Hepatitis C virus inhibitors

European).

EP 2 784 075

Hepatitis C virus inhibitors

REFERENCE TO RELATED APPLICATIONS

This application reserves the benefit of a provisional US application with serial number 60 / 836.996 filed August 11, 2006.

The disclosure relates generally to an antiviral compound for use in therapy. The compound may inhibit the function of the NS5A protein encoded by the hepatitis C virus (HCV). HCV is the main human pathogen that infects an estimated 170 million people around the world - about five times the number of human immunodeficiency virus-infected viruses. A significant proportion of those infected with HCV develop severe progressive liver disease, including cirrhosis and hepatocellular carcinoma.

The most effective HCV therapy currently uses a combination of alpha interferon and ribavirin, leading in 40% of patients to sustained efficacy. Recent clinical results show that monotherapy with pegylated interferon alfa is better than unmodified interferon alfa. However, even with an experimental therapeutic dose regimen, including combinations of pegylated interferon alfa and ribavirin, a significant fraction of patients do not achieve a permanent reduction in viral load. Thus, there is a clear and long-felt need to develop effective drugs for the treatment of HCV infection.

HCV is a positive polarity RNA virus. Based on a comparison of the deduced amino acid sequence and significant similarity in the non-coding 5 'region, HCV has been classified as a separate genus in the Flaviviridae family. All members of the Flaviviridae family have enveloped virions containing a positive polarity RNA genome coding for all known viral-specific proteins through the translation of a single, uninterrupted, open reading frame.

Within the nucleotide and encoded amino acid sequence, significant heterogeneity was found within the HCV genome. At least six major genotypes have been characterized and more than 50 subtypes have been described. The main genotypes of HCV differ in their distribution throughout the world and the clinical significance of HCV genetic heterogeneity remains undetermined, despite numerous studies on the possible impact of the genotype on pathogenesis and therapy.

The single-stranded HCV RNA genome has an approximate length of 9,500 nucleotides and has a single open reading frame (ORF) encoding a single, large polyprotein, about 3000 amino acids in size. In infected cells, this polyprotein is cleaved in many places by cellular and viral proteases to produce structural and nonstructural (NS) proteins. In the case of HCV, the production of mature non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) is the result of the action of two viral proteases. The former is thought to be a metalloprotease and cleaves at the NS2-NS3 junction site; the other is a serine protease contained within the N-terminal region of NS3 (also referred to herein as the NS3 protease) and mediates all subsequent cleavages below NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, for other sites NS4A-NS4B, NS4B-NS5A, NS5A-NS5B. The NS4A protein appears to perform many functions, acting as a co-factor for the NS3 protease and possibly helping in the membrane localization of NS3 and other components of the viral replication. The formation of the NS3 protein complex with NS4A seems to be necessary for processing activities, enhancing proteolytic efficiency in all places. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5B (also referred to herein as HCV polymerase) is an RNA-dependent RNA polymerase that is involved in HCV replication. The formation of the NS3 protein complex with NS4A seems to be necessary for processing activities, enhancing proteolytic efficiency in all places. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5B (also referred to herein as HCV polymerase) is an RNA-dependent RNA polymerase that is involved in HCV replication. The formation of the NS3 protein complex with NS4A seems to be necessary for processing activities, enhancing proteolytic efficiency in all places. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5B (also referred to herein as HCV polymerase) is an RNA-dependent RNA polymerase that is involved in HCV replication.

Compounds useful in the treatment of HCV-infected patients that selectively inhibit viral HCV replication are desirable. In particular, compounds that are effective in inhibiting the function of the NS5A protein are desirable. The HCV NS5A protein is described, for example, in Tan, S.-L., Katzel, MG Virology 2001, 284, 1-12; and in Park, K. -J .; Choi, S.-H, J. Biological Chemistry 2003.

In a first aspect, the present disclosure provides a compound that is ((1S) -1 - (((2S) -2- (5 (4 '- (2 - ((2S) -1 - ((2S) -2 - (( Methoxycarbonyl (amino) -3-methylbutanoyl) -2-pyrrolidinyl) -1H-imidazol-5-yl) -4-biphenylyl) -1H-imidazol-2-yl) -1-pyrrolidinyl) carbonyl) methyl 2-methylpropyl) carbamate Formula I:

<img file="PL2784075T3_D0001.tif" />

or a pharmaceutically acceptable salt thereof for use in therapy.

In a first embodiment of this aspect, the pharmaceutically acceptable salt is a dihydrochloride salt.

In another embodiment of this aspect, the use in therapy further comprises the use of one or two additional compounds with anti-HCV activity. In another embodiment, at least one of the additional compounds is interferon or ribavirin. In another embodiment, the interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, alpha 2A interferon and lymphoblastoid interferon tau.

In another embodiment of this aspect, the use in treatment further comprises the use of one or two additional compounds with anti-HCV activity, wherein at least one of the additional compounds is selected from interleukin 2, interleukin 6, interleukin 12, a compound that supports the development of helper T cell responses. type 1, interfering RNA, antisense RNA, imiquimod, ribavirin, inosine-5'-monophosphate dehydrogenase inhibitor, amantadine and rimantadine.

In another embodiment of this aspect, the use in treatment further comprises using one or two additional compounds with anti-HCV activity, wherein at least one of the additional compounds is effective in inhibiting a target function selected from HCV metalloprotease, HCV serine protease, HCV polymerase, helicase. HCV, HCV NS4B proteins, HCV entries, HCV assembly, HCV outputs, HCV NS5A proteins and IMPDH for the treatment of HCV infection.

In another embodiment of this aspect, the use in therapy further comprises administering one or two additional compounds having anti-HCV activity before, after or concurrently with the compound of general formula (I), or a pharmaceutically acceptable salt thereof.

Other embodiments of the present disclosure may comprise a suitable combination of two or more embodiments disclosed herein.

Still other embodiments and aspects of the invention will be apparent as described below.

The compounds of the present disclosure also exist as tautomers, therefore the disclosure also includes all tautomeric forms.

The description of this disclosure should be interpreted in accordance with the laws and principles of chemical bonding.

It should be understood that the compounds included in this disclosure are those that are suitably stable for use as a pharmaceutical.

All patents, patent applications and references cited in the specification are incorporated by reference in their entirety. In the event of non-compliance, this disclosure, including definitions, will be conclusive.

The terms used in this description have the meanings indicated below:

The singular forms "form" as used herein include references to the plural unless the context clearly indicates otherwise.

Unless otherwise indicated, all of the aryl, cycloalkyl, and heterocyclyl groups of the present disclosure can be substituted as described in each of their definitions. For example, the aryl part of an aralkyl group can be substituted as described in the definition of the term 'aryl'.

In the compounds of the present disclosure, there are asymmetric centers. These centers are marked with the symbols "R" or "S", depending on the configuration of the substituents around the chiral carbon atom. It is to be understood that the disclosure includes all stereochemically isomeric forms, or mixtures thereof, which have the ability to inhibit NS5A. The individual stereoisomers of the compound can be prepared artificially from commercially available starting materials containing chiral centers or by preparing mixtures of enantiomeric products followed by separation, such as conversion to a mixture of diastereoisomers, followed by separation or recrystallization, chromatographic techniques or direct separation of enantiomers on chiral chromatographic columns.

Certain compounds of the present disclosure may exist in various stable conformational forms that may be separated. Torsional asymmetry, resulting from the limited rotation around the asymmetric single bond, for example, due to steric hindrance or strain on the ring, may allow the separation of different conformers. The present disclosure includes each conformational isomer of these compounds and mixtures thereof.

The term "compounds of the present disclosure" and expressions taken together are meant to include a compound of Formula (I), and pharmaceutically acceptable enantiomers, diastereoisomers and salts thereof. Similarly, references to intermediates are intended to include their salts, if the context permits.

The compounds of the present disclosure may exist as pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" as used herein means the amphoteric salts or forms of the compounds of the present disclosure that are soluble in water or oil or dispersible or which are, within a reasonable medical judgment, suitable for use in contact with the patient's tissues without excessive toxicity, irritation, allergic response or other problem or complications commensurate with a reasonable profit / risk ratio and are effective in their intended use. Salts can be prepared during the final isolation and purification of the compound or separately by reacting the appropriate nitrogen with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, citrate, aspartame, benzoate, benzoate, bisulfate, butyrate, camphorate, camphorsulphonate, digluconate, dihydrobromide, dihydrochloride, dihydrochloride, glycerophosphate, hemisulphate, heptanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, mesylatesulfonate, methanesulfonate, naphthalenesulphonate, nicotinate, 2-naphthalenesulphonate, oxalate, palmate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, frofluoroacetate, phosphate, glutamate, bicarbonate, para-toluenesulfonate and undecanoate. Examples of acids that can be used to form pharmaceutically acceptable addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric and phosphoric acids and organic acids,

Base addition salts may be prepared during the final isolation and purification of the compounds by reacting the carboxyl group with a suitable base such as a metal cation hydroxide, carbonate or bicarbonate or with ammonia or a primary, secondary or tertiary organic amine. Pharmaceutically acceptable salt cations include lithium, sodium, potassium, calcium, magnesium and aluminum, as well as non-toxic quaternary amine cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N, N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N, N-dibenzylphenotinylamine and N, N'-dibenzyltylenediamine. Other, representative organic amines,

Where it is possible that a therapeutically effective amount of a compound of formula (I), as well as a pharmaceutically acceptable salt thereof, for use in therapy can be administered as a chemical raw material, it is possible to present the active ingredient as a pharmaceutical composition. Such pharmaceutical compositions include therapeutically effective amounts of a compound of formula (I) or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients. The term "therapeutically effective amount" as used herein refers to the total amount of each active ingredient that is sufficient to show a significant patient benefit, e.g., reduction of viral titers. With regard to a single active ingredient administered alone, the expression applies only to this component. In relation to the combination, the expression refers to the combined amounts of active ingredients that produce a therapeutic effect, whether administered in combination, in series or simultaneously. The compound of formula (I) and its pharmaceutically acceptable salts are as described above. The carrier (s), diluent (s) or excipient (s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The method for preparing a pharmaceutical formulation comprises adding admixtures of a compound of formula (I) or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable carriers, diluents or excipients.

Pharmaceutical formulations may be in unit dosage form containing a predetermined amount of active ingredient per unit dose. Dosage levels of the compound of the present disclosure between about 0.01 and about 250 milligrams per kilogram ("mg / kg") of body weight per day, preferably between about 0.05 and about 100 mg / kg of body weight per day are typical in monotherapy for the prevention and treatment of diseases dependent on HCV. Typically, the pharmaceutical composition of the present disclosure will be administered from about 1 to about 5 times a day or alternatively as a continuous infusion. Such administration can be used as a chronic or acute treatment. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the condition being treated, the severity of the disease state, the time of administration, the route of administration, the rate of elimination of the compound employed, the duration of treatment, and the age, sex, weight and condition of the patient. Preferred unit dosage formulations are those that contain a daily dose or sub-dose of the active ingredient as set out above, or a suitable portion thereof. Treatment can be started at low doses, significantly less than the optimal dose of the compound. The dosage is then increased by small increments until the optimum effect under the given conditions is reached. In general, it is most desirable to administer the compound at a concentrated level that generally provides antivirally effective results without causing any harmful or adverse side effects. the rate of excretion of the compound employed, the duration of treatment, and the age, sex, weight and condition of the patient. Preferred unit dosage formulations are those that contain a daily dose or sub-dose of the active ingredient as set out above, or a suitable portion thereof. Treatment can be started at low doses, significantly less than the optimal dose of the compound. The dosage is then increased by small increments until the optimum effect under the given conditions is reached. In general, it is most desirable to administer the compound at a concentrated level that generally provides antivirally effective results without causing any harmful or adverse side effects. the rate of excretion of the compound employed, the duration of treatment, and the age, sex, weight and condition of the patient. Preferred unit dosage formulations are those that contain a daily dose or sub-dose of the active ingredient as set out above, or a suitable portion thereof. Treatment can be started at low doses, significantly less than the optimal dose of the compound. The dosage is then increased by small increments until the optimum effect under the given conditions is reached. In general, it is most desirable to administer the compound at a concentrated level that generally provides antivirally effective results without causing any harmful or adverse side effects. which contain a daily dose or sub-dose of the active ingredient, as set out above, or a suitable portion thereof. Treatment can be started at low doses, significantly less than the optimal dose of the compound. The dosage is then increased by small increments until the optimum effect under the given conditions is reached. In general, it is most desirable to administer the compound at a concentrated level that generally provides antivirally effective results without causing any harmful or adverse side effects. which contain a daily dose or sub-dose of the active ingredient, as set out above, or a suitable portion thereof. Treatment can be started at low doses, significantly less than the optimal dose of the compound. The dosage is then increased by small increments until the optimum effect under the given conditions is reached. In general, it is most desirable to administer the compound at a concentrated level that generally provides antivirally effective results without causing any harmful or adverse side effects.

When the compositions of the present disclosure comprise a combination of a compound of the present disclosure and one or more additional therapeutic or prophylactic agents, both the compound and the additive are usually present at dosage levels between about 10 to 150%, and more preferably between about 10 to 80%. dosing normally administered in the monotherapy dose method.

The pharmaceutical formulations may be adapted for administration by any appropriate route, for example, orally (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intradermal, intramuscular or intraarticular injection or infusion). , intrasystemic, homeostatic, intrathecal, focal, intravenous or intradermal). Such formulations may be prepared by any method known in the art of the pharmaceutical art, for example by bringing into association the active ingredient with a carrier (s) or excipient (s). Oral administration or administration by injection is preferred.

Pharmaceutical formulations adapted for oral administration may exist as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in the form of aqueous or non-aqueous liquids; edible marshmallows or mousses; or liquid oil-in-water emulsions or water-in-oil emulsions.

For example, for oral administration in the form of a tablet or capsule, the active drug component may be combined with an oral, non-toxic, pharmacologically acceptable, inert carrier such as ethanol, glycerol, water and the like. Powders are prepared by comminuting the compound to a suitably fine size and mixing with a similarly comminuted pharmaceutical carrier such as an edible carbohydrate, such as, for example, starch or mannitol. Flavor, preservative, dispersing and coloring agents may also be present.

Capsules are produced by making a powder mix as described above and filling formed gelatin sheaths. Before the filling operation, lubricating and lubricating agents such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene glycol can be added to the powder mixture. To improve the availability of the drug after ingestion of the capsule, disintegrating or dissolving agents such as agaragar, calcium carbonate or sodium carbonate can also be added.

Furthermore, if necessary or necessary, suitable binders, lubricants, disintegrants and dyes may also be incorporated into the mixture. Suitable binders include starch, gelatine, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, and the like. Lubricants used in these dosage forms include sodium oleate, sodium chloride, and the like. Disintegrating agents include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like. The tablets are formulated, for example, by preparing a powder mixture, granulating or kneading, adding a lubricant and disintegrant, and extruding into a tablet form. The powder mixture is prepared by mixing the compound, a suitable disintegrator with a diluent or a base as described above, and optionally with a binder such as carboxymethyl cellulose, alginate, gelatin or polyvinylpyrrolidone, a solution retarding substance such as paraffin, an absorption accelerator such as a quaternary salt and / or an absorption agent, such as bentonite, kaolin or dicalcium phosphate. The powder mixture can be granulated by wetting with a binder such as syrup, starch paste, acacia adhesive or solutions of cellulosic or polymeric materials and through a sieve. As an alternative to granulating, the powder mixture can be passed through a tableting machine and the result is heterogeneously formed nuggets broken into granules. To prevent sticking to the tablet forming matrix, the granules can be coated with lubricants by the addition of stearic acid, a stearic salt, talc or mineral oil. Then, the mixture covered with a lubricant is compressed into tablets. The compounds of the present disclosure can also be combined with a readily flowing inert carrier and compressed into tablets directly, without going through granulation or kneading steps. A transparent or opaque coating may be provided, comprising a shellac sealing layer, a sugar coating or a polymeric material, and a wax-like shine coating. To distinguish different unit dosages, dyes can be added to these coatings. stearic salt, talc or mineral oil. Then, the mixture covered with a lubricant is compressed into tablets. The compounds of the present disclosure can also be combined with a readily flowing inert carrier and compressed into tablets directly, without going through granulation or kneading steps. A transparent or opaque coating may be provided, comprising a shellac sealing layer, a sugar coating or a polymeric material, and a wax-like shine coating. To distinguish different unit dosages, dyes can be added to these coatings. stearic salt, talc or mineral oil. Then, the mixture covered with a lubricant is compressed into tablets. The compounds of the present disclosure can also be combined with a readily flowing inert carrier and compressed into tablets directly, without going through granulation or kneading steps. A transparent or opaque coating may be provided, comprising a shellac sealing layer, a sugar coating or a polymeric material, and a wax-like shine coating. To distinguish different unit dosages, dyes can be added to these coatings. without going through the granulation or kneading steps. A transparent or opaque coating may be provided, comprising a shellac sealing layer, a sugar coating or a polymeric material, and a wax-like shine coating. To distinguish different unit dosages, dyes can be added to these coatings. without going through the granulation or kneading steps. A transparent or opaque coating may be provided, comprising a shellac sealing layer, a sugar coating or a polymeric material, and a wax-like shine coating. To distinguish different unit dosages, dyes can be added to these coatings.

Oral fluids such as solutions, syrups and elixirs may be prepared in unit dosage form such that the given size contains a specific amount of the compound. Syrups can be prepared by dissolving the compound in a suitable flavored aqueous solution, while the elixirs are made by using a non-toxic carrier. It is also possible to add solubilizing agents and emulsifiers, such as ethoxylated isostearyl alcohols and polyoxyethylene sorbitol esters, preservatives, flavors such as peppermint oil or natural sweeteners or saccharin or other artificial sweeteners and the like.

Where appropriate, unit dosage formulations for oral administration may be microencapsulated. The formulation may be made to prolong or sustain the release, for example, by coating or embedding the material in polymers, wax or the like.

The compound of formula (I), and its pharmaceutically acceptable salts, can also be administered in the form of liposomal delivery systems such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from various phospholipids, such as cholesterol, searylamine or phosphatidylcholines.

The compound of formula (I) and its pharmaceutically acceptable salts can also be delivered using monoclonal antibodies as specific carriers to which compound molecules are attached. The compound can also be combined with soluble polymers as targeted drug carriers. Such polymers may include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl methacrylamide phenol, polyhydroxyethylaspartamidophenol, or polyoxyethylene oxydopolysine substituted with palitoyl residues. In addition, the compound can be combined with a class of biodegradable polymers useful in controlled release of the drug, for example, polylactic acid, polyethersilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyranes,

Pharmaceutical formulations adapted for transdermal administration may be presented as separate patches to be in direct contact with the skin of the recipient for an extended period of time. For example, the active ingredient can be delivered from the patch by iontophoresis, as described generally in Pharmaceutical Research 1986, 3 (6), 318.

Pharmaceutical formulations adapted for topical administration may be formulated as ointments, creams, suspensions, liquids, powders, solutions, pastes, gels, spray liquids, aerosols or oils.

Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or enemas.

Pharmaceutical formulations adapted for nasal administration, where the carrier is in a solid form, include a coarse powder, with particle size, for example, in the range of 20 to 500 microns, administered by sniffing, i.e. by rapid inhalation through the nasal passages from a container with powder kept near nose. Formulations in which the carrier is a liquid, suitable for administration as a nasal nebulizer or nasal drops, include aqueous or oily solutions of the active ingredient.

Pharmaceutical formulations adapted for administration by inhalation include finely divided dusts or mists that can be generated by means of various types of aerosols delivering a metered dose under pressure, nebulizers or insufflators.

Pharmaceutical formulations adapted for vaginal administration may be present as pessaries, tampons, creams, gels, pastes, foams or in aerosol formulations.

Pharmaceutical formulations adapted for parenteral administration include sterile aqueous and non-aqueous injections, which may contain antioxidants, buffers, bacteriostats and solutes, providing the formulation with isotonicity to the blood of the recipient selected; and aqueous or non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be in unit dose containers or with multiple doses, e.g. airtight ampoules or vials, and may be stored in a lyophilized condition requiring only the addition of a sterile liquid vehicle, e.g. injection water, immediately before use. Solutions and suspensions prepared immediately prior to use can be made from sterile powders, granules and tablets.

It is to be understood that, in addition to the individual ingredients mentioned above, the formulations may contain other agents conventional in the art having reference to the type of formulation, e.g. those suitable for oral administration may include flavoring agents.

The term "patient" includes both humans and other mammals.

The term "treatment" refers to: (i) preventing the onset of a disease, disorder or condition in a patient who may be predisposed to the disease, disorder and / or condition, but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder or condition, i.e. arresting its development; and (iii) relieving the disease, disorder or condition, i.e., causing regression of the disease, disorder and / or condition.

The compounds of the present disclosure may also be administered with a cyclosporin, e.g. cyclosporin A. Cyclosporin A has been shown to have anti-HCV activity in clinical trials (Hepatology 2003, 38, 1282; Biochem. Biophys. Res. Commun. 2004, 313, 42; J. Gastroenterol 2003, 38, 567).

Table 1 below shows some illustrative examples of compounds that can be administered with the compounds of the present disclosure. The compounds of the present disclosure may be administered with other anti-HCV compounds in combination therapy, together or separately or by combining the compounds into a composition.

Table 1

<td>trade name</td><td>Physiological class</td><td>The type of inhibitor or target</td><td>The supplier company</td>

<td>NIM811</td><td></td><td>Cyclophilin Inhibitor</td><td>Novartis</td>

<td>Zadaxin</td><td></td><td>immunomodulator</td><td>Sciclone</td>

<td>Suvus</td><td></td><td>Methylene blue</td><td>Bioenvision</td>

<td>Actilon (CPG10101)</td><td></td><td>TLR9 agonist</td><td>Coley</td>

<td>Batabulin (T67)</td><td>antineoplastic</td><td>Β-tubulin inhibitor</td><td>Tularik Inc., Southern San Francisco, CA</td>

<td></td><td></td><td></td><td></td>

<td>ISIS 14803</td><td>antiviral</td><td>antisense</td><td>ISIS Pharmaceuticals Inc, Carlsbad, CA / Elan Phamaceuticals Inc., New York, NY</td>

<td>Summetrel</td><td>antiviral</td><td>antiviral</td><td>Endo Pharmaceuticals Holdings Inc., Chadds Ford, PA</td>

<td>GS-9132 (ACH-806)</td><td>antiviral</td><td>HCV inhibitor</td><td>Achillion / Gilead</td>

<td>Pyrrazopyrimidine compounds and salts from WO-2005047288</td><td>antiviral</td><td>HCV inhibitors</td><td>arrow Therapeutics Ltd.</td>

<td>May 26, 2005</td><td></td><td></td><td></td>

<td>levovirin</td><td>antiviral</td><td>IMPDH inhibitor</td><td>Ribapharm Inc., Costa Mesa, CA</td>

<td>Merimepodib (VX-497)</td><td>antiviral</td><td>IMPDH inhibitor</td><td>Vertex Pharmaceuticals Inc., Cambridge, MA</td>

<td>XTL-6865 (XTL-002)</td><td>antiviral</td><td>antibody monoclonal</td><td>XTL Biopharmaceuticals Ltd., Rehovot, Isreal</td>

<td>Telaprevir (VX-950, LY-570310)</td><td>antiviral</td><td>NS3 serine protease inhibitor</td><td>Vertex Pharmaceuticals Inc., Cambridge, MA / Eli Lilly and What. Inc., Indianapolis, ION</td>

<td>HCV-796</td><td>antiviral</td><td>Inhibitor of Replicase NS5B</td><td>Wyeth / ViroPharma</td>

<td>NM-283</td><td>antiviral</td><td>Inhibitor of Replicase NS5B</td><td>Idenix / Novartis</td>

<td>GL-59728</td><td>antiviral</td><td>Inhibitor of Replicase NS5B</td><td>Gene Labs / Novartis</td>

<td>GL-60667</td><td>antiviral</td><td>Inhibitor of Replicase NS5B</td><td>Gene Labs / Novartis</td>

<td>2'C MeA</td><td>antiviral</td><td>Inhibitor of Replicase NS5B</td><td>Gilead</td>

<td>PSI 6130</td><td>antiviral</td><td>Inhibitor of Replicase NS5B</td><td>Roche</td>

<td>R1626</td><td>antiviral</td><td>Inhibitor of Replicase NS5B</td><td>Roche</td>

<td>2'C Methyl-adenosine</td><td>antiviral</td><td>Inhibitor of Replicase NS5B</td><td>Merck</td>

<td>JTK-003</td><td>antiviral</td><td>RdRp inhibitor</td><td>Japan Tobacco Inc., Tokyo, Japan</td>

<td>levovirin</td><td>antiviral</td><td>ribavirin</td><td>ICN Pharmaceuticals, Costa Mesa, CA</td>

<td>ribavirin</td><td>antiviral</td><td>ribavirin</td><td>Schering-Plow Corporation, Kenilworth, NJ</td>

<td>viramidine</td><td>antiviral</td><td>Prolek of Ribavirin</td><td>Ribapharm Inc., Costa Mesa, CA</td>

<td>heptazyme</td><td>antiviral</td><td>ribozyme</td><td>Ribozyme Pharmaceuticals Inc., Boulder, CO</td>

<td>BILN-2061</td><td>antiviral</td><td>serine protease inhibitor</td><td>Boehringer Ingelheim Pharma</td>

<td></td><td></td><td></td><td>KG, Ingelheim, Germany</td>

<td>SCH 503034</td><td>antiviral</td><td>serine protease inhibitor</td><td>Schering Plow</td>

<td>Zadazim</td><td>immunomodulator</td><td>immunomodulator</td><td>SciClone Pharmaceuticals Inc., San Mateo, CA</td>

<td>Ceplene</td><td>Immunomodulalor</td><td>immunomodulator</td><td>Maxim Pharmaceuticals Inc., San Diego, CA</td>

<td>CellCept</td><td>Immunosupressa nt</td><td>immunosuppressant IgG HCV</td><td>F. Hoffmann- La Roche LTD, Basel, Switzerland</td>

<td>civacir</td><td>Immunosupressa nt</td><td>immunosuppressant IgG HCV</td><td>Nabi Biopharmaceuticals Inc., Boca Raton, FL</td>

<td>Albuferon-α</td><td>interferon</td><td>albumin IFN-a2b</td><td>Human Genome Sciences Inc., Rockville, MD</td>

<td>Infergen A</td><td>interferon</td><td>IFN alfacon-1</td><td>InlerMune Pharmaceuticals Inc., Brisbane, CA</td>

<td>Omega IFN</td><td>interferon</td><td>IFN-ω</td><td>Intarcia Therapeutics</td>

<td>IFN-β and EMZ701</td><td>interferon</td><td>IFN-β and EMZ701</td><td>Transition Therapeutics Inc., Ontario, Canada</td>

<td>Rebif</td><td>interferon</td><td>FN-βΙα</td><td>Serono, Geneva, Switzerland</td>

<td>Roferon A</td><td>interferon</td><td>IFN-a2a</td><td>F. Hoffmann- La Roche LTD, Basel, Switzerland</td>

<td>Intron A</td><td>interferon</td><td>IFN-a2b</td><td>Schering-Plow Corporation, Kenilworth, NJ</td>

<td>Intron A and Zadaxin</td><td>interferon</td><td>IFN? 2b /? 1-thymosin</td><td>RegeneRx Biopharmiceu ticals Inc., Belhesda, MD / SciClone Pharmaceuticals Inc, San Mateo, CA</td>

<td>Rebetron</td><td>interferon</td><td>IFN-a2b / ribavirin</td><td>Schering-Plow Corporation, Kenilworth, NJ</td>

<td>Actimmune®</td><td>interferon</td><td>INF-γ</td><td>InterMune Inc., Brisbane, CA</td>

<td>Interferon-β</td><td>interferon</td><td>Interferon-β-Ία</td><td>Serono</td>

<td>Multiferon</td><td>interferon</td><td>IFN long-acting</td><td>Viragen / Valentis</td>

<td>Wellferon</td><td>interferon</td><td>lymphoblastoid IFNani</td><td>GlaxoSmithkline plc, Uxbridge, UK</td>

<td>Oinniferon</td><td>interferon</td><td>natural IFN-α</td><td>Viragen Inc., Plantation, FL</td>

<td>Pegasys</td><td>interferon</td><td>PEGylated IFN-a2a</td><td>F. Hoffinann- La Roche LTD, Basel, Switzerland</td>

<td>Pegasys and Ceplene</td><td>interferon</td><td>PEGylated IFNa2a / immunomodulator</td><td>Maxim Phannaceuticals Inc., San Diego, CA</td>

<td>Pegasys and Rybavirin</td><td>interferon</td><td>PEGylated IFNa2a / ribavirin</td><td>F. Hoffmann- La Roche LTD, Basel, switzerland</td>

<td>PEG-Intron</td><td>interferon</td><td>PEGylated IFN-a2b</td><td>Schering-Plow Corporation, Kenilworth, NJ</td>

<td>PEG-Intron / Ribavirin</td><td>interferon</td><td>PEGylated IFNa2b / ribavirin</td><td>Schering-Plow Corporation, Kenilworth, NJ</td>

<td>IP-501</td><td>Liver protection</td><td>antithrombotic</td><td>Indevus Phannaceuticals Inc., Lexington, MA</td>

<td>IDN-6556</td><td>Liver protection</td><td>caspase inhibitor</td><td>Idun Pharmaceuticals Inc., San Diego, CA</td>

<td>ITMN-191 (R-7227)</td><td>antiviral</td><td>serine protease inhibitor</td><td>InterMune Pharmaceuticals Inc., Brisbane, CA</td>

<td>GL-59728</td><td>antiviral</td><td>Inhibitor of Replicase NS5B</td><td>Genelabs</td>

<td>ANA-971</td><td>antiviral</td><td>TLR-7 agonist</td><td>Anadys</td>

The compounds of the present disclosure may be used as laboratory reagents. The compounds can be helpful in providing research tools for the design of tests for viral replication assays, validation of animal systems of research and structural biology research, to further increase knowledge about the mechanisms of HCV diseases. In addition, the compounds of the present disclosure are useful in establishing or determining binding sites of other antiviral compounds, for example, by competitive inhibition.

The compounds of the present disclosure may also be used to treat or prevent virus infections of materials, reducing the risk of viral infection of laboratory or medical personnel or patients who have been in contact with such materials, e.g., blood, tissue, instruments and surgical clothing, laboratory tools and clothing, blood donation or transfusion apparatus and materials.

The disclosure is intended to include a compound of formula (I) when produced by synthetic processes or metabolic processes, including those occurring in the human or animal body (in vivo) or in vitro processes.

The abbreviations used in the application, including especially those in the following illustrative schemes and examples, are well known to those skilled in the art. Some of the abbreviations used are as follows: HATU for O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate; Boc or BOC for tert-butoxycarbonyl; NBS for N-bromosuccinimide; tBu or t-Bu for tert-butyl; SEM for (trimethylsilyl) ethoxymethyl; DMSO for dimethylsulfoxide; MeOH for methanol; TFA for trifluoroacetic acid; RT for room temperature or retention time (as will the context); tR for the retention time; EDCI for 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; DMAP for 4-dimethylaminopyridine; THF for tetrahydrofuran; DBU for 1,8-diazabicyclo [5.4.0] undec-7-ene; t-Bu; DEA for diethylamine; HMDS for hexamethyldisilazide; DMF for N, N-dimethylformamide; Bzl of benzyl; EtOH for ethanol; iPrOH or i-PrOH for isopropanol; Me2S for dimethyl sulphide; Et3N or TEA for triethylamine; Ph for phenyl; OAc for acetate; EtOAc for ethyl acetate; dppf for 1,1'-bis (diphenylphosphino) ferrocene; iPr2EtN or DIPEA for diisopropylethylamine; Cbz for carbobenzyloxy; n-BuLi for n-butyllithium; ACN for acetonitrile; h or h for hours; m or min for minutes; s for seconds; LiHMDS for lithium hexamethyldisilazide; DIBAL for diisobutylaluminum hydride; TBDMSCl for tert-butyldimethylsilyl chloride; Me for methyl; approx. for about; OAc for acetate; iPr for isopropyl; Et for ethyl; Bn for benzyl; and HOAT for 1-hydroxy-7-azabenzotriazole. bis (diphenylphosphino) ferrocene; iPr2EtN or DIPEA for diisopropylethylamine; Cbz for carbobenzyloxy; n-BuLi for n-butyllithium; ACN for acetonitrile; h or h for hours; m or min for minutes; s for seconds; LiHMDS for lithium hexamethyldisilazide; DIBAL for diisobutylaluminum hydride; TBDMSCl for tert-butyldimethylsilyl chloride; Me for methyl; approx. for about; OAc for acetate; iPr for isopropyl; Et for ethyl; Bn for benzyl; and HOAT for 1-hydroxy-7-azabenzotriazole. bis (diphenylphosphino) ferrocene; iPr2EtN or DIPEA for diisopropylethylamine; Cbz for carbobenzyloxy; n-BuLi for n-butyllithium; ACN for acetonitrile; h or h for hours; m or min for minutes; s for seconds; LiHMDS for lithium hexamethyldisilazide; DIBAL for diisobutylaluminum hydride; TBDMSCl for tert-butyldimethylsilyl chloride; Me for methyl; approx. for about; OAc for acetate; iPr for isopropyl; Et for ethyl; Bn for benzyl; and HOAT for 1-hydroxy-7-azabenzotriazole. iPr for isopropyl; Et for ethyl; Bn for benzyl; and HOAT for 1-hydroxy-7-azabenzotriazole. iPr for isopropyl; Et for ethyl; Bn for benzyl; and HOAT for 1-hydroxy-7-azabenzotriazole.

The abbreviations used in the application, including especially those in the following illustrative schemes and examples, are well known to those skilled in the art.

EXAMPLES

The present disclosure will now be described in connection with specific embodiments that are not intended to limit its scope. Thus, the following examples, including specific embodiments, will illustrate practicing the disclosure, it being understood that the examples are for illustrative purposes of individual embodiments and are presented to provide what is considered the most useful and broadly understood description of its procedures and aspects Framework.

The percentages of the solution express the mass-to-volume ratio, and the solution ratios are volume-to-volume, unless otherwise stated. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 300, 400, or 500 MHz spectrometer; chemical shifts (δ) were recorded in parts per million. Flash chromatography was performed on silica gel (SiO2) according to Still's flash chromatography technique (J. Org. Chem. 1978, 43, 2923).

The evaluation of purity and mass analysis in low resolution was carried out in the Shimadzu LC system, related to the Waters Micromass ZQ MS system. It should be noted that retention times may vary slightly between devices. The LC conditions used in determining the retention time (RT) were:

Condition 1

<td>Column</td><td>= Phenomenex-Luna 3.0X 50 mm S10</td>

<td>Initial% B</td><td>= 0</td>

<td>Final% B</td><td>= 100</td>

<td>Gradient time = 2 min</td><td></td>

<td>Stop time</td><td>= 3 min</td>

<td>Flow Rate</td><td>= 4 mL / min</td>

<td>Wavelength</td><td>= 220 nm</td>

<td>Solvent A</td><td>= 0.1% TFA in 10% methanol / 90% H2O</td>

<td>Solvent B</td><td>= 0.1% TFA in 90% methanol / 10% H2O</td>

<td></td><td>Condition 2</td>

<td>Column</td><td>= Phenomenex-Luna 4.6X50 mm S10</td>

<td>Initial% B</td><td>= 0</td>

<td>Final% B</td><td>= 100</td>

<td>Gradient time = 2 min</td><td></td>

<td>Stop time</td><td>= 3 min</td>

Flow Rate = 5 mL / min

Wavelength = 220 nm

Solvent A = 0.1% TFA in 10% methanol / 90% H2O

Solvent B = 0.1% TFA in 90% methanol / 10% H2O

Condition 3

Column = HPLC XTERRA C18 3.0 x 50mm S7

Initial% B

Final% B = 100

Gradient time = 3 min

Stop time = 4 min

Flow Rate = 4 mL / min

Wavelength = 220 nm

Solvent A = 0.1% TFA in 10% methanol / 90% H2O

Solvent B = 0.1% TFA in 90% methanol / 10% H2O

Method A: LCMS - Xterra MS C-18 3.0 x 50mm, 0 to 100% B for 30.0 minutes gradient, 1 minute hold, A = 5% acetonitrile, 95% water, 10mm ammonium acetate, B = 95% acetonitrile, 5% water, 10mm ammonium acetate.

Method B: HPLC - X - Terra C-18 4.6 x 50mm, 0 to 100% B for 10.0 minutes gradient, 1 minute hold, A = 10% water, 90% methanol, 0.1% TFA, B = 90% water, 10 % methanol, 0.1% TFA

Method C: HPLC - YMC C-18 4.6 x 50mm, 0 to 100% B for 10.0 minutes gradient, 1 minute hold, A = 10% methanol, 90% water, 0.2% H3PO4, B = 90% methanol, 10% water 0.2% H3PO4.

Method D: HPLC - Phenomenex C-18 4.6 x 150mm, 0 to 100% B for 10.0 minutes gradient, 1 minute hold, A = 10% methanol, 90% water, 0.2% H3PO4, B = 90% methanol, 10% water 0.2% H3PO4

Method E: LCMS - Gemini C-18 4.6 x 50mm, 0 to 100% B for 10.0 minutes gradient, 1 minute hold, A = 5% acetonitrile, 95% water, 10mm ammonium acetate, B = 95% acetonitrile, 5% water 10mm ammonium acetate.

Method F: LCMS - Luna C-18 3.0 x 50mm, 0 to 100% B for 7.0 minutes gradient, 1 minute hold, A = 5% acetonitrile, 95% water, 10mm ammonium acetate, B = 95% acetonitrile, 5% water 10mm ammonium acetate.

Example 1 (Reference Example) (1R, 1'R) -2,2 '- (4,4'-biphenyl diylbis (1H-imidazol-5,2-diyl (2S) -2,2-pyrrolidinyl)) bis (N , N-dimethyl-2-oxo-1-fenyloetanamina)

<img file="PL2784075T3_D0002.tif" />

Example 1, Step a

<img file="PL2784075T3_D0003.tif" />

N, N-Diisopropylethylamine (18 mL, 103.3 mmol) was added dropwise over 15 minutes to a heterogeneous mixture of N-Boc-L-proline (7.139 g, 33.17 mmol), HATU (13.324 g, 35.04 mmol), HCl 2 salt. amino-1- (4-bromophenyl) ethanone (8.127 g, 32.44 mmol), and DMF (105 mL), and stirred at ambient conditions for 55 minutes. Most of the volatile component was removed in vacuo, and the obtained residue was partitioned between ethyl acetate (300 mL) and water (200 mL). The organic layer was washed with water (200 mL) and brine, dried (MgSO4), filtered and concentrated in vacuo. Silica gel was obtained from the residue and subjected to flash chromatography (silica gel, 5060% ethyl acetate / hexanes) to provide ketoamid 1a as a white solid (12.8 g).¹H NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): δ 8.25-8.1 (m, 1H), 7.92 (br d, J = 8.0, 2H), 7.75 (br d, J = 8.6, 2H), 4.61 (dd, J = 18.3, 5.7, 1H), 4.53 (dd, J = 18.1, 5.6, 1H), 4.22-4.12 (m, 1H), 3.43-3.3 (m, 1H), 3.30-3.23 ( m, 1H), 2.18-2.20 (m, 1H), 1.90-1.70 (m, 3H), 1.40 / 1.34 (two app br s, 9H). LC (Condition 1): RT = 1.70 min; LC / MS: Anal. Calc. for [M + Na]⁺ C18H23BrN2NaO4: 433.07; found 433.09.

Example 1, Step b

<img file="PL2784075T3_D0004.tif" />

A mixture of ketoamide 1a (12.8 g, 31.12 mmol) and NH4OAc (12.0 g, 155.7 mmol) in xylenes (155 mL) was heated in a sealed tube at 140 ° C for 2 hours. The volatile component was removed in vacuo, and the residue was cautiously partitioned between ethyl acetate and water, with sufficiently saturated NaHCO3 solution added to make the pH of the aqueous phase slightly basic after shaking in a two-phase system. The layers were separated, and the aqueous layer was extracted with additional ethyl acetate. The combined organic phases were washed with brine, dried (MgSO4), filtered and concentrated in vacuo. The obtained material was recrystallized from ethyl acetate / hexanes to provide two projections of imidazole 1b as a light yellow thick solid, weighing 5.85 g. The mother liquor was concentrated in vacuo and subjected to flash chromatography (silica gel;¹H NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): δ 12.17 / 11.92 / 11.86 (m, 1H), 7.72-7.46 / 7.28 (m, 5H), 4.86-4.70 (m, 1H), 3.52 ( app br s, 1H), 3.36 (m, 1H), 2.30-1.75 (m, 4H), 1.40 / 1.15 (app br s, 9H). LC (Condition 1): RT = 1.71 min; &gt; 98% homogeneity index; LC / MS: Anal. Calc. for [M + H]⁺ C18H23BrN3O2: 392.10; found 391.96; HRMS: Anal. Calc. for [M + H]⁺ C 18 H 23 BrN 3 O 2: 392.0974; found 392.0959

The optical purity of both samples 1b was estimated using the chiral HPLC conditions indicated below (ee&gt; 99% for combined projections, ee = 96.7% for a sample from a flash chromatography column):

Column: Chiralpak AD, 10 μm, 4.6 x 50 mm 5 Solvent: 2% ethanol / heptane (isocratic)

Flow rate: 1 mL / min

Wavelength: either 220 or 254 nm

Relative retention time: 2.83 minutes (R), 5.34 minutes (S)

Example 1, Step c

<img file="PL2784075T3_D0005.tif" />

Pd (Ph3P) 4 (469 mg, 0.406 mmol) was added to a pressure tube containing a mixture of bromide 1b (4.008 g, 10.22 mmol), bis (pinacolane) diborane (5.422 g, 21.35 mmol), potassium acetate (2.573g, 26.21 mmol) and 1,4-dioxane (80 mL). The reaction flask was purged with nitrogen, closed and heated in an oil bath at 80 ° C for 16.5 hours. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The crude material was cautiously partitioned between CH2Cl2 (150 mL) and an aqueous medium (50 mL of water + 10 mL of saturated NaHCO3 solution). The aqueous layer was extracted with CH2Cl2, and the combined organic phases were dried (MgSO4), filtered and concentrated in vacuo. The obtained material was purified by flash chromatography (the sample was applied with a wash solvent, 20-35% ethyl acetate / CH2Cl2) to provide boronate 1c, contaminated with pinacol, as an off-white thick solid; the relative molar ratio of 1c to pinacol was about 10: 1 (¹H NMR). The sample weighed 3.925 g after -2.5 days of exposure to high vacuum.¹H NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 12.22 / 11.94 / 11.87 (m, 1H), 7.79-7.50 / 7.34- 7.27 (m, 5H), 4.86-4.70 (m, 1H), 3.52 (app br s, 1 H), 3.36 (m, 1H), 2.27-1.77 (m, 4H), 1.45-1.10 (m, 21H). LC (Condition 1): RT = 1.64 min;

LC / MS: Anal. Calc. for [M + H]⁺ C 24 H 15 BN 3 O 4: 440.27; found 440.23.

Example 1, step d (2S, 2'S) -2,2 '- (4,4'-biphenyl diylbis (1H-imidazol-5, 2-diyl)) di (1-pyrrolidinecarboxylate) di-tert-butyl

<img file="PL2784075T3_D0006.tif" />

Pd (Ph3P) 4 (59.9 mg, 0.0518 mmol) was added to a mixture of bromide 1b (576.1 mg, 1.469 mmol), boronate 1c (621.8 mg, 1.415 mmol), NaHCO3 (400.4 mg, 4.766 mmol) in 1,2-dimethoxyethane. (12 mL) and water (4 mL). The reaction mixture was flushed with nitrogen, heated in an oil bath at 80 ° C for 5.75 hours, and then the volatile component was removed in vacuo. The residue was partitioned between 20% methanol / CHCl 3 (60 mL) and water (30 mL), and the aqueous phase was extracted with 20% methanol / CHCl 3 (30 mL). The combined organic phases were washed with brine, dried (MgSO4), filtered and concentrated in vacuo. Silica gel mesh was obtained from the obtained crude material and subjected to flash chromatography (ethyl acetate) to give dimide 1d, contaminated with PH3PO, as an off-white solid (563 mg).¹H NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): δ 12.21-12- 16 / 11.95-11.78 (m, 2H), 7.85-7.48 / 7.32-7.25 (m, 10H), 4.90-4.71 (m , 2H), 3.60-3.32 (m, 4H), 2.30-1.79 (m, 8H), 1.46-1.10 (m, 18H). LC (Condition 1b): RT = 1.77 min; LC / MS: Anal. Calc. for [M + H]⁺ C36H45BN6O4: 625.35; found 625.48.

Example 1, Step e

5,5 '- (4,4'-bifenyldiylo) bis (2 - ((2S) -2-pyrrolidinyl) -1H-imidazole)

<img file="PL2784075T3_D0007.tif" />

A mixture of carbamate 1d (560 mg) and 25% TFA / CH 2 Cl 2 (9.0 mL) was stirred at ambient conditions for 3.2 hours. The volatile component was removed in vacuo, and the obtained material was converted to the free base using a MCX column (methanol washing, 2.0 M NH3 leaching / methanol) to provide pyrrolidine 1e as a matte yellow solid (340 mg).¹H NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): δ 11.83 (br s, 2H), 7.80 (d, J = 8.1, 4H), 7.66 (d, J = 8.3, 4H), 7.46 (br s, 2H), 4.16 (app t, J = 7.2, 2H), 2.99- 2.69 (m, 6H), 2.09-2.00 (m, 2H), 1.94-1.66 (m, 6H). LC (Condition 1): RT = 1.27 min; &gt; 98% homogeneity index; LC / MS: Anal. Calc. for [M + H]⁺ C26H29N6: 425.25; found 425.25; HRMS: Anal. Calc. for [M + H]⁺ C26H29N6: 425.2454; found 425.2448

Alternative Synthesis of Example 1, Step e

5,5 '- (4,4'-bifenyldiylo) bis (2 - ((2S) -2-pyrrolidinyl) -1H-imidazole)

<img file="PL2784075T3_D0008.tif" />

Example A-1e-1

<img file="PL2784075T3_D0009.tif" />

An L, 3-necked round bottom flask, equipped with a nitrogen line, stirrer and thermocouple, was charged with 20 g (83.9 mmol, 1 equiv.) Of 1,1 '- (biphenyl-4,4'-diyl) diethanone, 200 mL of CH 2 Cl 2 and 8.7 mL (27.1g, 169.3 mmol, 2.02 equivalent) bromine. The mixture was allowed to stir under nitrogen for about 20 h at ambient conditions. 200 mL of CH 2 Cl 2 was added to the resulting suspension and concentrated to about 150 mL by vacuum distillation. The solvent was then exchanged in the suspension

THF to a target volume of 200 mL by vacuum distillation. The suspension was cooled to 20-25 ° C in 1 h and allowed to stir at 20-25 ° C for an additional hour. The off-white crystalline solids were filtered and washed with 150 mL of CH 2 Cl 2. The product was dried under vacuum at 60 ° C to provide 27.4 g (69.2 mmol, 82%) of the desired product:¹1 H NMR (400 MHz, CDCl 3) δ 7.95-7.85 (m, 4 H), 7.60-7.50 (m, 4 H), 4.26 (s, 4 H); ¹³C NMR (100 MHz, CDCl3) δ 191.0, 145.1, 133.8, 129.9, 127.9, 30.8; IR (KBr, cm -1) 3007, 2950, ​​1691, 1599, 1199; Anal. Calc. for C16H12Br2O2: C, 48.52; H, 3.05; Br, 40.34. Found: C, 48.53; H, 3.03; Br, 40.53. HRMS calcd for C 16 H 12 Br 2 O 2 (M + H; DCI⁺): 394.9282. Found: 394.9292. mp 224-226 ° C.

Example A-1e-2

<img file="PL2784075T3_D0010.tif" />

A 500 ml jacketed flask equipped with a nitrogen line, a thermocouple and a stirrer was charged with 20 g (50.5 mmol, 1 eq.) Of Example A-1e-1, 22.8 g (105.9 moles, 2.10 eq.) 1- (tert-butoxycarbonyl) -L -proline, and 200 mL acetonitrile. The suspension was cooled to 20 ° C, then 18.2 mL (13.5 g, 104.4 mmol, 2.07 eq.) DIPEA was added. The suspension was warmed to 25 ° C and allowed to stir for 3h. The resulting clear organic solution was washed with 3 x 100 mL 13% wt. NaCl. The solvent rich in acetonitrile solution was exchanged into toluene (target volume = 215 mL) by vacuum distillation until less than 0.5 vol% acetonitrile remained.

Example A-1e-3

<img file="PL2784075T3_D0011.tif" />

To the above toluene solution from Example A-1e-2 was added 78 g (1.011 mole, 20 eq) of ammonium acetate and heated to 95-100 ° C. The mixture was allowed to stir at 95-100 ° C for 15h. After completion of the reaction, the mixture was cooled to 70-80 ° C and 7 mL of acetic acid, 40 mL of n-butanol, and 80 mL of 5% volume were added. aqueous acetic acid. The resulting biphasic solution was divided by maintaining the temperature at&gt; 50 ° C. 80 mL of 5% v / v was added to the rich organic phase. aqueous acetic acid, 30 mL acetic acid and 20 mL n-butanol maintaining the temperature&gt; 50 ° C. The resulting three-phase solution was divided by keeping the temperature&gt; 50 ° C and the rich organic phase was washed with an additional 80 mL of 5 vol%. aqueous acetic acid. In the rich organic phase, the solvent was then exchanged into toluene to a final volume of 215 mL by vacuum distillation. Maintaining the temperature&gt; 60 ° C, 64 mL of MeOH was added. The resulting suspension was heated to 70-75 ° C and aged for 1h. The suspension was cooled to 20-25 ° C over 1 h and aged at this temperature for an additional hour. The suspension was filtered and the dough was washed with 200 mL of 10: 3 toluene: MeOH. The product was dried in vacuo at 70 ° C, yielding 19.8 g (31.7 mmol, 63%) of the desired product:¹H NMR (400 MHz, DMSO-d6) δ 13.00-11.00 (s, 2H), 7.90-7.75 (m, 4H), 7.75-7.60 (m, 4H), 7.60-7.30 (s, 2H), 4.92-4.72 (m, 2H), 3.65-3.49 (m, 2H), 3.49-3.28 (m, 2H), 2.39-2.1 (m, 2H), 2.10-1.87 (m, 6H), 1.60-1.33 (s, 8H), 1.33-1.07 (s, 10H); ¹³C NMR (100 MHz, DMSO-d6) δ 154.1, 153.8, 137.5, 126.6, 125.0, 78.9, 78.5, 55.6, 55.0, 47.0, 46.7, 33.7, 32.2, 28.5, 28.2, 24.2, 23.5; IR (KBr, cm-1) 2975, 2876, 1663, 1407, 1156, 1125; HRMS calcd for C36H45N6O4 (M + H, ESI⁺): 625.3502. Found: 625.3502. mp 190-195 ° C (decomposition).

Example A-1e-4

<img file="PL2784075T3_D0012.tif" />

To a 250 mL reactor equipped with a nitrogen line and an external stirrer, 25.0 g of Example A-1e-3 (40.01 mmol, 1 eq) and 250 mL of methanol and 32.85 mL (400.1 mmol, 10 eq) of 6M aqueous hydrogen chloride solution were added. The temperature was increased to 50 ° C and stirred at 50 ° C for 5h. The resulting suspension was cooled to 20-25 ° C and kept under stirring for ca. 18h. Filtration of the slurry gave a solid which was washed successively with 100 mL 90% methanol / water (WV) and 2x100 mL methanol. The wet cake was dried in a vacuum oven at 50 ° C overnight to give 18.12 g (31.8 mmol, 79.4%) of the desired product.

Recrystallization of Example A-1e-4

To a 250 mL reactor equipped with a nitrogen line and an overhead stirrer, 17.8 g of crude Example A-1e-4 was added followed by 72 mL of methanol. The resulting suspension was stirred at 50 ° C for 4h, cooled to 20-25 ° C and kept under stirring at 20-25 ° C for 1 h. Filtration of the slurry gave a crystalline solid which was washed with 60 mL of methanol. The resulting wet cake was dried in a vacuum oven at 50 ° C for 4 days to give 14.7 g (25.7 mmol, 82.6%) of the desired product:¹H NMR (400 MHz, DMSO-d6) δ 10.5-10.25 (br, 2H), 10.1-9.75 (br, 2H), 8.19 (s, 2H), 7.05 (d, J = 8.4, 4H), 7.92 (d , J = 8.5, 4H), 5.06 (m, 2H), 3.5-3.35 (m, 4H), 2.6-2. (M, 4H), 2.25-2.15 (m, 2H), 2.18-1.96 (m, 2H); ¹³C NMR (100 MHz, DMSO-d6) δ 156.6, 142.5, 139.3, 128.1, 127.5, 126.1, 116.9, 53.2, 45.8, 29.8, 24.3; IR (KBr, cm^-1) 3429, 2627, 1636, 1567, 1493, 1428, 1028. Anal. Calc. for C26H32N6Cl4: C, 54.75; H, 5.65; Cl, 24.86; Adjusted for 1.9% water: C, 53.71; H, 5.76; N, 14.46; Cl, 24.39. Found: C, 53.74; H, 5.72; N, 14.50; Cl, 24.49; KF = 1.9. mp 240 ° C (decomposition)

<img file="PL2784075T3_D0013.tif" />

(1R, 1'R) -2,2 '- (4,4'-biphenyl diylbis (1H-imidazol-5,2-diyl (2S) -2,2-pyrrolidinyl)) bis (N, N-dimethyl-2-oxo) -1-fenyloetanamina)

HATU (44.6 mg, 0.17 mmol) was added to the pyrrolidine 1e mixture (22.9 mg, 0.054 mmol), diisopropylethylamine (45 μL, 0.259 mmol) and Cap-1 (28.1 mg, 0.13 mmol) in DMF (1.5 mL), and the obtained the mixture was stirred at ambient temperature for 90 minutes. The volatile component was removed in vacuo, and the residue was first purified by MCX (methanol washing, 2.0 M NH 3 leaching / methanol) and then by reverse phase HPLC (H 2 O / methanol / TFA) to provide the TFA salt of Example 1 as off white foam (44.1 mg). ).¹H NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): δ 10.25 (br s, 2H), 8.20-7.10 (m, 20H), 5.79-5.12 (m, 4H), 4.05-2.98 (m, 4H). ), 2.98-2.62 (m, 6H), 2.50-1.70 (m, 14H), [Note: the imidazole signal NH was too wide to be assigned a chemical shift]; LC (Condition 1): RT = 1.40 min; &gt; 98% homogeneity index; LC / MS: Anal. Calc. for [M + H]⁺ C46H51N8O2: 747.41; found 747.58

Solvent B = 0.1% TFA in 90% methanol / 10% H2O

Example 2

<img file="PL2784075T3_D0014.tif" />

((1S) -1 - (((2S) -2- (5- (4 '- (2 - ((2S) -1 - ((2S) -2 - ((methoxycarbonyl) amino) -3-methylbutanoyl) -2-pyrrolidinyl) -1H-imidazol-5-yl) -4-biphenylyl) -1H-imidazol-2-yl) -1-pyrrolidinyl) carbonyl) methyl 2- (methylpropyl) carbamate

To a 50 mL flask equipped with a stirrer was added successively 2.5 mL of acetonitrile, 0.344 g (2.25 mmol, 2.5 eq.) Of hydroxybenzotriazole hydrate, 0.374 g (2.13 mmol, 2.4 eq.) N (methoxycarbonyl) -L-valine, 0.400 g (2.09 mmol). , 2.4 eq.) 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and an additional 2.5 mL of acetonitrile. The resulting solution was stirred at 20 ° C for 1 hour and 0.501 g (0.88 mmol, 1 eq.) Of Example A-1e-4 was added. The suspension was cooled to about 0 ° C and 0.45 g (3.48 mmol, 4 equiv.) Of diisopropylethylamine was added over 30 minutes while maintaining the temperature below 10 ° C. The solution was slowly warmed to 15 ° C over 3 hours and held at 15 ° C for 16 hours. The temperature was increased to 20 ° C and stirred for 3.25 hours. 3.3 g of 13 wt.% Was added to the solution obtained. aqueous NaCl and heated to 50 ° C for 1 hour. After cooling to 20 ° C, 2.5 mL of isopropyl acetate is added. The rich organic phase was washed with 2 x 6.9 g of a 0.5 N NaOH solution containing 13 wt. NaCl, then 3.3 g 13 wt% NaCl. The solvent was replaced with isopropyl acetate by vacuum distillation to a 10 mL target volume. The resulting cloudy solution was cooled to 20 ° C and filtered through a 0.45 μm filter. In a clear solution, the solvent was then exchanged into ethanol by vacuum distillation with a target volume of 3 mL. 1.67 mL (2.02 mmol, 2.3 eq.) 1.21 M HCl in ethanol was added. The mixture was then stirred at 25 ° C for 15 hours. The resulting suspension was filtered and the wet cake was washed with 2.5 mL of 2: 1 acetone: ethanol. The solids were dried in a vacuum oven at 50 ° C to give 0.550 g (0.68 mmol, 77%) of the desired product. 5 mL of isopropyl acetate was added. The rich organic phase was washed with 2 x 6.9 g of a 0.5 N NaOH solution containing 13 wt. NaCl, then 3.3 g 13 wt% NaCl. The solvent was replaced with isopropyl acetate by vacuum distillation to a 10 mL target volume. The resulting cloudy solution was cooled to 20 ° C and filtered through a 0.45 μm filter. In a clear solution, the solvent was then exchanged into ethanol by vacuum distillation with a target volume of 3 mL. 1.67 mL (2.02 mmol, 2.3 eq.) 1.21 M HCl in ethanol was added. The mixture was then stirred at 25 ° C for 15 hours. The resulting suspension was filtered and the wet cake was washed with 2.5 mL of 2: 1 acetone: ethanol. The solids were dried in a vacuum oven at 50 ° C to give 0.550 g (0.68 mmol, 77%) of the desired product. 5 mL of isopropyl acetate was added. The rich organic phase was washed with 2 x 6.9 g of a 0.5 N NaOH solution containing 13 wt. NaCl, then 3.3 g 13 wt% NaCl. The solvent was replaced with isopropyl acetate by vacuum distillation to a 10 mL target volume. The resulting cloudy solution was cooled to 20 ° C and filtered through a 0.45 μm filter. In a clear solution, the solvent was then exchanged into ethanol by vacuum distillation with a target volume of 3 mL. 1.67 mL (2.02 mmol, 2.3 eq.) 1.21 M HCl in ethanol was added. The mixture was then stirred at 25 ° C for 15 hours. The resulting suspension was filtered and the wet cake was washed with 2.5 mL of 2: 1 acetone: ethanol. The solids were dried in a vacuum oven at 50 ° C to give 0.550 g (0.68 mmol, 77%) of the desired product. 5 N NaOH solution containing 13 wt.% NaCl, then 3.3 g 13 wt% NaCl. The solvent was replaced with isopropyl acetate by vacuum distillation to a 10 mL target volume. The resulting cloudy solution was cooled to 20 ° C and filtered through a 0.45 μm filter. In a clear solution, the solvent was then exchanged into ethanol by vacuum distillation with a target volume of 3 mL. 1.67 mL (2.02 mmol, 2.3 eq.) 1.21 M HCl in ethanol was added. The mixture was then stirred at 25 ° C for 15 hours. The resulting suspension was filtered and the wet cake was washed with 2.5 mL of 2: 1 acetone: ethanol. The solids were dried in a vacuum oven at 50 ° C to give 0.550 g (0.68 mmol, 77%) of the desired product. 5 N NaOH solution containing 13 wt.% NaCl, then 3.3 g 13 wt% NaCl. The solvent was replaced with isopropyl acetate by vacuum distillation to a 10 mL target volume. The resulting cloudy solution was cooled to 20 ° C and filtered through a 0.45 μm filter. In a clear solution, the solvent was then exchanged into ethanol by vacuum distillation with a target volume of 3 mL. 1.67 mL (2.02 mmol, 2.3 eq.) 1.21 M HCl in ethanol was added. The mixture was then stirred at 25 ° C for 15 hours. The resulting suspension was filtered and the wet cake was washed with 2.5 mL of 2: 1 acetone: ethanol. The solids were dried in a vacuum oven at 50 ° C to give 0.550 g (0.68 mmol, 77%) of the desired product. The solvent was replaced with isopropyl acetate by vacuum distillation to a 10 mL target volume. The resulting cloudy solution was cooled to 20 ° C and filtered through a 0.45 μm filter. In a clear solution, the solvent was then exchanged into ethanol by vacuum distillation with a target volume of 3 mL. 1.67 mL (2.02 mmol, 2.3 eq.) 1.21 M HCl in ethanol was added. The mixture was then stirred at 25 ° C for 15 hours. The resulting suspension was filtered and the wet cake was washed with 2.5 mL of 2: 1 acetone: ethanol. The solids were dried in a vacuum oven at 50 ° C to give 0.550 g (0.68 mmol, 77%) of the desired product. The solvent was replaced with isopropyl acetate by vacuum distillation to a 10 mL target volume. The resulting cloudy solution was cooled to 20 ° C and filtered through a 0.45 μm filter. In a clear solution, the solvent was then exchanged into ethanol by vacuum distillation with a target volume of 3 mL. 1.67 mL (2.02 mmol, 2.3 eq.) 1.21 M HCl in ethanol was added. The mixture was then stirred at 25 ° C for 15 hours. The resulting suspension was filtered and the wet cake was washed with 2.5 mL of 2: 1 acetone: ethanol. The solids were dried in a vacuum oven at 50 ° C to give 0.550 g (0.68 mmol, 77%) of the desired product. In a clear solution, the solvent was then exchanged into ethanol by vacuum distillation with a target volume of 3 mL. 1.67 mL (2.02 mmol, 2.3 eq.) 1.21 M HCl in ethanol was added. The mixture was then stirred at 25 ° C for 15 hours. The resulting suspension was filtered and the wet cake was washed with 2.5 mL of 2: 1 acetone: ethanol. The solids were dried in a vacuum oven at 50 ° C to give 0.550 g (0.68 mmol, 77%) of the desired product. In a clear solution, the solvent was then exchanged into ethanol by vacuum distillation with a target volume of 3 mL. 1.67 mL (2.02 mmol, 2.3 eq.) 1.21 M HCl in ethanol was added. The mixture was then stirred at 25 ° C for 15 hours. The resulting suspension was filtered and the wet cake was washed with 2.5 mL of 2: 1 acetone: ethanol. The solids were dried in a vacuum oven at 50 ° C to give 0.550 g (0.68 mmol, 77%) of the desired product.

Recrystallization of the compound of Example 2

A solution of the compound of Example 2 obtained above was obtained by dissolving 0.520 g of the above product in 3.65 mL of methanol. 0.078 g of Cuno Zeta type 3 loose carbon was then added to the solution and allowed to stir for 0.25 hours. The mixture was then filtered and washed with 6 ml of methanol. The solution rich in the product was concentrated to 2.6 mL by vacuum distillation. 7.8 mL of acetone was added and allowed to stir at 25 ° C for 15 h. The solids were filtered, washed with 2.5 mL of 2: 1 acetone: ethanol and dried in a vacuum oven at 70 ° C to give 0.406 g (57.0%) of the desired product in the form white crystals:¹1 H NMR (400 MHz, DMSO-d6, 80 ° C): 8.02 (d, J = 8.34 Hz, 4 H), 7.97 (s, 2 H), 7.86 (d, J = 8.34 Hz, 4 H), 6.75 (s, 2 H), 5.27 (t, J = 6.44 Hz, 2 H), 4.17 (t, J = 6.95 Hz, 2 H), 3.97 - 4.11 (m, 2 H), 3.74 - 3.90 (m, 2) H), 3.57 (s, 6 H), 2.32 - 2.46 (m, 2 H), 2.09 - 2.31 (m, 6 H), 1.91-2.07 (m, 2 H), 0.88 (d, J = 6.57 Hz, 6H), 0.79 (d, J = 6.32Hz, 6H); ¹³C NMR (75 MHz, DMSOd6): δ 170.9, 156.9, 149.3, 139.1, 131.7, 127.1, 126.5, 125.9, 115.0, 57.9, 52.8, 51.5, 47.2, 31.1, 28.9, 24.9, 19.6, 17.7; IR (pure, cm^-1): 3385, 2971, 2873, 2669, 1731, 1650. Anal. Calc. for C40H52N8O6Cl2: C, 59.18; H, 6.45; N, 13.80; Cl, 8.73. Found C, 59.98; H, 6.80; N, 13.68; Cl, 8.77. mp 267 ° C (decomposition). Characteristic positions of diffraction peaks (degrees 2θ + - 0.1) @ RT, based on a high quality pattern collected using a diffractometer (CuKa) with a rotating capillary 2θ calibrated by NIST, other relevant standards are: 10.3, 12.4, 12.8, 13.3, 13.6, 15.5, 20.3, 21.2, 22.4, 22.7, 23.7.

BIOLOGICAL ACTIVITY

In the present disclosure, the HCV replicon assay was used, and was obtained, conducted and validated as described in our joint PCT / US2006 / 022197 and in O'Boyle et. al. Antimicrob Agents Chemother. 2005 Apr; 49 (4): 1346-53.

HCV 1b-377-neo replicon cells were used to study the series of compounds described herein, and cells resistant to compound A due to the Y2065H mutation in NS5A (described in PCT / US2006 / 022197). The test compounds were determined to have over 10-fold less inhibitory activity in Compound A-resistant cells than wild-type cells, indicating an associated mechanism of action between the two series of compounds. Thus, the compounds of the present disclosure may be effective in inhibiting the function of HCV NS5A protein and are understood to be as effective in combinations as the compounds described in previous PCT / US2006 / 022197 and jointly owned WO / O4014852. In addition, the compounds of the present disclosure may be effective against the HCV 1b genotype. It is also to be understood that the compounds of the present disclosure may inhibit many HCV genotypes. Table 2 shows the EC50 values ​​of the representative compounds of the present disclosure against the HCV 1b genotype. In one embodiment, the compounds of the present disclosure are active with respect to the genotypes 1a, 1b, 2a, 2b, 3a, 4a and 5a. The EC 50 ranges against HCV 1b are as follows: A = 1-10 μΜ; B = 100-999 nM; C = 1-99 nM; and D = 10-999 pM.

The compounds of the present disclosure may inhibit HCV through additional or non-NS5A inhibitory mechanisms. In one embodiment, the compounds of the present disclosure of inhibiting HCV replicon, and in another embodiment, compounds of the disclosure inhibit NS5A.

Table 2

<td>Example</td><td>Range</td>

<td>1</td><td>D</td>

<td>2</td><td>D</td>

It is desirable to consider the examples in all respects as illustrative and non-limiting, reference is made to the attached claims, not the above examples.

The compounds of the present disclosure may inhibit HCV through additional mechanisms or other than inhibition of NS5A. In one embodiment, compounds of the present disclosure inhibit NS5A. The compounds of the present disclosure may inhibit many HCV genotypes.

LEGAL-PATENT LAW "BELLEPAT"

Izabela Szych ulska-Hawranek ul. Słowackiego 44, 37-700 Prz. '' Ł ł 'tel (016) 7c2-37-77 fax: (016) .176-02-87 mobile (0608) 503-081 e -maii <a href="mailto:fcellepat@op.pl">fcellepat@op.pl</a> NIP: 795-207-16-72 REGON: 1803505 (6

Proxy:

<img file="PL2784075T3_D0015.tif" />