# Patent

## Title
Piperazine derivatives useful as CCR5 antagonists

## Abstract

The use of CCR5 antagonists of the formula




or a pharmaceutically acceptable salt thereof, wherein

    
    
        R is optionally substituted phenyl, pyridyl, thiophenyl or naphthyl;
        R1 is hydrogen or alkyl;
        R2 is substituted phenyl, substituted heteroaryl, naphthyl, fluorenyl, diphenylmethyl or optionally substituted phenyl- or heteroaryl-alkyl;
        R3 is hydrogen, alkyl, alkoxyalkyl, cycloalkyl, cycloalkylalkyl, or optionally substituted phenyl, phenylalkyl, naphthyl, naphthylalkyl, heteroaryl or heteroarylalkyl;
        R4, R5 and R7 are hydrogen or alkyl;
        R6 is hydrogen, alkyl or alkenyl;

for the treatment of HIV, solid organ transplant rejection, graft v. host disease, arthritis, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, psoriasis, asthma, allergies or multiple sclerosis is disclosed, as well as novel compounds, pharmaceutical compositions comprising them, and the combination of CCR5 antagonists of the invention in combination with antiviral agents useful in the treatment of HIV or agents useful in the treatment of inflammatory diseases.

## Background
This application is a continuation-in-part (CIP) of U.S. Ser. No. 10/668,862 filed Sep. 23, 2003, which is a divisional of of U.S. Ser. No. 10/061,011, filed Jan. 30, 2002, now U.S. Pat. No. 6,689,765 B2, which is a divisional of U.S. Ser. No. 09/562,814, filed May 1, 2000, now U.S. Pat. No. 6,391,865 B1, which claims the benefit of U.S. Provisional Application No. 60/132,509, filed May 4, 1999. Each of the above-mentioned application is incorporated herein in its entirety by reference.


BACKGROUND
The present invention relates to piperazine derivatives useful as selective CCR5 antagonists, pharmaceutical compositions containing the compounds, and methods of treatment using the compounds. The invention also relates to the use of a combination of a CCR5 antagonist of this invention and one or more antiviral or other agents useful in the treatment of Human Immunodeficiency Virus (HIV). The invention further relates to the use of a CCR-5 antagonist of this invention, alone or in combination with another agent, in the treatment of solid organ transplant rejection, graft v. host disease, arthritis, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, psoriasis, asthma, allergies or multiple sclerosis.
The global health crisis caused by HIV, the causative agent of Acquired Immunodeficiency Syndrome (AIDS), is unquestioned, and while recent advances in drug therapies have been successful in slowing the progression of AIDS, there is still a need to find a safer, more efficient, less expensive way to control the virus.
It has been reported that the CCR5 gene plays a role in resistance to HIV infection. HIV infection begins by attachment of the virus to a target cell membrane through interaction with the cellular receptor CD4 and a secondary chemokine co-receptor molecule, and proceeds by replication and dissemination of infected cells through the blood and other tissue. There are various chemokine receptors, but for macrophage-tropic HIV, believed to be the key pathogenic strain that replicates in vivo in the early stages of infection, the principal chemokine receptor required for the entry of HIV into the cell is CCR5. Therefore, interfering with the interaction between the viral receptor CCR5 and HIV can block HIV entry into the cell. The present invention relates to small molecules which are CCR5 antagonists.
CCR-5 receptors have been reported to mediate cell transfer in inflammatory diseases such as arthritis, rheumatoid arthritis, atopic dermatitis, psoriasis, asthma and allergies, and inhibitors of such receptors are expected to be useful in the treatment of such diseases, and in the treatment of other inflammatory diseases or conditions such as inflammatory bowel disease, multiple sclerosis, solid organ transplant rejection and graft v. host disease.
Related piperazine derivatives which are muscarinic antagonists useful in the treatment of cognitive disorders such as Alzheimer's disease are disclosed in U.S. Pat. Nos. 5,883,096; 6,037,352; 5,889,006.
A-M. Vandamme et al., Antiviral Chemistry & Chemotherapy, 9:187-203 (1998) disclose current clinical treatments of HIV-1 infections in man including at least triple drug combinations or so-called Highly Active Antiretroviral Therapy (“HAART”); HAART involves various combinations of nucleoside reverse transcriptase inhibitors (“NRTI”), non-nucleoside reverse transcriptase inhibitors (“NNRTI”) and HIV protease inhibitors (“PI”). In compliant drug-naive patients, HAART is effective in reducing mortality and progression of HIV-1 to AIDS. However, these multidrug therapies do not eliminate HIV-1 and long-term treatment usually results in multidrug resistance. Development of new drug therapies to provide better HIV-1 treatment remains a priority.
SUMMARY OF THE INVENTION
The present invention relates to the treatment of HIV comprising administering to a mammal in need of such treatment an effective amount of a CCR5 antagonist represented by the structural formula I:




or a pharmaceutically acceptable salt thereof, wherein

R is R8-phenyl, R8-pyridyl, R8-thiophenyl or R8-naphthyl;
R1 is hydrogen or C1-C6 alkyl;
R2 is R9, R10, R11-phenyl; R9, R10, R11-substituted 6-membered heteroaryl; R9, R10, R11-substituted 6-membered heteroaryl N-oxide;
R12, R13-substituted 5-membered heteroaryl; naphthyl; fluorenyl; diphenylmethyl




R3 is hydrogen, C1-C6 alkyl, (C1-C6)alkoxy(C1-C6)alkyl, C3-C10 cycloalkyl, C3-C10 cycloalkyl(C1-C6)alkyl, R8-phenyl, R8-phenyl(C1-C6)alkyl, R8-naphthyl, R8-naphthyl(C1-C6)alkyl, R8-heteroaryl or R8-heteroaryl(C1-C6)alkyl;
R4, R5, R7 and R13 are independently selected from the group consisting of hydrogen and (C1-C6)-alkyl;
R6 is hydrogen, C1-C6 alkyl or C2-C6 alkenyl;
R8 is 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, —CF3, CF3O—, CH3C(O)—, —CN, CH3SO2—, CF3SO2—, R14-phenyl, R14-benzyl, CH3C(═NOCH3), CH3C(═NOCH2CH3),




—NH2, —NHCOCF3, —NHCONH(C1-C6 alkyl), —NHCO(C1-C6 alkyl), —NHSO2(C1-C6 alkyl), 5-membered heteroaryl and





wherein X is —O—, —NH— or —N(CH3)—;

R9 and R10 are independently selected from the group consisting of (C1-C6)alkyl, halogen, —NR17R18, —OH, —CF3, —OCH3, —O-acyl, —OCF3 and —Si(CH3)3;
R11 is R9, hydrogen, phenyl, —NO2, —CN, —CH2F, —CHF2, —CHO, —CH═NOR17, pyridyl, pyridyl N-oxide, pyrimidinyl, pyrazinyl, —N(R17)CONR18R19, —NHCONH(chloro-(C1-C6)alkyl), —NHCONH((C3-C1)cycloalkyl(C1-C6)alkyl), —NHCO(C1-C6)alkyl, —NHCOCF3, —NHSO2N((C1-C6)alkyl)2, —NHSO2(C1-C6)alkyl, —N(SO2CF3)2, —NHCO2(C1-C6)alkyl, C3-C10 cycloalkyl, —SR20, —SOR20, —SO2R20, —SO2NH(C1-C6 alkyl), —OSO2(C1-C6)alkyl, —OSO2CF3, hydroxy(C1-C6)alkyl, —CON R17R18, —CON(CH2CH2—O—CH3)2, —OCONH(C1-C6)alkyl, —CO2R17, —Si(CH3)3 or —B(OC(CH3)2)2;
R12 is (C1-C6)alkyl, —NH2 or R14-phenyl;
R14 is 1 to 3 substituents independently selected from the group consisting of hydrogen, (C1-C6) alkyl, —CF3, —CO2R17, —CN, (C1-C6)alkoxy and halogen;
R15 and R16 are independently selected from the group consisting of hydrogen and C1-C6 alkyl, or R15 and R16 together are a C2-C5 alkylene group and with the carbon to which they are attached form a spiro ring of 3 to 6 carbon atoms;
R17, R18 and R19 are independently selected from the group consisting of H and C1-C6 alkyl; and
R20 is C1-C6 alkyl or phenyl.
Preferred are compounds of formula I wherein R is R8-phenyl or R8-naphthyl, especially wherein R8 is a single substituent, and especially wherein the R8 substituent is in the 4-position. For R8-phenyl, preferred R8 substituents are —CF3, —OCF3, CH3SO2—, CH3CO—, CH3C(═NOCH3)—, Br and I. For R8-naphthyl, R8 is preferably C1-C6 alkoxy. Also preferred are compounds of formula I wherein R3 is hydrogen, (C1-C6) alkyl, R8-phenyl. R8-benzyl or R8-pyridyl; more preferred definitions for R3 are methyl, ethyl, phenyl, benzyl and pyridyl. R1 is preferably hydrogen. For compounds of formula I, R6 is preferably hydrogen or methyl, especially methyl. R4 is preferably methyl; R5 and R7 are each preferably hydrogen.
In compounds of formula I, R2 is preferably R9, R10, R11-phenyl, R9, R10, R11-pyridyl or an N-oxide thereof, or R9, R10, R11-pyrimidyl. When R2 is pyridyl, it is preferably 3- or 4-pyridyl, and when pyrimidyl, it is preferably 5-pyrimidyl. The R9 and R10 substituents are preferably attached to carbon ring members adjacent to the carbon joining the ring to the rest of the molecule and the R11 substituent can be attached to any of the remaining unsubstituted carbon ring members, for example as shown in the following structures:




Preferred R9 and R10 substituents are: (C1-C6)alkyl, especially methyl; halogen, especially chloro or bromo, —OH and —NH2. When R2 is phenyl, R11 is preferably hydrogen or —OH; when R2 is pyridyl, R11 is preferably hydrogen; and when R2 is pyrimidyl, R11 is preferably hydrogen, methyl or phenyl. Examples of particularly preferred R2 groups are as follows:




Also claimed are novel CCR5 antagonist compounds represented by the structural formula II




or a pharmaceutically acceptable salt thereof, wherein

(1) Ra is R8a-phenyl, R8b-pyridyl, R8b-thiophenyl or R8-naphthyl;

R1 is hydrogen or C1-C6 alkyl;
R2 is R9, R10, R11-phenyl; R9, R10, R11-substituted 6-membered heteroaryl;
R9, R10, R11-substituted 6-membered heteroaryl N-oxide;
R12, R13-substituted 5-membered heteroaryl; naphthyl; fluorenyl; diphenylmethyl,




R3 is hydrogen, C1-C6 alkyl, (C1-C6)alkoxy(C1-C6)alkyl, C3-C10 cycloalkyl, C3-C10 cycloalkyl(C1-C6)alkyl, R8-phenyl, R8-phenyl(C1-C6)alkyl, R8-naphthyl, R8-naphthyl(C1-C6)alkyl, R8-heteroaryl or R8-heteroaryl(C1-C6)alkyl;
R4, R5, R7 and R13 are independently selected from the group consisting of hydrogen and (C1-C6)-alkyl;
R6 is hydrogen, C1-C6 alkyl or C2-C6 alkenyl;
R8 is 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, —CF3, CF3O—, CH3C(O)—, —CN, CH3SO2—CF3SO2—, R14-phenyl, R14-benzyl, CH3C(═NOCH3), CH3C(═NOCH2CH3),




—NH2, —NHCOCF3, —NHCONH(C1-C6 alkyl), —NHCO(C1-C6 alkyl), —NHSO2(C1-C6 alkyl), 5-membered heteroaryl and





wherein X is —O—, —NH— or —N(CH3)—;

R8a is 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, —CF3, CF3O—, —CN, CF3SO2—, R14-phenyl, —NHCOCF3, 5 membered heteroaryl and




wherein X is as defined above;

R8b is 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, —CF3, CF3O—, CH3C(O)—, —CN, CF3SO2—, R14-benzyl, CH3C(═NOCH3), CH3C(═NOCH2CH3),




—NHCOCF3, 5-membered heteroaryl and





wherein X is as defined above;

R9 and R10 are independently selected from the group consisting of (C1-C6)alkyl, halogen, —NR17R18, —OH, —CF3, —OCH3, —O-acyl, —OCF3 and —Si(CH3)3;
R11 is R9, hydrogen, phenyl, —NO2, —CN, —CH2F, —CHF2, —CHO, —CH═NOR17, pyridyl, pyridyl N-oxide, pyrimidinyl, pyrazinyl, —N(R17)CONR18R19, —NHCONH(chloro-(C1-C6)alkyl), —NHCONH((C3-C1)cycloalkyl(C1-C6)alkyl), —NHCO(C1-C6)alkyl, —NHCOCF3, —NHSO2N((C1-C6)alkyl)2, —NHSO2(C1-C6)alkyl, —N(SO2CF3)2, —NHCO2(C1-C6)alkyl, C3-C10 cycloalkyl, —SR20, —SOR20, —SO2R20, —SO2NH(C1-C6 alkyl), —OSO2(C1-C6)alkyl, —OSO2CF3, hydroxy(C1-C6)alkyl, —CON R17R18, —CON(CH2CH2—O—CH3)2, —OCONH(C1-C6)alkyl, —CO2R17, —Si(CH3)3 or —B(OC(CH3)2)2;
R12 is (C1-C6)alkyl, —NH2 or R14-phenyl;
R14 is 1 to 3 substituents independently selected from the group consisting of hydrogen, (C1-C6) alkyl, —CF3, —CO2R17, —CN, (C1-C6)alkoxy and halogen;
R15 and R16 are independently selected from the group consisting of hydrogen and C1-C6 alkyl, or R15 and R16 together are a C2-C5 alkylene group and with the carbon to which they are attached form a spiro ring of 3 to 6 carbon atoms;
R17, R18 and R19 are independently selected from the group consisting of H and C1-C6 alkyl; and
R20 is C1-C6 alkyl or phenyl; or
(2) Ra is R8-phenyl, R8-pyridyl or R8-thiophenyl;
R2 is fluorenyl, diphenylmethyl,




and R1, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19 and R20 are as defined in (1).
Preferred compounds of formula II are those defined in (1).
More preferred are those of formula II(1) wherein Ra is R8a-phenyl or R8-naphthyl, wherein R8a is —CF3, CF3O— or halogen and R8 is C1-C6 alkoxy. The R8a or R8 substituent is preferably a single substituent; it is especially preferred that the R8a or R8 substituent is in the 4-position. Also preferred are compounds of formula II(1) wherein R3 is hydrogen, (C1-C6) alkyl, R8-phenyl. R8-benzyl or R8-pyridyl; more preferred definitions for R3 are methyl, ethyl, phenyl, benzyl and pyridyl. R1 is preferably hydrogen. For compounds of formula II(1), R6 is preferably hydrogen or methyl, especially methyl. R4 is preferably methyl; R5 and R7 are each preferably hydrogen.
R2 in formula II(1) is preferably as defined for formula I, i.e., R9, R10, R11-phenyl, R9, R10, R11-pyridyl or an N-oxide thereof, or R9, R10, R11-pyrimidyl, wherein the R9, R10, R11-substitution is as defined above for preferred compounds of formula I.
Another aspect of the invention is a pharmaceutical composition for treatment of HIV comprising an effective amount of a CCR5 antagonist of formula II in combination with a pharmaceutically acceptable carrier. Another aspect of the invention is a pharmaceutical composition for treatment of solid organ transplant rejection, graft v. host disease, arthritis, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, psoriasis, asthma, allergies or multiple sclerosis comprising an effective amount of a CCR5 antagonist of formula II in combination with a pharmaceutically acceptable carrier.
Yet another aspect of this invention is a method of treatment of HIV comprising administering to a human in need of such treatment an effective amount of a CCR5 antagonist compound of formula II. Another aspect of the invention is a method of treatment of solid organ transplant rejection, graft v. host disease, arthritis, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, psoriasis, asthma, allergies or multiple sclerosis comprising administering to a human in need of such treatment an effective amount of a CCR5 antagonist compound of formula I or II.
Still another aspect of this invention is the use of a CCR5 antagonist of formula I or II of this invention in combination with one or more antiviral or other agents useful in the treatment of Human Immunodeficiency Virus for the treatment of AIDS. Still another aspect of this invention is the use of a CCR5 antagonist of formula I or II of this invention in combination with one or more other agents useful in the treatment of solid organ transplant rejection, graft v. host disease, inflammatory bowel disease, rheumatoid arthritis or multiple sclerosis. The CCR5 and antiviral or other agents which are components of the combination can be administered in a single dosage form or they can be administered separately; a kit comprising separate dosage forms of the actives is also contemplated.



DESCRIPTION OF DRAWINGS
FIG. 1 shows biotransformation of Vicriviroc in human, monkey and rat following a single oral dose of 14C-VIC.
FIG. 2 shows comparison of representative radiochromatographic profiles of pooled plasma extract following a single oral administration of Vicriviroc to healthy male volunteers, male monkeys and rats
FIG. 3 shows comparison of representative radiochromatographic profiles of pooled urine following a single oral administration of Vicriviroc to healthy male volunteers, male monkeys and rats.
FIG. 4 shows comparison of representative radiochromatographic profiles of pooled fecal extract following a single oral administration of Vicriviroc to healthy male volunteers, male monkeys and rats.



DETAILED DESCRIPTION OF THE INVENTION
As used herein, the following terms are used as defined below unless otherwise indicated.
Alkyl represents straight and branched carbon chains and contains from one to six carbon atoms.
Alkenyl represents C2-C6 carbon chains having one or two unsaturated bonds, provided that two unsaturated bonds are not adjacent to each other.
Substituted phenyl means that the phenyl group can be substituted at any available position on the phenyl ring.
Acyl means a radical of a carboxylic acid having the formula alkyl-C(O)—, aryl-C(O)—, aralkyl-C(O)—, (C3-C7)cycloalkyl-C(O)—, (C3-C7)cycloalkyl-(C1-C6)alkyl-C(O)—, and heteroaryl-C(O)—, wherein alkyl and heteroaryl are as defined herein; aryl is R14-phenyl or R14-naphthyl; and aralkyl is aryl-(C1-C6)alkyl, wherein aryl is as defined above.
Heteroaryl represents cyclic aromatic groups of 5 or 6 atoms or bicyclic groups of 11 to 12 atoms having 1 or 2 heteroatoms independently selected from O, S or N, said heteroatom(s) interrupting a carbocyclic ring structure and having a sufficient number of delocalized pi electrons to provide aromatic character, provided that the rings do not contain adjacent oxygen and/or sulfur atoms. For 6-membered heteroaryl rings, carbon atoms can be substituted by R9, R10 or R11 groups. Nitrogen atoms can form an N-oxide. All regioisomers are contemplated, e.g., 2-pyridyl, 3-pyridyl and 4-pyridyl. Typical 6-membered heteroaryl groups are pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl and the N-oxides thereof. For 5-membered heteroaryl rings, carbon atoms can be substituted by R12 or R13 groups. Typical 5-membered heteroaryl rings are furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl and isoxazolyl. 5-Membered rings having one heteroatom can be joined through the 2- or 3-position; 5-membered rings having two heteroatoms are preferably joined through the 4-position. Bicyclic groups typically are benzo-fused ring systems derived from the heteroaryl groups named above, e.g. quinolyl, phthalazinyl, quinazolinyl, benzofuranyl, benzothienyl and indolyl.
Preferred points of substitution for 6-membered heteroaryl rings at R2 are described above. When R2 is a 5-membered heteroaryl group, the R12 and R13 substituents are preferably attached to carbon ring members adjacent to the carbon joining the ring to the rest of the molecule, and R12 is preferably alkyl; however, if a heteroatom is adjacent to the carbon joining the ring to the rest of the molecule (i.e., as in 2-pyrrolyl), R12 is preferably attached to a carbon ring member adjacent to the carbon joining the ring to the rest of the molecule.
Halogen represents fluoro, chloro, bromo and iodo.
Prodrugs and solvates of the compounds of the invention are also contemplated herein. The term “prodrug”, as employed herein, denotes a compound that is a drug precursor which, upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes to yield a compound of Formula 1 or a salt and/or solvate thereof. A discussion of prodrugs is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems (1987) Volume 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, (1987) Edward B. Roche, ed., American Pharmaceutical Association and Pergamon Press, both of which are incorporated herein by reference thereto.
“Solvate” means a physical association of a compound of this invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. “Solvate” encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like. “Hydrate” is a solvate wherein the solvent molecule is H2O. In general, the solvated forms are equivalent to the unsolvated forms and are intended to be encompassed within the scope of this invention.
Pharmaceutically acceptable esters of the present compounds include the following groups: (1) carboxylic acid esters obtained by esterification of the hydroxy groups, in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, acetyl, n-propyl, t-butyl, or n-butyl), alkoxyalkyl (for example, methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl optionally substituted with, for example, halogen, C1-4alkyl, or C1-4alkoxy or amino); (2) sulfonate esters, such as alkyl- or aralkylsulfonyl (for example, methanesulfonyl); (3) amino acid esters (for example, L-valyl or L-isoleucyl); (4) phosphonate esters and (5) mono-, di- or triphosphate esters. The phosphate esters may be further esterified by, for example, a C1-20 alcohol or reactive derivative thereof, or by a 2,3-di (C6-24)acyl glycerol.
One or more, preferably one to four, antiviral agents useful in anti-HIV-1 therapy may be used in combination with a CCR5 antagonist of the present invention. The antiviral agent or agents may be combined with the CCR5 antagonist in a single dosage form, or the CCR5 antagonist and the antiviral agent or agents may be administered simultaneously or sequentially as separate dosage forms. The antiviral agents contemplated for use in combination with the compounds of the present invention comprise nucleoside and nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors and other antiviral drugs listed below not falling within these classifications. In particular, the combinations known as HAART (Highly Active Antiretroviral Therapy) are contemplated for use in combination with the CCR5 antagonists of this invention.
The term “nucleoside and nucleotide reverse transcriptase inhibitors” (“NRTI” s) as used herein means nucleosides and nucleotides and analogues thereof that inhibit the activity of HIV-1 reverse transcriptase, the enzyme which catalyzes the conversion of viral genomic HIV-1 RNA into proviral HIV-1 DNA.
Typical suitable NRTIs include zidovudine (AZT) available under the RETROVIR tradename from Glaxo-Wellcome Inc., Research Triangle, N.C. 27709; didanosine (ddI) available under the VIDEX tradename from Bristol-Myers Squibb Co., Princeton, N.J. 08543; zalcitabine (ddC) available under the HIVID tradename from Roche Pharmaceuticals, Nutley, N.J. 07110; stavudine (d4T) available under the ZERIT trademark from Bristol-Myers Squibb Co., Princeton, N.J. 08543; Iamivudine (3TC) available under the EPIVIR tradename from Glaxo-Wellcome Research Triangle, N.C. 27709; abacavir (1592U89) disclosed in WO96/30025 and available under the ZIAGEN trademark from Glaxo-Wellcome Research Triangle, N.C. 27709; adefovir dipivoxil [bis(POM)-PMEA] available under the PREVON tradename from Gilead Sciences, Foster City, Calif. 94404; Iobucavir (BMS-180194), a nucleoside reverse transcriptase inhibitor disclosed in EP-0358154 and EP-0736533 and under development by Bristol-Myers Squibb, Princeton, N.J. 08543; BCH-10652, a reverse transcriptase inhibitor (in the form of a racemic mixture of BCH-10618 and BCH-10619) under development by Biochem Pharma, Laval, Quebec H7V, 4A7, Canada; emitricitabine [(−)-FTC] licensed from Emory University under Emory Univ. U.S. Pat. No. 5,814,639 and under development by Triangle Pharmaceuticals, Durham, N.C. 27707; beta-L-FD4 (also called beta-L-D4C and named beta-L-2′,3′-dideoxy-5-fluoro-cytidene) licensed by Yale University to Vion Pharmaceuticals, New Haven Conn. 06511; DAPD, the purine nucleoside, (−)-beta-D-2,6,-diamino-purine dioxolane disclosed in EP 0656778 and licensed by Emory University and the University of Georgia to Triangle Pharmaceuticals, Durham, N.C. 27707; and Iodenosine (FddA), 9-(2,3-dideoxy-2-fluoro-b-D-threo-pentofuranosyl)adenine, a acid stable purine-based reverse transcriptase inhibitor discovered by the NIH and under development by U.S. Bioscience Inc., West Conshohoken, Pa. 19428.
The term “non-nucleoside reverse transcriptase inhibitors” (“NNRTI”s) as used herein means non-nucleosides that inhibit the activity of HIV-1 reverse transcriptase.
Typical suitable NNRTIs include nevirapine (BI-RG-587) available under the VIRAMUNE tradename from Boehringer Ingelheim, the manufacturer for Roxane Laboratories, Columbus, Ohio 43216; delavirdine (BHAP, U-90152) available under the RESCRIPTOR tradename from Pharmacia & Upjohn Co., Bridgewater N.J. 08807; efavirenz (DMP-266) a benzoxazin-2-one disclosed in WO94/03440 and available under the SUSTIVA tradename from DuPont Pharmaceutical Co., Wilmington, Del. 19880-0723; PNU-142721, a furopyridine-thio-pyrimide under development by Pharmacia and Upjohn, Bridgewater N.J. 08807; AG-1549 (formerly Shionogi # S-1153); 5-(3,5-dichlorophenyl)-thio-4-isopropyl-1-(4-pyridyl)methyl-1H-imidazol-2-ylmethyl carbonate disclosed in WO 96/10019 and under clinical development by Agouron Pharmaceuticals, Inc., LaJolla Calif. 92037-1020; MKC-442 (1-(ethoxy-methyl)-5-(1-methylethyl)-6-(phenylmethyl)-(2,4(1H,3H)-pyrimidinedione) discovered by Mitsubishi Chemical Co. and under development by Triangle Pharmaceuticals, Durham, N.C. 27707; and (+)-calanolide A (NSC-675451) and B, coumarin derivatives disclosed in NIH U.S. Pat. No. 5,489,697, licensed to Med Chem Research, which is co-developing (+) calanolide A with Vita-invest as an orally administrable product.
The term “protease inhibitor” (“PI”) as used herein means inhibitors of the HIV-1 protease, an enzyme required for the proteolytic cleavage of viral polyprotein precursors (e.g., viral GAG and GAG Pol polyproteins), into the individual functional proteins found in infectious HIV-1. HIV protease inhibitors include compounds having a peptidomimetic structure, high molecular weight (7600 daltons) and substantial peptide character, e.g. CRIXIVAN(available from Merck) as well as nonpeptide protease inhibitors e.g., VIRACEPT (available from Agouron).
Typical suitable PIs include saquinavir (Ro 31-8959) available in hard gel capsules under the INVIRASE tradename and as soft gel capsules under the FORTOUASE tradename from Roche Pharmaceuticals, Nutley, N.J. 07110-1199; ritonavir (ABT-538) available under the NORVIR tradename from Abbott Laboratories, Abbott Park, Ill. 60064; indinavir (MK-639) available under the CRIXIVAN tradename from Merck & Co., Inc., West Point, Pa. 19486-0004; nelfinavir (AG-1343) available under the VIRACEPT tradename from Agouron Pharmaceuticals, Inc., LaJolla Calif. 92037-1020; amprenavir (141W94), tradename AGENERASE, a non-peptide protease inhibitor under development by Vertex Pharmaceuticals, Inc., Cambridge, Mass. 02139-4211 and available from Glaxo-Wellcome, Research Triangle, N.C. under an expanded access program; lasinavir (BMS-234475) available from Bristol-Myers Squibb, Princeton, N.J. 08543 (originally discovered by Novartis, Basel, Switzerland (CGP-61755); DMP-450, a cyclic urea discovered by Dupont and under development by Triangle Pharmaceuticals; BMS-2322623, an azapeptide under development by Bristol-Myers Squibb, Princeton, N.J. 08543, as a 2nd-generation HIV-1 PI; ABT-378 under development by Abbott, Abbott Park, Ill. 60064; and AG-1549 an orally active imidazole carbamate discovered by Shionogi (Shionogi #S-1153) and under development by Agouron Pharmaceuticals, Inc., LaJolla Calif. 92037-1020.
Other antiviral agents include hydroxyurea, ribavirin, IL-2, IL-12, pentafuside and Yissum Project No. 11607. Hydroxyurea (Droxia), a ribonucleoside triphosphate reductase inhibitor, the enzyme involved in the activation of T-cells, was discovered at the NCI is under development by Bristol-Myers Squibb; in preclinical studies, it was shown to have a synergistic effect on the activity of didanosine and has been studied with stavudine. IL-2 is disclosed in Ajinomoto EP-0142268, Takeda EP-0176299, and Chiron U.S. Pat. Nos. RE 33653, 4,530,787, 4,569,790, 4,604,377, 4,748,234, 4,752,585, and 4,949,314 is available under the PROLEUKIN (aldesleukin) tradename from Chiron Corp., Emeryville, Calif. 94608-2997 as a lyophilized powder for IV infusion or sc administration upon reconstitution and dilution with water; a dose of about 1 to about 20 million IU/day, sc is preferred; a dose of about 15 million IU/day, sc is more preferred. IL-12 is disclosed in WO96/25171 and is available from Roche Pharmaceuticals, Nutley, N.J. 07110-1199 and American Home Products, Madison, N.J. 07940; a dose of about 0.5 microgram/kg/day to about 10 microgram/kg/day, sc is preferred. Pentafuside (DP-178, T-20) a 36-amino acid synthetic peptide, disclosed in U.S. Pat. No. 5,464,933 licensed from Duke University to Trimeris which is developing pentafuside in collaboration with Duke University; pentafuside acts by inhibiting fusion of HIV-1 to target membranes. Pentafuside (3-100 mg/day) is given as a continuous sc infusion or injection together with efavirenz and 2 PI's to HIV-1 positive patients refractory to a triple combination therapy; use of 100 mg/day is preferred. Yissum Project No. 11607, a synthetic protein based on the HIV-1 Vif protein, is under preclinical development by Yissum Research Development Co., Jerusalem 91042, Israel. Ribavirin, 1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, is available from ICN Pharmaceuticals, Inc., Costa Mesa, Calif.; its manufacture and formulation are described in U.S. Pat. No. 4,211,771.
The term “anti-HIV-1 therapy” as used herein means any anti-HIV-1 drug found useful for treating HIV-1 infections in man alone, or as part of multidrug combination therapies, especially the HAART triple and quadruple combination therapies. Typical suitable known anti-HIV-1 therapies include, but are not limited to multidrug combination therapies such as (i) at least three anti-HIV-1 drugs selected from two NRTIs, one PI, a second PI, and one NNRTI; and (ii) at least two anti-HIV-1 drugs selected from, NNRTIs and P is. Typical suitable HAART—multidrug combination therapies include:
(a) triple combination therapies such as two NRTIs and one PI; or (b) two NRTIs and one NNRTI; and (c) quadruple combination therapies such as two NRTIs, one PI and a second PI or one NNRTI. In treatment of naive patients, it is preferred to start anti-HIV-1 treatment with the triple combination therapy; the use of two NRTIs and one PI is preferred unless there is intolerance to P is. Drug compliance is essential. The CD4+ and HIV-1-RNA plasma levels should be monitored every 3-6 months. Should viral load plateau, a fourth drug, e.g., one PI or one NNRTI could be added. See the table below wherein typical therapies are further described:
Anti-HIV-1 Multi Drug Combination Therapies
A. Triple Combination Therapies
1. Two NRTIs1+one PI2 
2. Two NRTIs1+one NNRTI3 
B. Quadruple Combination Therapies4 
Two NRTIs+one PI+a second PI or one NNRTI
C. Alternatives:5 
Two NRTI1 
One NRTI5+one PI2 
Two PIs6±one NRTI7 or NNRTI3 
One PI2+one NRTI7+one NNRTI3 
Footnotes to Table
1. One of the following: zidovudine+lamivudine; zidovudine+didanosine; stavudine+lamivudine; stavudine+didanosine; zidovudine+zalcitabine
2. Indinavir, nelfinavir, ritonavir or saquinavir soft gel capsules.
3. Nevirapine or delavirdine.
4. See A-M. Vandamne et al Antiviral Chemistry & Chemotherapy 9:187 at p 193-197 and FIGS. 1+2.
5. Alternative regimens are for patients unable to take a recommended regimen because of compliance problems or toxicity, and for those who fail or relapse on a recommended regimen. Double nucleoside combinations may lead to HIV-resistance and clinical failure in many patients.

6. Most data obtained with saquinavir and ritonavir (each 400 mg bid).

7. Zidovudine, stavudine or didanosine.

Agents known in the treatment of rheumatoid arthritis, transplant and graft v. host disease, inflammatory bowel disease and multiple sclerosis which can be administered in combination with the CCR5 antagonists of the present invention are as follows:
solid organ transplant rejection and graft v. host disease: immune suppressants such as cyclosporine and Interleukin-10 (IL-10), tacrolimus, antilymphocyte globulin, OKT-3 antibody, and steroids;
inflammatory bowel disease: IL-10 (see U.S. Pat. No. 5,368,854), steroids and azulfidine;
rheumatoid arthritis: methotrexate, azathioprine, cyclophosphamide, steroids and mycophenolate mofetil;
multiple sclerosis: interferon-beta, interferon-alpha, and steroids.
Certain compounds of the invention may exist in different isomeric forms (e.g., enantiomers, diastereoisomers, atropisomers and rotamers). The invention contemplates all such isomers both in pure form and in admixture, including racemic mixtures.
Certain compounds will be acidic in nature, e.g. those compounds which possess a carboxyl or phenolic hydroxyl group. These compounds may form pharmaceutically acceptable salts. Examples of such salts may include sodium, potassium, calcium, aluminum, gold and silver salts. Also contemplated are salts formed with pharmaceutically acceptable amines such as ammonia, alkyl amines, hydroxyalkylamines, N-methylglucamine and the like.
Certain basic compounds also form pharmaceutically acceptable salts, e.g., acid addition salts. For example, the pyrido-nitrogen atoms may form salts with strong acid, while compounds having basic substituents such as amino groups also form salts with weaker acids. Examples of suitable acids for salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic, methanesulfonic and other mineral and carboxylic acids well known to those in the art. The salts are prepared by contacting the free base form with a sufficient amount of the desired acid to produce a salt in the conventional manner. The free base forms may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate. The free base forms differ from their respective salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the acid and base salts are otherwise equivalent to their respective free base forms for purposes of the invention.
All such acid and base salts are intended to be pharmaceutically acceptable salts within the scope of the invention and all acid and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of the invention.
Compounds of the invention can be made by the procedures known in the art, for example by the procedures described in the following reaction schemes, by the methods described in the examples below, and by using the methods described in WO96/26196 and WO98/05292.
The following solvents and reagents may be referred to herein by the abbreviations indicated: tetrahydrofuran (THF); ethanol (EtOH); methanol (MeOH); acetic acid (HOAc or AcOH); ethyl acetate (EtOAc); N,N-dimethylformamide (DMF); trifluoroacetic acid (TFA); 1-hydroxy-benzotriazole (HOBT); m-chloroperbenzoic acid (MCPBA); triethylamine (Et3N); diethyl ether (Et2O); dimethylsulfoxide (DMSO); and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (DEC). RT is room temperature, and TLC is thin-layer chromatography. Me is methyl, Et is ethyl, Pr is propyl, Bu is butyl, Ph is phenyl, and Ac is acetyl.




Reagents and conditions: a: R4CH(OSO2CF3)CO2CH3, base (e.g., K2CO3); b: ClCH2COCl; c: NH3; d: NaBH4—BF3; e: N-Boc-4-piperidone, NaBH(OAc)3; f: CF3CO2H; g: acylation; h: N-Boc-4-piperidone, Ti(OPr-i)4, Et2AlCN; i: CH3MgBr.

In Scheme 1, a benzylamine (1), wherein R and R3 are as defined above and R1 is hydrogen, is converted via (2) and (3) to the diketopiperazine (4), wherein R4 is as defined above, which is reduced to the piperazine (5). Depending upon the desired R6 substituent, this is processed in two ways. Reductive amination gives (6), which can be deprotected to (7) and finally acylated to the compounds of formula IA wherein R5 and R6 are H; alternatively, a modified Strecker reaction on (5) gives the aminonitrile (8), which, after treatment with methyl Grignard to give (9), deprotection to (10) and final N-acylation affords the compounds of formula IB wherein R5 is H and R6 is methyl. Acylation of (7) and (10) is carried out under standard conditions, e.g., with a compound R2COOH and reagents such as DEC and HOBT. Use of a chiral compound of formula I, e.g., (S)-methyl 4-substituted benzylamine, and a chiral lactate in step a, e.g., methyl (R)-lactate triflate, will result in chiral compounds of formulas IA and IB.




Reagents: j: oxaborazolidine, BH3; k: CH3SO2Cl, base; I: CF3CO2H.

In Scheme 2, the compounds are prepared by an alkylation process on a pre-formed piperazine derivative. For example, preferred compounds with the S,S stereochemistry may be obtained in this way by chiral reduction of a ketone (11) to the alcohol (12), activation as the mesylate, and displacement with inversion by treatment with a suitable piperazine, which may either be mono-protected, in which case final elaboration requires deprotection followed by the steps described in (e)-(g) in Scheme 1 to obtain IC, or may be elaborated prior to the displacement step, in which case the final steps are (f) and (g) (deprotection and acylation) as in Scheme 1 to obtain ID.




For compounds where R3 and R1 are each H, either the alkylation route of Scheme 2 or a reductive amination method as typified in Scheme 3 can be used.




For diaryl compounds, wherein R and R3 are each aryl, an alkylation method as typified in Scheme 4 is preferred.




Piperazines of formula 14, especially those wherein R3 is C2-C6 alkyl or benzyl, may also be obtained by a process wherein the




portion is introduced as shown above by an alkylation-decyanation sequence. The reaction is exemplified for compounds wherein R is CF3O-phenyl, R1 is hydrogen, R3 is ethyl and R4 is methyl, but using appropriate starting materials, other compounds of formula 14 can be similarly prepared.





Reagents: m: BOC2O, base; n: R6MgBr; 0: CCl3CO2H, NaBH3CN; p: CF3CO2H; q: NaBH4, BF3.

As shown in Scheme 6, compounds bearing an additional alkyl group at R5 on the piperazine ring may be prepared from the diketopiperazine intermediates (4) of Scheme 1. (4) is activated by conversion to the N(t-butoxycarbonyl) compound (17); addition of a Grignard reagent and sequential reduction, deprotection and lactam reduction provides (21), which can be used to prepare compounds of formula I in the manner described for intermediate (5) in Scheme 1.




Many piperazines wherein R is R8-phenyl (or their Boc derivatives) shown in Scheme 1 can be obtained from a common intermediate, wherein R8 is I. Several examples are shown in the above scheme, wherein R8 is converted to Cl, CN, —C(O)NH2, H, Ph and p-ClC6H4CH2—. Detailed procedures for these conversions are provided in the examples below. The resultant piperazine or BOC-piperazine is then treated as shown in Scheme 1.




Some compounds of the invention may be obtained by a Mannich method, as shown in the specific example of Scheme 8.
Compounds useful in this invention are exemplified by the following preparative examples, which should not be construed to limit the scope of the disclosure. Alternative mechanistic pathways and analogous structures within the scope of the invention may be apparent to those skilled in the art.
Example 1




Step 1: Stir methyl R-lactate (5.0 g) in CH2Cl2 (40 ml) at −70° C. and add trfluoromethanesulfonic anhydride (7.6 ml), then 2,6-lutidine (7.8 ml). Remove the cooling, stir 0.5 h, wash with 2N HCl and add the organic solution to (S)-methyl 4-bromobenzylamine (9.0 g) and K2CO3 (11.2 g) in water (60 ml). Stir 20 h at RT, dry the organic phase over K2CO3, evaporate and chromatograph on silica gel with Et2O—CH2Cl2 to give the desired product (7.50 g) as a thick oil.

Step 2: Reflux the product of step 1 (7.5 g) in 1,2-dichloroethane (40 ml) and ClCH2COCl (5.0 ml) for 5 h, then evaporate and use the resultant residue directly in the next step.

Step 3: Stir the product of step 2 in DMSO (80 ml), water (10 ml) and NaI (8 g), cool in ice, add conc. NH4OH solution (15 ml) and stir to RT for 20 h. Add water (200 ml) dropwise, collect the solid, wash well with water and dry at 70° C./5 mm to give the diketopiperazine, suitable for the next step.

Step 4: Stir a mixture of the product of step 3 (6.8 g), 1,2-dimethoxyethane (60 ml) and NaBH4 (3.4 g) under N2, add BF3.OEt2 (6.8 ml) dropwise, then heat at 100° C. for 10 h. Cool and add CH3OH (20 ml) dropwise, followed by conc. HCl (30 ml). Heat at 100° C. for 1 h., cool, basify with excess 2N NaOH and extract with EtOAc. Dry over K2CO3 and evaporate to obtain the piperazine (5.85 g), suitable for the next step.

Step 5: Stir for 20 h. at RT a mixture of the product of step 4 (5.48 g), N-Boc-4-piperidinone (4.32 g), HOAc (1.15 ml), CH2Cl2 (80 ml) and sodium triacetoxy-borohydride (NaBH(OAc)3) (8.3 g). Add excess aqueous Na2CO3 solution slowly, stir for 0.5 h, separate and filter the organic phase through a pad of silica gel, washing with 10:1 CH2Cl2-Et2O to elute all of the product. Evaporate and dissolve the residue in Et2O (100 ml). Stir and add a 4M solution of HCl in 1,4-dioxane (10 ml) dropwise. Collect the solid, wash with Et2O, and stir with CH2Cl2 and excess aqueous NaOH. Dry the organic phase over K2CO3 and evaporate to obtain the desired product (5.45 g).

Step 6: Stir at RT for 2 h a mixture of the product of step 5 (1.5 g) and TFA (4 ml). Evaporate, dissolve in CH2Cl2 and wash with excess 1N NaOH solution. Dry over K2CO3 and evaporate to obtain the product (1.15 g).

Compound 1A: Following the standard procedure, react the product of step 6 with 2,6-dimethylbenzoyl chloride in CH2Cl2 and aqueous NaOH, and convert the product to the hydrochloride. Mp 185-192° C. (decomposition). HRMS found: 498.2130; MH+ Calc: 498.2120.

Compound 1B: Following the standard procedure, couple the product of step 6 with 2-amino-6-methylbenzoic acid using HOBT and DEC with diisopropylethylamine in DMF, purify the amide by preparative TLC and convert to the hydrochloride. Mp 188-196° C. (decomposition). HRMS found: 499.2069; MH+ Calc: 499.2072.

Compound 1C: Following the above method, couple the product of step 6 with 2-amino-6-chlorobenzoic acid and convert after purification to the hydrochloride. Mp 192-200° C. (decomposition). HRMS found: 519.1530; MH+ Calc: 519.1526.

Example 2




Step 1: Stir the product of Example 1, step 4 (1.00 g), N-t-butoxycarbonyl-4-piperidinone (0.77 g) and titanium (IV) isopropoxide (Ti(OiPr)4) (1.00 g) for 20 h at RT in CH2Cl2 (15 ml), reflux for 3 h and cool to RT. Add diethylaluminum cyanide (Et2AlCN) (4.2 ml of 1M toluene solution) and the stir for 5 days at RT under dry N2. Workup in CH2Cl2-aq. NaOH, dry and evaporate the organic phase and chromatograph on silica gel with CH2Cl2-CH3OH (100:1) to obtain the desired product (0.72 g).

Step 2: React the product of step 1 (0.70 g) in dry THF (15 ml) under N2 with CH3MgBr (4 ml of 3M Et2O solution) at RT for 20 h. Workup in EtOAc-water and filter the organic phase through silica gel, washing with EtOAc. Evaporate to obtain the desired product (0.65 g).

Step 3: Deprotect the product of step 2 with TFA according to the procedure described in Example 1, step 6.

Compound 2A: React the product of step 3 with dimethylbenzoyl chloride as described in Example 1 and convert to the HCl salt. Mp 180-187° C. (decomposition). HRMS Found: 512.2272; MH+ Calc: 512.2276.

Compound 2B: React the product of step 3 with 2-amino-6-chlorobenzoic acid as described in Example 1, purify the crude product by preparative TLC and convert to the HCl salt. Mp 195-200° C. (decomposition). HRMS Found: 535.1662; MH+ Calc: 535.1652.

Compound 2C: React the product of step 3 with 2-hydroxy-6-methylbenzoic acid as described in Example 1, purify the crude product by preparative TLC and convert to the HCl salt. Mp 206-210° C. (decomposition). HRMS Found: 514.2067; MH+ Calc: 514.2069.

Compound 2D: React the product of step 3 with 2-amino-6-methylbenzoic acid using a procedure similar to that described in Example 1, purify the crude product by preparative TLC and convert to the HCl salt. Mp 202-209° C. (decomposition). HRMS Found: 513.2227; MH+ Calc: 513.2229.

Example 3




Step 1: Reflux and stir a mixture of S-alanine methyl ester hydrochloride (14 g), anhydrous Na2CO3 (60 g), dry CH3CN (125 ml), chlorodiphenylmethane (22.3 g) and NaI (5 g) for 6 hr. Cool, add ice-H2O and extract with Et2O (350 ml, then 50 ml). Combine the Et2O extracts and wash with portions of 1N aq. HCl: 200 ml, 100 ml, then 4×10 ml. Combine the aqueous acid extracts, stir and add excess Na2CO3 in small portions until the mixture is basic. Extract with Et2O, dry over MgSO4 and evaporate to obtain the N-diphenylmethyl compound (23.2 g).

Step 2: Reflux all of the above compound with ClCH2COCl (10 ml) in dichloroethane (60 ml) for 4 h. Evaporate, and co-evaporate with toluene (20 ml). Dissolve the residue in CH2Cl2 (200 ml), stir for 0.5 h with activated carbon (10 g), filter and evaporate. Stir the residue with ice cooling in DMSO (200 ml) and gradually add concentrated aqueous NH3 (100 ml), then NaI (10 g). Stir at RT for 20 hr. Add iced water (500 ml), collect the solid, wash well with water, then with several small portions of a 10:1 hexane-Et2O mixture, and dry at 50° C. with high vacuum to obtain the solid diketopiperazine (15.5 g). Recrystallize a small sample from CH2Cl2-hexanes: mp 186-188° C.; [α]D20=+272.60.

Step 3: Stir the product of step 2 (4.0 g) in dimethoxyethane (40 ml) and NaBH4 (1.6 g) under N2 and add BF3.OEt2 (3.2 ml) slowly. Reflux for 20 h. Cool and add CH3OH (10 ml) dropwise, then conc. HCl (15 ml). Reflux for 2 h., and work up in excess 2N aq. NaOH and extract with CH2Cl2. Dry over K2CO3 and evaporate. Chromatograph on silica, eluting with CH2Cl2-CH3OH mixtures, and finally with 5:1:0.1 v/v/v CH2Cl2:CH3OH:NH4OH. Combine and evaporate the product fractions to obtain the desired product (1.95 g) as a pale yellow gum.

Step 4: Stir a mixture of the product of step 3 (0.50 g), N-allyloxycarbonyl-4-piperidone (0.40 g), CH2Cl2 (5 ml) and NaBH(OAc)3 (0.70 g) at RT for 20 h. Work up in CH2Cl2 and excess aq. NaOH, dry over MgSO4, evaporate and isolate the product by preparative TLC, eluting with 10% Et2O in CH2Cl2, to obtain the desired compound (0.80 g) as an oil, contaminated with a small amount of starting ketone, but suitable for the next step.

Step 5: Stir a mixture of the product of step 4 (0.80 g), CH3CN (20 ml), water (5 ml) and piperidine (1.5 ml). Add tri(4-sulfophenyl)phosphine (0.072 g) and palladium (II) acetate (0.02 g) and stir at RT under N2 for 2 h. Work up with aqueous NaOH, extract with 5:1 v/v toluene:CH2Cl2, dry over K2CO3 and evaporate to obtain a yellow oil, suitable for acylation.

Compound 3A: Stir and reflux a mixture of the product of step 5 (0.10 g), N-(2,6-dimethoxybenzoyl)-4-piperidinone (0.10 g), CH2Cl2 (2 ml) and NaBH(OAc)3 (0.15 g) for 2.5 h., cool, and work up with CH2Cl2 and aqueous NaOH. Dry over MgSO4, evaporate and isolate the major product by preparative TLC, eluting with 3:1 v/v Et2O:CH2Cl2. Precipitate the hydrochoride to obtain the desired compound as the HCl salt (0.13 g). Mp 173-177° C. (decomposition). HRMS Found: 482.3175; MH+ Calc: 482.3171.

Compound 3B: Couple the product of step 5 with 2-amino-6-chlorobenzoic acid using DEC-HOBT as described in Example 1, isolate the product by PTLC and precipitate the hydrochloride to give compound 3B. Mp 188-195° C. (decomposition). HRMS Found: 503.2567; MH+ Calc: 503.2578.

Compound 3C: Couple the product of step 5 with 2,4-dimethylnicotinic acid using DEC-HOBt as described above, isolate the product by PTLC and precipitate the hydrochloride to give compound 3C. Mp 180-188° C. (decomposition). HRMS Found: 483.3114; MH+ Calc: 483.3124.

Using procedures similar to those described above, the following compounds were prepared:




Example 4




Step 1: A solution of 4-trifluoromethyl acetophenone (1.88 g; 10 mmol) in dry THF (10 ml) was cooled in an ice bath and treated with freshly prepared solid (S)-2-methyl oxaborolidine (0.54 g; 2 mmol). After 10 min., a solution of 2M borane-methyl sulfide complex (3 ml; 6 mmol) in THF was added dropwise over 5 min. TLC at the end of 30 min. showed that the starting material had been converted to a more polar product. The reaction was quenched with about 5 ml of CH3OH carefully until effervescence stopped; volatiles were removed in vacuo. The residue was dissolved in CH2Cl2 and washed with 1N HCl, water, 10% NaHCO3 solution and brine. Concentration in vacuo gave 2 g of a yellow gum. Flash silica gel chromatography (FSGC) using 10-20% EtOAc in hexanes furnished the desired chiral alcohol (1.6 g; 84%) as a colorless oil.

TLC

Rf=0.6 in 25% EtOAc:hexanes.

Step 2: To a solution of the product of step 1 (1.55 g; 8.16 mmol) in 10 ml of CH2Cl2 cooled in an ice bath were added Et3N (2.3 ml; 16.32 mmol) and CH3SO2Cl (0.87 ml; 10.6 mmol) to form a turbid white solution. The reaction was quenched with water and the organic product was extracted with CH2Cl2, washing with water, 1N HCl, 10% NaHCO3 solution and brine. Concentration in vacuo gave the chiral mesylate (2.1 g; 96%) as a pale yellow oil. TLC Rf=0.6 in 25% EtOAc:hexanes.

Step 3: A solution of the product of step 2 (2.1 g; 7.8 mmol), the N—BOC protected 2(S)-methyl piperazine (1.56 g; 7.8 mmol—prepared from the reaction of commercial 2(S)-methyl piperazine with N-(tert-butoxy-carbonyloxy)phthalimide) and 2,2,6,6-tetramethyl piperidine (1.34 ml; 8 mmol) in 14 ml of dry CN3CN were heated at reflux until TLC indicated complete disappearance of the mesylate (16 h). The reaction mixture was cooled to RT, diluted with CH2Cl2 (50 ml) and washed with water (3×100 ml) and brine. The organic extract was dried over solid MgSO4 and then concentrated to obtain 2.8 g of a yellow gum. FSGC (20% EtOAc in hexanes) served to isolate the desired (S,S)-diastereomer (1.5 g; 52%) and its benzylic epimer, the (R,S)-diastereomer (0.5 g; 17%) for a combined 69% yield. TLC Rf=0.75 (S,S) and 0.56 (R,S) in 25% EtOAc:hexanes.

Step 4: TFA (6 ml) was added to a solution of the product of step 3 in 12 ml of CH2Cl2 and the resulting yellow-orange solution was stirred at RT for 8 h. The reaction was quenched by adding 1N NaOH solution to adjust the pH to 10. Extractive work up in CH2Cl2 gave 1.1 g of a yellow syrup. FSGC using 10% CH3OH in CH2Cl2 removed the less polar impurity and gradient elution with 1% Et3N in 10% CH3OH:CH2Cl2 was needed to elute the desired free amine of the (S,S) diastereomer. Yield=0.9 g (75%). TLC Rf=0.5 in 10% CH3OH:CH2Cl2.

Step 5: A colorless solution of the product of step 4 (0.9 g; 3.3 mmol), 4-piperidinone (0.86 g; 4.3 mmol), NaB(OAc)3H (1.05 g; 4.95 mmol) and glacial AcOH (80 μl) in 8 ml of CH2Cl2 was stirred at ambient temperature for a day. TLC indicated absence of starting material. The reaction mixture was diluted with 50 ml of CH2Cl2, washed with 1N NaOH solution, water (2×) and brine. The CH2Cl2 extract was dried over anhydrous MgSO4 and concentrated to obtain 1.7 g of yellow oil. FSGC (25% acetone in hexanes) was used to isolate the pure product (1.3 g; 86%) as a white foam. TLC Rf=0.6 in 25% acetone/hexanes.

Step 6: TFA (5 ml) was added to a solution of the product of step 5 (1.3 g; 2.87 mmol) in CH2Cl2 (10 ml) and the resulting yellow orange solution was stirred at RT for 7 h. The reaction was quenched with 1N NaOH solution and the pH was adjusted to 10. The organic product was extracted into 50 ml of CH2Cl2 and washed with water, then brine and dried over MgSO4. Concentration gave the free amine (0.98 g; 98%) as a yellow syrup. TLC Rf=0.1 in 25% acetone/hexanes.

Step 7: The product of step 6 (0.78 g; 2.21 mmol), DEC (0.65 g; 3.4 mmol), HOBT (0.46 g; 3.4 mmol) and 2-amino-6-chloro benzoic acid (0.51 g; 2.9 mmol) were dissolved in 8 ml of CH2Cl2 to which was added diisopropylethyl amine (0.7 ml) and the mixture was stirred at ambient temperature for 16 h. TLC analysis showed absence of starting material and formation of two over-lapping spots of medium polarity (rotomers of the hindered amide) as the major product. The crude product (1.3 g) was isolated by extractive work up and purified through FSGC using 25% acetone in CH2Cl2 as eluant to give the title compound (0.88 g; 80%) as a pale yellow foam. TLC Rf=0.45 and 0.5 in 25% acetone:CH2Cl2.

A solution of hydrogen chloride in Et2O (1M; 3 ml) was added to a solution of the title compound free base (0.76 g; 1.54 mmol) in CH2Cl2 (5 ml) to obtain an instantaneous white precipitate. After stirring at RT for 2 h, the volatiles were removed on a rotary evaporator and the white residue was suspended in dry toluene (3×10 ml) and azeotroped. The white solid thus obtained was suspended in dry Et2O containing 10% EtOAc, stirred for 30 min, filtered and washed with Et2O (100 ml). The HCl salt of the title compound was dried under high vacuum to yield an off-white solid (0.88 g; 95%). Mp: 205-210° C.
The product of step 6 was converted to other amides (4A-4E) as described in step 7 using the appropriate carboxylic acids. Physical data for compounds 4-4E having the following structures is as follows:




wherein R8 and R2 are as defined in the table:















Ex.
R8
R2
Mp (° C.)
HRMS (MH+)







4
CF3




205-210
509.2295


 


4A
CF3




192-195
489.2841


 


4B
CF3




203-206
490.2681


 


4C
CF3




186-190
488.2902


 


4D
CF3




207-210
489.2851


 


4E
CF3




152
505


 


4F
CF3




—
490.2796









Example 5




A solution of the racemic benzyl chloride 24 (1.26 g, 5.62 mmol) which was prepared freshly from the corresponding carbinol, the 2(S)-methyl piperazine (1.12 g, 5.62 mmol) and 2,2,6,6-tetramethyl piperidine (TMP) (1.9 ml, 11.2 mmol) were dissolved in dry DMF (2 ml) and heated to 100-110° C. (internal temp.) for 10 h. TLC analysis showed absence of 24 and formation of two well-separated products. The mixture was diluted with water and the organics were extracted into Et2O. The organic extract was washed with saturated NH4Cl and brine and concentrated in vacuo to obtain 2 g of crude product. Flash chromatography on silica gel and elution first with 25% Et2O-hexane followed by 25% EtOAc-hexane gave  ̃0.5 grams of 25a and  ̃0.5 grams of 25b respectively (−45% combined yield). TLC Rf=0.6 (for 25a) and 0.4 (for 25b) in 25% EtOAc-hexanes.
Purified 25a was treated as described previously to obtain the final products 5 to 5F having the formula.




wherein R2 is as defined in the table:

















Ex.
R2
mp (° C.)
HRMS









5




208-212
519.2958



 



5A




198-203
535.2913



 



5B




233 (sharp)
539.2390



 



5C




190
575.1800



 



5D




253
558.1887



 



5E




202
519.2964



 



5F




190
535.2901



 



5G




198-203
—



 



5H




205-210
—










Example 6




Step 1:





A mixture of the aldehyde 26 (3.9 g, 20.5 mmol), the 2(S)-methyl-N—BOC-piperazine (4.1 g, 20.5 mmol) and Ti(OiPr)4 (6.1 mL; 20.5 mmol) in 40 ml of CH2Cl2 was stirred at RT for 24 h. Et2AlCN was introduced and stirred for an additional day. The reaction mixture was processed as described before to obtain 4.71 grams (58%) of the cyano amine 27 after FSGC (TLC Rf=0.45/0.5 for diastereomers seen with 25% Et2O-hexanes as solvent).
Step 2: Sodium hexamethyldisilazide (1M; 3.1 ml) was added to a solution of 27 (1 g; 2.5 mmol) in dry THF cooled in a dry ice/acetone bath. The resulting bright yellow solution was treated with CH3CH21 (7.5 mmol; 0.6 ml). The dry ice bath was removed and the reaction was stirred at ambient temperature for 15 min. followed by gentle warming in a warm water bath (40° C.) for 30 min. TLC indicated two well-separated spots. Standard extractive work up and purification by FSGC gave two alkylated compounds (combined yield: 0.7 g; 70%). TLC Rf=0.6 and 0.4 (25% EtOAc/hexanes).

Step 3: The product of step 2 was stirred with NaBH(OAc)3 (2×) and MgBr2:OEt2 (1×) in CH3CN for a day. The reaction mixture was quenched with water, the organics were extracted into EtOAc and processed to obtain 0.8 grams of crude product. FSGC (25% EtOAc-hexanes) gave  ̃0.4 grams of each diastereomer (combined yield  ̃100%). TLC Rf=0.55 (28a) and 0.45 (28b) in 25% EtOAc-hexanes.

Step 4: Compound 28a (S,S-diastereomer) was processed through the usual 5 step sequence to complete the synthesis of compounds of Example 6, 6A and 6B with an ipso-methyl group as well as compounds 6C and 6D which lack the ipso-methyl group:



















Ex.
R6
R2
mp (° C.)
MS (MH+)







6
CH3




204
549.5


 


6A
CH3




253
589.4


 


6B
CH3




260
534.4


 


6C
H




225
520.4


 


6D
H




215
575.4









Example 7
The synthesis of compounds with an alkyl or arylsulfonyl R8 group at the para position started with the corresponding para-substituted acetophenone which was treated as in Example 4, steps 1-6 to obtain the sulfone containing compounds of Example 7 having the formula:




wherein R8 and R2 are as defined in the table:















Ex.
R8
R2
Mp (° C.)
HRMS (MH+)







7
H3CSO2—




220-225
498.2790


 


7A
H3CSO2—




212-215
519.2197


 


7B








190 (dec.)
604.2861


 


7C








178 (dec.)
625.2246


 


7D








170 (dec.)
605.2799


 


7E








170 (dec.)
609.2540


 


7F








200 (dec.)
612.2336


 


7G








158 (dec.)
644.1735


 


7H
H3CSO2—




197 (dec.)
514.2847









Example 8




Step 1: A solution of the product of Example 4, step 4 (1.25 g; 4.6 mmol), N—BOC-4-piperidinone (0.91 g; 4.6 mmol) and (Ti(OiPr)4) (1.4 ml; 4.6 mmol) in 10 ml of CH2Cl2 was stirred at ambient temperature for 24 h. The reaction mixture was then treated with Et2AlCN (5.5 ml; 1M solution in toluene) and stirring continued for 20 h. The reaction mixture was diluted with EtOAc and stirred with saturated NaHCO3 solution (10 min.) and the layers were separated as much as possible. The turbid (from inseparable aqueous layer) organic layer was treated with excess celite and filtered, washing the filtercake with EtOAc. The filtrate layers were separated and the organic layer was washed with water and brine, dried over anhydrous MgSO4 and concentrated to obtain 2.16 g (98%) of an amber gum.

Step 2: The Strecker amine from step 1 (2.16 g) was dissolved in dry THF, cooled in an ice bath and treated with CH3MgBr (7.5 ml of a 3M solution in Et2O). After 1 h, the ice bath was removed and the yellow, heterogeneous reaction mixture was stirred at RT for 18 h. The reaction was quenched with saturated NH4Cl solution, diluted with water and extracted with CH2Cl2. Concentration gave 2.2 g of a yellow gum which was purified by FSGC, eluting the major product away from more polar impurities using a 1:1 mixture of CH2Cl2:EtOAc. The ipso-methyl compound was isolated as a yellow gum (1.85 g; 88%). TLC Rf=0.5 in 1:1 Et2O:hexanes.

Step 3: TFA (6 ml) was added to a solution of the product of step 2 (1.5 g; 3.2 mmol) in 10 ml of CH2Cl2 and stirred at 25° C. for 2 h. The reaction was quenched with 1N NaOH solution to a pH of 9-10 and processed by extraction into CH2Cl2 to obtain 1.2 g of crude product. FSGC using 1:1 CH2Cl2:EtOAc removed all the less polar impurities and gradient elution with 10% CH3OH in CH2Cl2 and finally with 10% (ca. 7N—NH3) CH3OH in CH2Cl2 led to the isolation of the free piperidine as a yellow gum (1.07 g; 90%). TLC Rf=0.2 in 10% CH3OH:CH2Cl2.

Step 4: A solution of the product of step 3 (1.03 g; 2.8 mmol), 2,4-dimethyl nicotinic acid (0.42 g; 2.8 mmol), DEC (0.8 g; 4.2 mmol), HOBT (0.57 g; 4.2 mmol) and diisopropyl ethyl amine (1 ml; 5.6 mmol) in CH2Cl2 (15 ml) was stirred at 25° C. for 24 h. The reaction mixture was diluted with CH2Cl2 (25 ml), washed with water, 10% NaHCO3 solution and brine, then concentrated to obtain 1.6 g of crude oil. FSGC of this material using gradient elution with 10% acetone-CH2Cl2 followed by 2-5% CH3OH in CH2Cl2 gave the title compound (1.1 g; 80%) as a white foam. TLC Rf=0.45 in 5% CH3OH—CH2Cl2.

The free base of the title compound (1 g; 2 mmol) isolated above was dissolved in a 1:1 mixture of EtOAc:Et2O (8 ml) and a fresh solution of hydrogen chloride in Et2O (6.1 ml of a 1M solution) was added, instantly forming a white precipitate. After stirring at 25° C. for 1 h, the volatiles were removed in vacuo. The product was suspended in Et2O and filtered, washing the filtrate with Et2O. The HCl salt of the title compound thus obtained was dried in vacuo (1.1 g; mp. 213-215° C.). HRMS (MH+) 503.2997.
The following amides 8A-8E were prepared in a similar manner from the product of step 3 using appropriate acids, and compounds 8F-8H, wherein the R8-substituent is a p-methyl sulfonyl group were similarly prepared.




wherein R8 and R2 are as defined in the table:















Ex.
R8
R2
Mp (° C.)
HRMS (MH+)







8A
CF3




216
503.3021


 


8B
CF3




222-224
504.2850


 


8C
CF3




262-263
502.3039


 


8D
CF3




216-218
523.2466


 


8E
CF3




210-212
519.2970


 


8F
—SO2CH3




201-205
512.2955


 


8G
—SO2CH3




217-221
533.2355


 


8H
—SO2CH3




216-219
514.2736


 


8I
—CF3




195-198
—


 


8J
—CF3




250-255
528.1791


 


8K
—CF3




223-226
576.1562


 


8L
—CF3




>245
528.2439


 


8M
—CF3




176-181
570.1739


 


8N
—CF3




218-223
708.0040


 


8O
—CF3




215-220
522.2507


 


8P
—CF3




208-212
566.1987


 


8Q
—CF3




190-194
586.1442


 


8R
—CF3




255-257
526.2243









Using procedures described following the table, compounds 8S-8EE of the structure




were prepared, wherein R11 is defined in the table:

















HRMS


Ex.
R11
Mp (° C.)
(MH+)







8S
—OH
210-220
518.2997




(2 × HCl salt)


8T
—OC(O)NHCH2CH3
205-210
589.3374




(2 × HCl salt)


8U
—OSO2CH3
165-171
596.2757




(2 × HCl salt)


 


8V




199-204 (2 × HCl salt)
595.3254


 


8W
—CHO
88-92
530.2985


8X
—CH═NH—OCH3
202-205
559.3260




(2 × HCl salt)


8Y
—CHF2
>245 (dec)
552.3020




(2 × HCl salt)


8Z
—NH—C(O)—NH—CH2CH3
214-219
588.3521




(2 × HCl salt)


8AA
—NH2
92-98
517.3154


8BB
—NHSO2CH2CH3
205-211
609.3078




(2 × HCl salt)


8CC
—F
212-217
520.2949




(2 × HCl salt)


8DD
—Cl
235-238
536.2663




(2 × HCl salt)


8EE
—Br
237-240
580.2141




(2 × HCl salt)









8S: The tri-hydrochloride salt of the product of Example 8, step 3 (75 mg, 0.16 mmol), EDC (61 mg, 0.32 mmol), HOBT (49 mg, 0.32 mmol), iPr2NEt (0.16 ml, 0.96 mmol), and 2,6-dimethyl-4-hydroxy-benzoic acid (53 mg, 0.32 mmol) were taken up in CH2Cl2 and stirred at 25° C. for 20 h. The solution was concentrated. Purification via preparative TLC (EtOAc, SiO2) gave the title compound as a yellow oil. m.p. (2×HCl salt) 210-220° C. HRMS (MH+) calcd. for C29H39O2N3F3, 518.2994; Found, 518.2997.

8T: 8S (100 mg, 0.19 mmol), ethyl isocyanate (0.05 ml, 0.58 mmol), and Et3N (0.13 ml, 0.95 mmol) were taken up in CH2Cl2 and stirred at 25° C. for 16 h. The solution was diluted with CH2Cl2 and washed with 1 N NaOH. The organic layer was dried (Na2SO4), filtered, and concentrated. Purification via preparative TLC (2/1 EtOAc/hexanes, SiO2) gave the title compound as a yellow oil.

8U: 8S (250 mg, 0.48 mmol), methane sulfonyl anhydride (250 mg, 1.44 mmol), and NaH (38 mg, 60 wt % in oil) were taken up in THF and stirred at 25° C. for 20 h. The solution was diluted with EtOAc and washed with sat'd NaHCO3. The organic layer was dried (Na2SO4), filtered, and concentrated. Purification via preparative TLC (1/1 EtOAc/hexanes, SiO2) gave the title compound as a yellow oil (280 mg, 98%).

8V: The tri-hydrochloride salt of the product of Example 8, step 3 (50 mg, 0.1 mmol), EDC (38 mg, 0.2 mmol), HOBT (27 mg, 0.2 mmol), iPr2NEt (0.07 ml, 0.4 mmol), and 2,6-dimethyl-4-(4-pyridyl-N-oxide)-benzoic acid (73 mg, 0.3 mmol) (see preparation below) were taken up in CH2Cl2 and stirred at 25° C. for 19 h. The solution was concentrated. Purification via preparative TLC (2/1 acetone/hexanes, SiO2) gave 8V as a yellow oil (23 mg, 39%).

Preparation of 2,6-dimethyl-4-(4-pyridyl-N-oxide) benzoic acid




Step A: 4-Benzyloxy-2,6-dimethyl benzoic acid (8.7 g, 34 mmol; Thea, S. et al Journal of the American Chemical Society 1985, 50, 1867), MeI (3.2 ml, 51 mmol), and Cs2CO3 (17 g, 51 mmol) were allowed to stir in DMF at 25° C. for 17 h. The solution was filtered and partitioned between Et2O and water. The aqueous layer was extracted with Et2O. The combined Et2O layers were washed with H2O and brine. The organic layer was dried (MgSO4), filtered, and concentrated. Purification via flash chromatography (10/1 hexanes/Et2O, SiO2) gave 8.6 g (94%) of the methyl ester as a colorless oil.

Step B: The benzyl protected phenol (8.5 g, 32 mmol) and Pd/C (750 mg, 10 wt % Pd) were taken up in CH3OH. The solution was charged with 50 psi H2 and shaken in a Parr apparatus at 25° C. for 17 h. The solution was filtered (Celite). Concentration gave 5.6 g (98%) of the phenol as a white solid.

Step C: The phenol (3.5 g, 19.4 mmol) and iPr2NEt (3.76 g, 29.1 mmol) were dissolved in CH2Cl2 at 0° C. Triflic anhydride (Tf2O) (4.2 ml, 25.2 mmol) was added dropwise to the solution at 0° C. The solution was warmed to 25° C. and stirred at that temperature for 4.5 h. The solution was diluted with CH2Cl2 and washed with sat NaHCO3. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried over Na2SO4. Filtration and concentration gave the crude aryl triflate. Purification via flash chromatography (10/1, hexanes/Et2O, SIO2) gave 5.7 g (94%) of the triflate as a yellow oil.

Step D: The triflate (1 g, 3.2 mmol), 4-pyridyl boronic acid (1.2 g, 9.6 mmol), Pd(PPh3)4 (370 mg, 0.32 mmol), and Na2CO3 (1 g, 9.6 mmol) were taken up in DME/H2O (4/1, 25 ml). The solution was heated to 90° C. (oil bath) under N2 for 18 h. The solution was partitioned between EtOAc and H2O. The aqueous layer was extracted with EtOAc. The combined EtOAc layers were dried (Na2SO4). Filtration and concentration gave a dark brown oil. Purification via flash chromatography (3/1 hexanes/EtOAc, SiO2) gave 770 mg (100%) of the pyridyl derivative as an orange oil.

Step E: The pyridyl derivative (390 mg, 1.6 mmol) and mCPBA (550 mg, 3.2 mmol) were dissolved in CH2Cl2. The solution was stirred at 25° C. for 18 h. The solution was diluted with CH2Cl2 and washed with 1 N NaOH. The organic layer was dried (Na2SO4). Filtration and concentration gave 400 mg (97%) of the N-oxide as an orange oil. HRMS (MH+) calcd. for C15H16O3N, 258.1130; Found, 258.1131.

Step F: The methyl ester (400 mg, 1.6 mmol) was taken up in 5 ml of 3 N NaOH and 2 ml of EtOH. The solution was heated at reflux for 20 h. The solution was concentrated. The residue was treated with conc. HCl. The resulting solid was filtered and washed with water and brine. After drying under high vacuum, the free acid (377 mg, 100%) was obtained as a tan solid. m.p. >225° C. (decomp). HRMS (MH+) calcd. for C14H14O3N, 244.0974; Found, 244.0981.

8W: The tri-hydrochloride salt of the product of Example 8, step 3 (1.34 g, 2.8 mmol), 2,6-dimethyl-4-formyl benzoic acid (500 mg, 2.8 mmol) (see preparation below), EDC (1.1 g, 5.6 mmol), HOBT (760 mg, 5.6 mmol) and iPrNEt (2 ml, 11 mmol) were subjected to the standard coupling conditions. Purification via flash chromatography (2/1 hexanes/EtOAc, SiO2) gave 898 mg (61%) of 8W as a yellow foam.

Preparation of 2,6-dimethyl-4-formyl benzoic acid




Step A: 4-Hydroxy-2,6-dimethyl-benzoic acid, tert-butyl ester (6.4 g, 29 mmol) and iPr2NEt (5.6 g, 43 mmol) were taken up in CH2Cl2 and cooled to 0° C. Tf2O (5.8 ml, 34 mmol) was added slowly to the solution at 0° C. The solution was stirred at 0° C. for 3 h. The solution was partitioned between sat. NaHCO3 and CH2Cl2. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried (Na2SO4). Filtration and concentration gave a brown oil. Purification via flash chromatography (20/1 hexanes/Et2O, SiO2) gave 7.99 g (82%) of the triflate as a yellow solid.

Step B: The triflate (5 g, 15 mmol), LiCl (1.25 g, 30 mmol), Pd(PPh3)4 (340 mg, 0.3 mmol), and vinyl tributyl tin (4.5 ml, 16 mmol) were taken up in THF under N2. The solution was heated at 70° C. for 16 h. The solution was partitioned between EtOAc and sat. KF. The mixture was filtered. The organic layer was separated, and the aqueous layers were extracted with EtOAc. The combined organic layers were dried (MgSO4). Filtration and concentration gave a yellow oil. Purification via flash chromatography (20/1 hexanes/Et2O, SiO2) gave 1.96 g (57%) of the olefin as a yellow oil.

Step C: The olefin (0.6 g, 2.6 mmol) was taken up in CH2Cl2/MeOH (1/1). The solution was cooled to −78° C. Ozone was bubbled through the solution until a dark blue color persisted. The reaction was quenched with dimethyl sulfide. The reaction was concentrated to furnish the aldehyde as an oil.

Step D: The tert-butyl ester (650 mg, 2.8 mmol) and TFA (3 ml) were taken up in CH2Cl2 and stirred at 25° C. for 19 h. Concentration of the solution gave the acid as a beige solid.

8X: 8W (100 mg, 0.19 mmol), H2NOMe-HCl (28 mg, 0.34 mmol), NaOAc (32 mg, 0.46 mmol) were taken up in MeOH. The solution was stirred at 25° C. for 17 h. The solution was concentrated. The residue was partitioned between CH2Cl2 and 1 N NaOH. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried (Na2SO4). Filtration and concentration gave the crude product. Purification via preparative TLC (1/1 hexanes/EtOAc, SiO2) gave 85 mg (84%) of 8×.

8Y: The tri-hydrochloride salt of the product of Example 8, step 3 (75 mg, 0.16 mmol) and 4-difluoromethyl-2,6-dimethyl benzoic acid (32 mg, 0.16 mmol) were subjected to the standard coupling conditions (EDC/HOBT/iPr2NEt). Purification via preparative TLC (2/1 hexanes/EtOAc, SiO2) gave 64 mg (73%) of 8Y.

Preparation of 4-difluoromethyl-2,6-dimethyl benzoic acid




Step A: The aldehyde (400 mg, 1.7 mmol), [bis(2-methoxyethyl)amino]-sulfur trifluoride (640 mg, 2.9 mmol), and EtOH (0.02 ml, 0.34 mmol) were taken up 1,2-dichloroethane and stirred at 65° C. for 6 h and at 25° C. for 19 h. The solution was quenched with sat. NaHCO3. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried (NaSO2). Filtration and concentration gave the crude product. Purification via preparative TLC (10/1 hexanes/Et2O, SiO2) gave 210 mg (50%) of the difluoro derivative.

Step B: The tert-butyl ester (210 mg, 0.82 mmol) and HCl (2.1 ml of 4 M in dioxane, 8.2 mmol) were taken up in MeOH. The solution was stirred at 45° C. for 20 h. The solution was concentrated to obtain the acid as a white solid.

8Z: The tri-hydrochloride salt of the product of Example 8, step 3 (811 mg, 1.7 mmol) and 4-[(ethylamino)carbonylamino]-2,6-dimethyl benzoic acid (400 mg, 1.7 mmol) (see preparation below) were subjected to the standard coupling conditions (EDC/HOBT/iPr2NEt). Purification via flash chromatography (1/1 hexanes/acetone, SiO2) gave 803 mg (81%) of 8Z as a foam.

Preparation of 4-[(ethylamino)carbonylamino]-2,6-dimethyl benzoic acid




Step A: 3,5-Dimethyl aniline (18.5 ml, 149 mmol) was taken up in CH2Cl2. The solution was cooled in a water bath. Trifluoroacetic anhydride (29.5 ml, 209 mmol) was added slowly to the solution. After the addition, the solution was stirred at 25° C. for 15 minutes. Bromine (7.3 ml, 142 mmol) was added slowly to the solution while maintaining the RT water bath. The solution was stirred at 25° C. for 3.5 h. The solution was quenched with 10% Na2S2O3. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried (MgSO4), treated with activated carbon and filtered. Concentration gave an orange solid. Purification via recrystallization (hexanes/Et2O) gave two crops (34 g total, 77%) of the brominated derivative as a white solid.

Step B: The aryl bromide (17 g, 57 mmol) was taken up in THF and cooled to −78° C. under N2. Methyllithium/LiBr (54 ml of a 1.5 M solution in Et2O, 80 mmol) was added slowly to the solution at −78° C. After 5 min of stirring, sec-BuLi (62 ml of a 1.3 M in cyclohexane, 80 mmol) was added slowly to the reaction solution at −78° C. After 5 min, di-t-butyl dicarbonate (22.5 g, 103 mmol) in THF was added to the solution at −78° C. The solution was warmed to 25° C. After 30 min, the reaction mixture was partitioned between water and CH2Cl2. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried (MgSO4). Filtration and concentration gave a yellow solid. Purification via flash chromatography (1/1 to 1/4 hexanes/CH2Cl2, SiO2) gave 13.1 g (72%) of the tert-butyl ester as an off-white solid.

Step C: The trifluoro-acetamide (10 g, 31 mmol) and NaOH (2.5 g, 62 mmol) were taken up in MeOH/H2O (3/1) and heated at 60° C. for 3 h. The solution was partitioned between CH2Cl2 and water. The aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with water and dried (Na2SO4). Filtration and concentration gave 6.4 g (93%) of the aniline as an orange solid.

Step D: The aniline (1 g, 4.5 mmol), ethyl isocyanate (0.4 ml, 5 mmol), and CuCl (90 mg, 0.9 mmol) were taken up in DMF at 0° C. The solution was warmed to 25° C. and stirred at that temperature for 2 h. The solution was partitioned between EtOAc and 10% NH4OH. The aqueous layer was extracted with EtOAc. The combined layers were washed with brine and dried (MgSO4). Filtration and concentration gave a yellow solid. Purification via flash chromatography (3/1 to 1/1 hexanes/EtOAc, SiO2) gave 904 mg (69%) of the urea as a yellow solid.

Step E: The tert-butyl ester (900 mg, 3.1 mmol) and 4 M HCl in dioxane (3 ml) were taken up in iPrOH and heated at 45° C. for 3.5 h and at 25° C. for 16.5 h. The solution was concentrated under reduced pressure. The residue was partitioned between Et2O and 1 N NaOH. The aqueous, basic layer was extracted with Et2O. The aqueous layer was cooled to 0° C. and acidified with conc. HCl (pH=1-2). The aqueous layer was extracted with EtOAc. The combined EtOAc layers were dried (Na2SO4). Filtration and concentration gave the 400 mg (55%) of the acid as a white solid.

8AA: The tri-hydrochloride salt of the product of Example 8, step 3 (2 g, 4.3 mmol) and 4-amino-2,6-dimethyl benzoic acid (710 mg, 4.3 mmol) (see preparation below) were subjected to the standard coupling conditions (EDC/HOBT/iPr2NEt). Purification via flash chromatography (2/1 hexanes/acetone, SiO2) gave 1.16 g (52%) of 8AA as a yellow foam.

Preparation of 4-amino-2,6-dimethyl benzoic acid




The tert-butyl ester (950 mg, 4.3 mmol) and HCl (11 ml, 4 M in dioxane) were taken up in MeOH at heated at 45° C. for 20 h. The solution was concentrated to obtain the acid (710 mg) in quantitative yield.
8BB: 8AA (100 mg, 0.19 mmol) and ethane sulfonyl chloride (0.02 ml, 0.21 mmol) were taken up in pyridine and stirred at 25° C. for 19 h. The solution was concentrated. The residue was partitioned between 1 N NaOH and CH2Cl2. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried (Na2SO4). Filtration and concentration gave a brown oil. Purification via preparative TLC (2/1 hexanes/acetone, SIO2) gave 100 mg (86%) of 8BB as a colorless oil.

8CC: The trihydrochloride salt of the product of Example 8, step 3 (127 mg, 0.27 mmol) and 4-fluoro-2,6-dimethyl benzoic acid (58 mg, 0.35 mmol) (see preparation below) were coupled according to the general procedure (EDC/HOBT/iPr2NEt). Purification via preparative TLC (2/1 hexanes/EtOAc, SiO2) gave 8CC as a colorless oil (87 mg bis-HCl salt, 54%).

Preparation of 4-fluoro-2,6-dimethyl benzoic acid




4-Amino-2,6-dimethyl benzoic acid (200 mg, 1.1 mmol) and NOBF4 (196 mg, 1.7 mmol) were heated in 1,2-dichlorobenzene at 100° C. for 30 min. The solution was cooled and diluted with MeOH and water. A few pellets (2-3) of KOH were added, and the solution was heated at reflux for 16 h. The solution was concentrated. The residue was partitioned between Et2O and 1 N NaOH. The aqueous layer was extracted with Et2O. The aqueous layer was cooled to 0° C. and acidified with conc. HCl (pH=1-2). The aqueous layer was extracted with CH2Cl2. The organic layers were dried (Na2SO4). Filtration and concentration gave 58 mg (31%) of the acid as a tan solid.
8DD: The trihydrochloride salt of the product of Example 8, step 3 (150 mg, 0.31 mmol) and 4-chloro-2,6-dimethyl benzoic acid (76 mg, 0.41 mmol) (see preparation below) were coupled according to the general procedure (EDC/HOBT/iPr2NEt). Purification via preparative TLC (4/1 hexanes/acetone, SiO2) gave 8DD as a colorless oil.
Preparation of 4-chloro-2,6-dimethyl benzoic acid




4-Amino-2,6-dimethyl benzoic acid (172 mg, 0.96 mmol) and CuCl2 (155 mg, 1.15 mmol) were taken up in CH3CN at 0° C. Tert-butyl nitrite (0.17 ml, 1.4 mmol) was added to the solution at 0° C. The solution was warmed to 25° C. and then at 65° C. for 45 min. The solution was partitioned between Et2O and water. The aqueous layer was extracted with Et2O. The combined organic layers were washed with brine and dried (MgSO4). Filtration and concentration gave the methyl ester. The methyl ester was hydrolyzed as described above for the fluoro derivative (KOH). After extractive workup, 4-chloro-2,6-dimethyl benzoic acid (158 mg, 89%) was obtained as a yellow solid.
8EE: The trihydrochloride salt of the product of Example 8, step 3 (180 mg, 0.38 mmol) and 4-bromo-2,6-dimethyl benzoic acid (95 mg, 0.41 mmol) (see preparation below) were coupled according to the general procedure (EDC/HOBT/iPr2NEt). Purification via preparative TLC (4/1 hexanes/acetone, SiO2) gave 8EE as a colorless oil (140 mg bis-HCl salt, 56%).
Preparation of 4-bromo-2,6-dimethyl benzoic acid




Step A: The triflate (500 mg, 1.48 mmol), hexamethylditin (0.31 mmol, 1.48 mmol), LiCl (377 mg, 8.9 mmol), and Pd(PPh3)4 (171 mg, 0.15 mmol) were heated in THF (70° C.) under N2 for 21 h. The solution was partitioned between Et2O and pH=7 buffer (NH4OAc). The aqueous layer was extracted with Et2O. The combined Et2O layers were washed with brine and dried (Na2SO4). Filtration and concentration gave the crude aryl stannane as a yellow semisolid.

Step B: The aryl stannane (0.74 mmol) was taken up in CH2Cl2 at 0° C. Bromine (0.7 ml of 1 M Br2 in CH2Cl2) was added to the solution. The solution was stirred at 0° C. for 30 min. The solution was diluted with CH2Cl2 and washed with 10% Na2S2O3. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried (Na2SO4). The solution was filtered. TFA (2 ml) was added to the solution, and the solution was stirred at 25° C. for 17 h. The solution was concentrated. The residue was partitioned between Et2O and 1 N NaOH. The aqueous layer was extracted with Et2O. The aqueous layer was cooled to 0° C. and acidified with conc. HCl (pH=1-2). The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried (Na2SO4). Filtration and concentration gave 100 mg (59%) of the acid as a white solid.

Using procedures described following the table, compounds 8FF-8HH of the structure




were prepared, wherein R11 is defined in the table:














Ex.
R11
Mp (° C.)
HRMS (MH+)







8FF
—OCH3
217-220
572.2048




(2×HCl salt)


8GG
—OH
198-204
558.1898




(2×HCl salt)


 


8HH




200-205 (2×HCl salt)
635.2172









8FF: The trihydrochloride salt of the product of Example 8, step 3 (100 mg, 0.21 mmol) and 2,6-dichloro-4-methoxy-benzoic acid (140 mg, 0.63 mmol) were coupled according to the general procedure (EDC/HOBT/iPr2NEt). Purification via preparative TLC (3/1 hexanes/EtOAc, SiO2) gave 8FF as a colorless oil (27 mg, 23%).

8GG: The trihydrochloride salt of the product of Example 8, step 3 (330 mg, 0.7 mmol) and 2,6-dichloro-4-hydroxy-benzoic acid (290 mg, 1.4 mmol) (see preparation below) were coupled according to the general procedure (EDC/HOBT/iPr2NEt). Purification via preparative TLC (1/1 hexanes/EtOAc, SiO2) gave 8GG as a colorless oil (75 mg, 19%).

Preparation of 2,6-dichloro-4-hydroxy-benzoic acid




2,6-Dichloro-4-methoxy-benzoic acid (500 mg, 2.3 mmol) was taken up in CH2Cl2 and cooled to −78° C. BBr3 (6.9 ml of a 1 M solution in CH2Cl2) was added to the solution at −78° C. The solution was warmed to 25° C. and stirred at that temperature for 16 h. The solution was quenched with 3 N NaOH. The aqueous layer was extracted with CH2Cl2. The aqueous layer was cooled (0° C.) and acidified with conc. HCl (pH=1-2). The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried (Na2SO4). Filtration and concentration gave the crude phenol which was used without further purification.
8HH: The trihydrochloride salt of the product of Example 8, step 3 (96 mg, 0.2 mmol) and 2,6-dichloro-4-(4-pyridyl-N-oxide)-benzoic acid (55 mg, 0.2 mmol) (see preparation below) were coupled according to the general procedure (EDC/HOBT/iPr2NEt). Purification via preparative TLC (1/5 hexanes/acetone, SiO2) gave 8HH as a colorless oil (54 mg, 43%).
Preparation of 2,6-dichloro-4-(4-pyridyl-N-oxide) benzoic acid




2,4,6-Trichloro benzoic acid, tert-butyl ester (500 mg, 1.8 mmol), 4-pyridyl boronic acid (270 mg, 2.16 mmol), Pd(PCy3)2Cl2 (130 mg, 0.18 mmol), and CsF (540 mg, 3.6 mmol) were taken up in NMP and heated at 100° C. under N2 (16 h). The solution was partitioned between EtOAc and water. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with water and brine and dried (Na2SO4). Filtration and concentration gave the crude product. Purification via preparative TLC (1/1 hexanes/EtOAc, SiO2) gave 68 mg (12%) of the pyridyl ester. The tert-butyl ester was converted into the acid as done previously for the dimethyl derivative (a. mCPBA/b. TFA).
Using suitable starting materials and the procedures described for examples 8S to 8HH, the compounds of the following structure were prepared:




wherein R11 is defined in the table


















HRMS (MH+)
HRMS (MH+)


Ex.
R11
m.p. (° C.)
calc.
found







8II
—OCH3
236-240
532.3151
532.3166


8JJ
—CH3
>260
516.3202
516.3213


 


8KK




186-190
603.3522
603.3513


 


8LL




202-208
579.3311
579.3303


 


8MM




210-216
579.3311
579.3311


 


8NN




196-203
595.3260
595.3256


 


8OO




>230 (dec)
578.3358
578.3368


 


8PP




135-140
617.3679
617.3671


 


8QQ




205-215
602.3682
602.3722


 


8RR
CH2OH
>235 (dec)
532.3151
532.3124


 


8SS




206-212
580.3263
580.3258


 


8TT




198-204
579.3311
579.3315


 


8UU




231-236
580.3263
580.3252


 


8VV




201-207
613.2977
613.2981


 


8WW




215-220
650.2487
650.2497


 


8XX




198-201
545.3103
545.3098


 


8YY




210-214
595.2930
595..2921


 


8ZZ
CH2F
>245
534.3108
534.3117


 


8AB




202-205
624.3195
624.3204


 


8AC




208-213
559.3260
559.3263


 


8AD




215-220
560.3212
560.3220


 


8AE




215-220
573.3416
573.3424


 


8AF




215-220
559.3260
559.3257


 


8AG




205-209
602.3682
602.3672


 


8AH




186-192
574.3369
574.3378


 


8AI




200-206
616.3838
616.3844


 


8AJ




165-173
661.3941
661.3949


 


8AK
CN
240-250
527.2998
527.2991


 


8AL




211-215
622.3136
622.3129


 


8AM




170-174
616.3838
616.3836


 


8AN




192-196
614.3682
614.3690









All melting points were done on the bis hydrochloride salts (2×HCl) except 8PP was performed on the free base

Using derivatives of the triflate intermediate described in 8Z in procedures similar to those described above and following the table for 8AO-8AQ, the compounds of the following structure were prepared:




wherein R11 is defined in the table













Ex.
R11
m.p. (° C.)







8AO
—CN
240-250


8AP
—CONHEt
215-220


8AQ
-N(CH3)CONHEt
186-203


8AR
—CONH2
200-208


8AS
—CONHCH3
215-220


8AT
—CON(CH2CH2OCH3)2
165-173


8AU
—CON(Et)2
170-180


8AV
—N(CH3)CONHCH3
198-210


8AW
—NHCH3
190-200


8AX
—N(CH3)CONH2
190-220







8AO:


 








 













 




















Step 1: The triflate intermediate (see 8W) (0.4 g), Zn(CN)2 (0.2 g), Pd(PPh3)4 (0.3 g) and DMF (1.5 ml) were heated at 80° C. for 17 h. The reaction was cooled to RT, diluted with EtOAc and saturated aqueous NaHCO3. The EtOAc layer was removed, washed with water, dried with brine and evaporated to give a crude oil which was purified by preparative plate chromatography (2000 μM silica plates; 8:1 hexanes: EtOAc eluant), to give, after isolation of the appropriate band, the cyano intermediate (0.2 g) in 77% yield.

Step 2: The product of Step 1 (0.2 g) was dissolved in MeOH (1.5 ml) and HCl (4M solution in 1,4-dioxane; 2 ml) was added. The resulting solution was stirred at 50° C. for 3 h and evaporated. This crude intermediate (0.038 g) and the product of Example 8, Step 3 (65 mg; trihydrochloride form) were treated in the same fashion as Example 8, Step 4, using DMF (2 ml), HOBt (45 mg), DEC (60 mg) and diisopropyl ethyl amine (0.1 ml) to give, after isolation and purification, the free base form of 8AO, which was converted to its HCl salt (45 mg) in 95% yield.





Step 1: 2,6-Dimethyl-4-formyl benzoic acid (1.96 g) (see 8W) was dissolved in t-butanol (94 ml) and 2-methyl-2-butene (24 ml). A solution of NaClO2 (6.89 g), NaH2PO4 monohydrate (8.17 g) and water (45 ml) was added dropwise to the first solution. After complete addition, the pH was adjusted to 3 and two layers resulted. The organic layer was removed and evaporated to give intermediate acid (1.80 g) as a white crystalline solid, which was used without purification.

Step 2: To a solution of the product of Step 1 (0.62 g), CH2Cl2 (5 ml) and DMF (1 drop) was added oxalyl chloride (0.31 ml) and the resulting solution was stirred for 10 min, at which time a second portion of oxalyl chloride (0.30 ml) was added. The reaction was stirred for 10 min, toluene was added and the mixture was evaporated to dryness. CH2Cl2 (10 ml) and EtNH2 (1 ml) were added and the reaction was stirred for 2 days, then partitioned between brine and CH2Cl2. The CH2Cl2 layer was evaporated and HCl (4 ml of a 4 M solution in 1,4-dioxane) was added. The resulting solution was stirred for 3 h and evaporated to give a solid which was washed with Et2O and collected to give the amide intermediate (0.13 g) in 24% yield.

Step 3: The product of Example 8, Step 3 (60 mg; trihydrochloride form) and the product of step 2 (35 mg) were treated in the same fashion as Example 8, Step 4 to give, after work up and purification, 8AP as the free base form, which was converted to the HCl salt (50 mg) in 62% yield.





Step 1: To a solution of the amine intermediate (2 g) (see 8Z) was added NaH (0.4 g of a 60% oil dispersion). The resulting suspension was stirred for 15 min and Me2SO4 was added. After heating at reflux for 1.5 h, the reaction was cooled to RT, poured into saturated NH4Cl aqueous solution and extracted with Et2O. After evaporation, the crude reaction mixture was chromatographed on silica gel, eluting with 4:1 hexanes:EtOAc, to give, after evaporation of the appropriate fractions, the methylamine intermediate (0.8 g) in 38% yield.

Step 2: The product of Step 1 (0.12 g), THF (5 ml) and EtNCO (54 mg) were heated at reflux for 17 h. EtNCO (54 mg) and 1,4-dioxane (2 ml) were added and the resulting solution was heated in a sealed tube at 65° C. for 17 h. The solution was cooled, evaporated and purified by preparative plate chromatography (silica gel; 25% EtOAc:CH2Cl2), to give the desired product (0.1 g) as a crystalline solid in 64% yield.

Step 3: The product of Step 2 (0.1 g) was treated in the same fashion as Example 8, Step 3 (p 28) to give the desired intermediate (0.08 g) which was used directly in the next step.

Step 4: The product of Example 8, Step 3(75 mg; trihydrochloride form) and the product of Step 3 (0.04 g) were treated in the same fashion as Example 8, Step 4, to give, after work up and purification, 8AQ as the free base form, which was converted to the HCl salt (65 mg) in 62% yield.

Using procedures described above and employing commercially available acids, compounds 8AY-8BT of the structure




were prepared, wherein R10 and R11 are defined in the table:

















Ex.
R10
R11
Mp (° C.)









8AY
—CH3
H
205-208



8AZ
F
H
250-255



8BA
Cl
H
215-217



8BC
—CH3
Br
228-231



 



8BD
—CH3




194-198



 



8BE
Cl
Cl
240-241



8BF
Cl
F
268-270



8BG
Br
H
210-213



8BH
Cl
Br
213-217



8BI
Br
F
176-181



8BJ
I
H
184-190



8BK
—CF3
F
204-209



8BL
F
F
268-270



8BM
Cl
NH2
215-220



8BN
H
F
258-260



8BO
H
Br
238-240



8BP
H
Cl
235-240



8BQ
Br
Cl
190-194



8BR
CH3CH2—
H
211-214



8BS
—Si(CH3)3
H
230-240



8BT
Cl
NO2
275-280










Using procedures similar to those described above, the following compounds were prepared:




wherein R8, R3, R6 and R2 are as defined in the table:
















Ex.
R8
R3
R6
R2
Mp (° C.)







8BU
—CF3




—CH3




195-220


 


8BV
—CF3




—CH3




80-85


 


8BW
—CF3




—CH3




212-217


 


8BX
—CF3




—CH3




235-238


 


8BY
—CF3




—CH3




195-200


 


8BZ
—CF3




—CH3




237-240


 


8CA
—CF3




—CH2CH3




179-181


 


8CB
—CF3




—CH2CH3




200-202


 


8CD
—CF3




—CH2CH3




199-205


 


8CE








—CH3




206-210


 


8CF
—CF3




—CH3




235-239









Example 9




Step 1: A solution of 4-N—BOC-2(S)-methyl piperazine (1.5 g; 7.5 mmol), 4-methoxy-benzyl chloride (1.1 ml; 8.1 mmol) and diisopropyl ethyl amine (1.5 ml) in dry CH3CN were heated at reflux for 5 h. The reaction mixture was cooled to RT and volatiles were removed in vacuo. The residue was dissolved in CH2Cl2 (30 ml) and washed with water and brine. Concentration gave the crude product, which was purified by FSGC (10% EtOAc-hexanes) to obtain 2.1 g (88%) of product as a pale yellow liquid.

TFA (6 ml) was added to a solution of the above compound (2.1 g; 6.56 mmol) in 12 ml of CH2Cl2 and the mixture stirred at 25° C. for 1.5 h. The reaction was quenched with 1 N NaOH and adjusted to pH 10. Extractive work-up in CH2Cl2 furnished the desired product (1.4 g; 97%) as a colorless gum.
Step 2: A mixture of the product of step 1 (1.4 g; 6.36 mmol), N—BOC-4-piperidinone (1.27 g; 6.4 mmol) and Ti(OiPr)4 (1.9 ml; 6.4 mmol) was stirred at 25° C. for 24 h. A 1M solution of Et2AlCN in toluene (7.6 ml) was added to the reaction mixture and the mixture stirred at ambient temperature for another day. The Strecker amine thus formed was worked-up and isolated (2.7 g; 100%) as described in Example 8, step 2. TLC Rf=0.3 in 25% EtOAc—CH2Cl2.
The Strecker amine (2.7 g; 6.3 mmol) was dissolved in 15 ml of dry THF at 0° C. and CH3MgBr (3M in Et2O; 10.5 ml) was added to it. After 1 h, the ice bath was removed and the reaction allowed to proceed at RT for 15 h. TLC analysis of the heterogeneous reaction mixture showed no change from the starting material; the mixture was warmed at 60° C. for 5 h with no observed change in TLC behavior. The reaction mixture was quenched with saturated NH4Cl and organic products extracted into CH2Cl2. FSGC of the crude product (2.7 g) using 15% acetone-hexanes as the eluant provided the desired ipso-methyl compound as a colorless gum (2.3 g; 87%).
Step 3: The product of step 2 (1.7 g; 4.08 mmol), ammonium formate (1.4 g; 22 mmol) and 10% palladium on carbon (0.4 g) were mixed in 20 ml of CH3OH and heated at reflux for 5 h. The reaction mixture was filtered through celite and volatiles were removed. The residue was dissolved in CH2Cl2 and washed with 10% NaOH solution, water and brine. Concentration in vacuo gave 1.1 g (92%) of pale yellow gum.

Step 4: A solution of the product of step 3 (0.12 g; 0.4 mmol), p-trifluoro-methyl benzyl bromide (0.1 g; 0.4 mmol) and diisopropyl ethyl amine (0.1 ml) in dry CH3CN was gently warmed (60-70° C.) for 16 h. The mixture was cooled and organic product isolated via extractive work-up in CH2Cl2. FSGC (10-30% Et2O—CH2Cl2; Rf=0.4) yielded the major product as a colorless film (0.12 g; 68%).

Treatment of the above product (in CH2Cl2) with TFA (1 ml) for 1 h followed by basification and standard work-up provided the desired compound (0.09 g; 96%) as a colorless film.
Step 5: The product of step 4 (0.045 g; 0.13 mmol) and 6-chloro anthranilic acid (0.022 g; 0.13 mmol) were coupled as described in Example 1 and after work-up and FSGC (5% CH3OH in CH2Cl2) the title compound was isolated as a colorless film (0.058 g; 90%).
The HCl salt of the title compound was prepared in the usual manner by the reaction of the free base with 1M HCl-Et2O and processing the precipitate to obtain a beige solid (0.066 g).
Using a similar procedure, the product of step 3 was converted to other compounds, first by alkylation of the piperazine nitrogen with the appropriate halide, followed by deprotection and coupling of the piperidinyl portion with the appropriate acid to form the amides of general structure:




wherein R and R2 are as defined in the table:



















HRMS


Ex.
R
R2
Mp (° C.)
(MH+)







9A








246-249
509.2293


 


9B








204-208
488.2895


 


9C








247-249
546.1978


 


9D








249-251
567.1407


 


9E








206-209
504.2848


 


9F








244-247
525.2242


 


9G








201-204
484.2630


 


9H








222-226
505.2039


 


9I








226-229
451.3060


 


9J








229-232
472.2474


 


9K








268-271
455.2577


 


9L








212-216
476.1975


 


9M








229-232
450.3126


 


9N








246-251
434.3168


 


9O








192-205
—


 


9P








185-196
—


 


9Q








202-210
—


 


9R








203-206
—


 


9S








190-205
—


 


9T








180-205
—


 


9U








258-262
—









Using a similar procedure described below, compounds wherein R is 4-ethoxynaphthyl were also prepared:
Steps 1-3: See Example 9.
Step 4A: 4-Hydroxynaphthaldehyde (0.86 g) and K2CO3 (1.38 g, 2 equiv.) in CH3CN (35 ml) were treated with CH3CH21 (0.80 ml, 2 equiv.), and the resulting mixture was stirred at RT for 20 h. The reaction mixture was concentrated in vacuo, the residue treated with EtOAc, and the mixture filtered. The filtrate was partitioned with H2O. The dried (MgSO4) EtOAc was concentrated in vacuo to give an orange-brown residue (0.89 g). This residue was placed on preparative thin layer plates (10, 1000μ), and eluted with CH2Cl2 to give the title compound (0.82 g).

Step 4: Under argon, the products of step 3 (0.270 g; 0.95 mmol) and step 4A (0.571 g; 2.9 mmol) in CH2Cl2 (25 ml) were stirred at RT for 30 min. Na(OAc)3BH (0.506 g; 3.4 mmol) was added. After 19 h, the reaction mixture was quenched with dilute NaOH. The aqueous layer was washed with CH2Cl2 (3×). The combined CH2Cl2 solution was washed with H2O (3×) and then brine. The dried (MgSO4) CH2Cl2 solution was concentrated to −50 ml. Amberlyst 15 (4.5 meq/g: 2.4 g; 11.025 mmeq) was added. After 19 h, additional Amberlyst 15 (2.3 g) was added. After 7 h, the resin was washed with CH2Cl2 (5×), THF (5×), THF:H2O (5×), H2O (5×), CH3OH (5×) and CH2Cl2 (5×). The resin was eluted with 2M NH3 in CH3OH (300 ml) (3×), followed by concentration in vacuo to give an amber oil (0.215 g). The crude material was placed on preparative thin layer plates (4, 1000μ), and eluted with CH2Cl2:2M NH3 in CH3OH (9:1) to give an amber oil (0.125 g, 36%).

Step 5: Using the appropriate carboxylic acid in the procedure of Example 9, step 5, the following compounds were prepared:





LCMS found M+H=531; HPLC* Retention time 5.52 min.





LCMS found M+H=516; HPLC* Retention time 5.66 min.

*HPLC: VYDAC 218TP5405 column; gradient 5-95% B over 10 min hold 2 min; Soln A 0.1% TFA/H2O, Soln B 0.1% TFA/CH3CN at 245 nm.

Using a similar procedure wherein the starting piperazine does not have the methyl substituent, the following compound was prepared:




Example 10




Step 1: A solution of 4-N—BOC-2(S)-methyl piperazine (0.4 g; 2 mmol), p-iodobenzaldehyde (0.46 g; 2 mmol) and NaBH(OAc)3 (0.65 g; 3 mmol) in 6 ml of CH2Cl2 was heated at gentle reflux for 14 h. The contents were cooled, diluted with 30 ml of CH2Cl2 and washed with 1N NaOH solution, water and brine to isolate an yellow oil (0.8 g). FSGC (25% EtOAc-hexane) afforded the desired product (0.66 g; 79%) as a colorless film. TLC Rf=0.6 in 25% EtOAc-hexane

The BOC protecting group was removed from the product (0.66 g; 1.58 mmol) by treatment with TFA (1 ml) in CH2Cl2 (2 ml). Following standard work up, the mono-alkylated piperazine (0.5 g; 100%) was obtained as a colorless gum.
Step 2: NaBH(OAc)3 (0.63 g; 3 mmol) and two drops of AcOH were added to a solution of the product of step 1 (0.5 g; 1.58 mmol) and N—BOC-piperidinone (0.6 g; 3 mmol) in 5 ml of CH2Cl2 and the resulting solution was stirred at ambient temperature for 16 h. After the usual work up and FSGC, the desired product (0.6 g; 76%) was obtained as a colorless oil. TLC Rf=0.4 in 25% acetone-CH2Cl2.
The free piperidine (0.38 g; 79%) was prepared from the N—BOC protected compound (0.6 g; 1.2 mmol) by treatment with TFA (2 ml) in CH2Cl2 (5 ml).
Compound 10A: The coupling of 6-chloro anthranilic acid (0.065 g; 0.38 mmol) with the product of step 2 (0.127 g; 0.32 mmol) in the presence of DEC (0.092 g; 0.48 mmol), HOBT (0.065 g; 0.48 mmol) and diisopropylethyl amine (0.1 ml), followed by product isolation, were carried out as described previously. This procedure furnished the compound 10A (0.13 g; 73%) as a colorless film. TLC Rf=0.5/0.45 for a pair of rotomers in 2% CH3OH—CH2Cl2.
The HCl salt of the title compound was prepared in the usual manner. Mp: 198-202° C.; HRMS (MH+)=553.1231.
Compound 10B: Coupling the product of step 2 with 6-methyl anthranilic acid gave compound 10B (HCl salt) in 73% yield. Mp: 197-200° C.; HRMS (MH+)=533.1774.
Compound 10C: 2,6-Dimethyl benzoic acid was coupled to the product of step 2 to obtain the amide 10C(HCl salt) in 50% yield. Mp: 202-205° C.; HRMS (MH+)=532.1826.
Example 11




Step 1: (S)-Methylbenzylamine (27 ml, 0.2 mol) in CH2Cl2 (50 ml) was dropped into ice-cold trifluoroacetic anhydride (40 ml) in CH2Cl2 (200 ml) within 15 min. The mixture was stirred at RT for 1 h, then cooled in an ice water bath, iodine was added (27 g, 0.106 mol) and then [bis(trifluoro-acetoxy)iodo]-benzene (25 g, 0.058 mol). After being stirred at RT overnight in the dark, more [bis(trifluoroacetoxy)iodo]benzene (24 g, 0.056 mol) was added and the mixture was stirred at RT for one more day. The mixture was diluted with CH2Cl2 (500 ml) and ice-cold Na2SO3 (10% aqueous, 500 ml) and stirred for 0.5 h. The organic layer was separated and washed with NaHCO3, filtered through a short silica gel column and washed with CH2Cl2 (500 ml). After CH2Cl2 was evaporated, Et2O (125 ml) was added and the mixture stirred for 10 min. Hexanes (600 ml) was added gradually to the Et2O solution and the mixture was stirred for 0.5 h. The precipitate was collected and washed with hexanes. The white solid was dried at RT and iodo compound (36.5 g, 53% yield, Rf=0.7, EtOAc/hexanes, 1:3) was obtained.

Step 2: The product of step 1 (11.2 g, 0.033 mol) was dissolved in CH3OH (200 ml) and NaOH (15 g, 0.375 mol) in water (100 ml) was added dropwise. The mixture was stirred at RT for 2.5 h. After the CH3OH was evaporated, the aqueous layer was extracted with Et2O (3×100 ml) and the combined organic portion was washed with brine, dried over Na2SO4, filtered and concentrated to give a free amine.

Methyl-R-lactate (4.08 g, 0.039 mol) was dissolved in CH2Cl2 (40 ml) and the mixture was stirred and cooled in acetone-CO2 to −78° C. under N2 atmosphere. Trifluoromethane sulfonic anhydride (10.2 g, 0.036 mmol) and then 2,6-lutidine (6.27 g, 0.059 mol) were added and the mixture was stirred for 5 min at −78° C. The mixture was warmed to RT and stirred for 30 min. More CH2Cl2 was added to the mixture and the solution was washed with 2N HCl. The freshly prepared amine from above was added to the triflate solution followed by K2CO3 (18 g, 0.132 mol) in water (20 ml). The mixture was stirred at RT overnight. Extractive work-up with CH2Cl2 followed by silica gel column chromatography gave a secondary amine (8.27 g, 75% yield, Rf=0.65, hexanes/EtOAc, 3:1) as a yellow syrup.
Step 3: The amine of step 2 (17.3 g, 0.052 mol) was dissolved in dichloroethane (100 ml) and ClCH2COCl (117.2 g, 82 ml, 1.04 mol). The mixture was stirred under reflux condition for 3 h. Both the solvent and ClCH2COCl were removed under vacuum. The remaining yellow syrup was dissolved in DMSO (40 ml) at 0° C. and NaI (5.2 g, 0.035 mol) and NH4OH (56 ml, 1.04 mol) were added. The reaction mixture was stirred 0° C. for 30 min., warmed up to RT and stirred overnight. Water (100 ml) was added to the mixture and the precipitate was filtered and washed with water. The white solid obtained was dried in air to give the diketopiperazine (14.3 g, 77% yield, Rf=0.56, hexanes/EtOAc, 3:1).

Step 4: The diketopiperazine of step 3 (14.3 g, 0.04 mol) was dissolved in dimethoxy ethane (200 ml) and NaBH4 (15.1 g, 0.4 mol) and BF3.OEt2 (34 g, 29.5 ml, 0.24 mol) were added to the solution. The mixture was stirred under reflux conditions for 3 h and then cooled to about 0° C. on a ice bath. CH3OH (500 ml) and then concentrated HCl (300 ml) were added slowly to the mixture. The solution was stirred for 20 min. at RT and then under reflux conditions for 45 min. The mixture was concentrated and NaOH was added until the pH was more than 10. Extractive work up with EtOAc gave the desired piperazine as a yellow syrup (12.9 g, 98% yield).

Step 5: The product of step 4 (1.9 g, 5.79 mmol), N—BOC-4-piperidone (5.73 g, 28.8 mmol), NaBH(OAc)3 (6.1 g, 28.8 mmol) and 2M AcOH (5.76 ml, 11.52 mmol) were combined in CH2Cl2 (150 ml) and the mixture was stirred overnight. After the solvent was removed, NaOH (3N) was added and extractive work up with EtOAc followed by silica gel chromatography afforded pure piperazino-piperidine (2.21 g, 75% yield, Rf=0.18, hexanes/EtOAc, 1:1) as a syrup.

Step 6: The product of step 5 (1.9 g, 3.7 mmol) was dissolved in CH2Cl2 (10 ml) and TFA (10 ml) was added. The mixture was stirred at RT for 2 h. After the removal of the solvent and TFA under reduced pressure, NaOH solution (3N) was added to the remaining syrup and extractive work up with EtOAc gave the free piperazino-piperidine (1.3 g, 85% yield) as a yellow syrup. To a solution of the free piperazino-piperidine (200 mg, 0.484 mmol) in CH2Cl2 (2 ml) were added 2,6-dimethylbenzoic acid (150 mg, 0.99 mmol), DEC (191 mg, 0.99 mmol) and HOBT (135 mg, 0.99 mmol). The mixture was stirred at RT overnight and then the solvent was removed under reduced pressure. NaOH solution (3N) was added to the remaining syrup and extractive work up with EtOAc followed by column chromatography afforded the title compound (210 mg, 80% yield, Rf=0.37, CH2Cl2/CH3OH, 20:1). HRMS (as the HCl) calcd for C27H37N3OI (M+H+) 546.1981, found 546.1965. Mp: 190° C. (dec.).

Using a similar procedure, compounds of the formula




were prepared, wherein R9 and R10 are as defined in the table:





Example 12














Ex
R9
R10
Mp (° C.)
HRMS







11A
—CH3
—NH2
198 (dec.)
547.1928


11B
—Cl
—NH2
203 (dec.)
567.1395


11C
—OH
—OH
200 (dec.)
550.1555


11D
—OCH3
—OCH3
200 (dec.)
578.1860









Step 1: To the solution of the product of Example 11, step 4 (1.4 g, 4.2 mmol) and 1-tert-butoxycarbonyl-4-piperidone (0.93 g, 4.67 mmol) in CH2Cl2 was added Ti(OiPr)4 (1.19 g, 4.2 mmol) and the mixture was stirred at RT overnight. 1M Et2AlCN (5.04 ml, 5.04 mmol) was added, the mixture was stirred overnight at RT and the solvent was evaporated. Saturated NaHCO3 was added to the residue and extractive work up with EtOAc gave the Strecker amine as a yellow syrup. The syrup was dissolved in THF (40 ml) and 3M CH3MgBr (7 ml, 21 mmol) was added to the solution. The mixture was stirred at RT overnight, then cooled to 0° C. and saturated NH4Cl and water was added. Extractive work up with EtOAc followed by silica gel chromatography gave the piperazino-piperidine product (1.78 g, 81% yield, Rf=0.52, hexanes/EtOAc, 2:1).

Step 2: Treat the product of step 1 in the manner described in Example 11, Step 6, to obtain the title compound. Mp. 190° C. (dec.); HRMS (as the HCl salt): found 560.2145.

Using a similar procedure, compounds of the formula




were prepared, wherein R2 is as defined in the table:

















Ex
R2
mp (° C.)
HRMS









12A




145 (dec.)
581.1537



 



12B




150 (dec.)
561.2083



 



12C




208 (dec.)
561.2096



 



12D




206 (dec.)
562.1944



 



12E




190 (dec.)
577.2029



 



12F




245 (dec.)
601.1006



 



12G




218 (dec.)
577.2029



 



12H




195 (dec.)
617.0945



 



12I




116 (dec.)
562.2048










Example 13




Step 1: To a solution of the N—BOC protected product of Example 11, step 4 (250 mg, 0.581 mmol) in DMF (2.5 ml), CuCl (1 g, 10.1 mmol) was added. The suspension was stirred under N2 at 110° C. for 24 h. After the mixture was cooled to RT, NH4OH was added and the solution gradually turned bright blue. Extractive work up with EtOAc gave a mixture of the chloro-substituted piperazine and its BOC derivative. After treating the mixture with TFA (5 ml) in CH2Cl2 (2 ml) for 2 h, the solvent was evaporated and NaOH (3N) was added. Extractive work up with EtOAc afforded the pure piperazine (110 mg, 79%) as a yellow syrup.

Step 2: The product of step 1 was treated in a manner similar to Example 11, steps 5 and 6, to obtain the title compound. Mp. 180° C. (dec.); HRMS (as the HCl salt): found 454.2617.

Using a similar procedure, compounds of the formula




were prepared, wherein R9 and R10 are as defined in the table:















Ex
R9
R10
Mp (° C.)
HRMS







13A
—CH3
—NH2
200 (dec.)
455.2577


13B
—Cl
—NH2
200 (dec.)
475.2023


13C
—Cl
—Cl
187 (dec.)
494.1536









Using the product of step 1 in the procedure of Example 12, compounds of the formula




were prepared, wherein R2 is as defined in the table:

















Ex
R2
Mp (° C.)
HRMS









13D




197 (dec.)
468.2779



 



13E




205 (dec.)
489.2184



 



13F




210 (dec.)
469.2734



 



13G




195 (dec.)
470.2689



 



13H




260 (dec.)
509.1634



 



13I




200 (dec.)
485.2688










Example 14




Step 1: To a solution of the N—BOC protected product of Example 11, step 4 (5 g, 0.012 mol) in DMF (20 ml), CuCN (20.8 g, 0.23 mol) was added. The suspension was stirred under N2 at 110° C. for 22 h. After the mixture was cooled to RT, NH4OH was added and the solution gradually turned bright blue. Extractive work up with EtOAc followed by silica gel column chromatography gave the cyano derivative (2.29 g, 60% yield, Rf=0.5, hexanes/EtOAc, 4:1), the carboxamide derivative (0.95 g, 23.6% yield, Rf=0.2, CH2Cl2/CH3OH, 10:1) and the unsubstituted derivative (85 mg, 2.4% yield, Rf=0.75, hexanes/EtOAc, 2:1).

Step 2: The BOC group on the cyano compound of step 1 was first removed under acidic conditions and the resultant amine was converted to the title compound following the procedure of Example 11, steps 5 and 6. HRMS (as the HCl salt): found 445.4970.

Example 15




Step 1: To a solution of the N—BOC protected product of Example 11, step 4 (1.4 g, 3.26 mmol) and CuCl (1.61 g, 16.3 mmol) in CH3OH at 0° C. was added NaBH4 (3.69 g, 97.6 mmol) slowly. A black precipitate was formed. The mixture was warmed to RT and stirred overnight. The precipitate was removed by celite filtration and CH3OH was removed under vacuum. Extractive work up with EtOAc afforded the desired compound (1 g, 100% yield, Rf=0.55, hexanes/EtOAc, 5:1) as a syrup.

Step 2: The BOC group on the product of step 1 was removed under acidic conditions and the resultant amine was converted to the title compound following the procedure of Example 11, steps 5 and 6.

Mp. 195° C.; HRMS (as the HCl salt): found 420.3016.
Using a similar procedure, the following compound is prepared:




HRMS (as the HCl salt): found 441.2426

Example 16




Step 1: To a solution of the N—BOC protected product of Example 11, step 4 (2.5 g, 5.8 mmol) in benzene were added phenyl boric acid (1.68 g, 13.8 mmol), 2M Na2CO3 (14 ml) and tetrakis(tri-phenyl phosphine) palladium (0.67 g, 0.58 mmol). The mixture was stirred under reflux overnight. Extractive work up with EtOAc followed by silica gel column chromatography gave the phenyl derivative (1.37 g, 62% yield, Rf=0.5, hexane/EtOAc, 5:1) as a syrup.

Step 2: The BOC group on the product of step 1 was removed under acidic conditions and the resultant amine was converted to the title compound following the procedure of Example 11, steps 5 and 6.

Mp. 190° C.; HRMS (as the HCl salt): found 496.3319.
Using a similar procedure, compounds of the formula




were prepared, wherein R2 is as defined in the table:















Sch
Ex
R2
Mp (° C.)
HRMS







223254
16A




190 (dec.)
517.2754


 


223255
16B




65-70*
497.3287


 


2?5666
16C




190 (dec.)
498.3225





*free base






Example 17




Step 1: The N—BOC protected product of Example 11, step 4 (800 mg, 1.88 mmol) was dissolved in dry THF and the temperature was brought to −78° C. under N2. Butyl lithium (2.5 M solution, 0.832 ml, 2 mmol) was added and the mixture was stirred at −78° C. for 10 min. The solution then was dropped into p-chlorobenzyl aldehyde (234 mg, 2.07 mmol) in THF at −78° C. The mixture was stirred for 30 min. at −78° C., then gradually warmed up to RT. Saturated NH4Cl was added to the mixture and extractive work up with EtOAc followed by silica gel column chromatography gave the desired alcohol (30 mg, 3.6% yield, Rf=0.5, hexanes/EtOAc, 2:1) as a yellow syrup.

Step 2: A solution of alcohol of step 1 (40 mg, 0.090 mmol), triethylsilane (52 mg, 0.45 mmol) and TFA (5 ml) in CH2Cl2 (5 ml) was stirred under reflux conditions for 2 h. After CH2Cl2, triethylsilane and TFA were removed under reduced pressure, NaOH solution (3N) was added to the remaining syrup. Extractive work up with EtOAc afforded the chlorobenzyl derivative (20 mg, 68% yield) as a yellow syrup.

Step 3: The product of step 2 was converted to the title compound following the procedure of Example 11, steps 5 and 6. Mp. 170° C. (dec.); HRMS (as the HCl salt): found 544.3101.

Example 18




Step 1: To a solution of the N—BOC protected 4-piperidinyl derivative of the cyano compound of Example 14, step 1 (510 mg, 1.24 mmol) in Et2O (4 ml) was added 3M CH3MgBr (4 ml) in a dropwise manner. The mixture was stirred under reflux overnight. After the solution was cooled on ice-bath, 12N HCl (4 ml) was added and the mixture was stirred on a steam bath for 2 h. The solution was cooled to RT and solid NaOH pellets were added until the pH was more than 10. Extractive work up with EtOAc/CH3OH (3:1) afforded the desired methyl ketone (249 mg, 61% yield) as a syrup.

Step 2: The product of step 1 was treated according to the standard DEC peptide coupling procedures of Example 11, step 6, to obtain the title compound. Mp. 210° C.; HRMS (as the HCl salt): found 483.2522.

Using a similar procedure, the following compound is prepared:




Mp. 210° C. (dec.); HRMS (as the HCl salt): found 463.3088
Example 19




Step 1: To a solution of the product of Example 22 (140 mg, 0.29 mmol) in CH3OH (10 ml) and EtOH (1 ml) were added NH2OCH3—HCl (738 mg, 8.84 mmol) and NaOAc (725 mg, 8.84 mmol). The suspension was stirred at 40° C. overnight, the solvents were evaporated and water was added to the residue. Extractive work up with EtOAc followed by silica gel chromatography generated the title compound (99 mg, 68% yield, Rf=0.38, CH2Cl2/CH3OH, 20:1). HRMS (as the tartrate) calc'd. for C31H45N4O2 (M+H+) 505.3543; found 505.3542.

Using a similar procedure, compounds of the formula




were prepared, wherein R8, R6 and R2 are as defined in the table:
















Ex
R8
R6
R2
mp (° C.)
HRMS







19A




H




194 (dec.)
512.2785


 


19B




H




150 (dec.)
492.3344


 


19C




H




—
506.3494


 


19D




—CH3




180 (dec.)
508.3296


 


19E




—CH3




195 (dec.)
493.3291









Example 20




Dissolve the free piperazino-piperidine of Example 11, step 6 (1.7 g, 3.3 mmol) in CHCl3 (30 ml; =Stock solution A). Add 250 ul of stock solution A (0.027 mmol) to a slurry of 0.15 g ( ̃0.14 mmol) of resin bound carbodiimide (prepared by reacting Argopore-CI resin with 1-(3-dimethyl-aminopropyl)3-ethyl carbodiimide in DMF at 100° C. in DMF (1.5 ml) in a polyethylene SPE cartridge. To this mixture add 75 ul of a 1 M solution of 5-methyl-3-phenylisoxazole-4-carboxylic acid in DMF (0.075 mmol), and HOBT (24 ul of a 1M solution in DMF). Shake this mixture for 14 h, filter and add 0.1 g of Amberlyst-15 resin (0.47 mmol) to the filtrate. Shake for 1 to 2 h, filter and wash the resin twice with each of the following solvents THF, CH2Cl2 and CH3OH, then wash with THF and CH2Cl2. Treat the resin with 2M NH3 in CH3OH (1 time for 30 min, and 1 time for 5 min). Combine and concentrate the filtrates under reduced pressure to afford the title compound. LCMS found MH+=599.1 (calculated MW 598); TLC Rf=0.74 (CH2Cl2/CH3OH/NH4OH (95/5/0.5)).
Using the procedure above with the appropriate carboxylic acids gave the following compounds




wherein R2 is as defined in the table:
















LCMS
TLC


Ex.
R2
results
Rf values







20A




MH+ = 600.1 Rt = 6.56 min.
0.92


 


20B




MH+ = 601.1 Rt = 5.69 min.
0.63


 


20C




MH+ = 560.1 Rt = 5.77 min.
0.60


 


20D




MH+ = 588.1 Rt = 6.61 min.
0.66


 


20E




MH+ = 604.1 Rt = 5.60 min.
0.87


 


20F




MH+ = 658.2 Rt = 5.69 min.
0.86


 


20G




MH+ = 606.1 Rt = 6.17 min.
0.43


 


20H




MH+ = 568.1 Rt = 5.67 min.
0.57


 


20I




MH+ = 586.1 Rt = 6.02 min.
0.63


 


20J




MH+ = 558.1 Rt = 5.35 min.
0.33


 


20K




MH+ = 546.1 Rt = 5.37 min.
0.52









Example 21




Step 1: The BOC group on the cyano compound of Example 14, step 1, was first removed under acidic conditions and the resulting amine (1.59 g, 6.96 mmol), 1-tert-butoxycarbonyl-4-piperidone (1.66 g, 8.35 mmol) and Ti(OiPr)4 (2.18 g, 7.66 mmol) in CH2Cl2 were stirred at RT overnight. 1M Et2AlCN (8.35 ml, 8.35 mmol) was added, the mixture was stirred overnight at RT and the solvent was evaporated. Saturated NaHCO3 was added to the residue and extractive work up with EtOAc followed by column chromatography gave the Strecker amine as a yellow syrup (1.76 g, 58% yield, Rf=0.70, Hexanes/EtOAc, 2:1).

Step 2: The amine of Step 1 (200 mg, 0.46 mmol) was dissolved in anhydrous THF (2 ml) and 3M CH3MgBr (0.76 ml, 2.29 mmol) was added dropwise. The mixture was stirred at RT overnight and then cooled to 0° C. Saturated NH4Cl (10 ml) was added and a precipitate appeared. Water (40 ml) was added and the precipitate disappeared. Extractive work up with EtOAc followed by column chromatography gave the desired ipso-methyl derivative (169 mg, 86% yield, Rf=0.53, Hexanes/EtOAc, 2:1).

Step 3: The product of step 2 was treated in the manner described in Example 11, Step 6, to obtain the title compound. Dec. 198° C.; HRMS (as the HCl salt): found 460.3079.

Using a similar procedure, compounds of the formula




were prepared, wherein R2 is as defined in the table:

















Ex
R2
Mp (° C.)
HRMS









21A




205 (dec.)
480.2532



 



21B




 65-75*
476.3033



 



21C




250 (dec.)
500.1992



 



21D




195 (dec.)
461.3019







*Mp for the free amine






Example 22




Step 1: The Strecker amine from Example 21, step 1 (380 mg, 0.87 mmol) was treated with CH3MgBr (2.9 ml, 8.7 mmol) in Et2O (5 ml) under reflux conditions overnight. The mixture was cooled on ice and water (5 ml) was added dropwise. 12N HCl (6 ml) was added and the mixture was stirred on a steam bath for 2 h. After the mixture was cooled on ice, NaOH was added until the pH of the solution was above 10. Extractive work up with EtOAc afforded a free amine as a syrup (307 mg, 100% yield).

Step 2: The product of step 1 was converted to the title compound following the peptide coupling procedure described in Example 11, step 6. Mp. 80-85° C.; HRMS found 476.3271.

Using a similar procedure, compounds of the formula




were prepared, wherein R2 is as defined in the table:

















Ex.
R2
Mp (° C.)
HRMS









22A




195 (dec.)
493.3172



 



22B




200 (dec.)
478.3178










Example 23




Steps 1-3:





Step 1: Ethyl diacetoacetate (93.4 g), Cs2CO3 (185 g) and CH3CN (550 ml) were mixed together, using an overhead mechanical stirrer. CH3CN (50 ml) was added and the resulting mixture was cooled to 0° C. Methyl trifluoromethane sulfonate (88.6 g) was added dropwise and after addition, the cooling bath was removed. The mixture was stirred for 1 h at RT, filtered, and the salts were washed with Et2O (2×50 ml). The organic extracts were combined and Et2O (300 ml) was added. The resulting mixture was filtered, the filter cake was washed with Et2O (2×100 ml), the Et2O extracts were combined and evaporated to half volume. The solution was cooled in an ice bath and washed once with cooled (0° C.) 2 N NaOH (pH=11). The Et2O layer was dried over MgSO4, filtered and evaporated to give the desired product as a yellow liquid (64.7 g) in 65% yield, which was used directly in the next step.

Step 2: The product of step 1 (64.2 g), sodium ethoxide in ethanol (commercial solution; 21 wt %; 113 g) ethanol (587 ml) and formamidine acetate (36.2 g) were mixed together at RT. After refluxing for 4 h, the mixture was cooled to RT, the resulting precipitate was filtered off and the ethanol was removed under vacuum. The resulting liquid was partitioned between water and CH2Cl2 and the aqueous layer was extracted with CH2Cl2 (3×150 ml). The CH2Cl2 extracts were dried over MgSO4, filtered and evaporated to give a dark crude liquid (50.7 g) which was purified by silica gel chromatography (980 g; 4:1 hexanes:EtOAc as eluant). After evaporation of the appropriate fractions, the desired product (28.5 g) was isolated in 46% yield and used directly in the next step.

Step 3: The product of step 2 (28.1 g), NaOH (6.72 g), water (65 ml) and EtOH (130 ml) were mixed together at RT and heated at reflux for 1 h. The resulting solution was cooled to RT and the volatile materials were removed in vacuo until a thick paste resulted. Water (20 ml) was added, the mixture was cooled to 0° C. and conc. HCl (14.3 ml) was added dropwise with stirring. The resulting white precipitate was collected by filtration, washed with ice water (2×10 ml) and air dried with suction for 30 min. The resulting white solid was treated with toluene (2×20 ml), the solvent was removed in vacuo at 50° C. and then dried under vacuum (1 mm Hg) for 18 h. The desired product (14.9 g) was isolated as a white solid in 63% yield, mp: 176-178° C. Elemental analysis of C7H8N2O2: calc'd C 55.26%, H 5.30%, N 18.41%; found: C 55.13%, H 5.44%, N 18.18%.

A second crop of product was isolated by evaporation of the aqueous filtrate (from above) to dryness and addition of water (20 ml). The resulting mixture was stirred at RT for 5 min, cooled in an ice bath and the precipitate formed was collected by filtration. The resulting solid was washed with ice water (2×5 ml) and dried as described above to give the product (4.68 g) as a cream colored solid to give a combined yield of 83%.
Step 4: The product of Example 4, step 6 (trihydrochloride form; 5.4 g), DMF (11.3 ml), HOBt (3.07 g), diisopropyl ethyl amine (12.3 ml) and the product of step 3 (3.45 g) were mixed together and DEC (4.35 g) was added in portions over 15 min. The resulting mixture was heated at 45° C. for 18 h, cooled to RT, diluted with EtOAc (80 ml) and washed with 2 N NaOH (25 ml). The aqueous layer was extracted with EtOAc (3×25 ml), the organic extracts were combined, washed with brine, dried over Na2SO4, filtered and evaporated. The resulting crude oil was purified by silica gel chromatography (170 g; 76:19:5 hexanes:EtOAc:Et3N as eluant). After evaporation of the appropriate fractions, the free base form of the title compound (5.21 g) was isolated as a light colored foam in 91% yield.

Step 5: To a cooled (0° C.) solution of the free base of step 4 (2.00 g) and EtOAc (20 ml) was added HCl (3.0 ml of a 4.0 M solution in 1,4-dioxane). The resulting mixture was warmed to RT, diluted with Et2O (20 ml), filtered, washed with Et2O (2×20 ml), air dried with suction for 10 min and then under vacuum (1 mm Hg) at 90° C. for 5 h to give the title compound (2.30 g) as a white solid in 97% yield. mp: 159-162° C. Elemental analysis of C27H36N5OF3.2HCl.0.5H2O: calc'd: C 55.38%, H 6.71%, N 11.96%, Cl 12.11%; found: C 55.19%, H 6.69%, N 11.75%, Cl 11.45%.

Additional pyrimidine derivative-compounds were made using similar procedures:




Steps 1-2:





Step 1: The product of Example 23, step 1 was treated in the same manner as in Example 23, step 2, substituting acetamidine hydrochloride (2.03 g) for formamidine acetate. The amounts of the reagents were: product of Example 23, step 1 (4.0 g), ethanol (20 ml) and sodium ethoxide in ethanol (commercial solution; 21 wt %; 8.03 g). After extraction and purification as described above, the product was isolated (1.7 g) as a colorless liquid in 41% yield, which was used directly in the next step.

Step 2: The product of step 1 (1.7 g) was treated in the same manner as Example 23, step 3, using ethanol (5 ml), water (5 ml) and NaOH (1.0 g). After extraction and purification as described above, the product was isolated (0.12 g) as a white solid in 8% yield, which was used directly in the next step.

Step 3: The product of Example 4, step 6 (0.05 g), and the product of step 2 (immediately above) (0.028 g) were subjected to the same reaction conditions as in Example 23, step 4, using HOBt (20 mg), DEC (45 mg), diisopropyl ethylamine (40 mg) and DMF (1.5 ml). After extraction and purification as described above, the product was converted to its HCl salt using the procedure outlined for Example 23, step 5 to give the title compound (77 mg) as a white solid in 97% yield over the two steps. mp: 185-190° C.





Steps 1-2:





Step 1: The product of Example 23, step 1 was treated in the same as in Example 23, step 2, substituting benzamidine hydrochloride (3.35 g) for formamidine acetate. The amounts of the reagents were: product of Example 23, step 1 (4.0 g), ethanol (20 ml) and sodium ethoxide in ethanol (commercial solution; 21 wt %; 8.03 g). After extraction and purification as described above, the product was isolated (4.5 g) as a liquid in 82% yield which was used directly in the next step.

Step 2: The product of step 1 (4.5 g) was treated in the same manner as Example 23, step 3, using ethanol (10 ml), water (10 ml) and NaOH (2.0 g). After extraction and purification as described above, the product was isolated (3.0 g) as a white solid in 77% yield which was used directly in the next step.

Step 3: The product of Example 4, step 6 (75 mg), and the product of step 2 (immediately above) (39 mg) were subjected to the same reaction conditions as in Example 23, step 4, using HOBt (35 mg), DEC (53 mg), diisopropyl ethylamine (100 mg) and DMF (2 ml). After extraction and purification as described above, the product was converted to its HCl salt using the procedure outlined for Example 23, step 5 to give the title compound (98 mg) as a white solid in 96% yield over the two steps.

mp: 250-253° C.




Steps 1-2:





Step 1: The product of Example 23, step 2 (528 mg) was dissolved in CH2Cl2 (5.0 ml) and meta-chloroperbenzoic acid (mCPBA) (600 mg) was added in three portions at RT. The resulting mixture was stirred at RT for 24 h and CH2Cl2 (2 ml) and mCPBA (200 mg) were added. After 3 h, the mixture was poured onto a silica gel column (40 g) and eluted with 1:1 hexanes:EtOAc and then 10:1 CH2Cl2:CH3OH. After evaporation of the appropriate fractions, the product was isolated (512 mg) as a waxy white solid in 89% yield, which was used directly in the next step.

Step 2: The product of step 1 was dissolved in CH3OH (1.8 ml) and a solution of 1.0 M Na2CO3 (1.5 ml) was added. After stirring at RT for 36 h, the resulting mixture was evaporated to dryness, toluene (2 ml) was added and the mixture was evaporated to dryness. The resulting crude solid (153 mg) was used directly in the next step without purification.

Step 3: The product of Example 4, step 6 (94 mg), and the product of step 2 (immediately above) (76 mg) were subjected to the same reaction conditions as in Example 23, step 4, using HOBt (92 mg), DEC (130 mg), diisopropyl ethylamine (0.14 ml) and DMF (0.25 ml). After extraction and purification by preparative thin layer chromatography (1000 μM silica plate; 95:5 EtOAc:Et3N eluant), the free base form of the title compound was isolated (52 mg) as a foam in 40% yield. HRMS: calc'd: M′H+: C27H37N5O2F3: 520.2899; measured: 520.2908.

Step 4: The product of step 3 (52 mg) was subjected to the reaction conditions in Example 23, step 5, using EtOAc (1.0 ml) and HCl (4.0 M solution in 1,4-dioxane; 75 μl) to give, after work up, the title compound (44.5 mg) as a white solid in 76% yield. mp: decomposition above 161° C.

Using similar procedures, the compounds of the formula




were also prepared, wherein R8a and R11 are as defined in the table:

















Ex.
R8a
R11
m.p. (° C.)









23D
—CF3
—OH
175-185



23E
—CF3
—OCH3
169-173



23F
—CF3
—NH2
200-210



23G
—CF3
—NHCONHEt
184-190



23H
—CF3
—CF3
83-86



 



23I
—CF3




154-159



 



23J
—CF3
—SCH3
>176 (dec)



23K
—OCF3
—CH3
205-210



23L
—OCF3
Ph
239-242



23M
—OCF3
—OCH3
200-210



23N
—OCF3
—OH
185-191










Example 24
Arylcyclopropylamides




Step 1: To the stannane (0.39 g, 0.95 mmol) in DMF (10 ml) was added the 2-chloro-4-fluoroiodobenzene (0.73 g, 2.86 mmol), CuI (0.19 g, 1.05 mmol) and tetrakis(triphenylphosphine)palladium (0) (0.11 g, 0.095 mmol). The reaction was stirred at RT under N2 for 21 h. The reaction mixture was added to Et2O and the heterogeneous solution filtered through a bed of celite, washing with EtOAc. The filtrate was washed with water and brine and dried (MgSO4). Filtration and evaporation of the solvent in vacuo afforded a residue that was preabsorbed on silica gel. Purification by silica gel chromatography (4% EtOAc/hexane) yielded the arylacrylate (0.19 g, 78%), which was used directly in the next step.

Step 2: To trimethylsulfoxonium iodide (0.18 g, 0.81 mmol) in DMSO (1.6 ml) was added potassium tert-butoxide (0.09 g, 0.81 mmol). The reaction mixture was stirred at RT for 1 h, at which time the arylacrylate (0.19 g, 0.74 mmol) in DMSO (1.6 ml) was added. The reaction mixture was stirred at RT for 5 h and water was added. The mixture was extracted with EtOAc. The combined organic layers were washed with water and brine and dried (MgSO4). Filtration and evaporation of the solvent in vacuo afforded the arylcyclopropyl ester that was used directly by taking up into CH2Cl2 (3 ml) and adding TFA (0.5 ml). The reaction mixture was stirred at RT for 15 h and then concentrated in vacuo to afford the arylcyclopropylcarboxylic acid (0.14 g, 91%-2 steps). Without further purification, the carboxylic acid was coupled to the product of example 8, step 3, using the procedure of Example 8, step 4 to obtain 24A as the HCl salt. HRMS (M+H): found 566.2561.





To the 2-fluorophenylacetonitrile (0.80 g, 5.92 mmol), benzyltriethyl-ammonium chloride (0.03 g, 0.12 mmol), and 1-bromo-2-chloroethane (1.70 g, 11.9 mmol) was added 50% aqueous NaOH (3.5 ml). The reaction was stirred at 45° C. for 21 h and ethylene glycol was added (3 ml). The reaction was then warmed to 100° C. and stirred for 7 h. Upon cooling to RT, the reaction was diluted with water and washed with EtOAc. The aqueous layer was acidified to pH 2-3 with aqueous 6N HCl. The acidified solution was extracted with Et2O. The combined Et2O extracts were washed with water and brine and dried (MgSO4). Filtration and evaporation of the solvent in vacuo afforded a pale yellow solid (1.06 g, 99%). The arylcyclopropyl acid was coupled to the product of example 8, step 3, using the procedure of Example 8, step 4 to obtain 24B as the HCl salt. HRMS (M+H): found 532.2949.
Using similar procedures, the compounds of the formula




were prepared, wherein





is as defined in the table:














Ex.




HRMS (M + H)
m.p. (° C.)







24C




—
240-245


 


24D




—
>225


 


24E




—
172-176


 


24F




—
225-230


 


24G




—
>225


 


24H




544.3151
—


 


24I




592.2150
—


 


24J




532.2956
—


 


24K




539.3003
—


 


24L




558.2949
—


 


24M




572.3107
—


 


24N




582.2910
—


 


24O




582.2910
—


 


24P




520.2609
—


 


24Q




515.2991









Example 25




Step 1:





Cyclopropyl carboxaldehyde (3.4 ml), S-methyl N—BOC piperazine (8.28 g), CH2Cl2 (82 ml) and Ti(OiPr)4 (15.80 ml) were mixed together and stirred at RT for 23 h, then the resulting solution was cooled to 0° C. and Et2AlCN (1.0 M in toluene; 62.1 ml) was added. The solution was stirred for 5 h at RT. A mixture of KF (20 g) and Celite (10 g) was added, followed by cautious addition of EtOAc (120 ml) and water (120 ml). The resulting slurry was stirred for 15 min, filtered, washed with EtOAc (3×35 ml) and the EtOAc layer was removed, washed with brine, dried over Na2SO4, filtered and evaporated to give the desired intermediate (12.0 g) which was used directly in the next step.

Step 2:





To a 0° C. solution of 4-iodobenzotrifluoride (40 g) and THF (52 ml) was added isopropyl magnesium chloride (2.0 M in Et2O; 74 ml). The resulting solution was stirred at RT for 1 h and then added to a 0° C. solution of the product of step 1 (10.0 g) and THF (26 ml) over 10 min. The reaction solution was warmed to RT, stirred overnight and EtOAc (50 ml) was added. After stirring for 10 min, 2 N NaOH (50 ml) was added and the resulting mixture was stirred for 30 min, filtered and the salts were washed with EtOAc (3×20 ml). The combined EtOAc extracts were washed with brine, dried over Na2SO4, filtered and evaporated to give the crude product (28 g) as a gold oil which was chromatographed on silica gel (1 kg), eluting with hexanes:EtOAc (8:1). Two diastereomeric products were collected as a single fraction (15.9 g) and further purified by column chromatography as described above to give intermediate A (Rf=0.47 in 4:1 hexanes:EtOAc; 5.34 g), which was contaminated with an unidentified impurity. (The second diastereomer B (Rf=0.29 in 4:1 hexanes:EtOAc) was also collected.)
Step 3:




To a solution of A from Step 2 (3.96 g) and CH2Cl2 (120 ml) was added DOWEX 50X2-100 ion exchange resin (15 g) and the resulting mixture was shaken for 2.5 h at RT. The resin was filtered off and washed with CH2Cl2 (2×40 ml). The resin was treated with 7 N NH3 in CH3OH (30 ml), the resin was filtered off and this procedure was repeated two times. The CH3OH extracts were combined and evaporated. The resulting oil was treated with toluene:CH2Cl2 (1:1; 15 ml) and evaporated to give the piperazine intermediate (0.80 g) as a clear oil. HRMS: calc'd: M·H+: C16H21N2F3:299.1735; measured: 299.1748.
Step 4:




The product of Step 3 (0.57 g) was treated in the same fashion as Example 8, Step 1, using N—BOC 4-piperidone (0.42 g), CH2Cl2 (3.84 ml), Ti(OiPr)4 (3.39 ml), Et2AlCN (2.88 ml) and CH3MgBr (3.0 M in Et2O; 3.2 ml) to give the desired product (0.78 g) as a clear oil in 82% yield.
Step 5: The product of Step 4 (0.12 g) was treated with AcOH:CH2Cl2 (3:1, v:v; 1.4 ml) followed by BF3.Et2O (0.14 ml). After stirring for 1 h, the resulting solution was diluted with CH2Cl2 (10 ml), cooled to 0° C. and the pH was adjusted to 10 with solid NaOH. Water (2 ml) was added and the CH2Cl2 layer was removed. After further extraction (2×10 ml) with CH2Cl2, the organic layer was washed with water, brine, dried over Na2SO4, filtered and evaporated to give the free piperidine (80 mg) in 81% yield.

Step 6: The product of Step 5 (57 mg) was treated in the same fashion as in Example 8, Step 4, using DMF (0.30 ml), HOBt (41 mg), DEC (57 mg), diisopropyl ethyl amine (0.08 ml) and 4,6-dimethyl 5-pyrimidine carboxylic acid (43 mg); the reaction was stirred at 45° C. for 5 h. Purification of the crude oil was carried out by preparative plate chromatography (silica adsorbent; 2000 μM; 76:19:5 EtOAc:hexanes:Et3N as eluant) to give, after elution of the desired band (1:1 CH2Cl2:MeOH) and concentration of solvent, the title compound (70 mg) as a clear oil in 93% yield. The HCl salt was prepared as described for Example 8, Step 4 (78 mg) in 100% yield. mp: 147-149° C.

Using a similar procedure, the following compound was prepared:




Example 26




Step 1:





The desired compound was prepared in a manner similar to Example 25, Step 1, using p-trifluoromethyl benzaldehyde (20 g) instead of cyclopropyl carboxaldehyde, to give, after work up, a mixture of diastereomers (22.7 g) in 59% yield.
Step 2:




To a −70° C. solution of the product of step 1 (1.9 g) and THF (15 ml) was added NaHMDS (1.0 M in THF; 7.5 ml) followed by benzyl bromide (2 ml). The cooling bath was removed and the resulting solution was stirred for 45 min. Concentrated NH4OH (10 ml) was added and the reaction was stirred for 30 min. The resulting mixture was partitioned between water and CH2Cl2, the CH2Cl2 extracts were removed and evaporated and the crude oil was purified by column chromatography (silica gel; 2:1 hexanes:CH2Cl2; 10:1 to 7:1 hexanes:EtOAc as eluant) to give, after evaporation of the appropriate fractions, a mixture of intermediates (1.92 g) as a yellow foam.
Step 3:




The mixture of Step 2 (1.91 g), CH3CN (35 ml), sodium triacetoxy borohydride (4.0 g) and magnesium bromide etherate (2.25 g) were mixed and stirred at RT for 70 h. Water (25 ml) was added and then, gradually, a solution of Na2CO3 (10 g) in water (50 ml). After extraction with EtOAc (2×50 ml), drying and evaporation of the organic layer, the resulting oil was purified by preparative plate chromatography (5×2000 mM silica plates; 6:1 hexanes:EtOAc as eluant). The less polar band was removed, treated with 1:1 methanol:CH2Cl2, filtered and evaporated to give intermediate A (0.84 g) as a white foam. HRMS: calc'd: M·H+: C25H29O2N2F3:449.2407; measured: 4492416.
Step 4: The product of Step 3 (0.81 g) was treated in the same fashion as in Example 8, Step 3, using TFA (5 ml) and CH2Cl2 (10 ml), to give, after work up, the free piperazine (0.60 g) as a clear gum. HRMS: calc'd: M·H+: C20H23N2F3: 349.1892; measured: 349.1894.

Step 5: The product of Step 4 (0.39 g) was treated in the same fashion as in Example 8, Step 1, using N—BOC 4-piperidone (0.25 g), CH2Cl2 (8 ml), Ti(OiPr)4 (0.40 mg), Et2AlCN (2 ml) and CH3MgBr (3.0 M in Et2O; 1.5 ml) to give the desired BOC-protected piperidinyl intermediate (0.44 g) as a clear oil in 72% yield. HRMS: calc'd: M·H+: C31H42O2N3F3:546.3307; measured: 546.3315.

Step 6: The product of step 5 (0.43 g) was treated in the same fashion as in Example 8, Step 3, using TFA (3 ml), CH2Cl2 (2 ml) and water (0.2 ml) to give, after work up, the free piperidinyl intermediate (0.37 g) as a clear oil.

Step 7: The product of step 6 (50 mg) was treated in the same fashion as in Example 8, Step 4, using CH2Cl2 (3 ml), HOBt (28 mg), DEC (40 mg), diisopropyl ethyl amine (42 mg) and 4,6-dimethyl 5-pyrimidine carboxylic acid (24 mg); the reaction was stirred at RT for 2 days. Using the procedure described in Example 8, Step 4, the HCl salt of the title compound was prepared (59 mg) in 91% yield (from the product of Step 5). M.p: 187-196° C. HRMS: calc'd: M·H+: C33H40ON5F3:580.3263; measured: 580.3263.

Using a similar procedure, compounds of the formula




were prepared, wherein R8a, R3 and R2 are as defined in the table:















Ex.
R8a
R3
R2
Mp (° C.)







26B
—CF3








86-92


 


26C
—CF3








83-90


 


26D
—CF3








195-205


 


26E
—CF3








118-125


 


26F
—OCF3








175-185


 


26G
—OCF3








180-190


 


26H
—OCF3








220-230


 


26I
—OCF3








195-210


 


26J
—OCF3








190-200


 


26K
—OCF3








180-205


 


26L
—OCF3








230-240


 


26M
—OCF3








60-65


 


26N
—OCF3








65-68


 


26O
—OCF3








60-62


 


26P
—CF3








256-258


 


26Q
—CF3








254-256 (dec)


 


26R
—CF3








249-250 (dec)









Example 27




Step 1:





4′-Trifluoromethyl)propiophenone (2.02 g, 0.01 mol) and (S)-2-methyl-CBS-oxazaborolidine (1M in THF) (2.0 ml, 0.002 mol) in THF (10 ml) was cooled in an ice-bath and borane-methyl sulfide complex (2M in THF) (3 ml, 0.006 mol) was added dropwise to the mixture. The mixture was stirred for 30 min at 0° C. and CH3OH was added slowly until no bubbles appeared. The solvents were removed under reduced pressure and HCl solution (1N) was added to the mixture. EtOAc extractive work up followed by silica gel chromatography afforded the alcohol (1.47 g) in 72% yield.
Step 2: A solution of the product of Step 1 (4.32 g, 0.021 mol) and Et3N (5.9 ml, 0.042 mol) in CH2Cl2 (20 ml) was cooled to 0° C. in ice bath and CH3SO2Cl (2.13 ml, 0.028 mol) was added dropwise. The mixture was stirred at 0° C. for 1 h and the ice bath was removed. Water was added to the mixture and CH2Cl2 extractive work up afforded the mesylate (5.99 g) in quantitative yield.

Step 3: The product of Step 2 (5.93 g, 0.021 mol) and 1-tert-butoxy-carbonyl-3S-methyl piperazine (4.2 g, 0.021 mol) were dissolved in anhydrous CH3CN (20 ml) and oven-dry K2CO3 (4.35 g, 0.032 mol) was added to the solution. The mixture was stirred under reflux for 2 days, then diluted with water. EtOAc extractive work up followed by silica gel chromatography gave the desired product (3.16 g) in 39% yield.

Step 4: TFA (10 ml) was added to a solution of the product of Step 3(1.15 g, 2.59 mmol) in CH2Cl2 (5 ml) and the mixture was stirred at RT for 2 h, then concentrated under reduced pressure. NaOH (3N) was added to the residue and extractive work up with EtOAc gave the desired amine in quantitative yield.

Step 5: The product of Step 4 and 1-tert-butoxycarbonyl-4-piperidone (0.94 g, 4.74 mmol) were treated with Ti(OiPr)4, Et2AlCN and CH3MgBr in a manner similar to that described in Example 8, step 1, to obtain the desired product (1.09 g) in 87% yield (from the amine of Step 4).

Step 6: TFA (4 ml) was added to a solution of the product of Step 5 (0.76 mg, 1.57 mmol) in CH2Cl2 (2 ml) and the mixture was stirred at RT for 2 h before it was concentrated under reduced pressure. NaOH (3N) was added to the residue and extractive work up with EtOAc gave the desired amine in quantitative yield.

Step 7: The amine of Step 6 and 4,6-dimethylpyrimidine 5-carboxylic acid (0.36 g, 2.35 mmol), were coupled as described in Example 8, Step 4, to obtain the title compound (0.58 g) in 72% yield. M.p. 160; HRMS (MH+) found: 518.3123.

Using a similar procedure, compounds of the formula




were prepared wherein Z, R3, R6 and R2 are as defined in the table below:

















Ex.
Z
R3
R6
R2
Dec.(0° C.)
HRMS







27A
N
Me
H




185
491.2744


 


27B
N
Me
H




190
506.2729


 


27C
N
Me
Me




190
505.2898


 


27D
N
Me
Me




200
520.2902


 


27E
CH
Et
Me




197
533.3097


 


27F
CH
Et
Me




215
532.3147


 


27G
CH
Et
Me




230
627.3145


 


27H
CH
Et
Me




210
602.3678


 


27I
CH
Et
Me




215
531.3305


 


27J
CH
Et
Me




215
593.3470


 


27K
CH
Et
Me




195
609.3424


 


27L
CH
Et
Me




170
745.2308


 


27M
N
n-Pr
Me




204
533.3207


 


27N
N
n-Pr
Me




210
617.3798


 


27O
N
n-Pr
Me




202
531.3304


 


27P
N
n-Pr
Me




165
543.3311


 


27Q
N
n-Pr
Me




225
584.3205


 


27R
N
n-Pr
Me




195
548.3217









Using similar procedures, the following compounds were also prepared:




Example 28




Steps 1-4:





Step 1: The cyano amine was prepared from p-trifluoromethyl benzaldehyde and 2(S)-methyl-4-(tert-butoxycarbonyl) piperazine exactly as described in Example 6, Step 1.

Step 2: A solution of the cyano amine 2 (2.5 g; 6.53 mmol) in 30 ml of dry THF was placed under a blanket of N2 and cooled to −78° C. This solution was treated with a solution of sodium hexa-methyl disilazide in THF (1M; 26 ml) followed after 5 min with neat allyl bromide (6 ml). Upon removal of the bath and letting the reaction mixture warm to RT ( ̃1 h), it changed from a yellow solution to dark reddish brown solution. The reaction was quenched with saturated NH4Cl solution and the product extracted with EtOAc, washed with water, brine and dried. Concentration in vacuo afforded a brown semi solid. FSGC of this material using 25% Et2O in hexane as eluant gave 2.5 grams (92%) of the desired product as an amber gum (TLC Rf=0.65, 0.6 for two overlapping spots).

Step 3: A solution of the product of Step 2 (2.4 g) in CH3OH was treated with 10% Pd/C (0.2 g) and placed under a balloon of H2 gas. After stirring at RT for 4 h, the catalyst was removed via filtration through celite. Concentration of the filtrate yielded an amber gum.

The α-propyl nitrile obtained above was dissolved in CH3CN (12 ml). Magnesium bromide etherate (2.1 g; 8.14 mmol) and sodium triacetoxy borohydride (3.44 g; 16.2 mmol) were added and the reaction mixture was stirred at RT overnight. The reaction was quenched with water and rendered basic with saturated NaHCO3. The organic products were extracted with EtOAc and processed to obtain  ̃2 g of crude material. FSGC (10-25% Et2O in hexane) served to isolate two diasteromeric products (1.7 g total; 79% for two steps):
(S,S)-Diastereomer (A): TLC Rf=0.6 (25% Et2O-Hexane). 0.9 g of a colorless gum.
(R,S)-Diastereomer (B): TLC Rf=0.5 (25% Et2O-Hexane). 0.8 g of a colorless gum.
Step 4: Removal of the BOC-protecting group from the intermediate A was accomplished by treatment with TFA in CH2Cl2. The isolated free piperazine (0.68 g; 2.3 mmol), N-(tert-butoxycarbonyl)-4-piperidinone (0.45 g; 2.3 mmol) and Ti(OiPr)4 (0.7 mL; 2.5 mmol) were dissolved in 10 ml of CH2Cl2 and stirred overnight. Et2AlCN (1M in toluene; 2.7 ml) was introduced into the reaction mixture and the resultant solution was stirred for a day. The reaction was diluted with EtOAc and quenched with water. Celite was added to aid in the filtration of titanium and aluminum salts. The biphasic filtrate was washed with water, brine and dried. Concentration in vacuo yielded 1.1 g of a yellow gum (TLC Rf=0.55 in 25% EtOAc-hexane).
The resultant ipso-cyano compound was dissolved in dry THF (8 ml) and treated with a solution of CH3MgBr (3M in Et2O; 6 ml) and stirred overnight at RT. The reaction flask was placed in a cold water bath and carefully quenched with saturated NH4Cl solution. The organic product was extracted with EtOAc and washed with water and brine. Concentration to a crude product which was purified by rapid FSGC (10-25% EtOAc in hexane) gave the BOC-piperidinyl compound as a pale yellow gum (1.1 g; 100%). TLC Rf=0.6 in 25% EtOAc-hexane.
Step 5: The BOC-protecting group on the piperidine nitrogen in the product of Step 4 was removed by treatment with TFA in CH2Cl2. Basification with I M NaOH and processing in CH2Cl2 afforded the unprotected piperidine in 90% yield. This intermediate was coupled (EDCI, HOBt) to aryl and heteroaryl carboxylic acids to obtain the amides exemplified in the following table:




wherein R2 is as defined in the table:














Ex.
R2
Mp (° C.)
HRMS (MH+)


















28A




249
Calculated: 532.3263 Found: 532.3268


 


28B




59
Calculated: 547.3260 Found: 547.3278


 


28C




246
Calculated: 530.3358 Found: 530.3372


 


28D




239
Calculated: 542.3358 Found: 542.3361


 


28E




258
Calculated: 583.3260 Found: 583.3272


 


28F




102
Calculated: 623.3573 Found: 623.3572


 


28G




216
Calculated: 545.3467 Found: 545.3459


 


28H




217
Calculated: 546.3307 Found: 546.3309


 


28I




223
Calculated: 616.3838 Found: 616.3848









Using similar procedures, the following compounds were prepared:




wherein R8, R3 and R2 are as defined in the table:















Ex.
R8
R3
R2
Mp (° C.)







28J
—CF3








195-220


 


28K
—CF3








105-115


 


28L
CH3CONH—








177-180


 


28M
—CF3








224-232









Using 3-fluoro benzyl bromide or chloride in place of benzyl bromide in the procedure of Example 28, steps 1-4 (processing isomer B in step 3), then using the process of Example 1, step 5, followed by the process of Example 26, steps 6-7, the following compound was prepared (HCl salt):




Example 29




Steps 1-3:





Step 1: Solid m-CPBA was added to a solution of p-trifluoromethyl styrene (3 g; 17.4 mmol) in 30 ml of CH2Cl2 and stirred at RT for 20 h. About 20 ml of a saturated solution of NaHCO3 was added and stirred at RT for 2 h. The mixture was diluted with 20 ml of CH2Cl2 and the organic product extracted into the CH2Cl2 layer. The organic extract was processed to obtain the crude product. FSGC gave 3 g (90%) of the desired epoxide as a colorless oil. TLC Rf=0.8 (25% EtOAc in hexane).

Step 2: Freshly prepared NaOCH3 (0.6 g; 10.6 mmol) was added to a solution of the product of Step 1 (2 g; 10.6 mmol) in 20 ml of anhydrous CH3OH. After stirring at RT for a day, CH3OH was removed in vacuo. The residue was dissolved in CH2Cl2 and washed with water and brine. Concentration, followed by FSGC, furnished 1.3 g (55%) of the carbinol as a colorless oil (Rf=0.3 50% Et2O in hexane).

Step 3: The carbinol of Step 2 (1.3 g; 5.9 mmol) was dissolved in CH2Cl2 and cooled in an ice bath. Sequential treatment with Et3N (1.7 ml; 12 mmol) and CH3SO2Cl (0.6 ml; 7.7 mmol) and stirring for 30 min formed the mesylate. The product was extracted by standard work up (yield=100%).

The mesylate (1.76 g; 5.9 mmol) and 2(S)-methyl-4-(tert-butoxycarbonyl) piperazine (2.4 g; 12 mmol) were dissolved in 5 ml of CH3CN and heated to reflux for 19 h. The reaction mixture was cooled to RT and directly subjected to flash chromatography on silica gel. Eluting with 25%, then 50% Et2O in hexane served to isolate the diastereomeric products A and B (Total yield=86%).
A: Rf=0.5 (50% Et2O in hexane). Light yellow gum (0.9 g; 42%)
B: Rf=0.4 (50% Et2O in hexane). Amber gum (1.13 g; 44%)
Step 4: Reductive amination of the free piperazine derived from A (0.9 g; 2.2 mmol) with N—BOC-piperidin-4-one with the installation of the ipso-methyl group was carried out as described in Example 1, step 4, to obtain the BOC-protected piperidinyl compound (0.87 g; 92%). Rf=0.3 (50% EtOAc in hexane).

Step 5: The BOC protecting group was removed from the piperidine nitrogen via TFA, and the resultant compound was coupled with acids using the EDCI/HOBt method as described in Example 8, step 4, to obtain the compounds shown in the following table:





wherein R2 is as shown in the table:














Ex.
R2
Mp (° C.)
HRMS (MH+)







29A




163
Calculated: 534.3056 Found: 534.3050


 


29B




208
Calculated: 548.3100 Found: 548.3092


 


29C




101
Calculated: 549.3053 Found: 549.3057


 


29D




192
Calculated: 618.3631 Found: 618.3638









Example 29E




The piperidine A (130 mg), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (130 mg), 1-hydroxybenzotriazole (92 mg), and diisopropylethylamine (0.3 mL) were taken up in CH2Cl2 and stirred at 25° C. for 19 h. The solution was diluted with CH2Cl2 and washed with 1 N NaOH (aq.). The aqueous layer was extracted with CH2Cl2 and dried over MgSO4. Filtration and concentration gave a yellow oil. Purification via preparative thin-layer chromatography (10/1 acetone/hexanes, SiO2) gave 95 mg (51%) of compound 29E as a colorless oil. HRMS calcd (MH+): 550.3005; Found: 550.3000.
A was prepared according to Steps 1-5 for Example 29 set forth above.
The pyrimidine acid was prepared according to the procedure outlined for Example 23C Steps 1 and 2 set forth above.
Compound 29E can also be isolated as the metabolite of compound 29A in plasma, urine, bile or fecal sample of a patient who has been administered compound 29A as set forth below in Example 29F. In addition to compound 29E, other compounds that can be isolated as metabolites in human or other animal species include the following:




Example 29F
Isolation of Metabolites
Chemicals: 14C-Vicriviroc [1-[(4,6-dimethyl-5-pyrimidinyl)carbonyl]-4-[4-[2-methoxy-1 (R)-[4-(trifluoromethyl)phenyl]ethyl]-3(S)-methyl-1-piperazinyl]-4-methylpiperidine, i.e., 14C-compound 29A shown below]




was synthesized at Schering-Plough Research Institute (Kenilworth, N.J.) and had >97% radiochemical purity. All other compounds/reference standards were obtained from the Department of Chemical Research at Schering-Plough Research Institute. HPLC grade acetonitrile and methanol were from Burdick and Jackson (Muskegon, Mich.). Water was purified using the Millipore Milli-Qplus water purification system (Bedford, Mass.).

Test Species:
















Weight or





Body Mass


Species
Age
Index (BMI)
Oral Dose







Human (M)
8-50 yr
BMI = 19-29
50 mg 14C-compoud


(n = 8)

kg/m2
29A maleate





(100 μCi) in water


Monkey (M & F)
2-5 yrs
2-5 kg
5 mg/kg (25.8 μCi/mg)


(n = 4)


14C-compound


(Strain:


29LA in water


Cynomolgus


macaque)


Rat (M & F)
7-10 wk
175-270 g
6 mg/kg (12.3 μCi/mg)


(n = 3)


14C-compound 29A


(Strain:


in 0.4% methylcellulose


Sprague


Dawley)





M = Male;


F = Female;


n = number of animals/subjects per gender






Sample Collection: Urine and feces over selected intervals and blood at selected time points were collected from healthy male volunteers, monkeys and rats through 336-hr, 432-hr and 168-hr post dose, respectively.

Radioactivity: Total radioactivity was measured using liquid scintillation spectrometer (LSS).

Sample Processing for Profiling and Characterization of Metabolites:

Sample Pooling:

For each species, plasma samples were pooled across subjects/animals by time point. All the other matrices were first pooled for a desired collection interval within each subject/animal and then across subjects/animals to obtain a composite sample containing >90% of the radioactivity excreted in each respective matrix.














Species
Plasma (hr)
Urine (hr)
Feces (hr)







Human
Pre-dosea, 4, 8 & 24
0-96
0-264


Monkey
Pre-dose, 1 & 4
0-168
0-72


Rat
Pre-dose, 2, 8, 12 & 24
0-48 (M) &
0-72




0-72 (F)





aPre-dose plasma was used for optimizing extraction procedure/conditions


M = Male and F = Female






Sample Processing:















Matrix
Methods









Plasma
SPE using Oasis HLB cartridges (Waters




Corp., Milford, MA) or Solvent extraction




with protein precipitation using acetonitrile



Urine
SPE using Oasis HLB cartridges (Waters




Corp., Milford, MA) or direct Injection



Feces
Solvent extraction using methanol










Mobile Phase and HPLC Conditions:

HPLC column was maintained at room temperature for all LC-MS and LC-MSn experiments. The mobile phase, which consisted of 95% 10-mM ammonium acetate (pH 6.0) containing 5% acetonitrile (A) and 95% acetonitrile and 5% water (B), was maintained at a constant flow rate (1 mL/min). For all LC-MS experiments, the column effluent was split to divert 20-25% into TSQ Quantum (ThermoElectron, San Jose, Calif.) mass spectrometer and the balance into a Flow Scintillation Analyzer (FSA) analyzer.
Mobile Phase Gradient:
Separation of metabolites was achieved using programmed linear changes in mobile phase composition as summarized in the following table:












Time (min)
% A
% B

















0.0
90
10


10.0
70
30


40.0
30
70


40.1
10
90


50.0
10
90


50.1
90
10


60.0
90
10









HPLC and FSA System:












Equipment
Model and Vendor







HPLC Pump, Controller,
Alliance Model 2690 (Waters Corp.,


Degasser, Column Oven
Milford, MA)


and Autosampler


Flow Scintillation
Model 500TR (Packard Instrument Co.,


Analyzer (FSA)
Meriden, CT)


Flow Scintillation
250 or 500 μL (Packard Instrument


Analyzer Cell Volume
Co., Meriden, CT)


Scintillation Fluid
Ultima Flo M at 2.4 mL/min (Packard



Instrument Co., Meriden, CT)


Column
Luna Phenyl-Hexyl 250 × 4.6 mm, 5-μm



particle size (Phenomenex, Torrance, CA).


Guard Column
MetaGuard Polaris C18-A, 5-μm



particle size (Metachem Technologies,



Torrance, CA).









Mass Spectrometer:

All LC-MS and LC-MS/MS experiments were performed using a TSQ mass spectrometer (ThermoElectron, San Jose, Calif.) nominally operated under the conditions listed below:














Parameter
Setting









Ionization Source
Electrospray Ionization (ESI)



Ionization Mode
Positive



Spray Needle Voltage
4.0-4.5 kV



Capillary Temperature
250-270° C.



Sample Flow rate
0.20-0.25 mL/min after splitting



Sheath Gas
Nitrogen (25-50)



Auxiliary Gas
Nitrogen (4-15)










Radiochromatograms from study samples were examined to locate radioactive peaks corresponding to metabolites. After correcting for the delay time (0.2-0.5 min), each radiolabelled peak was examined for possible molecular ions related to the drug and/or its putative metabolites. Based on the elution order, metabolite peak labels were assigned as M1 to M48 where M48 is the first eluting compound and M1 is the last to elute from the column. (see FIGS. 1-3 below). When available, synthetic reference standards were used to confirm the structural assignment.
Results
As shown in Table 1, following 50 mg single oral administration of VIC to healthy male volunteers, the dose was near equally eliminated in urine and feces. By contrast, a majority (53-71%) of the dose was recovered in the feces from all non-clinical species investigated. As shown in FIG. 1, VIC was rapidly and extensively metabolized in human, monkey and rat after a single 50 mg, 6-mg/kg, and 5-mg/kg oral administration of 14C-VIC, respectively. Since there were no sex-related qualitative differences in the metabolism of VIC, only the profiles for each matrix from male rats and monkeys are shown in FIGS. 2-4.







TABLE 1







Excretion of radioactivity as % of dose in humans (50 mg


dose), monkeys (5 mg/kg dose) and rats (6 mg/kg dose) following


a single oral administration of 14C-Vicriviroc.











Human (n = 8)
Monkey (n = 4)
Rat (n = 3)














Urine
Feces
Urine
Feces
Urine
Feces

















Time (h)
M
M
M
F
M
F
M
F
M
F





Totalb
47.1
45.1
26.3
25.8
48.6
56.4
18.0
15.6
69.1
72.9





aNumber of animals


bUrine and fecal samples were collected for 0-336, 0-432 and 0-168 hr from humans, monkeys and rats respectively.






FIG. 1 shows the biotransformation of Vicriviroc in Human, Monkey and Rat following a single oral dose of 14C-VIC.

FIG. 2 shows a comparison of representative radiochromatographic Profiles of Pooled Plasma Extract Following a Single Oral Administration of Vicriviroc to Healthy Male Volunteers, Male Monkeys and Rats.

The following table describes the distribution of compound 29A (vicriviroc) and its metabolites in plasma in human, monkey and rat species.














VIC and Metabolites (Plasma)










Species
Major
Minor
Trace





Human
Vicriviroc
VIC-N-Oxide (M2/M3).
M4 (m/z 550), M7



(VIC)
O-desmethyl-VIC
(m/z 538), M10 (m/z




(M15), O-
508), M14 (m/z 550),




desmethyl-VIC-
M16 (m/z 494),




glucuronide (M35),
M18/M19 (m/z 536),




monooxy-O-
M20/M20a (m/z 534),




desmethyl-VIC-
M21/M22 (m/z 536),




glucuronide (M37)
M25 (m/z 520),




& N-desalkyl-VIC
M25e/M25f/M25g (m/z




(M41)
649), M30/M31 (m/z





536), M35b/M37a





(534) & M36 (m/z 712)


Monkey
Vicriviroc
VIC-N-Oxide (M2/M3).
M1 (m/z 400), M4



(VIC)
VIC-hydroxylamine
(m/z 550), M6 (m/z




(M7) O-desmethyl-
518), M16 (m/z 494),




VIC (M15), O-
M18/M19 (m/z 536),




desmethyl-VIC-
M21/M22 (m/z 536),




glucuronide (M35),
M25 (m/z 520),




VIC-hydroxylamine-
M25e/M25f/M25g (m/z




glucuronide (M26),
649), M28 (m/z




O-desmethyl-VIC-
480) & M36 (m/z 712)




glucuronide (M35),




VIC-carboxylic




acid (M35b/M37a),




monooxy-O-




desmethyl-VIC-




glucuronide (M37),




N-desalkyl-VIC




(M41) & monooxy-




N-desalkyl-VIC




(M45/M46/M47)




VIC-N-Oxide
M10 (m/z 508), M14




(M2/M3). monooxy-
(m/z 550),




VIC (M4),


Rat
Vicriviroc
O-desmethyl-VIC
M16 (m/z 494),



(VIC)
(M15), O-
M18/M19 (m/z 536),




desmethyl-VIC
M20/M20a (m/z 534),




(M25), N-desalkyl-
M22 (m/z 536), M27




VIC (M41) &
(m/z 536), M30/M31




monooxy-N-
(m/z 536), M35 (m/z




desalkyl-VIC
696) & M36/M37 (m/z




(M45/M46/M47)
712)





Major: Components with ≧20% of the total chromatographic radioactivity (TCR);


Minor: Components between 3 and 20% of the TCR;


Trace: Components with <3% of the TCR and/or only detected with a mass spectrometer






Based on the above, the following observations can be made:

    
    
        Qualitatively similar profiles were observed in plasma from human, monkey and rat.
        The major circulating drug derived component in human, monkey and rat was VIC.
        While glucuronide conjugate of O-desmethyl-VIC (M35) was a prominent circulating metabolite in human and monkey plasma, in rats this metabolite was only detected in trace quantities.
        There was no human specific circulating metabolite detected.

FIG. 3 shows a Comparison of Representative Radiochromatographic Profiles of Pooled Urine Following a Single Oral Administration of Vicriviroc to Healthy Male Volunteers, Male Monkeys and Rats.

The following table describes the distribution of compound 29A (vicriviroc) and its metabolites in urine of human, monkey and rat species.

    
    















VIC and Metabolites (Urine)










species
Major
Minor
Trace





Human
O-desmethyl-
VIC, VIC-N-
M1 (m/z 400), M4



VIC-
Oxide (M2/M3).
(m/z 550), M7 (m/z



glucuronide
O-desmethyl-
538), M10 (m/z



(M35) & N-
VIC (M15),
508), M14 (m/z 550),



desalkyl-
monooxy-O-
M16 (m/z 494),



VIC (M41)
desmethyl-
M18/M19 (m/z 536),




VIC (M18/
M19a/M19b (m/z




M19), M20/
534), M21/M22 (m/z




M20a (m/z
536), M22a/M22b




534), monooxy-
(m/z 550), M25




O-desmethyl-
(m/z 520),




VIC-
M25e/M25f/M25g




glucuronide
(m/z 649), M28




(M37) & monooxy-
(480), M34a/M34b/




N-desalkyl-
M34c (m/z 712),




VIC (M45/M46/
M30/M31 (m/z 536),




M47)
M35b/M37a (534) &





M36 (m/z 712)


Monkey
O-desmethyl-
VIC, VIC-N-
M1 (m/z 400), M4



VIC-
Oxide (M2/M3),
(m/z 550), M6 (m/z



glucuronide
VIC-
518), M10 (m/z



(M35), N-
hydroxylamine
508), M15 (m/z



desalkyl-
(M7), monooxy-
536), M16 (m/z



VIC (M41)
O-desmethyl-
494), M23 (m/z



& monooxy-
VIC (M18/M19),
494), M25 (m/z



N-desalkyl-
M20/M20a (m/z
520), M25e/M25f/



VIC (M45/
534), monooxy-
M25g (m/z 649),



M46/M47)
O-desmethyl-
M28 (480) & M36




VIC (M21/M22),
(m/z 712)




VIC-




hydroxylamine-




glucuronide




(M26) &




monooxy-O-




desmethyl-VIC-




glucuronide




(M37)


Rat
N-desalkyl-
VIC, VIC-N-
M1 (m/z 400), M4



VIC (M41)
Oxide (M2/M3),
(m/z 550), M6 (m/z




O-desmethyl-
518), M10 (m/z




VIC (M15),
508), M20/M20a




N,N-desalkyl-
(m/z 534) &




VIC (M16),
M35b/M37a (m/z




monooxy-O-
534)




desmethyl-




VIC (M18/M19),




monooxy-O-




desmethyl-




VIC (M21/




M22), O-




desmethyl-




VIC (M25),




N,N-dealkyl-




O-desmethyl-




VIC (M28), O-




desmethyl-




VIC-glucuronide




(M35) & monooxy-




N-desalkyl-VIC




(M45/M46/M47)





Major: Components with ≧3% of the administered dose.


Minor: Components between 0.5 and 3% of the administered dose.


Trace: Components with <0.5% of the administered dose and/or only detected with a mass spectrometer






Based on the above, the following observations can be made:

    
    
        Major urinary metabolites N-desalkyl-VIC (M41) and O-desmethyl-VIC-glucuronide (M35) collectively accounted for 21%, 8% and 4% of the dose in human, monkey and rat, respectively.
        While M35 contributed to 11% and 3% of the dose in urine, this metabolite accounted for less than 1% in the urine from rat.

FIG. 4 shows a comparison of representative radiochromatographic profiles of pooled fecal extract following a single oral administration of Vicriviroc to healthy male volunteers, male monkeys and rats.

The following table describes the distribution of compound 29A (vicriviroc) and its metabolites in feces of human, monkey and rat species.

    
    















VIC and Metabolites (Feces)










Species
Major
Minor
Trace





Human
N-desalkyl-
VIC, Monooxy-VIC
M6 (m/z 518), M7



VIC (M41) &
(M14). O-desmethyl-
(m/z 538), M10 (m/z



M20/M20a
VIC (M15), N,N-
508), M14 (m/z



(m/z 534)
desalkyl-VIC
550), M19a/M19b




(M16), N,N-
(m/z 534), M23




desalkyl-VIC
(m/z 494), M25




(M16a), monooxy-
(m/z 520), M27 (m/z




O-desmethyl-
536) & M28 (480)




VIC (M19),




M25e/M25f/M25g




(m/z 649),




VIC-carboxylic




acid (M35b/




M37a) & monooxy-




N-desalkyl-VIC




(M45/M46/M47)


Monkey
N-desalkyl-
VIC, VIC-
M1 (m/z 400), M6



VIC (M41) &
hydroxylamine
(m/z 518), M10



M20/M20a
(M7), N,N-
(m/z 508), M15



(m/z 534)
desalkyl-VIC
(m/z 520), M16




(M16a), N,N-
(m/z 494) & M18/




desalkyl-VIC
M19 (m/z 536) &




(M23), monooxy-
M21/M22 (m/z 536)




VIC (M22a),




M25e/M25f?M25g




(m/z 649), N,N-




desalkyl-O-




desmethyl-VIC




(M28), monooxy-




VIC (M33), VIC-




carboxylic acid




(M35b/M37a) &




monooxy-N-




desalkyl-VIC




(M45/M46/M47)


Rat
N-desalkyl-
VIC, monooxy-
M1 (m/z 400),



VIC (M41) &
VIC (M4), VIC-
M2/M3 (m/z 550),



O-desmethyl-
hydroxylamine
M5a/M5b (m/z



VIC (M15)
(M7), N,N-
548), M6 (m/z




desalkyl-VIC
518), M10 (m/z




(M16), monooxy-
508), M20/M20a




O-desmethyl-
(m/z 534) & M25d




VIC (M19), O-
(m/z 536)




desmethyl-VIC




(M25), M25e/




M25f/M25g (m/z




649), N,N-




desalkyl-O-




desmethyl-VIC




(M28), M35a1




(m/z 600), VIC-




carboxylic acid




(M35b/M37a) &




monooxy-N-




desalkyl-VIC




(M45/M46/M47)





Major: Components with ≧5% of the administered dose.


Minor: Components between 1 and 5% of the administered dose.


Trace: Components with <1% of the administered dose and/or only detected with a mass spectrometer






Based on the above, the following observation can be made:

    
    
        Major fecal metabolites which collectively accounted for 16-35% of the administered dose in human, monkey and rat included N-desalkyl-VIC (M41), O-demethyl-VIC(M15) and a oxidative product of M15 (M20/M20a at m/z of 534).

The overall conclusion from the metabolite studies are as follows:

        Following a single oral 50 mg administration to healthy volunteers, Vicriviroc (VIC, compound 29A) and its metabolites were near equally excreted in feces and urine. By contrast, following a single 5 mg/kg and 6 mg/kg oral administration of VIC to rats and monkeys, respectively, radioactivity was predominantly eliminated in the feces.
        In all species investigated, the primary biotransformation of VIC involved O-demethylation, N-dealkylation, oxidation and glucuronidation.
        No human specific metabolites were observed following a single 50 mg oral administration of VIC to healthy male volunteers.
    
    


Example 30




Step 1:





A solution of p-trifluoromethoxy benzaldehyde (0.48 ml, 3.36 mmol), the piperidino-pipiperazine (1.00 g, 3.36 mmol) and benzotriazole (0.48 g, 4.00 mmol) in dry toluene were heated at reflux for 6 h. The reaction mixture was cooled to RT and the solvent was removed in vacuo. Following NMR verification of the formation of the product, the product was used without further purification in the next step.
Step 2:




To a solution of the product of Step 1 (1.16 g, 1.97 mmol) in 20 ml of toluene was added a solution of n-propyl magnesium bromide (2M in Et2O, 1.1 ml) and the mixture stirred at RT for 15 h. The reaction mixture was quenched by pouring onto ice and saturated aqueous NH4Cl solution. The aqueous layer was extracted with EtOAc, washed with 1M NaOH solution, water and brine. Concentration and purification by FSGC (20% EtOAc-hexane) provided the desired product A. Further elution with 30% EtOAc in hexane gave the (R,S) diastereomer B.
Step 3: The amine A was treated with TFA in CH2Cl2 to remove the BOC-protecting group. Coupling of the free piperidine with acids using EDCI/HOBt provided compounds 30-30B in the following table; similar methods were used to prepare compounds 30C-I.
















 

















HRMS (MH+)


Ex.
R8a
R3
R2
MP (° C.)
found





30
—OCF3
n-Pr




237
546.3314


 


30A
—OCF3
n-Pr




241
548.3217


 


30B
—OCF3
n-Pr




219
632.3779


 


30C
H








175-178
—


 


30D
H








177-189
—


 


30E
H








84-90
—


 


30F
—CF3








180-192
—


 


30G*
—CF3








180-186
—


 


30H
H








178-188
—


 


30I*
—OCF3








165-175
—





*Mixture of diastereomers






Example 31




A solution of the product of Example 12, step 2 (150 mg, 0.27 mmol), imidazole (27.4 mg, 0.403 mmol), 1,10-phenanthroline (48 mg, 0.27 mmol), trans,trans-dibenzylideneacetone (6.28 mg, 0.027 mmol), copper (II) trifluoromethanesulfonate benzene complex (15 mg, 0.027 mmol) and Cs2CO3 (96.1 mg, 0.30 mmol) in xylene (2 ml) was stirred at 110° C. for 5 days. The reaction mixture was cooled to RT and saturated NaHCO3 was added. Extractive EtOAc work up followed by silica gel chromatography gave the title compound (70 mg, 52% yield). Dec. 215° C. (HCl salt).
HRMS calcd for C29H39ClN3OS (M+H+) 500.3389, found 500.3396.
The following assays can be used to determine the CCR5 antagonistic activity of the compounds of the invention.
CCR5 Membrane Binding Assay:
A high throughput screen utilizing a CCR5 membrane binding assay identifies inhibitors of RANTES binding. This assay utilizes membranes prepared from NIH 3T3 cells expressing the human CCR5 chemokine receptor which have the ability to bind to RANTES, a natural ligand for the receptor. Using a 96-well plate format, membrane preparations are incubated with 125I-RANTES in the presence or absence of compound for one hour. Compounds are serially diluted over a wide range of 0.001 ug/ml to 1 ug/ml and tested in triplicates. Reaction cocktails are harvested through glass fiber filters, and washed thoroughly. Total counts for replicates are averaged and data reported as the concentration required to inhibit 50 percent of total 125I-RANTES binding. Compounds with potent activity in the membrane binding assay are further characterized in secondary cell-based HIV-1 entry and replication assays.
HIV-1 Entry Assay:
Replication defective HIV-1 reporter virions are generated by cotransfection of a plasmid encoding the NL4-3 strain of HIV-1 (which has been modified by mutation of the envelope gene and introduction of a luciferase reporter plasmid) along with a plasmid encoding one of several HIV-1 envelope genes as described by Connor et al, Virology, 206 (1995), p. 935-944. Following transfection of the two plasmids by calcium phosphate precipitation, the viral supernatants are harvested on day 3 and a functional viral titer determined. These stocks are then used to infect U87 cells stably expressing CD4 and the chemokine receptor CCR5 which have been preincubated with or without test compound. Infections are carried out for 2 hours at 37° C., the cells washed and media replaced with fresh media containing compound. The cells are incubated for 3 days, lysed and luciferase activity determined. Results are reported as the concentration of compound required to inhibit 50% of the luciferase activity in the control cultures.
HIV-1 Replication Assay:
This assay uses primary peripheral blood mononuclear cells or the stable U87-CCR5 cell line to determine the effect of anti-CCR5 compounds to block infection of primary HIV-1 strains. The primary lymphocytes are purified from normal healthy donors and stimulated in vitro with PHA and IL-2 three days prior to infection. Using a 96-well plate format, cells are pretreated with drug for 1 hour at 37° C. and subsequently infected with an M-tropic HIV-1 isolates. Following infection, the cells are washed to remove residual inoculum and cultured in the presence of compound for 4 days. Culture supernatants are harvested and viral replication measured by determination of viral p24 antigen concentration.
Calcium Flux Assay:
Cells expressing the HIV coreceptor CCR5 are loaded with calcium sensitive dyes prior to addition of compound or the natural CCR5 ligand. Compounds with agonist properties will induce a calcium flux signal in the cell, while CCR5 antagonists are identified as compounds which do not induce signaling by themselves but are capable of blocking signaling by the natural ligand RANTES.
GTPγS Binding Assay:
A GTPγS binding assay measures receptor activation by CCR5 ligands. This assay measures the binding of 35S labeled-GTP to receptor coupled G-proteins that occurs as a result of receptor activation by an appropriate ligand. In this assay, the CCR5 ligand, RANTES, is incubated with membranes from CCR5 expressing cells and binding to the receptor activation (or binding) is determined by assaying for bound 35S label. The assay quantitatively determines if compounds exhibit agonist characteristics by inducing activation of the receptor or alternatively antagonist properties by measuring inhibition of RANTES binding in a competitive or non-competitive fashion.
Chemotaxis Assay:
The chemotaxis assay is a functional assay which characterizes the agonist vs. antagonist properties of the test compounds. The assay measures the ability of a non-adherent murine cell line expressing human CCR5 (BaF-550) to migrate across a membrane in response to either test compounds or natural ligands (i.e., RANTES, MIP-1β). Cells migrate across the permeable membrane towards compounds with agonist activity. Compounds that are antagonists not only fail to induce chemotaxis, but are also capable of inhibiting cell migration in response to known CCR5 ligands.
The role of CC chemokine receptors such as CCR-5 receptors in inflammatory conditions has been reported in such publications as Immunology Letters, 57, (1997), 117-120 (arthritis); Clinical & Experimental Rheumatology, 17 (4) (1999), p. 419-425 (rheumatoid arthritis); Clinical & Experimental Immunology, 117 (2) (1999), p. 237-243 (atopic dermatitis); International Journal of Immunopharmacology, 20 (11) (1998), p. 661-7 (psoriasis); Journal of Allergy & Clinical Immunology, 100 (6, Pt 2) (1997), p. S52-5 (asthma); and Journal of Immunology, 159 (6) (1997), p. 2962-72 (allergies).
In the assay to determine inhibition of RANTES binding, compounds of the invention range in activity from a Ki of about 0.5 to about 1500 nM, with preferred compounds having a range of activity from about 0.5 to about 750 nM, more preferably about 0.5 to 300 nM, and most preferably about 0.5 to 50 nM. The results for preferred and representative compounds of formulas I and II in the test to determine inhibition of RANTES binding are given in the table below. In the table, “Ex. No.” stands for “Example Number” and “nM” stands for “nanomolar.”















Ki (nM)




Inhibition of



Ex. No.
RANTES binding



















3C
9.97



6C
30.0



6E
1.43



11
10.5



16
60



20A
1300



23
2.95










For preparing pharmaceutical compositions of the CCR5 antagonist compounds described by this invention, inert, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, cachets and suppositories. The powders and tablets may be comprised of from about 5 to about 95 percent active ingredient. Suitable solid carriers are known in the art, e.g. magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, cachets and capsules can be used as solid dosage forms suitable for oral administration. Examples of pharmaceutically acceptable carriers and methods of manufacture for various compositions may be found in A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition, (1990), Mack Publishing Co., Easton, Pa.
Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for parenteral injection or addition of sweeteners and opacifiers for oral solutions, suspensions and emulsions. Liquid form preparations may also include solutions for intranasal administration.
Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier, such as an inert compressed gas, e.g. nitrogen.
Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions and emulsions.
The CCR5 antagonist compounds of the invention may also be deliverable transdermally. The transdermal compositions can take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.
Preferably the CCR5 antagonist compound is administered orally.
Preferably, the pharmaceutical preparation is in a unit dosage form. In such form, the preparation is subdivided into suitably sized unit doses containing appropriate quantities of the active component, e.g., an effective amount to achieve the desired purpose.
The quantity of active compound in a unit dose of preparation may be varied or adjusted from about 10 mg to about 500 mg, preferably from about 25 mg to about 300 mg, more preferably from about 50 mg to about 250 mg, and most preferably from about 55 mg to about 200 mg, according to the particular application.
The actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage regimen for a particular situation is within the skill of the art. For convenience, the total daily dosage may be divided and administered in portions during the day as required.
The amount and frequency of administration of the CCR5 antagonist compounds of the invention and/or the pharmaceutically acceptable salts thereof will be regulated according to the judgment of the attending clinician considering such factors as age, condition and size of the patient as well as severity of the symptoms being treated. A typical recommended daily dosage regimen for oral administration can range from about 100 mg/day to about 300 mg/day, preferably 150 mg/day to 250 mg/day, more preferably about 200 mg/day, in two to four divided doses.
The doses and dosage regimen of the NRTIs, NNRTIs, PIs and other agents will be determined by attending clinician in view of the approved doses and dosage regimen in the package insert or as set forth in the protocol taking into consideration the age, sex and condition of the patient and the severity of the HIV-1 infection.
While the present invention has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications and variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are intended to fall within the spirit and scope of the present invention.

## Claims

1. A compound in pure and isolated form, said compound being




or a pharmaceutically acceptable salt or ester thereof.

2. A pharmaceutical composition comprising an effective amount of a compound of claim 1 or a pharmaceutically acceptable salt or ester thereof in combination with a pharmaceutically acceptable carrier.

3. A method of treating Human Immunodeficiency Virus comprising administering to a human in need of such treatment a therapeutically effective amount of the compound of claim 1 or a pharmaceutically acceptable salt, or ester thereof.

4. The method of claim 3 further comprising administering one or more antiviral or other agents useful in the treatment of Human Immuno-deficiency Virus in combination with the compound of claim 1 or a pharmaceutically acceptable salt, solvate or ester thereof.

5. The method of claim 4 wherein the antiviral agent is selected from the group consisting of nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitors.

6. The method of claim 5 wherein the antiviral agent is selected from the group consisting of zidovudine, lamivudine, zalcitabine, didanosine, stavudine, abacavir, adefovir dipivoxil, Iobucavir, BCH-10652, emitricitabine, beta-L-FD4, DAPD, Iodenosine, nevirapine, delaviridine, efavirenz, PNU-142721, AG-1549, MKC-442, (+)-calanolide A and B, saquinavir, indinavir, ritonavir, nelfinavir, lasinavir, DMP-450, BMS-2322623, ABT-378, amprenavir, hydroxyurea, ribavirin, IL-2, IL-12, pentafuside, Yissum No. 11607 and AG-1549.

7. A method treating solid organ transplant rejection, graft v. host disease, arthritis, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, psoriasis, asthma, allergies or multiple sclerosis, comprising administering to a human in need of such treatment a therapeutically effective amount of the compound of claim 1 or a pharmaceutically acceptable salt, or ester thereof.

8. The method of claim 7 for the treatment of solid organ transplant rejection, graft v. host disease, arthritis, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, psoriasis, asthma, allergies or multiple sclerosis, further comprising one or more other agents useful in the treatment of said diseases.