Imidazo 4,5-c pyridine compounds and methods of antiviral treatment

FIELD OF THE INVENTION

The present invention relates to a series of novel imidazo[4,5-c]pyridine compounds, processes for their preparation, their use to treat or prevent viral infections and their use to manufacture a medicine to treat or prevent viral infections, particularly infections with viruses belonging to the family of the Flaviviridae and Picornaviridae and more preferably infections with hepatitis-C-virus (HCV).

BACKGROUND OF THE INVENTION

The family of the Flaviviridae consists of 3 genera, the pestiviruses, the flaviviruses and the hepaciviruses and also contains the hepatitis G virus (HGV/GBV-C) that has not yet been assigned to a genus. Pestiviruses such as the Classical Swine Fever Virus (CSFV), the Bovine Viral Diarrhea Virus (BVDV) and the Border Disease Virus (BDV) cause infections of domestic livestock (respectively pigs, cattle and sheep) and are responsible for significant economic losses world-wide. BVDV, the prototypic representative of the pestivirus genus is ubiquitous and causes a range of clinical manifestations, including abortion, teratogenesis, respiratory problems, chronic wasting disease, immune system dysfunction, and predisposition to secondary viral and bacterial infections and may also cause acute fatal disease. Fetuses of cattle can be infected persistently with BVDV, these animals remain viremic throughout life and serve as a continuous source for virus spread in herds.

Vaccines are used in some countries with varying degrees of success to control pestivirus disease. In other countries, animal culling and slaughter are used to contain pestivirus disease outbreaks.

The World Health Organization estimates that world-wide 170 million people (3% of the world's population) are chronically infected with HCV. These chronic carriers are at risk of developing cirrhosis and/or liver cancer. In studies with a 10 to 20 year follow-up, cirrhosis developed in 20-30% of the patients, 1 to 5% of who may develop liver cancer during the next then years. The only treatment option available today is the use of interferon α-2 (or its pegylated from) either alone or combined with ribavirin. However, sustained response is only observed in about 40% of the patients and treatment is associated with serious adverse effects. There is thus an urgent need for potent and selective inhibitors of the replication of the HCV in order to treat infections with HCV. Furthermore, the study of specific inhibitors of HCV replication has been hampered by the fact that it is not possible to propagate HCV (efficiently) in cell culture. Since HCV and pestiviruses belong to the same virus family and share many similarities (organization of the genome, analogous gene products and replication cycle), pestiviruses have been adopted as a model and surrogate for HCV. For example, BVDV is closely related to hepatitis C virus (HCV) and used as a surrogate virus in drug development for HCV infection.

The compound 3-[((2-dipropylamino)ethyl)thio]-5H-1,2,4-triazino[5,6-b]indole has been reported to selectively inhibit the replication of BVDV and other pestiviruses (Baginski S G et al., Proc. Natl. Acad. Sci. U.S.A. 2000 Jul. 5; 97(14):7981-6). Currently, there is no treatment strategy available for controlling infections caused by pestiviruses.

Coxsackie viruses belong to the group of the enteroviruses, family of the Picornaviridae. They cause a heterogeneous group of infections including herpangina, aseptic meningitis, a common-cold-like syndrome, a non-paralytic poliomyelitis-like syndrome, epidemic pleurodynia (an acute, febrile, infectious disease generally occurring in epidemics), hand-foot-mouth syndrome, pediatric and adult pancreatitis and serious myocarditis.

Currently only pleconaril (3-13,5-dimethyl-4-[[3-methyl-5-isoxazolyl)propyl]phenyl]-5-(trifluoromethyl-1,2,4-oxadiazole)) and enviroxime (2-amino-1-(isopropylsulfonyl)-6-benzimidazole phenyl ketone oxime) have been studied clinically for the treatment of infections with enteroviruses. Pleconaril is a so called “capsid function-inhibitor”; enviroxime prevents the formation of the RNA replicative intermediate. Enviroxime resulted in only modest clinical and virological benefit in some studies and no benefits in others. Clinical response with pleconaril has been observed in some studies, but the compound has not been approved by the Food and Drug Administration (hearing of Mar. 18, 2002).

Relevant disclosures include U.S. Pat. Nos. 4,914,108; 4,988,707; 4,990,518; 5,137,896; 5,208,242; 5,227,384; 5,302,601; 5,374,638; 5,405,964; 5,438,063; 5,486,525; 6,479,508; and U.S. Patent Publication No. US2003/0108862 A1, Canadian Patent No. 2423800 A1, German Patent Nos. 4211474 A1, 4236026, 4309969, 4318813, European Patent Nos. EP 0 138 552 A2, EP 0 706 795 A2, EP 1 132 381 A1, Great Britain Patent No. 2158440 A, PCT Patent Publication Nos. WO 00/20416, WO 00/39127, WO 00/40583, WO 03/007945 A1, WO 03/010140 A2, WO 03/010141 A2, WO 93/02080, WO 93/14072, WO 96/11192, WO 96/12703, WO 99/27929, Akamatsu, et al., New Efficient Route for Solid-Phase Synthesis of Benzimidazole Derivatives”, 4:475-483, J. COMB. CHEM, 2002, Cleve et al., “Derivate des Imidazo[4,5-b]- und Imidazo[4,5-c]pyridins”, 747:158-171, JUSTUS LIEBIGS AANALEN DER CHEMICA, 1971, Kiyama, et al., “Synthesis and Evaluation of Novel Nonpeptide Angiotensin II Receptor Antagonists: Imidazo[4,5-c]pyridine Derivatives with an Aromatic Substituent”, 43(3):450-60, CHEM PHARM BULL, 1995, Mederski et al., “Synthesis and Structural Assignment of Some N-substituted Imidazopyridine Derivatives”, 48(48):10549-58, TETRAHEDRON, 1992, Yutilov et al., 23(1):56-9, KHIMIKO-FARMATSEVTICHESKII ZHURNAL, 1989. The disclosures of all citations set forth herein are expressly incorporated by reference to the extent such disclosures are relevant to the contents herein.

A need exists for compounds having antiviral and other desirable properties, such as bioavailability, efficacy, nontoxicity, optimal clearance, potency and the like. In particular, a need exists for compounds having selective activity against viruses belonging to the family of Flaviviridae including hepatitis C virus, and against viruses belonging to the family of Picornaviridae. These and other objects of this invention will be apparent to one skilled in the art from consideration of this specification as a whole.

SUMMARY OF THE INVENTION

An embodiment of the present invention provides compounds having the general formula (A),

embedded image

wherein:

the dotted lines represent an optional double bond, provided that no two double bonds are adjacent to one another, and that the dotted lines represent at least 3, optionally 4 double bonds;

R¹is selected from hydrogen, aryl, heterocyclic, C₁-C₁₀alkoxy, C₁-C₁₀thioalkyl, C₁-C₁₀alkyl-amino, C₁-C₁₀dialkyl-amino, C_3-10cycloalkyl, C_4-10cycloalkenyl, and C_4-10cycloalkynyl, wherein each are optionally substituted with 1 or more R⁶;

Y is selected from single bond, O, S(O)_m, NR¹¹, or C_1-10alkylene, C_2-10alkenylene, C_2-10alkynylene, wherein each may optionally include 1 to 3 heteroatoms selected from O, S or N;

R²and R⁴are independently selected from hydrogen, C_1-18alkyl, C_2-18alkenyl, C_2-18alkynyl, C_1-18alkoxy, C_1-18alkylthio, halogen, —OH, —CN, —NO₂, —NR⁷R⁸, haloalkyloxy, haloalkyl, —C(═O)R⁹, —C(═S)R⁹, SH, aryl, aryloxy, arylthio, arylalkyl, C_1-18hydroxyalkyl, C_3-10cycloalkyl, C_3-10cycloalkyloxy, C_3-10cycloalkylthio, C_3-10cycloalkenyl, C_7-10cycloalkynyl, or heterocyclic, provided that when one of R²⁵or R²⁶is present, then either R²or R⁴is selected from (═O), (═S), and ═NR²⁷;

X is selected from C₁-C₁₀alkylene, C_2-10alkenylene or C_2-10alkynylene, where each may include one or more heteroatoms selected from O, S, or N, provided any such heteroatom is not adjacent to the N in the ring;

m is any integer from 0 to 2;

R³is selected from aryl, aryloxy, arylthio, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl-N(R¹⁰)—, or heterocyclic, where each said substituent may be optionally substituted with at least one R¹⁷, provided that for cycloalkenyl the double bond is not adjacent to a nitrogen, and provided R³-M-Q is not biphenyl;

R⁵is selected from hydrogen; C_1-18alkyl, C_2-18alkenyl, C_2-18alkynyl, C_1-18alkoxy, C_1-18alkylthio, halogen, —OH, —CN, —NO₂, —NR⁷R⁸, haloalkyloxy, haloalkyl, —C(═O)R⁹, —C(═O)OR⁹, —C(═S)R⁹, SH, aryl, aryloxy, arylthio, arylalkyl, C_1-18hydroxyalkyl, C_3-10cycloalkyl, C_3-10cycloalkyloxy, C_3-10cycloalkylthio, C_3-10cycloalkenyl, C_7-10cycloalkynyl, or heterocyclic;

R⁶is selected from hydrogen, C_1-18alkyl, C_2-18alkenyl, C_2-18alkynyl, C_1-18alkoxy, C_1-18alkylthio, C_1-18alkylsulfoxide, C_1-18alkylsulfone, C_1-18halo-alkyl, C_2-18halo-alkenyl, C_2-18halo-alkynyl, C_1-18halo-alkoxy, C_1-18halo-alkylthio, C_3-10cycloalkyl, C_3-10cycloalkenyl, C_7-10cycloalkynyl, halogen, OH, CN, cyanoalkyl, —CO₂R¹⁸, NO₂, —NR⁷R⁸, C_1-18haloalkyl, C(═O)R¹⁸, C(═S)R¹⁸, SH, aryl, aryloxy, arylthio, arylsulfoxide, arylsulfone, arylsulfonamide, aryl(C_1-18)alkyl, aryl(C_1-18)alkyloxy, aryl(C_1-18)alkylthio, heterocyclic, C_1-18hydroxyalkyl, where each may be optionally substituted with at least 1 R¹⁹;

R⁷and R⁸are independently selected from hydrogen, C_1-18alkyl, C_1-18alkenyl, aryl, C_3-10cycloalkyl, C_4-10cycloalkenyl, heterocyclic, —C(═O)R¹²; —C(═S)R¹², an amino acid residue linked through a carboxyl group thereof, or where R⁷and R⁸together with the nitrogen form a heterocyclic;

R⁹and R¹⁸are independently selected from hydrogen, OH, C_1-18alkyl, C_2-18alkenyl, C_3-10cycloalkyl, C_4-10cycloalkenyl, C_1-18alkoxy, —NR¹⁵R¹⁶, aryl, an amino acid residue linked through an amino group of the amino acid, CH₂OCH(═O)R^9a, or CH₂OC(═O)OR^9awhere R^9ais C₁-C₁₂alkyl, C₆-C₂₀aryl, C₆-C₂₀alkylaryl or C₆-C₂₀aralkyl;

R¹⁰and R¹¹are independently selected from the group consisting of hydrogen, C_1-18alkyl, C_2-18alkenyl, C_3-10cycloalkyl, C_4-10cycloalkenyl, aryl, —C(═O)R¹², heterocyclic, or an amino acid residue;

R¹²is selected from the group consisting of hydrogen, C_1-18alkyl, C_2-18alkenyl, aryl, C_3-10cycloalkyl, C_4-10cycloalkenyl, or an amino acid residue;

R¹³and R¹⁴are independently selected from hydrogen, C_1-18alkyl, C_2-18alkenyl, aryl, C_3-10cycloalkyl, C_4-10cycloalkenyl, —C(═O)R¹², —C(═S)R¹², or an amino acid residue;

R¹⁵and R¹⁶are independently selected from hydrogen, C_1-18alkyl, C_2-18alkenyl, C_2-18alkynyl, aryl, C_3-10cycloalkyl, C_4-10cycloalkenyl, or an amino acid residue;

R¹⁷is independently M-Q- wherein M is a ring optionally substituted with 1 or more R¹⁹, and Q is a bond or a linking group connecting M to R³having 1 to 10 atoms and optionally substituted with 1 or more R¹⁹;

R¹⁹is selected from hydrogen, C_1-18alkyl, C_2-18alkenyl, C_2-18alkynyl, C_1-18alkoxy, C_2-18alkenyloxy, C_2-18alkynyloxy, C_1-18alkylthio, C_3-10cycloalkyl, C_4-10cycloalkenyl, C_4-10cycloalkynyl, halogen, —OH, —CN, cyanoalkyl, —NO₂, —NR²⁰R²¹, C_1-18haloalkyl, C_1-18haloalkyloxy, —C(═O)R¹⁸, —C(═O)OR¹⁸, —OalkenylC(═O)OR¹⁸, —OalkylC(═O)NR²⁰R²¹, —OalkylOC(═O)R¹⁸, —C(═S)R¹⁸, SH, —C(═O)N(C_1-6alkyl), —N(H)S(O)(O)(C_1-6alkyl), aryl, heterocyclic, C_1-18alkylsulfone, arylsulfoxide, arylsulfonamide, aryl(C_1-18)alkyloxy, aryloxy, aryl(C_1-18alkyl)oxy, arylthio, aryl(C_1-18)alkylthio or aryl(C_1-18)alkyl, where each may be optionally substituted with 1 or more ═O, NR²⁰R²¹, CN, C_1-18alkoxy, heterocyclic, C_1-18haloalkyl, heterocyclic alkyl, heterocyclic connected to R¹⁷by alkyl, alkoxyalkoxy or halogen;

R²⁰and R²¹are independently selected from hydrogen, C_1-18alkyl, C_2-18alkenyl, C_2-18alkynyl, aryl, C_3-10cycloalkyl, C_4-10cycloalkenyl, —C(═O)R¹², or —C(═S)R¹²;

R²²is selected from hydrogen, —OH, C_1-18alkyl, C_2-18alkenyl, C_1-18alkoxy, —NR²³R²⁴, aryl, C_3-10cycloalkyl, and C_4-10cycloalkenyl;

R²³and R²⁴are independently selected from hydrogen, C_1-18alkyl, or a heterocyclic formed by taking C_2-3alkyl together with N of R²², which heterocyclic is optionally substituted with OH or aryl, or an amino acid residue linked through a carboxyl group of the amino acid;

R²⁵and R²⁶are not present, or are independently selected from hydrogen, C_1-18alkyl, C_3-10cycloalkyl, aryl, heterocyclic, where each is optionally independently substituted with 1 to 4 of C_1-6alkyl, C_1-6alkoxy, halo, CH₂OH, benzyloxy, and OH; and

R²⁷is selected from hydrogen, C_1-18alkyl, C_3-10cycloalkyl, (C_3-10cycloalkyl)-C_1-6alkyl, aryl, and aryl C_1-18alkyl, and

salts, tautomers, isomers and solvates thereof.

Another embodiment of the present invention provides compounds having the general formula (A),

embedded image

wherein:

the dotted lines represent an optional double bond, provided that no two double bonds are adjacent to one another, and that the dotted lines represent at least 3, optionally 4 double bonds;

Y is selected from single bond, O, S(O)_m, NR¹¹, or C_1-10alkylene, C_2-10alkenylene, C_2-10alkynylene, wherein each may optionally include 1 to 3 heteroatoms selected from O, S or N;

m is any integer from 0 to 2;

R³is a heterocycle optionally substituted with at least one R¹⁷provided, however, that R³optionally substituted with at least one R¹⁷is not pyridinyl or 5-chlorothienyl, provided that R³-MQ is not biphenyl;

R⁶is selected from hydrogen, C_1-18alkyl, C_2-18alkenyl, C_2-18alkynyl, heterocyclic, C_1-18alkoxy, C_1-18alkylthio, C_1-18alkylsulfoxide, C_1-18alkylsulfone, C_1-18halo-alkyl, C_2-18halo-alkenyl, C_2-18halo-alkynyl, C_1-18halo-alkoxy, C_1-18halo-alkylthio, C_3-10cycloalkyl, C_3-10cycloalkenyl, C_7-10cycloalkynyl, halogen, OH, CN, cyanoalkyl, —CO₂R¹⁸, NO₂, —NR⁷R⁸, C_1-18haloalkyl, C(═O)R¹⁸, C(═S)R¹⁸, SH, aryl, aryloxy, arylthio, arylsulfoxide, arylsulfone, arylsulfonamide, aryl(C_1-18)alkyl, aryl(C_1-18)alkyloxy, aryl(C_1-18)alkylthio, C_1-18hydroxyalkyl, where each may be optionally substituted with at least 1 R¹⁹;

R¹²is selected from the group consisting of hydrogen, C_1-18alkyl, C_2-18alkenyl, aryl, C_3-10cycloalkyl, C_4-10cycloalkenyl, or an amino acid residue;

R¹³and R¹⁴are independently selected from hydrogen, C_1-18alkyl, C_2-18alkenyl, aryl, C_3-10cycloalkyl, C_4-10cycloalkenyl, —C(═O)R¹², —C(═S)R¹², or an amino acid residue;

R¹⁵and R¹⁶are independently selected from hydrogen, C_1-18alkyl, C_2-18alkenyl, C_2-18alkynyl, aryl, C_3-10cycloalkyl, C_4-10cycloalkenyl, or an amino acid residue;

R¹⁷is independently selected from the group consisting of hydrogen, C_1-18alkyl, C_2-18alkenyl, C_2-18alkynyl, C_1-18alkoxy, C_1-18alkylthio, C_1-18alkylsulfoxide, C_1-18alkylsulfone, C_1-18halogenated alkyl, C_2-18halogenated alkenyl, C_2-18halogenated alkynyl, C_1-18halogenated alkoxy, C_1-18halogenated alkylthio, C_3-10cycloalkyl, C_3-10cycloalkenyl, C_7-10cycloalkynyl, halogen, OH, CN, CO₂H, CO₂R¹⁸, NO₂, NR⁷R⁸, haloalkyl, C(═O)R¹⁸, C(═S)R¹⁸, SH, aryl, aryloxy, arylthio, arylsulfoxide, arylsulfone, arylsulfonamide, arylalkyl, arylalkyloxy, arylalkylthio, heterocyclic, C_1-18hydroxyalkyl, where each of said aryl, aryloxy, arylthio, arylsulfoxide, arylsulfone, arylsulfonamide, arylalkyl, arylalkyloxy, arylalkylthio, heterocycle, or C_1-18hydroxyalkyl is optionally substituted with 1 or more R¹⁹;

R²⁰and R²¹are independently selected from hydrogen, C_1-18alkyl, C_2-18alkenyl, C_2-18alkynyl, aryl, C_3-10cycloalkyl, C_4-10cycloalkenyl, —C(═O)R¹², carboxylester-substituted heterocyclic or —C(═S)R¹²;

R²²is selected from hydrogen, —OH, C_1-18alkyl, C_2-18alkenyl, C_1-18alkoxy, —NR²³R²⁴, aryl, C_3-10cycloalkyl, and C_4-10cycloalkenyl;

R²⁷is selected from hydrogen, C_1-18alkyl, C_3-10cycloalkyl, (C_3-10cycloalkyl)-C_1-6alkyl, aryl, and aryl C_1-18alkyl, and

the salts, tautomers, isomers and solvates thereof.

An embodiment of the present invention provides compounds having the general formula (A),

embedded image

wherein:

the dotted lines represent an optional double bond, provided that no two double bonds are adjacent to one another, and that the dotted lines represent at least 3, optionally 4 double bonds;

Y is selected from single bond, O, S(O)_m, NR¹¹, or C_1-10alkylene, C_2-10alkenylene, C_2-10alkynylene, wherein each may optionally include 1 to 3 heteroatoms selected from O, S or N;

m is any integer from 0 to 2;

R³is a heterocycle optionally substituted with at least one R¹⁷, provided R³-M-Q is not biphenyl;

R⁹and R¹⁸are independently selected from hydrogen, OH, C_1-18alkyl, C_2-18alkenyl, C_3-10cycloalkyl, C_4-10cycloalkenyl, C_1-18alkoxy, —NR¹⁵R¹⁶, aryl, an amino acid residue linked through an amino group of the amino acid, CH₂OCH(═O)R^9a, or CH₂C(═O)OR^9awhere R^9ais C₁-C₁₂alkyl, C₆-C₂₀aryl, C₆-C₂₀alkylaryl or C₆-C₂₀aralkyl;

R¹²is selected from the group consisting of hydrogen, C_1-18alkyl, C_2-18alkenyl, aryl, C_3-10cycloalkyl, C_4-10cycloalkenyl, or an amino acid residue;

R¹³and R¹⁴are independently selected from hydrogen, C_1-18alkyl, C_2-18alkenyl, aryl, C_3-10cycloalkyl, C_4-10cycloalkenyl, —C(═O)R¹², —C(═S)R¹², or an amino acid residue;

R¹⁵and R¹⁶are independently selected from hydrogen, C_1-18alkyl, C_2-18alkenyl, C_2-18alkynyl, aryl, C_3-10cycloalkyl, C_4-10cycloalkenyl, or an amino acid residue;

R¹⁷is M-Q-, wherein M is a C_3-10cycloalkyl optionally substituted with 1 or more R¹⁹, and Q is a bond, or C_1-10alkyl optionally substituted with 1 or more R¹⁹;

R²⁰and R²¹are independently selected from hydrogen, C_1-18alkyl, C_2-18alkenyl, C_2-18alkynyl, aryl, C_3-10cycloalkyl, C_4-10cycloalkenyl, —C(═O)R¹², or —C(═S)R¹²;

R²²is selected from hydrogen, —OH, C_1-18alkyl, C_2-18alkenyl, C_1-18alkoxy, —NR²³R²⁴, aryl, C_3-10cycloalkyl, and C_4-10cycloalkenyl;

R²³and R¹⁴are independently selected from hydrogen, C_1-18alkyl, or a heterocyclic formed by taking C_2-3alkyl together with N of R²², which heterocyclic is optionally substituted with OH or aryl, or an amino acid residue linked through a carboxyl group of the amino acid;

R²⁷is selected from hydrogen, C_1-18alkyl, C_3-10cycloalkyl, (C_3-10cycloalkyl)-C_1-6alkyl, aryl, and aryl C_1-18alkyl, and

the salts, tautomers, isomers and solvates thereof.

Yet another embodiment of the present invention provides compounds having the formula (B),

embedded image

wherein:

the dotted lines represent an optional double bond, provided that no two double bonds are adjacent to one another, and that the dotted lines represent at least 3, optionally 4 double bonds; and R¹, R², R³, R⁴, R⁵, R²⁵, R²⁶, X and Y are as disclosed above.

An embodiment of the present invention provides compounds of the formula (B) wherein Y is a single bond, and R¹is aryl.

Another embodiment of the present invention provides compounds of formula (B) wherein X is C₁-C₁₀alkylene, C_2-10alkenylene or C_2-10alkynylene.

Another embodiment of the present invention provides compounds of formula (B) wherein R³is heterocylic.

Another embodiment of the present invention provides compounds of formula (B) wherein R³is heterocyclic substituted with R¹⁷where Q is a bond and M is aryl.

Another embodiment of the present invention provides compounds of formula (B) wherein Y is a single bond, and R¹is phenyl.

Another embodiment of the present invention provides compounds of formula (B) wherein R³is isoxazole substituted with R¹⁷where Q is a bond and M is aryl.

Another embodiment of the present invention provides compounds of formula (B) wherein R³is isoxazole substituted with R¹⁷where Q is a bond and M is phenyl.

Yet another embodiment of the present invention provides compounds having the formula (C),

embedded image

wherein R¹, R², R³, R⁴, R⁵, R²⁵, R²⁶, X and Y are as disclosed above.

An embodiment of the present invention provides compounds of the formula (C) wherein Y is a single bond, and R¹is aryl.

Another embodiment of the present invention provides compounds of formula (C) wherein X is C₁-C₁₀alkylene, C_2-10alkenylene or C_2-10alkynylene.

Another embodiment of the present invention provides compounds of formula (C) wherein R³is heterocylic.

Another embodiment of the present invention provides compounds of formula (C) wherein R³is heterocyclic substituted with R¹⁷where Q is a bond and M is aryl.

Another embodiment of the present invention provides compounds of formula (C) wherein Y is a single bond, and R¹is phenyl.

Another embodiment of the present invention provides compounds of formula (C) wherein R³is isoxazole substituted with R¹⁷where Q is a bond and M is aryl.

Another embodiment of the present invention provides compounds of formula (C) wherein R³is isoxazole substituted with R¹⁷where Q is a bond and M is phenyl.

The compounds of formula (A) are optionally combined with pharmacologically acceptable excipients.

The compounds of formula (A) are administered in therapeutically effective amounts to subjects (humans or animals) in need of antiviral therapy, in particular for inhibiting the infection, growth or replication of Flaviviridae and Picornaviridae, especially BVDV, HCV and Coxsackie virus.

The invention further relates to a method of screening antiviral compounds which comprises providing a compound of formula (A) and determining the anti-viral activity of said compound.

Also within the scope of the invention is a metabolite of the compounds of formula (A) made by the process of administering a compound of formula (A) to a subject and recovering the metabolite from the subject.

The invention also comprises a method for structure-activity determination of analogues of formula (A) compounds

embedded image

wherein the substituents are defined in WO 2004/005286, comprising

- (A) preparing a compound of formula (A) in which at least one substituent is not disclosed by WO 2004/005286; and
- (B) determining the anti-HCV activity of the compound of step (a).

DETAILED DESCRIPTION OF THE INVENTION

“Alkyl” means saturated hydrocarbon moiety where the moiety may be acyclic, cyclic or a combination of acyclic and cyclic portions. The acyclic portion may contain 1 to 3 carbon atoms, and each ring may contain 3 to 6 carbon atoms (for example, 3-methylcyclohexyl). Within this definition, the term “cycloalkyl” refers to the saturated hydrocarbon moieties that are cyclic. Examples of “alkyl” include methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl (i-Bu), 2-butyl (s-Bu) 2-methyl-2-propyl (t-Bu), 1-pentyl (n-pentyl), 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclooctyl and the like, or a C_7-10polycyclic saturated hydrocarbon radical having from 7 to 10 carbon atoms such as, for instance, norbornyl, fenchyl, trimethyltricycloheptyl or adamantyl.

“Alkenyl” means a hydrocarbon moiety with at least one site of double bond unsaturation where the moiety may be acyclic, cyclic or a combination of acyclic and cyclic portions. The acyclic portion may contain 1 to 3 carbon atoms, and each cyclic portion may contain 3 to 6 carbon atoms. A site of double bond unsaturation may be in a acyclic portion, a cyclic portion. In the instance of a moiety having a combination of acyclic and cyclic portions, there may be a site of double bond unsaturation in each of the portions. Within this definition, the term “cycloalkenyl” refers to the double bond unsaturated hydrocarbon moieties that are cyclic. Examples the term “alkenyl” include, but are not limited to, ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl (—C₅H₇), 5-hexenyl (—CH₂CH₂CH₂CH₂CH═CH₂), 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and 1-cyclohex-3-enyl. The double bond optionally is in the cis or trans configuration.

“Alkynyl” means a hydrocarbon moiety with a least one site of triple bond unsaturation where the moiety may be acyclic, cyclic or a combination of acyclic and cyclic portions. The acyclic portion may contain 1 to 3 carbon atoms, and each cyclic portion may contain 7 or more carbon atoms. Within this definition, the term “cycloalkynl” refers to triple bond unsaturated hydrocarbon moieties that are cyclic. Examples of the term “alkynyl” include, but are not limited to, —C≡CH, —CH₂C≡CH, —CH₂C≡C-cyclohexyl, or —CH₂-cycloheptynyl.

The suffix “-ene” used in connection with alkyl, alkenyl and alkynyl groups refers to such groups with at least 2 sites of substitution. Such polyvalent hydrocarbon radicals include, but are not limited to, methylene (—CH₂—) 1,2-ethylene (—CH₂CH₂—), 1,3-propylene (—CH₂CH₂CH₂—), 1,4-butylene (—CH₂CH₂CH₂CH₂—), 1,2-ethylene (—CH═CH—), —C□C—, propargyl (—CH₂C□C—), and 4-pentynyl (—CH₂CH₂CH₂C□CH—).

“Aryl” means an aromatic hydrocarbon containing 1 or more rings, generally 1, 2 or 3, with 4 to 6 carbon atoms in each, ordinarily 5 or 6 carbon atoms.

“Arylalkyl,” “arylalkenyl” and “arylalkynyl” means an alkyl, alkenyl or alkynyl radical, respectively, in which one of the hydrogen atoms, typically a terminal or sp3 carbon atom, is replaced with an aryl radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like.

As noted, carbocycles optionally are found as single rings or multiple ring systems. Ordinarily the hydrocarbons of the compounds of formula (A) are single rings. Monocyclic carbocycles generally have 3 to 6 ring atoms, still more typically 5 or 6 ring atoms. Bicyclic carbocycles typically have 7 to 12 ring atoms, e.g. arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms arranged as a bicyclo [5,6] or [6,6] system.

If the number of carbon atoms is unspecified for a hydrocarbon, typically the number of carbon atoms will range from 1 to 18, except that the number of carbons typically will range from 2 to 18 for unsaturated hydrocarbons and from 6 to 10 for aryl.

“Heterocyclic” or “heterocycle” means any 4, 5, 6, 7, 8 or 9 membered single or fused ring system containing one or more heteroatoms selected from the group consisting of O, N or S. Heterocycles optionally are entirely aromatic, entirely saturated, or contain 1 or more intra-ring sites of unsaturation, typically double bonds. Multiple heterocyclic rings (one or more of which contains a heteroatom) are bridged or spiro. Generally, the heterocyclic rings will be aromatic, and usually they are single rings. Examples of heterocycles include oxazacyloalkyl, morpholinyl, dioxacycloalkyl, thiacycloalkenyl, pyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl, furanyl, thienyl, pyrrolyl, pyranyl, pyrazolyl, pyrazolidinyl, pyrazolinyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, piperazinyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isothiazoledinyl, isoxazolyl, oxazolinyl, pyrazinyl, pyridazinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazoly, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, benzothienyl, benzothiazolyl and isatinoyl. Other suitable heterocycles are exemplified in Rigaudy et al., Nomenclature of Organic Chemistry, Sections A-H (1979) at pp. 53-76 and Fletcher et al., Nomenclature of Organic Compounds, Adv. Chem. Ser. 126 (1974) at pp 49-64.

The location on the heterocycle which provides the point of attachment(s) to the rest of the compound of this invention is not critical, but those skilled in the art will recognize substitution sites that are optimal for compound stability and/or ease of synthesis. Carbon bonded heterocycles typically are bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. Still more typically, carbon bonded heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

Nitrogen containing heterocycles are bonded at nitrogen or a carbon, typically a carbon atom. These include, for example, position 1 of aziridine, 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, 1-piperidinyl, 2-pyrroline, 3-pyrroline, 2-imidazoline, 3-imidazoline, 9-carbazole, 4-morpholine, 9-alpha or β-carboline, 2-isoindole, 2-pyrazoline and 3-pyrazoline, and by analogy, azetidine, pyrrole, pyrrolidine piperidine, piperazine, indole, pyrazoline, indoline, imidazole, imidazolidine, 1H-indazole and isoindoline. These and other N-containing heterocycles are well-known to those skilled in the art, and their linkage sites are a matter of discretion.

Sulfur containing heterocycles are bonded through carbon or sulfur. They include oxidized states such as —S(═O)(═O). In general, they are linked in the compounds of formula (A) analogous to N-containing heterocycles.

“Alkoxy”, “cycloalkoxy”, “aryloxy”, “arylalkyloxy”, “oxy heterocycle”, “thioalkyl”, “thiocycloalkyl”, “arylthio”, and “arylalkylthio” means substituents wherein an alkyl, cycloalkyl, aryl, or arylalkyl, respectively, are attached to an oxygen atom or a sulfur atom through a single bond, such as but not limited to methoxy, ethoxy, propoxy, butoxy, thioethyl, thiomethyl, phenyloxy, benzyloxy, mercaptobenzyl and the like.

“Halogen” means any atom selected from the group consisting of fluorine, chlorine, bromine and iodine.

Any substituent designation that is found in more than one site in a compound of this invention shall be independently selected.

When a group is stated to be substituted with “one or more” of another group, this typically means 1 to 3 substituents, ordinarily 1, 2 or 3 substitutents.

Those of skill in the art will also recognize that the compounds of the invention may exist in many different protonation states, depending on, among other things, the pH of their environment. While the structural formulae provided herein depict the compounds in only one of several possible protonation states, it will be understood that these structures are illustrative only, and that the invention is not limited to any particular protonation state—any and all protonated forms of the compounds are intended to fall within the scope of the invention.

Amino Acids

“Amino-acid” refers to a radical derived from a molecule having the chemical formula H₂N—CHR²⁸—COOH, wherein R²⁸is a side group of a naturally-occurring or known synthetic amino-acid. The amino acids optionally are substituted with hydrocarbon typically of 1 to 8 carbons at one or more carboxyl or amino groups, whether those groups are on the side chain or are free after linking the amino acid to the remainder of the compound of this invention.

Optionally the amino acid residue is a hydrophobic residue such as mono- or di-alkyl or aryl amino acids, cycloalkylamino acids and the like. Optionally, the residue does not contain a sulfhydryl or guanidino substituent.

Naturally-occurring amino acid residues are those residues found naturally in plants, animals or microbes, especially proteins thereof. Polypeptides most typically will be substantially composed of such naturally-occurring amino acid residues. These amino acids are glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, glutamic acid, aspartic acid, lysine, hydroxylysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, asparagine, glutamine and hydroxyproline. Additionally, unnatural amino acids, for example, valanine, phenylglycine and homoarginine are also included.

Generally, only one of any site in the parental molecule is substituted with an amino acid, although it is within the scope of this invention to introduce amino acids at more than one permitted site. In general, the α-amino or α-carboxyl group of the amino acid are bonded to the remainder of the molecule, i.e., carboxyl or amino groups in the amino acid side chains generally are not used to form the amide bonds with the parental compound (although these groups may need to be protected during synthesis of the conjugates).

The amino acid esters optionally are hydrolyzable in vivo or in vitro under acidic (pH<3) or basic (pH>10) conditions. Optionally, they are substantially stable in the gastrointestinal tract of humans but are hydrolyzed enzymatically in blood or in intracellular environments.

R²⁸usually is C₁-C₆alkyl or C₁-C₆alkyl substituted with amino, carboxyl, amide, carboxyl (as well as esters, as noted above), hydroxyl, C₆-C₇aryl, guanidinyl, imidazolyl, indolyl, sulfhydryl, sulfoxide, and/or alkylphosphate. R²⁸also is nitrogen to form a proline residue taken together with the amino acid α-However, R²⁸is generally the side group of the naturally-occurring amino acid disclosed above, for example H, —CH₃, —CH(CH₃)₂, —CH₂—CH(CH₃)₂, —CHCH₃—CH₂—CH₃, —CH₂—C₆H₅, —CH₂CH₂—S—CH₃, —CH₂OH, —CH(OH)—CH₃, —CH₂—SH, —CH₂—C₆H₄OH, —CH₂—CO—NH₂, —CH₂—CH₂—CO—NH₂, —CH₂—COOH, —CH₂—CH₂—COOH, —(CH₂)₄—NH₂and —(CH₂)₃—NH—C(NH₂)—NH₂. R²⁸also includes 1-guanidinoprop-3-yl, benzyl, 4-hydroxybenzyl, imidazol-4-yl, indol-3-yl, methoxyphenyl and ethoxyphenyl.

Exemplary Embodiments

R¹is generally aryl or aromatic heterocyle substituted with 1, 2 or 3 R⁶wherein R⁶is halogen, C_1-18alkoxy; or C_1-18haloalkyl. Typically, R¹is phenyl substituted with 1, 2 or 3 halogens, usually fluoro.

Y generally is a single bond, O, C_1-6alkylene, C_2-6alkenylene, C_2-6alkynylene or one of said groups containing 1 to 3, usually 1, heteroatoms selected from O, S or NR¹¹. Examples include —O(CH₂)_1-5—, —(CH₂)_1-4—O—(CH₂)_1-4—, —S—(CH₂)_1-5—, —(CH₂)_1-4—S—(CH₂)_1-4—, —NR¹¹—(CH₂)_1-5—, —(CH₂)_1-4—NR¹¹—(CH₂)_1-4or C_3-10cycloalkylidene. Typically, Y is —OCH₂—, —CH₂O—, C_1-2alkylene, C_2-3alkenylene, C_2-3alkynylene, O or a bond, but usually a bond.

In general, YR¹is not any one of H, an unsubstituted C_3-10cycloalkyl or C1-C6 alkyl. Typically YR¹is halo or halomethyl-substituted (typically trihalomethyl)phenyl (and usually 1 to 2 substituents in ortho or meta).

X usually is alkylene, alkynylene or alkenylene, typically alkylene, or said hydrocarbons having an intrachain heteroatom, typically O or S. Examples include —CH₂—, —CH(CH₃)—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂, —(CH₂)_2-4—O—(CH₂)_2-4—, —(CH₂)_2-4—S—(CH₂)_2-4—, —(CH₂)_2-4—NR¹⁰—(CH₂)_2-4—, C_3-10cycloalkylidene, C_2-6alkenylene (such as —CH═CH—CH₂—) and C_2-6alkynylene. Usually, X is methylene.

R³generally is aryl or a heterocycle, typically an aromatic heterocycle. The heterocycle generally will contain 1, 2 or 3 N, S or O atoms in the ring, usually is linked to X through a ring carbon atom and typically contains 4 to 6, usually 5, total ring atoms. The R³aryl or heterocycle ordinarily is substituted with 1, 2 or 3, usually 1, R¹⁷. R³optionally is not indolyl.

When R³is substituted with R¹⁷then R¹⁷typically is aryl or a heterocycle further substituted with 1 or more, usually 1, 2 or 3, R¹⁹.

R¹⁷is M-Q in some embodiments of the invention. M is a ring. This means any cyclic organic structure, whether carbocyclic or heterocyclic, and whether saturated, unsaturated or aromatic or single or fused ring systems. M is chosen from rings that are structurally stable in biological systems. In general, M is a aryl or aromatic heterocycle where heterocycle is defined above.

Q is a spacer group, and is not critical. Typically it is not cyclic and contains from no to 3 atoms, generally C, O or S, usually C or O.

R¹⁷typically is selected from the group consisting of C_3-10cycloalkyl, C_3-10cycloalkenyl, C_7-10cycloalkynyl, halogen, aryl, aryloxy, arylthio, arylsulfoxide, arylsulfone, arylsulfonamide, arylalkyl; arylalkyloxy (optionally an benzyloxy); arylalkylthio (optionally a benzylthio); a heterocycle; C_1-18hydroxyalkyl, but typically is an aryl or a heterocycle, and where each of said aryl, aryloxy, arylthio, arylsulfoxide, arylsulfone, arylsulfonamide, arylalkyl, arylalkyloxy, arylalkylthio, or heterocycle is optionally substituted with 1 or more R¹⁹. R¹⁷generally is positioned distally to X. Optionally, R¹⁷is not C(O)R¹⁸.

R⁹and R¹⁸typically are H, OH or alkyl. R¹⁸optionally is not NR¹⁵R¹⁶.

R⁵typically is H.

R⁶generally is halogen. Optionally, R⁶is not C(O)R¹⁸.

R⁷, R⁸, R¹⁰, R¹¹, R¹³, R¹⁴, R¹⁵, R¹⁶, R²⁰, R²¹, R²³and R²⁴typically are independently H or C_1-18alkyl.

R¹²and R²²typically are independently OH or alkyl.

R¹⁹usually is H; C_1-18alkyl; C_2-18alkenyl; C_2-18alkynyl; C_1-18alkoxy; alkenyloxy; alkynyloxy; C_1-18alkylthio; C_3-10cycloalkyl; C_4-10cycloalkenyl; C_4-10cycloalkynyl; halogen; OH; CN; cyanoalkyl; NO₂; NR²⁰R²¹; haloalkyl; haloalkyloxy; C(═O)R¹⁸; C(═O)OR¹⁸; OalkenylC(═O)OR¹⁸; —OalkylC(═O)NR²⁰R²¹; aryl; heterocycle; —OalkylOC(═O)R¹⁸; C(═O)N(C_1-6alkyl), N(H)S(O)(O)(C_1-6alkyl); arylalkyloxy; aryloxy; arylalkyloxy; and arylalkyl; each of which is unsubstituted or substituted with 1 or more ═O; NR²⁰R²¹; CN; alkoxy; heterocycle; haloalkyl- or alkyl-substituted heterocycle; heterocycle linked to R¹⁷by alkyl; alkoxyalkoxy or halogen. R¹⁸as a substituent in here is generally not H. R¹⁹typically is independently halogen, N(R²⁰R²¹), alkoxy or halo-substituted alkyl or alkoxy.

R²⁵and R²⁶usually are not present but if they are then typically they are cyclopentyl or cyclohexyl. If the compound is substituted at R²⁵or R²⁶, either R²or R⁴is selected from (═O), (═S), and (═NR²⁷), usually ═O.

M typically is an aromatic ring, usually single or two fused rings, and containing 4 to 10 atoms. Usually, M is hydrocarbon, but also optionally comprises 1 to 3 N, O and/or S heteroatoms.

Q usually is a hydrocarbon chain, typically a normal or secondary alkylene, which optionally comprises at least one oxy or thio ester. Generally Q is 1 to 6 atoms, usually 1 to 3. Q typically is not substituted with R¹⁹, but if it is then typically it is substituted with one R¹⁹. R¹⁹as substituted on Q usually is halogen, nitro or cyano. Substituents optionally are designated with or without bonds. Regardless of bond indications, if a substituent is polyvalent (based on its position in the structure referred to), then any and all possible orientations of the substituent are intended.

Haloalkyl or haloalkyloxy typically are —CF3 or —OCF3.

The present invention provides a compound of Formula (A) of the following the structure,

embedded image

having antiviral activity as determined following the procedures taught throughout the

Specification, such as in Part B “Methodology For Determination Of Antiviral And Cytostatic Activity” in the Examples Section. Preparation of this compound is taught throughout the Specification, such as in Example 6.

The present invention further provides a compound of Formula (A) of the following structure,

embedded image

having antiviral activity as determined following the procedures taught throughout the Specification, such as in Part B “Methodology For Determination Of Antiviral And Cytostatic Activity” in the Examples Section. Preparation of this compound is taught throughout the Specification, such as in Example 8A.

Formula (A) depicts optional single or double bonds. It will be understood that the bonds are present such that the aromatic nature of the nucleus of formula (A) is preserved, i.e., these formulas are intended to embrace all possible tautomers. For example R²⁵or R²⁶will be absent if the ring N to which they are bonded as indicated in the formula is linked to a flanking ring carbon atom by a double bond. On the other hand, R²⁵or R²⁶may be present when the N atom to which it is bonded as indicated in the formula is linked to its flanking carbon atoms by single bonds only; in this case aromaticity is accommodated by other substituents, e.g. where R²or R⁴is oxo.

The term “prodrug” as used herein refers to any compound that when administered to a biological system generates the drug substance, i.e. active ingredient, as a result of spontaneous chemical reaction(s), enzyme catalyzed chemical reaction(s), photolysis, and/or metabolic chemical reaction(s). A prodrug is thus a covalently modified analog or latent form of a therapeutically-active compound.

Prodrugs

Certain of the compounds herein when substituted with appropriate selected functionalities are capable of acting as prodrugs. These are labile functional groups which separate from an active inhibitory compound during metabolism, systemically, inside a cell, by hydrolysis, enzymatic cleavage, or by some other process (Bundgaard, Hans, “Design and Application of Prodrugs” in Textbook of Drug Design and Development (1991), P. Krogsgaard-Larsen and H. Bundgaard, Eds. Harwood Academic Publishers, pp. 113-191). These prodrug moieties can serve to enhance solubility, absorption and lipophilicity to optimize drug delivery, bioavailability and efficacy. A “prodrug” is thus a covalently modified analog of a therapeutically-active compound. A prodrug moiety of course can be therapeutically active in its own right.

Exemplary prodrug moieties include the hydrolytically sensitive or labile esters (—CO₂R′) of carboxylic acids (—CO₂H) or other functional groups with an acidic proton which is bound to the imidazo[4,5-c]pyridine compounds of the invention. The R′ group of such hydrolytically sensitive or labile esters may include: (i) acyloxymethyl esters —CH₂C(═O)R^9a; and (ii) acyloxymethyl carbonates —CH₂C(═O)OR^9awhere R^9ais C₁-C₆alkyl, C₁-C₆substituted alkyl, C₆-C₂₀aryl or C₆-C₂₀substituted aryl. A close variant of the acyloxyalkyl ester, the alkoxycarbonyloxyalkyl ester (carbonate), may also enhance oral bioavailability as a prodrug moiety in the compounds of the invention. An exemplary acyloxymethyl ester R group is pivaloyloxymethoxy, (POM) —CH₂C(═O)C(CH₃)₃. An exemplary acyloxymethyl carbonate prodrug moiety is pivaloyloxymethylcarbonate (POC) —CH₂OC(═O)OC(CH₃)₃. Cleavable moieties capable of acting as prodrug functionalities are optionally linked at any tolerant site on the compound of this invention, for example R³and any of its substituents.

Excluded Compounds

The present invention excludes all compounds expressly disclosed in any prior art reference (to the extent the reference is effective as novelty- or inventive step/obviousness-defeating as the case may be) set forth in this application (as well as any compounds disclosed in any reference patent family member) and any other compounds over which the claims of this application are not novel or do not posses an inventive step or are obvious under applicable law.

The present invention excludes, as required, compounds according to the general formula (A) where

(a) Any of the substituents X, Y, R¹, R², R³, R⁴, R⁵are a cephalosporin or wherein the substituents X, Y, R¹, R², R³, R⁴, R⁵are an azabicyclo group, more particularly 5-Thia-1-aza-bicyclo[4.2.0]oct-2-en-8-one;

(b) The compound is 5-(2-piperidin-1-yl-ethyl)-2-(4-hydroxyphenyl)-1H-imidazo[4,5-c]pyridin-5-ium bromide (X=ethyl, Y=bond, R¹=phenyl substituted in para with OH, R²=H, R³=piperidinyl, and R⁴, R⁵=H) (as disclosed in example 52 of EP 1132381);

(c) The compound is 4-[5-(2-{4-[Bis-(4-fluorophenyl)-methyl]-piperazin-1-yl}-ethyl)-5H-imidazo[4,5-c]pyridin-2-yl]phenol (X=ethyl, Y=bond, R¹=phenyl substituted in para with OH, R²=H, R³=heterocycle with 2 N heteroatoms, wherein one N is substituted with an arylalkyl consisting of CH(Phenyl)₂, wherein each phenyl carries an F in para) (as disclosed in example 54 of EP 1132381);

(d) The compound is 4-[5-(3-{4-[Bis-(4-fluorophenyl)-methyl]-piperazin-1-yl}-propyl)5H-imidazo[4,5-c]pyridin-2-yl]phenol (X=butyl, Y=bond, R¹=phenyl substituted in para with OH, R²=H, R³=heterocycle with 2 N heteroatoms, wherein one N is substituted with an arylalkyl consisting of CH(Phenyl)₂, wherein each phenyl carries an F in para) (as disclosed in example 55 of EP 1132381);

(e) The compound is 5-(phenylmethyl)-5H-imidazo[4,5-c]pyridine wherein phenyl is substituted with CONR¹⁵R¹⁶and R¹⁵is a branched C3 alkyl and R¹⁶is phenyl (X=—CH₂—; Y=bond; R¹=hydrogen; R²=H; R³=phenyl substituted with 1 C(═O)R¹⁸, wherein R¹⁸is NR¹⁵R¹⁶, with R¹⁵and R¹⁶a branched C₆alkyl; R⁴=H) (as disclosed in example 35 of U.S. Pat. No. 5,302,601);

(f) The compound is 6-(5H-imidazo[4,5-c]pyridin-5-yl-methyl)-N-(1methylethyl)-N-phenyl-3-pyridinecarboxamide (X=—CH₂—; Y=bond; R¹=hydrogen; R²=H, R³=pyridine substituted with 1 R⁶, wherein R⁶=1 C=0 R¹⁸, wherein R¹⁸is NR¹⁵R¹⁶, wherein R¹⁵=isopropyl and R¹⁶=phenyl) (as disclosed in example 6 of U.S. Pat. No. 4,990,518);

(g) The compound is a compound wherein X=—CH²—; Y=bond; R¹=hydrogen; R²=H, R³=5-6 membered heterocycle, in particular a pyridinyl or furanyl, substituted with 1 R¹⁷wherein R¹⁷=C(═O)R¹⁸, and wherein R¹⁸=NR¹⁵R¹⁶and R¹⁵and R¹⁶are either a C_1-18alkyl, in particular methyl, ethyl or isopropyl, C_2-18alkenyl, in particular 2-methyl allyl, or a C_3-10cycloalkyl, in particular cyclopentyl or cyclohexyl (as disclosed in U.S. Pat. No. 4,990,518);

(h) The compound is a compound wherein X=—CH²—; Y=bond; R¹=hydrogen; R²=H, R³=5-6 membered heterocycle, in particular a pyridinyl or furanyl, substituted with 1 R¹⁷wherein R¹⁷=C(═O)R¹⁸, and wherein R¹⁸=C_3-10cycloalkyl or C_4-10cycloalkenyl.

(i) The compound is 2,6-bis(1,1,-dimethylethyl)-4-[[2-(5H-imidazo-[4,5-c]pyridin-5-yl)ethyl]thio]-phenol hydrate and/or 2,6-bis(1,1,-dimethylethyl)-4-[[2-(5H-imidazo-[4,5-c]pyridin-5-yl)propyl]thio]-phenol hydrate (X=CH²—CH²—; Y=bond; R¹=hydrogen, R²=H, R³=thioaryl substituted with three R⁶, wherein R⁶=2 branched C₄alkyl in meta and OH in para) (as disclosed in example 6 of WO96/12703);

(j) The compound is 5-[2-(Biphenyl-4-yloxy)-ethyl]-5H-imidazo[4,5-c]pyridine (X=CH₂CH₂, Y=bond, R¹=hydrogen, R²=H, R³=phenoxy substituted with 1 R¹⁷in para, wherein R¹⁷=benzyl; R⁴=H) (as disclosed in WO96/11192);

(k) The compound is 5-[2-(4-Phenoxy-phenoxy)-ethyl]-5H-imidazo[4,5-c]pyridine (X=CH₂CH₂, Y=bond, R¹=hydrogen, R²=H, R³=phenoxy substituted with 1 R¹⁷in para, wherein R¹⁷=phenoxy; R⁴=H) (as disclosed in WO96/11192);

(l) The compound is [5-(4-Fluorobenzyl)-5H-imidazo[4,5-c]pyridin-2-yl]-methylamine (X=CH₂, Y=NR¹¹, wherein R¹¹=methyl, R¹=R²=H, R³=phenyl substituted with 1 R¹⁷in para, wherein R⁶is F, R⁴=H, R⁵=H) (as disclosed in EP76530);

(m) The compound is 2,6-bis(1,1,-dimethylethyl)-4-[[3-(5H-imidazo-[4,5-c]pyridin-5-yl)propyl]thio]-phenol hydrate (X=CH₂—CH₂—CH₂, Y=bond; R¹=hydrogen, R²=H, R³=thiophenyl substituted with 3 R⁶, wherein R⁶=2 branched C4 alkyl in meta and OH in para) (as disclosed in WO96/12703);

(n) The compound is 5-[2-(4-Phenylmethyloxy-phenoxy)-ethyl]-5H-imidazo[4,5-c]pyridine (X=CH₂CH₂, Y=bond, R¹=hydrogen, R²=H, R³=phenoxy substituted with 1 R¹⁷in para, wherein R¹⁷=benzyl oxy) (as disclosed in WO96/11192);

(o) The compound is 5-[3-(4-Phenoxy-phenoxy)-propyl]-5H-imidazo[4,5-c]pyridine (X=CH₂CH₂CH₂, Y=bond, R¹=hydrogen, R²=H, R³=phenoxy substituted with 1 R⁶in para, wherein R⁶=phenoxy substituted in para with F; R⁴=H) (as disclosed in WO96/11192);

(p) The compound is 5-{2-[4-(4-Fluorophenoxy)-phenoxy]-ethyl}-5H-imidazo[4,5-c]pyridine (X=CH₂CH₂, Y=bond, R¹=hydrogen, R²=H, R³=phenoxy substituted with 1 R⁶in para, wherein R⁶=phenoxy, substituted in para with F; R⁴=H) (as disclosed in WO96/11192);

(q) The compound is 5-[3-(4-Phenylmethyl-phenoxy)-propyl]-5H-imidazo[4,5-c]pyridine (X=CH₂CH₂CH₂, Y=bond, R¹=hydrogen, R²=H, R³=phenoxy substituted with 1 R⁶in para, wherein R⁶=benzyl; R⁴=H) (as disclosed in WO96/11192);

(r) The compound is (1H-Indol-3-yl)-[3-(2-methyl-5H-imidazo[4,5-c]pyridine-5-carbonyl)-phenyl]-methanone (X=—(C═O)— or SO₂, Y=CH₂, R¹=H, R²=H, R³=phenyl substituted with 1 R⁶, wherein R⁶is C(═O)R¹⁸, wherein R¹⁸is indole) (as disclosed in U.S. Pat. No. 5,486,525);

(s) The compound is 4 or 3-[(2-methyl-5H-imidazo[4,5-c]pyridin-5-yl)methyl]-benzoic acid alkylester or 5-[4 or 3-(alkoxycarbonyl-phenyl)-methyl]-2-methyl-5H-imidazo[4,5-c]pyridine, in particular 4 or 3-[(2-methyl-5H-imidazo[4,5-c]pyridin-5-yl)methyl]-methyl ester (X=CH₂, Y=CH₂, R¹=H, R²=H, R³=phenyl substituted at the para or meta position with one R¹⁷, wherein R¹⁷is (C═O)R¹⁸, wherein R¹⁸=alkoxy) (as disclosed in U.S. Pat. No. 5,486,525)

(t) The compound is 5-[(fluorophenyl)methyl]-2-amino-5-H-imidazo[4,5-c]-pyridine (XR³=fluorobenzyl, Y=NR¹¹with R¹¹=methyl, R¹=H, R², R³, R⁴=H) (as disclosed in U.S. Pat. No. 5,137,896);

(u) The compound is ((5-[4-(Fluorophenyl)methyl]-5-H-imidazo[4,5-c]-pyridine-2-yl)methyl)-carbamate, methyl ester (XR³=fluorobenzyl, Y═C(═O)R¹²with R¹²=methyl, R¹=H, R², R³, R⁴=H) (as disclosed in U.S. Pat. No. 5,137,896);

(v) The compound is 5-(4-Chlorophenylmethyl)-2-(piperidin-1-ylmethyl)-5H-imidazo[4,5-c]pyridine and its dihydrochloride salt (XR³=chlorobenzyl, Y=—CH₂—, R¹=piperidinyl) (as disclosed in Justus Liebigs Annalen der Chemie (1971), 747, 158-171);

(w) The compound is 5-(4-Chlorophenylmethyl)-2-(4-methyl-piperazin-1-ylmethyl)-5H-imidazo[4,5-c]pyridine (XR³=chlorobenzyl, Y=—CH₂—, R¹=piperazinyl, R⁶=methyl) (as disclosed in Journal of the Chemical Society [section B]: Physical Organic (1966), 4, 285-291);

(x) Compounds, particularly compound 9 on page 160, Cleve et al. “Liebigs Ann. Chem. 747:158-171 (1971);

(y) Compounds, particularly compounds 19 and 20, of Kiyama et al. “Chem. Pharm. Bull. 43(3):450-460 (1995); and

(z) Compounds, particularly compound 14, of Medereski et al. “Tet. Lt.”48(48):10549-10558 (1992)

The compounds of the invention optionally exclude those compounds according to the general formula (A) as described above, wherein (a) YR¹is not phenyl para substituted with OH, or (b) is H, an unsubstituted C_3-10cycloalkyl, or C_1-6alkyl.

The compounds of the invention optionally exclude those compounds according to the general formula (A) as described above, wherein R¹is not H, Y is not NR¹¹with R¹¹C_1-6alkyl or methyl, and/or YR¹is not monomethylamino.

The compounds of the invention optionally exclude those compounds according to the general formula (A) as described above, wherein R¹is a phenyl substituted with 1R⁶, R⁶is C(═O)R¹⁸and R¹⁸is t-butoxy.

The compounds of the invention optionally exclude those compounds according to the general formula (A) as described above, wherein R¹is not piperidinyl and is not piperazinyl substituted with methyl.

The compounds of this invention exclude those compounds disclosed by WO 2004/005286, in particular the compounds in table 8 thereof.

The compounds of this invention Optionally exclude those in which XR³is the definitional equivalent to the substructure —(CH₂)n-Y—C(O)—N(R1)(R2) set forth on column 1, line 49 to column 2 line 38 of U.S. Pat. No. 5,302,601 and the comparable disclosure in any member of the patent family of U.S. Pat. No. 5,302,601, which disclosure is herewith expressly incorporated by reference.

The compounds of this invention optionally exclude those in which R⁵contains any of the substituents designated as <<Ar>> in WO 00/39127, in particular aryl, aryl phenoxy, or benzyl.

The compounds of this invention optionally do not include the compounds of Example 35 of U.S. Pat. No. 5,302,601, Example 6 of U.S. Pat. No. 4,990,518, Examples 1 to 5 of U.S. Pat. No. 4,988,707, Examples 1-5 of U.S. Pat. No. 5,208,241, Example 39 of U.S. Pat. No. 5,137,896, the azabenzimidazole compound of WO 99/27929, Examples 1-20 and 45 of U.S. Pat. No. 5,227,384, Examples 3 and/or 11 of WO 96/12703 and/or compounds 340A, 347C, 349C, 351C, 355C and/or 356 C of WO 96/11192.

The compounds of this invention optionally exclude those in which XR³is equivalent to the substructure —(CH₂)n-Het-C(O)—N(R¹)(R²) set forth on column 1, line 41 to column 2 line 24 of U.S. Pat. No. 4,990,518.

The compounds of this invention do not include the compounds expressly disclosed in the patents listed in the Background of the Invention above, in Chemical Abstracts acc no. 1987:18435 and in Chemical Abstracts acc no. 1983:594812.

The compounds of this invention do not include the compounds expressly disclosed in Justus Liebigs Annalen der Chemie (1971), 747, 158-171 or in the Journal of the Chemical Society [section B]: Physical Organic (1966), 4, 285-291.

Optionally, the compounds of this invention exclude those compounds wherein YR¹is one of the substituents designated R¹³in column 5, lines 22-38 of U.S. Pat. No. 5,486,525 and/or R²and/or R⁵are one of the substituents collectively designated R¹⁴and R¹⁵in column 5, lines 38-53 of U.S. Pat. No. 5,486,525.

Optionally, the compounds of this invention exclude the compounds found in any patent family member of any published or issued patent specifically recited in this application.

Finally, the compounds of this invention optionally also exclude the methylene homologues of the foregoing known compounds excluded from the scope of this invention. It is understood that a compound optionally excluded also includes the salts thereof.

Utilities

The compounds of this invention, or the metabolites produced from these compounds in vivo, have a large number of uses. They are useful in immunology, chromatography, diagnostics and therapeutics, among other fields.

The compounds of formula (A) are conjugated to immunogenic polypeptides as a reagent for eliciting antibodies capable of binding specifically to the polypeptide, to the compounds or to their metabolic products which retain immunologically recognized epitopes (sites of antibody binding). These immunogenic compositions therefore are useful as intermediates in the preparation of antibodies for use in diagnostics, quality control, or the like, or in assays for the compounds of formula (A) or their novel metabolic products. The compounds are useful for raising antibodies against otherwise non-immunogenic polypeptides, in that the compounds serve as haptenic sites stimulating an immune response which cross-reacts with the unmodified conjugated protein.

Conjugates of the compounds of formula (A) with immunogenic polypeptides such as albumin or keyhole limpet hemocyanin generally are useful as immunogens. The polypeptides are conjugated at the same sites denoted for amino acids. The metabolic products described above may retain a substantial degree of immunological cross reactivity with the compounds of the invention. Thus, the antibodies of this invention will be capable of binding to the unprotected compounds of the invention without binding to the protected compounds. Alternatively the metabolic products will be capable of binding to the protected compounds and/or the metabolitic products without binding to the protected compounds of the invention, or will be capable of binding specifically to any one or all three. The antibodies desirably will not substantially cross-react with naturally-occurring materials. Substantial cross-reactivity is reactivity under specific assay conditions for specific analytes sufficient to interfere with the assay results.

The immunogens of this invention contain the compound of this invention presenting the desired epitope in association with an immunogenic substance. Within the context of the invention such association means covalent bonding to form an immunogenic conjugate (when applicable) or a mixture of non-covalently bonded materials, or a combination of the above. Immunogenic substances include adjuvants such as Freund's adjuvant, immunogenic proteins such as viral, bacterial, yeast, plant and animal polypeptides, in particular keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor, and immunogenic polysaccharides. Typically, the compound having the structure of the desired epitope is covalently conjugated to an immunogenic polypeptide or polysaccharide by the use of a polyfunctional (ordinarily bifunctional) cross-linking agent. Methods for the manufacture of hapten immunogens are conventional per se, and any of the methods used heretofore for conjugating haptens to immunogenic polypeptides or the like are suitably employed here as well, taking into account the functional groups on the precursors or hydrolytic products which are available for cross-linking and the likelihood of producing antibodies specific to the epitope in question as opposed to the immunogenic substance.

Typically the polypeptide is conjugated to a site on the compound of the invention distant from the epitope to be recognized.

The conjugates are prepared in conventional fashion. For example, the cross-linking agents N-hydroxysuccinimide, succinic anhydride or alkN═C═Nalk are useful in preparing the conjugates of this invention. The conjugates comprise a compound of the invention attached by a bond or a linking group of 1-100, typically, 1-25, more typically 1-10 carbon atoms to the immunogenic substance. The conjugates are separated from starting materials and by products using chromatography or the like, and then are sterile filtered and vialed for storage.

Animals are typically immunized against the immunogenic conjugates or derivatives and antisera or monoclonal antibodies prepared in conventional fashion.

The compounds of this invention are useful as linkers, spacers or affinity (typically hydrophobic) moieties in preparing affinity absorption matrices. The compounds of the invention optionally are bound covalently to an insoluble matrix and used for affinity chromatography separations, depending on the nature of the groups of the compounds, for example compounds with pendant aryl groups are useful in making hydrophobic affinity columns.

They also are useful as linkers and spacers in preparing immobilized enzymes for process control, or in making immunoassay reagents. The compounds herein contain functional groups that are suitable as sites for cross-linking desired substances. For example, it is conventional to link affinity reagents such as hormones, peptides, antibodies, drugs, and the like to insoluble substrates. These insolubilized reagents are employed in known fashion to absorb binding partners for the affinity reagents from manufactured preparations, diagnostic samples and other impure mixtures. Similarly, immobilized enzymes are used to perform catalytic conversions with facile recovery of enzyme. Bifunctional compounds are commonly used to link analytes to detectable groups in preparing diagnostic reagents.

The compounds of this invention are labeled with detectable moieties such biotin, radioisotopes, enzymes and the like for diagnostic purposes. Suitable techniques for accomplishing the labeling of the compounds of formula (A) are well known and will be apparent to the artisan from consideration of this specification as a whole. For example, one suitable site for labeling is R17 or R19.

More typically, however, the compounds of the invention are employed for the treatment or prophylaxis of viral infections such as yellow fever virus, Dengue virus, hepatitis B virus, hepatitis G virus, Classical Swine Fever virus or the Border Disease Virus, but more particularly flaviviral or picornaviral infections, in particular, HCV and BVDV.

The therapeutic compound(s) of this invention are administered to a subject mammal (including a human) by any means well known in the art, i.e. orally, intranasally, subcutaneously, intramuscularly, intradermally, intravenously, intra-arterially, parenterally or by catheterization. The therapeutically effective amount of the compound(s) is a flaviviral or picornaviral growth inhibiting amount. More preferably, it is a flaviviral or picornaviral replication inhibiting amount or a flaviviral or picornaviral enzyme inhibiting amount of the compounds of formula (A). This is believed to correspond to an amount which ensures a plasma level of between about 1 μg/ml and 100 mg/ml, optionally of 10 mg/ml. This optionally is achieved by administration of a dosage of in the range of 0.001 mg to 60 mg, preferably 0.01 mg to 10 mg, preferably 0.1 mg to 1 mg per day per kg bodyweight for humans. These are starting points for determining the optimal dosage of the compound of this invention. The actual amount will depend upon many factors known to the artisan, including bioavailability of the compound, whether it contains a prodrug functionality, its metabolism and distribution in the subject and its potency, among others. It typically is necessary to determine the proper dosing in the clinical setting, and this is well within the skill of the ordinary artisan. The therapeutically effective amount of the compound(s) of this invention optionally are divided into several sub-units per day or are administered at daily or more than one day intervals, depending upon the pathologic condition to be treated, the patient's condition and the nature of the compound of this invention.

As is conventional in the art, the evaluation of a synergistic effect in a drug combination may be made by analyzing the quantification of the interactions between individual drugs, using the median effect principle described by Chou et al. in Adv. Enzyme Reg. (1984) 22:27 or tests such as, but not limited to, the isobologram method, as previously described by Elion et al. in J. Biol. Chem. (1954) 208:477-488 and by Baba et al. in Antimicrob. Agents Chemother. (1984) 25:515-517, using EC₅₀for calculating the fractional inhibitory concentration.

Suitable anti-viral agents for inclusion in combination antiviral compositions or for coadministration in a course of therapy include, for instance, interferon alpha, ribavirin, a compound falling within the scope of disclosure of EP 1162196, WO 03/010141, WO 03/007945 and WO 03/010140, a compound falling within the scope of disclosure of WO 00/204425, and other patents or patent applications within their patent families, in amounts of 1 to 99.9% by weight compound of this invention, preferably from 1 to 99% by weight, more preferably from 5 to 95% by weight as can be readily determined by one skilled in the art. Such co-administered agents need not be formulated in the same dosage form as the compound of the invention. They optionally are simply administered to the subject in the course of treatment along with a course of treatment with a compound of formula (A).

The present invention further provides veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier therefore, for example in the treatment of BVDV. Veterinary carriers are materials useful for the purpose of administering the composition and are excipients which are otherwise inert or acceptable in the veterinary art and are compatible with the compound of this invention. These veterinary compositions may be administered orally, parenterally or by any other desired route.

Salts

The term “pharmaceutically acceptable salts” as used herein means the therapeutically active non-toxic salt forms formed by the compounds of formula (A). Such salts may include those derived by combination of appropriate cations such as alkali and alkaline earth metal ions or ammonium and quaternary amino ions with an acid anion moiety, typically a carboxylic acid.

The compounds of the invention may bear multiple positive or negative charges. The net charge of the compounds of the invention may be either positive or negative. Any associated counter ions are typically dictated by the synthesis and/or isolation methods by which the compounds are obtained. Typical counter ions include, but are not limited to ammonium, sodium, potassium, lithium, halides, acetate, trifluoroacetate, etc., and mixtures thereof. It will be understood that the identity of any associated counter ion is not a critical feature of the invention, and that the invention encompasses the compounds in association with any type of counter ion. Moreover, as the compounds can exist in a variety of different forms, the invention is intended to encompass not only forms of the compounds that are in association with counter ions (e.g., dry salts), but also forms that are not in association with counter ions (e.g., aqueous or organic solutions).

Metal salts typically are prepared by reacting the metal hydroxide with a compound of this invention. Examples of metal salts which are prepared in this way are salts containing Li+, Na+, Ca+2 and Mg+2 and K+. A less soluble metal salt can be precipitated from the solution of a more soluble salt by addition of the suitable metal compound. In addition, salts may be formed from acid addition of certain organic and inorganic acids to basic centers, typically amines, or to acidic groups. Examples of such appropriate acids include, for instance, inorganic acids such as hydrohalogen acids, e.g. hydrochloric or hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, benzoic, 2-hydroxypropanoic, 2-oxopropanoic, lactic, fumaric, tartaric, pyruvic, maleic, malonic, malic, salicylic (i.e. 2-hydroxybenzoic), p-aminosalicylic, isethionic, lactobionic, succinic oxalic and citric acids; organic sulfonic acids, such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids; and inorganic acids, such as hydrochloric, sulfuric, phosphoric and sulfamic acids, C1-C6 alkylsulfonic, benzenesulfonic, p-toluenesulfonic, cyclohexanesulfamic, and the like. Preferred salts include mesylate and HCl.

The compounds of this invention include the solvates formed with the compounds of formula (A) and their salts, such as for example hydrates, alcoholates and the like. The compositions herein comprise compounds of the invention in their un-ionized, as well as zwitterionic form, and combinations with stoichiometric amounts of water as in hydrates.

Also included within the scope of this invention are the salts of the compounds of formula (A) with one or more amino acids as described above. The amino acid typically is one bearing a side chain with a basic or acidic group, e.g., lysine, arginine or glutamic acid, or a neutral group such as glycine, serine, threonine, alanine, isoleucine, or leucine.

Salts of acids or bases which are not physiologically acceptable may also find use, for example, in the preparation or purification of a compound of formula (A). All salts, whether or not derived form a physiologically acceptable acid or base, are within the scope of the present invention.

Isomers

The term “isomers” as used herein means all possible isomeric forms, including tautomeric and stereochemical forms, which the compounds of formula (A) may possess, but not including position isomers. Typically, the structures shown herein exemplify only one tautomeric or resonance form of the compounds, but the corresponding alternative configurations are contemplated as well. Unless otherwise stated, the chemical designation of compounds denotes the mixture of all possible stereochemically isomeric forms, said mixtures containing all diastereomers and enantiomers (since the compounds of formula (A) may have one or more chiral centers), as well as the stereochemically pure or enriched isomers. More particularly, stereogenic centers may have either the R- or S-configuration, and double or triple bonds optionally are in either the cis- or trans-configuration.

Enriched isomeric forms of a compound of this invention are defined as a single isomer substantially free of the compound's other enantiomers or diastereomers. In particular, the term “stereoisomerically enriched” or “chirally enriched” relates to compounds having a single stereoisomeric proportion of at least about 80% (i.e. at least 90% of one isomer and at most 10% of the other possible isomers), preferably at least 90%, more preferably at least 94% and most preferably at least 97%. The terms “enantiomerically pure” and “diastereomerically pure” contain undetectable levels of any other isomer.

Separation of stereoisomers is accomplished by standard methods known to those in the art. One enantiomer of a compound of the invention can be separated substantially free of its opposing enantiomer by a method such as formation of diastereomers using optically active resolving agents (“Stereochemistry of Carbon Compounds,” (1962) by E. L. Eliel, McGraw Hill; Lochmuller, C. H., (1975) J. Chromatogr., 113:(3) 283-302). Separation of isomers in a mixture can be accomplished by any suitable method, including: (1) formation of ionic, diastereomeric salts with chiral compounds and separation by fractional crystallization or other methods, (2) formation of diastereomeric compounds with chiral derivatizing reagents, separation of the diastereomers, and conversion to the pure enantiomers, or (3) enantiomers can be separated directly under chiral conditions. Under method (1), diastereomeric salts can be formed by reaction of enantiomerically pure chiral bases such as brucine, quinine, ephedrine, strychnine, a-methyl-b-phenylethylamine (amphetamine), and the like with asymmetric compounds bearing an acidic functionality, such as carboxylic acid and sulfonic acid.

The diastereomeric salts optionally are induced to separate by fractional crystallization or ionic chromatography. For separation of the optical isomers of amino compounds, addition of chiral carboxylic or sulfonic acids, such as camphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid can result in formation of the diastereomeric salts. Alternatively, by method (2), the substrate to be resolved may be reacted with one enantiomer of a chiral compound to form a diastereomeric pair (Eliel, E. and Wilen, S. (1994). Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., p. 322). Diastereomeric compounds can be formed by reacting asymmetric compounds with enantiomerically pure chiral derivatizing reagents, such as menthyl derivatives, followed by separation of the diastereomers and hydrolysis to yield the free, enantiomerically enriched xanthene. A method of determining optical purity involves making chiral esters, such as a menthyl ester or Mosher ester, a-methoxy-a-(trifluoromethyl)phenyl acetate (Jacob III. (1982) J. Org. Chem. 47:4165), of the racemic mixture, and analyzing the NMR spectrum for the presence of the two atropisomeric diastereomers. Stable diastereomers can be separated and isolated by normal- and reverse-phase chromatography following methods for separation of atropisomeric naphthyl-isoquinolines (Hoye, T., WO 96/15111). Under method (3), a racemic mixture of two asymmetric enantiomers is separated by chromatography using a chiral stationary phase. Suitable chiral stationary phases are, for example, polysaccharides, in particular cellulose or amylose derivatives. Commercially available polysaccharide based chiral stationary phases are ChiralCeI™ CA, OA, OB5, OC5, OD, OF, OG, OJ and OK, and Chiralpak™ AD, AS, OP(+) and OT(+). Appropriate eluents or mobile phases for use in combination with said polysaccharide chiral stationary phases are hexane and the like, modified with an alcohol such as ethanol, isopropanol and the like. (“Chiral Liquid Chromatography” (1989) W. J. Lough, Ed. Chapman and Hall, New York; Okamoto, (1990). “Optical resolution of dihydropyridine enantiomers by High-performance liquid chromatography using phenylcarbamates of polysaccharides as a chiral stationary phase”, J. of Chromatogr. 513:375-378).

Metabolites

The present invention also provides the in vivo metabolic products of the compounds described herein, to the extent such products are novel and unobvious over the prior art. Such products may result for example from the oxidation, reduction, hydrolysis, amidation, esterification and the like of the administered compound, primarily due to enzymatic processes. Accordingly, the invention includes novel and unobvious compounds produced by a process comprising contacting a compound of this invention with a mammal for a period of time sufficient to yield a metabolic product thereof. Such products typically are identified by preparing a radiolabelled (e.g. C14 or H3) compound of the invention, administering it parenterally in a detectable dose (e.g. greater than about 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, or to man, allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion products from the urine, blood or other biological samples. These products are easily isolated since they are labeled (others are isolated by the use of antibodies capable of binding epitopes surviving in the metabolite). The metabolite structures are determined in conventional fashion, e.g. by MS or NMR analysis. In general, analysis of metabolites is done in the same way as conventional drug metabolism studies well-known to those skilled in the art. The conversion products, so long as they are not otherwise found in vivo, are useful in diagnostic assays for therapeutic dosing of the compounds of the invention even if they possess no antiviral activity of their own.

Formulations

The compounds of the invention optionally are formulated with conventional pharmaceutical carriers and excipients, which will be selected in accord with ordinary practice. Tablets will contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic. Formulations optionally contain excipients such as those set forth in the “Handbook of Pharmaceutical Excipients” (1986) and include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like.

Subsequently, the term “pharmaceutically acceptable carrier” as used herein means any material or substance with which the active ingredient is formulated in order to facilitate its application or dissemination to the locus to be treated, for instance by dissolving, dispersing or diffusing the said composition, and/or to facilitate its storage, transport or handling without impairing its effectiveness. The pharmaceutically acceptable carrier may be a solid or a liquid or a gas which has been compressed to form a liquid, i.e. the compositions of this invention can suitably be used as concentrates, emulsions, solutions, granulates, dusts, sprays, aerosols, suspensions, ointments, creams, tablets, pellets or powders.

Suitable pharmaceutical carriers for use in the said pharmaceutical compositions and their formulation are well known to those skilled in the art, and there is no particular restriction to their selection within the present invention. They may also include additives such as wetting agents, dispersing agents, stickers, adhesives, emulsifying agents, solvents, coatings, antibacterial and antifungal agents (for example phenol, sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodium chloride) and the like, provided the same are consistent with pharmaceutical practice, i.e. carriers and additives which do not create permanent damage to mammals. The pharmaceutical compositions of the present invention may be prepared in any known manner, for instance by homogeneously mixing, coating and/or grinding the active ingredients, in a one-step or multi-steps procedure, with the selected carrier material and, where appropriate, the other additives such as surface-active agents. may also be prepared by micronisation, for instance in view to obtain them in the form of microspheres usually having a diameter of about 1 to 10 gm, namely for the manufacture of microcapsules for controlled or sustained release of the active ingredients.

Suitable surface-active agents, also known as emulgent or emulsifier, to be used in the pharmaceutical compositions of the present invention are non-ionic, cationic and/or anionic materials having good emulsifying, dispersing and/or wetting properties. Suitable anionic surfactants include both water-soluble soaps and water-soluble synthetic surface-active agents. Suitable soaps are alkaline or alkaline-earth metal salts, unsubstituted or substituted ammonium salts of higher fatty acids (C₁₀-C₂₂), e.g. the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures obtainable form coconut oil or tallow oil. Synthetic surfactants include sodium or calcium salts of polyacrylic acids; fatty sulphonates and sulphates; sulphonated benzimidazole derivatives and alkylarylsulphonates. Fatty sulphonates or sulphates are usually in the form of alkaline or alkaline-earth metal salts, unsubstituted ammonium salts or ammonium salts substituted with an alkyl or acyl radical having from 8 to 22 carbon atoms, e.g. the sodium or calcium salt of lignosulphonic acid or dodecylsulphonic acid or a mixture of fatty alcohol sulphates obtained from natural fatty acids, alkaline or alkaline-earth metal salts of sulphuric or sulphonic acid esters (such as sodium lauryl sulphate) and sulphonic acids of fatty alcohol/ethylene oxide adducts. Suitable sulphonated benzimidazole derivatives preferably contain 8 to 22 carbon atoms. Examples of alkylarylsulphonates are the sodium, calcium or alcoholamine salts of dodecylbenzene sulphonic acid or dibutyl-naphthalenesulphonic acid or a naphthalene-sulphonic acid/formaldehyde condensation product. Also suitable are the corresponding phosphates, e.g. salts of phosphoric acid ester and an adduct of p-nonylphenol with ethylene and/or propylene oxide, or phospholipids. Suitable phospholipids for this purpose are the natural (originating from animal or plant cells) or synthetic phospholipids of the cephalin or lecithin type such as e.g. phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerine, lysolecithin, cardiolipin, dioctanylphosphatidyl-choline, dipalmitoylphoshatidyl-choline and their mixtures.

Suitable non-ionic surfactants include polyethoxylated and polypropoxylated derivatives of alkylphenols, fatty alcohols, fatty acids, aliphatic amines or amides containing at least 12 carbon atoms in the molecule, alkylarenesulphonates and dialkylsulphosuccinates, such as polyglycol ether derivatives of aliphatic and cycloaliphatic alcohols, saturated and unsaturated fatty acids and alkylphenols, said derivatives preferably containing 3 to 10 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenol. Further suitable non-ionic surfactants are water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylenediaminopolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethyleneglycol ether groups and/or 10 to 100 propyleneglycol ether groups. Such compounds usually contain from 1 to 5 ethyleneglycol units per propyleneglycol unit. Representative examples of non-ionic surfactants are nonylphenol-polyethoxyethanol, castor oil polyglycolic ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethyleneglycol and octylphenoxypolyethoxyethanol. Fatty acid esters of polyethylene sorbitan (such as polyoxyethylene sorbitan trioleate), glycerol, sorbitan, sucrose and pentaerythritol are also suitable non-ionic surfactants.

Suitable cationic surfactants include quaternary ammonium salts, particularly halides, having 4 hydrocarbon radicals optionally substituted with halo, phenyl, substituted phenyl or hydroxy; for instance quaternary ammonium salts containing as N-substituent at least one C8C22 alkyl radical (e.g. cetyl, lauryl, palmityl, myristyl, oleyl and the like) and, as further substituents, unsubstituted or halogenated lower alkyl, benzyl and/or hydroxy-lower alkyl radicals.

A more detailed description of surface-active agents suitable for this purpose may be found for instance in “McCutcheon's Detergents and Emulsifiers Annual” (MC Publishing Crop., Ridgewood, N.J., 1981), “Tensid-Taschenbucw’, 2 d ed. (Hanser Verlag, Vienna, 1981) and “Encyclopaedia of Surfactants, (Chemical Publishing Co., New York, 1981).

Compounds of the invention and their physiologically acceptable salts (hereafter collectively referred to as the active ingredients) may be administered by any route appropriate to the condition to be treated, suitable routes including oral, rectal, nasal, topical (including ocular, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural). The preferred route of administration may vary with for example the condition of the recipient.

While it is possible for the active ingredients to be administered alone it is preferable to present them as pharmaceutical formulations. The formulations, both for veterinary and for human use, of the present invention comprise at least one active ingredient, as above described, together with one or more pharmaceutically acceptable carriers therefore and optionally other. therapeutic ingredients. The carrier(s) optimally are “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. For infections of the eye or other external tissues e.g. mouth and skin, the formulations are optionally applied as a topical ointment or cream containing the active ingredient(s) in an amount of, for example, 0.075 to 20% w/w (including active ingredient(s) in a range between 0.1% and 20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc), preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. When formulated in an ointment, the active ingredients may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least 30% w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG400) and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogs.

The oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Optionally, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.

The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low. Thus the cream should optionally be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient. The active ingredient is optionally present in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10% particularly about 1.5% w/w. Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate. Formulations suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns (including particle sizes in a range between 20 and 500 microns in increments of 5 microns such as 30 microns, 35 microns, etc), which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as for example a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol administration may be prepared according to conventional methods and may be delivered with other therapeutic agents.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

Compounds of the invention can be used to provide controlled release pharmaceutical formulations containing as active ingredient one or more compounds of the invention (“controlled release formulations”) in which the release of the active ingredient can be controlled and regulated to allow less frequency dosing or to improve the pharmacokinetic or toxicity profile of a given invention compound. Controlled release formulations adapted for oral administration in which discrete units comprising one or more compounds of the invention can be prepared according to conventional methods.

Additional ingredients may be included in order to control the duration of action of the active ingredient in the composition. Control release compositions may thus be achieved by selecting appropriate polymer carriers such as for example polyesters, polyamino acids, polyvinyl pyrrolidone, ethylene-vinyl acetate copolymers, methylcellulose, carboxymethylcellulose, protamine sulfate and the like. The rate of drug release and duration of action may also be controlled by incorporating the active ingredient into particles, e.g. microcapsules, of a polymeric substance such as hydrogels, polylactic acid, hydroxymethylcellulose, polymethyl methacrylate and the other above-described polymers. Such methods include colloid drug delivery systems like liposomes, microspheres, microemulsions, nanoparticles, nanocapsules and so on. Depending on the route of administration, the pharmaceutical composition may require protective coatings. Pharmaceutical forms suitable for injectionable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation thereof. Typical carriers for this purpose therefore include biocompatible aqueous buffers, ethanol, glycerol, propylene glycol, polyethylene glycol and the like and mixtures thereof.

In view of the fact that, when several active ingredients are used in combination, they do not necessarily bring out their joint therapeutic effect directly at the same time in the mammal to be treated, the corresponding composition may also be in the form of a medical kit or package containing the two ingredients in separate but adjacent repositories or compartments. In the latter context, each active ingredient may therefore be formulated in a way suitable for an administration route different from that of the other ingredient, e.g. one of them may be in the form of an oral or parenteral formulation whereas the other is in the form of an ampoule for intravenous injection or an aerosol.

Synthetic Methods

The compounds of formula (A) are prepared using a series of chemical reactions well known to those skilled in the art, altogether making up the process for preparing said compounds and exemplified further. The processes described further are only meant as examples and by no means are meant to limit the scope of the present invention.

The invention also relates to methods of making the compositions of the invention. The compositions are prepared by any of the applicable techniques of organic synthesis. Many such techniques are well known in the art. However, many of the known techniques are elaborated in “Compendium of Organic Synthetic Methods” (John Wiley & Sons, New York), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade, Jr., 1980; Vol. 5, Leroy G. Wade, Jr., 1984; and Vol. 6, Michael B. Smith; as well as March, J., “Advanced Organic Chemistry, Third Edition”, (John Wiley & Sons, New York, 1985), “Comprehensive Organic Synthesis. Selectivity, Strategy & Efficiency in Modern Organic Chemistry. In 9 Volumes”, Barry M. Trost, Editor-in-Chief (Pergamon Press, New York, 1993 printing).

Exemplary methods for the preparation of the compositions of the invention are provided below. These methods are intended to illustrate the nature of such preparations, and are not intended to limit the scope of applicable methods.

Generally, the reaction conditions such as temperature, reaction time, solvents, workup procedures, and the like, will be those common in the art for the particular reaction to be performed. The cited reference material, together with material cited therein, contains detailed descriptions of such conditions. Typically the temperatures will be −100° C. to 200° C., solvents will be aprotic or protic, and reaction times will be 10 seconds to 10 days. Workup typically consists of quenching any unreacted reagents followed by partition between a water/organic layer system (extraction) and separating the layer containing the product.

Oxidation and reduction reactions are typically carried out at temperatures near room temperature (about 20° C.), although for metal hydride reductions frequently the temperature is reduced to 0° C. to −100° C., solvents are typically aprotic for reductions and may be either protic or aprotic for oxidations. Reaction times are adjusted to achieve desired conversions.

Condensation reactions are typically carried out at temperatures near room temperature, although for non-equilibrating, kinetically controlled condensations reduced temperatures (0° C. to −100° C.) are also common. Solvents can be either protic (common in equilibrating reactions) or aprotic (common in kinetically controlled reactions).

Standard synthetic techniques such as azeotropic removal of reaction by-products and use of anhydrous reaction conditions (e.g. inert gas environments) are common in the art and will be applied when applicable.

General aspects of these exemplary methods are described below. Each of the products of the following processes is optionally separated, isolated, and/or purified prior to its use in subsequent processes.

The terms “treated”, “treating”, “treatment”, and the like, mean contacting, mixing, reacting, allowing to react, bringing into contact, and other terms common in the art for indicating that one or more chemical entities is treated in such a manner as to convert it to one or more other chemical entities. This means that “treating compound one with compound two” is synonymous with “allowing compound one to react with compound two”, “contacting compound one with compound two”, “reacting compound one with compound two”, and other expressions common in the art of organic synthesis for reasonably indicating that compound one was “treated”, “reacted”, “allowed to react”, etc., with compound two.

“Treating” indicates the reasonable and usual manner in which organic chemicals are allowed to react. Normal concentrations (0.01M to 10M, typically 0.1M to 1M), temperatures (−100° C. to 250° C., typically −78° C. to 150° C., more typically −78° C. to 100° C., still more typically 0° C. to 100° C.), reaction vessels (typically glass, plastic, metal), solvents, pressures, atmospheres (typically air for oxygen and water insensitive reactions or nitrogen or argon for oxygen or water sensitive), etc., are intended unless otherwise indicated. The knowledge of similar reactions known in the art of organic synthesis is used in selecting the conditions and apparatus for “treating” in a given process. In particular, one of ordinary skill in the art of organic synthesis selects conditions and apparatus reasonably expected to successfully carry out the chemical reactions of the described processes based on the knowledge in the art.

Modification of the exemplified schemes and examples leads to various analogs of the specific exemplary materials produced above. The above citations describing suitable methods of organic synthesis are applicable to such modifications.

In the exemplary schemes it may be advantageous to separate reaction products from one another and/or from starting materials. The desired products of each step or series of steps is separated and/or purified (hereinafter separated) to the desired degree of homogeneity by the techniques common in the art. Typically such separations involve multiphase extraction, crystallization from a solvent or solvent mixture, distillation, sublimation, or chromatography. Chromatography can involve any number of methods including, for example, size exclusion or ion exchange chromatography, high, medium, or low pressure liquid chromatography, small scale and preparative thin or thick layer chromatography, as well as techniques of small scale thin layer and flash chromatography.

Another class of separation methods involves treatment of a mixture with a reagent selected to bind to or render otherwise separable a desired product, unreacted starting material, reaction by product, or the like. Such reagents include adsorbents or absorbents such as activated carbon, molecular sieves, ion exchange media, or the like. Alternatively, the reagents can be acids in the case of a basic material, bases in the case of an acidic material, binding reagents such as antibodies, binding proteins, selective chelators such as crown ethers, liquid/liquid ion extraction reagents (LIX), or the like.

Selection of appropriate methods of separation depends on the nature of the materials involved. For example, boiling point, and molecular weight in distillation and sublimation, presence or absence of polar functional groups in chromatography, stability of materials in acidic and basic media in multiphase extraction, and the like. One skilled in the art will apply techniques most likely to achieve the desired separation.

Suitable methods for making the compounds of this invention also are found in WO 2004/005286, in particular schemes 1-13 therein.

Another synthetic route to 5-benzyl-2-phenyl-5H-imidazo[4,5-c]pyridine and analogues is shown in scheme 1.

embedded image

The following list includes carboxylic acid reactants which may be employed in the condensation, ring closure reaction of Scheme 1. The compounds so produced will bear the residue of the acid at the site of YR¹. Optionally, the remainder of the molecule will be as in any of the compounds of examples 2-7.

Acid
MW

embedded image

258.117

158.103

174.558

191.013

214.219

152.148

244.22

164.159

190.12

191.013

165.191

214.219

206.118

194.229

164.159

178.23

125.126

112.084

128.151

124.099

200.213

201.201

112.088

124.099

174.158

240.257

166.175

137.137

204.267

141.101

154.139

173.17

178.21

187.197

173.17

154.139

112.088

192.169

187.197

179.199

124.099

156.204

189.173

113.072

146.144

137.137

174.158

200.213

252.272

250.256

216.21

277.116

215.251

126.114

129.139

143.165

124.099

127.099

126.114

222.238

174.158

230.266

221.279

257.107

223.614

140.141

176.17

188.128

262.21

187.197

178.15

176.17

157.556

234.2

170.138

The following list includes alkylating reagents which may be employed in the pyridyl alkylation reaction of Scheme 1. Here, the residue of the alkylating agent is located at the XR³site of the compound of this invention. Optionally, the remainder of the compound will be as found in any of the compounds of examples 2-7.

Alkylating reagent
MW

embedded image

195.475

168.666

154.639

145.588

190.672

338.832

205.039

325.225

262.579

228.721

207.016

307.03

199.09

175.057

154.639

216.663

218.682

275.144

198.648

294.907

244.144

222.084

152.623

118.523

255.138

328.828

202.611

281.123

170.638

257.023

255.032

174.63

186.637

247.134

190.672

204.676

257.023

262.579

224.646

194.62

262.617

237.042

275.144

186.637

169.035

229.065

250.727

324.526

261.569

262.617

273.699

249.127

131.561

210.581

303.154

223.471

179.02

273.478

257.023

241.12

166.61

205.995

188.613

277.696

133.602

208.647

272.144

219.052

229.065

209.699

226.648

132.613

207.659

223.471

276.549

168.047

162.566

224.646

186.637

154.639

277.16

263.133

231.088

200.648

215.727

291.469

273.478

237.498

223.471

203.053

223.471

253.109

203.053

273.478

269.059

223.471

289.478

269.059

253.06

221.043

285.567

344.203

261.569

212.078

195.57

299.113

228.077

193.632

223.471

255.961

252.11

190.039

176.012

237.099

238.087

239.623

198.648

350.235

252.11

236.111

320.206

228.995

273.478

203.053

214.036

203.053

214.036

285.913

241.462

283.251

177.064

267.922

350.235

196.7

199.09

253.06

258.103

331.052

88.5365

132.988

102.563

144.644

144.044

296.239

172.098

158.071

170.082

186.081

184.109

149.663

248.195

313.064

158.071

186.124

230.133

129.589

167.038

Scheme 2 shows a synthetic route to 5-biarylmethyl-2-phenyl-5H-imidazo[4,5-c]pyridines and 5-benzyl-2-biaryl-5H-imidazo[4,5-c]pyridines.

embedded image

Scheme 3 shows a synthetic route to 5-(alkoxybenzyl)-2-phenyl-5H-imidazo[4,5-c]pyridines and 5-benzyl-2-alkoxybenzyl-5H-imidazo[4,5-c]pyridines. R, R′, and R″ can be any alkyl, benzylic or heterobenzylic groups.

embedded image

Analogous compounds may be synthesized in the same fashion as in the foregoing schemes by varying the starting materials, intermediates, solvents and conditions as will be known by those skilled in the art.

EXAMPLES
Part A
Compound Synthesis
Example 1
2-(2,3-difluorophenyl)-3H-imidazo[4,5-c]pyridine

embedded image

Phosphorous pentoxide (24.56 g) was dissolved in methanesulfonic acid (165.8 mL) at 50° C. with stirring. To the solution, 3,4-diaminopyridine (12.3 g, 0_—11 moles) and 2,3-difluorobenzoic acid (19.4 g, 0.12 moles) were added. The reaction mixture was heated to 190° C. for 3 hours. The reaction was done three times. The reaction mixtures was cooled to 50° C. and poured into ice with stirring. At this stage, all three batches were combined. The reaction mixture was neutralized by the addition of NaOH with stirring until the pH is 8. Solid material precipitated out of solution, was collected by filtration and air-dried. The final product was re-crystallized from ethanol/water twice to yield 36 g of 2-(2,3-difluorophenyl)-3H-imidazo[4,5-c]pyridine. 1H 300 Mhz (CD₃OD) sigma 7.3-7.42 (m, 1p); 7.43-7.58 (m, 1p); 7.70 (d, 1p); 8.0 (m, 1p); 8.34 (d, 1p); and 8.95 (s, 1p). LC/MS data M/z=232.

Following the above taught procedure and substituting 2-fluorobenzoic acid in place of 2,3-difluorobenzoic acid, the compound 2-(2-fluorophenyl)-3H-imidazo[4,5-c]pyridine can be prepared.

Example 2
5-((3-(4-chlorophenyl)isoxazol-5-yl)methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine

embedded image

To a suspension of 2-(2-fluorophenyl)-3H-imidazo[4,5-c]pyridine (11.0 g, 50.0 mmoles) in DMF was added a 10% (w/v) solution of aqueous NaOH. To this solution, 5-(chloromethyl)-3-(4-chlorophenyl)isoxazole (13.68 g, 60.0 mmoles) dissolved in DMF was added. The reaction mixture was stirred at room temperature and monitored every half hour by LCMS. The reaction was stopped at 4 hours, after LCMS showed no progress between at 2 hour and 4 hour monitor points. The reaction product was triturated with first with water and then with EtoAc (3×). The material was crystallized by dissolving the material in MeOH with heat, followed by precipitation with water. This crystallization process was then repeated yielding 5-((3-(4-chlorophenyl)isoxazol-5-yl)methyl)-2-(2-fluorophenyl)-5H-imdazo[4,5-c]pyridine (15.385 g, 38 mmole) as white crystal at a yield of 74%. 1H 300 Mhz (d₆-DMSO) sigma 6.02 (s, 2p); 7.13 (s, 1p); 7.26-7.35 (m, 2p); 7.43-7.52 (m, 1p); 7.56 (d, 2p); 7.84 (d, 1); 7.89 (d, 2p); 8.24 (d, 1); 8.28-8.36 (m, 1p); and 9.19 (s, 1p). LCMS data M/Z=405.31

Example 3A
5-(4-(trifluoromethoxy)benzyl)-2-(2,3-difluorophenyl)-5H-imidazo[4,5-c]pyridine

embedded image

First, 2-(2,3-difluorophenyl)-3H-imidazo[4,5-c]pyridine (20 g, 86.6 mmole) was added to 430 mL of DMF. Some of the solid material did not dissolve. To this solution was added 43 mL of a 10% NaOH (w/v) solution. With vigorous stirring, the un-dissolved material went into solution. The resulting solution was divided into 30 equal portions of 16.3 mL, 3 mmole of 2-(2,3-difluorophenyl)-3H-imidazo[4,5-c]pyridine so as to fit into a microwave reaction vessel. To each reaction vessel was added of 1-(chloromethyl)-4-(trifluoromethoxy)benzene (693 mg, 3 mmole). Each reaction mixture was microwaved for 1 minute at 110° C. Following the completion of all the microwave reactions, all of the reaction vessels were combined (one was lost due to breakage of the vessel) into three batches for workup. For each batch, DMF was removed by vacuum, and the resulting material washed three times with deionized water. The resulting crude material was dissolved in CH₂Cl₂, Purified using a 330 g SiO₂column (Redisep (Isco) 0% to 0%/5 min to 10% B/30 min to 20%/5 min), and the resulting material was re-crystallized from ethanol/H₂O. The three batches yielded 14 g, 33.5 mmole of 5-(4-(trifluoromethoxy)benzyl)-2-(2,3-difluorophenyl)-5H-imidazo[4,5-c]pyridine. 1H 300 Mhz (CD₃OD) sigma 5.79 (s, 2p); 7.25-7.35 (m, 1p); 7.37 (d, 2p); 7.38-7.42 (m, 2p); 7.55 (d, 2p); 7.88-7.95 (m, 1p); 8.25 (d, 1p); and 9.05 (s, 1p). LC/MS M/z=406.23.

Example 3B

Following the above-taught procedure, and substituting 1-(chloromethyl)-2,4 difluorobenzene in place of 1-(chloromethyl)-4-(trifluoromethoxy)benzene, the compound 5-(4-iodobenzyl)-2-(2,3-difluorophenyl)-5H-imidazo[4,5-c]pyridine can be prepared.

Example 4
5-(2,4-difluoro-biphenyl)methyl-2-(2,3-difluorophenyl)-5H-imidazo[4,5-c]pyridine

embedded image

2,4-difluorophenylboronic acid (196 mg, 1.24 mmole) was added to a solution of 5-(4-iodobenzyl)-2-(2,3-difluorophenyl)-5H-imidazo[4,5-c]pyridine (460 mg, 1.03 mmole) in DMF (10 mL). Na₂CO₃was dissolved in H₂O, added to the DMF solution and stirred. Pd(PPh3)₄was then added to the DMF reaction mixture. The reaction mixture was heated in a microwave at 200° C. for 2 minutes. After extractive work-up using ethyl acetate/water, the crude product was purified in two batches using an Isco 40 g SiO₂column (0 to 10% B/20 min, A=CH₂Cl₂, B=MeOH, flow rate=40 ml/min) for each purification. The pure product fractions were combined and concentrated. The resulting solid was re-crystallized from CH₂Cl/hexane. The collected crystals were dried under high vacuum overnight resulting in 5-(2,4-difluoro-biphenyl)methyl-2-(2,3-difluorophenyl)-5H-imidazo[4,5-c]pyridine (223 mg, 0.515 mmole) at 50% yield. 1H 300 Mhz (CD3OD) sigma 5.8 (s, 2p); 7.0-7.1 (m, 2p); 7.25-7.35 (m, 1p); 7.35-7.45 (m, 1p); 7.45-7.60 (m, 5p); 7.85 (d, 1p); 7.85-8.0 (m, 1p); 8.3 (d, 1p); and 9.10 (s, 1p). LC/MS data M/z=434.18.

Example 5
5-((3-(4-chlorophenyl)isoxazol-5-yl)methyl)-2-(2,3-difluorophenyl)-5H-imidazo[4,5-c]pyridine

embedded image

To a solution of azabelizimidazole (10 g, 43.3 mmole) in DMF was added 10% (w/v) aqueous NaOH followed by a solution of 5-(chloromethyl)-3-(4-chlorophenyl)-isoxazole (11.8 g, 51.9 mmole) in DMF. The reaction mixture was stirred at room temperature for 7 hours, and then concentrated. The solid material was treated with EtOAc/H₂O, and collected by filtering. The solid material was then tritrated with H₂O and EtoAc, and air-dried. The solid was further purified by re-crystallization from MeOH to obtain 5-((3-(4-chlorophenyl)isoxazol-5-yl)methyl)-2-(2,3-difluorophenyl)-5H-imidazo[4,5-c]pyridine (8.5 g, 20.1 mmole) at 46.6% yield. 1H 300 Mhz (DMSO-d6) sigma 6.03 (s, 2p); 7.12 (s, 1p); 7.25-7.35 (m, 1p); 7.44-7.53 (m, 1p); 7.55 (d, 2p); 7.88 (d, 3p); 8.11-8.18 (m, 1p); 8.24-8.29 (dd, 1p); and 9.23 (s, 1p). LC/MS data M/z=423.34, 425.22

Example 6
5-((3-(2,4-trifluoromethyphenyl)isoxazol-5-yl)methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine

embedded image

2,4-(bis-trifluoromethyl)benzaldoxime

To aromatic aldehyde (0.021 mol) suspended in EtOH/H₂O (1:2, 230 mL, 0.09 M) was added hydroxylamine hydrochloride (1.58 g, 0.023 mol) and cooled to 4° C. To this solution was added aqueous NaOH 50% w/w (4.13 mL, 0.052 mol) dropwise. After stirring for 1.5 h at room temperature, the reaction mixture was acidified with 2N aqueous HCl and extracted with CH₂Cl₂(3×50 mL). The organic solution was washed with saturated aqueous NaCl and dried over sodium sulfate. Removal of solvent gave crude oxime (5.3 g, quant.) that was used directly in the next step.

2,4-(bis-trifluoromethyl)phenyl chloromethyl isoxazole

2,4-(bis-trifluoromethyl)benzaldoxime (9.75 g, 0.038 mol) was suspended in CH₂Cl₂(45 mL, 0.85 M) and cooled to 4° C. Propargyl chloride (2.72 mL, 0.038 mol) was added to the reaction solution followed by dropwise addition of NaOCl (10-13% free chlorine, 37.6 mL, 0.061 mol). The reaction mixture was stirred at 4° C. for 15 min then heated to reflux for 3 h. After cooling to room temperature, the reaction was partitioned between CH₂Cl₂and H₂O. The organic layer was separated, washed with saturated aqueous NaCl, and dried over sodium sulfate. After removal of solvent, the crude product chloromethylisoxazole was purified by column chromatography on silica (10% CH₂Cl₂/hexanes) (6.5 g, 0.020 mol).

5-((3-(2,4-trifluoromethyphenyl)isoxazol-5-yl)methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine

To imidazopyridine (14.28 g, 0.067 mol) suspended in DMF (40 mL) was added aqueous NaOH 10% w/w (32.2 mL, 0.080 mol) dropwise followed by addition of the chloromethyl isoxazole from the previous step (26.3 g, 0.080 mol) in DMF (16 mL). After stirring for 12 h at room temperature, solvents were evaporated to give crude product as a tan solid. The crude solid was triturated with H₂O (7×) and crystallized (2×) from MeOH/H₂O (2:1) to provide pure title product.

NMR; 300 Mhz D₆MSO

Chemical shift, multiplicity, # of protons:

6.1, s, 2

7.0, s, 1

7.3, t, 2

7.4-7.5, m, 1

7.8-7.9, d, 1

7.9-8.0, d, 1

8.2-8.4, m, 4

9.2, s, 1

Example 7
5-((3-(4-trifluoromethyl-2-fluorophenyl)isoxazol-5-yl)methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine

Isoxazole Synthesis

embedded image

Compound
MW
Amount
Moles
Equivalents

A
207.13
9.3
g
0.044
1

NaOCl (10% free Cl)
74.44
43.0
mL
0.44
1.6

Propargyl chloride
74.51
3.14
mL
0.044
1

Dichloromethane

48.7
mL

“A” was suspended in dichloromethane at 0° C. and NaOCl was added at 0° C. with vigorous stirring, followed by propargyl chloride. Reaction stirred at 0° C. for 5 min and then heated to reflux for 2 h. It was then cooled to room temperature, washed with water, dried over sodium sulfate and concentrated in vacuo to obtain a yellow solid. It was purified on the combiflash on a silica gel column, eluting with 3-50% ethyl acetate-hexanes. 4.5 g of shiny white solid obtained.

embedded image

Compound
MW
Amount
mMoles
Equivalents

A
279.62
2.0
g
7.6
1.2

B
213.21
1.373
g
6.4
1

10% w/v

2.26
mL

aq NaOH

DMF

13.73 mL + 6.56 mL

“B” was suspended in 13.73 mL DMF and 10% (w/v) aq. NaOH was added to it. “A” was dissolved in 6.56 mL DMF and this solution was added to the above with stirring. The reaction was stirred at room temperature for 5 hours. DMF was removed by concentrating in vacuo and the solid obtained was triturated with water two times and then with ethyl acetate. The solid thus obtained was recrystallized from methanol-water to obtain 533 mg of the desired compound.

NMR (DMSO) Data:

Chemical shift, multiplicity, # of protons:

6.14, s, 2

7.18, d, 1

7.28-7.36, m, 2

7.44-7.54, m, 1

7.70-7.76, d, 1

7.86-7.90, d, 1

7.90-7.96, d, 1

8.08-8.16, t, 1

8.28-8.36, t, 2

9.24, s, 1

Example 8A
5-((3-(2-trifluoromethyl-4-fluorophenyl)isoxazol-5-yl)methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine

embedded image

To a solution of azabenzimidazole (12.7 g, 59.6 mmole) in DMF (120 mL) was added 10% (w/v) aqueous NaOH (30.5 mL, 76.6 mmole) followed by a solution of 5-(chloromethyl)-3-(2-trifluoromethyl-4-fluorophenyl)-isoxazole (21.3 g, 76.6 mmole) in DMF (60 mL). The reaction mixture was stirred at room temperature for 18 hours, and then concentrated. The material was precipitated from MeOH/H₂O, and collected by filtering. The solid material was recrystallized from EtoAc/hexanes to obtain 5-((3-(2-trifluoromethy-4-fluorophenyl)isoxazol-5-yl)methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine in 69% yield.

NMR Data 300 Mhz D₆MSO

Chemical shift, multiplicity, #of protons:

6.15, s, 2

6.91, s, 1

7.3, t, 2

7.42-7.52, m, 1

7.65-7.9, m, 2

7.84-7.9, m, 2

8.22-8.45, m, 2

9.19, S, 1

Example 8B

Salts of 5-((3-(2-trifluoromethyl-4-fluorophenyl)isoxazol-5-yl)methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine

Methanesulfonic Acid Salt

5-((3-(2-trifluoromethyl-4-fluorophenyl)isoxazol-5-yl)methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine free base (200 mg) was slurried in 2.0 mL acetone. Methanesulfonic acid (42.6 mg) was added and the mixture was warmed to ˜60° C. Water was added in small increments until a solution was formed (110 μL required). The solution was cooled to ambient temperature and stirred overnight. The slurry was cooled in an ice bath before being filtered and washed with acetone. The solid obtained was dried at 40° C. to give 149 mg of the desired salt. DSC endotherm 213.1° C. NMR was consistent with the desired structure.

HCl Salt

5-((3-(2-trifluoromethyl-4-fluorophenyl)isoxazol-5-yl)methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine free base (200 mg) was slurried in 2.0 mL acetone. Concentrated hydrochloric acid (46 mg) was added and the mixture was warmed to ˜60° C. Water was added to the thick slurry in small increments until a solution was formed (100 μL required). The solution was cooled to ambient temperature and stirred overnight. The slurry was cooled in an ice bath before being filtered and washed with acetone. The solid obtained was dried at 40° C. to give 80 mg of the desired salt. DSC endotherm 241.5° C. NMR consistent with the desired structure.

Example 8B
Formulation of 5-((3-(2-trifluoromethyl-4-fluorophenyl)isoxazol-5-yl)methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine salts

Either salt of Example 7B was mixed 1:1 by weight in dry pregelatinized starch. 100 mg of the mixture was loaded into a hard gel capsule.

Additional compounds of this invention were made by the methods of procedures A, C, D, E and F.

Procedure A; Alkylation

embedded image

For compounds prepared in an array format, 100 um of the scaffold (in this case 2-(2,3-Difluoro-phenyl)-3H-imidazo[4,5-c]pyridine) was used for each reaction. The total amount of 2-(2,3-Difluoro-phenyl)-3H-imidazo[4,5-c]pyridine was dissolved in enough DMF to give 500 ul/reaction. To each solution was added 60 μL of 10% (w/v)NaOH/H₂O. The alkylating agents were dissolved in DMF at a concentration 480 μmole/mL and 250 μL of these solutions were added to the respective reaction. Each reaction was then heated to 110° C. for 1 min using microwave irradiation. After cooling, the reactions were filtered through a 0.45 um filter. Each compound was then purified by mass based fractionation on a C-18 reverse phase column using 0.1% TFA/H₂O and 0.1% TFA/Acetonitrile as the eluting solvents. Each compound was identified by its mass spectrum and purity was determined by UV absorbance at 254 nm. The HPLC fractions were concentrated by centrifugal evaporation and weighed to determine quantity collected.

Procedure C; Suzuki Boronic Acid

embedded image

The aryl boronic acid (1.2 eq.) was added to a solution of 5-(4-iodobenzyl)-2-(2,3-difluorophenyl)-5H-imidazo[4,5-c]pyridine (1 eq.) in DMF. Na₂CO₃(2 eq) was dissolved in H₂O, added to the DMF solution and stirred. Pd(PPh3)4 (5 mole %) was then added to the DMF reaction mixture. The reaction mixture was heated in a microwave at 200° C. for 2 minutes. The reaction mixture was applied to a 1 g solid phase extraction cartridge (C-18) and the column washed with 3×2 mL of methanol. The eluents were filtered through a 0.45 um filter and then concentrated to dryness. The resulting material was redissolved in DMF, and purified by reverse phase HPLC/MS.

Procedure D

General Procedure for Oxime Formation

To aromatic aldehyde suspended in EtOH/H₂O (1:2) was added hydroxylamine hydrochloride (1.1 equiv.) and cooled to 4° C. To this solution was added aqueous NaOH 50% w/w (2.5 equiv.) dropwise. After stirring for 1.5 h at room temperature, the reaction mixture was acidified with 2N aqueous HCl and extracted with CH₂Cl₂. The organic solution washed with saturated aqueous NaCl and dried over sodium sulfate. Removal of solvent gave crude oxime that was used directly in the next step.

General Procedure for Cycloaddition

Oxime was suspended in CH₂Cl₂and cooled to 4° C. Propargyl chloride (1 equiv.) was added to the reaction solution followed by dropwise addition of NaOCl (10-13% free chlorine, 1 equiv.). The reaction mixture was stirred at 4° C. for 15 min then heated to reflux for 3 h. After cooling to room temperature, the reaction was partitioned between CH₂Cl₂and H₂O. The organic layer was separated, washed with saturated aqueous NaCl, and dried over sodium sulfate. After removal of solvent, the crude product was purified by trituration (hexanes) or by column chromatography on silica (10% CH₂Cl₂/hexanes).

General Procedure for Alkylation

To imidazopyridine suspended in DMF was added aqueous NaOH 10% w/w (1.2 equiv.) dropwise followed by addition of chloromethyl isoxazole (1.2 equiv.) in DMF. After stirring for 12 h at room temperature, solvents were evaporated to give crude product as a tan solid. The crude solid was triturated with H₂O and crystallized from MeOH/H₂O (2:1) to provide pure final product.

Procedure E; Suzuki Bromides

embedded image

The aryl bromide (1.2 eq.) was added to a solution of 4-((2-(2,3-difluorophenyl)-5H-imidazo[4,5-c]pyridin-5-yl)methyl)phenylboronic acid (1 eq.) in DMF. Na₂CO₃(2 eq) was dissolved in H₂O, added to the DMF solution and stirred. Pd(PPh₃)₄(5 mole %) was then added to the DMF reaction mixture. The reaction mixture was heated in a microwave at 200° C. for 2 minutes. The reaction mixture was applied to a 1 g solid phase extraction cartridge (C_1-18) and the column washed with 3×2 mL of methanol. The eluents were filtered through a 0.45 um filter and then concentrated to dryness. The resulting material was redissolved in DMF, and purified by reverse phase HPLC/MS.

Procedure F

Preparation of Biphenyl Array

embedded image

The appropriately substituted 4′-[2-(2,3-Difluoro-phenyl)-imidazo[4,5-c]pyridin-5-ylmethyl]-biphenyl-4-ol (5) scaffold was prepared by first treating 2-(2,3-Difluoro-phenyl)-3H-imidazo[4,5-c]pyridine) (1) with 1-bromomethyl-4-iodobenzene (2) in DMF using aqueous sodium hydroxide as base. The resulting 2-(2,3-Difluoro-phenyl)-5-(4-iodo-benzyl)-5H-imidazo[4,5-c]pyridine (3) (1 equivalent) was treated with three different substituted 4-hydroxyphenyl boronic acids ((4-hydroxyphenyl)boronic acid, 4-Hydroxy-2-(trifluoromethyl)phenyl boronic acid and (4-hydroxy-2-methylphenyl)boronic acid) and (4-Fluoro-2-hydroxy)phenylboronic acid (1.1 equivalents) under Suzuki coupling conditions (sodium carbonate, water, palladium tetrakis(triphenyl)phosphine) to afford the appropriately substituted 4′-[2-(2,3-Difluoro-phenyl)-imidazo[4,5-c]pyridin-5-ylmethyl]-biphenyl-4-ol or -[2-(2,3-Difluoro-phenyl)-imidazo[4,5-c]pyridin-5-ylmethyl]-biphenyl-2-ol. The products were precipitated in ethyl acetate and filtered over a medium frit followed by washing with water to afford the pure product (5).

For compounds prepared in array format of the general type (7), 50 μM of the scaffold (5) in 250 μL DMF was used for each reaction. To each reaction was added 1.4 equivalents of Cesium Carbonate. The alkylating agents (6) were added as a 0.4M solution (0.05 mMoles) in DMF. The reactions were shaken at 60° C. for 4 hours and monitored by analytical LC/MS. Each reaction was filtered through a 0.45 μM filter and purified by mass-based fractionation on a C-18 reverse phase column using 0.1% TFA/water and 0.1% TFA/acetonitrile as the eluting solvents. Each compound was identified by its mass spectrum and purity was determined by its UV absorbance at 254 nm. The HPLC fractions were concentrated in vacuo and weighed to afford the product (7) as its trifluoroacetate salt.

The compounds produced according to these procedures and examples, and certain of their properties, are described in the Table below. The substituent designated “C” is methyl.

Structures
Purity
MW
Obs. MW
Method

Example 9
95
387.340
388.340
A

embedded image

Example 10
90
395.440
396.440
A

embedded image

Example 11
90
413.431
414.431
A

embedded image

Example 12
92
404.451
405.451
A

embedded image

Example 13
95
407.451
408.451
A

embedded image

Example 14
85
405.479
406.479
A

embedded image

Example 15
90
389.331
390.331
A

embedded image

Example 16
90
431.418
432.418
A

embedded image

Example 17
93
404.834
405.834
A

embedded image

Example 18
90
370.389
371.389
A

embedded image

Example 19
95
389.331
390.331
A

embedded image

Example 20
95
389.331
390.331
A

embedded image

Example 21
95
389.331
390.331
A

embedded image

Example 22
97
355.777
356.777
A

embedded image

Example 23
90
407.451
408.451
A

embedded image

Example 24
90
461.134
462.134
A

embedded image

Example 25
90
401.404
402.404
A

embedded image

Example 26
95
371.377
372.377
A

embedded image

Example 27
95
439.375
440.375
A

embedded image

Example 28
90
405.822
406.822
A

embedded image

Example 29
90
427.485
428.485
A

embedded image

Example 30
85
456.833
457.833
A

embedded image

Example 31
95
439.375
440.375
A

embedded image

Example 32
90
386.454
387.454
A

embedded image

Example 33
90
392.480
393.480
A

embedded image

Example 34
95
361.301
362.301
A

embedded image

Example 35
92
369.804
370.804
A

embedded image

Example 36
90
405.785
406.785
A

embedded image

Example 37
92
421.785
422.785
A

embedded image

Example 38
90
401.367
402.367
A

embedded image

Example 39
90
351.814
352.814
A

embedded image

Example 40
92
423.812
424.812
A

embedded image

Example 41
98
339.391
340.391
A

embedded image

Example 42
92
449.408
450.408
A

embedded image

Example 43
95
422.825
423.825
A

embedded image

Example 44
93
388.380
389.380
A

embedded image

Example 45
95
479.124
480.124
A

embedded image

Example 46
97
419.394
420.394
A

embedded image

Example 47
94
389.367
390.367
A

embedded image

Example 48
92
457.366
458.366
A

embedded image

Example 49
90
363.778
364.778
A

embedded image

Example 50
92
445.476
446.476
A

embedded image

Example 51
95
457.366
458.366
A

embedded image

Example 52
95
472.443
473.443
A

embedded image

Example 53
95
404.444
405.444
A

embedded image

Example 54
95
395.393
396.393
A

embedded image

Example 55
90
410.470
411.470
A

embedded image

Example 56
92
457.329
458.329
A

embedded image

Example 57
93
353.350
354.350
A

embedded image

Example 58
95
423.776
424.776
A

embedded image

Example 59
95
439.775
440.775
A

embedded image

Example 60
92
419.357
420.357
A

embedded image

Example 61
90
390.222
391.222
A

embedded image

Example 62
90
405.330
406.330
A

embedded image

Example 63
90
431.421
432.421
A

embedded image

Example 64
0
422.441
423.441
A

embedded image

Example 65
90
425.442
426.442
A

embedded image

Example 66
95
431.876
432.876
C

embedded image

Example 67
95
442.429
443.429
C

embedded image

Example 68
95
411.458
412.458
C

embedded image

Example 69
95
411.458
412.458
C

embedded image

Example 70
95
411.458
412.458
C

embedded image

Example 71
95
415.422
416.422
C

embedded image

Example 72
95
403.457
404.457
C

embedded image

Example 73
90
441.485
442.485
C

embedded image

Example 74
95
465.430
466.430
C

embedded image

Example 75
95
431.876
432.876
C

embedded image

Example 76
95
415.422
416.422
C

embedded image

Example 77
95
441.485
442.485
C

embedded image

Example 78
95
441.485
442.485
C

embedded image

Example 79
95
443.522
444.522
C

embedded image

Example 80
95
387.392
388.392
C

embedded image

Example 81
95
433.412
434.412
C

embedded image

Example 82
95
439.469
440.469
C

embedded image

Example 83
95
439.469
440.469
C

embedded image

Example 84
95
425.485
426.485
C

embedded image

Example 85
95
465.430
466.430
C

embedded image

Example 86
95
465.430
466.430
C

embedded image

Example 87
95
415.422
416.422
C

embedded image

Example 88
95
466.321
467.321
C

embedded image

Example 89
95
433.412
434.412
C

embedded image

Example 90
95
533.428
534.428
C

embedded image

Example 91
95
466.321
467.321
C

embedded image

Example 92
90
425.485
426.485
C

embedded image

Example 93
90
457.484
458.484
C

embedded image

Example 94
90
447.492
448.492
C

embedded image

Example 95
90
489.529
490.529
C

embedded image

Example 96
90
457.484
458.484
C

embedded image

Example 97
90
425.485
426.485
C

embedded image

Example 98
90
425.485
426.485
C

embedded image

Example 99
90
440.500
441.500
C

embedded image

Example 100
90
429.449
430.449
C

embedded image

Example 101
90
437.902
438.902
C

embedded image

Example 102
90
437.453
438.453
C

embedded image

Example 103
90
453.517
454.517
C

embedded image

Example 104
90
481.429
482.429
C

embedded image

Example 105
90
481.429
482.429
C

embedded image

Example 106
90
453.517
454.517
C

embedded image

Example 107
90
449.867
450.867
C

embedded image

Example 108
90
466.321
467.321
C

embedded image

Example 109
90
422.441
423.441
C

embedded image

Example 110
90
433.412
434.412
C

embedded image

Example 111
90
442.429
443.429
C

embedded image

Example 112
90
427.458
428.458
C

embedded image

Example 113
90
427.458
428.458
C

embedded image

Example 114
90
425.485
426.485
C

embedded image

Example 115
90
439.512
440.512
C

embedded image

Example 116
90
466.321
467.321
C

embedded image

Example 117
90
503.556
504.556
C

embedded image

Example 118
90
443.522
444.522
C

embedded image

Example 119
90
422.441
423.441
C

embedded image

Example 120
90
475.521
476.521
C

embedded image

Example 121
90
433.412
434.412
C

embedded image

Example 122
90
503.556
504.556
C

embedded image

Example 123
90
429.449
430.449
C

embedded image

Example 124
90
453.540
454.540
C

embedded image

Example 125
90
466.321
467.321
C

embedded image

Example 126
90
456.456
457.456
C

embedded image

Example 127
90
481.429
482.429
C

embedded image

Example 128
90
483.522
484.522
C

embedded image

Example 129
90
445.448
446.448
C

embedded image

Example 130
90
392.870
393.870
A

embedded image

Example 131
90
358.425
359.425
A

embedded image

Example 132
90
392.486
393.486
A

embedded image

Example 133
90
453.540
454.540
C

embedded image

Example 134
90
546.582
547.582
C

embedded image

Example 135
90
445.448
446.448
C

embedded image

Example 136
0
456.378
457.378
A

embedded image

Example 137
95
472.378
473.378
A

embedded image

Example 138
95
423.812
424.812
A

embedded image

Example 139
99
457.270
458.270
D

embedded image

Example 140
98
472.378
473.378
D

embedded image

Example 141
98
524.377
525.377
D

embedded image

Example 142
0
474.369
475.369
D

embedded image

Example 143
99
454.387
455.387
D

embedded image

Example 144
98
474.369
475.369
D

embedded image

Example 145
98
485.266
486.266
D

embedded image

Example 146
95
442.351
443.351
D

embedded image

Example 147
90
420.397
421.397
D

embedded image

Example 148
90
402.407
403.407
D

embedded image

Example 149
98
448.433
449.433
D

embedded image

Example 150
98
474.369
475.369
D

embedded image

Example 151
96
439.280
440.280
D

embedded image

Example 152
98
454.387
455.387
D

embedded image

Example 153
98
506.386
507.386
D

embedded image

Example 154
98
456.378
457.378
D

embedded image

Example 155
98
436.397
437.397
D

embedded image

Example 156
98
456.378
457.378
D

embedded image

Example 157
98
467.276
468.276
D

embedded image

Example 158
98
430.442
431.442
D

embedded image

Example 159
98
456.378
457.378
D

embedded image

Example 160
85
473.725
474.725
D

embedded image

Example 161
98
488.832
489.832
D

embedded image

Example 162
98
540.831
541.831
D

embedded image

Example 163
98
490.823
491.823
D

embedded image

Example 164
98
470.842
471.842
D

embedded image

Example 165
98
490.823
491.823
D

embedded image

Example 166
98
501.721
502.721
D

embedded image

Example 167
90
415.422
416.422
C

embedded image

Example 168
90
483.420
484.420
C

embedded image

Example 169
90
499.419
500.419
C

embedded image

Example 170
90
445.448
446.448
C

embedded image

Example 171
90
461.513
462.513
C

embedded image

Example 172
90
451.402
452.402
C

embedded image

Example 173
90
465.430
466.430
C

embedded image

Example 174
90
481.429
482.429
C

embedded image

Example 175
90
427.458
428.458
C

embedded image

Example 176
90
443.522
444.522
C

embedded image

Example 177
90
433.412
434.412
C

embedded image

Example 178
90
431.876
432.876
C

embedded image

Example 179
90
515.874
516.874
C

embedded image

Example 180
90
461.903
462.903
C

embedded image

Example 181
90
477.967
478.967
C

embedded image

Example 182
90
467.857
468.857
C

embedded image

Example 183
90
501.410
502.410
C

embedded image

Example 184
90
397.431
398.431
C

embedded image

Example 185
95
479.556
480.556
E

embedded image

Example 186
95
423.469
424.469
E

embedded image

Example 187
95
441.485
442.485
E

embedded image

Example 188
95
455.468
456.468
E

embedded image

Example 189
95
469.495
470.495
E

embedded image

Example 190
95
483.522
484.522
E

embedded image

Example 192
95
436.468
437.468
E

embedded image

Example 193
95
475.475
476.475
E

embedded image

Example 194
95
453.496
454.496
E

embedded image

Example 195
95
463.438
464.438
E

embedded image

Example 196
95
464.479
465.479
E

embedded image

Example 199
90
415.422
416.422
C

embedded image

Example 200
90
483.420
484.420
C

embedded image

Example 201
90
499.419
500.419
C

embedded image

Example 202
90
445.448
446.448
C

embedded image

Example 203
90
461.513
462.513
C

embedded image

Example 204
90
451.402
452.402
C

embedded image

Example 205
90
397.431
398.431
C

embedded image

Example 206
90
465.430
466.430
C

embedded image

Example 207
90
481.429
482.429
C

embedded image

Example 208
90
427.458
428.458
C

embedded image

Example 209
90
443.522
444.522
C

embedded image

Example 210
90
433.412
434.412
C

embedded image

Example 211
90
431.876
432.876
C

embedded image

Example 212
90
499.875
500.875
C

embedded image

Example 213
90
477.967
478.967
C

embedded image

Example 214
95
462.506
463.506
E

embedded image

Example 215
95
421.840
422.840
A

embedded image

Example 216
95
403.850
404.850
A

embedded image

Example 217
95
417.422
418.422
A

embedded image

Example 218
95
399.431
400.431
A

embedded image

Example 219
95
434.400
435.400
A

embedded image

Example 220
95
416.409
417.409
A

embedded image

Example 221
98
424.361
425.361
D

embedded image

Example 222
85
406.370
407.370
D

embedded image

Example 223
98
440.815
441.815
D

embedded image

Example 224
90
553.493
554.493
F

embedded image

Example 225
90
567.520
568.520
F

embedded image

Example 226
90
579.575
580.575
F

embedded image

Example 227
84
551.521
552.521
F

embedded image

Example 228
100
537.537
538.537
F

embedded image

Example 229
92
551.564
552.564
F

embedded image

Example 230
100
593.646
594.646
F

embedded image

Example 231
81
567.520
568.520
F

embedded image

Example 232
78
539.510
540.510
F

embedded image

Example 233
77
583.563
584.563
F

embedded image

Example 234
85
549.548
550.548
F

embedded image

Example 235
85
579.531
580.531
F

embedded image

Example 236
82
593.558
594.558
F

embedded image

Example 237
90
535.521
536.521
F

embedded image

Example 238
85
551.564
552.564
F

embedded image

Example 239
85
595.574
596.574
F

embedded image

Example 240
80
551.564
552.564
F

embedded image

Example 241
85
535.521
536.521
F

embedded image

Example 242
85
577.603
578.603
F

embedded image

Example 243
100
595.574
596.574
F

embedded image

Example 244
83
533.505
534.505
F

embedded image

Example 245
90
487.529
488.529
F

embedded image

Example 246
90
501.556
502.556
F

embedded image

Example 247
90
501.556
502.556
F

embedded image

Example 248
90
501.556
502.556
F

embedded image

Example 249
90
539.485
540.485
F

embedded image

Example 250
90
483.497
484.497
F

embedded image

Example 251
90
483.566
484.566
F

embedded image

Example 252
90
513.549
514.549
F

embedded image

Example 253
90
485.538
486.538
F

embedded image

Example 254
90
529.592
530.592
F

embedded image

Example 255
90
525.560
526.560
F

embedded image

Example 256
90
481.550
482.550
F

embedded image

Example 257
90
541.603
542.603
F

embedded image

Example 258
90
481.550
482.550
F

embedded image

Example 259
90
541.603
542.603
F

embedded image

Example 260
90
479.534
480.534
F

embedded image

Example 261
90
522.559
523.559
F

embedded image

Example 262
90
469.539
470.539
F

embedded image

Example 263
90
471.511
472.511
F

embedded image

Example 264
90
515.565
516.565
F

embedded image

Example 265
90
511.533
512.533
F

embedded image

Example 266
90
467.523
468.523
F

embedded image

Example 267
90
527.576
528.576
F

embedded image

Example 268
90
521.494
522.494
F

embedded image

Example 269
80
465.507
466.507
F

embedded image

Example 270
90
453.496
454.496
F

embedded image

Example 271
90
470.483
471.483
F

embedded image

Example 272
90
495.577
496.577
F

embedded image

Example 273
90
481.550
482.550
F

embedded image

Example 274
90
610.644
611.644
F

embedded image

Example 275
90
506.560
507.560
F

embedded image

Example 276
90
485.538
486.538
F

embedded image

Example 277
90
595.574
596.574
F

embedded image

Example 278
90
521.494
522.494
F

embedded image

Example 279
90
538.481
539.481
F

embedded image

Example 280
90
563.576
564.576
F

embedded image

Example 281
85
565.548
566.548
F

embedded image

Example 282
90
580.606
581.606
F

embedded image

Example 283
90
549.548
550.548
F

embedded image

Example 284
85
678.643
679.643
F

embedded image

Example 285
90
574.558
575.558
F

embedded image

Example 286
90
588.585
589.585
F

embedded image

Example 287
90
659.599
660.599
F

embedded image

Example 288
90
617.547
618.547
F

embedded image

Example 289
90
572.542
573.542
F

embedded image

Example 290
90
553.537
554.537
F

embedded image

Example 291
90
629.514
630.514
F

embedded image

Example 292
85
571.502
572.502
F

embedded image

Example 293
80
545.566
546.566
F

embedded image

Example 294
90
471.486
472.486
F

embedded image

Example 295
90
488.473
489.473
F

embedded image

Example 296
90
628.635
629.635
F

embedded image

Example 297
90
524.550
525.550
F

embedded image

Example 298
90
538.577
539.577
F

embedded image

Example 300
90
503.529
504.529
F

embedded image

Example 301
90
579.506
580.506
F

embedded image

Example 302
90
521.494
522.494
F

embedded image

Example 303
90
541.603
542.603
F

embedded image

Example 304
90
467.523
468.523
F

embedded image

Example 305
90
483.566
484.566
F

embedded image

Example 306
90
509.604
510.604
F

embedded image

Example 307
90
511.577
512.577
F

embedded image

Example 308
90
495.577
496.577
F

embedded image

Example 309
90
520.587
521.587
F

embedded image

Example 310
90
534.614
535.614
F

embedded image

Example 311
90
605.627
606.627
F

embedded image

Example 312
90
563.576
564.576
F

embedded image

Example 313
90
499.565
500.565
F

embedded image

Example 314
90
575.543
576.543
F

embedded image

Example 315
90
791.891
792.891
F

embedded image

Example 316
95
486.405
487.405
D

embedded image

Example 317
90
417.397
418.397
A

embedded image

Example 318
90
396.787
397.787
A

embedded image

Example 319
90
387.34

A

embedded image

Example 320
90
371.34

A

embedded image

Example 321
90
400.23

A

embedded image

Example 322
90
401.37

A

embedded image

Example 323
90
405.33

A

embedded image

Example 324
90
345.42

A

embedded image

Example 325
90
409.47

A

embedded image

Example 326
90
403.40

A

embedded image

Example 327
90
363.41

A

embedded image

Example 328
90
389.33

A

embedded image

Example 329
90
359.45

A

embedded image

Example 330
90
385.37

A

embedded image

Example 331
90
417.37

A

embedded image

Example 332
90
421.78

A

embedded image

Example 333
90
466.24

A

embedded image

Example 334
90
393.90

A

embedded image

Example 335
90
416.68

A

embedded image

Example 336
90
405.79

A

embedded image

Example 337
90
463.68

A

embedded image

Example 338
90
379.87

A

embedded image

Example 339
90
423.81

embedded image

Example 340
90
419.39

embedded image

Example 341
90
403.39

embedded image

Example 342
90
418.41

D

embedded image

Example 343
90
402.41

D

embedded image

Example 344
90
378.34

D

embedded image

Example 345
90
394.41

D

embedded image

Example 346
90
431.45

D

embedded image

Example 347
90
464.48

D

embedded image

Example 348
90
467.28

D

embedded image

Example 349
90
494.51

D

embedded image

Example 350
90
434.47

D

embedded image

Example 351
90
432.43

D

embedded image

Example 352
90
432.43

D

embedded image

Example 353
90
436.40

D

embedded image

Example 354
90
440.82

D

embedded image

Example 355
90
446.46

D

embedded image

Example 356
90
480.48

D

embedded image

Example 357
90
391.38

D

embedded image

Example 358
90
446.46

D

embedded image

Example 359
90
440.82

D

embedded image

Example 360
90
478.48

D

embedded image

Example 361
90
428.85

D

embedded image

Example 362
90
460.49

D

embedded image

Example 363
90
428.47

D

embedded image

Example 364
90
444.44

D

embedded image

Example 365
90
474.51

D

embedded image

Example 366
90
473.30

D

embedded image

Example 367
90
378.34

D

embedded image

Example 368
90
500.55

D

embedded image

Example 369
90
500.55

D

embedded image

Example 370
90
488.54
D

embedded image

Example 371
90
488.53
D

embedded image

Example 372
90
487.4
D

embedded image

Example 373
90
417.4
C

embedded image

Example 374
Isoxazole Analogues

embedded image

Synthesis of 374a:

embedded image

To a stirred solution of 4-ethoxy-benzaldehyde (3.000 g) in 50% ethanol (7 mL) ice (10 g) and hydroxylamine hydrochloride (2.100 g) were added, followed by 30% aqueous sodium hydroxide solution (3.5 mL). After completion of the reaction (1 h) hydrochloric acid was added to adjust pH to 1 and the suspension was cooled on an ice bath and filtered. The crude oxime can be used for the next step without purification. Alternatively, it can be recrystallized from a mixture of diisopropyl ether and ethyl acetate. Yield: 71%.

To a solution of propargyl chloride (655 mg, 1 equ.) and triethylamine (35 mg, 0.1 equ.) in dichloromethane (9.5 mL) were subsequently added with cooling 10% aqueous sodium hypochlorite solution (9.5 mL, 1.5 equ.) and then a solution of the oxime (1.40 g, ˜1.3 M in dichloromethane) over a period of 15 minutes and stirring was continued for an additional hour. The reaction was monitored by TLC (silicagel, eluent: 5% MeOH in dichloromethane). After completion the reaction mixture was extracted 3 times with 30 mL dichloromethane. The combined organic phases were dried over anhydrous sodium sulphate and evaporated under reduced pressure. The crude 5-(chloromethyl)-3-(4-ethoxyphenyl)-isoxazole was purified by column chromatography (silicagel, ethyl acetate/petroleum ether=1:9). Yield: 1.1 g.

A mixture of 3,4-diaminopyridine (2.00 g), 2,3-difluorobenzoic acid (1 equivalent) and polyphosphoric acid (50 g) was heated at 180° C. for 4 h with stirring. Then the mixture was cooled to ambient temperature and poured into ice/water. The resulting mixture was neutralized by addition of solid NaOH. The crude 2-(2,3-difluorophenyl)-1(3)H-imidazo[4,5-c]pyridine was collected by filtration, washed with water and dried. It was used in the next step without further purification. Yield: 88%.

2-(2,3-Difluorophenyl)-1(3)H-imidazo[4,5-c]pyridine (0.500 g) was dissolved in dry DMF (5 mL) and the resulting solution was cooled to 0° C. Aqueous 50% sodium hydroxide (1.5 equivalents) was added and the mixture was stirred for 15 min. Then 5-(chloromethyl)-3-(4-ethoxyphenyl)-isoxazole (1.2 equivalents) was added and the resulting mixture was stirred for 24 h at room temperature. Finally, water (50 mL) was added, the precipitate was collected by filtration and dried to give the crude product.

Recrystallized from ethyl acetate; colorless crystals; yield: 35%

¹H NMR (200 MHz, DMSO-d₆) δ 9.24 (d, 1H, H4, J=1.2 Hz), 8.28 (dd, 1H, H6, J=6.6, 1.2 Hz), 8.15 (m, 1H, phenyl-H), 7.89 (d, 1H, H7, J=6.6 Hz), 7.77 (AA′BB′, 2H, benzyl-H), 7.49 (m, 1H, phenyl-H), 7.31 (m, 1H, phenyl-H), 7.07-7.00 (m, 3H, arom. H), 6.02 (s, 2H, CH₂), 4.06 (q, 2H, OCH₂, J=6.9 Hz), 1.32 (t, 3H, CH₃, J=6.9 Hz).

The following examples were prepared by analogy to the above procedure:

374b

embedded image

Starting from 4-methoxybenzaldehyde.

¹H NMR (200 MHz, DMSO-d₆) δ 9.23 (d, 1H, H4, J=1.2 Hz), 8.28 (dd, 1H, H6, J=6.6, 1.2 Hz), 8.15 (m, 1H, phenyl-H), 7.88 (d, 1H, H7, J=6.6 Hz), 7.79 (AA′BB′, 2H, benzyl-H), 7.49 (m, 1H, phenyl-H), 7.31 (m, 1H, phenyl-H), 7.09-7.00 (m, 3H, arom. H), 6.01 (s, 2H, CH₂), 3.80 (s, 3H, OCH₃).

374c

embedded image

Starting from 4-methylbenzaldehyde.

¹H NMR (200 MHz, DMSO-d₆) δ 9.24 (br s, 1H, H4), 8.29 (d, 1H, H6, J=6.7 Hz), 8.14 (m, 1H, phenyl-H), 7.88 (d, 1H, H7, J=6.7 Hz), 7.58-7.27 (m, 6H, arom. H), 7.00 (s, 1H, isoxazole-H), 6.04 (s, 2H, CH₂), 2.41 (s, 3H, CH₃).

374d

embedded image

Starting from 2-furaldehyde

¹H NMR (200 MHz, DMSO-d₆) δ 9.24 (br s, 1H, H4), 8.28 (d, 1H, H6, J=6.7 Hz), 8.15 (m, 1H, phenyl-H), 7.90-7.87 (m, 2H, arom. H), 7.51 (m, 1H, phenyl-H), 7.32 (m, 1H, phenyl-H), 7.15 (d, 1H, furane-H, J=3.6 Hz), 6.96 (s, 1H, isoxazole-H), 6.68 (m, 1H, furane-H), 6.02 (s, 2H, CH₂).

374e

embedded image

Starting from thiophene-2-carboxaldehyde

¹H NMR (200 MHz, DMSO-d₆) δ 9.23 (d, 1H, H4, J=1.6 Hz), 8.27 (dd, 1H, H6, J=7.0, 1.6 Hz), 8.14 (m, 1H, phenyl-H), 7.88 (d, 1H, H7, J=7.0 Hz), 7.75-7.70 (m, 2H, arom. H), 7.50 (m, 1H, phenyl-H), 7.31 (m, 1H, phenyl-H), 7.19 (dd, 1H, thiophene-H), 7.06 (s, 1H, isoxazole-H), 6.02 (s, 2H, CH₂).

374f

embedded image

Starting from 4-dimethylanainobenzaldehyde

¹H NMR (200 MHz, DMSO-d₆) δ 9.23 (d, 1H, H4, J=1.6 Hz), 8.27 (dd, 1H, H6, J=7.0, 1.6 Hz), 8.14 (m, 1H, phenyl-H), 7.88 (d, 1H, H7, J=7.0 Hz), 7.65 (AA′BB′, 2H, benzyl-H), 7.49 (m, 1H, phenyl-H), 7.31 (m, 1H, phenyl-H), 6.98 (s, 111, isoxazole-H), 6.76 (AA′BB′, 2H, benzyl-H), 5.75 (s, 2H, CH₂), 2.95 (s, 6H, N(CH₃)₂).

374g

embedded image

Starting from 4-biphenylcarboxaldehyde

¹H NMR (200 MHz, DMSO-d₆) δ 9.25 (d, 1H, H4, J=1.6 Hz), 8.30 (dd, 1H, H6, J=7.0, 1.6 Hz), 8.16 (m, 1H, phenyl-H), 7.98-7.70 (m, 7H, arom. H), 7.57-7.26 (m, 5H, arom. H), 7.18 (s, 1H, isoxazole-H), 6.05 (s, 2H, CH₂).

374h

embedded image

Starting from 4-bromobenzaldehyde

¹H NMR (200 MHz, DMSO-d₆) δ 9.23 (d, 1H, H4, J=1.6 Hz), 8.28 (dd, 1H, H6, J=7.0, 1.6 Hz), 8.16 (m, 1H, phenyl-H), 7.90-7.68 (m, 4H, arom. H), 7.51 (m, 1H, phenyl-H), 7.31 (m, 1H, phenyl-H), 7.15 (s, 1H, isoxazole-H), 6.05 (s, 2H, CH₂).

374i

embedded image

Starting from 4-benzyloxybenzaldehyde

¹H NMR (200 MHz, DMSO-d₆) δ 9.23 (d, 1H, H4, J=1.6 Hz), 8.27 (dd, 1H, H6, J=7.0, 1.6 Hz), 8.15 (m, 1H, phenyl-H), 7.90-7.76 (m, 3H, arom. H), 7.57-7.26 (m, 7H, arom. H), 7.15-7.05 (m, 3H, arom. H), 6.01 (s, 2H, N—CH₂), 5.16 (s, 2H, O—CH₂).

374j

embedded image

Starting from 4-(methylthio)benzaldehyde

¹H NMR (200 MHz, DMSO-d₆) δ 9.23 (d, 1H, H4, J=1.2 Hz), 8.28 (dd, 1H, H6, J=6.6, 1.2 Hz), 8.15 (m, 1H, phenyl-H), 7.88 (d, 1H, H7, J=6.6 Hz), 7.79 (AA′BB′, 2H, benzyl-H), 7.50 (m, 1H, phenyl-H), 7.38-7.25 (m, 3H, arom. H), 7.10 (s, 1H, isoxazole-H), 6.03 (s, 2H, CH₂), 2.51 (s, 3H, SCH₃).

374k

embedded image

Starting from 2-fluoro-4-methoxybenzaldehyde

¹H NMR (200 MHz, DMSO-d₆) δ 9.26 (d, 1H, H4, J=1.2 Hz), 8.30 (dd, 1H, H6, J=6.6, 1.2 Hz), 8.14 (m, 1H, phenyl-H), 7.88 (d, 1H, H7, J=6.6 Hz), 7.80 (m, 1H, benzyl-H), 7.49 (m, 1H, phenyl-H), 7.31 (m, 1H, phenyl-H), 7.04-6.71 (m, 3H, arom. H), 6.03 (s, 2H, CH₂), 3.82 (s, 3H, OCH₃).

374w

embedded image

Prepared as described above, starting from 4-chloro-2-fluorobenzaldehyde.

¹H NMR (200 MHz, DMSO-d₆) □ 9.26 (d, 1H, H4, J=1.4 Hz), 8.30 (dd, 1H, H6, J=6.8, 1.4 Hz), 8.14 (m, 1H, phenyl-H), 7.90-7.87 (m, 2H, arom. H), 7.66 (dd, 1H, arom. H, J=10.8, 1.8 Hz), 7.53-7.41 (m, 2H, arom. H), 7.31 (m, 1H, phenyl-H), 7.10 (d, 1H, isoxazole-H, J=2.7 Hz), 6.06 (s, 2H, CH₂).

374l

embedded image

Starting from 4-propoxybenzaldehyde

¹H NMR (200 MHz, DMSO-d₆) δ 9.23 (d, 1H, H4, J=1.2 Hz), 8.29 (dd, 1H, H6, J=6.6, 1.2 Hz), 8.14 (m, 1H, phenyl-H), 7.88 (d, 1H, H7, J=6.6 Hz), 7.78 (AA′BB′, 2H, benzyl-H), 7.49 (m, 1H, phenyl-H), 7.31 (m, 1H, phenyl-H), 7.06-7.00 (m, 3H, arom. H), 6.01 (s, 2H, CH₂), 3.97 (t, 2H, OCH₂, J=6.5 Hz), 1.73 (hex, 2H, CH₂), 0.97 (t, 3H, CH₃, J=7.3 Hz).

374m

embedded image

Starting from 4-phenoxybenzaldehyde

¹H NMR (200 MHz, DMSO-d₆) δ 9.25 (d, 1H, H4, J=1.2 Hz), 8.29 (dd, 1H, H6, J=6.6, 1.2 Hz), 8.16 (m, 1H, phenyl-H), 7.92-7.83 (m, 3H, arom. H), 7.58-7.05 (m, 10H, arom. H), 6.04 (s, 2H, CH₂).

374n

embedded image

Starting from 1-methylpyrrole-2-carboxaldehyde

¹H NMR (200 MHz, DMSO-d₆) δ 9.24 (d, 1H, H4, J=1.2 Hz), 8.28 (dd, 1H, H6, J=6.8, 1.2 Hz), 8.16 (m, 1H, phenyl-H), 7.89 (d, 1H, H7, J=6.8 Hz), 7.50 (m, 1H, phenyl-H), 7.31 (m, 1H, phenyl-H), 6.98 (dd, 1H, pyrrole-H), 6.92 (s, 1H, isoxazole-H), 6.68 (dd, 1H, pyrrole-H), 6.12 (dd, 1H, pyrrole-H), 6.00 (s, 2H, CH₂).

374o

embedded image

Starting from 4-isopropoxybenzaldehyde.

¹H NMR (200 MHz, DMSO-d₆) δ 9.24 (d, 1H, H4, J=1.4 Hz), 8.28 (dd, 1H, H6, J=7.0, 1.4 Hz), 8.15 (m, 1H, phenyl-H), 7.89 (d, 1H, H7, J=7.0 Hz), 7.76 (AA′BB′, 2H, benzyl-H), 7.50 (m, 1H, phenyl-H), 7.31 (m, 1H, phenyl-H), 7.05-6.98 (m, 3H, arom. H), 6.01 (s, 2H, CH₂), 4.67 (hept, 1H, OCH, J=6.2 Hz), 1.26 (d, 6H, (CH₃)₂, J=6.2 Hz).

374p

embedded image

Synthesized as described above, starting from 5-chlorothiophene-2-carboxaldehyde.

¹H NMR (200 MHz, DMSO-d₆) δ 9.23 (d, 1H, H4, J=1.4 Hz), 8.27 (dd, 1H, H6, J=7.0, 1.4 Hz), 8.14 (m, 1H, phenyl-H), 7.89 (d, 1H, H7, J=7.0 Hz), 7.63 (d, 1H, thiophene-H, J=4.0 Hz), 7.51 (m, 1H, phenyl-H), 7.37-7.24 (m, 2H, arom. H), 7.07 (s, 1H, isoxazole-H), 6.03 (s, 2H, CH₂).

374q

embedded image

Synthesized as described above, starting from 5-bromothiophene-2-carboxaldehyde.

¹H NMR (200 MHz, DMSO-d₆) δ 9.23 (d, 1H, H4, J=1.4 Hz), 8.27 (dd, 1H, H6, J=6.6, 1.4 Hz), 8.14 (m, 1H, phenyl-H), 7.89 (d, 1H, H7, J=6.6 Hz), 7.59 (d, 1H, thiophene-H, J=3.6 Hz), 7.50 (m, 1H, phenyl-H), 7.36-7.27 (m, 2H, arom. H), 7.06 (s, 1H, isoxazole-H), 6.02 (s, 2H, CH₂).

374r

embedded image

Synthesized as described above, starting from 4-butoxybenzaldehyde (prepared by alklylation of 4-hydroxybenzaldehyde).

¹H NMR (200 MHz, DMSO-d₆) δ 9.24 (d, 1H, H4, J=1.2 Hz), 8.28 (dd, 1H, H6, J=6.6, 1.2 Hz), 8.15 (m, 1H, phenyl-H), 7.89 (d, 1H, H7, J=6.6 Hz), 7.78 (AA′BB′, 2H, benzyl-H), 7.50 (m, 1H, phenyl-H), 7.31 (m, 1H, phenyl-H), 7.07-7.01 (m, 3H, arom. H), 6.01 (s, 2H, CH₂), 4.01 (t, 2H, OCH₂, J=6.5 Hz), 1.72 (m, 2H, CH₂), 1.42 (m, 2H, CH₂), 0.93 (t, 3H, CH₃, J=7.2 Hz).

374s

embedded image

Synthesized as described above, starting from 4-propoxybenzaldehyde and using 2-(2-fluorophenyl)-1(3)H-imidazo[4,5-c]pyridine instead of 2-(2,3-difluorophenyl)-1(3)H-imidazo[4,5-c]pyridine.

¹H NMR (200 MHz, DMSO-d₆) □ 9.18 (d, 1H, H4, J=1.2 Hz), 8.38-8.23 (m, 2H, arom. H), 7.85 (d, 1H, H7, J=6.6 Hz), 7.78 (AA′BB′, 2H, benzyl-H), 7.54-7.25 (m, 3H, phenyl-H), 7.06-7.00 (m, 3H, arom. H), 6.00 (s, 2H, CH₂), 3.98 (t, 2H, OCH₂, J=6.6 Hz), 1.73 (hex, 2H, CH₂), 0.97 (t, 3H, CH₃, J=7.3 Hz).

374t

embedded image

Starting from 4-allyloxybenzaldehyde

¹H NMR (200 MHz, DMSO-d₆) δ 9.23 (d, 1H, H4, J=1.2 Hz), 8.27 (dd, 1H, H6, J=6.7, 1.2 Hz), 8.14 (m, 1H, phenyl-H), 7.89 (d, 1H, H7, J=6.7 Hz), 7.79 (AA′BB′, 2H, benzyl-H), 7.50 (m, 1H, phenyl-H), 7.31 (m, 1H, phenyl-H), 7.09-7.00 (m, 3H, arom. H), 6.15-5.98 (m, 3H), 5.45-5.24 (m, 2H), 4.62 (d, 2H, J=4.8 Hz).

374u

embedded image

A mixture of 5-(chloromethyl)-3-(4-chlorophenyl)-isoxazole (2.00 g), NCS (11.75 g, 10 equivalents), glacial acetic acid (35 mL) and 20 drops of concentrated sulphuric acid is heated to reflux for 3 days. After cooling to room temperature dichloromethane (100 mL) is added, and the resulting mixture is extracted with water (2×100 mL) and saturated aqueous sodium bicarbonate solution (2×100 mL). Then the organic phase was dried over anhydrous sodium sulphate and evaporated. The crude product, thus obtained, was purified by column chromatography (silica gel, eluent: petroleum ether/ethyl acetate=19/1) to give 1.14 g.

The final step was performed as described above. Recrystallized from a mixture of ethyl acetate and ethanol. Yield: 60%.

¹H NMR (200 MHz, DMSO-d₆) □ 9.20 (d, 1H, H4, J=1.4 Hz), 8.25 (dd, 1H, H6, J=6.8, 1.4 Hz), 8.15 (m, 1H, phenyl-H), 7.89 (d, 1H, H7, J=6.8 Hz), 7.83 (AA′BB′, 2H, benzyl-H), 7.66 (AA′BB′, 2H, benzyl-H), 7.51 (m, 1H, phenyl-H), 7.31 (m, 1H, phenyl-H), 6.14 (s, 2H, CH₂).

374v

embedded image

¹H NMR (200 MHz, DMSO-d₆) δ 9.18 (d, 1H, H4, J=1.4 Hz), 8.22 (dd, 1H, H6, J=6.8, 1.4 Hz), 8.14 (m, 1H, phenyl-H), 7.89 (d, 1H, H7, J=6.8 Hz), 7.80 (AA′BB′, 2H, benzyl-H), 7.65 (AA′BB′, 2H, benzyl-H), 7.49 (m, 1H, phenyl-H), 7.30 (m, 1H, phenyl-H), 6.11 (s, 2H, CH₂).

Synthesized in analogy to the chloroisoxazole derivative 374u: 4 equ. NBS, 2.5 h reflux, yield: 91%.

Example 375

embedded image

To a solution of 500 mg 3-methyl-1-phenylpyrazole in 4 mL carbontetrachloride is added in portions at 70° C. a mixture of 678 mg (1.2 equi.) NBS and AIBN (62.3 mg, 0.12 equ.). The resulting mixture is heated at reflux for an additional 15 minutes and then cooled to room temperature. The precipitate is filtered off and the filtrate is concentrated to precipitate the crude product (380 mg), which—after collecting by filtration and drying—was used in the next step without further purification

The final step was performed as described above. Recrystallized from ethyl acetate. Yield: 35%.

Example 376
imidazo[4,5-c]pyridin-4-one Analogues

embedded image

A mixture of 4-chloro-3-nitro-pyridin-2-one (1.00 g), 4-(trifluoromethyl)benzyl chloride (1.226 g), anhydrous potassium carbonate (0.871 g) and anhydrous DMF (10 mL) was stirred at ambient temperature for 24 hours. Then water (100 mL) was added and the resulting precipitate was collected by filtration, washed with water and dried. Yield: 58.2% 4-chloro-3-nitro-1-(4-trifluoromethyl)benzyl-pyridin-2-one.

4-Chloro-3-nitro-1-(4-trifluoromethyl)benzyl-pyridin-2-one (500 mg) was dissolved in anhydrous THF (10 mL). Then concentrated aqueous ammonia (7.5 mL) was added and the resulting mixture was stirred at room temperature for 24 hours. Water (50 mL) was added and the resulting precipitate was collected by filtration, washed with water and dried. Yield: 45.8% of 4-amino-3-nitro-1-(4-trifluoromethyl)benzyl-pyridin-2-one.

A mixture of 4-amino-3-nitro-1-(4-trifluoromethyl)benzyl-pyridin-2-one (1.400 g), saturated aqueous ammoniumchloride solution (9.4 mL), zink powder (1.400 g) and methanol (235 mL) was stirred at room temperature for 1 hour. Then additional zink powder (1.400 g) was added and the resulting mixture was stirred for an additional 23 hours. After evaporation of the solvent water (30 mL) was added and the pH was adjusted to 8-9 by addition of 2N NaOH. The resulting mixture was extracted with ethyl acetate (3×30 mL) and the combined organic phases were washed with water (30 mL), dried over anhydrous sodium sulphate and evaporated. Yield: 53.4% 3,4-diamino-1-(4-trifluoromethyl)benzyl-pyridin-2-one.

A mixture of 3,4-diamino-1-(4-trifluoromethyl)benzyl-pyridin-2-one (0.200 mg), 2,3-difluorobenzaldehyde (100 mg), sodium pyrosulfite (0.134 g) and N,N-dimethylacetamide (4.6 mL) was heated at 130° C. for 24 hours. Then water (30 mL) was added and the resulting precipitate was collected by filtration, washed with water and dried. The crude product was purified by column chromatography (silicagel, eluent: dichloromethane/methanol=12/1) and then recrystallized from a mixture of diisopropyl ether and ethyl acetate. Yield: 16.8%.

Melting point: 279-283° C.

¹H NMR (200 MHz, DMSO-d₆) δ 13.05 (br s, 1H, NH), 7.88 (m, 1H, phenyl-H), 7.74-7.32 (m, 7H, arom. H), 6.69 (br d, 1H, H7, J=6.0 Hz), 5.34 (s, 2H, CH₂).

Example 377
Synthesis of the 4-methyl analogue 377

embedded image

A mixture of 2-(2,3-difluorophenyl)-1(3)H-imidazo[4,5-c]pyridine (2.00 g), 50 mg methyltrioxorhenium, 100 mL methanol and 30% aqueous hydrogen peroxide (4 mL) was stirred at room temperature for 4 days. Then, additional 50 mg of methyltrioxorhenium and 30% hydrogen peroxide (4 mL) were added and the resulting mixture was stirred for another 2 days. After evaporation of the methanol water (200 mL) was added and the pH was adjusted to 9 by addition of 2N NaOH. The resulting precipitate was filtered, dried and recrystallized from a mixture of ethyl acetate (20 mL) and ethanol (53 mL) to give 1.208 g (56.5%) of 2-(2,3-difluorophenyl)-1(3)H-imidazo[4,5-c]pyridine 5-oxide.

2-(2,3-Difluorophenyl)-1(3)H-imidazo[4,5-c]pyridine 5-oxide (1.00 g) was dissolved in dry tetrahydrofurane (100 mL) and MeMgBr-solution (14 mL, 3M in diethyl ether) was added dropwise under argon. The resulting mixture was stirred for 1.5 hours at ambient temperature. Then water (100 mL) was added slowly and the pH was adjusted to 8.5. Extraction with ethyl acetate (3×70 mL), drying of the combined organic phases over anhydrous sodium sulphate and evaporation of the solvent afforded 0.630 g (60%) of crude 2-(2,3-difluorophenyl)-4-methyl-[(3)H-imidazo[4,5-c]pyridine. Recrystallization from a mixture of diisopropyl ether (20 mL) and ethyl acetate (34 mL) gave 240 mg (24.2%) of pure 2-(2,3-difluorophenyl)-4-methyl-1(3)H-imidazo[4,5-c]pyridine.

The final step was performed as described above. Purification by column chromatography (silica gel, eluent: dichloromethane/methanol=20/1). Yield: 22.4%.

¹H NMR (200 MHz, DMSO-d₆) δ 8.25 (d, 1H, H6, J=6.8 Hz), 8.11 (m, 1H, phenyl-H), 7.89 (AA′BB′, 2H, benzyl-H), 7.77 (d, 1H, H7, J=6.8 Hz), 7.60-7.41 (m, 3H, arom. H), 7.30 (m, 1H, phenyl-H), 7.12 (s, 1H, isoxazole-H), 6.05 (s, 2H, CH₂), 3.05 (s, 3H, CH₃).

Example 378
Synthesis of 7-Substituted Analogues

embedded image

378a

embedded image

3-Methyl-4-nitropyridine 1-oxide (5.85 g) was dissolved in glacial acetic acid (115 mL) and hydrogenated in a Parr hydrogenation apparatus (catalyst: 220 mg PtO₂×2H₂O, 50 psi) at ambient temperature for 2.5 h. Then the catalyst was filtered off and the solvent was evaporated. After addition of 150 mL of water the pH was adjusted to 12 by addition of 2N NaOH. The resulting solution was extracted 10 times with 100 mL of dichloromethane (containing 5% methanol). The combined organic phases were dried over anhydrous sodium sulphate and evaporated to give 3.81 g (83.6%) of 4-amino-3-methylpyridine.

4-Amino-3-methylpyridine (3.00 g) was dissolved with icecooling in concentrated sulfuric acid (36 mL). Then, fuming nitric acid (4.72 g) was added dropwise. After stirring at room temperature for 1 h, the solution was heated at 60° C. for 14 hours. After cooling to ambient temperature, the reaction mixture was poured on ice and the resulting solution was adjusted to pH 13 by addition of solid KOH. The precipitate was filtered off, washed with water and dried. Yield: 1.198 g (31.3%) 4-amino-3-methyl-5-nitropyridine.

A mixture of 4-amino-3-methyl-5-nitropyridine (1.198 g), iron powder (1.748 g), ethanol (52 mL) and hydrochloric acid (13 mL) was heated to reflux for 3 hours. After cooling to room temperature the ethanol was distilled off and the resulting suspension was diluted with water to 50 mL and the pH was adjusted to 13 by addition of 2N NaOH. Extraction with ethyl acetate (3×70 mL), drying of the combined organic phases of anhydrous sodium sulphate and evaporation of the solvent afforded 0.579 g (60%) of 3,4-diamino-5-methylpyridine.

The cyclization with 2,3-difluorbenzoic acid in PPA was performed as described above. Purified by column chromatography (silica gel, eluent: dichloromethan/methanol=12/1). Yield: 22.2%.

The final step was performed as described above. Recrystallized from a mixture of ethyl acetate and ethanol. Yield: 42.9% 378a.

¹H NMR (200 MHz, DMSO-d₆) δ 9.14 (d, 1H, H4, J=1.2 Hz), 8.17-8.10 (m, 2H, arom. H), 7.90 (AA′BB′, 2H, benzyl-H), 7.60-7.42 (m, 3H, arom. H), 7.32 (m, 1H, phenyl-H), 7.15 (s, 1H, isoxazole-H), 5.99 (s, 2H, CH₂), 2.58 (s, 3H, CH₃).

The following compounds were prepared in analogy to the above procedures:

378b

embedded image

¹H NMR (200 MHz, DMSO-d₆) δ 9.32 (d, 1H, H4, J=1.4 Hz), 8.67 (d, 1H, H6, J=1.4 Hz), 8.16 (m, 1H, phenyl-H), 7.78 (AA′BB′, 2H, benzyl-H), 7.54 (m, 1H, phenyl-H), 7.34 (m, 1H, phenyl-H), 7.07-7.00 (m, 3H, arom. H), 6.00 (s, 2H, CH₂), 4.07 (q, 2H, OCH₂, J=7.0 Hz), 1.33 (t, 3H, CH₃, J=7.0 Hz).

378c

embedded image

¹H NMR (200 MHz, DMSO-d₆) δ 9.47 (d, 1H, H4, J=1.4 Hz), 8.94 (d, 1H, H6, J=1.4 Hz), 8.16 (m, 1H, phenyl-H), 7.89 (AA′BB′, 2H, benzyl-H), 7.63-7.50 (m, 3H, arom. H), 7.35 (m, 1H, phenyl-H), 7.16 (s, 1H, isoxazole-H), 6.10 (s, 2H, CH₂).

378d

embedded image

¹H NMR (200 MHz, DMSO-d₆) δ 9.30 (br s, 1H, H4), 8.66 (dd, 1H, H6, J=7.4, 1.4 Hz), 8.15 (m, 1H, phenyl-H), 7.89 (AA′BB′, 2H, benzyl-H), 7.61-7.47 (m, 3H, arom. H), 7.33 (m, 1H, phenyl-H), 7.16 (s, 1H, isoxazole-H), 6.04 (s, 2H, CH₂).

Example 379
1,2,4-oxadiazoles

379a

embedded image

A mixture of 4-methoxybenzonitrile (1.00 g), hydroxylamine hydrochloride (0.785 g), KOH (0.640 g) and methanol (20 mL) was heated to reflux for 3 hours. After cooling to room temperature the precipitate was filtered off and the filtrate was evaporated. The resulting residue was dissolved in 1N HCl (100 mL) and the resulting solution was extracted with diethyl ether (100 mL). The aqueous phase was neutralized by addition of solid NaHCO₃and extracted with diethyl ether (2×100 mL). The combined organic phases were dried over anhydrous sodium sulphate and evaporated to give 450 mg of the desired amidoxime, which was used without further purification.

A solution of 700 mg of (4-methoxyphenyl)amidoxime and 1.08 g (1.5 equivalents) chloroacetic anhydride in toluene (30 mL) was heated to reflux for 3 hours. After cooling to ambient temperature the reaction mixture was extracted subsequently with water (twice 50 mL), saturated sodium bicarbonate solution (twice 50 mL) and water (50 mL). Finally, the toluene phase was dried over anhydrous sodium sulphate and evaporated to give 660 mg of the desired oxadiazole, which was used without further purification.

The final step was performed as described above (see, for example, isoxazole analogues). Recrystallized from a mixture of ethyl acetate and ethanol. Yield: 35%

¹H NMR (200 MHz, DMSO-d₆) δ 9.23 (d, 1H, H4, J=1.4 Hz), 8.28 (dd, 1H, H6, J=6.8, 1.4 Hz), 8.15 (m, 1H, phenyl-H), 7.92-7.77 (m, 3H, arom. H), 7.49 (m, 1H, phenyl-H), 7.33 (m, 1H, phenyl-H), 7.08-7.00 (m, 3H, arom. H), 6.01 (s, 2H, CH₂), 3.80 (s, 3H, OCH₃).

379b

embedded image

Prepared as described above, starting from 4-methylbenzonitrile.

¹H NMR (200 MHz, DMSO-d₆) δ 9.23 (d, 1H, H4, J=1.4 Hz), 8.31 (dd, 1H, H6, J=6.8, 1.4 Hz), 8.14 (m, 1H, phenyl-H), 7.93-7.78 (m, 3H, arom. H), 7.50 (m, 1H, phenyl-H), 7.35-7.27 (m, 3H, arom. H), 6.25 (s, 2H, CH₂), 2.35 (s, 3H, CH₃).

379c

embedded image

Prepared as described above, starting from pyridine-2-carbonitrile.

¹H NMR (200 MHz, DMSO-d₆) δ 9.24 (d, 1H, H4, J=1.4 Hz), 8.72 (ddd, 1H, pyridine-H), 8.32 (dd, 1H, H6, J=6.8, 1.4 Hz), 8.15 (m, 1H, phenyl-H), 8.00-7.90 (m, 3H, arom. H), 7.64-7.27 (m, 3H, arom. H), 6.30 (s, 2H, CH₂).

379d

embedded image

Prepared as described above, starting from 4-chlorobenzonitrile.

¹H NMR (200 MHz, DMSO-d₆) δ 9.24 (d, 1H, H4, J=1.4 Hz), 8.31 (dd, 1H, H6, J=6.8, 1.4 Hz), 8.16 (m, 1H, phenyl-H), 7.96-7.90 (m, 3H, arom. H), 7.60 (AA′BB′, 2H, benzyl-H), 7.49 (m, 1H, phenyl-H), 7.34 (m, 1H, phenyl-H), 6.28 (s, 2H, CH₂).

379e

embedded image

Prepared as described above, starting from pyridine-4-carbonitrile.

¹H NMR (200 MHz, DMSO-d₆) δ 9.24 (d, 1H, H4, J=1.4 Hz), 8.77 (AA′BB′, 2H, pyridine-H2/6), 8.32 (dd, 1H, H6, J=7.0, 1.4 Hz), 8.15 (m, 1H, phenyl-H), 7.93 (d, 1H, H7, J=7.0 Hz), 7.86 (AA′BB′, 2H, pyridine-H3/5), 7.51 (m, 1H, phenyl-H), 7.32 (m, 1H, phenyl-H), 6.32 (s, 2H, CH₂).

Part B
Methodology for Determination of Antiviral and Cytostatic Activity

Cells and Viruses

Madin-Darbey Bovine Kidney (MDBK) cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with BVDV-free 5% fetal calf serum (DMEME-FCS) at 37° C. in a humidified, 5% CO₂atmosphere. BVDV-1 (strain PE515) was used to assess the antiviral activity in MDBK cells.

Anti-BVDV Assay

Ninety-six-well cell culture plates were seeded with MDBK cells in DMEM-FCS so that cells reached 24 hr later confluency. Then medium was removed and serial 5-fold dilutions of the test compounds were added in a total volume of 100 μL, after which the virus inoculum (100 μL) was added to each well. The virus inoculum used resulted in a greater than 90% destruction of the cell monolayer after 5 days incubation at 37° C. Uninfected cells and cells receiving virus without compound were included in each assay plate. After 5 days, medium was removed and 90 μL of DMEM-FCS and 10 μL of MTS/PMS solution (Promega) was added to each well. Following a 2 hr incubation period at 37° C. the optical density of the wells was read at 498 nm in a microplate reader. The 50% effective concentration (EC₅₀) value was defined as the concentration of compound that protects 50% of the cell monolayer from virus-induced cytopathic effect.

Anti-HCV Assay/Replicon Assay—1

Huh-5-2 cells [a cell line with a persistent HCV replicon I389luc-ubi-neo/NS3-3′/5.1; replicon with firefly luciferase-ubiquitin-neomycin phosphotransferase fusion protein and EMCV-IRES driven NS3-5B HCV polyprotein] was cultured in RPMI medium (Gibco) supplemented with 10% fetal calf serum, 2 mM L-glutamine (Life Technologies), 1× non-essential amino acids (Life Technologies); 100 IU/mL penicillin and 100 μg/ml streptomycin and 250 μg/mL G418 (Geneticin, Life Technologies). Cells were seeded at a density of 7000 cells per well in 96 well View Plate™ (Packard) in medium containing the same components as described above, except for G418. Cells were allowed to adhere and proliferate for 24 hr. At that time, culture medium was removed and serial dilutions of the test compounds were added in culture medium lacking G418. Interferon alfa 2a (500 IU) was included as a positive control. Plates were further incubated at 37° C. and 5% CO₂for 72 hours. Replication of the HCV replicon in Huh-5 cells results in luciferase activity in the cells. Luciferase activity is measured by adding 50 μL of 1× Glo-lysis buffer (Promega) for 15 minutes followed by 50 μL of the Steady-Glo Luciferase assay reagent (Promega). Luciferase activity is measured with a luminometer and the signal in each individual well is expressed as a percentage of the untreated cultures. Parallel cultures of Huh-5-2 cells, seeded at a density of 7000 cells/well of classical 96-well cell culture plates (Becton-Dickinson) are treated in a similar fashion except that no Glo-lysis buffer or Steady-Glo Luciferase reagent is added. Instead the density of the culture is measured by means of the MTS method (Promega).

Quantitative Analysis of HCV RNA by Tagman Real-Time RT-PCR

Replicon cells were plated at 7.5×10³cells per well in a 96-well plate plates at 37° C. and 5% CO₂in Dulbecco's modified essential medium containing 10% fetal calf serum, 1% nonessential amino acids and 1 mg/ml Geneticin. After allowing 24 h for cell attachment, different dilutions of compound were added to the cultures. Plates were incubated for 5 days, at which time RNA was extracted using the Qiamp Rneazyi Kit (Qiagen, Hilden, Gemiany). A 50 μL PCR reaction contained TaqMan EZ buffer (50 mmol/L Bicine, 115 mmol/L potassium acetate, 0.01 mmol/L EDTA, 60 mmol/L 6-carboxy-X-rhodamine, and 8% glycerol, pH 8.2; Perkin Elmer Corp./Applied Biosystems), 300 μmol/L deoxyadenosine triphosphate, 300 μmol/L deoxyguanosine triphosphate, 300 μmol/L deoxycytidine triphosphate, 600 μmol/L deoxyuridine triphosphate, 200 μmol/L forward primer [5′-ccg gcT Acc Tgc ccA TTc], 200 μmol/L reverse primer [ccA GaT cAT ccT gAT cgA cAA G], 100 μmol/L TaqMan probe [6-FAM-AcA Tcg cAT cgA gcg Agc Acg TAc-TAMRA], 3 mmol/L manganese acetate, 0.5 U AmpErase uracil-N-glycosylase, 7.5 U rTth DNA polymerase, and 10 μl of RNA elution. After initial activation of uracil-N-glycosylase at 50° C. for 2 minutes, RT was performed at 60° C. for 30 minutes, followed by inactivation of uracil-N-glycosylase at 95° C. for 5 minutes. Subsequent PCR amplification consisted of 40 cycles of denaturation at 94° C. for 20 seconds and annealing and extension at 62° C. for 1 minute in an ABI 7700 sequence detector. For each PCR run, negative template and positive template samples were used. The cycle threshold value (Ct-value) is defined as the number of PCR cycles for which the signal exceeds the baseline, which defines a positive value. The sample was considered to be positive if the Ct-value Was <50. Results are expressed as genomic equivalents (GE).

Anti-HCV Assay/Replicon Assay—2

HCV Replicon Media

DMEM w/High Glucose (or MEM)

1× Glutamine

1× Sodium Pyruvate

10% Heat Inactivated FBS

1× Antibiotics

Cell Culture Preparation

1. Unthaw frozen stock in 10-12 mls of Media

2. Allow cells to attach before adding G418 (4-6 hrs)

3. Add G418 for a final concentration of 200 μg/mL (higher amounts are possible but cells grow slowly)

4. Split cells 1:4 to 1:6 for optimal growth

5. In-house replicon seems to maintain Luciferase signal for ˜20 passages

HCV Replicon Assay

1. Dilute compounds in 100 uL of HCV Replicon Media (without G418). If compounds are diluted in DMSO add DMSO to media (Final DMSO concentration should be <1%)

2. Once cells have reached 80-90% confluency, trypsinize with 1× Trypsin

3. Do not over trypsinize. These cells tend to clump if over trypsinized

4. For 96 well format add 6,000-8,000 cells per well (G418 is withheld during compound testing)

5. Incubate for 3 days at 37° C. Cells should be very close to confluent.

6. Remove media and wash cells with 1×PBS

7. Remove PBS and add 100 μL of 1× Promega Lysis Buffer

8. Incubate cells at Room Temperature for 5-20 minutes

9. Add 100 μL of room temperature Luciferase Substrate Solution (Promega) to Microfluor Black Plate (VWR)

10. Thoroughly Mix Cell lysate (pipet up and down) before adding to Luciferase substrate

11. Add 75 μL of lysate to the Luciferase substrate solution

12. Read Plate On Top Count (FusionLucB program ˜5 second read)

13. Left over lysate can be frozen and used for later analysis

Determination of Cytostatic Effect on MDBK Cells

The effect of the drugs on exponentially growing MDBK cells was assessed as follows. Cells were seeded at a density of 5000 cell/well in 96 well plates in MEM medium (Gibco) supplemented with 10% fetal calf serum, 2 mM L-glutamine (Life Technologies) and bicarbonate (Life Technologies). Cells were cultured for 24 hr after which serial dilutions of the test compounds were added. Cultures were then again further incubated for 3 days after which the effect on cell growth was quantified by means of the MTS method (Promega). The concentration that results in 50% inhibition of cell growth is defined as the 50% cytostatic concentration (CC₅₀)

HCV CC50 Assay Protocol

HCV Replicon Media

DMEM w/High Glucose (or MEM)

1× Glutamine

1× Sodium Pyruvate

10% Heat Inactivated FBS

1× Antibiotics

Cell Culture Preparation

1. Unthaw frozen stock in 10-12 mls of Media

2. Allow cells to attach before adding G418 (4-6 hrs)

3. Add G418 for a final concentration of 200 μg/ml (higher amounts are possible but cells grow slowly)

4. Split cells 1:4 to 1:6 for optimal growth

5. In-house replicon seems to maintain Luciferase signal for ˜20 passages

HCV Replicon Assay

1. Dilute compounds in 100 μL of HCV Replicon Media (without G418). If compounds are diluted in DMSO add DMSO to media (Final DMSO concentration should be <1%)

2. Once cells have reached 80-90% confluency, trypsinize with 1× Trypsin

3. Do not over trypsinize. These cells tend to clump if over trypsinized

4. For 96 well format add 6,000-8,000 cells per well (G418 is withheld during compound testing)

5. Incubate for 3 days at 37° C. Cells should be very close to confluent.

6. Remove media and add 200 μL of a 0.2 mg/mL MTT solution prepared in media.

7. Incubate for 1.5 hours to 2 hours.

8. Remove media and add 150 μL of DMSO

9. Mix and incubate for 5 mins at room temperature

10. Read plate at 530 nm in the plate reader.

Results

The compounds of Examples 2, 3A, 4 and 5 were found to have an EC50 in Replicon assay 2 of, respectively in micromoles, 0.01, 0.02, 0.01 and 0.0039, and to have a CC50 in the CC50 assay protocol of, respectively in micromoles, 26, 34, 19 and 10.8 (replicate 13.4).

Substantially all of the compounds in Table 1 demonstrated activity of at least 1 micromolar in an anti-HCV/Replicon assay system. In addition, a number of the compounds also exhibited anti-BVDV activity.

Number	Name	Date	Kind
2191978	Balle et al.	Feb 1940	A
2411662	Martin et al.	Nov 1946	A
2516674	Havertown et al.	Jul 1950	A
2548863	Havertown et al.	Apr 1951	A
3985891	Kutter et al.	Oct 1976	A
4358387	Zoleski et al.	Nov 1982	A
4565816	Neumann	Jan 1986	A
4692443	Katner	Sep 1987	A
4804658	Manley et al.	Feb 1989	A
4914108	Khanna et al.	Apr 1990	A
4988707	Stealey et al.	Jan 1991	A
4990518	Khanna et al.	Feb 1991	A
5011832	Dininno et al.	Apr 1991	A
5019581	Khanna et al.	May 1991	A
5057517	Johnston et al.	Oct 1991	A
5137896	Van Daele et al.	Aug 1992	A
5208242	Khanna et al.	May 1993	A
5227384	Khanna et al.	Jul 1993	A
5302601	Khannal et al.	Apr 1994	A
5332744	Chakravarty et al.	Jul 1994	A
5374638	Dhanoa et al.	Dec 1994	A
5405964	Mederski et al.	Apr 1995	A
5438063	Osswald et al.	Aug 1995	A
5446032	Whittaker et al.	Aug 1995	A
5486525	Summers, Jr. et al.	Jan 1996	A
5585492	Chandrakumar et al.	Dec 1996	A
5587372	Aszodi et al.	Dec 1996	A
5607944	Linz et al.	Mar 1997	A
5719306	Chandrakumar et al.	Feb 1998	A
5723492	Chandrakumar et al.	Mar 1998	A
5854265	Anthony	Dec 1998	A
5859035	Anthony et al.	Jan 1999	A
5872136	Anthony et al.	Feb 1999	A
5874452	Anthony	Feb 1999	A
5880140	Anthony	Mar 1999	A
5883105	Anthony	Mar 1999	A
5939557	Anthony et al.	Aug 1999	A
6051574	Anthony	Apr 2000	A
6063930	Dinsmore et al.	May 2000	A
6080870	Anthony et al.	Jun 2000	A
6329381	Kurimoto et al.	Dec 2001	B1
6376515	Zhu et al.	Apr 2002	B2
6479508	Beaulieu et al.	Nov 2002	B1
6492384	Mederski et al.	Dec 2002	B1
6627651	Shiraishi	Sep 2003	B1
6767654	Tamao et al.	Jul 2004	B2
6770666	Hashimoto et al.	Aug 2004	B2
6803374	Priestley et al.	Oct 2004	B2
6835739	Zhu et al.	Dec 2004	B2
6844367	Zhu et al.	Jan 2005	B1
7026051	Schauer et al.	Apr 2006	B2
7098231	Poupart et al.	Aug 2006	B2
7112600	Hashimoto et al.	Sep 2006	B1
7223785	Beaulieu et al.	May 2007	B2
7285551	Hashimoto et al.	Oct 2007	B2
7294457	Kukolj et al.	Nov 2007	B2
20030073836	Priepke et al.	Apr 2003	A1
20030108862	Kukolj et al.	Jun 2003	A1
20040082635	Hashimoto et al.	Apr 2004	A1
20040097438	Hashimoto et al.	May 2004	A1
20040097574	Marshall	May 2004	A1
20040171626	Beaulieu et al.	Sep 2004	A1
20040186125	Poupart et al.	Sep 2004	A1
20050026921	Eckhardt et al.	Feb 2005	A1
20050096337	Ackermann et al.	May 2005	A1
20050222198	Bondy et al.	Oct 2005	A1
20050239821	Neyts et al.	Oct 2005	A1
20060052602	Kim et al.	Mar 2006	A1
20060229336	Kazmierski et al.	Oct 2006	A1
20060252791	Bondy et al.	Nov 2006	A1
20070021472	Zhu et al.	Jan 2007	A1
20070032497	Hashimoto et al.	Feb 2007	A1
20080188516	Bondy et al.	Aug 2008	A1
20080199427	Bondy	Aug 2008	A1
20090036460	Dowdy et al.	Feb 2009	A1

Number	Date	Country
643289	Jun 1991	AU
2093290	Jul 1993	CA
2158996	Mar 1994	CA
2357771	Jul 2000	CA
2471566	Jan 2003	CA
2423800	Mar 2003	CA
2496249	Aug 2003	CA
4211474	Oct 1993	DE
4230464	Mar 1994	DE
4236026	Jun 1994	DE
4309969	Sep 1994	DE
4318813	Dec 1994	DE
0 076 530	Apr 1983	EP
0138552	Apr 1985	EP
0228845	Jul 1987	EP
0232937	Aug 1987	EP
0300726	Jan 1989	EP
0 344 414	Dec 1989	EP
0 417 745	Mar 1991	EP
0462009	Dec 1991	EP
0510260	Oct 1992	EP
0605836	Jul 1994	EP
0706795	Apr 1996	EP
1 132 381	Sep 2001	EP
1162196	Dec 2001	EP
1386923	Feb 2004	EP
1400241	Feb 2004	EP
2 158 440	Nov 1985	GB
2264115	Aug 1993	GB
78019	May 1999	HU
89588	Mar 1989	IL
813921	Dec 1986	SU
1048742	Dec 1986	SU
851940	Apr 1988	SU
860463	May 1998	SU
WO-9222556	Dec 1992	WO
WO-9302080	Feb 1993	WO
WO-9314072	Jul 1993	WO
WO-9316075	Aug 1993	WO
WO-9412461	Jun 1994	WO
WO-9429321	Dec 1994	WO
WO-9502597	Jan 1995	WO
WO-9516687	Jun 1995	WO
WO-9611192	Apr 1996	WO
WO 9612703	May 1996	WO
WO-9615111	May 1996	WO
WO 9927929	Jun 1999	WO
WO-0020400	Apr 2000	WO
WO-0020416	Apr 2000	WO
WO-0020425	Apr 2000	WO
WO-0020445	Apr 2000	WO
WO-0039127	Jul 2000	WO
WO-0040583	Jul 2000	WO
WO-0040586	Jul 2000	WO
WO-0073307	Dec 2000	WO
WO-0160315	Aug 2001	WO
WO-0166526	Sep 2001	WO
WO-0185172	Nov 2001	WO
WO-0195910	Dec 2001	WO
WO-0204425	Jan 2002	WO
WO-02057425	Jul 2002	WO
WO-02067942	Sep 2002	WO
WO-03000254	Jan 2003	WO
WO-03004020	Jan 2003	WO
WO-03007945	Jan 2003	WO
WO-03010140	Feb 2003	WO
WO-03010141	Feb 2003	WO
WO-03014229	Feb 2003	WO
WO-03026587	Apr 2003	WO
WO-03057205	Jul 2003	WO
2004005286	Jan 2004	WO
WO 2004005286	Jan 2004	WO
WO-2004018468	Mar 2004	WO
WO-2004019935	Mar 2004	WO
WO-2004033455	Apr 2004	WO
WO-2004043913	May 2004	WO
WO-2004054974	Jul 2004	WO
2004067516	Aug 2004	WO
WO-2004072243	Aug 2004	WO
WO-2005063744	Jul 2005	WO
WO-2006029966	Mar 2006	WO
WO-2006033703	Mar 2006	WO
WO-2006069193	Jun 2006	WO
WO-2008005519	Jan 2008	WO
WO-2009009001	Jan 2009	WO

Number	Date	Country
60590990	Jul 2004	US
60590989	Jul 2004	US
60591024	Jul 2004	US
60591069	Jul 2004	US
60533963	Jan 2004	US
60532292	Dec 2003	US

Imidazo 4,5-c pyridine compounds and methods of antiviral treatment

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Term Extension

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

PCT Information

US Referenced Citations (75)

Foreign Referenced Citations (85)

Related Publications (1)

Provisional Applications (6)