NZ617102B2 - Substituted aliphanes, cyclophanes, heteraphanes, heterophanes and hetero-heteraphanes useful for treating hcv infections - Google Patents

Abstract

The disclosure relates to substituted aliphanes, cyclophanes, heteraphanes, heterophanes and hetero-heteraphanes, of the formula shown in the abstract figure useful as antiviral agents. Certain substituted aliphanes, cyclophanes, heteraphanes, heterophanes and hetero-heteraphanes disclosed herein are potent and/ or selective inhibitors of viral replication, particularly Hepatitis C virus replication. Pharmaceutical compositions/ and combinations containing one or more substituted aliphanes, cyclophanes, heteraphanes, heterophanes and hetero-heteraphanes and a pharmaceutically acceptable carrier are also provided by this disclosure. Their use for treating viral infections, including Hepatitis C viral infections are provided by the disclosure. e potent and/ or selective inhibitors of viral replication, particularly Hepatitis C virus replication. Pharmaceutical compositions/ and combinations containing one or more substituted aliphanes, cyclophanes, heteraphanes, heterophanes and hetero-heteraphanes and a pharmaceutically acceptable carrier are also provided by this disclosure. Their use for treating viral infections, including Hepatitis C viral infections are provided by the disclosure.

Description

SUBSTITUTED NES, CYCLOPHANES, HETERAPHANES, HETEROPHANES AND HETERO-HETERAPHANES USEFUL FOR TREATING HCV INFECTIONS PRIORITY INFORMATION This application claims priority from US Provisional Application Nos. ,881 filed May 27, 2011, 61/504,905, filed July 6, 2011, and 61/567,216, filed er 6, 2011 all of which are hereby incorporated by reference in their entirety.

FIELD OF THE DISCLOSURE The present disclosure provides substituted aliphanes, cyclophanes, heteraphanes, heterophanes, hetero-heteraphanes and metallocenes, useful as antiviral .

Certain substituted aliphanes, cyclophanes, heteraphanes, heterophanes, hetero-heteraphanes and metallocenes disclosed herein are potent and/or selective inhibitors of viral replication, particularly Hepatitis C virus replication. Pharmaceutical compositions and combinations containing one or more substituted aliphanes, cyclophanes, heteraphanes, heterophanes, heteroheteraphanes and metallocenes and a pharmaceutically acceptable carrier are also provided by this disclosure. Methods for treating viral infections, including Hepatitis C viral infections are provided by the disclosure.

BACKGROUND An estimated 3% of the s population is infected with the hepatitis C virus.

Of those exposed to HCV, 80% to 85% become cally infected, at least 30% develop cirrhosis of the liver and 1-4% develop hepatocellular carcinoma. tis C Virus (HCV) is one of the most prevalent causes of chronic liver disease in the United States, reportedly accounting for about 15 percent of acute viral hepatitis, 60 to 70 percent of chronic tis, and up to 50 percent of cirrhosis, end-stage liver e, and liver cancer. c HCV infection is the most common cause of liver transplantation in the U.S., Australia, and most of Europe. Hepatitis C causes an ted 10,000 to 12,000 deaths annually in the United States.

While the acute phase of HCV infection is usually associated with mild symptoms, some evidence suggests that only about 15% to 20% of infected people will spontaneously clear HCV.

HCV is an enveloped, single—stranded RNA virus that contains a ve— stranded genome of about 9.6 kb. HCV is classified as a member of the Hepacivirus genus of the family iridae. At least 4 strains of HCV, GT—l — GT—4, have been characterized.

The HCV lifecycle includes entry into host cells; translation of the HCV genome, polyprotein processing, and replicase complex assembly; RNA replication, and virion assembly and release. Translation of the HCV RNA genome yields a more than 3000 amino acid long polyprotein that is processed by at least two cellular and two viral proteases.

The HCV polyprotein is: NH2-C-E1-E2-p7-NSZ—NS3—NS4A—NS4B—N85A—NS5B—COOH.

The cellular signal peptidase and signal peptide peptidase have been ed to be responsible for cleavage of the inal third of the polyprotein E2—p7) from the uctural proteins (NSZ—NS3—NS4A—NS4B—NS5A—NS5B). The NSZ—NS3 protease mediates a first cis cleavage at the NSZ—NS3 site. The NS3—NS4A protease then mediates a second cis—cleavage at the NS3—NS4A junction. The NS3—NS4A complex then cleaves at three downstream sites to separate the remaining nonstructural proteins. Accurate processing of the polyprotein is asserted to be essential for forming an active HCV replicase complex.

Once the polyprotein has been cleaved, the replicase complex comprising at least the NS3—NS5B nonstructural proteins assembles. The replicase complex is cytoplasmic and membrane—associated. Major enzymatic activities in the replicase complex include serine protease activity and NTPase helicase activity in N83, and pendent RNA polymerase activity of NS5B. In the RNA replication s, a complementary negative strand copy of the genomic RNA is produced. The negative strand copy is used as a template to synthesize additional positive strand c RNAs that may participate in translation, replication, packaging, or any combination thereof to produce progeny virus. Assembly of a functional replicase complex has been described as a component of the HCV replication ism.

US Provisional ation No. 60/669,872 “Pharmaceutical Compositions and Methods of Inhibiting HCV Replication” filed April 11, 2005, is hereby incorporated by reference in its entirety for its disclosure d to assembly of the replicase complex. t treatment of hepatitis C infection typically includes administration of an interferon, such as pegylated interferon (IFN), in combination with ribavirin. The success of current therapies as measured by sustained virologic response (SVR) depends on the strain of HCV with which the patient is infected and the patient’s adherence to the ent n. Only 50% of ts infected with HCV strain GT—l exhibit a sustained virological response. Direct acting ral agents such as ACH—l625, telaprevir, EMS—790052, and 0032 are in clinical development for treatment of chronic HCV. Due to lack of effective therapies for treatment for certain HCV strains and the high mutation rate of HCV, new therapies are needed. The present disclosure fulfills this need and es additional advantages, which are described herein.

SUMMARY tuted aliphanes, cyclophanes, heteraphanes, phanes, hetero— heteraphanes and metallocenes of Formula I are provided herein. The compounds of Formula I provided in this disclosure posses antiviral ty.

The disclosure provides compounds of Formula I that are potent and/ or selective inhibitors of Hepatitis C virus replication. Without being bound to any particular theory it is believed the t compounds are potent and selective inhibitors of HCV NSSa. ceutical compositions containing one or more compounds of Formula I, or a salt of such nds, and one or more pharmaceutically acceptable carriers are also provided herein. Pharmaceutical ations containing one or more compounds of Formula I, or a salt of such compounds, at least one additional active agent, and one or more ceutically acceptable carriers are also provided herein.

Also disclosed are methods of treating patients suffering from certain viral ions, particularly HCV infections, by providing to such patients an amount of a compound of Formula I effective to reduce signs or symptoms of the viral infection.

Methods of treatment include providing a compound of Formula I as a single active agent or providing a compound of Formula I in combination with one or more other therapeutic active agents.

In a first aspect the disclosure includes compounds of Formula I D—M—D (Formula I) or a pharmaceutically acceptable salt thereof.

Within Formula I, the variables D and M carry the ing definitions.

D is T—R— where M is covalently bound to R.

M is -P-A-P-.

P is —J—W— where J is covalently bound to R and W is covalently bound to A, P is —J— where J is covalently bound to R and A.

J is independently chosen at each occurrence and is J where i is an integer from 1 to 2.

T is independently chosen at each occurrence and is Tk where k is an integer from 1 to 2; T1 is —Y—Z, where Y is covalently bound to R and Y is a bond, C1—C4alkylene optionally substituted with oxo; and Z is a 5 or 6—membered cyclic group, each of which T1 is substituted with (i) at least one substituent selected from —(C=O)OH, — (C=O)NH2, —(C=O)H, —C1—C4alkoxy, C2—C4alkanoyl, C1—C4alkylester, C1—C4alkenylester, mono— or di—Cl—C4alkylcarboxamide and (ii) optionally substituted with one or more tuents independently chosen from halogen, hydroxyl, C1—C2alkyl, and C1—C2alkoxy; and T2 is independently chosen at each occurrence from C2—C6alkanoyl, C1—C6alkylester, C1— C6alkenylester, C1—C6alkylsulfonamide, C1—C6alkylsulfonyl, C2—C6alkanoyl tuted with mono— or C6hydrocarbylcarbamate, C2—C6alkanoyl substituted with urea or mono— or di— C1—C6alkylurea, and C2—C6alkanoyl substituted with mono— or di—C1—C6alkylcarboxamide, each of which T is optionally tuted with l or more substituents independently chosen from amino, cyano, yl, halogen, (C1—C4alkoxy)C0—C4alkyl, (mono— and di— C1— lamino)C0—C4alkyl, C1—C6alkyl, thioalkyl)C0—C4alkyl, C3—C7cycloalkyl, phenyl, C1—C4haloalkyl, and C1—C4haloalkoxy; R is independently chosen at each occurrence from (a) 4— to 6—membered rings containing one or two nitrogen atoms with remaining ring atoms being carbon, which R is saturated or contains 1 unsaturated bond and is optionally bridged with an methylene or ethylene bridge, fused to a phenyl or 5— to 6—membered heteroaryl ring; and (b) 6— to 10—membered fused or spiro bicyclic ring systems containing one or two nitrogen atoms with remaining ring atoms being carbon, which 6— to 10—membered bicyclic ring is saturated or contains 1 unsaturated bond; each R is optionally substituted with one or more substituents independently chosen from cyano, hydroxyl, halogen, C1— Czalkyl, lkoxy, C1—C2haloalkyl, C1—C2haloalkyl, aloalkylene, and C1— Czalkylsulfonyl.

J1 is phenyl or a 5— to 6—membered heteroaryl group containing 1 to 3 heteroatoms independently chosen from N, O, and S, where each J1 is optionally substituted with one or more substituents ndently chosen from amino, cyano, hydroxyl, halogen, C1—C4alkyl, C1—C4alkoxy, mono— and di— C1—C4alkylamino, C1—C2haloalkyl, and C1— Cghaloalkoxy.

J is an 8— to 10—membered heteroaryl group containing 1 to 4 heteroatoms independently chosen from N, O, and S, wherein J is optionally substituted with one or more substituents independently chosen from amino, cyano, hydroxyl, halogen, lkyl, C1— C4alkoxy, mono— and di— C1—C4alkylamino, C1—C2haloalkyl, and C1—C2haloalkoxy.

W is independently chosen at each occurrence and is a phenyl, pyridyl or l group, optionally tuted with one or more substituents independently chosen from amino, cyano, yl, halogen, C1—C4alkyl, C1—C4alkoxy, mono— and di— C1— C4alkylamino, aloalkyl, and C1—C2haloalkoxy.

A is [j.k]—cyclophane, hetera—phane, [j.k]—hetero—phane, [j.k]—hetero— —phane, or [j.k]—aliphane; where j is an integer from 1 to 4, k is an integer from 0 to 4, the difference between j and k is not more than 2, and each j and k linker optionally contains a heteroatom selected from N, O, and S, and is optionally substituted with l oxo group, and one or more tuents independently chosen from halogen, hydroxy, amino, C1—C2alkyl, and C1—C2alkoxy; or A is a [j.k.j’.k’]—cyclophane, where j, j’, k, and k’ are integers from 1 to 4, the difference between j and k or k’ is not more than 2, the difference between j’ and k or k’ is not more than 2, and each j, j’, k, and k’ linker optionally contains a heteroatom selected from N, O, and S, and is optionally substituted with l oxo group, and one or more substituents independently chosen from halogen, hydroxy, amino, C1—C2alkyl, and C1—C2alkoxy; ; or ©g ©‘i ('3 Q A is a group of the formula 2—@ or i—@ wherein Q is a neutral or cationic metal, each of which A is optionally substituted with one or more substituents independently chosen from halogen, C1—C2alkyl, and C1—C2alkoxy. In n embodiments Q is chosen from Fe, Co, Cr, Ni, V, Li, Rb, and K; or A is a group of the formula £63: which A is optionally substituted with one or more substituents independently chosen from halogen, C1—C2alkyl, and C1— Czalkoxy.

Pharmaceutical itions and combinations containing a compound of Formula I, together with a pharmaceutically acceptable carrier are provided by the disclosure.

The compositions and combinations provided by this disclosure may include a compound of Formula I as the only active agent or may e one ore more additional active agents. In certain embodiments the additional active agent is an NS3a protease inhibitor. This disclosure also includes a method of treating hepatitis C infection in a t, comprising providing a therapeutically effective amount of one or more compounds of Formula I to the patient. The compound of Formula I may be provided as the only active agent or may be provided er with one or more additional active agents such as an NS3a protease inhibitor. n compounds of Formula I disclosed herein exhibit good activity in an HCV replication assay, such as the HCV replicon assay set forth in Example 9, which follows. Preferred compounds of Formula I exhibit an EC50 of about 10 micromolar or less, or more preferably an EC50 of about 1 micromolar or less; or still more preferably an EC50 of about 100 nanomolar or less in an HCV replicon replication assay.

DETAILED DESCRIPTION CHEMICAL DESCRIPTION AND TERMINOLOGY Prior to setting forth the invention in detail, it may be l to provide definitions of certain terms to be used in this disclosure. Compounds are described using standard nomenclature. Unless defined otherwise, all cal and ific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention s. Unless clearly contraindicated by the context each compound name includes the free acid or free base form of the compound as well as all pharmaceutically acceptable salts of the compound.

The term “compounds of Formula I” encompasses all compounds that satisfy Formula I, including any enantiomers, racemates and stereoisomers, as well as all pharmaceutically acceptable salts of such compounds. The phrase “a nd of Formula I” includes all subgeneric groups of Formula I, and also includes pharmaceutically acceptable salts of a compound of Formula I, unless clearly contraindicated by the context in which this phrase is used.

“Formula II” encompasses all compounds that satisfy a II, including any enantiomers, racemates and stereoisomers, as well as all pharmaceutically acceptable salts of such compounds. The phrase “a compound of Formula II” includes all eric groups of a II, and also includes pharmaceutically acceptable salts of a compound of Formula II, unless clearly indicated by the context in which this phrase is used.

The terms “a” and “an” do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. The term “or” means “and/or”.

The open—ended transitional phrase “comprising” encompasses the intermediate tional phrase “consisting essentially of” and the close—ended phrase “consisting of.” Claims reciting one of these three transitional phrases, or with an alternate transitional phrase such as “containing” or “including” can be written with any other transitional phrase unless y precluded by the context or art. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are ed within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise ted herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No ge in the specification should be construed as indicating any non—claimed t as essential to the ce of the invention as used . Unless defined ise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

An “active agent” means a compound (including a compound disclosed herein), element, or mixture that when administered to a patient, alone or in combination with another compound, element, or mixture, confers, directly or indirectly, a physiological effect on the patient. The indirect physiological effect may occur via a metabolite or other indirect mechanism.

A dash ("—") that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, —(C=O)NH2 is ed through carbon of the keto (C20) group.

An ane” as used herein is a group composed of one or two cycloalkyl rings, with at least one aliphatic bridge between two non—adjacent positions of the single cycloalkyl ring or n the two cycloalkyl rings. The aliphatic bridges contain between 1 and 4 carbon atoms. The aliphatic bridges are optionally substituted with an oxo group.

“Alkanoyl” is an alkyl group as defined herein, ntly bound to the group it substitutes by a keto (—(C=O)—) bridge. Alkanoyl groups have the indicated number of carbon atoms, with the carbon of the keto group being ed in the numbered carbon atoms. For example a Cgalkanoyl group is an acetyl group having the a CH3(C=O)—.

“Alkyl” is a branched or straight chain saturated aliphatic hydrocarbon group, having the specified number of carbon atoms, generally from 1 to about 12 carbon atoms.

The term C1—C6alkyl as used herein indicates an alkyl group having from 1, 2, 3, 4, 5, or 6 carbon atoms. Other embodiments include alkyl groups having from 1 to 8 carbon atoms, 1 to 4 carbon atoms or 1 or 2 carbon atoms, e.g. lkyl, C1—C4alkyl, and C1—C2alkyl. When Co—Cn alkyl is used herein in conjunction with another group, for example, (C1—C4alkoxy)C0— C4 alkyl, the indicated group, in this case alkoxy, is either directly bound by a single covalent bond yl), or attached by an alkyl chain having the specified number of carbon atoms, in this case 1, 2, 3, or 4 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n—propyl, isopropyl, l, 3—methylbutyl, l, n—pentyl, and sec—pentyl.

“Alkynyl” is a branched or straight chain aliphatic hydrocarbon group having one or more triple carbon—carbon bonds that may occur at any stable point along the chain, having the specified number of carbon atoms. Examples of alkynyl include, but are not limited to, ethynyl and propynyl.

“Alkoxy” is an alkyl group as defined above with the indicated number of carbon atoms covalently bound to the group it substitutes by an oxygen bridge (—O—).

Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n—propoxy, i—propoxy, n—butoxy, 2—butoxy, t—butoxy, n—pentoxy, 2—pentoxy, 3— pentoxy, isopentoxy, neopentoxy, n— hexoxy, 2—hexoxy, 3—hexoxy, and 3— methylpentoxy.

The term ester” indicates an alkyl group as defined herein covalently bound to the group it substitutes by an ester linkage. The ester linkage may be in either orientation, e. g., a group of the formula —O(C=O)alkyl or a group of the formula — (C=O)Oalkyl.

“Alkylsulfonyl” is a group of the formula —SOZalkyl, where the alkyl group s the definition set forth .

“Cycloalkyl” is a saturated hydrocarbon ring group, having the specified number of carbon atoms. Monocyclic cycloalkyl groups typically have from 3 to about 8 carbon ring atoms or from 3 to 7 (3, 4, 5, 6, or 7) carbon ring atoms. Cycloalkyl substituents may be pendant from a substituted nitrogen or carbon atom, or a tuted carbon atom that may have two substituents may have a cycloalkyl group, which is attached as a spiro group. es of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and exyl.

A “cyclophane” as used herein is a group composed of one or two aromatic rings, usually benzene rings, with at least one aliphatic bridge between two non—adjacent positions of the single aromatic ring or between the two aromatic rings. The aliphatic s are in the meta or para or meta, para (meta on one aromatic ring and para on the other) orientation and contain between 1 and 4 carbon atoms. The aliphatic bridges are optionally substituted with an oxo group.

“Haloalkyl” indicates both branched and straight—chain alkyl groups having the specified number of carbon atoms, substituted with l or more halogen atoms, up to the maximum allowable number of n atoms. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2—fluoroethyl, and penta—fluoroethyl. lkoxy” indicates a kyl group as defined herein attached through an oxygen bridge (oxygen of an alcohol radical).

“Halo” or “halogen” indicates any of fluoro, chloro, bromo, and iodo.

“Heteroaryl” indicates a stable monocyclic aromatic ring haVing the indicated number of ring atoms which contains from 1 to 3, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon, or a stable bicyclic or tricyclic system containing at least one 5— to 7—membered ic ring which ns from 1 to 3, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon. clic aryl groups typically have from 5 to 7 ring atoms. In some embodiments bicyclic heteroaryl groups are 9— to 10— membered heteroaryl groups, that is, groups containing 9 or 10 ring atoms in which one 5— to 7—member aromatic ring is fused to a second aromatic or non—aromatic ring. When the total number of S and O atoms in the heteroaryl group exceeds 1, these heteroatoms are not adjacent to one another. It is preferred that the total number of S and O atoms in the heteroaryl group is not more than 2. It is particularly preferred that the total number of S and O atoms in the aromatic heterocycle is not more than 1. Examples of aryl groups include, but are not limited to, oxazolyl, pyranyl, pyrazinyl, pyrazolopyrimidinyl, pyrazolyl, pyridizinyl, pyridyl, pyrimidinyl, pyrrolyl, quinolinyl, tetrazolyl, thiazolyl, thienylpyrazolyl, thiophenyl, triazolyl, benzo[d]oxazolyl, benzofuranyl, benzothiazolyl, benzothiophenyl, benzoxadiazolyl, dihydrobenzodioxynyl, furanyl, imidazolyl, indolyl, and isoxazolyl.

“Heteroaryloxy” is a heteroaryl group as described bound to the group it substituted Via an oxygen bridge.

A “hetera—phane” as used herein is a group composed of one or two aromatic rings, y benzene rings, with at least one aliphatic bridge between two non—adjacent ons of the single aromatic ring or between the two ic rings that contains a heteroatom. The aliphatic bridges are in the meta or para or meta, para (meta on one ic ring and para on the other) orientation and contain between 1 and 4 atoms, at least one of which is a heteroatom with the remaining atoms being carbon atoms. The aliphatic bridges are optionally substituted with an oxo group.

A “hetero—phane” as used herein is a group composed of one or two ic rings, wherein at least one aromatic ring is heteroaryl, with at least one aliphatic bridge between two non—adjacent positions of the single aromatic ring or between the two aromatic rings. The aliphatic bridges are in the meta or para or meta, para (meta on one aromatic ring and para on the other) orientation and contain between 1 and 4 carbon atoms. The aliphatic s are optionally substituted with an oxo group.

A “hetero—hetera—phane” as used herein is a group composed of one or two aromatic rings, wherein at least one aromatic ring is heteroaryl, with at least one aliphatic bridge between two non—adjacent positions of the single aromatic ring or between the two aromatic rings that contains a hetero atom chosen from N, O, or S, and one aliphatic bridge between two non—adjacent positions of the single aromatic ring or n the two aromatic rings that does not contain a heteroatom. The aliphatic bridges are in the meta or para or meta, para (meta on one aromatic ring and para on the other) orientation and contain between 1 and 4 atoms, at least one of which is a heteroatom with the remaining atoms being carbon atoms. The aliphatic bridges are optionally substituted with an oxo group.

“Hydrocarbyl” is a saturated or unsaturated aliphatic group containing the ted number of carbon atoms. “Hydrocarbyl may be used in conjunction with other groups, such as carbamate, as in “mono— or di— hydrocarbylcarbamate.” Mono— or di— arbylcarbamate include groups of the formula (alkyll)NH(C=O)O— and (alkyll)N(alkylz)(C=O)O— as well as groups in which one or both of the alkyl groups are replaced by an arbon group containing unsaturated carbon—carbon bonds.

A “metallocene” is a compound consisting of two 5— or 6—membered ic carbocyclic groups bound to a metal center, where the metal is neutral or cationic. An example of metallocene includes, but is not limited to, ferrocene.

The term “mono— and/ or di—alkylamino” indicates secondary or tertiary alkyl amino groups, wherein the alkyl groups are ndently chosen alkyl groups, as defined herein, haVing the indicated number of carbon atoms. The point of ment of the mino group is on the en. es of mono— and di—alkylamino groups include ethylamino, dimethylamino, and methyl—propyl—amino.

“Mono— and/or di—alkylcarbamate” includes mono—alkylcarbamate groups of formula l)O(C=O)NH— or a dialkylcarboxamide groups of the formula (alkyll)O(C=O)N(alkylz)— in which the point of attachment of the mono— or dialkylcarboxamide substituent to the molecule it substitutes is on the nitrogen of the carbamate amino. The term “mono and/ or di—alkylcarbamate” also includes groups of the formula (alkyll)NH(C=O)O— and (alkyll)N(alkylz)(C=O)O— in which the carbamate is covalently bound to the group it tutes by its non—keto oxygen atom. The groups alkyll and alkylg are independently chosen alkyl groups, carrying the alkyl definition set forth in this disclosure and having the indicated number of carbon atoms.

“Mono— and/ or di—alkylcarboxamide” indicates a mono—alkylcarboxamide group of formula (alkyll)—NH—(C=O)— or a dialkylcarboxamide group of the formula (alkyll)(alky12)—N—(C=O)— in which the point of attachment of the mono— or lcarboxamide substituent to the molecule it substitutes is on the carbon of the carbonyl group. The term “mono and/ or ylcarboxamide” also includes groups of the formula (alkyll)(C=O)NH— and l)(C=O) (alky12)N— in which the point of ment is the nitrogen atom. The groups alkyll and alkylg are independently chosen alkyl groups having the indicated number of carbon atoms. Likewise “mono— and/or di—alkyl amide” is any of a mono—alkylsulfonamide group of formula (alkyll)—NH—(SOZ)—, a mono—alkylsulfonamide group of formula l)—(SOZ)—NH—, a dialkylsulfonamide group of the formula (alkyll)(alkylg)—N—(SOZ)—, and a group of the formula (alkyll)—(SOz)—(alkylg)N—.

“Thioalkyl” is an alkyl group as defined above with the indicated number of carbon atoms covalent bound to the group it substitutes by an sulfur bridge (—S—).

The term “substituted”, as used herein, means that any one or more hydrogens on the designated atom or group is replaced with a selection from the ted group, provided that the designated atom's normal valence is not exceeded. When the substituent is oxo (i.e., =0) then 2 hydrogens on the atom are replaced. When an oxo group substitutes aromatic moieties, the corresponding partially unsaturated ring replaces the aromatic ring.

For example a pyridyl group substituted by oxo is a pyridone. Combinations of substituents and/or variables are sible only if such combinations result in stable compounds or useful synthetic intermediates. A stable compound or stable structure is meant to imply a compound that is sufficiently robust to survive ion from a reaction mixture, and subsequent formulation into an effective therapeutic agent. Unless otherwise specified substituents are named into the core structure. For e, it is to be understood that when lkyl is listed as a possible substituent the point of attachment of this substituent to the core structure is in the alkyl portion.

Suitable groups that may be present on a “substituted” position include, but are not limited to, e.g., halogen; cyano; hydroxyl; nitro; azido; alkanoyl (such as a C2—C6 alkanoyl group); carboxamide; alkyl groups (including cycloalkyl groups) having 1 to about 8 carbon atoms, or 1 to about 6 carbon atoms; alkenyl and alkynyl groups including groups having one or more unsaturated linkages and from 2 to about 8, or 2 to about 6 carbon atoms; alkoxy groups having one or more oxygen linkages and from 1 to about 8, or from 1 to about 6 carbon atoms; aryloxy such as phenoxy; alkylthio groups including those having one or more thioether linkages and from 1 to about 8 carbon atoms, or from 1 to about 6 carbon atoms; alkylsulfinyl groups including those having one or more sulfinyl es and from 1 to about 8 carbon atoms, or from 1 to about 6 carbon atoms; alkylsulfonyl groups including those having one or more sulfonyl linkages and from 1 to about 8 carbon atoms, or from 1 to about 6 carbon atoms; aminoalkyl groups including groups having one or more N atoms and from 1 to about 8, or from 1 to about 6 carbon atoms; aryl having 6 or more carbons and one or more rings, (e.g., phenyl, biphenyl, naphthyl, or the like, each ring either tuted or unsubstituted aromatic); kyl having 1 to 3 separate or fused rings and from 6 to about 18 ring carbon atoms, with benzyl being an exemplary arylalkyl group; arylalkoxy having 1 to 3 separate or fused rings and from 6 to about 18 ring carbon atoms, with benzyloxy being an exemplary arylalkoxy group; or a saturated, unsaturated, or aromatic heterocyclic group having 1 to 3 separate or fused rings with 3 to about 8 members per ring and one or more N, O or S atoms, e.g. coumarinyl, quinolinyl, isoquinolinyl, quinazolinyl, pyridyl, nyl, pyrimidinyl, l, pyrrolyl, thienyl, thiazolyl, nyl, oxazolyl, isoxazolyl, imidazolyl, l, benzofuranyl, benzothiazolyl, tetrahydrofuranyl, ydropyranyl, piperidinyl, morpholinyl, piperazinyl, and pyrrolidinyl. Such heterocyclic groups may be further substituted, e.g. with hydroxy, alkyl, alkoxy, halogen and amino.

A “dosage form” means a unit of administration of an active agent. es of dosage forms include tablets, capsules, injections, suspensions, liquids, emulsions, creams, ointments, suppositories, inhalable forms, transdermal forms, and the like. aceutical compositions” are compositions comprising at least one active agent, such as a compound or salt of a I, and at least one other substance, such as a carrier. Pharmaceutical compositions al contain one or more additional ative agents. When specified, pharmaceutical compositions meet the U.S. FDA’s GMP (good manufacturing practice) standards for human or man drugs. “Pharmaceutical combinations” are combinations of at least two active agents which may be combined in a single dosage form or provided together in separate dosage forms with instructions that the active agents are to be used together to treat a disorder, such as hepatitis C.

“Pharmaceutically acceptable salts” includes derivatives of the disclosed compounds in which the parent compound is modified by making inorganic and organic, non— toxic, acid or base addition salts thereof. The salts of the present compounds can be synthesized from a parent compound that contains a basic or acidic moiety by tional chemical methods. lly, such salts can be prepared by reacting free acid forms of these nds with a iometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such ons are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, non— aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred, where practicable. Salts of the present compounds further include solvates of the compounds and of the compound salts. es of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as ; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non—toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non—toxic inorganic or organic acids. For example, conventional non—toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, , hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2—acetoxybenzoic, fumaric, toluenesulfonic, esulfonic, ethane disulfonic, oxalic, isethionic, HOOC—(CH2)n—COOH where n is 0—4, and the like. Lists of additional suitable salts may be found, e.g., in Remington '5 Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).

The term “carrier” applied to pharmaceutical compositions/ combinations of the invention refers to a diluent, excipient, or e with which an active compound is provided.

A “pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition/ combination that is generally safe, xic and neither biologically nor otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as human pharmaceutical use. A aceutically acceptable excipient” as used in the present application includes both one and more than one such excipient.

A “patient” is a human or non—human animal in need of medical treatment. l treatment can e treatment of an existing condition, such as a disease or WO 66716 disorder, prophylactic or preventative treatment, or diagnostic treatment. In some embodiments the patient is a human patient.

“Providing” means giving, administering, selling, distributing, transferring (for profit or not), cturing, compounding, or dispensing.

“Providing a compound of Formula I with at least one additional active agent” means the compound of Formula I and the additional active agent(s) are provided simultaneously in a single dosage form, provided concomitantly in separate dosage forms, or provided in separate dosage forms for stration separated by some amount of time that is within the time in which both the compound of a I and the at least one additional active agent are within the blood stream of a t. In certain embodiments the nd of Formula I and the additional active agent need not be prescribed for a patient by the same medical care worker. In certain embodiments the additional active agent or agents need not require a prescription. Administration of the compound of Formula I or the at least one additional active agent can occur via any appropriate route, for example, oral tablets, oral capsules, oral liquids, inhalation, injection, suppositories or topical contact.

“Treatment,” as used herein includes ing a compound of Formula I, either as the only active agent or together with at least one onal active agent sufficient to: (a) prevent a disease or a symptom of a disease from occurring in a patient who may be posed to the disease but has not yet been diagnosed as having it (e.g. including diseases that may be associated with or caused by a primary disease (as in liver fibrosis that can result in the context of chronic HCV infection); (b) inhibiting the disease, i.e. arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. “Treating” and “treatment” also means providing a therapeutically effective amount of a compound of Formula I, as the only active agent or together with at least one additional active agent to a patient having or susceptible to a hepatitis C ion.

A “therapeutically effective amount” of a pharmaceutical composition/ combination of this ion means an amount effective, when administered to a patient, to provide a therapeutic benefit such as an amelioration of symptoms, e.g., an amount effective to decrease the ms of a hepatitis C infection. For example a patient infected with a hepatitis C virus may present elevated levels of n liver enzymes, including AST and ALT. Normal levels of AST are from 5 to 40 units per liter of serum (the liquid part of the blood) and normal levels of ALT are from 7 to 56 units per liter of serum. A therapeutically effect amount is thus an amount sufficient to provide a significant reduction in ed AST and ALT levels or an amount sufficient to provide a return of AST and ALT levels to the 2012/039835 normal range. A therapeutically effective amount is also an amount sufficient to prevent a significant increase or significantly reduce the detectable level of virus or viral antibodies in the patient’s blood, serum, or tissues. One method of determining treatment efficacy includes measuring HCV RNA levels by a tional method for determining viral RNA levels such as the Roche TaqMan assay. In certain preferred embodiments treatment reduces HCV RNA levels below the limit of quantitation (30 IU/mL, as measured by the Roche (R) assay) or more preferably below the limit of detection (10 IU/mL, Roche ).

A significant increase or reduction in the detectable level of virus or viral antibodies is any detectable change that is statistically significant in a standard parametric test of statistical significance such as Student’s T—test, where p < 0.05.

CHEMICAL DESCRIPTION a I and Formula II (shown below) include all subformulae thereof. In certain situations, the compounds of Formula I or Formula II may n one or more asymmetric elements such as stereogenic centers, stereogenic axes and the like, e.g. asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms.

These compounds can be, for example, racemates or optically active forms. For compounds with two or more asymmetric elements, these nds can additionally be mixtures of diastereomers. For nds having asymmetric centers, it should be understood that all of the optical isomers and mixtures thereof are encompassed. In addition, compounds with carbon—carbon double bonds may occur in Z— and s, with all isomeric forms of the compounds being included in the present invention. In these situations, single enantiomers, i.e., optically active forms, can be obtained by asymmetric synthesis, synthesis from optically pure precursors, or by resolution of the racemates. Resolution of the racemates can also be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example using a chiral HPLC column.

Where a compound exists in s tautomeric forms, the invention is not limited to any one of the specific tautomers, but rather includes all tautomeric forms.

The t invention is intended to include all es of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium and isotopes of carbon e 11C, 13C, and 14C. n compounds are described herein using a general a that includes variables, e.g. D, M, A, P, J, and W. Unless otherwise specified, each variable within such a formula is defined independently of other variables. Thus, if a group is said to be tuted, e. g. with 0—2 R*, then the group may be substituted with up to two R”< groups and R”< at each occurrence is selected independently from the definition of R*. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

In addition the compounds and salts of Formula I discussed in the SUMMARY n, the disclosure includes nds and salt of Formula I, and ceutical compositions/ combinations of such compounds in which the variables meet any of the following conditions. (i) D—M—D is any of T—R—Jl—W—A—W—Jl—R—T; T—R—Jl—A—Jl—R—T; T—R—Jz—A—Jz—R—T; T—R—Jl—W—A—Jl—R—T; T—R—Jl—W—A—JZ—R—T; and T—R—Jl—A—Jz—R—T. (ii) Ais (iii) (iv) (v) Ais WO 66716 (Vi) Ais (Vii) Ais (Viii) Ais (ix) Ais (X) Ais! s i.

(Xi) A is (Xii) A is (xiii) A is (xiV) A is '3‘@7 (xv) At least one P is J1—W; and W is , optionally substituted with one or more substituents independently chosen from halogen, C1—C2alkyl, and C1—C2alkoxy. (xvi) P is J—W or J, wherein at least one J is J1 and J1 is (xvii) At least one P is J1 and J1 is “N02- 2 2 (xviii) At least one P is J and J is a benzimidazole group, optionally substituted with one or more substituents independently chosen from halogen, lkyl, and C1—C2alkoxy. (xix) R is independently chosen from :1 4} Q} o 2' 9‘ fiz— 5‘2— <L“>2— §2— 3&- N f! ’ .' [N152 EN)? $0” 2' ‘2‘ each of which is optionally substituted with one or more substituents independently chosen from halogen, C1—C4alkyl, and C1—C4alkoxy. (xx) R is independently chosen from ”in fiand (XXi) T is independently chosen C2—C6alkanoyl substituted with mono— and di— C1—C6alkylcarbamate, each of which T is optionally substituted with (C1—C4thioalkyl)C0— C4alkyl.

(XXii) Also included are nds and salts of the formula T—R—Jz—A—Jz—R—T.

A, in the formula T—R—Jz—A—Jz—R—T, is a group of the formula J is an 8— to 10—membered heteroaryl group containing lor 2 heteroatoms independently chosen from N, O, and S, wherein J is ally substituted with one or more substituents independently chosen from amino, cyano, hydroxyl, halogen, C1—C4alkyl, C1— C4alkoxy, mono— and di— C1—C4alkylamino, C1—C2haloalkyl, and C1—C2haloalkoxy.

Each R is an independently chosen 8— to 10—membered bicyclic ring s containing one or two nitrogen atoms with remaining ring atoms being carbon, which 8— to —membered ic ring is saturated or contains 1 unsaturated bond; and R is optionally substituted with one or more substituents independently chosen from cyano, hydroxyl, halogen, C1—C2alkyl, C1—C2alkoxy, C1—C2haloalkyl, C1—C2haloalkyl, C1—C2haloalkylene, and C1—C2alkylsulfonyl.

T2 is independently chosen at each occurrence from C2—C6alkanoyl, C1— C6alkylester, C1—C6alkenylester, C1—C6alkylsulfonamide, lkylsulfonyl, C2—C6alkanoyl substituted with mono— or di—C1—C6hydrocarbylcarbamate, C2—C6alkanoyl substituted with urea or mono— or di— C1—C6alkylurea, and C2—C6alkanoyl substituted with mono— or di—C1— C6alkylcarboxamide, each of which T2 is optionally substituted with 1 or more tuents independently chosen from amino, cyano, hydroxyl, halogen, alkoxy)C0—C4alkyl, (mono— and di— C1—C4alkylamino)C0—C4alkyl, C1—C6alkyl, thioalkyl)C0—C4alkyl, C3— C7cycloalkyl, , C1—C2haloalkyl, and C1—C2haloalkoxy.

In certain embodiments of XXii, it is preferred that —J2—R— is a group of the formula 2,11% or more particularly112% —J2—R— may be unsubstituted or substituted with one or more substituents independently chosen from amino, cyano, hydroxyl, halogen, C1—C4alkyl, C1—C4alkoxy, mono— and di— C1—C4alkylamino, C1—C2haloalkyl, and C1—C2haloalkoxy.

In certain embodiments of XXii, it is also preferred that T2 is C2—C6alkanoyl substituted with mono— or di—C1—C6hydrocarbylcarbamate, which T2 is optionally substituted with 1 or more substituents independently chosen from amino, cyano, hydroxyl, halogen, (C1— C4alkoxy)C0—C4alkyl, (mono— and di— lkylamino)C0—C4alkyl, C1—C6alkyl, (C1— alkyl)C0—C4alkyl, C3—C7cycloalkyl, phenyl, C1—C2haloalkyl, and C1—C2haloalkoxy.

Any of the ing conditions for compounds of Formula I may be used together to define a subgeneric formula of Formula I so long as a stable compound results.

All such subgeneric formulas are included in this disclosure.

In addition the nds and salts of Formula I discussed in the SUMMARY section, the disclosure includes compositions and combinations of Formula I and Formula II, wherein Formula II is: Formula II Within a II the variables Rl’, RZ’, R3’, R4’, R6’, R7’, Rg’, R16, and T’ carry the definitions set forth below.

R1, and R2, are joined to form a 5— to 7— membered heterocycloalkyl ring containing 1 or 2 heteroatoms independently chosen from N, O, and S which ring is ally fused to a phenyl or 5— or 6— membered heteroaryl to form a bicyclic ring system, each of which 5— to 7— membered heterocycloalkyl ring or bicyclic ring system is optionally substituted.

For the variables R3’ — Rg’ one of the following ions is met.

R3,, R4,, R5,, and R6, are independently hydrogen, C1—C4alkyl, or (C3—C7cycloalkyl)C0— C4alkyl; and R7, and R8, are joined to form an ally tuted 3— to 7— membered cycloalkyl ring.

R3,, R4,, and R6, are independently hydrogen, lkyl, or (C3— C7cycloalkyl)C0—C4alkyl; and R8’ is en or C1—C4alkyl; and R5, is joined to R7, by a C6— C10 saturated or rated hydrocarbon chain.

R3,, R4,, and R6, are independently hydrogen, C1—C4alkyl, or (C3— C7cycloalkyl)C0—C4alkyl; and R7, and R8, are joined to form an optionally substituted 3— to 7— membered cycloalkyl ring; and R5, is joined to the 3— to 7— membered optionally substituted cycloalkyl ring formed by R7 and R8 by a C6—C10 saturated, partially unsaturated or unsaturated arbon chain.

T, is a group of the formula: [2%, R9 is hydroxyl, amino, —COOH, —NR10R11, —OR12, —SR12, —NR10(S=O)R11, or —NR10802R11. R10, R11, and R12 are independently at each occurrence hydrogen, or C1— C6alkyl, C2—C6alkenyl, (aryl)C0—C2alkyl, (C3—C7cycloalkyl)C0—C2alkyl, (heterocycloalkyl)C0— Cgalkyl, or (5— to 10—membered heteroaryl)C0—C2alkyl, each of which is optionally substituted with 1 to 3 substituents independently chosen from halogen, hydroxyl, oxo, C1—C2alkyl, C1— Czalkoxy, trifluoromethyl, and trifluoromethoxy: Z’ is Where X1, X2, X3, X4, and X5 are independently N or CH and no more than two of X1— X5 are N.

R21 represents from 0 to 3 groups independently chosen from halogen, hydroxyl, amino, cyano, —CONH2, —COOH, C1—C4alkyl, C2—C4alkanoyl, C1—C4alkoxy, C1— C4alkylthio, mono— and di—Cl—C4alkylamino, C1—C2haloalkyl, and C1—C2haloalkoxy.

R22 is hydrogen, halogen, hydroxyl, amino, cyano, —CONH2, —COOH, C1— C4alkyl, C2—C4alkanoyl, C1—C4alkoxy, lkylthio, mono— and di—C1—C4alkylamino, C1— C4alkylester, C1—C2haloalkyl, and C1—C2haloalkoxy; or R22 is (C3—C7cycloalkyl)C0—C2alkyl, (phenyl)C0—C2alkyl, (phenyl)C0—C2alkoxy, (5— or ered heteroaryl)C0—C2alkyl, (5— or 6— ed heteroaryl)C0—C2alkoxy, naphthyl, indanyl, (5— or 6—membered 2012/039835 heterocycloalkyl)C0—C2alkyl, or 9— or 10 membered bicyclic heteroaryl, each of which is substituted with 0, l, or 2 substituents independently chosen from (i) halogen, yl, amino, cyano, nitro, —COOH, —CONH2, CH3(C=O)NH—, C1— C4alkyl, C1—C4alkoxy, C1—C4hydroxyalkyl, mono— and C4alkylamino, -NR8802R11, —C(O)OR11, R11, O)OR11, trifluoromethyl, trifluoromethoxy, and (ii) phenyl and 5— or 6—membered heteroaryl, each of which is tuted with 0 or 1 or more of halogen, hydroxyl, C1—C4alkyl, and C1—C2alkoxy; wherein R8 is is hydrogen, C1— C4alkyl, or C3C6cycloalkyl, and R11 is as defined above.

R16 represents 0 to 4 substituents is independently chosen at from n, C1— Czalkyl, and C1—C2alkoxy.

Any of the preceding conditions for compounds of Formula I may be used together to define a subgeneric formula of Formula I so long as a stable compound results and all such subgeneric formulas are included in this disclosure.

This disclosure also includes pharmaceutical compositions and combinations comprising a compound of Formula I and a compound of Formula II. As well as methods of treatment comprising administering such compositions/ combinations to a patient infected with hepatitis C.

For example the disclosure includes compositions and combinations in which the nd of Formula II is /OCI N O\/ CNNHN_ O This disclosure also includes pharmaceutical compositions and combinations comprising a compound of Formula I and a compound of Formula 11. As well as methods of treatment comprising administering such compositions/ combinations to a patient infected with hepatitis C.

For example the disclosure includes compositions and combinations in which the nd of Formula II is NS3a se inhibitors of Formula II, useful in the pharmaceutical compositions and combinations described here have been disclosed previously. US Pat. No. 7,906,619, issued March 15, 2011, is hereby incorporated by reference in its entirety for its teachings regarding o—4—oxobutanoyl peptides. The ‘619 patent is particularly incorporated by nce at the Examples section beginning in column 50 and extending to column 85 which discloses compounds useful in compositions/ combination with nds of Formula I described here.

Published US Pat. Appl. No. 2010-0216725, published August 26, 2010, is hereby incorporated by reference in its ty for its teachings regarding 4—amino—4— oxobutanoyl peptides. The ‘725 application is ularly incorporated by reference at the es section beginning at page 22 and extending to page 100 which discloses compounds useful in compositions/ combination with Compounds of a I described here.

Published US Pat. Appl. No. 2010-0152103, published June 17, 2010, is hereby incorporated by reference in its entirety for its teachings regarding o—4— oxobutanoyl e cyclic analogues. The ‘103 application is particularly incorporated by reference at the Examples section beginning at page 19 and extending to page 60 which discloses nds useful in compositions/ combination with Compounds of Formula I described here.

PHARMACEUTICAL PREPARATIONS Compounds sed herein can be administered as the neat chemical, but are preferably administered as a pharmaceutical composition. Accordingly, the disclosure provides pharmaceutical compositions comprising a compound or pharmaceutically acceptable salt of Formula I, together with at least one pharmaceutically acceptable carrier.

The pharmaceutical composition/ combination may contain a compound or salt of Formula I as the only active agent, but is preferably contains at least one additional active agent. In certain embodiments it is preferred that the onal active agent is an NS3a protease inhibitor, such as a compound of salt of Formula II. In certain embodiments the pharmaceutical composition is in a dosage form that ns from about 0.1 mg to about 2000 mg, from about 10 mg to about 1000 mg, from about 100 mg to about 800 mg, or from about 200 mg to about 600 mg of a compound of a I and optionally from about 0.1 mg to about 2000 mg, from about 10 mg to about 1000 mg, from about 100 mg to about 800 mg, or from about 200 mg to about 600 mg of an additional active agent in a unit dosage form.

The pharmaceutical composition may also include a molar ratio of a compound of a I and an onal active agent. For example the pharmaceutical composition may contain a molar ratio of about 0.5 :1, about 1:1, about 2:1, about 3:1 or from about 1.5 :1 to about 4:1 of an NS3a protease inhibitor of Formula II to NSSa inhibitor of Formula I. nds disclosed herein may be administered orally, topically, parenterally, by inhalation or spray, sublingually, transdermally, via buccal administration, rectally, as an ophthalmic solution, or by other means, in dosage unit formulations containing conventional ceutically acceptable carriers. The pharmaceutical composition may be formulated as any pharmaceutically useful form, e.g., as an aerosol, a cream, a gel, a pill, a capsule, a tablet, a syrup, a transdermal patch, or an ophthalmic solution. Some dosage forms, such as s and capsules, are subdivided into suitably sized unit doses containing appropriate quantities of the active components, e.g., an ive amount to achieve the desired purpose. rs e excipients and diluents and must be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the patient being treated. The carrier can be inert or it can possess pharmaceutical benefits of its own. The amount of carrier employed in conjunction with the compound is sufficient to provide a practical quantity of material for administration per unit dose of the compound.

Classes of rs include, but are not limited to binders, buffering agents, coloring agents, diluents, disintegrants, emulsifiers, flavorants, glidents, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. Some carriers may be listed in more than one class, for example vegetable oil may be used as a lubricant in some formulations and a diluent in others. Exemplary pharmaceutically able carriers include sugars, starches, celluloses, ed tragacanth, malt, gelatin; talc, and vegetable oils. Optional active agents may be included in a pharmaceutical composition, which do not ntially interfere with the activity of the nd of the present invention.

The pharmaceutical compositions/ combinations can be formulated for oral administration. These compositions contain between 0.1 and 99 weight % (wt.%) of a compound of a I and usually at least about 5 wt.% of a nd of Formula. Some embodiments contain from about 25 wt.% to about 50 wt. % or from about 5 wt.% to about 75 wt.% of the compound of Formula.

METHODS OF TREATMENT The pharmaceutical compositions/ combinations disclosed herein are useful for treating hepatitis C infections in patients.

This disclosure provides methods of treating viral infections, including hepatitis C infections, by providing an effective amount of a compound or pharmaceutically acceptable salt of Formula I to patient infected with a hepatitis C virus. A compound or salt of Formula I may be provided as the only active agent or may be provided together with one or more additional active . In certain embodiments the nd or salt of Formula I is administered together with a compound or salt of Formula II or other NS3a protease inhibitor. In certain embodiment the ceutical composition contains a nd of Formula I together with an NSSb inhibitor, and ally an additional active agent.

An ive amount of a pharmaceutical composition/ combination of the invention may be an amount sufficient to (a) inhibit the progression of hepatitis C; (b) cause a regression of the hepatitis C infection; or (c) cause a cure of a hepatitis C infection such that HCV virus or HCV antibodies can no longer be ed in a previously infected patient’s blood or plasma. An amount of a pharmaceutical composition/ combination effective to inhibit the progress or cause a regression of hepatitis C includes an amount effective to stop the worsening of symptoms of hepatitis C or reduce the symptoms experienced by a t infected with the hepatitis C virus. Alternatively a halt in progression or regression of hepatitis C may be indicated by any of several s for the disease. For example, a lack of increase or reduction in the hepatitis C viral load or a lack of increase or reduction in the number of circulating HCV antibodies in a patient’s blood are markers of a halt in progression or regression of hepatitis C infection. Other tis C disease markers include aminotransferase levels, particularly levels of the liver enzymes AST and ALT. Normal levels of AST are from 5 to 40 units per liter of serum (the liquid part of the blood) and normal levels of ALT are from 7 to 56 units per liter of serum. These levels will typically be elevated in a HCV infected patient. Disease regression is usually marked by the return of AST and ALT levels to the normal range.

Symptoms of tis C that may be affected by an ive amount of a pharmaceutical composition/ combination of the invention include decreased liver function, fatigue, flu—like symptoms: fever, chills, muscle aches, joint pain, and headaches, nausea, aversion to certain foods, unexplained weight loss, psychological disorders including depression, tenderness in the n, and jaundice.

“Liver on” refers to a normal function of the liver, including, but not limited to, a tic function including synthesis of proteins such as serum proteins (e.g., albumin, clotting factors, ne phosphatase, aminotransferases (e.g., alanine transaminase, aspartate transaminase), 5'—nucleosidase, y glutaminyltranspeptidase, etc.), synthesis of bilirubin, synthesis of cholesterol, and synthesis of bile acids; a liver metabolic function, including carbohydrate lism, amino acid and ammonia metabolism, hormone metabolism, and lipid metabolism; detoxification of exogenous drugs; and a hemodynamic function, including splanchnic and portal hemodynamics.

An effective amount of a pharmaceutical composition/ combination described herein will also provide a sufficient tration of the active agents in the concentration when administered to a patient. A sufficient tration of an active agent is a concentration of the agent in the patient’ s body necessary to prevent or combat the infection.

Such an amount may be ascertained experimentally, for example by ng blood concentration of the agent, or theoretically, by calculating bioavailability. The amount of an active agent sufficient to inhibit viral infection in vitro may be determined with a conventional assay for viral infectivity such as a replicon based assay, which has been described in the literature.

Pharmaceutical compositions/ combinations and methods of treatment in which a compound or salt of Formula I is provided together with one or more additional active agents are included herein. In preferred embodiments a compound of Formula I is ed together with an NS3a protease inhibitor, either in a single pharmaceutical composition or a in separate dosage forms with instructions to the patient to use the compound of Formula I and onal active agent er. Compounds of a II and compounds disclosed in US Pat. No. 7,906,619, US Pat. Appl. No. 2010—0216725, and US Pat. Appl. No. 2010—0152103, most of which are within the scope of Formula II, are suitable NS3a protease inhibitors for use in combination with compounds and salts of Formula I. In certain embodiments the active agent (or agents) is an HCV protease inhibitor or HCV polymerase inhibitor. For example the protease inhibitor may be telaprevir (VX—950) and the polymerase inhibitor may be valopicitabine, or NM 107, the active agent which valopicitabine is converted into in vivo. In certain embodiments the at least one additional active agent is ribavirin, interferon, or Peg—interferon alpha conjugate. In certain embodiments the at least one additional active agent is 25 or ACH—2684.

According to the methods of the ion, the compound or pharmaceutically acceptable salt of Formula I and at least one additional active agent may be: (1) co— formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by any other combination therapy n known in the art. When delivered in alternation therapy, the methods of the ion may comprise administering or delivering the compound or salt of Formula I and an additional active agent sequentially, e.g., in separate solution, emulsion, suspension, tablets, pills or capsules, or by different ions in separate syringes. In general, during alternation y, an ive dosage of each active ingredient is administered sequentially, i.e., serially, whereas in simultaneous therapy, ive dosages of two or more active ingredients are administered together. Various sequences of intermittent combination therapy may also be used.

Methods of treatment and pharmaceutical combinations ing compounds or pharmaceutically able salts of Formula I bed herein together with any one or combination of the following compounds and substances as an onal active agent are provided by the disclosure: Anti—fibrotics: IP—501 (InterMune) Caspase Inhibitors: IDN—6556 (Idun Pharmaceuticals) and GS—9450 (Gilead) hilin Inhibitors: for example, NIM811 (Novartis), 5 (Scynexis), and DEBIO—025 (Debiopharm); C v‘tochrome P450 mom‘soxvoenase inhibitors: ritonavir, ketoconazole, troleandomycin, 4—methyl pyrazole, cyclosporin, clomethiazole, cimetidine, itraconazole, fluconazole, miconazole, fluvoxamine, fluoxetine, nefazodone, sertraline, indinavir, nelfinavir, amprenavir, fosamprenavir, saquinavir, vir, rdine, erythromycin, VX— 944, and VX—497 ebodib). Preferred CYP inhibitors include ritonavir, ketoconazole, troleandomycin, 4—methyl pyrazole, cyclosporin, and clomethiazole; orticoids: hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, paramethasone, betamethasone, and dexamethasone.

HCV se tors: for example ACH—1625 and ACH—2684. US patent number 7,906,619 is hereby incorporated for its teachings regarding CV compounds. US patent application nos. 12/635,270 at pages 52—167 and 12/635,049 at pages 43—92 are hereby incorporated by reference for their ngs regarding anti—HCV nds, ACH 1625 and ACH 2684 (Achillion), ABT-450 (Abbott), ACL-181 and AVL-192 (Avila), BI—335 (Boehringer Ingelheim), BMS—032 (Bristol Meyers Squibb), boceprevir (Merck), TMC—435, 2 (Merck), GS-9256 (Gilead), GS-9451 (Gilead), R7227 (Intermune), VX-500 (Vertex), VX—950 revir, Vertex), VX—985 (Vertex), TMC—435 (Tibotec), GW—433908 (prodrug of Amprenavir, Glaxo/ Vertex), indinavir (CRIXIVAN, , ITMN—191 (Intermune/ Array Biopharma), BILN 2061 (Boehringer—Ingelheim), TMC435350 (Tibotec/Medivir), BI 201335 (Boehringer Ingelheim), PHX—1766 mix), MK—7009 (Merck), narlaprevir (SCH900518, Schering) Hematopoietins: hematopoietin—1 and poietin—2. Other members of the hematopoietin superfamily such as the various colony stimulating factors (e.g. (e.g. G—CSF, GM—CSF, M—CSF), Epo, and SCF (stem cell factor) Homeopathic Therapies: Milk Thistle, silymarin, ginseng, glycyrrhizin, licorice root, schisandra, vitamin C, n E, beta carotene, and selenium Immunomodulatory compounds: thalidomide, IL—2, hematopoietins, IMPDH inhibitors, for example Merimepodib (Vertex Pharmaceuticals Inc.), interferon, including natural interferon (such as OMNIFERON, Viragen and SUMIFERON, Sumitomo, a blend of natural interferons), natural interferon alpha ON, Hemispherx Biopharma, Inc.), interferon alpha nl from lymphblastoid cells (WELLFERON, Glaxo Wellcome), oral alpha interferon, Peg—interferon, Peg—interferon alfa 2a (PEGASYS, Roche), recombinant interferon alfa 2a (ROFERON, Roche), inhaled interferon alpha 2b (AERX, m), Peg—interferon alpha 2b (ALBUFERON, Human Genome Sciences/ Novartis, PEGINTRON, Schering), recombinant interferon alfa 2b (INTRON A, Schering), pegylated interferon alfa 2b (PEG— INTRON, Schering, VIRAFERONPEG, Schering) ,interferon beta—la , Ares—Serono, Inc. and Pfizer), consensus interferon alpha (INFERGEN, Intermune), interferon gamma—lb (ACTIMMUNE, Intermune, Inc.), un—pegylated interferon alpha, alpha interferon, and its analogs, and synthetic thymosin alpha 1 (ZADAXIN, SciClone Pharmaceuticals Inc.), and lamdba interferon (BMS) Immunosupressants: sirolimus (RAPAMUNE, Wyeth) Interleukins: (IL-1, IL-3, IL-4, IL-5, IL-6, IL-10, IL-11, IL-12), LIF, TGF-beta, TNF- alpha) and other low molecular weight factors (e.g. AcSDKP, pEEDCK, thymic es, and minicytokines) Interferon Enhancers: EMZ702 (Transition Therapeutics) IRES inhibitors: VGX—410C (VGX Pharma) Monoclonal and Polyclonal antibodies: XTL—6865 (HEPX—C, XTL), HuMax—HepC b), Hepatitis C Immune Globin (human) (CIVACIR, Nabi Biopharmceuticals), XTL— 002 (XTL), Rituximab (RITUXAN, Genentech/ IDEC) Nucleoside analogues: IDX—l84 (Idenix), PSI—7977 and PSI—938 (Pharmasset), INX— 189 (Inhibitex), R7128 (Roche), R7348 ), GS-6620 (Gilead), TMC—649 (Tibotec), Lamivudine (EPIVIR, 3TC, GlaxoSmithKline), 8 (Merck), zalcitabine (HIVID, Roche US Pharmaceuticals), ribaVirin (including COPEGUS (Roche), REBETOL (Schering), VILONA (ICN Pharmaceuticals, and VIRAZOLE (ICN ceuticals), isatoribine (Anadys Pharmaceuticals), ANA971(Anadys Pharmaceuticals), ANA245 (Anadys Pharmaceuticals), and Viramidine (ICN), an e prodrug of rin. Combinations of nucleoside analogues may also be employed.

Non—nucleoside inhibitors: 30 (Roche/ Pharmasset), ABT—333 and ABT—072 t), delaViridine (RESCRIPTOR, ), PF-868554 (Pfizer), GSK-852 SmithKline), 5 (Idenix), 8 (Anadys), VX—222 and VX—759 (Vertex), MK—328l (Merck), BI—127 (Boehringer Ingelheim), EMS—325 (Bristol Meyers), and HCV— 796 (Viropharm) NS4a inhibitors: for example ACH—1095. US patent application no. /0004711 is hereby incorporated by reference in its entirety for its teachings regarding HCV tors and US patent application no. 12/125,554 at pages 45—90 is hereby incorporated by reference for its teachings regarding HCV inhibitors.

NS4b inhibitors: clemizole (Arrow Therapeutics) NS5a inhibitors: A—382 (Arrow Therapeutics), BMS—790052 (BMS) NS5b tors: INX-181, IDX-375, MK-3281, PSI-7977, PSI-7851, PSI-938, RG— 9190, VX—222 (Vertex), and BMS—791325 (Bristol Meyers Squibb).

P7 protein inhibitor: amantadine (SYMMETREL, Endo Pharmaceuticals, Inc.) ase inhibitors: Filibuvir (PF—00868554, Pfizer), NM283 (valopicitabine) (IdeniX), JTK 003 (AKROS Pharma), HCV—796 (ViroPharma/ Wyeth), IDX184 (IdeniX),VCH-916 (VertX), R7128 (P816130, Roche), R1626 (Roche), MK-3281 ), PSI—7851 (Pharmasset), ANA598 (Anadys), BI207127 (Boehringer—Ingelheim), GS 9190 (Gilead).

RNA interference: 034 RNAi (Sirna Therapeutics) and 181 14803 (Isis Pharmaceutical/ Elan) Therapeutic Vaccines: IC41 (Intercell), IMN—0101 (Imnogenetics), GI 5005 immune), Chronvac—C (Tripep/ Inovio), ED—002 (Imnogenetics), HepavaXX C (ViReX Medical) TNF agonists: adalimumab (HUMIRA, Abbott), rcept (ENBREL, Amgen and Wyeth), infliXimab (REMICADE, Centocor, Inc.) Tubulin inhibitors: Colchicine Sphingosine—l—phosphate receptor modulators: FTY720 (Novartis) TLR ts: ANA—975 (Anadys Pharmaceuticals), TLR7 agonist (Anadys Pharmaceuticals), CPG10101(Coley), andTLR9 agonists including CPG 7909 (Coley).

Vaccines: HCV/MF59 (Chiron), IC41 (Intercell), E—1 (Innogenetics) Patients receiving hepatitis C medications are typically given interferon together with another active agent. Thus methods of treatment and pharmaceutical combinations in which a compound of The invention is provided together with an interferon, such as pegylated interferon alfa 2a, as the additional active agents are included as embodiments. Similarly methods and ceutical combinations in which ribavirin is an additional active agent are provided .

Methods of ting HCV replication in vivo comprising providing a compound or pharmaceutically acceptable salt of Formula I to a patient infected with HCV, a concentration of the compound or salt of a I sufficient to inhibit HCV replicon replication in vitro are included herein. In this instance the concentration includes an in vivo concentration, such as a blood or plasma tration. The concentration of compound sufficient to inhibit HCV replicon replication in vitro may be determined from an assay of replicon replication such as the assay provided in Example 9, herein.

Methods of treatment include providing certain dosage amounts of a compound or ceutically acceptable salt of Formula I to a patient. Dosage levels of each active agent of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above—indicated conditions (about 0.5 mg to about 7 g per patient per day). The amount of active ingredient that may be combined with the carrier materials to produce a single unit dosage form will vary ing upon the t treated and the particular mode of stration. In certain ments about 0.1 mg to about 2000 mg, from about 10 mg to about 1500 mg, from about 100 mg to about 1000 mg, from about 200 mg to about 800 mg, or from about 300 to about 600 mg of a nd of Formula I and optionally from about 0.1 mg to about 2000 mg, from about 10 mg to about 1500 mg, from about 100mg to about 1000 mg, from about 200 mg to about 800 mg, or from about 300 to about 600 mg of a compound of an additional active agent, for example an NS3a protease inhibitor such as a compound of Formula II are ed daily to a patient. It is preferred that each unit dosage form contains less than 1200 mg of active agent in total.

Frequency of dosage may also vary depending on the compound used and the particular disease treated. However, for treatment of most infectious disorders, a dosage regimen of 4 times daily or less is preferred and a dosage regimen of 1 or 2 times daily is particularly preferred.

It will be tood, however, that the ic dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound ed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease in the patient undergoing therapy.

PACKAGED FORMULATIONS Methods comprising providing a compound or salt of Formula I in a container together with instructions for using the compound to treat a t suffering from Hepatitis C infection are included herein.

Packaged pharmaceutical compositions/ ations are also included herein. Such packaged combinations include a compound of Formula I in a container 2012/039835 together with instructions for using the combination to treat or prevent a viral infection, such as a hepatitis C infection, in a patient.

The packaged pharmaceutical composition/ combination may include one or more additional active agents. In certain embodiments the additional active agent is an NS3a protease inhibitor, such as a compound of a II.

The packaged pharmaceutical ation may include a compound or pharmaceutically acceptable salt of Formula I and the additional active agent provided simultaneously in a single dosage form, concomitantly in separate dosage forms, or provided in separate dosage forms for administration separated by some amount of time that is within the time in which both the compound of Formula I and the additional active agent are within the tream of the patient.

The packaged pharmaceutical combination may include a compound or pharmaceutically acceptable salt of Formula I provided in a container with an additional active agent provided in the same or te container, with instructions for using the combination to treat an HCV infection in a patient.

EXAMPLES ABBREVIATIONS The ing abbreviations are used in the reaction schemes and synthetic examples, which follow. This list in not meant to be an all—inclusive list of abbreviations used in the application as additional rd abbreviations, which are readily tood by those skilled in the art of organic synthesis, may also be used in the synthetic schemes and examples.

Ac acetyl ACN acetonitrile aq. s BOC t—butoxycarbonyl DCM dichloromethane DIEA N,N-diisopropylethylamine DlPEA N,N—diisopropylethylamine DME 1,2—dimethoxyethane DMF N,N—dimethylformamide dppf 1,1 ’ —bis(diphenylphosphino)ferrocene EDCI N-(3 -Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride Et ethyl EtZO diethyl ether FCC flash column chromatography HATU 0—(7—azabenzotriazol—1—yl)—N,N,N',N'—tetramethyluronium hexafluorophosphate MTBE methyl tert—butyl ether PTLC preparative thin layer chromatography rt room temperature TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TPP triphenylphosphine GENERAL CONSIDERATIONS All nonaqueous reactions were performed under an atmosphere of dry argon gas using oven—dried glassware and anhydrous solvents. The progress of reactions and the purity of target compounds were determined using one of the following two HPLC methods: (1) Waters AQUITY UPLC BEH C18 1.7 pm 2.1 X 50 mm column with an tic n of 0.24 min at 90:10 acetonitrile containing 0.05% formic acid followed by a in linear gradient elution from 90:10 to 10:90 at a flow rate of 1.0 mL/min with UV (PDA), ELS, and MS (SQ in APCI mode) ion (method 1); and (2) Waters AQUITY UPLC BEH C18 1.7 pm 2.1 X 50 mm column with an isocratic elution of 0.31 min at 95:5 acetonitrile containing 0.05% formic acid followed by a 17.47—min linear gradient elution from 95:5 to 5:95 at a flow rate of 0.4 mL/min with UV (PDA), ELS, and MS (SQ in APCI mode) detection (method 2).

Target compounds were purified via preparative reverse—phase HPLC using a YMC Pack Pro C18 5 pm 150 x 20 mm column with an isocratic elution of 0.35 min at 95:5 water:acetonitrile containing 0.1% trifluoroacetic acid followed by a 23.3—min linear gradient elution from 95:5 to 5:95 at a flow rate of 18.9 mL/min with UV and mass—based fraction collection.

GENERAL SYNTHETIC SCHEME ,Boc HO N DIPEA e, A “W“ P06 , BQ—CNH HATU, DIPEA i, 0‘ ’0 3—3 8 HN I \ / NH o :0 oi N N KOAc, cat. PdClzdppf.CH2C|2 dioxane, A 0’ 0Br iqB 3* O / NH ‘3;ng K3PO4, cat. PdClzdppf.CH2C|2 dioxa ne/wate r, A ’0 11 EXAMPLE 1. SYNTHESIS OF COMPOUND 10 Compound 10 was prepared Via bromination of [2.2]paracyclophane as outlined previously , H. J .; Cram, D. J. J. Am. Chem. Soc. 1969, 91, 3527-3533; Reich, H. J.; Cram, D. J. J. Am. Chem. Soc. 1969, 9], 3534—3543). Compounds 1, 2, 6, 8, and can be obtained from commercial sources. Compounds 3—7 and 9 were prepared using general synthetic methods known in the art.

EXAMPLE 2. SYNTHESIS OF COMPOUND 11 A deoxygenated (argon) mixture of 9 (284.2 mg), 10 (52.3 mg), K3PO4 (248.1 mg), and PdClzdppf-CH2C12 (7.4 mg) in dioxane/water (5.5. mL/0.55 mL) was irradiated in a microwave for 2 h at 80 OC. The resulting mixture was evaporated under reduced pressure and the remaining solid was extracted with DCM. This crude material was ed by PTLC (20 cm x 20 cm x 2000 um glass ; eluted with 45:50:5 v/v/v DCMzEtOAczMeOH, Rf 0.28) to give 75.3 mg of 11. The purity of 11 was ined via analytical reverse—phase HPLC using a 35—min gradient elution of increasing concentrations of ACN in water (10— 90%) containing 0.05% formic acid with a flow rate of 1.0 mL/min on a Waters AQUITY UPLC BEH C18 1.7 pm 2.1 x 50 mm column with UV (PDA), ELS, and MS (SQ in APCI mode) detection. HPLC: IR 1.57 min (98% purity). MS m/z calculated for C56H64N806 ([M]+), 945; found, 946 ([M + 1]+).

E 3. SYNTHESIS OF DIMETHYL ((25,25)—((25,2'S,3AS,3A'S,7AS,7A'S)—2,2'—(5,5'— (TRICYCLo[8.2.2.24’7]HEXADECA—4,6,10,12,13,15—HEXAENE—5,11—D1YL)BIS(1H— BENZO[D]IMIDAZOLE—S,2—DIYL))BIS(OCTAHYDRO— 1H—1NDOLE—2, 1 —DIYL))BIS(3—METHYL— 1 — ANE—Z,1—DIYL))DICARBAMATE (20) 8312 1‘. 0 rice 0 12 13 To a stirred solution of (2R,3aS,7aS)—octahydro—1H—indole—2—carboxylic acid (250 g, 1.0 equiv) (12) in THF (3 L) and water (1.5 L) at 00 C, was added dropwise a cooled aq solution of 2.5 M NaOH (1 L). The reaction mixture was d for 15 min at the same temperature. Then di—tert—butyl dicarbonate (1.3 equiv) was added dropwise, maintaining the temperature at 0 OC. The ing reaction mixture was stirred at rt for 12 h. The reaction mixture was waShed with MTBE (3 times). The aq phase was acidified with 1 M aq citric acid and extracted with ethyl acetate (3 times). The combined organic layers were dried over sodium sulphate and concentrated to s to give (2R,3aS,7aS)—1—(tert— butoxycarbonyl)octahydro—1H—indole—2—carboxylic acid (368 g) (13).

Step2 JCENH 03*“+ —> Br NHZ O WW . 1“! )\Br Boc 0 13 14 +Is0mer To a stirred solution of o—1,2—diaminobenzene (23.1 g, 1.2 equiv), (2R,3aS,7aS)—1—(tert—butoxycarbonyl)octahydro—1H—indole—2—carboxylic acid (26.9 g, 1.0 equiv) 13, and EDCI (23.6g, 1.2 equiv) in ACN (600 mL) at 0 0C was added DIEA (21.5 mL, 1.3 equiv) dropwise. The reaction mixture was stirred for additional 1 h after completion of addition. Water (1.2 L) was added and the reaction mixture was stirred overnight. The solid powder (14) was collected, washed with water, and dried for use in the next step without further purification (39.1 g).

Step 3 H2N H H N N I) N Br N \ Br \ O Boc 14 + Isomer An isomeric mixture (14) of (2S,3aS,7aS)—tert—butyl 2—((2—amino—4— bromophenyl)carbamoyl)octahydro—1H—indole—1—carboxylate and (2S,3aS,7aS)—tert—butyl 2— ((2—amino—5—bromophenyl)carbamoyl)octahydro—1H—indole—1—carboxylate (160 g, 0.36 mol) was dissolved in acetic acid (480 mL) and the reaction mixture was stirred at 65 0C until the ng materials were consumed (as judged by LC—MS analysis). The reaction was cooled to rt and the solvent was d under vacuum. The remaining residue was dissolved in ethyl acetate (500 mL) and aq ammonia (100 mL) was added carefully. Additional water (100 mL) was added and the organic layer was separated and collected. The aq phase was extracted with ethyl acetate (2 X 300 mL). The combined organic phase was washed with water (200 mL), followed by brine (200 mL), and dried over MgSO4. The solution was concentrated and the remaining e was purified by silica gel column chromatography (hexanes/ethyl acetate) to afford (2S,3aS,7aS)—tert—butyl romo—1H—benzo[d]imidazol—2— yl)octahydro—1H—indole—1—carboxylate (15) (140 g).

Step 4 H H (1%I:N N N CENHI:N ‘Boc Br ‘Boc T’00% 16 Under an atmosphere of argon, a mixture of S,7aS)—tert—butyl 2—(6— bromo—1H—benzo[d]imidazol—2—yl)octahydro—1H—indole—1—carboxylate (15) (30 g, 1.0 equiv), bis(pinacolato)diborane (27.2 g, 1.5 equiv), potassium acetate (21 g, 3.0 equiv), and Pd(dppf)Clz (5.7 g, 0.098 equiv) in anhydrous 1,4—dioxane (300 mL) was heated at 80—90 0C for ~4 h (until the reaction was complete as judged by LC—MS). The cooled (rt) reaction mixture was diluted with ethyl acetate (300 mL), d with activated carbon (60 g) for 1 h, and filtered h a pad of Celite. The filtrate was concentrated under reduce pressure and the resulting brown foam was ed by silica gel column chromatography (hexanes/ethyl acetate, 5:1 —> 1:2 v/v) to give (2S,3aS,7aS)—tert—butyl 2—(6—(4,4,5,5—tetramethyl—1,3,2— dioxaborolan—2—yl)—1H—benzo[d]imidazol—2—yl)octahydro—1H—indole—1—carboxylate as an off— white solid ) (16).

Step5 0 O —» O 0 A mixture of B12 (82.8 g) and iron powder (4.6 g) in DCM (1.6 L) was stirred at rt for 1 h. To this mixture was added, in one portion, a slurry of [2.2]paracyclophane (200 g, 1.0 equiv) in DCM. The resulting e was heated to reflux, and to it was added slowly a solution of B12 (228 g) in DCM (400 mL) over a 3—h period. After this addition was complete, the reaction mixture continued to reflux for 3 h, was d to cool to rt with stirring overnight, was washed with 5% w/v aq Na28203 (2 L) and water (2 L), dried (MgSO4), and evaporated to dryness. The isolated crude solid was dissolved in hot toluene (1.2 L, ~100 OC), allowed to cool slowly ght to rt with stirring, and further cooled to 5 0C for 3 h. The resulting solid was collected and washed with cold toluene (~100 mL) to afford 4,16—dibromo[2.2]paracyclophane (17) (83 g). 1H NMR (300 MHz, CDCl3, rt): 5 2.79—3.00 (m, 4H), 3.10—3.21 (m, 2H), 3.44—3.54 (m, 2H), 6.44 (d, J: 8.0 Hz, 2H), 6.51 (d, J = 2.0 Hz, 2H), 7.14 (dd, J = 8.0 Hz, 2.0 Hz, 2H).

Step 6 NBoc Under an atmosphere of argon, a mixture of 4,16—dibromo[2.2]paracyclophane (17) (20 g, 1.0 equiv), (2S,3aS,7aS)—tert—butyl 2—(6—(4,4,5,5—tetramethyl—1,3,2—dioxaborolan—2— yl)—1H—benzo[d]imidazol—2—yl)octahydro—1H—indole—1—carboxylate (64 g, 2.5 equiv), Cs2C03 (16) (44.5 g, 2.5equiv), Pd(PPh3)4 (3.16 g, 0.05 equiv), DMF (500 mL), and water (25 mL) was heated at 130 0C for ~2—3 h (until the reaction was complete as judged by LC—MS). The reaction mixture was allowed to cool to rt and filtered through a pad of silica gel (30 g) layered with Celite. This pad was washed with DMF (2 X 50 mL) and the combined tes were added to stirred water (2.5 L) to give a pale yellow precipitate. This solid was collected by filtration, washed with water (1 L) and ACN (500 mL), and dissolved in a mixture of DCM (250 mL) and MeOH (25 mL). To this solution was added ACN (250 mL) to generate a fine slurry, which was then concentrated under reduce pressure at 30—35 0C to remove ~150 mL of solvent. Another portion of ACN (500 mL) was added and additional solvent (~100 mL) was removed under reduce re at 40—45 0C. The solid was ted by filtration and dried under vacuum to give (2S,2'S,3aS,3a'S,7aS,7a'S)—di—tert—butyl 5,5'— (tricyclo[8.2.2.24’7]hexadeca—4,6,10,12,13,15—hexaene—5,11—diyl)bis(1H—benzo[d]imidazole— ,2—diyl))bis(octahydro—1H—indole—1—carboxylate) as a pale yellow powder (33.8 g).

To a cooled (0 °C) solution of (2S,2'S,3aS,3a'S,7aS,7a'S)—di—tert—butyl 2,2'— (5,5'—(tricyclo[8.2.2.24’7]hexadeca—4,6,10,12,13,15—hexaene—5,11—diyl)bis(1H— benzo[d]imidazole—S,2—diyl))bis(octahydro—1H—indole—1—carboxylate) (18) (20.33 g, 1.0 equiv) in DCM/MeOH (4/1 v/v, 200 mL) was added a 4 N HCl/dioxane solution (100 mL).

The reaction mixture was d at rt for 30 min and concentrated under reduce pressure to give a pale yellow powder (19) (21.7 g). The solid obtained was dried under vacuum until residual MeOH was undetectable by 1H NMR spectroscopic analysis. This thoroughly dried material was used directly in the next step.

Step 8 To a mixture of ((methoxycarbonyl)amino)—3—methylbutanoic acid (10 g, 2.3 equiv), HOBt monohydrate (8.8 g, 2.3 equiv), and ACN (50 mL) at rt was added EDCI (11.14 g, 2.3 equiv). After stirring 5 min, this activated acid mixture was added to a solution of the hloride salt (19) from above (21.7 g) and DIEA (32 mL, 7.2 equiv) in DMF (250 mL). The reaction mixture was stirred at rt until the reaction was judged as te by LC— MS analysis (~4 h) and then poured into water (1.2 L) with stirring. The precipitate was collected by filtration, stirred in ACN/water (4:1 v/v, 500 mL) overnight, collected again by filtration, and dried in vacuo to give dimethyl ((2S,2'S)—((2S,2'S,3aS,3a'S,7aS,7a'S)-2,2'—(5,5'— clo[8.2.2.24’7]hexadeca—4,6,10,12,13,15—hexaene—5,11—diyl)bis(1H—benzo[d]imidazole— ,2—diyl))bis(octahydro— ole—2, 1 )bis(3 —methyl—1—oxobutane—2,1—diyl))dicarbamate (20) (22.62 g). 1H NMR (300 MHz, DMSO—d6, 120 °C): 50.87 (d, J: 6.5 Hz, 6H), 0.92 (d, J = 6.5 Hz, 6H), 1.20—1.60 (m, 6H), 1.65—2.10 (m, 12H), 2.31 (m, 2H), 2.38—2.52 (m, 2H), 2.54—2.76 (m, 4H), 2.85 (m, 2H), 3.07 (m, 2H), 3.45 (m, 2H), 3.57 (br s, 6H), 4.07 (t, J: 8.0 Hz, 2H), 4.34 (m, 2H), 5.28 (t, J: 8.5 Hz, 2H), 6.51 (br, 2H), 6.57 (d, J: 8.0 Hz, 2H), 6.73 (s, 2H), 6.76 (d, J: 8.0 Hz, 2H), 7.33 (d, J: 8.5 Hz, 2H), 7.64 (d, J: 8.5 Hz, 2H), 7.72 (s, 2H).

EXAMPLE 4. SYNTHESIS OF DIMETHYL ((25,2'5,3R,3'R)—((25,2'5,3AS,3A'S,7AS,7A'S)—2,2'—(5 ,5 '— (TRICYCLO[8.2.2.24’7]HEXADECA—4,6,10,12,13,15—HEXAENE—5,1l—DIYL)BIS(1H— BENZO[D]IMIDAZOLE—S,2—DIYL))BIS(OCTAHYDRO— 1H—1NDOLE—2, 1 —DIYL))BIS(3—METHOXY— 1 — OXOBUTANE—Z,l—DIYL))DICARBAMATE (22) Step 1 H2N,, o I N, OH _> / T 1, 'I/O/ 0 O—Methyl—L—threonine (25 g, 0.19 mol) was dissolved in 1,4—dioxane (125 mL) and cooled to 0 0C. An aq 2 M NaOH (22.5 g, 0.56 mol, 3 equiv) solution was then added to the reaction e followed by methyl chloroformate (17.4 mL, 0.22 mol, 1.2 equiv) at the same temperature. The reaction mixture was warmed to rt and stirred for 16 h.

The reaction mixture was washed with ethyl acetate (500 mL). The aq layer was acidified with 3 N HCl (up to pH 2) and extracted with ethyl acetate (3 X 250mL). The combined organic layers were dried over Na2$O4 and trated to afford the crude product (23 g).

Ethyl acetate (46 mL) was added to the crude product and heated at 80 0C to obtain a clear solution. This solution was then cooled to 0 OC. The solid obtained was filtered and dried to afford the d pure product (21) (18.75 g).

Step 2 05H”H 0 N N O H H . o N,, H + OH N\ N 19 ON (,0/ H 21 4HCl H \ 03—6“ 3~NH N \N O O §8\.¢O\ —> O oflun. o N N / N 22 H DIEA (32.8 mL, 0.192 mol) was added dropwise to a mixture of the hydrochloride salt from above (32.0 g, 0.0384 mol), Moc—O—methyl—L—threonine (18.3 g, 0.0960 mol), and HATU (36.5 g, 4 mol) in DMF (160 mL) at 0 OC. The reaction mixture was allowed to warm to rt, stirred for 16 h, and poured into water (1.6 L). The ing solid was collected by filtration and dissolved in DCM (500 mL). This solution was washed with water (100 mL), washed with brine (50 mL), dried over sodium sulphate, and concentrated under reduced pressure. The e was purified by silica gel column chromatography to give dimethyl ((2S,2'S,3R,3'R)—((2S,2'S,3aS,3a'S,7aS,7a'S)—2,2'—(5 ,5 '— (tricyclo[8.2.2.24’7]hexadeca—4,6,10,12,13,15—hexaene—5,11—diyl)bis(1H—benzo[d]imidazole— ,2—diyl))bis(octahydro— 1H—indole—2, 1 )bis(3—methoxy— 1 —oxobutane—2, 1 — diyl))dicarbamate (22) (30 g). 1H NMR (400 MHz, DMSO—d6, rt): 50.91—1.04 (m, 6H), 1.21—1.59 (m, 6H), 1.64—1.88 (m, 6H), 1.90—2.07 (m, 4H), 2.23—2.49 (m, 6H), 2.62 (m, 2H), 2.85 (m, 2H), 3.08 (m, 2H), 3.19 (s, 3H), 3.20 (s, 3H), 3.42 (m, 2H), 3.57 (s, 6H), 4.15 ent t, J: 8.0 Hz, 2H), 4.47 (m, 2H), 5.16 (apparent t, J: 8.5 Hz, 2H), 6.52 (apparent t, J: 8.5 Hz, 2H), 6.75 (s, 2H), 6.82 (m, 2H), 7.41 (m, 2H), 7.71 (apparent t, J: 6.0 Hz, 2H), 7.64—7.79 (m, 4H).

EXAMPLE 5. SYNTHESIS OF DIMETHYL ((25,2'5,3R,3'R)—((252'S,3AS,3A'S,7AS,7A'S)—2,2'— (5 ,5'—(TRICYCLO[8.2.2.24’7]HEXADECA—4,6,10,12,13,15—HEXAENE—5 ,1 l—DIYL)BIS(1H— BENZO[D]IMIDAZOLE—S,2—DIYL))BIS(OCTAHYDRO— 1H—1NDOLE—2, 1 —DIYL))BIS(3—METHOXY— 1 — OXOBUTANE—Z,l—DIYL))DICARBAMATE (23) 2012/039835 CFH” \O / )‘NH N 0 $04 HN Q or“?H . 0 /0 ”OH oW Dimethyl ((2S,2'S,3R,3'R)—((2S,2'S,3aS,3a'S,7aS,7a'S)—2,2'—(5,5'— (tricyclo[8.2.2.24’7]hexadeca—4,6,10,12,13,15—hexaene—5,11—diyl)bis(1H—benzo[d]imidazole— ,2—diyl))bis(octahydro— 1H—indole—2, 1 —diyl))bis(3 —hydroxy—1—oxobutane—2, 1— diyl))dicarbamate was prepared in a manner analogous to that described above for the synthesis of dimethyl ((2S,2'S,3R,3'R)—((2S,2'S,3aS,3a'S,7aS,7a'S)—2,2'—(5,5'— (tricyclo[8.2.2.24’7]hexadeca—4,6,10,12,13,15—hexaene—5,11—diyl)bis(1H—benzo[d]imidazole— ,2—diyl))bis(octahydro— 1H—indole—2, 1 —diyl))bis(3—methoxy— 1 —oxobutane—2, 1 — diyl))dicarbamate. 1H NMR (400 MHz, DMSO—d6, rt): 1H NMR (400 MHz, 6, rt): 6 0.95—1.04 (m, 6H), 1.20—1.56 (m, 6H), 1.64—1.85 (m, 6H), 1.90—2.09 (m, 4H), 2.25 (m, 2H), 2.32—2.49 (m, 4H), 2.63 (m, 2H), 2.83 (m, 2H), 3.06 (m, 2H), 3.39 (m, 2H), 3.57 (s, 6H), 3.69 (m, 2H), 4.06 (apparent t, J: 7.5 Hz, 2H), 4.46 (m, 2H), 4.72 (m, 2H), 5.13 (m, 2H), 6.44—6.57 (m, 2H), 6.68—6.87 (m, 4H), 7.23—7.35 (m, 4H), 7.53—7.78 (m, 4H).

E 6. SYNTHESIS OF COMPOUND 29 VIA THE TETRAAMINE SYNTHETIC ROUTE Step 1 : :NHZ —> Br NH2 Br : :NHBoc NHBoc 1,2—Diamino—4—bromobenzene (10 g, 0.053 mol) was dissolved in DCM (150 mL) and cooled to 0 0C. A solution of NaOH (50 mL, 2.5 mol) was added se at the same temperature. After 15 min, di—tert—butyl dicarbonate (58 g, 0.26 mol) was added dropwise at the same temperature. Then the reaction e was d to warm to room temperature, stirred for 16 h, diluted with DCM (100 mL), and washed with water (100 mL).

The organic layer was separated, dried over Na2804, and concentrated. The crude material was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 1:1 V/V) to afford the desired product (18 g). wo 2012/166716 2012/039835 NHBoc /©:NHBoc —> 0, /©: NHB°° Br NHBoc )§<'13 Under an atmosphere of argon, a mixture of di—tert—butyl (4—bromo—1,2— phenylene)dicarbamate (24) (18 g, 0.046 mol), bis(pinacolato)diboron (17.7 g, 0.070 mol), potassium acetate (13.66 g, 0.14 mol), and Pd(dppf)Clz (3.8 g, 0.0046 mol) in 1,4—dioxane (360 mL) was heated at 85 0C for 16 h. The reaction mixture was then diluted with ethyl acetate (200 mL) and filtered through a bed of Celite. The filtrate was concentrated under reduced pressure and the remaining al was purified by silica gel column chromatography (petroleum ethyl acetate, 80:20 V/V) to afford the desired t (25) (16.0 g).

Step 3 BocHN NHBoc BocHN O + C)‘B NHBat: NHBoc Br I NHBoc 17 25 26 Under an atmosphere of argon, a mixture of 4,16—dibromo[2.2]paracyclophane (17) (6 g, 0.014 mol), di—tert—butyl (4—(4,4,5,5—tetramethyl—1,3,2—dioxaborolan—2—yl)—1,2— phenylene)dicarbamate (17.78 g, 0.032 mol), aq Cs2C03 solution (15.98 g, 0.049mol, in 66 mL water), and Pd(PPh3)4 (1.33 g, 0.0016 mol) was heated in a sealed tube at 80 0C for 16 h.

The reaction mixture was poured into water (250 mL) and the resulting precipitate was collected by filtration and washed with water. This crude material was purified by column chromatography (petroleum ether/ethyl acetate, 6:4 V/V) to afford the desired product (26) (5.0 g).

Step 4 BocHN HZN O BocHN O HZN ONHBoc O””2 NHBoc NH2 26 4TFA The tetra Boc—protected product from above (26) (10 g) was added to TFA (100 mL) at 0—5 0C. After completion of the addition, the reaction mixture was warmed to rt and stirred for 3 h. The reaction mixture was then concentrated and co—evaporated with DCM (3 X 50 mL). The crude material (27) was used directly in the next step.

Step 5 DIEA (1 mL, 0.0125 mol) was added dropwise to a cooled (0 OC) e of (tert—butoxycarbonyl)—5—azaspiro[2.4]heptane—6—carboxylic acid (0.5 g, 0.00057 mol), HOBt (0.35 g, 0.0026 mol), EDCI (0.5 g, 0.0026 mol), and the TFA salt (27) from above (0.343 g, 0.0014 mol) in DMF (5 mL) at 0 0C. After the addition was complete, the reaction mixture was allowed to warm to rt and stirred for 16 h. The on mixture was poured into water and the precipitate (28) was collected by filtration and purified by silica gel column chromatography to give the desired product (0.32 g).

Step 6 moN N Boc / “N AER 0 50¢ HN O “2“ O O . 0 O Boc 0 NH}?N N O NH2 H Acetic acid (5 mL) was added to the diamide product from above (28) (0.32g, 0.00035 mol) and heated at 45 0C for 4 h. The reaction mixture was evaporated and the residue was diluted with ethyl acetate (95 mL), then washed with aq NaHCO3 (2 X 25 mL) and water (2 X 30 mL). The organic layer was separated, dried over NaZSO4, and evaporated under reduced pressure. The crude material was purified by column chromatography to give the desired product (29) (0.2 g).

AWN” 0“maxAM: 0$1ij 4HCl The boc—protected product from above (0.09 g) was dissolved in DCM (0.9 mL) and cooled at 0 OC. Then 4 N HCl/dioxane (0.9 mL) was added dropwise. The reaction mixture was warmed to rt and stirred for 3 h. Then the volatiles were removed under vacuum and co—evaporated with DCM (3 X 50 mL). The remaining crude material (30) was used directly in the next step t further cation.

Step 8 u H/N O WO :3qu i / 4H(,l The hydrochloride salt (30) from above (0.011 g, 0.0000141 mol, 1.0 equiv) was dissolved in DMF (1 mL) and cooled to 0 0C. To this cooled on were added (S)—2— ((methoxycarbonyl)amino)—3—methylbutanoic acid (0.0062 g, 0.000033 mol, 2.5 equiv), HOBt (0.0044 g, 33 mol, 2.5 equiv) and EDCI 3 g, 0.000033 mol, 2.5 equiv).

DIEA (0.02 mL, 0.00013 mol, 10 equiv) was then added dropwise at the same temperature.

The reaction mixture was allowed to warm to rt and stirred for 16 h. The on mixture was then poured into water (25 mL) and the precipitated solid was collected by filtration, dried, and purified by silica gel column chromatography to give the desired product (31) (3 mg). 1H NMR (400 MHz, DMSO-d6, rt): 50.91—104 (m, 6H), 1.21—1.59 (m, 6H), 1.64—1.88 (m, 6H), 1.90—2.07 (m, 4H), 2.23—2.49 (m, 6H), 2.62 (m, 2H), 2.85 (m, 2H), 3.08 (m, 2H), 3.19 (s, 3H), 3.20 (s, 3H), 3.42 (m, 2H), 3.57 (s, 6H), 4.15 (apparent t, J: 8.0 Hz, 2H), 4.47 (m, 2H), 5.16 (apparent t, J: 8.5 Hz, 2H), 6.52 (apparent t, J: 8.5 Hz, 2H), 6.75 (s, 2H), 6.82 (m, 2H), 7.41 (m, 2H), 7.71 (apparent t, J = 6.0 Hz, 2H), 7.64—7.79 (m, 4H).

E 7. SYNTHESIS OF FERROCENE NSSA INHIBITORS (32) HN’£ : O M @3427 doFe / N 0 O /0 32 Ferrocene compounds are prepared by the method discussed in Butler, 1. R., et al., “A Convenient Preparation of Iodoferrocenes,” Polyhedron (1993) 12: 129—131.To a stirred solution of 1,1’—diiodoferrocene (220 mg, 0.5 mmol) in 1,4—dioxane (10 mL) was added methyl ((S)—3—methyl—1—oxo—1—((2S,3aS,7aS)—2—(5 —(4,4,5 ramethyl—1,3,2— dioxaborolan—2—yl)— 1H—benzo [d]imidazol—2—yl)octahydro—1H—indol— 1 —yl)butan—2—yl)carbamate (1.1 g, 4 equiv), K3PO4 (853 mg, 2 M aq solution, 8 equiv), and PdClzdppf (49 mg, 12 mol %) under an atmosphere of argon. The resulting mixture was subjected to microwave irradiation (CEM Discover System) at 80 0C for 1 h. The on mixture was allowed to cool to room temperature, ed, and concentrated in vacuo. The remaining residue was purified by preparative HPLC to give the d product as the trifluoroacetate salt (43 mg). 1H NMR (400 MHz, DMSO-d6, 27 °C): 50.63 (d, J: 6.5 Hz, 6H), 0.78 (d, J: 6.5 Hz, 6H), 1.13—1.49 (m, 6H), 1.55—1.87 (m, 10H), 1.95 (m, 2H), 2.26 (m, 2H), 2.35 (m, 2H), 2.42 (m, 2H), 3.48 (s, 6H), 3.83 (t, J: 8.0 Hz, 2H), 4.18 (m, 4H), 4.37 (m, 2H), 4.67 (m, 4H), 5.10 (dd, J: 10.0 Hz, 7.5 Hz, 2H), 7.39 (d, J: 9.0 Hz, 2H), 7.42 (d, J: 9.0 Hz, 2H), 7.45 (d, J: 8.0 Hz, 2H), 7.53 (s, 2H). 2012/039835 mg mmo mvo oi: H552: H H H Dw— IEA 2N 9:: £<mmz $80.0 Snood 33:3me flow E £<mmz Nfioooo. Nmoodv Nmoodv @052: H NE <AD§OE 8333 m0 8a onsausbm mQZDOmEOU qusomaoo A<ZOELHQQ< mEBozfl .w F28 mimzaflvmm .oZ ow in Nv WO 66716 m2 Hoe mew mow 3m: 352: 9:: £<mmz flow £<mmz mwmood wovood Nmoodv 3:83am -3238?.aHHoioéfiQumnméanmmvvaanmm: afioéu -H“N-DEEBEV$15223“méoafiéi3292223293:55” 33832333$33333-afioaémBQEu .oZ mw mw WO 66716 Ohm Ohm :w 3m: 352: 9:: £<mmz flow £<mmz Smoodv Nomood Smoodv 3:83am .oZ ow in WO 66716 mom 5mm mww 3m: 352: 9:: £<mmz flow £<mmz :uwood 3:83am .62 av Hm 2012/039835 m<a vHoH moofi Nvo DE: 352: 9:: £<mmz flow £<mmz mmmOOAv 3:83am .62 mm wm WO 66716 woo m8 Noe 3m: 352: 9:: £<mmz flow £<mmz $30.0 3:83am .62 mm mm WO 66716 woo coca 3m: 352: 9:: £<mmz flow £<mmz 3:83am .62 mm mm WO 66716 m<a vmofi mafia NNHH DE: 352: 9:: £<mmz flow £<mmz 33035 .62 oo No WO 66716 m2 Hmofi 3m: 352: 9:: £<mmz flow £<mmz 3:83am .62 mo we 2012/039835 DE: 352: 9:: £<mmz flow £<mmz moooood 3:83am .62 we we 2012/039835 (”M) EC50 Structure N0. 69 70 71 WO 66716 m<a whoa mvfifi DE: 352: 9:: £<mmz flow £<mmz 3:83am .62 mm mm vb WO 66716 DE: 352: 9:: £<mmz flow £<mmz 3:83am .62 mm on 55 WO 66716 3m: 352: 9:: £<mmz flow £<mmz 3:83am .62 mm mm Ow WO 66716 (”M) EC50 Structure N0. 81 82 83 WO 66716 DE: 352: 9:: £<mmz flow £<mmz 33035 .oZ ww mm cm WO 66716 0mm mom 3m: 352: 9:: £<mmz $20.0 wfiood flow £<mmz mmoood mvoood 3:83am .62 mm mm mm WO 66716 3m: 352: 9:: £<mmz flow £<mmz 33035 .62 oo 5 No WO 66716 DE: 352: 9:: £<mmz flow £<mmz 3:83am .62 mm mm WO 66716 m<a mNoH ooofi DE: 352: 9:: £<mmz flow £<mmz 33035 .62 cm no WO 66716 m2 mm: 3m: 352: 9:: £<mmz flow £<mmz @HSaUH—H—m WO 66716 m2 wNoH omofi mmo 3m: 352: 9:: £<mmz Cud flow £<mmz Smoodv @HSaUH—H—m 2012/039835 com www 3m: 352: 9:: £<mmz flow £<mmz @HSaUH—H—m WO 66716 m5 omm 05—: 352: H H 3 .SA au<mmz m: 9:: 5.0 flow au<mmz mwwood 3:83am .62 he we 08 WO 66716 DE: 352: 9:: £<mmz flow £<mmz 0000006 800006 33035 .62 o: H: N: M: WO 66716 DE: 352: 9:: £<mmz flow £<mmz 33035 .62 v: m: o: b: 2012/039835 DE: 352: 9:: £<mmz flow £<mmz @HSaUH—H—m WO 66716 DE: 352: 9:: £<mmz flow £<mmz 3:83am .oZ HNH NNH mg WO 66716 2 o ‘02 S w—< O (\l O\ w—< w—< (”M) EC50 Structure WO 66716 mmo coca 3m: 352: 9:: £<mmz flow £<mmz woooood @HSaUH—H—m WO 66716 m2 32 DE: 352: 9:: £<mmz flow £<mmz moooood @HSaUH—H—m 2012/039835 DE: 352: 9:: £<mmz flow £<mmz @HSaUH—H—m WO 66716 DE: 352: 9:: £<mmz flow £<mmz 3:83am .62 02 hmfi me WO 66716 (”M) EC50 Structure N0 139 140 WO 66716 (”M) EC50 Structure N0 141 142 WO 66716 3m: 352: 9:: £<mmz flow £<mmz 33035 .62 mi El mi WO 66716 (”M) EC50 Structure WO 66716 (”M) EC50 Structure N0 149 150 151 WO 66716 DE: 352: 9:: £<mmz flow £<mmz @HSaUH—H—m WO 66716 3m: 352: 9:: £<mmz flow £<mmz 3:83am .oZ m2 02 EXAMPLE 9. ASSAY FOR IDENTIFYING NDS WHICH INHIBIT HCV REPLICATION Compounds claimed herein are tested for the ability to t viral ation of the Hepatitis C replicon in cultured cells in which the HCV replicon uct has been incorporated. The HCV replicon system was described by Bartenschlager, et. al (Science, 285, pp. 110—1 13 (1999)). The replicon system is predictive of in vivo anti—HCV activity; compounds that are active in humans uniformly evidence activity in the replicon assay.

In this assay HCV replicon containing cells are treated with different concentrations of the test compound to ascertain the ability of the test compound to ss replication of the HCV replicon. As a positive control, HCV on—containing cells are treated with different concentrations of interferon alpha, a known inhibitor of HCV replication. The replicon assay system includes in Phosphotransferase (NPT) as a component of the replicon itself in order to detect the transcription of replicon gene ts in the host cell. Cells in which the HCV replicon is actively replicating have high levels of NPT; the level of NPT is proportional to HCV replication. Cells in which the HCV replicon is not replicating also have low levels of NPT and thus do not survive when treated with Neomycin. The NPT level of each sample is measured using a captured ELISA.

A protocol for testing compounds for the ability to inhibit viral replication of the Hepatitis C replicon cultured cells in which the replicon construct has been incorporated, 9A. HCV Replicon and Replicon Expression The HCV genome consists of a single ORF that encodes a 3000 amino acid polyprotein. The ORF is flanked on the 5' side by an untranslated region that serves as an internal me entry site (IRES) and at the 3' side by a highly conserved ce necessary for viral replication (3'—NTR). The structural proteins, necessary for viral infection, are located near the 5' end of the ORF. The non—structural proteins, designated NS2 to NSSB comprise the remainder of the ORF.

The HCV replicon contains, 5'—3', the HCV—IRES, the neomycin phosphotransferase (neo) gene, the IRES of encephalomyocarditis virus, which s translation of HCV sequences NS3 to NSSB, and the 3'—NTR. The sequence of the HCV replicon has been deposited in GenBank (Accession no. AJ242652).

W0 2012/166716 The on is transfected into Huh—7 cells using standard s such as electroporation. 9B. Cell Maintenance The equipment and materials include, but are not limited to, Huh—7 HCV replicon— containing cells, maintenance media (DMEM (Dulbecco's modified Eagle media) supplemented with 10% FBS, L—glutamine, non—essential amino acids, penicillin (100 units/ml), streptomycin (100 micrograms/ml), and 500 micrograms/ml of Geneticin (G418), screening media (DMEM mented with 10% FBS, L—glutamine, non—essential amino acids, penicillin (100 units/ml) and streptomycin (100 rams/ml)), 96 well tissue culture plates (flat bottom), 96 well plates (U bottom for drug dilution), Interferon alpha for positive control, fixation reagent (such as methanol: acetone), primary antibody (rabbit anti—NPTII), secondary antibody: Eu—N1 l, and enhancement solution.

HCV replicon—containing cells support high levels of viral RNA replicon replication when their density is suitable. Over—confluency causes decreased viral RNA replication. Therefore, cells must be kept growing in log phase in the presence of 500 micrograms/ml of G418. Generally, cells should be passed twice a week at 1: 4—6 on. Cell maintenance is conducted as follows: HCV replicon—containing cells are examined under a microscope to ensure that cells growing well. Cells are rinsed once with PBS and 2 ml trypsin is added. The cell/ trypsin mixture is incubated at 37 0C in a C02 incubator for 3—5 minutes. After incubation 10 ml of complete media is added to stop the nization reaction. Cells are blown gently, put into a 15 ml tube, and spun at 1200 rpm for 4 s. The trypsin/ medium solution is removed.

Medium (5 ml) is added and the cells are mixed carefully. The cells are counted.

The cells are then seeded onto 96—well plates at a density of 6000—7500 cells/100 microliters/ well (6—7.5 x 105 cells/10 ml/plate). The plates are then ted at 37 0C in a 5% CO2 incubator.

Cells are ed under a microscope approximated 24 hours after seeding and prior to adding drugs. If counting and dilution were performed correctly, cells are 60—70% nt and nearly all cells should attach and spread evenly in the well. 6C. ent of HCV—replicon containing cells with Test Compound HCV replicon—containing cells are rinsed with once PBS once; 2 mls of trypsin W0 2012/166716 are then added. Cells are incubated at 37°C in a 5% CO2 incubator for 3—5 minutes. 10 mls of complete medium is added to stop the reaction. Cells are blown gently, put into a 15 ml tube, and spun at 1200 rpm for four minutes. The trypsin/medium solution is removed and 5 mls of medium (500 ml DMEM (high glucose)) from BRL catalog #12430—054; 50 mls 10% PBS, 5% Geneticin G418 (50 mg/ml, BRL catalog #10131—035), 5 ml MEM non—essential amino acids (100x BRL #11140—050) and 5 ml pen—strep (BRL #15140—148) is added. The cells and media are mixed carefully Cells are plated with ing medium (500 ml DMEM (BRL #21063—029), 50 ml FBS (BRL #10082—147) and 5 ml MEM sential amino acid (BRL #11140—050) at 6000—7500 fcells/100 ul/well of 96 well plate (6—7.5x105 cells/10 te). Plates are placed into 37°C 5% C02 incubator overnight. 4D. Assay The following morning, drugs (test compounds or interferon alpha) are diluted in 96 well U bottom plates with media or DMSO/media, depending on the final concentration chosen for screening. lly for 6 concentrations of each test compounds ranging from 10 micromolar to 0.03 micromolar are applied. 100 ul of the test compound dilution is placed in wells of the 96 well plate containing the HCV replicon cells. Media without drug is added to some wells as a ve controls. DMSO is known to affect cell growth. Therefore, if drugs diluted in DMSO are used, all wells, including negative control (media only) and positive control (interferon alpha) wells, must n the same concentration of DMSO, for single dose screening. The plates are incubated at 37°C in a humidified 5% CO2 environment for three days.

On day four, the NTPII assay is quantitated. The medium is poured from the plates and the plates are washed once in 200 ul of PBS. The PBS is then decanted and the plates tapped in a paper towel to remove any remaining PBS. Cells are fixed in situ with 100 ul/well of pre—cooled (—20°C) methanol: e (1:1) and the plates are placed at —20°C for 30 minutes.

The fixing solution is poured from the plates and the plates d to air—dry completely (approximately one hour). The appearance of the dried cell layer is recorded and the density of the cells in the toxic wells is scored with the naked eye. Alternatively cell viability may be assessed using the MTS assay described below.

The wells are blocked with 200 ul of blocking solution (10% PBS; 3% NGS in PBS) for 30 minutes at room ature. The blocking on is removed and 100 ul of rabbit PTII diluted 1:1000 in blocking solution is added to each well. The plates are then ted 45—60 minutes at room temperature. After incubation, wells are washed six times with PBS—0.05% Tween—20 solution. 100 ul of 1:15 ,000 diluted Europium onjugated goat anti— rabbit in blocking buffer is added to each well and incubated at room temperature for 30—45 minutes. The plates are washed again and 100 ul of enhancement solution (Perkin Elmer #4001— 0010) is added to each well. Each plate is shaken x. 30 rpm) in a plate shaker for three minutes. 95 ul is transferred from each well to a black plate; the EU signal is tated in a —Elmer VICTOR plate reader (EU—Lance).

When tested in this assay Compounds 11, 16, 25, 33, 38, 39, and 40 t EC50 values of about 10 micromolar or less.

EXAMPLE 10. CYTOTOXICITY ASSAYS To insure that the decrease in replicon replication is due to compound activity against the HCV replicon rather than nonspecific toxicity assays are used to quantitate compound cytotoxicity. 10A. Cellular protein albumin assay for cytotoxicity Cellular protein albumin measurements provide one marker of cytotoxicity. The protein levels obtained from cellular albumin assays may also be used to provide a normalization reference for antiviral activity of compounds. In the protein albumin assay HCV replicon— containing cells are treated for three days with different concentrations of helioxanthin; a compound that is known to be cytotoxic at high concentrations. The cells are lysed and the cell lysate used to bind plate—bound goat anti—albumin antibody at room temperature (25 0C to 28 0C) for 3 hours. The plate is then washed 6 times with 1X PBS. After washing away the unbound proteins, mouse monoclonal uman serum albumin is applied to bind the albumin on the plate. The complex is then detected using atase—labeled anti—mouse IgG as a second antibody. 10B. MTS Assay for Cytotoxicity Cell viability may also be determined by CELLTITER 96 AQUEOUS ONE Solution Cell eration Assay (Promega, Madison WI), a colorimetric assay for ining the number of viable cells. In this method, before fixing the cells, 10—20 ul MTS reagent is added to each well according to manufacturer's instructions, plates are incubated at 37°C and read at OD 490 nm. During the incubation period living cells covert the MTS reagent to a formazan product which absorbs at 490 nm. Thus the 490 nm absorbance is directly proportional to the number of living cells in culture.

A direct comparison of the Cellular Albumin and MTS methods for determining cytotoxicity may be obtained as s: Cells are d with ent concentrations of test compound or Helioxanthin for a three day—period. Prior to lysis for detection albumin as described above, the MTS reagent is added according to manufacturer's instruction to each well and incubate at 37 0C and read at OD 490 nm. The cellular albumin quantitation is then performed as described above.

Claims (19)

What we claim is:

1. A compound of the a: T-R-J1-W-A-W-J1-R-T; T-R-J1-A-J1-R-T; T-R-J2-A-J2-R-T; T-R-J1-W-A-J1-R-T; T-R-J1-W-A-J2-R-T; or T-R-J1-A-J2-R-T; wherein T is independently chosen at each occurrence from T1 and T2; T1 is –Y-Z, where Y is covalently bound to R and Y is a bond or C1-C4alkylene optionally substituted with oxo; and Z is a 5 or 6-membered heterocyclic group, each of which T1 is substituted with (i) at least one tuent selected from -(C=O)OH, -(C=O)NH2, -(C=O)H, alkoxy, C2-C4alkanoyl, C1-C4alkylester, C1-C4alkenylester, and mono- or di-C1-C4alkylcarboxamide and (ii) optionally substituted with one or more substituents independently chosen from halogen, hydroxyl, lkyl, and C1-C2alkoxy; T2 is independently chosen at each occurrence from C2-C6alkanoyl, lkylester, C1-C6alkenylester, C1-C6alkylsulfonamide, C1-C6alkylsulfonyl, C2-C6alkanoyl substituted with mono- or C6hydrocarbylcarbamate, lkanoyl substituted with urea or mono- or di-C1-C6alkylurea, and C2-C6alkanoyl tuted with mono- or di-C1-C6alkylcarboxamide, each of which T2 is optionally substituted with 1 or more tuents independently chosen from amino, cyano, hydroxyl, halogen, (C1-C4alkoxy)C0-C4alkyl, (mono- and di-C1-C4alkylamino)C0-C4alkyl, C1-C6alkyl, (C1-C4thioalkyl)C0-C4alkyl, C3-C7cycloalkyl, phenyl, C1-C2haloalkyl, and C1-C2haloalkoxy; R is independently chosen at each occurrence from 4- to 6-membered rings containing one or two nitrogen atoms with remaining ring atoms being carbon, which R is saturated or contains 1 rated bond and is optionally bridged with an methylene or ethylene bridge, or fused to a phenyl or 5- to 6-membered heteroaryl ring; and 6- to 10-membered fused or spiro bicyclic ring systems containing one or two nitrogen atoms with remaining ring atoms being carbon, which 6- to 10-membered bicyclic ring is saturated or contains 1 unsaturated bond; each R is optionally substituted with one or more substituents independently chosen from cyano, hydroxyl, halogen, C1-C2alkyl, C1-C2alkoxy, C1-C2haloalkyl, C1-C2haloalkyl, C1-C2haloalkylene, and C1-C2alkylsulfonyl; J1 is a group, where each J1 is optionally substituted with amino, cyano, hydroxyl, halogen, C1-C4alkyl, C1-C4alkoxy, mono- and di-C1-C4alkylamino, C1-C2haloalkyl, or C1-C2haloalkoxy; J2 is a benzimidazole group, wherein J2 is optionally substituted with one or more substituents ndently chosen from amino, cyano, hydroxyl, halogen, C1-C4alkyl, C1- C4alkoxy, mono- and di-C1-C4alkylamino, C1-C2haloalkyl, and C1-C2haloalkoxy; W is independently chosen at each occurrence and is a phenyl, l or alkynyl group, ally substituted with one or more substituents independently chosen from amino, cyano, hydroxyl, halogen, C1-C4alkyl, C1-C4alkoxy, mono- and di- C1-C4alkylamino, C1-C2haloalkyl, and C1-C2haloalkoxy; and A is a [2.2]-cyclophane, where each 2 atome linker of the [2.2]-cyclophane ally ns a heteroatom selected from N, O, and S and is optionally substituted with 1 oxo group, and one or more substituents independently chosen from halogen, hydroxy, amino, C1-C2alkyl, and C1-C2alkoxy.

2. A compound or salt of Claim 1, wherein A is any one of ; ; or .

3. A compound or salt of Claim 1 or Claim 2 wherein W is phenyl, optionally substituted with one or more substituents independently chosen from halogen, C1-C2alkyl, and C1-C2alkoxy.

4. A compound or salt of any one of Claims 1 to 3, wherein J1 is

5. A compound or salt of any one of Claims 1 to 4 wherein J2 is a benzimidazole group, ally substituted with one or more substituents independently chosen from n, C1-C2alkyl, and C1-C2alkoxy.

6. A nd or salt of any one of Claims 1 to 5 wherein each R is independently chosen from , , , , , , , , , , , , , , each of which is optionally substituted with one or more substituents independently chosen from halogen, C1-C4alkyl, and C1-C4alkoxy.

7. A compound or salt of Claim 6 wherein each R is ndently chosen from , , and .

8. A compound or salt of any one of Claims 1 to 7 wherein T is independently chosen from C2-C6alkanoyl substituted with mono- or di-C1-C6alkylcarbamate, each of which T is optionally substituted with (C1-C4thioalkyl)C0-C4alkyl.

9. A compound or salt of Claim 1, of the formula T-R-J2-A-J2-R-T; where A is a group of the formula each R is an ndently chosen 8- to 10-membered bicyclic ring systems containing one or two nitrogen atoms with remaining ring atoms being carbon, which 8- to 10-membered bicyclic ring is saturated or contains 1 unsaturated bond; each R is optionally substituted with one or more substituents independently chosen from cyano, hydroxyl, halogen, lkyl, C1-C2alkoxy, C1-C2haloalkyl, C1-C2haloalkyl, C1-C2haloalkylene, and C1-C2alkylsulfonyl; and T is T2.

10. A nd or pharmaceutically acceptable salt thereof of Claim 1, wherein the compound is , or

11. A compound of Claim 1 of the formula or a pharmaceutically able salt thereof.

12. A compound of Claim 1 of the formula or a pharmaceutically able salt thereof.

13. A pharmaceutical composition comprising a compound or salt of any one of Claims 1 to 12, together with a pharmaceutically acceptable carrier.

14. The pharmaceutical composition of Claim 13, wherein the composition comprises at least one additional active agent.

15. Use of a compound or salt thereof of any one of Claims 1 to 12, in the manufacture of a medicament for treating a tis C infection.

16. A compound of the a T-R-J1-W-A-W-J1-R-T; T-R-J1-A-J1-R-T; -A-J2-R-T; T-R-J1-W-A-J1-R-T; T-R-J1-W-A-J2-R-T; or T-R-J1-A-J2-R-T; as defined in Claim 1, and substantially as hereinbefore described with reference to the accompanying examples.

17. A pharmaceutical composition comprising a compound or salt of Claim 16, together with a pharmaceutically acceptable carrier.

18. The ceutical composition of Claim 17, wherein the composition comprises at least one additional active agent.

19. Use of a compound or salt thereof of Claim 16, in the manufacture of a medicament for treating a hepatitis C infection. Achillion Pharmaceuticals, Inc. by the patent attorneys for the applicant CULLENS