381.11; found 382.2. ¹ H NMR (DMSO-d ₆ , δ = 2.50 ppm, 300 MHz): δ 12.46-12.27 (m, 1 H), 7.82-7.65 (m, 1 H), 7.59-7.41 (m, 1 H), 7.29 (dt, J = 1.9, 8.5 Hz, 1 H), 7.12-6.90 (m, 1 H), 4.64 (d, J = 9.8 Hz, 1 H), 1.44-1.27 (m, 9 H), 0.88 (br.s., 9 H).

(S)-tert−ブチル(1-(6-ブロモ-1H-ベンゾ[d]イミダゾール−2−イル)-2,2-ジメチルプロピル)カルバメート(1.57 g, 4.11 mmol)/ジオキサン(25 mL)の溶液に、ビス(ピナコラト)ジボロン(1.564 g, 6.16 mmol)および酢酸カリウム(1.209 g, 12.32 mmol)を加えた。該反応混合溶液を、アルゴンで10分間パージして、次いでPdCl₂(dppf)(0.150 g, 0.205 mmol)を上記反応混合溶液に加えて、再度アルゴンで5分間パージした。反応混合溶液を、90℃に終夜加熱した。反応混合溶液を、水(15 ml)で希釈して、EtOAc(2 x 25 ml)で抽出した。有機相を合わせて、塩水で洗い、Na₂SO₄で乾燥して、真空で濃縮した。粗製物質を、40 g Redisep column, 溶離液としてヘキサン/酢酸エチルを用いるISCOにより精製して、(S)-tert−ブチル (2,2-ジメチル-1-(6-(4,4,5,5-テトラメチル-1,3,2-ジオキサボロラン-2−イル)-1H-ベンゾ[d]イミダゾール−2−イル)プロピル)カルバメート(1.35 g)を黄色の固体として得た。LC (条件1)：R_t = 2.21分. LC/MS：Anal. Calcd. for [M+H]⁺ C₂₃H₃₇BN₃O₄ ：430.29；実測値430.4. ¹H NMR (CD₃OD, δ = 3.34 ppm, 400 MHz)：δ 7.98 (s, 1 H), 7.65 (dd, J = 1.0, 8.5 Hz, 1 H), 7.53(d, J = 8.5 Hz, 1 H), 4.73 (br. s., 1 H), 1.37 (s, 12 H), 1.24 (m, 9 H), 1.01 (s, 9 H). Example B-1 Step c

(S) -tert-butyl (1- (6-bromo-1H-benzo [d] imidazol-2-yl) -2,2-dimethylpropyl) carbamate (1.57 g, 4.11 mmol) / dioxane (25 mL) To the solution was added bis (pinacolato) diboron (1.564 g, 6.16 mmol) and potassium acetate (1.209 g, 12.32 mmol). The reaction mixture was purged with argon for 10 minutes, then PdCl ₂ (dppf) (0.150 g, 0.205 mmol) was added to the reaction mixture and again purged with argon for 5 minutes. The reaction mixture was heated to 90 ° C. overnight. The reaction mixture was diluted with water (15 ml) and extracted with EtOAc (2 × 25 ml). The organic phases were combined, washed with brine, dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by ISCO using 40 g Redisep column, hexane / ethyl acetate as eluent to give (S) -tert-butyl (2,2-dimethyl-1- (6- (4,4,5, 5-Tetramethyl-1,3,2-dioxaborolan-2-yl) -1H-benzo [d] imidazol-2-yl) propyl) carbamate (1.35 g) was obtained as a yellow solid. LC (condition 1): R _t = 2.21 min. LC / MS: Anal. Calcd. For [M + H] ⁺ C ₂₃ H ₃₇ BN ₃ O ₄ : 430.29; found 430.4. ¹ H NMR (CD ₃ OD, (δ = 3.34 ppm, 400 MHz): δ 7.98 (s, 1 H), 7.65 (dd, J = 1.0, 8.5 Hz, 1 H), 7.53 (d, J = 8.5 Hz, 1 H), 4.73 (br. s., 1 H), 1.37 (s, 12 H), 1.24 (m, 9 H), 1.01 (s, 9 H).

ジオキサン(10 mL)中の(S)-tert−ブチル(2,2-ジメチル-1-(6-(4,4,5,5-テトラメチル-1,3,2-ジオキサボロラン-2−イル)-1H-ベンゾ[d]イミダゾール−2−イル)プロピル)カルバメート(1.114 g, 2.59 mmol)および4,16-ジブロモ[2,2]パラシクロファン(0.38g, 1.038 mmol)の溶液に、Cs₂CO₃(0.845 g, 2.59 mmol)/水(2 mL)を加えて、10分間脱気した。PdCl₂(dppf)(0.038 g, 0.052 mmol)を、上記反応混合溶液に加えて、再度5分間脱気した。該反応混合溶液を、90℃に12時間加熱した。次いで、反応混合溶液を、濾過して、実施例B-1 工程dを得て、これをさらなる精製をせずに次工程に用いた。LC(条件1)：R_t = 2.54分. LC/MS：[M+H]⁺ C₅₀H₆₃N₆O₄について計算：計算値；811.49；実測値811.6. ¹H NMR (DMSO-d₆, δ = 2.50 ppm, 300 MHz)：δ 12.36 (br. s., 2 H), 7.85 - 7.52 (m, 4 H), 7.32 (d, J = 7.9 Hz, 2 H), 7.05 (br. s., 2 H), 6.89 - 6.67 (m, 4 H), 6.54 (br. s., 2 H), 4.72 (d, J = 8.7 Hz, 2 H), 3.57 - 3.44 (m, 2 H), 3.07 (br. s., 2 H), 2.83 (br. s., 2 H), 2.65 (br. s., 2 H), 1.36 (s, 18 H), 1.08 - 0.91 (m, 18 H). Example B-1 Step d

(S) -tert-butyl (2,2-dimethyl-1- (6- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) in dioxane (10 mL) -1H-benzo [d] imidazol-2-yl) propyl) carbamate (1.114 g, 2.59 mmol) and 4,16-dibromo [2,2] paracyclophane (0.38 g, 1.038 mmol) in Cs ₂ CO ₃ (0.845 g, 2.59 mmol) / water (2 mL) was added and degassed for 10 minutes. PdCl ₂ (dppf) (0.038 g, 0.052 mmol) was added to the reaction mixture and degassed again for 5 minutes. The reaction mixture was heated to 90 ° C. for 12 hours. The reaction mixture was then filtered to give Example B-1 step d, which was used in the next step without further purification. LC (Condition 1): R _t = 2.54 min. LC / MS: Calculated for [M + H] ⁺ C ₅₀ H ₆₃ N ₆ O ₄ : Calculated; 811.49; Found 811.6. ¹ H NMR (DMSO-d ₆ , δ = 2.50 ppm, 300 MHz): δ 12.36 (br. s., 2 H), 7.85-7.52 (m, 4 H), 7.32 (d, J = 7.9 Hz, 2 H), 7.05 (br. s ., 2 H), 6.89-6.67 (m, 4 H), 6.54 (br. S., 2 H), 4.72 (d, J = 8.7 Hz, 2 H), 3.57-3.44 (m, 2 H), 3.07 (br. S., 2 H), 2.83 (br. S., 2 H), 2.65 (br. S., 2 H), 1.36 (s, 18 H), 1.08-0.91 (m, 18 H) .

HCl/ジオキサン(4 mL，24.00 mmol)を、実施例B-1 工程d(0.1 g, 0.102 mmol)に加えて、該反応混合溶液を、RTで2時間撹拌させた。反応の完了を、LCMSによりモニターした。揮発性成分を、真空にて除去して、残留物を、ジエチルエーテルで洗浄して、乾燥させて、実施例B-1 工程e(0.07 g)を黄色の固体として得た。LC(条件1)：R_t = 2.54分. LC/MS：[M+H]⁺ C₄₀H₄₇N₆について計算：計算値：611.39；実測値611.4. ¹H NMR (CD₃OD, δ = 3.34 ppm, 400 MHz)：δ 7.90 (d, J = 13.1 Hz, 2 H), 7.83 (d, J = 8.5 Hz, 2 H), 7.61 (d, J = 8.5 Hz, 2 H), 6.84 (d, J = 6.5 Hz, 2 H), 6.78 (s, 2 H), 6.70 - 6.65 (m, 2 H), 4.54 (d, J = 1.0 Hz, 2 H), 3.54 - 3.46 (m, 2 H), 3.18 - 3.10 (m, 2 H), 2.98 - 2.86 (m, 2 H), 2.71 (br. s., 2 H), 1.25 - 1.22 (m, 18 H). Example B-1 Step e

HCl / dioxane (4 mL, 24.00 mmol) was added to Example B-1 step d (0.1 g, 0.102 mmol) and the reaction mixture was allowed to stir at RT for 2 h. The completion of the reaction was monitored by LCMS. The volatile components were removed in vacuo and the residue was washed with diethyl ether and dried to give Example B-1 step e (0.07 g) as a yellow solid. LC (Condition 1): R _t = 2.54 min. LC / MS: Calculated for [M + H] ⁺ C ₄₀ H ₄₇ N ₆ : Calculated: 611.39; Found 611.4. ¹ H NMR (CD ₃ OD, δ = 3.34 ppm, 400 MHz): δ 7.90 (d, J = 13.1 Hz, 2 H), 7.83 (d, J = 8.5 Hz, 2 H), 7.61 (d, J = 8.5 Hz, 2 H), 6.84 (d , J = 6.5 Hz, 2 H), 6.78 (s, 2 H), 6.70-6.65 (m, 2 H), 4.54 (d, J = 1.0 Hz, 2 H), 3.54-3.46 (m, 2 H) , 3.18-3.10 (m, 2 H), 2.98-2.86 (m, 2 H), 2.71 (br. S., 2 H), 1.25-1.22 (m, 18 H).

Example B-1 A solution of step e (0.04 g, 0.053 mmol) / DMF (5 mL) was added to 4,4-difluorocyclohexanecarboxylic acid (0.017 g, 0.106 mmol), DIPEA (0.055 mL, 0.317 mmol) and HATU. (0.030 g, 0.079 mmol) was added. After stirring for 2 hours at room temperature, the volatile components were removed in vacuo and the residue was dissolved in DCM (10 mL) and washed with NH ₄ Cl saturated solution, 10% NaHCO ₃ solution, brine. , Dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by reverse phase HPLC purification to give Example B-1 as a white solid. LC (condition 1): R _t = 2.37 min. LC / MS: Anal. Calcd. For [M + H] ⁺ C ₅₄ H ₆₃ F ₄ N ₆ O ₂ : 903.49; found 903.4. ¹ H NMR (DMSO- d ₆ , δ = 2.50 ppm, 400 MHz): δ 12.53-12.32 (m, 2 H), 8.41-8.21 (m, 2 H), 7.84-7.50 (m, 4 H), 7.43-7.24 (m, 2 H), 6.90-6.67 (m, 4 H), 6.60-6.44 (m, 2 H), 5.14-4.97 (m, 2 H), 3.44 (br. S., 2 H), 3.08 (br. S. , 2 H), 2.93-2.77 (m, 2 H), 2.73-2.56 (m, 4 H), 2.20-1.98 (m, 3 H), 1.96-1.49 (m, 13 H), 1.02 (s, 18 H).

The following examples were prepared from Example B-1 step e and the appropriate acid precursor using the method described for the synthesis of Example B-1. The resulting product was purified by preparative HPLC (CH ₃ CN / H ₂ O / NH ₄ OAc).

工程a:
ジオキサン(8 mL)中のボロネートY-1a (0.80 g, 1.93 mmol)、シクロファンY-1b(0.25 g, 0.683 mmol)の溶液に、Cs₂CO₃(0.70 g, 2.148 mmol)/水(2 mL)を加えて、該混合溶液を、10分間脱気して、PdCl₂(dppf)(0.025 g, 0.034 mmol)を上記反応混合物に加えて、再度5分間脱気した。反応混合溶液を、90℃で18時間加熱して、周囲温度まで冷却して、EtOAcで希釈し、濾過して、乾燥させて、Y-1c(0.45g)を灰色の固体として得た。LC(条件3)：R_t = 2.79分. LC/MS：C₄₈H₅₈N₆O₄として計算：計算値；782.45；実測値783.61 [M+H]⁺. Examples Y-1 to Y-5

Process a:
To a solution of boronate Y-1a (0.80 g, 1.93 mmol), cyclophane Y-1b (0.25 g, 0.683 mmol) in dioxane (8 mL) was added Cs ₂ CO ₃ (0.70 g, 2.148 mmol) / water (2 mL) was added, the mixture was degassed for 10 min, PdCl ₂ (dppf) (0.025 g, 0.034 mmol) was added to the reaction mixture and degassed again for 5 min. The reaction mixture was heated at 90 ° C. for 18 hours, cooled to ambient temperature, diluted with EtOAc, filtered and dried to give Y-1c (0.45 g) as a gray solid. LC (condition 3): R _t = 2.79 min. LC / MS: calculated as C ₄₈ H ₅₈ N ₆ O ₄ : calculated; 782.45; found 783.61 [M + H] ⁺ .

Process b:
Bis-carbamate Y-1c was deprotected to Y-1d (HCl salt) according to the method described in Example 1, step e. LC (Condition 3): R _t = 2.79 min. LC / MS: Calculated as C ₃₈ H ₄₂ N ₆ · 4HCl: Calculated; 582.78; Found 583.45 [M + H] ⁺ .

Process c:
To a mixed solution of Y-1d (HCl salt) (30 mg, 0.041 mmol) and 4,4-difluorocyclohexanecarboxylic acid (18 mg, 0.110 mmol) in DCM (1 mL) and acetonitrile (1 mL) was added DIPEA ( 0.1 mL, 0.573 mmol) and TBTU (30 mg, 0.093 mmol) were added. The reaction mixture was stirred at RT for 45 min, diluted with MeOH (1 mL), concentrated and purified by preparative HPLC (H ₂ O / CH ₃ CN / NH ₄ OAc). Y-1 was obtained. Examples Y-2 to Y-5 were similarly prepared from the appropriate carboxylic acid precursor. This synthesis has been reported in the art. Note that the acid precursors used in the preparation of Example Y-4 were racemic mixtures containing each of their hydroxyl and carboxyl reactive groups cis.

(Biological activity)
The NS5A synergistic inhibitory effect of a test compound can be determined by titrating a second compound of interest with various amounts of NS5A targeted compound. Both the NS5A targeting compound and the second compound of interest are essentially inactive or weakly active when tested separately against the HCV variant, but against the HCV variant When tested in combination, it has been found to regain synergistic inhibitory potency (more than 3 fold inhibition). In one embodiment, the compound BMS-790052 as an NS5A targeting compound can be kept constant at a fixed concentration of 200 nM before titration of the test compound with a variant of HCV. In one embodiment, the genotype of the HCV may be genotype 1a containing a change from glutamine to glutamic acid at amino acid position 30 of the NS5A protein. The test compound may be selected from the above compounds or other compounds described in the literature. One skilled in the art can readily test compounds in an HCV replicon cell-based assay as previously described in the art, and at concentrations effective for 50% inhibition of specific compounds (EC ₅₀ ) Can be easily determined.

Illustratively, the compound P-55 shown below can be titrated in an HCV replicon cell-based assay consisting of a genotype-1a mutant in which the 30th glutamine is changed to glutamic acid in the NS5A protein. Titration with BMS-790052 alone gives EC ₅₀ values up to 200 nM, while titration with P-55 alone gives EC ₅₀ values> 200 nM. Titration of P-55 in the presence of a fixed amount of BMS-790052 of 200 nM gave an EC ₅₀ value of ~ 2 nM for P-55, indicating a synergistic inhibitory effect (> 100-fold) with the combination Yes. Similarly, titration of BMS-790052 in the presence of a fixed amount of P-55 of 200 nM gave an EC ₅₀ value of ˜2 nM for BMS-790052, indicating that the combination had a synergistic inhibitory effect (˜100 (PCT / US2011 / 043785 filed on July 13, 2011), Table 3). Additional compounds could be tested in a similar manner and ranking of synergist activity was determined; these rankings for genotype 1a Q → E mutants are shown in the table below for selected compounds .

As shown in applicant's common WO2012 / 009394, the genotype is not limited to genotype 1a mutants, but all HCV genotype mutants (but not limited to 1b, 2a, 3a, 4a, It is understood that 5a, 6a HCV variants may be included). It is also understood that the synergistic effect is not limited to the combination of BMS-790052 or P-55 and can be obtained from other combinations of NS5A targeted compounds that have reduced or no efficacy against HCV variants alone. Is done.

  It will be apparent to those skilled in the art that the present invention is not limited to the foregoing exemplary embodiments and may be embodied in other specific forms without departing from the essential characteristics thereof. Accordingly, the examples are to be considered in all respects only as illustrative and not restrictive, and reference should be made to the claims rather than to the foregoing examples, and therefore the claims It is desirable to include all modifications that fall within the meaning and range equivalent to

Claims (12)

A combination comprising an NS5A targeting compound and an NS5A synergist, which when administered results in synergistic anti-HCV activity against a mutant comprising a mutation that confers resistance only to the NS5A targeting compound; The NS5A synergist has the formula (I):

[Where
R ¹ ′ and R ¹ ′ are independently selected from alkoxyalkyl, alkyl, cycloalkyl and pyranyl, wherein the cycloalkyl and pyranyl are optionally independently of alkyl, halo, haloalkyl, hydroxy and hydroxyalkyl. Optionally substituted by 1, 2 or 3 selected substituents; and
R ² and R ² ′ are the same or different alkyl groups]
Or a pharmaceutically acceptable salt thereof. The compound of formula (I) is

The combination according to claim 1, which is a compound of formula (I) selected from: or a pharmaceutically acceptable salt thereof.   A composition comprising the combination of claim 1 and one or more pharmaceutically acceptable carriers.   4. The composition of claim 3, further comprising one or two other compounds having anti-HCV activity.   5. The composition of claim 4, wherein at least one of the other compounds is interferon or ribavirin.   6. The composition according to claim 5, wherein the interferon is selected from interferon α2B, pegylated interferon α, pegylated interferon λ, consensus interferon, interferon α2A or lymphoblastoid interferon τ.   At least one other compound is a target selected from HCV protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein or IMPDH for the treatment of HCV infection 5. The composition of claim 4, which is effective to inhibit function.   A method of treating HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of a combination according to claim 1 or a pharmaceutically acceptable salt thereof.   9. The method of claim 8, further characterized in that one or two other compounds having anti-HCV activity are administered before, after or simultaneously with the combination or a pharmaceutically acceptable salt thereof.   10. The method of claim 9, wherein at least one of the other compounds is interferon or ribavirin.   11. The method of claim 10, wherein the interferon is selected from interferon α2B, pegylated interferon α, pegylated interferon λ, consensus interferon, interferon α2A, or lymphoblastoid interferon τ.   At least one other compound is a target selected from HCV protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein or IMPDH for the treatment of HCV infection 10. A method according to claim 9, which is effective to inhibit function.