WO2005051410A1 - Hepatitis c virus inhibitors - Google Patents

Hepatitis c virus inhibitors Download PDF

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Publication number
WO2005051410A1
WO2005051410A1 PCT/US2004/039331 US2004039331W WO2005051410A1 WO 2005051410 A1 WO2005051410 A1 WO 2005051410A1 US 2004039331 W US2004039331 W US 2004039331W WO 2005051410 A1 WO2005051410 A1 WO 2005051410A1
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Prior art keywords
compound
alkyl
hcv
optionally substituted
alkoxy
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PCT/US2004/039331
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French (fr)
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WO2005051410A9 (en
WO2005051410B1 (en
Inventor
Paul Michael Scola
Jeffrey Allen Campbell
Ny Sin
Li-Qiang Sun
Xiangdong Alan Wang
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Bristol-Myers Squibb Company
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Application filed by Bristol-Myers Squibb Company filed Critical Bristol-Myers Squibb Company
Priority to EP04811956.4A priority Critical patent/EP1684787B1/en
Priority to JP2006541656A priority patent/JP4688815B2/en
Publication of WO2005051410A1 publication Critical patent/WO2005051410A1/en
Publication of WO2005051410A9 publication Critical patent/WO2005051410A9/en
Publication of WO2005051410B1 publication Critical patent/WO2005051410B1/en
Priority to IS8476A priority patent/IS8476A/en
Priority to NO20062339A priority patent/NO20062339L/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is generally directed to antiviral compounds, and more specifically directed to compounds which hihibit the functioning ofthe NS3 protease (also refened to herein as "serine protease") encoded by Hepatitis C virus (HCV), compositions comprising such compounds and methods for inhibiting the functioning ofthe NS3 protease.
  • HCV Hepatitis C virus
  • HCV is a major human pathogen, infecting an estimated 170 million persons worldwide - roughly five times the number infected by human hnmunodef ⁇ ciency virus type 1. A substantial fraction of these HCV infected individuals develop serious progressive liver disease, including cinhosis and hepatoceliular carcinoma. (Lauer, G. M.; Walker, B. D. N. Engl. J. Med. (2001), 345, 41-52).
  • HCV is a positive-stranded R ⁇ A viras. Based on a comparison ofthe deduced amino acid sequence and the extensive similarity hi the 5' untranslated region, HCV has been classified as a separate genus in the Flaviviridae family. All members ofthe Flaviviridae family have enveloped virions that contain a positive stranded RNA genome encoding all known viras- specific proteins via translation of a single, uninterrupted, open reading frame.
  • the single strand HCV RNA genome is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the stractural and non- structural (NS) proteins. In the case of HCV, the generation of mature non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases.
  • ORF open reading frame
  • the first one is believed to cleave at the NS2-NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, for the remaining NS4A- NS4B, NS4B-NS5A, NS5A-NS5B sites.
  • the NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components. The complex formation ofthe NS3 protein with NS4A seems necessary to the processing events, enhancing the proteolytic efficiency at all ofthe sites.
  • the NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities.
  • NS5B is a RNA-dependent RNA polymerase that is involved in the replication of HCV.
  • the present invention provides compounds of formula I;
  • Ri is C 3-7 cycloalkyl, C 4-7 cycloalkenyl; C 6- ⁇ o aryl; C 7-1 alkylaryl; C 6- j .0 aryloxy; C- ⁇ 4 alkylaryloxy; C 8 -i 5 alkylarylester; Het; or C 1-8 alkyl optionally substituted with C ⁇ -6 alkoxy, hydroxy, halo, C -1 o alkenyl, C 2-10 alkynyl, C 3- cycloalkyl, C 4-7 cycloalkenyl, C 6-10 aryl, C 7-14 alkylaryl, C ⁇ -io aryloxy, C 7-14 alkylaryloxy, C 8-15 alkylarylester or Het; or Ri is trialkylsilane or halogen, provided q is 0;
  • R 3 is C ⁇ - 8 alkyl optionally substituted with halo, cyano, amino, C ⁇ -6 dialkylamino, C 6- ⁇ o aryl, C 7- ⁇ alkylaryl, C ⁇ -6 alkoxy, carboxy, hydroxy, aryloxy, C .
  • Y is H, phenyl substituted with nitro, pyridyl substituted with nitro, or C 1-6 alkyl optionally substituted with cyano, OH or C 3-7 cycloalkyl; provided that if Rt or R is H then Yis H;
  • R 4 is (i) C ⁇ - 10 alkyl optionally substituted with phenyl, carboxyl, C 1-6 alkanoyl, 1-3 halogen, hydroxy, -OC(O)C 1-6 alkyl, C 1-6 alkoxy, amino optionally substituted with C 1-6 alkyl, amido, or (lower alkyl) amido; (ii) C 3-7 cycloalkyl, C 3-7 cycloalkoxy, or C 4-10 alkylcycloalklyl, each optionally substituted with hydroxy, carboxyl, (C 1-6 alkoxy)carbonyl, amino optionally substituted with C ⁇ -6 alkyl, amido, or (lower alkyl) amido; (iii) C 6- ⁇ o aryl or C -16 arylalkyl, each optionally substituted with C 1-6 alkyl, halogen, nitro, hydroxy, amido, (lower alkyl) amido, or amino optionally substituted with C ⁇
  • R 5 is H; C 1-6 alkyl optionally substituted with 1-3 halogens; or C 1-6 alkoxy provided 4 is C ⁇ io alkyl;
  • X is O, S, SO, SO 2 , OCH 2 , CH 2 O or NH;
  • R' is Het, C 6- ⁇ o aryl or C 7-1 4 alkylaryl, each optionally substituted with
  • R a is C ⁇ -6 alkyl, C 3-7 cycloalkyl, C ⁇ -6 alkoxy, C 3-7 cycloalkoxy, halo-Ci- 6 alkyl, CF 3 , mono-or di- halo-C ⁇ -6 alkoxy, cyano, halo, thioalkyl, hydroxy, alkanoyl, NO 2 , SH, , amino, C 1-6 alkylamino, di (Ci- 6 ) alkylaniino, di (C ⁇ - 6 ) alkylamide, carboxyl, (C ⁇ - 6 ) carboxyester, C 1-6 alkylsulfone, C 1-6 alkylsulfonamide, di (Ci- ⁇ ) alkyl(alkoxy)amine, C 6- 10 aryl, C 7-1 alkylaryl, or a 5-7 membered monocyclic heterocycle; and
  • R 6 and R are each independently H; or C 1-6 alkyl, C 2-10 alkenyl or C 6- ⁇ o aryl, each of which may be optionally substituted with halo, cyano, nitro, C 1-6 alkoxy, amido, amino or phenyl;
  • the present invention also provides compositions comprising the compounds or pharmaceutically acceptable salts, solvates or prodrugs thereof and a pharmaceutically acceptable canier.
  • the present invention provides pharmaceutical compositions useful for inhibiting HC"V NS3 comprising a therapeutically effective amount of a compound ofthe present invention, or a pharmaceutically acceptable salt, solvate or prodrag thereof, and a pharmaceutically acceptable canier.
  • the present invention further provides methods for treating patients infected with HCV, comprising administering to the patient a therapeutically effective amount of a compound ofthe present invention, or a pharmaceutically acceptable salt, solvate or prodrag thereof. Additionally, the present invention provides methods of inhibiting HCV NS3 protease by contacting the NS3 protease with a compound ofthe present invention.
  • the present invention provides peptide compounds that can inhibit the functioning ofthe NS3 protease, e.g., in combination with the NS4A protease. Further, the present invention makes it possible to administer combination therapy to a patient whereby a compound in accordance with the present invention, which is effective to inhibit the HCV NS3 protease, can be administered with another compound having anti-HCV activity, e.g.
  • a compound which is effective to inhibit the function of a target selected from the group consisting of HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH and a nucleoside analog for the treatment of an HCV infection.
  • d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory and (+) or d, meaning the compound, is dextrorotatory.
  • these compounds called stereoisomers, are identical except that they are minor images of one another.
  • a specific stereoisomer of a minor image pair may also be refened to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture.
  • (R) or (S) it is to designate the absolute configuration of a substituent in context to the whole compound and not in context to the substituent alone.
  • racemic mixture and “racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
  • chiral refers to molecules which have the property of non-superimposability ofthe mirror image partner, while the term “achiral” refers to molecules which are superimposable on their mirror image partner.
  • stereoisomers refers to compounds which have identical chemical composition, but differ with regard to the anangement ofthe atoms or groups in space.
  • diastereomer refers to a stereoisomer which is not an enantiomer, e.g., a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another.
  • Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography.
  • enantiomers refers to two stereoisomers of a compound which are non-superimposable mirror images of one another.
  • salts are intended to mclude nontoxic salts synthesized from a compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base fonns of these compounds with a stoichiometric amount ofthe appropriate base or acid in water or in an organic solvent, or in a mixture ofthe two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are prefened. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445.
  • the compounds ofthe present invention are useful in the form ofthe free base or acid or in the form of a pharmaceutically acceptable salt thereof. All forms are within the scope ofthe invention.
  • the term "therapeutically effective amount” means the total amount of each active component that is sufficient to show a meaningful patient benefit, e.g., a sustained reduction in viral load.
  • the tenn refers to that ingredient alone.
  • the term refers to combined amounts ofthe active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • derivative means a chemically modified compound wherein the modification is considered routine by the ordinary skilled chemist, such as an ester or an amide of an acid, protecting groups, such as a benzyl group for an alcohol or thiol, and tert-butoxycarbonyl group for an amine.
  • solvate means a physical association of a compound of this invention with one or more solvent molecules, whether organic or inorganic. This physical association includes hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are inco ⁇ orated in the crystal lattice ofthe crystalline solid. "Solvate” encompasses both solution-phase and isolable solvates. Exemplary solvates include hydrates, ethanolates, methanolates, isopropanolates and the like.
  • prodrag means derivatives ofthe compounds ofthe invention which have chemically or metabolically cleavable groups and become, by solvolysis or under physiological conditions, the compounds ofthe invention which are pharmaceutically active in vivo.
  • a prodrag of a compound may be formed in a conventional manner with a functional group ofthe compounds such as with an amino, hydroxy or carboxy group when present.
  • the prodrag derivative form often offers advantages of solubility, tissue compatibility, or delayed release in a mammalian organism (see, Bundgard, H., Design of Prodrags, pp. 7-9, 21-24, Elsevier, Amsterdam 1985).
  • Prodrags include acid derivatives well known to practitioners ofthe art, such as, for example, esters prepared by reaction ofthe parent acidic compound with a suitable alcohol, or amides prepared by reaction ofthe parent acid compound with a suitable amine.
  • patient includes both human and other mammals.
  • composition means a composition comprising a compound ofthe invention in combination with at least one additional pharmaceutical canier, i.e., adjuvant, excipient or vehicle, such as diluents, preserving agents, fillers, flow regulating agents, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature ofthe mode of administration and dosage fonns.
  • additional pharmaceutical canier i.e., adjuvant, excipient or vehicle, such as diluents, preserving agents, fillers, flow regulating agents, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature ofthe mode of administration and dosage fonns.
  • diluents
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable risk/benefit ratio.
  • treating refers to: (i) preventing a disease, disorder or condition from occurring in a patient which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder or condition, i.e., anesthig its development; and (iii) relieving the disease, disorder or condition, i.e., causing regression ofthe disease, disorder and/or condition.
  • substituted includes substitution at from one to the maximum number of possible binding sites on the core, e.p., organic radical, to which the subsitutent is bonded, e.g., mono-, di-, tri- or tetra- substituted, unless otherwise specifically stated.
  • organic radicals e.g., hydrocarbons and substituted hydrocarbons
  • groups e.g., alkylalkoxyamine or arylalkyl
  • groups include all possible stable configurations, unless otherwise specifically stated. Certain radicals and combinations are defined below for purposes of illustration.
  • halo as used herein means a halogen substituent selected from bromo, chloro, fluoro or iodo.
  • haloalkyl means an alkyl group that in substituted with one or more halo substituents.
  • alkyl as used herein means acyclic, straight or branched chain, alkyl substituents having the specified number of carbon atoms and includes, for example, methyl, ethyl, propyl, butyl, tert-butyl, hexyl, 1-methylethyl, 1- methylpropyl, 2-methypropyl, 1,1-dimethylethyl.
  • C ⁇ -6 alkyl refers to an alkyl group having from one to six carbon atoms.
  • lower alkyl means an alkyl group having from one to six, preferably from one to four carbon atoms.
  • alkylester means an alkyl group additionally containing on ester group. Generally, a stated carbon number range, e.g., C -6 alkylester, includes all ofthe carbon atoms in the radical.
  • alkenyl as used herein means an alkyl radical containing at least one double bond, e.g., ethenyl (vinyl) and alkyl.
  • alkoxy as used herein means an alkyl group with the indicated number of carbon atoms attached to an oxygen atom. Alkoxy includes, for example, methoxy, ethoxy, propoxy, 1-methylethoxy, " butoxy and 1,1-dimethylethoxy. The latter radical is refened to in the art as tert-butoxy.
  • alkoxycarbonyi means an alkoxy group additionally containing a carbonyl group.
  • cycloalkyl as used herein means a cycloalkyl substituent containing the indicated number of carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and spiro cyclic groups such as spirocyclopropyl as spirocyclobutyl.
  • cycloalkoxy as used herein means a cycloalkyl group linked to an oxygen atom, such as, for example, cyclobutyloxy or cyclopropyloxy.
  • alkylcycloalkyl means a cycloalkyl group linked to an alkyl group.
  • the stated carbon number range includes the total number of carbons in the radical, unless otherwise specfically stated.
  • This a C 4- ⁇ 0 alkylcycloalkyl may contain from 1-7 carbon atoms in the alkyl group and from 3-9 carbon atoms in the ring, e.g., cyclopropylmethyl or cyclohexylethyl.
  • aryl as used herein means an aromatic moiety containing the indicated number of carbon atoms, such as, but not limited to phenyl, indanyl or naphthyl.
  • C ⁇ -io aryl refers to an aromatic moiety having from six to ten carbon atoms which may be in the form of a monocyclic or bicyclic stracture.
  • haloaryl refers to an aryl mono, di or tri substituted with one or more halogen atoms.
  • alkylaryl arylalkyl
  • aralalkyl mean an aryl group substituted with one or more alkyl groups.
  • a C 7-14 alkylaryl group many have from 1-8 carbon atoms in the alkyl group for a monocyclic aromatic and from 1-4 carbon atoms in the alkyl group for a fused aromatic.
  • the attachment ofthe group to bonding site on the molecule can be either at the aryl group or the alkyl group.
  • aryl radicals include those substituted with typical substituents known to those skilled in the art, e.g., halogen, hydroxy, carboxy, carbonyl, nitro, sulfo, amino, cyano, dialkylamino haloalkyl, CF 3 , haloalkoxy, thioalkyl, alkanoyl, SH, alkylamino, alkylamide, dialkylamide, carboxyester, alkylsulfone, alkylsulfonamide and alkyl(alkoxy)amine.
  • typical substituents e.g., halogen, hydroxy, carboxy, carbonyl, nitro, sulfo, amino, cyano, dialkylamino haloalkyl, CF 3 , haloalkoxy, thioalkyl, alkanoyl, SH, alkylamino, alkylamide, dialkylamide, carboxyester, alkylsulfone, al
  • alkylaryl groups examples include benzyl, butylphenyl and 1-naphthylmethyl.
  • alkanoyl as used herein means straight or branched 1-oxoalkyl radicals containing the indicated number of carbon atoms and includes, for example, fonnyl, acetyl, 1-oxopropyl (propionyl), 2-methyl-l-oxopropyl, 1-oxohexyl and the like.
  • alkylamide as used herein means an amide mono-substituted with an alkyl, such as
  • heterocycle also refened to as “Het”, as used herein means 7-12 membered bicyclic heterocycles and 5-9 membered monocyclic heterocycles.
  • Prefened bicyclic heterocycles are 7-12 membered fused bicyclic ring systems (both rings share an adjacent pair of atoms) containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur, wherein one or both rings of the heterocycle can be saturated, partially saturated or fully unsaturated ring system (this latter subset also herein refened to as unsaturated heteroaromatic).
  • the nitrogen and sulfur heteroatoms atoms may be optionally oxidized.
  • the bicyclic heterocycle may contain the heteroatoms in one or both rings.
  • the heterocycles include those substituted with typical substituents known to those skilled in the art.
  • the bicyclic heterocycle may also contain substituents on any ofthe ring carbon atoms, e.g., one to three substituents. Examples of suitable substituents include C 1-6 alkyl, C 3-7 cycloalkyl,
  • bicyclic heterocycle When two substituents are attached to vicinal carbon atoms ofthe bicyclic heterocycle, they can join to form a ring, e.g., a five, six or seven membered ring system containing up to two heteroatoms selecting from oxygen and nitrogen.
  • the bicyclic heterocycle may be attached to the molecule, e.g. Ri in formula I, at any atom in the ring and preferably carbon.
  • bicyclic heterocycles include, but are not limited to, the following ring systems:
  • Prefened monocyclic heterocycles are 5-9 membered saturated, partially saturated or fully unsaturated ring system (this latter subset also herein refened to as unsaturated heteroaromatic) containing in the ring from one to four heteroatoms selected from nitrogen, oxygen and sulfur, wherein the sulfur and nitrogen heteroatoms may be optionally oxidized.
  • the heterocycles include those substituted with typical substituents known to those skilled in the art.
  • the monocyclic heterocycle may also contain substituents on any ofthe ring atoms, e.g., one to three substituents.
  • suitable substituents include C ⁇ -6 alkyl, C 3-7 cycloalkyl, C ⁇ -6 alkoxy, C 3-7 cycloalkoxy, halo-Ci- 6 alkyl, CF 3 , mono-or di- halo-C ⁇ -6 alkoxy, cyano, halo, thioalkyl, hydroxy, alkanoyl, NO 2 , SH, , amino, C 1-6 alkylamino, di (C ⁇ - 6 ) alkylamino, di (C ⁇ - 6 ) alkylamide, carboxyl, (Ci- 6 ) carboxyester, Cj -6 alkylsulfone, C ⁇ - 6 alkylsulfoxide, C 1-6 alkylsulfonamide, di (C ⁇ - 6 ) alkyl(alkoxy)amine,
  • monocyclic heterocycles include, but are not limited to, the following (and their tautomers):
  • heterocycles used in the compounds ofthe present invention should be stable.
  • stable compounds are those which can be synthesized, isolated and formulated using techniques known the those skilled in the art without degradation ofthe compound.
  • substituted with reference to an amino acid or amino acid derivative means a radical derived from the conesponding ⁇ -amino acid.
  • substituents methyl, iso-propyl, and phenyl represent the amino acids alanine, valine, and phenyl glycine, respectively.
  • PI ', PI, P2, P3 and P4" map the relative positions ofthe amino acid residues of a protease inhibitor binding relative to the binding ofthe natural peptide cleavage substrate. Cleavage occurs in the natural substrate between PI and PI ' where the nonprime positions designate amino acids starting from the C-terminus end ofthe peptide natural cleavage site extending towards the N-terminus; whereas, the prime positions emanate from the N-terminus end ofthe cleavage site designation and extend towards the C-terminus.
  • PI' refers to the first position away from the right hand end ofthe C-terminus ofthe cleavage site (ie.
  • A&ca 1-aminocyclopropyl-carboxylic acid
  • tert-butylglycine refers to a compound ofthe formula:
  • residue with reference to an amino acid or amino acid derivative means a radical derived from the conesponding -amino acid by eliminatirig the hydroxyl ofthe carboxy group and one hydrogen ofthe ⁇ -amino acid group.
  • the terms Gin, Ala, Gly, He, Arg, Asp, Phe, Ser, Leu, Cys, Asn, Sar and Tyr represent the "residues" of E-glutamine, E-alanine, glycine, -isoleucine, L— arginine, Z-aspartic acid, E-phenylalanine, X-serine, J-leucine, E-cysteine, -asparagine, sarcosine and E-tyrosine, respectively.
  • side chain with reference to an amino acid or amino aci ⁇ l residue means a group attached to the ⁇ -carbon atom ofthe ⁇ -amino acid.
  • R-group side chain for glycine is hydrogen, for alanine it is methyl, for valine it is isopropyl.
  • specific R-groups or side chains ofthe ⁇ -amino acids reference is made to A.L. Lehninger's text on Biochemistry (see chapter 4).
  • O o o o z is x u -l * , L " 0 ⁇ , -h c -o ⁇ — c " - or NR 6 R 7 p is 1, 2 or 3; q is 0 or 1;
  • Ri is C 3-7 cycloalkyl, C 4-7 cycloalkenyl; C 6- ⁇ 0 aryl; C 7- ⁇ 4 alkylaryl; C 6- ⁇ o aryloxy; C 7- ⁇ 4 alkylaryloxy; C 8-15 alkylarylester; Het; or C ⁇ -8 alkyl optionally substituted with C ⁇ -6 alkoxy, hydroxy, halo, C -10 alkenyl,
  • Ri is trialkylsilane or halogen, provided q is 0;
  • R 3 is C ⁇ - 8 alkyl optionally substituted with halo, cyano, amino, C ⁇ -6 diall ⁇ lamino, C 6- ⁇ 0 aryl, C - ⁇ 4 alkylaryl, C 1-6 alkoxy, carboxy, hydroxy, aryloxy, C 7- ⁇ 4 alkylaryloxy, C 2-6 alkylester or C 8- ⁇ 5 alkylarylester; C 3- ⁇ 2 alkenyl; C 3-7 cycloalkyl or C 4- ⁇ o alkylcycloalkyl, wherein the cycloalkyl or alkylcycloalkyl are optionally substituted with hydroxy, C ⁇ -6 alkyl, C 2-6 alkenyl or C ⁇ -6 alkoxy; or R 3 together with the carbon atom to which it is attached forms a C 3-7 cycloalkyl group optionally substituted with C 2-6 alkenyl;
  • Y is H, phenyl substituted with nitro, pyridyl substituted with nitro, or C ⁇ -6 alkyl optionally substituted with cyano, OH or C 3-7 cycloalt yl; provided that if R 4 or R 5 is H then Yis H;
  • (h) i is (i) Ci-io alkyl optionally substituted with phenyl, carboxyl.. C ⁇ -6 alkanoyl, 1-3 halogen, hydroxy, -OC(O)C ⁇ -6 alkyl, C ⁇ -6 alkoxy, amino optionally substituted with C 1-6 alkyl, amido, or (lower alkyl) amido; (ii) C 3-7 cycloalkyl, C 3- cycloalkoxy, or C 4-10 alkylcycloalklyl, each optionally substituted with hydroxy, carboxyl, (C ⁇ -6 alkoxy)carbonyl, amino optionally substituted with C ⁇ -6 alkyl, amido, or (lower alkyl) amido; (iii) C 6-1 o aryl or C .
  • R 5 is H; C ⁇ - alkyl optionally substituted with 1-3 halogens; or C 1-6 alkoxy provided R 4 is C HO alkyl;
  • X is O, S, SO, SO 2 , OCH 2 , CH 2 O or NH;
  • R 1 is Het, C 6-10 aryl or C - ⁇ 4 alkylaryl, each optionally substitutedL with
  • R a is C1. 6 alkyl, C 3-7 cycloalkyl, C ⁇ -6 alkoxy, C 3 . 7 cycloalkoxy, halo-Ci- ⁇ alkyl, CF 3 , mono-or di- halo-C ⁇ -6 alkoxy, cyano, halo, thioalkyL, hydroxy, alkanoyl, NO 2 , SH, , amino, C ⁇ -6 alkylamino, di (Ci- 6 ) alkylamino, di (C ⁇ - 6 ) alkylamide, carboxyl, (C ⁇ - 6 ) carboxyester, C 1-6 alkylsulfone, C 1-6 alkylsulfonamide, di (C ⁇ - 6 ) alkyl(alkoxy)amine, C 6- ⁇ o aryl, C - ⁇ 4 alkylaryl, or a 5-7 membered monocyclic heterocycle; and
  • R 6 and R 7 are each mdependently H; or C ⁇ - alkyl, C 2- ⁇ 0 alkenyl or C 6- ⁇ o aryl, each of which may be optionally substituted with halo, cyano, nitro, C ⁇ -6 alkoxy, amido, amino or phenyl;
  • A is
  • o p o z is - ⁇ - C — 5 - ⁇ - C — O — or - C — NR 6 R 7 .
  • p is 1, 2 or 3;
  • q is O or 1;
  • Ri is C 3-7 cycloalkyl, C 4-7 cycloalkenyl; C 7- ⁇ 4 alkylaryl; C - ⁇ 4 alkylaryloxy; C 8- ⁇ alkylarylester; or C] -8 alkyl optionally substituted with C ⁇ -6 alkoxy, hydroxy, halo, C - ⁇ o alkenyl, C - ⁇ o alkynyl, C 3-7 cycloalkyl, C 4-7 cycloalkenyl, C 6- ⁇ 0 aryl, C 6- ⁇ 0 aryloxy, C 8- ⁇ 5 alkylarylester or Het; or Ri is trialkylsilane or halogen, provided q is
  • R and R 7 are each independently H; or C ⁇ -6 alkyl, C 2- ⁇ 0 alkenyl or C 6- !0 aryl, each of which may be optionally substituted with halo, cyano, nitro, C ⁇ -6 alkoxy, amido, amino or phenyl.
  • R 2 is C 1-6 alkyl, C -6 alkenyl or C 3-7 cycloalkyl. More preferably, R 2 is C -6 alkenyl.
  • R 3 is C ⁇ -8 alkyl optionally substituted with C 6 aryl, C ⁇ -6 alkoxy, carboxy, hydroxy, aryloxy, C - ⁇ 4 alkylaryloxy, C -6 alkylester or C 8 - ⁇ 5 alkylarylester; C - ⁇ 2 alkenyl; C 3-7 cycloalkyl; or C 4- ⁇ 0 alkylcycloalkyl. More preferably, R 3 is C ⁇ -8 alkyl optionally substituted with C 1-6 alkoxy; or C 3-7 cycloalkyl.
  • Y is H.
  • j is (i) C ⁇ - ⁇ 0 alkyl optionally substituted with phenyl, carboxyl, C 1-6 alkanoyl, 1-3 halogen, hydroxy, C 1-6 alkoxy; (ii) C 3-7 cycloalkyl, C 3-7 cycloalkoxy, or C 4-10 alkylcycloalklyl; or (iii) C 6 - 10 aryl or C 7- ⁇ 6 arylalkyl, each optionally substituted with C ⁇ -6 alkyl or halogen.
  • i is (i) Ci-io alkyl optionally substituted with 1-3 halogen or C ⁇ -6 alkoxy; or (ii) C 3-7 cycloalkyl or C 4- ⁇ 0 alkylcycloalkyl.
  • R 5 is H or C ⁇ -6 alkyl optionally substituted with 1-3 halogens. More preferably, R 5 is H.
  • X is O or NH.
  • R' is Het; or C 6- ⁇ o aryl optionally substituted with R a . More preferably, R' is Het.
  • the heterocycle contains 1 or 2 nitrogen atoms and optionally a sulfur atom or an oxygen atom in the ring.
  • the heterocycle is substituted with at least one of C 1-6 alkyl, C 1-6 alkoxy, halo, C 6- ⁇ 0 aryl, C 7- ⁇ alkylaryl, or a 5-7 membered monocyclic heterocycle.
  • R a is C ⁇ -6 alkyl, C 3-7 cycloalkyl, C ⁇ -6 alkoxy, halo-C ⁇ - alkyl, halo, amino, C 6 aryl, or a 5-7 membered monocyclic heterocycle.
  • substituents from each grouping may be selected individually and combined in any combination which provides a stable compound in accordance with the present invention. Also, more than one substituent from each group may be substituted on the core group provided there are sufficient available binding sites. For example, each ofthe following R 6; R , R 8 or R 9 substituents, C ⁇ -6 alkoxy, C 6 aryl and a 5-7 membered monocyclic heterocycle, may be substituted on a bicyclic heterocycle. ⁇ In a prefened aspect, the compounds ofthe present invention have the structure of Formula II:
  • Ri is C 3-7 cycloalkyl, C 4-7 cycloalkenyl; C 7 _ ⁇ 4 alkylaryl; C . 1 alkylaryloxy; C 8- ⁇ 5 alkylarylester; or C ⁇ -8 alkyl optionally substituted with Ci- 6 alkoxy, hydroxy, halo, C - ⁇ o alkenyl, C 2-10 alkynyl, C 3-7 cycloalkyl, C 4-7 cycloalkenyl, C 6- ⁇ o aryl, C 6- ⁇ o aryloxy, C 8- ⁇ alkylarylester or Het; or Ri is trialkylsilane or halogen, provided q is
  • R is C ⁇ -6 alkyl, C 2-6 alkenyl or C 3-7 cycloalkyl;
  • R 3 is C ⁇ -8 alkyl optionally substituted with C 6 aryl, C ⁇ -6 alkoxy, carboxy, hydroxy, aryloxy, C 7- ⁇ 4 alkylaryloxy, C -6 alkylester, C 8- ⁇ alkylarylester; C 3- ⁇ alkenyl, C 3-7 cycloalkyl, or C 4- ⁇ 0 alkylcycloalkyl;
  • Y is H;
  • R4 is (i) Ci_io alkyl optionally substituted with phenyl, carboxyl, C ⁇ -6 alkanoyl, 1-3 halogen, hydroxy, C ⁇ -6 alkoxy; (ii) C 3-7 cycloalkyl, C 3-7 cycloalkoxy, or C 4- ⁇ o alkylcycloalklyl; or (iii) C 6 - ⁇ o aryl or C 7- ⁇ 6 arylalkyl, each optionally substituted with C ⁇ -6 alkyl or halogen; (g) R 5 is H or C ⁇ -6 alkyl optionally substituted with 1-3 halogens;
  • R' is Het; or C 6- ⁇ o aryl optionally substituted with R a ;
  • R a is C ⁇ -6 alkyl, C 3-7 cycloalkyl, C ⁇ -6 alkoxy, halo-C ⁇ - 6 alkyl, halo, amino, C 6 aryl, or a 5-7 membered monocyclic heterocycle; and (k) R 6 and R 7 are each independently H; or C ⁇ -6 alkyl, C 2- ⁇ o alkenyl or C 6- 10 aryl, each of which may be optionally substituted with halo, cyano, nitro, C ⁇ -6 alkoxy, amido, amino or phenyl;
  • R' is a bicyclic heterocycle.
  • the heterocycle contains 1 or 2 nitrogen atoms and optionally a sulfur atom or an oxygen atom in the ring. More preferably, the heterocycle is substituted with at least one of C ⁇ -6 alkyl, C ⁇ -6 alkoxy, halo, C 6 aryl, and a 5-7 membered monocyclic heterocycle.
  • R' is a bicyclic heterocycle containing 1 nitrogen atom and substituted with methoxy and at least one of a C 6 aryl and a 5-7 membered monocyclic heterocycle.
  • R' is a monocyclic heterocycle.
  • the heterocycle contains 1 or 2 nitrogen atoms and optionally a sulfur atom or an oxygen atom in the ring.
  • the heterocycle is substituted with at least one of C ⁇ -6 alkyl, C ⁇ -6 alkoxy, halo, C 6- ⁇ o aryl, C 7- ⁇ 4 alkylaryl, or a 5-7 membered monocyclic heterocycle.
  • R' is a monoyclic heterocycle containing 1 or 2 nitrogen atoms and substituted with methoxy and at least one of a C 6 aryl and a 5-7 membered monocyclic heterocycle.
  • the compounds have the structure of Formula III
  • p 1, 2 or 3;
  • Ri is C 7-14 alkylaryl; C ⁇ -8 alkyl optionally substituted with C ⁇ -6 alkoxy,
  • R 2 is C 2-6 alkenyl
  • R 3 is C ⁇ -8 alkyl
  • (e) j is Ci-io alkyl
  • R' is a bicyclic heterocycle optionally substituted with R a ;
  • R a is C ⁇ -6 alkyl, C ⁇ -6 alkoxy, halo, C 6 aryl, or a 5-7 membered monocyclic heterocycle; or a pharmaceutically acceptable enantiomer, diastereomer salt, solvate or prodrag thereof.
  • Ri is cyclopropyl or cyclobutyl
  • R is vinyl
  • R 3 is t-butyl
  • R is t-butyl
  • R' is quinoline or isoquinoline optionally substituted with R a
  • R a is C ⁇ -6 alkoxy. More preferably, R a further includes at least one of C 6 aryl or a 5-7 membered monocyclic heterocycle.
  • the compounds ofthe present invention which are substituted with a basic group, can form salts by the addition of a pharmaceutically acceptable acid.
  • the acid addition salts are formed from a compound of Formula I and a pharmaceutically acceptable inorganic acid, including but not limited to hydrochloric, hydrobromic, hydroiodic, sulfuric, phosphoric, or organic acid such as /j-toluenesulfonic, methanesulfonic, acetic, benzoic, citric, malonic, fumaric, maleic, oxalic, succinic, sulfamic, or tartaric.
  • a pharmaceutically acceptable inorganic acid including but not limited to hydrochloric, hydrobromic, hydroiodic, sulfuric, phosphoric, or organic acid such as /j-toluenesulfonic, methanesulfonic, acetic, benzoic, citric, malonic, fumaric, maleic, oxalic, succinic, sulfa
  • examples of such pharmaceutically acceptable salts include chloride, bromide, iodide, sulfate, phosphate, methanesulfonate, citrate, acetate, malonate, fumarate, sulfamate, and tartrate.
  • Salts of an amine group may also comprise quaternary ammonium salts in which the amino nitrogen canies a suitable organic group such as an alkyl, alkenyl, alkynyl or aralkyl moiety.
  • Base addition salts include those derived from inorganic bases which include, for example, alkali metal salts (e.g. sodium and potassium), alkaline earth metal salts (e.g. calcium and magnesium), aluminum salts and ammonium salts.
  • alkali metal salts e.g. sodium and potassium
  • alkaline earth metal salts e.g. calcium and magnesium
  • aluminum salts e.g. aluminum salts and ammonium salts.
  • suitable base addition salts include salts of physiologically acceptable organic bases such as trimethylamine, triethylamine, mo ⁇ holine, pyridine, piperidine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amrne, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, N-benzyl- ⁇ -phenethylamine, dehydroabietylamine, N,N' -bishydroabietylamine, glucamine, N-methylglucamine, collidine, quinme, quinoline, ethylenediamine, ornithine, choline, N,N'-benzylphenethylamine, chloroprocaine, diethanolamine, diethylamine, piperazine, fris(hydroxymethyl)aminomethane and tetramethylammonium hydroxide and
  • Certain compounds ofthe present invention, and their salts may also exist in the form of solvates with water, for example hydrates, or with organic solvents such as methanol, ethanol or acetonitrile to form, respectively, a methanolate, ethanolate or acetonitrilate.
  • the present invention includes each solvate and mixtures thereof.
  • compounds ofthe present invention may exhibit polymo ⁇ hism.
  • the present invention also encompasses any such polymo ⁇ hic form.
  • the compounds ofthe present invention also contain two or more chiral centers.
  • the compounds may include PI cyclopropyl element of formula
  • C 2 each represent an asymmetric carbon atom at positions 1 and 2 of the cyclopropyl ring.
  • the presence of these two asymmetric centers means that the compounds can exist as racemic mixtures of diastereomers, such as the diastereomers wherein R 2 is configured either syn to the amide or syn to the carbonyl as shown below.
  • R 2 is syn to carbonyl
  • R 2 is syn to carbonyl
  • R 2 is syn to amide
  • R 2 is syn to amide
  • the present invention includes both enantiomers and mixtures of enantiomers such as racemic mixtures.
  • the enantiomers may be resolved by methods known to those skilled in the art, for example, by formation of diastereoisomeric salts which may be separated by crystallization, gas-liquid or liquid chromatography, selective reaction of one enantiomer with an enantiomer-specific reagent. It will be appreciated that where the desired enantiomer is converted into another chemical entity by a separation technique, then an additional step is required to form the desired enantiomeric form. Alternatively, specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.
  • the compounds ofthe present invention may be in the form of a prodrag.
  • Simple aliphatic or aromatic esters derived from, when present, acidic groups pendent on the compounds of this invention are prefened prodrugs.
  • double ester type prodrags such as (acyloxy) alkyl esters or (alkoxycarbonyl)oxy)alkyl esters.
  • Certain compounds ofthe present invention may also exist in different stable conformational forms which may be separable. Torsional asymmetry due to restricted rotation about an asymmetric single bond, for example because of steric hindrance or ring strain, may permit separation of different conformers.
  • the present invention mcludes each conformational isomer of these compounds and mixtures thereof.
  • Certain compounds ofthe present invention may exist in zwitterionic form and the present invention includes each zwitterionic form of these compounds and mixtures thereof.
  • the starting materials useful to synthesize the compounds ofthe present invention are known to those skilled in the art and can be readily manufactured or are commercially available.
  • the compounds ofthe present invention can be manufactured by methods known to those skilled in the art, see e.p., US Patent No. 6,323, 180 and US Patent Appl. 20020111313 Al.
  • the following methods set forth below are provided for illustrative purposes and are not intended to limit the scope ofthe claimed mvention. It will be recognized that it may be prefened or necessary to prepare such a compound in which a functional group is protected using a conventional protecting group then to remove the protecting group to provide a compound ofthe present invention.
  • the details concerning the use of protecting groups in accordance with the present invention are known to those skilled in the art.
  • the compounds ofthe present invention may, for example, be synthesized according to a general process as illustrated in Scheme I (wherein CPG is a carboxyl protecting group and APG is an amino protecting group): Scheme I
  • the PI, P2, and P3 can be linked by well known peptide coupling techniques.
  • the PI, P2, and P3 groups may be linked together in any order as long as the final compound conesponds to peptides ofthe invention.
  • P3 can be linked to P2-P1; or PI linked to P3-P2.
  • peptides are elongated by deprotecting the ⁇ -amino group ofthe N-terminal residue and coupling the unprotected carboxyl group ofthe next suitably N-protected amino acid through a peptide linkage using the methods described. This deprotection and coupling procedure is repeated until the desired sequence is obtained. This coupling can be performed with the constituent amino acids in stepwise fashion, as depicted in Scheme I.
  • Coupling between two amino acids, an amino acid and a peptide, or two peptide fragments can be canied out using standard coupling procedures such as the azide method, mixed carbonic-carboxylic acid anhydride (isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-soluble carbodiimide) method, active ester (p-nitrophenyl ester, N-hydroxysuccinic imido ester) method, Woodward reagent K-method, - o-
  • the coupling step involves the dehydrative coupling of a free carboxyl of one reactant with the free amino group ofthe other reactant in the present of a coupling agent to form a linking amide bond.
  • coupling agents are found in general textbooks on peptide chemistry, for example, M. Bodanszky, "Peptide Chemistry", 2 nd rev ed., Springer- Verlag, Berlin, Germany, (1993).
  • suitable coupling agents are N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole in the presence of N,N'-dicyclohexylcarbodiimide or N-ethyl-N'-[(3-dimethylamino)propyl]carbodiimide.
  • a practical and useful coupling agent is the commercially available (benzotriazol- 1 -yloxy)tris-(dimethylamino)phosphonium hexafluorophosphate, either by itself or in the present of 1-hydroxybenzotriazole or 4-DMAP.
  • Another practical and useful coupling agent is commercially available 2-(lH-benzotriazol-l-yl)-N, N, N', N'-tetramethyluronium tetrafluoroborate. Still another practical and useful coupling agent is commercially available O-(7-azabenzotrizol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate.
  • the coupling reaction is conducted in an inert solvent, e.g. dichloromethane, acetonitrile or dimethylfomiamide.
  • An excess of a tertiary amine e.g.
  • reaction temperature usually ranges between 0 °C and 50 °C and the reaction time usually ranges between
  • the functional groups ofthe constituent amino acids generally must be protected during the coupling reactions to avoid formation of undesired bonds.
  • Protecting groups that can be used are listed, for example, in Greene, "Protective
  • APG ⁇ -amino group of each amino acid to be coupled to the growing peptide chain
  • acyl groups such as formyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl
  • aromatic carbamate groups such as benzyloxycarbonyl (Cbz or Z) and substituted bensyloxycarbonyls, and 9-fluorenyhnethyloxycarbonyl (Fmoc)
  • 3) aliphatic carbamate groups such as tert-butyloxycarbonyl (Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl
  • cyclic alkyl carbamate groups such as cyclopentyloxycarbonyl and adamantyloxycarbonyl
  • alkyl groups such as triphenylmethyl and benzyl
  • 6)trialkylsilyl such as trimethylsilyl
  • thiol containing groups such as phenylthiocarbony
  • the prefened ⁇ -amino protecting group is either Boc or Fmoc. Many amino acid derivatives suitably protected for peptide synthesis are commercially available.
  • the ⁇ -amino protecting group ofthe newly added amino acid residue is cleaved prior to the coupling ofthe next amino acid.
  • the Boc group is used, the methods of choice are trifluoroacetic acid, neat or in dichloromethane, or HCI in dioxane or in ethyl acetate.
  • the resulting ammonium salt is then neutralized either prior to the coupling or in situ with basic solutions such as aqueous buffers, or tertiary amines in dichloromethane or acetonitrile or dimethylformamide.
  • the reagents of choice are piperidine or substituted piperidine in dimethylformamide, but any secondary amine can be used.
  • the deprotection is canied out at a temperature between 0°C and room temperature (rt or RT) usually 20-22°C.
  • any ofthe amino acids having side chain functionalities must be protected during the preparation ofthe peptide using any ofthe above-described groups.
  • Those skilled in the art will appreciate that the selection and use of appropriate protecting groups for these side chain functionalities depend upon the amino acid and presence of other protectmg groups in the peptide. The selection of such protecting groups is important in that the group must not be removed during the deprotection and coupling ofthe ⁇ -amino group.
  • Boc when used as the ⁇ -amino protecting group, the following side chain protecting group are suitable: ⁇ toluenesulfbnyl (tosyl) moieties can be used to protect the amino side chain of amino acids such as Lys and Arg; acetamidomethyl, benzyl (Bn), or tert-butylsulfonyl moieties can be used to protect the sulfide containing side chain of cysteine; bencyl (Bn) ethers can be used to protect the hydroxy containing side chains of serine, threonine or hydroxyproline; and benzyl esters can be used to protect the carboxy containing side chains of aspartic acid and glutamic acid.
  • toluenesulfbnyl (tosyl) moieties can be used to protect the amino side chain of amino acids such as Lys and Arg
  • Fmoc is chosen for the ⁇ -amine protection
  • usually tert-butyl based protecting groups are acceptable.
  • Boc can be used for lysine and arginine, tert-butyl ether for serine, threonine and hydroxyproline, and tert-butyl ester for aspartic acid and glutamic acid.
  • Triphenylmethyl (Trityl) moiety can be used to protect the sulfide containing side chain of cysteine.
  • Cyclopentanol is treated with phosgene to furnish the conesponding chloroformate.
  • the chloroformate is treated with the desired NH -tripeptide in the presence of a base such as triethylamine to afford the cyclopentylcarba ate.
  • the ⁇ -carboxyl group ofthe C-terminal residue is usually protected as an ester (CPG) that can be cleaved to give the carboxylic acid.
  • CPG ester
  • Protecting groups that can be used include: 1) alkyl esters such as methyl, trimethylsilylethyl and t-butyl, 2) aralkyl esters such as benzyl and substituted benzyl, or 3) esters that can be cleaved by mild base treatment or mild reductive means such as trichloroethyl and phenacyl esters.
  • the resulting ⁇ -carboxylic acid (resulting from cleavage by mild acid, mild base treatment or mild reductive means) is coupled with a ASO 2 NH 2 as described herein.
  • Scheme II further shows the general process wherein compounds of Formula I are constructed by the coupling of tripeptide carboxylic acid intermediate (1) with a P 1 ⁇ sulfonamide.
  • Said coupling reaction requires treatment of carboxylic acid (1) with a coupling reagent such as carbonyl diimidazole in a solvent such as THF, which can be heated to reflux, followed by the addition ofthe formed derivative of (1), to the PL sulfonamide, in a solvent such as THF or methylene chloride in the presence of a base such as DBU.
  • TFA amine salt can be directly employed in the subsequent coupling reaction or as an alternative the TFA amine salt can be first converted to the HCI amine salt, and this HCI amine salt is used in said coupling reaction as shown in Scheme III.
  • the coupling of said HCI amine salt (3) with the carboxyl terminus a P4-P3-P2 intermediate can be achieved using coupling reagents, such as HATU, in solvents such as dichloromethane to provide compounds of Formula I (4).
  • the resulting trifluoroacetic acid salt can be coupled with the carboxyl terminus ofthe P4-P3 element using standard coupling agents such as PyBop in the presence of base such as diisopropyl amine, and using solvents such methylene chloride to provide compounds of Formula I (4).
  • the P4-P3-P2 intermediate utilized in the above schemes can be constructed as previously described with a further description of this process shown in general Scheme V.
  • the free carboxyl terminus ofthe P4-P3 intermediate (1) can be coupled to the an ⁇ ino terminus ofthe P2 element to provide the P4-P3-P2 dipeptide (2).
  • the carboxyl terminus ofthe P4-P3-P2 intermediate can be deprotected by saponification ofthe ester group to provide P4-P3-P2 as the free carboxylic acid (3).
  • Intermediates like (3) can be converted to compounds of Formula I using the methods described herein.
  • intermediate (2) can be used as starting materials for the preparation of compounds of Fonnula I wherein the P3 group is capped with an amide or a sulfonamide, or thiourea, or a sulfamide.
  • the construction of said compounds of Formula I can be achieved using standard conditions for the formation of said P4 functionalities from amines.
  • the PL terminus is inco ⁇ orated into the molecules using one ofthe general processes outlined above and described in more detail below.
  • the PL elements that is the substituted cycloalkyl- sulfonamides are commercially available or can be prepared from the conesponding alkyl- or cycloalkyl-sulfonyl chloride by treating said sulfonyl chloride with ammonia .
  • these sulfonamides can be synthesized using the general process outline in Scheme VII.
  • the PI elements utilized in generating compounds of Formula I are in some cases commercially available, but are otherwise synthesized ushig the methods described herein and subsequently inco ⁇ orated into compounds of Formula I using the methods described herein.
  • the substituted PI cyclopropylamino acids can be synthesized following the general process outline in Scheme VIII.
  • this reaction is selective in that one ofthe enantiomers undergoes the reaction at a much greater rate than its mirror image providing for a kinetic resolution ofthe intermediate racemate.
  • the more prefened stereoisomer for integration into compounds of Formula I is 5a which houses the ( IR, 2S) stereochemistry.
  • this enantiomer does not undergo ester cleavage and thereby this enantiomer 5 a is recovered from the reaction mixture.
  • the less prefened enantiomer ,5b with houses the (IS, 2R) stereochemistry undergoes ester cleavage, i.e., hydrolysis, to provide the free acid 6.
  • the ester 5a can be separated from the acid product 6 by routine methods such as, for example, aqueous extraction methods or chromotography.
  • Scheme IX shows the coupling of an N-protected C4-hydroxyproline moiety with a heterocycle to form intermediate (4) and the subsequent modification of said intermediate (4) to a compound of Formula I by the process of peptide elongation as described herein.
  • a base is employed in the first step.
  • this coupling can be done using bases such as potassium tert-butoxide, or sodium hydride, in solvent such as DMF or DMSO or THF.
  • This coupling to the isoquinoline ring system occurs at the CI position (numbering for isoquinoline ring system shown in intermediate 2 of Scheme IX) and is directed by the chloro group which is displaced in this process.
  • the alternative leaving groups can be utilized at this position such as a fluoro as shown in the Scheme.
  • Said fluoro intermediates (3) are available from the conesponding chloro compound using literature procedures described herein.
  • the position of the leaving group (chloro or fluoro) in a given ring system can vary as shown in Scheme X, wherein the leaving group (fluoro in this example) is in the C6 position ofthe isoquinoline ring system of intermediate (2).
  • isoquinolines are inco ⁇ orated into the final compounds and specifically into the P2 region of said compounds.
  • One skilled in the art would recognize that a number of general methods are available for the synthesis of isoquinolines. Moreoever, said isoquinolines generated by these methods can be readily inco ⁇ orated into final compounds of Formula I using the processes described herein.
  • One general methodology for the synthesis of isoquinolines is shown in Scheme XII, wherein cinnamic acid derivatives, shown in general form as stracture (2) are
  • acylazides from carboxylic acids as for example said carboxylic acid can be treated with diphenylphosphorylazide (DPP A) in an aprotic solvent such as methylene chloride in the presence of a base, hi the next step ofthe reaction sequence the acylazide (3) is coverted to the corresponding isoquinolone (4) as shown in the Scheme.
  • DPP A diphenylphosphorylazide
  • aprotic solvent such as methylene chloride
  • the acylazide (3) is coverted to the corresponding isoquinolone (4) as shown in the Scheme.
  • the acylazide is heated to a temperature of approximately 190 degress celcius in a high boiling solvent such a diphenylmethane.
  • This reaction is general and provides moderate to good yields of substituted isoquinolone from the conesponding cinnamic acid derivatives.
  • said cinnamic acid derivatives are available commercially or can be obtained from the conesponding benzaldehyde (1) derivative by direct condensation with malonic acid or derivatives thereof and also by employing a Wittig reaction.
  • the intermediate isoquinolones (4) of Scheme XII can be converted to the conesponding 1- 1 chloroisoquinoline by treatment with phosphorous oxychloride. This reaction is general and can be applied to any ofthe isoquinolones, quinolones or additional heterocycles as shown herein to covert a hydroxy substituent to the conesponding chloro compound when said hydroxy is in conjugation with a nitrogen atom in said heterocylic ring systems.
  • the first step in this sequence is the formation ofthe isoquinoline N-oxide(4) by treatment of isoquinoline (3) with meta-chloroperbenzoic acid in an aprotic solvent such as dichloromethane.
  • Intermediate (4) can be converted to the conesponding 1-chloroquinoline by treatment with phosphorous oxychloroide in refluxing chloroform.
  • This two step process is general and can be employed to procure chloroisoquinolines and chloroquinolines from the conesponding isoquinolines and quinolines respectively.
  • Scheme XIV Another method for the synthesis ofthe isoquinoline ring system is shown in Scheme XIV. In this process an ortho-alkylbenzamide derivative (1) is treated with a strong Scheme XIV
  • keto imine (2) can be converted to the conesponding isoquinoline by condensation with ammonium acetate at elevated temperatures.
  • This method is general and can be used for the synthesis of substituted isoquinolines.
  • Said isoquinolines can be converted to the conesponding l-chloroquinoline by the methods described herein.
  • 6-fluoro-isoquinoline to the conesponding l-chloro-6-alkoxy-isoquinoline species by treatment of (1) of (eq.1) with a sodium or potassium alkoxide species in the alcohol solvent from which the alkoxide is derived at room temperature. In some cases it may be necessary to heat the reaction to drive it to completion.
  • Said chloroquinoline can be inco ⁇ orated into a compound of Formula I using the art described herein. Modifications of a P2 heterocyclic element can also be done after it's inco ⁇ oration into compounds of Formula I as shown in (eq.2) of Scheme VXII. Specifically compounds such as (1) in (eq.2) of Scheme VXII. Specifically compounds such as (1) in (eq.2)
  • reaction which contain a leaving group in the pthalazine nucleus can be displaced by a nucleophile such as an alkoxide in solvents such as the conesponding alcohol from which the alkoxide is derived.
  • a nucleophile such as an alkoxide in solvents such as the conesponding alcohol from which the alkoxide is derived.
  • Scheme XVIII provides a general example for the modification of heterocycles as defined herein by employing palladium mediated coupling reactions.
  • Said couplings can be employed to functionalize a heterocycle at each position ofthe ring system providing said ring is suitably activated or functionalized, as for example with a chloride as shown in the Scheme.
  • This sequence begins with 1-chloroisoquinoline (1) which upon treatment with metachloroperbenzoic acid can be converted to the conesponding N-oxide (2).
  • Said intermediate (2) can be converted to the conesponding 1,3-dichloroisoquinoline (3) by treatment with phosphorous oxychloride in refluxing chloroform.
  • Intermediate (3) can be coupled with N-Boc-4- hydroxyproline by the methods described herein to provide intermediate (5) as shown in the Scheme.
  • Intermediate (5) can undergo a Suzuki coupling with an aryl boronic acid, in the presence of a palladium reagent and base, and in a solvent such as THF or toluene or DMF to provide the C3-arylisoquinoline intermediate (6).
  • Heteroarylboronic acids can also be employed in this Pd mediated coupling process to provide C3-heteroarylisoquinolines.
  • Intermediate (6) can be converted into final compounds of Formula I by the methods described herein.
  • the isoquinoline ring system of intermediate (3) can be further functionalized by employing either Suzuki couplings (Process 1: subjecting (3) to heteroaryl or aryl boronic acids in the presence of a palladium catalyst such as palladium teixa(triphenylphosphine) and a base such as cesium carbonate in solvents such as DMF) or Stille couplings (Process 2: subjecting (3) to heteraryl or aryl tin dervatives in " the presence of palladium catalyst such as palladium tetra(triphenylphosphine in solvents such as toluene).
  • Suzuki couplings Process 1: subjecting (3) to heteroaryl or aryl boronic acids in the presence of a palladium catalyst such as palladium teixa(triphenylphosphine) and a base such as cesium carbonate in solvents such as DMF
  • Stille couplings Process 2: subjecting (3) to heteraryl or
  • Palladium reactions can also be employed to couple C4-amino proline elements with functionalized heterocycles.
  • Scheme XX shows intermediate (1) coupling with a functionalized isoquinoline in the presence of a palladium catalyst and a base in a solvent such as toluene.
  • Intermediates like (3) can be converted to compounds of Formula I using the methods described herein.
  • the present invention also provides compositions comprising a compound of the present invention, or a pharmaceutically acceptable enantiomer, diastereomer, salt, solvate or prodrag thereof, and a pharmaceutically acceptable canier.
  • compositions ofthe present invention comprise a therapeutically effective amount of a compound ofthe invention, or a pharmaceutically acceptable enantiomer, diastereomer, salt, solvate or prodrag thereof, and a pharmaceutically acceptable canier, with a pharmaceutically acceptable canier, e.g., excipient, or vehicle diluent.
  • a pharmaceutically acceptable canier e.g., excipient, or vehicle diluent.
  • the active ingredient, i.e., compound, in such compositions typically comprises from 0.1 weight percent to 99.9 percent by weight ofthe composition, and often comprises from about 5 to 95 weight percent.
  • a composition comprising the compound of formula 1 and a pharmaceutically acceptable canier.
  • the composition further comprises a compound having anti-HCV activity.
  • anti-HCV activity means the compound is effective to inhibit the function of a target selected from the group consisting of HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH and a nucleoside analog for the treatment of an HCV infection.
  • the other compound having anti-HCV activity is effective to inhibit the function of target in the HCV life cycle other than the HCV NS3 protease protein.
  • the compound having anti-HCV activity is an interferon.
  • the interferon is selected from the group consisting of interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, lymphoblastiod interferon tau.
  • the compound having anti-HCV activity is selected from the group consisting of interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, hiterfering RNA, anti-sense RNA, Imiqimod, ribavirin, an inosine 5'-monophospate dehydrogenase inhibitor, amantadine, and rimantadine.
  • the composition comprises a compound ofthe invention, an interferon and ribavirin.
  • the compound having anti-HCV activity is a small molecule compound.
  • small molecule compound means a compound having a molecular weight of less than 1,500 daltons, preferably less than 1000 daltons.
  • the small molecule compound is effective to inhibit the function of a target selected from the group consisting of HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, inosine monophophate dehydrogenase ("IMPDH”) and a nucleoside analog for the treatment of an HCV infection.
  • IMPDH inosine monophophate dehydrogenase
  • Table 1 lists some illustrative examples of compounds that can be administered with the compounds of this invention.
  • the compounds of the invention can be administered with other anti-HCV activity compounds in combination therapy, either jointly or separately, or by combining the compounds into a composition.
  • compositions of this invention maybe administered orally, parenterally or via an implanted reservoir. Oral administration or administration by injection are prefened. In some cases, the pH ofthe formul-ation may be adjusted with pharmaceutically acceptable acids, bases or buffers to ejnhance the stability ofthe formulated compound or its delivery form.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, mtramusculax, intra- articular, intrasynovial, intrasternal, intrathecal, and intralesional injection or infusion techniques.
  • the pharmaceutical compositions of this invention may be administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, caniers which are commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried com starch.
  • the active ingredient is combined with emulsifying and suspending agents.
  • certain sweetening and/or flavoring and/or coloring agents may be added.
  • Other suitable caniers for the above noted compositions can be found in standard pharmaceutical texts, e.g. in "Remington's Pharmaceutical Sciences", 19th ed., Mack Publishing Company, Easton, Penn., 1995.
  • compositions of this invention can be prepared by known procedures using well-known and readily available ingredients.
  • the compositions of this invention may be formulated so as to provide quick, sustained or delayed release ofthe active ingredient after administration to the patient by employing procedures well known in the art.
  • the active ingredient will usually be admixed with a canier, or diluted by a canier, or enclosed within a canier which may be in the form of a capsule, sachet, paper or other container.
  • the canier serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, excipient or medium for the active ingredient.
  • compositions can be in the fonn of tablets, pills, powders, beadlets, lozenges, sachets, elixirs, suspensions, emulsions, solutions, syraps, aerosols, (as a solid or in a liquid medium), soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders and the like. Further details concerning the design and preparation of suitable delivery forms ofthe pharmaceutical compositions ofthe invention are known to those skilled in the art.
  • Dosage levels of between about 0.01 and about 1000 milligram per kilogram (“mg/kg”) body weight per day, preferably between about 0.5 and about 250 mg/kg body weight per day ofthe compounds ofthe invention are typical in a monotherapy for the prevention and treatment of HCV mediated disease.
  • the pharmaceutical compositions of this invention will be administered from about 1 to about 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy.
  • the amount of active ingredient that may be combined with the canier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • compositions of this invention comprise a combination of a compound ofthe invention and one or more additional therapeutic or prophylactic agent
  • both the compound and the additional agent are usually present at dosage levels of between about 10 to 100%, and more preferably between about 10 and 80% ofthe dosage normally administered in a monotherapy regimen.
  • compositions may be administered in vivo to mammals, such as man, to inhibit HCV NS3 protease or to treat or prevent HCV viras infection.
  • another aspect of this invention provides methods of inhibiting HCV NS3 protease activity in patients by administering a compound ofthe present invention or a pharmaceutically acceptable enantiomer, diastereomer, salt or solvate thereof.
  • a method of treating an HCV infection in a patient comprising administering to the patient a therapeutically effective amount ofthe compound ofthe invention, or a pharmaceutically acceptable enantiomer, diastereomer, solvate, prodrag or salt thereof.
  • the method of administering the compound is effective to inhibit the function ofthe HCV NS3 protease protein.
  • the method further comprises administering another compound having anti-HCV activity (as described above) prior to, after or concunently with a compound ofthe invention.
  • the compounds ofthe invention may also be used as laboratory reagents.
  • Compounds may be instrumental in providing research tools for designing of viral replication assays, validation of animal assay systems and stractural biology studies to further enhance knowledge ofthe HCV disease mechanisms. Further, the compounds ofthe present invention are useful in establishing or determining the binding site of other antiviral compounds, for example, by competitive inhibition.
  • the compounds of this invention may also be used to treat or prevent viral contamination of materials and therefore reduce the risk of viral infection of laboratory or medical personnel or patients who come in contact with such materials, e.g., blood, tissue, surgical instruments and garments, laboratory instruments and garments, and blood collection or transfusion apparatuses and materials.
  • materials e.g., blood, tissue, surgical instruments and garments, laboratory instruments and garments, and blood collection or transfusion apparatuses and materials.
  • Solution percentages express a weight to volume relationship, and solution ratios express a volume to volume relationship, unless stated otherwise.
  • Nuclear magnetic resonance (NMR) spectra were recorded either on a Braker 300, 400 or 500 MHz spectrometer; the chemical shifts (5) are reported in parts per million. Flash chromatography was canied out on silica gel (SiO ) according to Still's flash chromatography technique (W.C. Still et al., J. Org. Chem., (1978), 43, 2923).
  • Method B - YMC ODS-A S7 C18 3.0x50 mm
  • Method C - YMC S7 C18 3.0x50 mm
  • Method D - YMC Xtena ODS S7 3.0x50 mm
  • Method E - YMC Xtena ODS S7 3.0x50 mm
  • Method F - YMC ODS-A S7 C 18 3.0x50 mm
  • Preparation of Intennediates Preparation of PI Intermediates: The PI intennediates described in this section can be used to prepare compounds of Formula I by the methods described herein.
  • Glycine ethyl ester hydrochloride (303.8 g, 2.16 mole) was suspended in tert- butylmethyl ether (1.6 L). Benzaldehyde (231 g, 2.16 mole) and anhydrous sodium sulfate (154.6 g, 1.09 mole) were added and the mixture cooled to 0 °C using an ice- water bath. Triethylamine (455 mL, 3.26 mole) was added dropwise over 30 min and the mixture stined for 48 h at rt. The reaction was then quenched by addition of ice- cold water (1 L) and the organic layer was separated.
  • the aqueous phase was extracted with tert-butyhnethyl ether (0.5 L) and the combined organic phases washed with a mixture of saturated aqueous NaHCO 3 (1 L) and brine (1 L). The solution was dried over MgSO 4 , concentrated in vacuo to afford 392.4 g ofthe N- benzyl imine product as a thick yellow oil that was used directly in the next step.
  • the organic phases were combined, 1 ⁇ HCI (1 L) was added and the mixture stined at room temperature for 2 h. The organic phase was separated and extracted with water (0.8 L). The aqueous phases were then combined, saturated with salt (700 g), TBME (1 L) was added and the mixture cooled to 0 °C. The stined mixture was then basif ⁇ ed to pH 14 by the dropwise addition of 10 N NaOH, the organic layer separated, and the aqueous phase extracted with TBME (2 x 500 mL). The combined organic extracts were dried (MgSO ) and concentrated to a volume of IL.
  • N-Boc-(lR,2S)/(lS,2R)-l -amino-2-vinylcyclopropane carboxylic acid ethyl ester (9.39 g, 36.8 mmol) was dissolved in 4 ⁇ HCl dioxane (90 ml, 360 mmol) and was stined for 2 h at rt. The reaction mixture was concentrated to supply (lR,2S)/(lS,2R)-l-amino-2-vinylcyclopropane carboxylic acid ethyl ester hydrochloride in quantitative yield (7 g, 100%).
  • the aqueous layer from the extraction process was then acidified to pH 2 with 50% H 2 SO 4 and extracted with MTBE (2 x 2 L).
  • the MTBE extract was washed with water (3 x 100 mL) and evaporated to give the acid as light yellow solid (42.74 g; purity: 99% @ 210 nm, containing no ester).
  • enantio-excess ofthe ester was determined to be 44.3% as following: 0.1 mL ofthe reaction mixture was removed and mixed well with 1 mL ethanol; after centrifugation, 10 microliter (" ⁇ l") ofthe supernatant was analyzed with the chiral HPLC. To the remaining reaction mixture, 0.1 mL of DMSO was added, and the plate was incubated for additional 3 days at 250 ⁇ m at 40°C, after which four L of ethanol was added to the well. After centrifugation, 10 ⁇ l ofthe supernatant was analyzed with the chiral HPLC and enantio-excess ofthe ester was determined to be 100%.
  • enantio-excess ofthe ester was determined to be 39.6% as following: 0.1 mL ofthe reaction mixture was removed and mixed well with 1 mL ethanol; after cemifugation, 10 ⁇ l ofthe supernatant was analyzed with the chiral HPLC. To the remaining reaction mixture, 0.1 mL of DMSO was added, and the plate was incubated for additional 3 days at 250 ⁇ m at 40°C, after which four mL of ethanol was added to the well. After centrifugation, 10 ⁇ l ofthe supernatant was analyzed with the chiral HPLC and enantio-excess ofthe ester was determined to be 100%.
  • Step 1 Preparation of 2-Ethylcyclopropane- 1 , 1 -dicarboxylic acid di-tert-butyl ester, shown below.
  • Step 2 Preparation of racemic 2-Ethylcyclopropane- 1,1 -dicarboxylic acid tert-butyl ester, shown below.
  • Step 1 The product of Step 1 (18.3 g, 67.8 mmol) was added to a suspension of potassium tert-butoxide (33.55 g, 299.0 mmol) in dry ether (500 mL) at 0 °C, followed by H 2 0 (1.35 mL, 75.0 mmol) and was vigorously stined overnight at rt.
  • the reaction rnixture was poured in a mixture of ice and water and washed with ether (3x).
  • the aqueous layer was acidified with a 10% aq. citric acid solution at 0°C and extracted with EtOAc (3x).
  • Step 3 Preparation of (1R,2R)/(1S,2S) 2-Ethyl-l-(2- trimethylsilanylethoxycarbonylamino)cyclopropane-carboxylic acid tert-butyl ester, shown below.
  • reaction mixture was filtered, diluted with Et 2 O, washed with a 10% aqueous citric acid solution, water, saturated aqueous NaHCO 3 , water (2x), brine (2X), dried (MgSO 4 ) and concentrated in vacuo.
  • Step 3 Preparation ofthe titled product, l-amino-spiro[2.3]hexane-l -carboxylic acid methyl ester hydrochloride salt.
  • Step 1 Spiro[2.4]heptane- 1,1 -dicarboxylic acid dimethyl ester, shown below, was prepared as follows.
  • Step 2 Preparation of Spiro[2.4]heptane-l,l-dicarboxylic acid methyl ester, shown below, was prepared as follows.
  • Step 3 Preparation of l-Arnino-spiro[2.4]heptane-l -carboxylic acid methyl ester hydrochloride salt, shown below, was prepared as follows.
  • Step 1 Spiro[2.2]pentane-l,l-dicarboxylic acid dimethyl ester, shown below, was prepared as follows.
  • Step 3 l-Amino-spiro[2.2]pentane-l -carboxylic acid methyl ester hydrochloride salt, shown below, was prepared as follows.
  • Isoquinoline (1) and substituted analogues thereof can be inco ⁇ orated into P2 elements using the two methods outline above and described in detail herein.
  • Said P2 elements (3) can then be converted into compounds of Formula I using procedures analogous to those described herein for similar isoquinoline analogues.
  • Isoxazole and oxazole heterocycle (1) and analogues thereof can be prepared using know chemistry and inco ⁇ orated into compounds of Formula I using the chemistry described herein for similar isoxazolepyridine intermediates as shown in section B.
  • P 1 prime elements prepared below can be used to prepare compounds of Formula I by using the methods described herein.
  • Step 1 Preparation of N-tert-Butyl-(3-chloro)propylsulfonamide tert-Bixtylamine (3.0 mol, 315.3 mL) was dissolved in THF (2.5 L). The solution was cooled to — 20°C. 3-Chloropropanesulfonyl chloride (1.5 mol, 182.4 mL) was added slowly. The reaction mixture was allowed to warm to rt and stined for 24 h. The mixture was filtered, and the filtrate was concentrated in vacuo. The residue was dissolved in CH C1 2 (2.0 L).
  • N-tert-Butyl-(l-methyl)cyclopropyI-sulf onamide A solution of N-tert-Butyl-(3-chloro)propylsulfonamide (4.3 g, 20 mmol) was dissolved in dry THF (100 mL) and cooled to - 78 °C. To this solution was added n- BuLi (17.6 mL, 44 mmol, 2.5 M in hexane) slowly. The dry ice bath was removed and the reaction mixture was allowed to warm to rt over a period of 1.5 h.
  • Example 3 l-methylcyclopropylsulfonamide.
  • a solution of N-tert-Butyl-(l-methyl)cyclopropylsulfonamide (1.91 g, 10 mmol) was dissolved in TFA (30 mL), and the reaction mixture stined at rt for 16 h.
  • Example 4 1-allylcyclopropylsulfonamide, was obtained in 40% yield from N-tert- butyl-(l-allyl)cyclopropylsulfonamide according to the procedure described in the synthesis of 1-Methylcyclopropylsulfonamide. The compound was purified by column chromotography over SiO using 2% MeOH in CH 2 C1 2 as the eluent: !
  • Sections B through I Preparation of Compounds Section B: Preparation of Compounds 100-113
  • Compound 101 was prepared by following Steps 1 through 5 described in Example 100 except that the following modifications were made:
  • Step 1-3
  • Step 1 A solution of ⁇ -chlorocinnamic acid (11.0 g, 60 mmol), diphenylphosphoryl azide (15.7 g, 57 mmol) ), and triethylamine (10 ml) in benzene (80 ml) was stined for 1 h.
  • the reaction mixture was concentrated in vacuo at ⁇ 50°C and purified by flash column chromatograph (Biotage Flash 40M) eluted with 10% EtOAc in hexane to give the desired product (4.1 g, 34%).
  • Example 103 preparation of compound 103
  • Step 1-2
  • Step 3 5-trifluoromethoxy-2H-isoquinolin-l -one (Product of Step 2, 4.58 g) was used as starting material and 4.35 g (88%) of product was obtained.
  • Step 4 l-Chloro-5-trifluoromethoxy-isoquinoline (Product of Step 3, 25 mg) was used as starting material and 28 mg (35%) of compound 103 was obtained.
  • Step 1-2 2-Trifluormethylcinnamic acid (10.0 g) was used as starting material and 5.0 g (50%) of product was obtained.
  • Step 4 l-Chloro-5-trifluoromethyl-isoquinoline (Product of Step 3, 23 mg) was used as starting material and 24 mg (31%) of compound 104 was obtained.
  • H NMR 400 MHz, CD 3 OD
  • Step 1-2
  • Step 4 l,5-Dichloro-6-methoxy-isoquinoline (Product of Step 3, 23 mg) was used as starting material and 19 mg (25%>) of compound 105 was obtained.
  • 1H NMR 400 MHz, CD 3 OD
  • Step 1-2
  • Step 4 l,6-Dichloro-5-methoxy-isoquinoline (Product of Step 3, 23 mg) was used as starting material and 28 mg (36%) of compound 106 was obtained.
  • H NMR 400 MHz, CD 3 OD
  • Step 1-2 Compound 107 was prepared by following Steps 1 through 4 described in Example 102 except that the following modifications were made: Step 1-2:
  • Step 3 4-Chloro-6-methoxy-2H-isoquinolin-l-one (Product of Step 2, 500 mg) was used as starting material and 400 mg of product was obtained.
  • Step 4 l,4-Dichloro-6-methoxy- isoquinoline (Product of Step 3, 42 mg) was used as starting material and 70 mg (45%) of compound 107 was obtained.
  • 1H NMR 400 MHz, CD 3 OD
  • Step 1-2
  • Step 3 2,3-Dihydro-7H-l,4-dioxa-7-aza-phenanthren-8-one (Product of Step 2, 2.05 g) was used as starting material and 1.5 g (68%) of product was obtained.
  • Example 109 and 110 Preparation of compound 109 and 110
  • Step 1-2
  • Step 4 l-Chloro-7-fluoro-6-methoxy-isoquinoline (Product of step 3, 21 mg) was used as 0 starting material and 9 mg (12%) of compound 109 and 24 mg (32%) of compound 110 were obtained.
  • Compound 111 was prepared by following Steps 1 through 5 described in Example 100 except that, in step 5, 1,5-dichloro-isoquinoline (20 mg) and (1(S)- ⁇ 2(S)-[1(R)- (l-ethyl-cyclopropanesulfonylaminocarbonyl)-2(S)-vinyl-cyclopropylcarbamoyl]-4- hydroxy-pynolidine-1 -carbonyl ⁇ -2,2 -dimethyl-propyl)-carbamic acid isopropyl ester (57 mg) were used as starting materials and 20 mg (27%) of compound 111 was obtained.
  • Compound 112 was prepared by followmg Steps 1 through 4 described in Example 102 except that, in step 4, (l(S)- ⁇ 2(S)-[l(R)-(l-ethyl-cyclopropanesulfonylamino- carbonyl)-2(S)-vinyl-cyclopropylcarbamoyl]-4-hydroxy-pynolidine-l-carbonyl ⁇ -2,2- dimethyl-propyl)-carbamic acid isopropyl ester (57 mg) was used as starting material and 14 mg (19%) of compound 112 was obtained.
  • Step 1-4 l-Clxloro-5-methoxy-isoquinoline was prepared by the same Steps 1 through 4 described in Example 100.
  • Step 7 A soltxtion of l,3-dichloro-5-methoxy-isoquinoline (Product of Step 6, 23 mg, 0.1 mmol) and 2(S)-[ 1 (R)-(l -ethyl-cyclopropanesulfonylaminocarbonyl)-2(S)-vinyl- cyclopropylcarbamoyi] -N- [N-Boc-amino-(S)-t-butyl-acetyl] -4(R)-hydroxy- pynolidine (58 mg, 0.1 mmol) in DMF (1 ml) was cooled to -78°C and t-BuOK (1.0 M in THF, 0.75 ml) was added.
  • Step 3 A solution of 1 (R ⁇ -tert-butoxycarbonylamino-2(S -vinyl-cyclopropanecarboxylic acid (4.45 g, 19.6 mmol) and 1,1 '-carbonyldiimidazole (3.97 g, 24.5 mmol) in dry THF (60 mL) was heated to boiling under reflux for 90 min. Upon cooling to rt, the mixture was treated sequentially with the product from Example 200, Step 2 (5.17 g, 24.5 mmol) and l,8-diazabicyclo[5.4.0]undec-7-ene (6.26 g, 41.1 mmol).
  • the aqueous solution was washed successively with diethyl ether (3 x 250 mL) and then with ethyl acetate (2 x 200 mL).
  • Example 200 The product of Example 200, Step 4 (3.00 g, 8.41 mmol) was combined with the product of Example 2O0, Step 5 (3.21 g, 8.41 mmol), HATU (3.84 g, 10.1 mmol), DIPEA (3.26 g, 25.2 mmol) and DMF (75 mL) and the resulting solution was stined at rt for 4.5 h.
  • Example 200 The product of Example 200, Step 6 (4.00 g, 6.05 mmol) was dissolved in 1,4- dioxane (50 mL) and the solution was treated with 4.0M HCI in 1,4-dioxane (15 mL). The mixture was stirred overnight at rt. The mixture was concentrated in vacuo and the resulting reddish-brown powder and placed under high vacuum.
  • Example 200 The product of Example 200, Step 7 (1.90 g, 3.00 mmol) was combined with N-Boc- L-t ⁇ rt-leucine (0.763 g, 3.30 mmol), HATU (1.48 g, 3.90 mmol), DIPEA (1.55 g, 12.0 mmol) and DMF (50 mL) and the resulting solution was stined at rt for 18 h.
  • Step 9 A solution ofthe product from Example 200, Step 8 (1.50g, 1.94 mmol) in DCM (50 mL) and trifluoroacetic acid (50 mL) was stined for 3 h at rt. The mixture was concentrated in vacuo to a viscous residue, and was then dissolved in 1,2- dichloroethane and again concentrated in vacuo to give the desired bis-trifluoroacetic acid salt product as an off-white glassy solid (quantitative). The material was used directly in the next step without purification: MS m/z 674 (MH+).
  • Compound 201 was prepared by following Step 10 of Example 200 except that phenyl chloroformate was used in place of />-tolyl chloroformate.
  • Compound 202 was prepared by following Step 10 of Example 200 except that 4- fluorophenyl chloroformate was used in place of /?-tolyl chloroformate.
  • Compound 203 was prepared by following Step 10 of Example 200 except that 4- methoxyphenyl chloroformate was used in place ofp-tolyl chloroformate.
  • Compound 204 was prepared by following Step 10 of Example 200 except that chloroformic acid 2-methoxyethyl ester was used in place ofp-tolyl chloroformate.
  • Compound 205 was prepared by following Step 10 of Example 200 except that neopentyl chloroformate was used in place of z tolyl chloroformate.
  • Compound 206 was prepared by following Step 10 of Example 200 except that 2- fluoroethyl chloroformate was used in place ofp-tolyl chloroformate.
  • Compound 207 was prepared by following Step 10 of Example 2O0 except that 2- methoxyphenyl chloroformate was used in place ofp-tolyl chloroformate.
  • Compound 208 was prepared by following Step 10 of Example 200 except that 3- trifluoromethylphenyl chloroformate was used in place of z tolyl chloroformate.
  • Compound 209 was prepared by following Step 10 of Example 200 except that 2-(- )- 7R -menthyl chloroformate was used in place ofp-tolyl chloroformate. Step 10:
  • Compound 211 was prepared by following Step 1 of Example 210 except that methoxyacetic acid was used in place of tert-butyl acetic acid.
  • Compound 212 was prepared by following Step 1 of Example 210 except that methoxypropionic acid was used in place of tert-butyl acetic acid.
  • Compound 213 was prepared by following Step 1 of Example 210 except that (S)- 1 ,4-benzodioxane-2-carboxylic acid was used in place of tert-but ⁇ yl acetic acid.
  • Compound 218 was prepared by the same methods as Compound 217 with the following modifications: Modifications: cyclopentylamine was used as a starting material to give Compound 218 (57.5 mg, 53% yield): MS m/z 739 (MH + ).
  • Compound 220 was prepared by the same methods as Compound 217 with the following modifications:
  • Method B - Xtena S7 3.0x50 mm
  • Method C - Xtena S7 C18 3.0x50 mm
  • Solvent A 10% MeOH - 90% H 2 O - 0.1%
  • Solvent B 90% MeOH - 10% H 2 O - 0.1% TFA
  • Example 300 Preparation of Compound 300 ⁇ l-[2-[l-(l-Ethyl- cyclopropanesulfonylaminocarbonyl)-2-vinyl-cyclopropylcarbamoyl]-4-(6-methoxy- isoquinolin-l-yloxy)-pynolidine-l-carbonyl]-2,2-dimethyl-propyl ⁇ -carbamic acid tert-butyl ester
  • step 1
  • Example 301 Preparation of Compound 301; (l- ⁇ 4-(6-Methoxy-isoquinolin-l- yloxy)-2-[l-(l-methyl-cyclopropanesulfonylaminocarbonyl)-2-vinyl- cyclopropylcarbamoyl] -pynolidine- 1 -carbonyl ⁇ -2,2-dimethyl-propyl)-carbamic acid tert-butyl ester
  • Compound 301 was prepared in the same procedure as described in Step 3 of Example 300 in preparation of Compound 300 instead of 1-methyl- cyclopropanesulfonamide (0.039 g, 0.26 mmol), was used in the place of 1-ethyl- cyclopropanesulfonamide, but purified by combination of Prep-HPLC (solvent B: 40% to 100%) and P-TLC (MeOH/CH 2 Cl 2 : 0% to 5%) as a white foam (0.0721g ).
  • Prep-HPLC solvent B: 40% to 100%
  • P-TLC MeOH/CH 2 Cl 2 : 0% to 5%
  • Example 302 Preparation of Compound 302; (l- ⁇ 4-(6-Methoxy-isoquinolin-l- yloxy)-2- [ 1 -( 1 -propyl-cyclopropanesulfonylaminocarbonyl)-2-vinyl- cyclopropylcarbamoyl]-pynolidine-l-carbonyl ⁇ -2,2-dimethyl-propyl)-carbamic acid tert-butyl ester
  • Compound 302 was prepared in the same procedure as described in Step 3 of Example 300 in preparation of Compound 300 instead of 1-methyl- cyclopropanesulfonamide (0.039 g, 0.26 mmol), was used in the place of 1-ethyl- cyclopropanesulfonamide, and purified by Prep-HPLC (solvent B: 50% to 100%) as a white foam (0.0628g ).
  • Example 303 Preparation of Compound 303; ⁇ l-[2-[l-(l-Benzyl- cyclopropanesulfonylaminocarbonyl)-2-vinyl-cyclopropylcarbamoyl]-4-(6-methoxy- iso qumolm-l-yloxy)-pynolidine-l-carbonyl]-2,2-dimethyl-propyl ⁇ -carbamic acid tert- butyl ester
  • Compound 303 was prepared in the same procedure as described in Step 3 of Example 300 in preparation of Compound 300 instead of 1-benzyl- cyclopropanesulfonamide (0.055 g, 0.26 mmol), was used in the place of 1-ethyl- cyclopropanesulfonamide, and purified by Prep-HPLC (solvent B: 50% to 100%) as a white foam (0.070g ).
  • Example 304 Preparation of Compound 304; ⁇ l-[2-[l-(l-Chloro- cyclopropanesulfonylaminocarbonyl)-2-vinyl-cyclopropylcarbamoyl]-4-(6-methoxy- isoquinolm-l-yloxy)-pynolidine-l-carbonyl]-2,2-dimethyl-propyl ⁇ -carbamic acid tert-butyl ester
  • Step 2 0 A mixture of cyclopropylsulfonylamhie tert-butyl carbamate 0.148g 0.58 mmol) and TFA (lmL) was stirred for 2.5 h at rt. Removed the solvent in vacuo to provide the product yield (0.09 g, 100%) as a light brown solid: 1H NMR (500 MHz, Methanol- d t ) ⁇ ppm 1.38 (m, 2 H), 1.70 (m, 2 H).
  • N-BOC-3-(R)-hydroxy-L-proline (231 mg, 1.0 mmol) in DMSO (10 mL) at the> ambient temperature was added potassium tert-butoxide (336 mg, 3.0 mmol) in one portion.
  • the formed suspension was stirred at this temperature for 30 min before being cooled to 10°C.
  • 1-Chloro-isoquinoline 180 mg, 1.1 mmol was added as solid in one portion and the final mixture was stined at the ambient temperature for 12 h. Quenched with iced 5% citric acid (aq), extracted with EtO C (10O L). The aqueous phase was extracted with EtOAC again.
  • Step 1 114 mg, 0.32 mmol
  • HATU hydroxy-3-(2-aminoethyl)
  • Step XX 107 mg, 0.33 mmol
  • CH 2 C1 2 5 mL
  • DIPEA 129 mg, 1.0 mmol
  • the formed solution was diluted with CH C1 (5 mL), washed with iced 5% citric acid (aq).
  • the organic layer was washed with 5% citric acid (aq) and brine respectively, dried over MgSO 4 , and filtered. The filtrate was evaporated in vacuo to dryness.
  • Step 3 A solution ofthe product of Example 400, Step 2 (77 mg, 0.12 mmol) in DCM (1 mL) and TFA (1 mL) was stined at room temperature for 1.5 h. The volatiles were removed in vacuo and the residue suspended in IN HCI in diethyl ether (5 mL) and concentrated in vacuo. This procedure was repeated once. The resulting mixture was triturated from pentane and filtered to give the desired compound as a hygroscopic, off-white solid (65 mg, 91%).
  • Step 4 To a mixture ofthe product of Example 400, Step 3 (65 mg, 0.12 mmol), HATU (66 mg, 0.17 mmol), and j -Boc-t-Butyl-L-glycine (32 mg, 0.14 mmol) in CH 2 C1 2 (2 mL) was added DIPEA (39 mg, 0.35 mmol) at 0°C. After stining at the ambient temperature for 12 h, the formed solution was diluted with CH 2 C1 2 (5 mL), washed with iced 5% citric acid (aq).
  • Example 401 Preparation of Compound 401.
  • Step 4 except using the product of Example 401, Step 2 instead.
  • Example 500 Preparation of Compound 500.
  • Step 1
  • Step 4 (1.01 g, 2.93 mmol) in DMSO (30 mL) was added potassium tert-butoxide (1.02 g, 9.08 mmol). The formed solution was stirred at the ambient temperature for 1 h before addition of 7-chloro-3- methyl-5-phenyl-isoxazolo[4,5-b]pyridine (0.75 g, 3.08 mrrxol). The final solution was stined for 12 h. Then was quenched with iced water, acidified with IM HCI to pH 4, extracted with EtOAc (two 200 mL portions). The organic layers were washed with brine, dried over MgSO 4 , filtered, evaporated. The residue was purified by prep- HPLC (60%B— 100%B, 15 min gradient) to afford 305 mg (19%) ofthe desired product as a pale yellow solid.
  • Step 4 To a mixture ofthe product of Example 500, Step 5 (82 mg, 0.15 mmol), HATU (84 mg, 0.22 mmol), and the product of Example 200, Step 4 (5S mg, 0.16 mmol) in CH 2 C1 2 (5 mL) was added DIPEA (50 mg, 0.44 mmol) at 0°C. After stining at the ambient temperature for 12 h, the formed solution was diluted with CH 2 C1 2 (15 mL), washed with iced 5% citric acid (aq). The organic layer was washed with 5% citric acid (aq) and brine respectively, dried over MgSO 4 , and filtered. The filtrate was evaporated in vacuo to dryness. The residue was purified by prep-HPLC to yield 42 mg (33%>) of Compound 500 as an off-white solid.
  • Example 501 Preparation of Compound 501.
  • Compound 501 was prepared by the same procedure as described in Example 500, Step 6.
  • Compound 502 was prepared by the same procedure as described in Example 500, Step 6,..
  • Step 5 a).
  • the product from step 4 of Example 600 (4.05 g, 9.01 mmol) was dissolved in 50% TFA in DCM (45 mL) and stined at rt for 20 min. The solvent was concentrated and the resulting brown viscous oil was dried in vctcuo overnight. The product was used directly for the next reaction.
  • Step 7 To a solution ofthe product from step 6 of Example 600 (0.250 g, 0.456 mmol) and DIEA (0.177 g, 1.37 mmol) in DCM (5 mL) were added the product from step 2 of Example 600 (0.182 g, 0.456 mmol), HATU (0.225 g, 0.592 mmol). After stining at rt for 14 h, the reaction mixture was wahed with 5% aqueous NaHCO 3 (5 mL), and 5% aqueous citric acid (5mL). DCM (25 mL) was used to extrated the two aqueous layer, started with the NaHCO 3 layer.
  • Compound 601 was prepared by the same methods as Compound 600 with the following modifications:
  • Stepl To a solution of Example 600 (0.245 g, 0.301 mmol) in DCM (1.5 mL) was a-dded TFA (1.5 L). After stirring rt for 15 min, reaction mixture was concentrated, and dried under vacuum to give a light brow solid product (0.281 g, 99% yield). The product was used as crade: MS m/z 715 (MH*).
  • Example 603 Preparation of Example 603
  • Compound 605 was prepared by the same methods as Compound 600 with the following modifications:
  • Compounds 700 ans 701 were prepared using the processes described herein. The preparation ofthe functionalized P2 Proline intermediate employed in the constraction of Compound 700 and Compound 701 is described in CT-2723
  • Compound 800 was prepared by the same procedure as described in Example 500, Step 6, except using the product of Example 800, Step 4 instead.

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Abstract

Hepatitis C virus inhibitors are disclosed having the general formula (I) wherein A, R2, R3, R', B and Y are described in the description. Compositions comprising the compounds and methods for using the compounds to inhibit HCV are also disclosed.

Description

HEPATITIS C VIRUS INHIBITORS
FIELD OF THE INVENTION
The present invention is generally directed to antiviral compounds, and more specifically directed to compounds which hihibit the functioning ofthe NS3 protease (also refened to herein as "serine protease") encoded by Hepatitis C virus (HCV), compositions comprising such compounds and methods for inhibiting the functioning ofthe NS3 protease.
BACKGROUND OF THE INVENTION
HCV is a major human pathogen, infecting an estimated 170 million persons worldwide - roughly five times the number infected by human hnmunodefϊciency virus type 1. A substantial fraction of these HCV infected individuals develop serious progressive liver disease, including cinhosis and hepatoceliular carcinoma. (Lauer, G. M.; Walker, B. D. N. Engl. J. Med. (2001), 345, 41-52).
Presently, the most effective HCV therapy employs a combination of alpha- interferon and ribavirin, leading to sustained efficacy in 40% of patients. (Poynard, T. et al. Lancet (1998), 352, 1426-1432). Recent clinical results demonstrate that pegylated alpha-interferon is superior to unmodified alpha-interferon as monotherapy (Zeuzem, S. et al. N. Engl. J. Med. (2000), 343, 1666-1672). However, even with experimental therapeutic regimens involving combinations of pegylated alpha- interferon and ribavirin, a substantial fraction of patients do not have a sustained reduction in viral load. Thus, there is a clear and long-felt need to develop effective therapeutics for treatment of HCV infection.
HCV is a positive-stranded RΝA viras. Based on a comparison ofthe deduced amino acid sequence and the extensive similarity hi the 5' untranslated region, HCV has been classified as a separate genus in the Flaviviridae family. All members ofthe Flaviviridae family have enveloped virions that contain a positive stranded RNA genome encoding all known viras- specific proteins via translation of a single, uninterrupted, open reading frame.
Considerable heterogeneity is found within the nucleotide and encoded amino acid sequence throughout the HCV genome. At least six major genotypes have been characterized, and more than 50 subtypes have been described. The major genotypes of HCV differ in their distribution worldwide, and the clinical significance ofthe genetic heterogeneity of HCV remains elusive despite numerous studies ofthe possible effect of genotypes on pathogenesis and therapy.
The single strand HCV RNA genome is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the stractural and non- structural (NS) proteins. In the case of HCV, the generation of mature non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. The first one is believed to cleave at the NS2-NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, for the remaining NS4A- NS4B, NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components. The complex formation ofthe NS3 protein with NS4A seems necessary to the processing events, enhancing the proteolytic efficiency at all ofthe sites. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities.
NS5B is a RNA-dependent RNA polymerase that is involved in the replication of HCV.
Among the compounds that have demonstrated efficacy in inhibiting HCV replication, as selective HCV serine protease inhibitors, are the peptide compounds disclosed in U.S. Patent No. 6,323,180. SUMMARY OF THE INVENTION
The present invention provides compounds of formula I;
Figure imgf000006_0001
wherein:
Figure imgf000006_0002
O O O O
II zis^-C — ,-^-C-O- -O- c- or -^-c- NR6R7 p is 1, 2 or 3; q is 0 or 1 ;
Ri is C3-7 cycloalkyl, C4-7 cycloalkenyl; C6-ιo aryl; C7-1 alkylaryl; C6- j.0 aryloxy; C-ι4 alkylaryloxy; C8-i5 alkylarylester; Het; or C1-8 alkyl optionally substituted with Cι-6 alkoxy, hydroxy, halo, C-1o alkenyl, C2-10 alkynyl, C3- cycloalkyl, C4-7 cycloalkenyl, C6-10 aryl, C7-14 alkylaryl, Cβ-io aryloxy, C7-14 alkylaryloxy, C8-15 alkylarylester or Het; or Ri is trialkylsilane or halogen, provided q is 0;
(b) m is 1 or 2; (c) n is 1 or 2; (d) R2 is H, C1-6 alkyl, C2-6 alkenyl or C3-7 cycloalkyl, each optionally substituted with halogen;
(e) R3 is Cι-8 alkyl optionally substituted with halo, cyano, amino, Cι-6 dialkylamino, C6-ιo aryl, C7-ι alkylaryl, Cι-6 alkoxy, carboxy, hydroxy, aryloxy, C .14 alkylaryloxy, C2-6 alkylester or Cs-15 alkylarylester; C3-ι2 alkenyl; C3-7 cycloalkyl or C4-ι0 alkylcycloalkyl, wherein the cycloalkyl or alkylcycloalkyl are optionally substituted with hydroxy, C1-6 alkyl, C2-6 alkenyl or Cι-6 alkoxy; or R3 together with the carbon atom to which it is attached fonns a C3-7 cycloalkyl group optionally substituted with C2-6 alkenyl;
(f) Y is H, phenyl substituted with nitro, pyridyl substituted with nitro, or C1-6 alkyl optionally substituted with cyano, OH or C3-7 cycloalkyl; provided that if Rt or R is H then Yis H;
(g) B is H, Cι.6 alkyl, R -(C=0 , R4θ(C=0)-, R4-N(R5)-C(=0)-, R4-N(R5)-C(=S)-, R4SO2-5 or R4-N(R5)-SO2-;
(h) R4 is (i) Cι-10 alkyl optionally substituted with phenyl, carboxyl, C1-6 alkanoyl, 1-3 halogen, hydroxy, -OC(O)C1-6 alkyl, C1-6 alkoxy, amino optionally substituted with C1-6 alkyl, amido, or (lower alkyl) amido; (ii) C3-7 cycloalkyl, C3-7 cycloalkoxy, or C4-10 alkylcycloalklyl, each optionally substituted with hydroxy, carboxyl, (C1-6 alkoxy)carbonyl, amino optionally substituted with Cι-6 alkyl, amido, or (lower alkyl) amido; (iii) C6-ιo aryl or C -16 arylalkyl, each optionally substituted with C1-6 alkyl, halogen, nitro, hydroxy, amido, (lower alkyl) amido, or amino optionally substituted with Cι-6 alkyl; (iv) Het; (v) bicyclo(l .1. l)pentane; or (vi) -C(O)OC1-6 alkyl, C2-6alkenyl or C2-6 alkynyl; (i) R5 is H; C1-6 alkyl optionally substituted with 1-3 halogens; or C1-6 alkoxy provided 4 is Cμio alkyl; (j) X is O, S, SO, SO2, OCH2, CH2O or NH; (k) R' is Het, C6-ιo aryl or C7-14 alkylaryl, each optionally substituted with
Ra; and (1) Ra is Cι-6 alkyl, C3-7 cycloalkyl, Cι-6 alkoxy, C3-7 cycloalkoxy, halo-Ci- 6 alkyl, CF3, mono-or di- halo-Cι-6 alkoxy, cyano, halo, thioalkyl, hydroxy, alkanoyl, NO2, SH, , amino, C1-6 alkylamino, di (Ci-6) alkylaniino, di (Cι-6) alkylamide, carboxyl, (Cι-6) carboxyester, C1-6 alkylsulfone, C1-6 alkylsulfonamide, di (Ci-δ) alkyl(alkoxy)amine, C6- 10 aryl, C7-1 alkylaryl, or a 5-7 membered monocyclic heterocycle; and
(m) R6 and R are each independently H; or C1-6 alkyl, C2-10 alkenyl or C6- \o aryl, each of which may be optionally substituted with halo, cyano, nitro, C1-6 alkoxy, amido, amino or phenyl;
or a pharmaceutically acceptable enantiomer, diastereomer, salt, solvate or prodrag thereof.
The present invention also provides compositions comprising the compounds or pharmaceutically acceptable salts, solvates or prodrugs thereof and a pharmaceutically acceptable canier. In particular, the present invention provides pharmaceutical compositions useful for inhibiting HC"V NS3 comprising a therapeutically effective amount of a compound ofthe present invention, or a pharmaceutically acceptable salt, solvate or prodrag thereof, and a pharmaceutically acceptable canier.
The present invention further provides methods for treating patients infected with HCV, comprising administering to the patient a therapeutically effective amount of a compound ofthe present invention, or a pharmaceutically acceptable salt, solvate or prodrag thereof. Additionally, the present invention provides methods of inhibiting HCV NS3 protease by contacting the NS3 protease with a compound ofthe present invention.
By virate ofthe present invention, it is now possible to provide improved drugs comprising the compounds ofthe invention which can be effective in the treatment of patients infected with HCV. Specifically, the present invention provides peptide compounds that can inhibit the functioning ofthe NS3 protease, e.g., in combination with the NS4A protease. Further, the present invention makes it possible to administer combination therapy to a patient whereby a compound in accordance with the present invention, which is effective to inhibit the HCV NS3 protease, can be administered with another compound having anti-HCV activity, e.g. a compound which is effective to inhibit the function of a target selected from the group consisting of HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH and a nucleoside analog for the treatment of an HCV infection.
DETAILED DESCRIPTION OF THE INVENTION
Stereochemical definitions and conventions used herein generally follow McGraw-Hill Dictionary of Chemical Terms, S. P. Parker, Ed., McGraw-Hill Book Company, New York (1984) and Stereochemistry of Organic Compounds, Eliel, E. and Wilen, S., John Wiley & Sons, Inc., New York (1994). Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration ofthe molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory and (+) or d, meaning the compound, is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are minor images of one another. A specific stereoisomer of a minor image pair may also be refened to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. With reference to the instances where (R) or (S) is used, it is to designate the absolute configuration of a substituent in context to the whole compound and not in context to the substituent alone.
Unless otherwise specifically noted herein, the terms set forth below will have the following definitions. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
The term "chiral" refers to molecules which have the property of non- superimposability ofthe mirror image partner, while the term "achiral" refers to molecules which are superimposable on their mirror image partner.
The term "stereoisomers" refers to compounds which have identical chemical composition, but differ with regard to the anangement ofthe atoms or groups in space.
The term "diastereomer" refers to a stereoisomer which is not an enantiomer, e.g., a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography.
The tenn "enantiomers" refers to two stereoisomers of a compound which are non-superimposable mirror images of one another.
The term "pharmaceutically acceptable salt" is intended to mclude nontoxic salts synthesized from a compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base fonns of these compounds with a stoichiometric amount ofthe appropriate base or acid in water or in an organic solvent, or in a mixture ofthe two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are prefened. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445. The compounds ofthe present invention are useful in the form ofthe free base or acid or in the form of a pharmaceutically acceptable salt thereof. All forms are within the scope ofthe invention. The term "therapeutically effective amount" means the total amount of each active component that is sufficient to show a meaningful patient benefit, e.g., a sustained reduction in viral load. When applied to an individual active ingredient, administered alone, the tenn refers to that ingredient alone. When applied to a combination, the term refers to combined amounts ofthe active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
The term "compounds ofthe invention", and equivalent expressions, are meant to embrace compounds of formula I, and pharmaceutically acceptable enantiomer, diastereomer salts, and solvates, e.g. hydrates, and prodrugs. Similarly, references to intermediates, are meant to embrace their salts, and solvates, where the context so permits. References to the compound ofthe invention also include the prefened compounds, e.g. formula II and A-M.
The term "derivative" means a chemically modified compound wherein the modification is considered routine by the ordinary skilled chemist, such as an ester or an amide of an acid, protecting groups, such as a benzyl group for an alcohol or thiol, and tert-butoxycarbonyl group for an amine.
The term "solvate" means a physical association of a compound of this invention with one or more solvent molecules, whether organic or inorganic. This physical association includes hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incoφorated in the crystal lattice ofthe crystalline solid. "Solvate" encompasses both solution-phase and isolable solvates. Exemplary solvates include hydrates, ethanolates, methanolates, isopropanolates and the like.
The term "prodrag" as used herein means derivatives ofthe compounds ofthe invention which have chemically or metabolically cleavable groups and become, by solvolysis or under physiological conditions, the compounds ofthe invention which are pharmaceutically active in vivo. A prodrag of a compound may be formed in a conventional manner with a functional group ofthe compounds such as with an amino, hydroxy or carboxy group when present. The prodrag derivative form often offers advantages of solubility, tissue compatibility, or delayed release in a mammalian organism (see, Bundgard, H., Design of Prodrags, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrags include acid derivatives well known to practitioners ofthe art, such as, for example, esters prepared by reaction ofthe parent acidic compound with a suitable alcohol, or amides prepared by reaction ofthe parent acid compound with a suitable amine.
The term "patient" includes both human and other mammals.
The term "pharmaceutical composition" means a composition comprising a compound ofthe invention in combination with at least one additional pharmaceutical canier, i.e., adjuvant, excipient or vehicle, such as diluents, preserving agents, fillers, flow regulating agents, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature ofthe mode of administration and dosage fonns. Ingredients listed in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA (1999) for example, maybe used.
The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable risk/benefit ratio.
The term "treating" refers to: (i) preventing a disease, disorder or condition from occurring in a patient which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder or condition, i.e., anesthig its development; and (iii) relieving the disease, disorder or condition, i.e., causing regression ofthe disease, disorder and/or condition. The term "substituted" as used herein includes substitution at from one to the maximum number of possible binding sites on the core, e.p., organic radical, to which the subsitutent is bonded, e.g., mono-, di-, tri- or tetra- substituted, unless otherwise specifically stated.
The nomenclature used to describe organic radicals, e.g., hydrocarbons and substituted hydrocarbons, generally follows standard nomenclature known in the art, unless otherwise specifically defined. Combinations of groups, e.g., alkylalkoxyamine or arylalkyl, include all possible stable configurations, unless otherwise specifically stated. Certain radicals and combinations are defined below for purposes of illustration.
The term "halo" as used herein means a halogen substituent selected from bromo, chloro, fluoro or iodo. The term "haloalkyl" means an alkyl group that in substituted with one or more halo substituents.
The term "alkyl" as used herein means acyclic, straight or branched chain, alkyl substituents having the specified number of carbon atoms and includes, for example, methyl, ethyl, propyl, butyl, tert-butyl, hexyl, 1-methylethyl, 1- methylpropyl, 2-methypropyl, 1,1-dimethylethyl. Thus, Cι-6 alkyl refers to an alkyl group having from one to six carbon atoms. The term "lower alkyl" means an alkyl group having from one to six, preferably from one to four carbon atoms. The term "alkylester" means an alkyl group additionally containing on ester group. Generally, a stated carbon number range, e.g., C -6 alkylester, includes all ofthe carbon atoms in the radical.
The temi "alkenyl" as used herein means an alkyl radical containing at least one double bond, e.g., ethenyl (vinyl) and alkyl.
The term "alkoxy" as used herein means an alkyl group with the indicated number of carbon atoms attached to an oxygen atom. Alkoxy includes, for example, methoxy, ethoxy, propoxy, 1-methylethoxy, "butoxy and 1,1-dimethylethoxy. The latter radical is refened to in the art as tert-butoxy. The term "alkoxycarbonyi" means an alkoxy group additionally containing a carbonyl group.
The term "cycloalkyl" as used herein means a cycloalkyl substituent containing the indicated number of carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and spiro cyclic groups such as spirocyclopropyl as spirocyclobutyl. The term "cycloalkoxy" as used herein means a cycloalkyl group linked to an oxygen atom, such as, for example, cyclobutyloxy or cyclopropyloxy. The term "alkylcycloalkyl" means a cycloalkyl group linked to an alkyl group. The stated carbon number range includes the total number of carbons in the radical, unless otherwise specfically stated. This a C4-ι0 alkylcycloalkyl may contain from 1-7 carbon atoms in the alkyl group and from 3-9 carbon atoms in the ring, e.g., cyclopropylmethyl or cyclohexylethyl.
The term "aryl" as used herein means an aromatic moiety containing the indicated number of carbon atoms, such as, but not limited to phenyl, indanyl or naphthyl. For example, Cβ-io aryl refers to an aromatic moiety having from six to ten carbon atoms which may be in the form of a monocyclic or bicyclic stracture. The term "haloaryl" as used herein refers to an aryl mono, di or tri substituted with one or more halogen atoms. The terms "alkylaryl", "arylalkyl" and "aralalkyl" mean an aryl group substituted with one or more alkyl groups. Unless the carbon range of each group is specified, the stated range applies to the entire substituent. Thus, a C7-14 alkylaryl group many have from 1-8 carbon atoms in the alkyl group for a monocyclic aromatic and from 1-4 carbon atoms in the alkyl group for a fused aromatic. The attachment ofthe group to bonding site on the molecule can be either at the aryl group or the alkyl group. Unless a specific aryl radical is specified, e.g., fluoro-phenyl, or the radical is stated to be unsubstituted, the aryl radicals include those substituted with typical substituents known to those skilled in the art, e.g., halogen, hydroxy, carboxy, carbonyl, nitro, sulfo, amino, cyano, dialkylamino haloalkyl, CF3, haloalkoxy, thioalkyl, alkanoyl, SH, alkylamino, alkylamide, dialkylamide, carboxyester, alkylsulfone, alkylsulfonamide and alkyl(alkoxy)amine. Examples of alkylaryl groups include benzyl, butylphenyl and 1-naphthylmethyl. The term "alkanoyl" as used herein means straight or branched 1-oxoalkyl radicals containing the indicated number of carbon atoms and includes, for example, fonnyl, acetyl, 1-oxopropyl (propionyl), 2-methyl-l-oxopropyl, 1-oxohexyl and the like.
The term "alkylamide" as used herein means an amide mono-substituted with an alkyl, such as
Figure imgf000015_0001
The term "heterocycle" , also refened to as "Het", as used herein means 7-12 membered bicyclic heterocycles and 5-9 membered monocyclic heterocycles.
Prefened bicyclic heterocycles are 7-12 membered fused bicyclic ring systems (both rings share an adjacent pair of atoms) containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur, wherein one or both rings of the heterocycle can be saturated, partially saturated or fully unsaturated ring system (this latter subset also herein refened to as unsaturated heteroaromatic). The nitrogen and sulfur heteroatoms atoms may be optionally oxidized. The bicyclic heterocycle may contain the heteroatoms in one or both rings. Unless a specific heterocycle is specified, e.g., a fluorinated 7-12 membered bicyclic heterocycle, or the heterocycle is stated to be unsubstituted, the heterocycles include those substituted with typical substituents known to those skilled in the art. For example, the bicyclic heterocycle may also contain substituents on any ofthe ring carbon atoms, e.g., one to three substituents. Examples of suitable substituents include C1-6 alkyl, C3-7 cycloalkyl,
Cι_6 alkoxy, C3.7 cycloalkoxy, halo-Cι-6 alkyl, CF3, mono-or di- halo-C1-6 alkoxy, cyano, halo, thioalkyl, hydroxy, alkanoyl, NO2, SH, , amino, Cι-6 al lainino, di (Ci-6) alkylamino, di (Cι-6) alkylamide, carboxyl, (Ci-6) carboxyester, C1-6 alkylsulfone, C1-6 alkylsulfonamide, Cι-6 alkylsulf oxide, di
Figure imgf000016_0001
alkyl(alkoxy)amine, Cβ-io aryl, C -ι4 alkylaryl, and a 5-7 membered monocyclic heterocycle. When two substituents are attached to vicinal carbon atoms ofthe bicyclic heterocycle, they can join to form a ring, e.g., a five, six or seven membered ring system containing up to two heteroatoms selecting from oxygen and nitrogen. The bicyclic heterocycle may be attached to the molecule, e.g. Ri in formula I, at any atom in the ring and preferably carbon.
Examples of bicyclic heterocycles include, but are not limited to, the following ring systems:
Figure imgf000016_0002
i Prefened monocyclic heterocycles are 5-9 membered saturated, partially saturated or fully unsaturated ring system (this latter subset also herein refened to as unsaturated heteroaromatic) containing in the ring from one to four heteroatoms selected from nitrogen, oxygen and sulfur, wherein the sulfur and nitrogen heteroatoms may be optionally oxidized. Unless a specific heterocycle is specified, e.g., a C1-6 alkoxy substituted 5-7 membered monocyclic heterocycle, or the heterocycle is stated to be unsubstituted, the heterocycles include those substituted with typical substituents known to those skilled in the art. For example, the monocyclic heterocycle may also contain substituents on any ofthe ring atoms, e.g., one to three substituents. Examples of suitable substituents include Cι-6 alkyl, C3-7 cycloalkyl, Cι-6 alkoxy, C3-7 cycloalkoxy, halo-Ci-6 alkyl, CF3, mono-or di- halo-Cι-6 alkoxy, cyano, halo, thioalkyl, hydroxy, alkanoyl, NO2, SH, , amino, C1-6 alkylamino, di (Cι-6) alkylamino, di (Cι-6) alkylamide, carboxyl, (Ci-6) carboxyester, Cj-6 alkylsulfone, Cι-6 alkylsulfoxide, C1-6 alkylsulfonamide, di (Cι-6) alkyl(alkoxy)amine, C6-ιo aryl, C7-ι4 alkylaryl and an additional 5-7 membered monocyclic heterocycle. The monocyclic heterocycle may be attached to the molecule, e.g. Ri in formula I, at any atom in the ring.
Examples of monocyclic heterocycles include, but are not limited to, the following (and their tautomers):
Figure imgf000017_0001
Those skilled in the art will recognize that the heterocycles used in the compounds ofthe present invention should be stable. Generally, stable compounds are those which can be synthesized, isolated and formulated using techniques known the those skilled in the art without degradation ofthe compound.
The term "substituent" with reference to an amino acid or amino acid derivative means a radical derived from the conesponding α-amino acid. For instance, the substituents methyl, iso-propyl, and phenyl represent the amino acids alanine, valine, and phenyl glycine, respectively.
Where used in naming compounds ofthe present invention, the designations "PI ', PI, P2, P3 and P4", as used herein, map the relative positions ofthe amino acid residues of a protease inhibitor binding relative to the binding ofthe natural peptide cleavage substrate. Cleavage occurs in the natural substrate between PI and PI ' where the nonprime positions designate amino acids starting from the C-terminus end ofthe peptide natural cleavage site extending towards the N-terminus; whereas, the prime positions emanate from the N-terminus end ofthe cleavage site designation and extend towards the C-terminus. For example, PI' refers to the first position away from the right hand end ofthe C-terminus ofthe cleavage site (ie. N-terminus first position); whereas PI starts the numbering from the left hand side ofthe C-terminus cleavage site, P2: second position from the C-terminus, etc.)(see Berger A. & Schechter I., Transactions ofthe Royal Society London series (1970 , B257. 249-264].
As used herein the term "1-aminocyclopropyl-carboxylic acid" (A&ca) refers to a compound of formula:
Figure imgf000018_0001
As used herein the term "tert-butylglycine" refers to a compound ofthe formula:
Figure imgf000018_0002
The term "residue" with reference to an amino acid or amino acid derivative means a radical derived from the conesponding -amino acid by eliminatirig the hydroxyl ofthe carboxy group and one hydrogen ofthe α-amino acid group. For instance, the terms Gin, Ala, Gly, He, Arg, Asp, Phe, Ser, Leu, Cys, Asn, Sar and Tyr represent the "residues" of E-glutamine, E-alanine, glycine, -isoleucine, L— arginine, Z-aspartic acid, E-phenylalanine, X-serine, J-leucine, E-cysteine, -asparagine, sarcosine and E-tyrosine, respectively.
The term "side chain" with reference to an amino acid or amino aciόl residue means a group attached to the α-carbon atom ofthe α-amino acid. For example, the R-group side chain for glycine is hydrogen, for alanine it is methyl, for valine it is isopropyl. For the specific R-groups or side chains ofthe α-amino acids reference is made to A.L. Lehninger's text on Biochemistry (see chapter 4).
The compounds ofthe present invention have the structure of Formula I:
Figure imgf000019_0001
wherein:
Figure imgf000019_0002
O o o o z is x u-l * , L " 0 ~ , -h c-o ^— c " - or NR6R7 p is 1, 2 or 3; q is 0 or 1;
Ri is C3-7 cycloalkyl, C4-7 cycloalkenyl; C6-ι0 aryl; C7-ι4 alkylaryl; C6- ιo aryloxy; C7-ι4 alkylaryloxy; C8-15 alkylarylester; Het; or Cι-8 alkyl optionally substituted with Cι-6 alkoxy, hydroxy, halo, C -10 alkenyl,
C2-1o alkynyl, C3-7 cycloalkyl, C4-7 cycloalkenyl, C6_ιo aryl, C7-ι4 alkylaryl, C6-ι0 aryloxy, C7-14 alkylaryloxy, C8-ι5 alkylarylester or Het; or Ri is trialkylsilane or halogen, provided q is 0;
(b) m is 1 or 2; (c) n is 1 or 2; (d) R is H, Ci-6 alkyl, C2-6 alkenyl or C3-7 cycloalkyl, each optionally substituted with halogen;
(e) R3 is Cι-8 alkyl optionally substituted with halo, cyano, amino, Cι-6 diall^lamino, C6-ι0 aryl, C -ι4 alkylaryl, C1-6 alkoxy, carboxy, hydroxy, aryloxy, C7-ι4 alkylaryloxy, C2-6 alkylester or C8-ι5 alkylarylester; C3-ι2 alkenyl; C3-7 cycloalkyl or C4-ιo alkylcycloalkyl, wherein the cycloalkyl or alkylcycloalkyl are optionally substituted with hydroxy, Cι-6 alkyl, C2-6 alkenyl or Cι-6 alkoxy; or R3 together with the carbon atom to which it is attached forms a C3-7 cycloalkyl group optionally substituted with C2-6 alkenyl;
(f) Y is H, phenyl substituted with nitro, pyridyl substituted with nitro, or Cι-6 alkyl optionally substituted with cyano, OH or C3-7 cycloalt yl; provided that if R4 or R5 is H then Yis H;
(g) B is H, Ci-6 alkyl, R4-(C=O , R4θ(C=O)-, R4-N(R5)-C(=O)-, R4-N(R5)-C(=S)-, R4SO2-, or R4-N(R5)-SO2-;
(h) i is (i) Ci-io alkyl optionally substituted with phenyl, carboxyl.. Cι-6 alkanoyl, 1-3 halogen, hydroxy, -OC(O)Cι-6 alkyl, Cι-6 alkoxy, amino optionally substituted with C1-6 alkyl, amido, or (lower alkyl) amido; (ii) C3-7 cycloalkyl, C3- cycloalkoxy, or C4-10 alkylcycloalklyl, each optionally substituted with hydroxy, carboxyl, (Cι-6 alkoxy)carbonyl, amino optionally substituted with Cι-6 alkyl, amido, or (lower alkyl) amido; (iii) C6-1o aryl or C .16 arylalkyl, each optionally substituted with Cι-6 alkyl, halogen, nitro, hydroxy, amido, (lower alkyl) amido, or amino optionally substituted with Cι-6 alkyl; (iv) Het; (v) bicyclo(l .1. l)pentane; or (vi) -C(O)OCι_6 alkyl, C2-6alkenyl or C -6 alkynyl; (i) R5 is H; Cι- alkyl optionally substituted with 1-3 halogens; or C 1-6 alkoxy provided R4 is CHO alkyl; (j) X is O, S, SO, SO2, OCH2, CH2O or NH; (k) R1 is Het, C6-10 aryl or C -ι4 alkylaryl, each optionally substitutedL with
Ra; and (1) Ra is C1.6 alkyl, C3-7 cycloalkyl, Cι-6 alkoxy, C3.7 cycloalkoxy, halo-Ci- β alkyl, CF3, mono-or di- halo-Cι-6 alkoxy, cyano, halo, thioalkyL, hydroxy, alkanoyl, NO2, SH, , amino, Cι-6 alkylamino, di (Ci-6) alkylamino, di (Cι-6) alkylamide, carboxyl, (Cι-6) carboxyester, C1-6 alkylsulfone, C1-6 alkylsulfonamide, di (Cι-6) alkyl(alkoxy)amine, C6- ιo aryl, C -ι4 alkylaryl, or a 5-7 membered monocyclic heterocycle; and
(m) R6 and R7 are each mdependently H; or Cι- alkyl, C2-ι0 alkenyl or C6- ιo aryl, each of which may be optionally substituted with halo, cyano, nitro, Cι-6 alkoxy, amido, amino or phenyl;
or a pharmaceutically acceptable enantiomer, diastereomer, salt, solvate or prodrag thereof.
In a prefened aspect ofthe invention, A is
Figure imgf000021_0001
o p o z is -^- C — 5 -^- C — O — or - C — NR6R7. p is 1, 2 or 3; q is O or 1;
Ri is C3-7 cycloalkyl, C4-7 cycloalkenyl; C7-ι4 alkylaryl; C -ι4 alkylaryloxy; C8-ι alkylarylester; or C]-8 alkyl optionally substituted with Cι-6 alkoxy, hydroxy, halo, C -ιo alkenyl, C -ιo alkynyl, C3-7 cycloalkyl, C4-7 cycloalkenyl, C6-ι0 aryl, C6-ι0 aryloxy, C8-ι5 alkylarylester or Het; or Ri is trialkylsilane or halogen, provided q is
0; and R and R7 are each independently H; or Cι-6 alkyl, C2-ι0 alkenyl or C6- !0 aryl, each of which may be optionally substituted with halo, cyano, nitro, Cι-6 alkoxy, amido, amino or phenyl. Preferably, R2 is C1-6 alkyl, C -6 alkenyl or C3-7 cycloalkyl. More preferably, R2 is C -6 alkenyl. Preferably, R3 is Cι-8 alkyl optionally substituted with C6aryl, Cι-6 alkoxy, carboxy, hydroxy, aryloxy, C -ι4 alkylaryloxy, C -6 alkylester or C8-ι5 alkylarylester; C -ι2 alkenyl; C3-7 cycloalkyl; or C4-ι0 alkylcycloalkyl. More preferably, R3 is Cι-8 alkyl optionally substituted with C1-6 alkoxy; or C3-7 cycloalkyl.
Preferably, Y is H. Preferably, B is H, Cw alkyl, R4-(C=O , R4O(C=O)-, R4-N(R5)-C(=O)-, R4-N(R5)-C(=S)-, jSOz-, or R4-N(R5)-SO2-. More preferably, B is R4-(C=O)-, R4θ(C=O)-, or R4-N(R5)-C(=O)-. Even more preferably, B is R4O(C=O)- and J is Cι-6 alkyl. Preferably, j is (i) Cι-ι0 alkyl optionally substituted with phenyl, carboxyl, C1-6 alkanoyl, 1-3 halogen, hydroxy, C1-6 alkoxy; (ii) C3-7 cycloalkyl, C3-7 cycloalkoxy, or C4-10 alkylcycloalklyl; or (iii) C6-10 aryl or C7-ι6 arylalkyl, each optionally substituted with Cι-6 alkyl or halogen. More preferably, i is (i) Ci-io alkyl optionally substituted with 1-3 halogen or Cι-6 alkoxy; or (ii) C3-7 cycloalkyl or C4-ι0 alkylcycloalkyl. Preferably, R5 is H or Cι-6 alkyl optionally substituted with 1-3 halogens. More preferably, R5 is H.
Preferably, X is O or NH. Preferably, R' is Het; or C6-ιo aryl optionally substituted with Ra. More preferably, R' is Het. Preferably, the heterocycle contains 1 or 2 nitrogen atoms and optionally a sulfur atom or an oxygen atom in the ring.
More preferably, the heterocycle is substituted with at least one of C1-6 alkyl, C1-6 alkoxy, halo, C6-ι0 aryl, C7-ι alkylaryl, or a 5-7 membered monocyclic heterocycle. Preferably, Rais Cι-6 alkyl, C3-7 cycloalkyl, Cι-6 alkoxy, halo-Cι- alkyl, halo, amino, C6 aryl, or a 5-7 membered monocyclic heterocycle.
The substituents from each grouping may be selected individually and combined in any combination which provides a stable compound in accordance with the present invention. Also, more than one substituent from each group may be substituted on the core group provided there are sufficient available binding sites. For example, each ofthe following R6; R , R8 or R9 substituents, Cι-6 alkoxy, C6 aryl and a 5-7 membered monocyclic heterocycle, may be substituted on a bicyclic heterocycle. ι In a prefened aspect, the compounds ofthe present invention have the structure of Formula II:
Figure imgf000023_0001
wherein:
Figure imgf000023_0002
O O O
II II z IS -$ ,- c ll — o or C — NR6R7. p is 1, 2 or 3; q is 0 or l;and
Ri is C3-7 cycloalkyl, C4-7 cycloalkenyl; C7_ι4 alkylaryl; C .1 alkylaryloxy; C8-ι5 alkylarylester; or Cι-8 alkyl optionally substituted with Ci-6 alkoxy, hydroxy, halo, C -ιo alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C4-7 cycloalkenyl, C6-ιo aryl, C6-ιo aryloxy, C8-ι alkylarylester or Het; or Ri is trialkylsilane or halogen, provided q is
0;
(b) R is Cι-6 alkyl, C2-6 alkenyl or C3-7 cycloalkyl; (c) R3 is Cι-8 alkyl optionally substituted with C6aryl, Cι-6 alkoxy, carboxy, hydroxy, aryloxy, C7-ι4 alkylaryloxy, C -6 alkylester, C8-ι alkylarylester; C3-ι alkenyl, C3-7 cycloalkyl, or C4-ι0 alkylcycloalkyl;
(d) Y is H; (e) B is H, Cι-6 alkyl, R C=O)-, R4θ(C=0)-, R4-N(R5)-C(=O)-,
Figure imgf000023_0003
R4SO2-, or R4-N(R5)-SO2-; " l"
(f) R4 is (i) Ci_io alkyl optionally substituted with phenyl, carboxyl, Cι-6 alkanoyl, 1-3 halogen, hydroxy, Cι-6 alkoxy; (ii) C3-7 cycloalkyl, C3-7 cycloalkoxy, or C4-ιo alkylcycloalklyl; or (iii) C6-ιo aryl or C7-ι6 arylalkyl, each optionally substituted with Cι-6 alkyl or halogen; (g) R5 is H or Cι-6 alkyl optionally substituted with 1-3 halogens;
(h) X is O orNH;
(i) R' is Het; or C6-ιo aryl optionally substituted with Ra;
(j) Ra is Cι-6 alkyl, C3-7 cycloalkyl, Cι-6 alkoxy, halo-Cι-6 alkyl, halo, amino, C6 aryl, or a 5-7 membered monocyclic heterocycle; and (k) R6 and R7 are each independently H; or Cι-6 alkyl, C2-ιo alkenyl or C6- 10 aryl, each of which may be optionally substituted with halo, cyano, nitro, Cι-6 alkoxy, amido, amino or phenyl;
or a pharmaceutically acceptable enantiomer, diastereomer salt, solvate or prodrag thereof.
Preferably, R' is a bicyclic heterocycle. Preferably, the heterocycle contains 1 or 2 nitrogen atoms and optionally a sulfur atom or an oxygen atom in the ring. More preferably, the heterocycle is substituted with at least one of Cι-6 alkyl, Cι-6 alkoxy, halo, C6 aryl, and a 5-7 membered monocyclic heterocycle.
In one aspect ofthe invention, R' is a bicyclic heterocycle containing 1 nitrogen atom and substituted with methoxy and at least one of a C6 aryl and a 5-7 membered monocyclic heterocycle.
In another aspect of theinvention, R' is a monocyclic heterocycle. Preferably, the heterocycle contains 1 or 2 nitrogen atoms and optionally a sulfur atom or an oxygen atom in the ring. Even more preferably, the heterocycle is substituted with at least one of Cι-6 alkyl, Cι-6 alkoxy, halo, C6-ιo aryl, C7-ι4 alkylaryl, or a 5-7 membered monocyclic heterocycle. In an especially prefened aspect ofthe invention, R' is a monoyclic heterocycle containing 1 or 2 nitrogen atoms and substituted with methoxy and at least one of a C6 aryl and a 5-7 membered monocyclic heterocycle. In a more prefened aspect ofthe invention, the compounds have the structure of Formula III
Figure imgf000025_0001
(III)
wherein:
Figure imgf000025_0002
p is 1, 2 or 3;
Ri is C7-14 alkylaryl; Cι-8 alkyl optionally substituted with Cι-6 alkoxy,
C -ιo alkenyl or C4-ιo alkylcycloalkyl; or Ri is trialkylsilane or halogen;
(b) R2 is C2-6 alkenyl;
(c) R3 is Cι-8 alkyl;
(d) B is ΪUO^O)-, or R4-N(H)-C(=O)-;
(e) j is Ci-io alkyl;
(f) R' is a bicyclic heterocycle optionally substituted with Ra; and
(g) Ra is Cι-6 alkyl, Cι-6 alkoxy, halo, C6 aryl, or a 5-7 membered monocyclic heterocycle; or a pharmaceutically acceptable enantiomer, diastereomer salt, solvate or prodrag thereof.
Preferably, Ri is cyclopropyl or cyclobutyl, R is vinyl, R3 is t-butyl and R is t-butyl. Preferably, R' is quinoline or isoquinoline optionally substituted with Ra. Preferbly, Ra is Cι-6 alkoxy. More preferably, Ra further includes at least one of C6 aryl or a 5-7 membered monocyclic heterocycle.
The compounds ofthe present invention, which are substituted with a basic group, can form salts by the addition of a pharmaceutically acceptable acid. The acid addition salts are formed from a compound of Formula I and a pharmaceutically acceptable inorganic acid, including but not limited to hydrochloric, hydrobromic, hydroiodic, sulfuric, phosphoric, or organic acid such as /j-toluenesulfonic, methanesulfonic, acetic, benzoic, citric, malonic, fumaric, maleic, oxalic, succinic, sulfamic, or tartaric. Thus, examples of such pharmaceutically acceptable salts include chloride, bromide, iodide, sulfate, phosphate, methanesulfonate, citrate, acetate, malonate, fumarate, sulfamate, and tartrate.
Salts of an amine group may also comprise quaternary ammonium salts in which the amino nitrogen canies a suitable organic group such as an alkyl, alkenyl, alkynyl or aralkyl moiety.
Compounds ofthe present invention, which are substituted with an acidic group, may exist as salts formed through base addition. Such base addition salts include those derived from inorganic bases which include, for example, alkali metal salts (e.g. sodium and potassium), alkaline earth metal salts (e.g. calcium and magnesium), aluminum salts and ammonium salts. In addition, suitable base addition salts include salts of physiologically acceptable organic bases such as trimethylamine, triethylamine, moφholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amrne, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, N-benzyl-β-phenethylamine, dehydroabietylamine, N,N' -bishydroabietylamine, glucamine, N-methylglucamine, collidine, quinme, quinoline, ethylenediamine, ornithine, choline, N,N'-benzylphenethylamine, chloroprocaine, diethanolamine, diethylamine, piperazine, fris(hydroxymethyl)aminomethane and tetramethylammonium hydroxide and basic amino acids such as lysine, arginine and N-methylglutamine. These salts may be prepared by methods known to those skilled in the art.
Certain compounds ofthe present invention, and their salts, may also exist in the form of solvates with water, for example hydrates, or with organic solvents such as methanol, ethanol or acetonitrile to form, respectively, a methanolate, ethanolate or acetonitrilate. The present invention includes each solvate and mixtures thereof.
In addition, compounds ofthe present invention, or a salt or solvate thereof, may exhibit polymoφhism. The present invention also encompasses any such polymoφhic form.
The compounds ofthe present invention also contain two or more chiral centers. For example, the compounds may include PI cyclopropyl element of formula
Figure imgf000027_0001
PI
wherein and C2 each represent an asymmetric carbon atom at positions 1 and 2 of the cyclopropyl ring. Not withstanding other possible asymmetric centers at other segments ofthe compounds, the presence of these two asymmetric centers means that the compounds can exist as racemic mixtures of diastereomers, such as the diastereomers wherein R2 is configured either syn to the amide or syn to the carbonyl as shown below.
Figure imgf000028_0001
(1R. 2S) (1S. 2R)
R2 is syn to carbonyl R2 is syn to carbonyl
Figure imgf000028_0002
(1R, 2R) (IS, 2S)
R2 is syn to amide R2 is syn to amide
The present invention includes both enantiomers and mixtures of enantiomers such as racemic mixtures.
The enantiomers may be resolved by methods known to those skilled in the art, for example, by formation of diastereoisomeric salts which may be separated by crystallization, gas-liquid or liquid chromatography, selective reaction of one enantiomer with an enantiomer-specific reagent. It will be appreciated that where the desired enantiomer is converted into another chemical entity by a separation technique, then an additional step is required to form the desired enantiomeric form. Alternatively, specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.
The compounds ofthe present invention may be in the form of a prodrag. Simple aliphatic or aromatic esters derived from, when present, acidic groups pendent on the compounds of this invention are prefened prodrugs. In some cases it is desirable to prepare double ester type prodrags such as (acyloxy) alkyl esters or (alkoxycarbonyl)oxy)alkyl esters.
Certain compounds ofthe present invention may also exist in different stable conformational forms which may be separable. Torsional asymmetry due to restricted rotation about an asymmetric single bond, for example because of steric hindrance or ring strain, may permit separation of different conformers. The present invention mcludes each conformational isomer of these compounds and mixtures thereof.
Certain compounds ofthe present invention may exist in zwitterionic form and the present invention includes each zwitterionic form of these compounds and mixtures thereof.
The starting materials useful to synthesize the compounds ofthe present invention are known to those skilled in the art and can be readily manufactured or are commercially available.
The compounds ofthe present invention can be manufactured by methods known to those skilled in the art, see e.p., US Patent No. 6,323, 180 and US Patent Appl. 20020111313 Al. The following methods set forth below are provided for illustrative purposes and are not intended to limit the scope ofthe claimed mvention. It will be recognized that it may be prefened or necessary to prepare such a compound in which a functional group is protected using a conventional protecting group then to remove the protecting group to provide a compound ofthe present invention. The details concerning the use of protecting groups in accordance with the present invention are known to those skilled in the art.
The compounds ofthe present invention may, for example, be synthesized according to a general process as illustrated in Scheme I (wherein CPG is a carboxyl protecting group and APG is an amino protecting group): Scheme I
PI Pl-CPG + APG-P2 APG-P2-P1-CPG
APG-P3-P2-P1-CPG P2-P1-CPG + APG-P3
Figure imgf000030_0001
B-P3-P2-P1 B-P3-P2-P1-P1'
Briefly, the PI, P2, and P3 can be linked by well known peptide coupling techniques. The PI, P2, and P3 groups may be linked together in any order as long as the final compound conesponds to peptides ofthe invention. For example, P3 can be linked to P2-P1; or PI linked to P3-P2.
Generally, peptides are elongated by deprotecting the α-amino group ofthe N-terminal residue and coupling the unprotected carboxyl group ofthe next suitably N-protected amino acid through a peptide linkage using the methods described. This deprotection and coupling procedure is repeated until the desired sequence is obtained. This coupling can be performed with the constituent amino acids in stepwise fashion, as depicted in Scheme I.
Coupling between two amino acids, an amino acid and a peptide, or two peptide fragments can be canied out using standard coupling procedures such as the azide method, mixed carbonic-carboxylic acid anhydride (isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-soluble carbodiimide) method, active ester (p-nitrophenyl ester, N-hydroxysuccinic imido ester) method, Woodward reagent K-method, - o-
carbonyldiimidazole method, phosphorus reagents or oxidation-reduction methods. Some of these methods (especially the carbodiimide method) can be enhanced by adding 1-hydroxybenzotriazole or 4-DMAP. These coupling reactions can be performed in either solution (liquid phase) or solid phase.
More explicitly, the coupling step involves the dehydrative coupling of a free carboxyl of one reactant with the free amino group ofthe other reactant in the present of a coupling agent to form a linking amide bond. Descriptions of such coupling agents are found in general textbooks on peptide chemistry, for example, M. Bodanszky, "Peptide Chemistry", 2nd rev ed., Springer- Verlag, Berlin, Germany, (1993). Examples of suitable coupling agents are N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole in the presence of N,N'-dicyclohexylcarbodiimide or N-ethyl-N'-[(3-dimethylamino)propyl]carbodiimide. A practical and useful coupling agent is the commercially available (benzotriazol- 1 -yloxy)tris-(dimethylamino)phosphonium hexafluorophosphate, either by itself or in the present of 1-hydroxybenzotriazole or 4-DMAP. Another practical and useful coupling agent is commercially available 2-(lH-benzotriazol-l-yl)-N, N, N', N'-tetramethyluronium tetrafluoroborate. Still another practical and useful coupling agent is commercially available O-(7-azabenzotrizol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate. The coupling reaction is conducted in an inert solvent, e.g. dichloromethane, acetonitrile or dimethylfomiamide. An excess of a tertiary amine, e.g. diisopropylethylamine, N-methylmoφholine, N-methylpynolidine or 4-DMAP is added to maintain the reaction mixture at a pH of about 8. The reaction temperature usually ranges between 0 °C and 50 °C and the reaction time usually ranges between
15 min and 24 h.
The functional groups ofthe constituent amino acids generally must be protected during the coupling reactions to avoid formation of undesired bonds. Protecting groups that can be used are listed, for example, in Greene, "Protective
Groups in Organic Chemistry", John Wiley & Sons, New York (1981) and "The Peptides: Analysis, Synthesis, Biology", Vol. 3, Academic Press, New York (1981), the disclosures of which are hereby incoφorated by reference. The α-amino group of each amino acid to be coupled to the growing peptide chain must be protected (APG). Any protecting group known in the art can be used. Examples of such groups include: 1) acyl groups such as formyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl; 2) aromatic carbamate groups such as benzyloxycarbonyl (Cbz or Z) and substituted bensyloxycarbonyls, and 9-fluorenyhnethyloxycarbonyl (Fmoc); 3) aliphatic carbamate groups such as tert-butyloxycarbonyl (Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl; 4) cyclic alkyl carbamate groups such as cyclopentyloxycarbonyl and adamantyloxycarbonyl; 5) alkyl groups such as triphenylmethyl and benzyl; 6)trialkylsilyl such as trimethylsilyl; and 7) thiol containing groups such as phenylthiocarbonyl and dithiasuccinoyl.
The prefened α-amino protecting group is either Boc or Fmoc. Many amino acid derivatives suitably protected for peptide synthesis are commercially available. The α-amino protecting group ofthe newly added amino acid residue is cleaved prior to the coupling ofthe next amino acid. When the Boc group is used, the methods of choice are trifluoroacetic acid, neat or in dichloromethane, or HCI in dioxane or in ethyl acetate. The resulting ammonium salt is then neutralized either prior to the coupling or in situ with basic solutions such as aqueous buffers, or tertiary amines in dichloromethane or acetonitrile or dimethylformamide. When the Fmoc group is used, the reagents of choice are piperidine or substituted piperidine in dimethylformamide, but any secondary amine can be used. The deprotection is canied out at a temperature between 0°C and room temperature (rt or RT) usually 20-22°C.
Any ofthe amino acids having side chain functionalities must be protected during the preparation ofthe peptide using any ofthe above-described groups. Those skilled in the art will appreciate that the selection and use of appropriate protecting groups for these side chain functionalities depend upon the amino acid and presence of other protectmg groups in the peptide. The selection of such protecting groups is important in that the group must not be removed during the deprotection and coupling ofthe α-amino group. For example, when Boc is used as the α-amino protecting group, the following side chain protecting group are suitable: τ toluenesulfbnyl (tosyl) moieties can be used to protect the amino side chain of amino acids such as Lys and Arg; acetamidomethyl, benzyl (Bn), or tert-butylsulfonyl moieties can be used to protect the sulfide containing side chain of cysteine; bencyl (Bn) ethers can be used to protect the hydroxy containing side chains of serine, threonine or hydroxyproline; and benzyl esters can be used to protect the carboxy containing side chains of aspartic acid and glutamic acid.
When Fmoc is chosen for the α-amine protection, usually tert-butyl based protecting groups are acceptable. For instance, Boc can be used for lysine and arginine, tert-butyl ether for serine, threonine and hydroxyproline, and tert-butyl ester for aspartic acid and glutamic acid. Triphenylmethyl (Trityl) moiety can be used to protect the sulfide containing side chain of cysteine.
Once the elongation ofthe peptide is completed all ofthe protecting groups are removed. When a liquid phase synthesis is used, the protecting groups are removed in whatever manner is dictated by the choice of protecting groups. These procedures are well known to those skilled in the art.
Further, the following guidance may be followed in the preparation of compounds ofthe present invention. For example, to form a compound where R4-C(O)-, Rt-S(O)2„ a protected P3 or the whole peptide or a peptide segment is coupled to an appropriate acyl chloride or sulfonyl chloride respectively, that is either commercially available or for which the synthesis is well known in the art. In preparing a compound where R4θ-C(O)-, a protected P3 or the whole peptide or a peptide segment is coupled to an appropriate chloroformate that is either commercially available or for which the synthesis is well known in the art. For Boc-derivatives (Boc)2O is used.
For example: + H2N-P3- [P2-P1] -COOEt
Figure imgf000034_0001
Figure imgf000034_0002
[P2-Pι] -COOEt
Cyclopentanol is treated with phosgene to furnish the conesponding chloroformate.
The chloroformate is treated with the desired NH -tripeptide in the presence of a base such as triethylamine to afford the cyclopentylcarba ate.
In preparing a compound where R4-N(R5)-C(O)-, or R4-NH-C(S)-, a protected P3 or the whole peptide or a peptide segment is treated with phosgene followed by amine as described in SynLett. Feb 1995; (2); 142-144 or is reacted with the cornmercially available isocyanate and a suitable base such as triethylamine.
In preparing a compound where R4-N(R5)-S(O2), a protected P3 or the whole peptide or a peptide segment is treated with either a freshly prepared or cornmercially available sulfamyl chloride followed by amine as described in patent Ger. Offen.(1998), 84 pp. DE 19802350 or WO 98/32748.
The α-carboxyl group ofthe C-terminal residue is usually protected as an ester (CPG) that can be cleaved to give the carboxylic acid. Protecting groups that can be used include: 1) alkyl esters such as methyl, trimethylsilylethyl and t-butyl, 2) aralkyl esters such as benzyl and substituted benzyl, or 3) esters that can be cleaved by mild base treatment or mild reductive means such as trichloroethyl and phenacyl esters. The resulting α-carboxylic acid (resulting from cleavage by mild acid, mild base treatment or mild reductive means) is coupled with a ASO2NH2 as described herein.
Furthermore, if the P3 protecting group APG is removed and replaced with a B moiety by the methods described above, and the resulting α-carboxylic acid resulting from cleavage (resulting from cleavage by mild acid, mild base treatment or mild reductive means) is coupled with a ASO2NH2.
Compounds ofthe present invention can be prepared by many methods including those described in the examples, below, and as described in U.S. Patent No. 6,323,180 and U.S. Patent Application No. 10/001,850 filed on November 20, 2001. The teachings of U.S. Patent No. 6,323,180 and U.S. Patent Application No. 10/001,850 are incoφorated herein, in their entirety, by reference.
Scheme II further shows the general process wherein compounds of Formula I are constructed by the coupling of tripeptide carboxylic acid intermediate (1) with a P 1 Λ sulfonamide. (It should be noted that the groups R6, R > R8; R9, Rio, Ri ι as shown below represent substituents ofthe heterocyclic system.) Said coupling reaction requires treatment of carboxylic acid (1) with a coupling reagent such as carbonyl diimidazole in a solvent such as THF, which can be heated to reflux, followed by the addition ofthe formed derivative of (1), to the PL sulfonamide, in a solvent such as THF or methylene chloride in the presence of a base such as DBU.
Scheme II
Process P4-P3-P2-P1 P1 — *- P4-P3-P2-P1-PT
Figure imgf000035_0001
An alternative process for the construction of compounds of Foπnula I is shown in Scheme III. Therein the PL sulfonamide element is coupled to the PI element using the process employed in Scheme 1. The resulting PI -PL moiety can then be deprotected at it's amino terminus. In this general example a Boc protecting group is employed but one skilled in the art would recognize that a number of suitable amino protecting groups could be employed in this process. Said Boc protecting group can be removed using acid such as trifluoroacetic acid in a solvent such as dichloroethane to provide the deprotected amine as the TFA salt. Said TFA amine salt can be directly employed in the subsequent coupling reaction or as an alternative the TFA amine salt can be first converted to the HCI amine salt, and this HCI amine salt is used in said coupling reaction as shown in Scheme III. The coupling of said HCI amine salt (3) with the carboxyl terminus a P4-P3-P2 intermediate can be achieved using coupling reagents, such as HATU, in solvents such as dichloromethane to provide compounds of Formula I (4).
Scheme III
Process P1 p >. Pl-Pr P4-P3-P2 f -p3.p2-pι.p
Figure imgf000036_0001
Compounds fo Formula I An alternative process for the construction of compounds of Formula I is shown in Scheme IV. Herein the hydrochloride salt ofthe Pl-PT terminal amine (1) is coupled to the free carboxyl group ofthe P2 element using coupling agents such as PyBOP, in the presence of a base such as diisopropyl amine, and in a solvent such as methylene chloride. The resulting P2-P1-PT intermediate can be converted to compounds of Formula I in a two step process wherein the first step is deprotection ofthe P2 amine terminus using an acid such as TFA hi a solvent such as methylene chloride. The resulting trifluoroacetic acid salt can be coupled with the carboxyl terminus ofthe P4-P3 element using standard coupling agents such as PyBop in the presence of base such as diisopropyl amine, and using solvents such methylene chloride to provide compounds of Formula I (4). Scheme IV
P9 PΛ-P^
Process P1-P1- T-£ ^ P2-P1-P1' -X-L →. P4-P3-P2-P1-P1*
Deprotection
Figure imgf000037_0001
Figure imgf000037_0002
The P4-P3-P2 intermediate utilized in the above schemes can be constructed as previously described with a further description of this process shown in general Scheme V. Therein the free carboxyl terminus ofthe P4-P3 intermediate (1), can be coupled to the anαino terminus ofthe P2 element to provide the P4-P3-P2 dipeptide (2). The carboxyl terminus ofthe P4-P3-P2 intermediate can be deprotected by saponification ofthe ester group to provide P4-P3-P2 as the free carboxylic acid (3). Intermediates like (3) can be converted to compounds of Formula I using the methods described herein. Scheme V
Figure imgf000038_0001
Compounds of Formula 1 can also be converted into other compounds of Formula I as described herein. An example of such a process is shown in Scheme VI wherein a compound of Formula 1 (1) which bears a Boc group at the P4 position is converted in a compound of Formula I (3) wherein said compound bears a urea group at the P4 position. The conversion of (1) to (3) can be canied out in a two step process the first of which is the conversion of (1) to amine (2) by treatment of (1) with an acid such as TFA in a solvent such as methylene chloride. The resulting amine TFA salt can be treated with an isocyanate in the presence of one equivalent of base to provide a compound of Formula I (3) wherein the P3 moiety is capped with a urea. As previously noted one skilled in the art will recognize that intermediate (2) can be used as starting materials for the preparation of compounds of Fonnula I wherein the P3 group is capped with an amide or a sulfonamide, or thiourea, or a sulfamide. The construction of said compounds of Formula I can be achieved using standard conditions for the formation of said P4 functionalities from amines.
Scheme VI
Process P4-P3-P2-P1-P1' P3-P2-P1-P1" P4-P3-P2-P1-P1*
Figure imgf000039_0001
Compounds fo'Formula I
Figure imgf000039_0002
Compounds fo Formula I
In the construction of compounds of Formula I, the PL terminus is incoφorated into the molecules using one ofthe general processes outlined above and described in more detail below. In some examples the PL elements, that is the substituted cycloalkyl- sulfonamides are commercially available or can be prepared from the conesponding alkyl- or cycloalkyl-sulfonyl chloride by treating said sulfonyl chloride with ammonia . Alternatively, these sulfonamides can be synthesized using the general process outline in Scheme VII. Therein commercially available 3-chloro- propylsulfonyl chloride (1) is converted to a suitable protected sulfonamide as for example by treatment with tert-butyl amine. The sulfonamide obtained (2) is then converted to the conesponding substituted cycloalkylsulfonamide by treatment with a base such as butyl lithium in a solvent such as THF at low temperature followed by the addition of an electrophile. The resulting substituted cycloalkylsulfonamide can be deprotected by treatment with an acid to provide the desired unprotected substituted cycloalkylsulfonamide. Scheme VII
eg. Mel
Figure imgf000040_0001
Coupling to P1 acid Acid followed by elogation Λ , , _ »- » Compounds of Formula I
Figure imgf000040_0002
Figure imgf000040_0003
The PI elements utilized in generating compounds of Formula I are in some cases commercially available, but are otherwise synthesized ushig the methods described herein and subsequently incoφorated into compounds of Formula I using the methods described herein. The substituted PI cyclopropylamino acids can be synthesized following the general process outline in Scheme VIII.
Treatment of commercially available or easily synthesized imine (1) with 1,4- dihalobutene (2) in presence of a base produces, provides the resulting imine (3). Acid hydrolysis of 3 then provides 4, which has an allyl substituent syn to the carboxyl group as a maj or product. The amine moiety of 4 can protected using a Boc group to provide the fully protected amino acid 5. This intermediate is a racemate which can be resolved by an enzymatic process wherein the ester moiety of 5 is cleaved by a protease to provide the conesponding carboxylic acid. Without being bound to any particular theory, it is believed that this reaction is selective in that one ofthe enantiomers undergoes the reaction at a much greater rate than its mirror image providing for a kinetic resolution ofthe intermediate racemate. In the examples cited herein, the more prefened stereoisomer for integration into compounds of Formula I is 5a which houses the ( IR, 2S) stereochemistry. In the presence ofthe enzyme, this enantiomer does not undergo ester cleavage and thereby this enantiomer 5 a is recovered from the reaction mixture. However, the less prefened enantiomer ,5b with houses the (IS, 2R) stereochemistry undergoes ester cleavage, i.e., hydrolysis, to provide the free acid 6. Upon completion of this reaction, the ester 5a can be separated from the acid product 6 by routine methods such as, for example, aqueous extraction methods or chromotography. Scheme VIII
Figure imgf000041_0001
Procedures for making P2 intermediates and compounds of Formula I are shown in the Schemes below. It should be noted that in many cases reactions are depicted for only one position of an intermediate. However, it is to be understood that such reactions could be used to impart modifications to other positions within this intermediate. Moreover, said intermediates, reaction conditions and methods given in the specific examples are broadly applicable to compounds with other substitation patterns. The general Schemes outlined below are followed with examples herein. Both general and specific examples are non-limiting, as for example the isoquinoline nucleus is shown as part ofthe general scheme, Scheme IX, however, this pathway represents a viable process for the construction of alternate heterocycle substituents as replacements for the isoquinoline element, such as quinolines, or pyridines.
Figure imgf000042_0001
Scheme IX shows the coupling of an N-protected C4-hydroxyproline moiety with a heterocycle to form intermediate (4) and the subsequent modification of said intermediate (4) to a compound of Formula I by the process of peptide elongation as described herein. It should be noted that in the first step, that is the coupling ofthe C4-hydroxy proline group with the heteroaryl element, a base is employed. One skilled in the art would recognized that this coupling can be done using bases such as potassium tert-butoxide, or sodium hydride, in solvent such as DMF or DMSO or THF. This coupling to the isoquinoline ring system occurs at the CI position (numbering for isoquinoline ring system shown in intermediate 2 of Scheme IX) and is directed by the chloro group which is displaced in this process. It should be noted that the alternative leaving groups can be utilized at this position such as a fluoro as shown in the Scheme. Said fluoro intermediates (3) are available from the conesponding chloro compound using literature procedures described herein. It should also be noted that the position of the leaving group (chloro or fluoro) in a given ring system can vary as shown in Scheme X, wherein the leaving group (fluoro in this example) is in the C6 position ofthe isoquinoline ring system of intermediate (2).
Figure imgf000042_0002
It should be further noted that the position ofthe ring heteroatom(s) in intermediates like (2) of Scheme IX and Scheme X is also variable, as defined by the term heterocycle described herein. In Scheme X intermediate (2) can be coupled to a C4 hydroxy proline derivative to provide the P2 element (3). This Cό-substitoted isoquinoline derivative can be converted to compounds of Formula I using the methods described herein.
An alternative to the method described above for the coupling ofthe C4- hydroxyproline to aromatics and heteroaromatics, is provided in the Mitsunobu reaction as depicted in Scheme XI
Figure imgf000043_0001
step 1 of Scheme XI. In this general reaction Scheme a C4-hydroxy proline derivative is coupled to a quinazoline ring system. This reaction makes use of reagents such as triphenylphosphine and DEAD (diethylazodicarboxylate) in aprotic solvents such as THF or dioxane and can be used for the formation of aryl and heteroaryl ethers. Note that in the course of this coupling reaction the stereochemistry ofthe C4 chiral center in the C4-hydroxyproline derivative is inverted and thereby it is necessary to use the C4-hydroxyproline derivative housing the (S) stereochemistry at the C4 position as starting material, (as shown in Scheme XI). It should be noted that numerous modifications and improvements ofthe Mitsunobu reaction have been described in the literature, the teachings of which are incoφorated herein.
In a subset of examples herein, isoquinolines are incoφorated into the final compounds and specifically into the P2 region of said compounds. One skilled in the art would recognize that a number of general methods are available for the synthesis of isoquinolines. Moreoever, said isoquinolines generated by these methods can be readily incoφorated into final compounds of Formula I using the processes described herein. One general methodology for the synthesis of isoquinolines is shown in Scheme XII, wherein cinnamic acid derivatives, shown in general form as stracture (2) are
Scheme XII
Figure imgf000044_0001
Reference: N. Briet at al, Tetrahedron, 2002, 5761 converted to 1-chloroisoquinolines in a four step process. Said chloroisoquinolines can be subsequently used in coupling reactions to C4-hydroxyproline derivatives as described herein. The conversion of cinnamic acids to chloroquinolines begins with the treatment of cinnamic acid with an alkylcholorformate in the presence of a base. The resulting anhydride is then treated with sodium azide which results in the formation of an acylazide (3) as shown in the Scheme. Alternate methods are available for the formation of acylazides from carboxylic acids as for example said carboxylic acid can be treated with diphenylphosphorylazide (DPP A) in an aprotic solvent such as methylene chloride in the presence of a base, hi the next step ofthe reaction sequence the acylazide (3) is coverted to the corresponding isoquinolone (4) as shown in the Scheme. In the event the acylazide is heated to a temperature of approximately 190 degress celcius in a high boiling solvent such a diphenylmethane. This reaction is general and provides moderate to good yields of substituted isoquinolone from the conesponding cinnamic acid derivatives. It should noted that said cinnamic acid derivatives are available commercially or can be obtained from the conesponding benzaldehyde (1) derivative by direct condensation with malonic acid or derivatives thereof and also by employing a Wittig reaction. The intermediate isoquinolones (4) of Scheme XII can be converted to the conesponding 1- 1 chloroisoquinoline by treatment with phosphorous oxychloride. This reaction is general and can be applied to any ofthe isoquinolones, quinolones or additional heterocycles as shown herein to covert a hydroxy substituent to the conesponding chloro compound when said hydroxy is in conjugation with a nitrogen atom in said heterocylic ring systems.
An alternative method for the synthesis ofthe isoquinoline ring system is the Pomeranz-Fritsh procedure. This general method is outlined in Scheme XIII. The process begins with the conversion of a benzaldehyde derivative (1) to a functionalized imine (2). Said imine is then converted to the isoquinoline ring system by treatment with acid at elevated Scheme XIII
Figure imgf000045_0001
Pomeranz-Fritsch synthesis
K. Hirao, R. Tsuchiya, Y. Yano, H. Tsue, Heterocycles 42(1) 1996, 415-422 temperature. This isoquinoline synthesis of Scheme XIII is general, and it should be noted that this process is particularly useful hi procuring isoquinoline intermediates that are substituted at the C8 position (note: in intermediate (3) of Scheme XIII the C8 position ofthe isoquinoline ring is substituted with substutuent Rn). The intermediate isoquinolines (3) can be converted to the conesponding 1- chloroquinolines (5) in a two step process as shown. The first step in this sequence is the formation ofthe isoquinoline N-oxide(4) by treatment of isoquinoline (3) with meta-chloroperbenzoic acid in an aprotic solvent such as dichloromethane. Intermediate (4) can be converted to the conesponding 1-chloroquinoline by treatment with phosphorous oxychloroide in refluxing chloroform. Note this two step process is general and can be employed to procure chloroisoquinolines and chloroquinolines from the conesponding isoquinolines and quinolines respectively. Another method for the synthesis ofthe isoquinoline ring system is shown in Scheme XIV. In this process an ortho-alkylbenzamide derivative (1) is treated with a strong Scheme XIV
Figure imgf000046_0001
base such as tert-butyl lithium hi a solvent such as THF at low temperature. To this reaction mixture is then added a nitrile derivative, which undergoes an addition reaction with the anion derived from deprotonation of (1), resulting in the formation of (2). This reaction is general and can be used for the formation of substituted isoquinolines. Intermediate (2) of Scheme XTV can be converted to the conesponding 1 -chloroquinoline by the methods described herein.
An additional method for the synthesis of isoquinolines is shown in Scheme XV. The deprotonation of intermediate (1) using tert-butyl lithium is described above. In the present method however, said intermediate anion is trapped by an ester, resulting in the formation of intermediate (2) as shown below. In a subsequent reaction ketone (2) is condensed with ammoniumn acetate at elevated temperature providing for the formation of quinolone (3). This reaction is general and can be applied for the construction of substituted isoquinolones which can then be converted to the conesponding 1-chloroisoquinolines as described herein. Scheme XV
Figure imgf000046_0002
Yet an additional method for the construction of isoquinolines is found in Scheme XVI. In the first step of this process an ortho-alky larylimine derivatives such as (1) is subjected to deprotonation conditions (sec-butyl lithium, THF) and the resulting anion is quenched by
Figure imgf000047_0001
L Flippin, J. Muchowski, JOC, 1993, 2631-2632 the addition of an activated carboxylic acid derivative such as a Weinreb amide. The resulting keto imine (2) can be converted to the conesponding isoquinoline by condensation with ammonium acetate at elevated temperatures. This method is general and can be used for the synthesis of substituted isoquinolines. Said isoquinolines can be converted to the conesponding l-chloroquinoline by the methods described herein.
The heterocycles described herein, and which are incoφorated into the compounds of Formula I can be further functionalized. It is obvious to one skilled in the art that additional functionalization of said heterocycles can be done either before or after incoφoration of these functionalities into compounds of Formula I. The following Schemes illustrate this point. For example Scheme XVII shows the conversion of a 1-chloro-
Scheme XVII
Figure imgf000047_0002
6-fluoro-isoquinoline to the conesponding l-chloro-6-alkoxy-isoquinoline species, by treatment of (1) of (eq.1) with a sodium or potassium alkoxide species in the alcohol solvent from which the alkoxide is derived at room temperature. In some cases it may be necessary to heat the reaction to drive it to completion. Said chloroquinoline can be incoφorated into a compound of Formula I using the art described herein. Modifications of a P2 heterocyclic element can also be done after it's incoφoration into compounds of Formula I as shown in (eq.2) of Scheme VXII. Specifically compounds such as (1) in (eq. 2) which contain a leaving group in the pthalazine nucleus can be displaced by a nucleophile such as an alkoxide in solvents such as the conesponding alcohol from which the alkoxide is derived. These reaction scan be conducted at room temperature but in some cases it may be necessary to heat the reaction to drive it to completion.
Scheme XVIII provides a general example for the modification of heterocycles as defined herein by employing palladium mediated coupling reactions. Said couplings can be employed to functionalize a heterocycle at each position ofthe ring system providing said ring is suitably activated or functionalized, as for example with a chloride as shown in the Scheme. This sequence begins with 1-chloroisoquinoline (1) which upon treatment with metachloroperbenzoic acid can be converted to the conesponding N-oxide (2). Said intermediate (2) can be converted to the conesponding 1,3-dichloroisoquinoline (3) by treatment with phosphorous oxychloride in refluxing chloroform. Intermediate (3) can be coupled with N-Boc-4- hydroxyproline by the methods described herein to provide intermediate (5) as shown in the Scheme. Intermediate (5) can undergo a Suzuki coupling with an aryl boronic acid, in the presence of a palladium reagent and base, and in a solvent such as THF or toluene or DMF to provide the C3-arylisoquinoline intermediate (6). Heteroarylboronic acids can also be employed in this Pd mediated coupling process to provide C3-heteroarylisoquinolines. Intermediate (6) can be converted into final compounds of Formula I by the methods described herein.
Scheme XVIII
Figure imgf000049_0001
Palladium mediated couplings of heteroaryl systems with aryl or heteroaryl elements can also be employed at a later synthetic stage in the construction of compounds of Fonnula I as shown in Scheme IXX. Therein tripeptide acylsulfonamide intermediate (1) is coupled to a l-chloro-3-bromoisocuinoline (2) using the previously described process of alkoxide displacement of an heteroarylhalo moiety to provide intermediate (3). The coupling of (1) and (2) is most efficient in the presence of a catalyst such as lanthanum chloride as described herein. The isoquinoline ring system of intermediate (3) can be further functionalized by employing either Suzuki couplings (Process 1: subjecting (3) to heteroaryl or aryl boronic acids in the presence of a palladium catalyst such as palladium teixa(triphenylphosphine) and a base such as cesium carbonate in solvents such as DMF) or Stille couplings (Process 2: subjecting (3) to heteraryl or aryl tin dervatives in "the presence of palladium catalyst such as palladium tetra(triphenylphosphine in solvents such as toluene).
Scheme IXX
Figure imgf000050_0001
Palladium reactions can also be employed to couple C4-amino proline elements with functionalized heterocycles. Scheme XX shows intermediate (1) coupling with a functionalized isoquinoline in the presence of a palladium catalyst and a base in a solvent such as toluene. Intermediates like (3) can be converted to compounds of Formula I using the methods described herein.
Figure imgf000050_0002
The construction of functionalized isoquinoline ring systems is also possible employing [4+2] cycloaddition reactions. For example (Scheme XXI) the use of vinyl isocyantes (1) in cycloaddition reactions with benzyne precusors (2) provides functionalized isoquinolones (3). Said isoquinolines can be incoφorated into compounds of Formula I using the methods described herein. Scheme XXI
Figure imgf000051_0001
Compounds ofthe invention can also be prepared by utilizing methods known to thise skilled in the art, such as, for example, the methods described in patent application WO 03/099274, published December 3 , 2003 , and WO 2004/043339, published May 27, 2004.
The present invention also provides compositions comprising a compound of the present invention, or a pharmaceutically acceptable enantiomer, diastereomer, salt, solvate or prodrag thereof, and a pharmaceutically acceptable canier.
Pharmaceutical compositions ofthe present invention comprise a therapeutically effective amount of a compound ofthe invention, or a pharmaceutically acceptable enantiomer, diastereomer, salt, solvate or prodrag thereof, and a pharmaceutically acceptable canier, with a pharmaceutically acceptable canier, e.g., excipient, or vehicle diluent.
The active ingredient, i.e., compound, in such compositions typically comprises from 0.1 weight percent to 99.9 percent by weight ofthe composition, and often comprises from about 5 to 95 weight percent.
Thus, in one aspect ofthe invention, there is provided a composition comprising the compound of formula 1 and a pharmaceutically acceptable canier. Preferably, the composition further comprises a compound having anti-HCV activity. As used herein, the term "anti-HCV activity" means the compound is effective to inhibit the function of a target selected from the group consisting of HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH and a nucleoside analog for the treatment of an HCV infection. Often, the other compound having anti-HCV activity is effective to inhibit the function of target in the HCV life cycle other than the HCV NS3 protease protein.
In one prefened aspect, the compound having anti-HCV activity is an interferon. Preferably, the interferon is selected from the group consisting of interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, lymphoblastiod interferon tau.
In another aspect ofthe invention, the compound having anti-HCV activity is selected from the group consisting of interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, hiterfering RNA, anti-sense RNA, Imiqimod, ribavirin, an inosine 5'-monophospate dehydrogenase inhibitor, amantadine, and rimantadine.
In one prefened aspect ofthe invention, the composition comprises a compound ofthe invention, an interferon and ribavirin.
In another prefened aspect ofthe invention, the compound having anti-HCV activity is a small molecule compound. As used herein, the term "small molecule compound" means a compound having a molecular weight of less than 1,500 daltons, preferably less than 1000 daltons. Preferably, the small molecule compound is effective to inhibit the function of a target selected from the group consisting of HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, inosine monophophate dehydrogenase ("IMPDH") and a nucleoside analog for the treatment of an HCV infection.
Certain illustrative HCV inhibitor compounds which can be administered with the compounds ofthe present invention include those disclosed in the following publications: WO 02/04425 A2 published January 17, 2002, WO 03/007945 Al published January 30, 2003, WO 03/010141 A2 published February 6, 2003, WO 03/010142 A2 published February 6, 2003, WO 03/010143 Al published February 6, 2003, WO 03/000254 Al published January 3, 2003, WO 01/32153 A2 published May 10, 2001, WO 00/06529 published February 10, 2000, WO 00/18231 published April 6, 2000, WO 00/10573 published March 2, 2000, WO 00/13708 published March 16, 2000, WO 01/85172 Al published November 15, 2001, WO 03/037893 Al published May 8, 2003, WO 03/037894 Al published May 8, 2003, WO 03/037895 Al published May 8, 2003, WO 02/100851 A2 published December 19, 2002, WO 02/100846 Al published December 19, 2002, EP 1256628 A2 published November 13, 2002, WO 99/01582 published January 14, 1999, WO 00/09543 published February 24, 2000.
Table 1 below lists some illustrative examples of compounds that can be administered with the compounds of this invention. The compounds of the invention can be administered with other anti-HCV activity compounds in combination therapy, either jointly or separately, or by combining the compounds into a composition.
TABLE 1
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
The pharmaceutical compositions of this invention maybe administered orally, parenterally or via an implanted reservoir. Oral administration or administration by injection are prefened. In some cases, the pH ofthe formul-ation may be adjusted with pharmaceutically acceptable acids, bases or buffers to ejnhance the stability ofthe formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, mtramusculax, intra- articular, intrasynovial, intrasternal, intrathecal, and intralesional injection or infusion techniques. When orally administered, the pharmaceutical compositions of this invention may be administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, caniers which are commonly used include lactose and corn starch.
Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried com starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added. Other suitable caniers for the above noted compositions can be found in standard pharmaceutical texts, e.g. in "Remington's Pharmaceutical Sciences", 19th ed., Mack Publishing Company, Easton, Penn., 1995.
The pharmaceutical compositions can be prepared by known procedures using well-known and readily available ingredients. The compositions of this invention may be formulated so as to provide quick, sustained or delayed release ofthe active ingredient after administration to the patient by employing procedures well known in the art. In making the compositions ofthe present invention, the active ingredient will usually be admixed with a canier, or diluted by a canier, or enclosed within a canier which may be in the form of a capsule, sachet, paper or other container. When the canier serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, excipient or medium for the active ingredient. Thus, the compositions can be in the fonn of tablets, pills, powders, beadlets, lozenges, sachets, elixirs, suspensions, emulsions, solutions, syraps, aerosols, (as a solid or in a liquid medium), soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders and the like. Further details concerning the design and preparation of suitable delivery forms ofthe pharmaceutical compositions ofthe invention are known to those skilled in the art.
Dosage levels of between about 0.01 and about 1000 milligram per kilogram ("mg/kg") body weight per day, preferably between about 0.5 and about 250 mg/kg body weight per day ofthe compounds ofthe invention are typical in a monotherapy for the prevention and treatment of HCV mediated disease. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the canier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
As the skilled artisan will appreciate, lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course ofthe infection, the patient's disposition to the infection and the judgment ofthe treating physician. Generally, treatment is initiated with small dosages substantially less than the optimum dose ofthe peptide. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. In general, the compound is most desirably administered at a concentration level that will generally afford antivirally effective results without causing any harmful or deleterious side effects.
When the compositions of this invention comprise a combination of a compound ofthe invention and one or more additional therapeutic or prophylactic agent, both the compound and the additional agent are usually present at dosage levels of between about 10 to 100%, and more preferably between about 10 and 80% ofthe dosage normally administered in a monotherapy regimen.
When these compounds or their pharmaceutically acceptable enantiomers, diastereomers, salts, solvates or prodrugs are formulated together with a pharmaceutically acceptable canier, the resulting composition may be administered in vivo to mammals, such as man, to inhibit HCV NS3 protease or to treat or prevent HCV viras infection. Accordingly, another aspect of this invention provides methods of inhibiting HCV NS3 protease activity in patients by administering a compound ofthe present invention or a pharmaceutically acceptable enantiomer, diastereomer, salt or solvate thereof.
In one aspect ofthe invention, there is provided a method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount ofthe compound ofthe invention, or a pharmaceutically acceptable enantiomer, diastereomer, solvate, prodrag or salt thereof.
Preferably, the method of administering the compound is effective to inhibit the function ofthe HCV NS3 protease protein. In a prefened aspect, the method further comprises administering another compound having anti-HCV activity (as described above) prior to, after or concunently with a compound ofthe invention.
The compounds ofthe invention may also be used as laboratory reagents. Compounds may be instrumental in providing research tools for designing of viral replication assays, validation of animal assay systems and stractural biology studies to further enhance knowledge ofthe HCV disease mechanisms. Further, the compounds ofthe present invention are useful in establishing or determining the binding site of other antiviral compounds, for example, by competitive inhibition.
The compounds of this invention may also be used to treat or prevent viral contamination of materials and therefore reduce the risk of viral infection of laboratory or medical personnel or patients who come in contact with such materials, e.g., blood, tissue, surgical instruments and garments, laboratory instruments and garments, and blood collection or transfusion apparatuses and materials.
Further, the compounds and compositions ofthe invention can be used for the manufacture of a medicament for treating HCV infection in a patient. EXAMPLES
The specific examples that follow illustrate the syntheses ofthe compounds of the instant invention, and are not to be construed as limiting the invention in sphere or scope. The methods may be adapted to variations in order to produce compounds embraced by this invention but not specifically disclosed. Further, variations ofthe methods to produce the same compounds in somewhat different manner will also be evident to one skilled in the art.
Solution percentages express a weight to volume relationship, and solution ratios express a volume to volume relationship, unless stated otherwise. Nuclear magnetic resonance (NMR) spectra were recorded either on a Braker 300, 400 or 500 MHz spectrometer; the chemical shifts (5) are reported in parts per million. Flash chromatography was canied out on silica gel (SiO ) according to Still's flash chromatography technique (W.C. Still et al., J. Org. Chem., (1978), 43, 2923).
All Liquid Chromatography (LC) data were recorded on a Shimadzu LC-10AS liquid chromatograph using a SPD-10AV UV-Vis detector and Mass Spectrometry (MS) data were determined with a Micromass Platform for LC in electrospray mode (ES+).
Unless otherwise noted, in the following examples each compound was analyzed by LC/MS, using one of seven methodologies, having the following conditions. Columns: (Method A) - YMC ODS S7 CI 8 3.0x50 mm
(Method B) - YMC ODS-A S7 C18 3.0x50 mm (Method C) - YMC S7 C18 3.0x50 mm (Method D) - YMC Xtena ODS S7 3.0x50 mm (Method E) - YMC Xtena ODS S7 3.0x50 mm (Method F) - YMC ODS-A S7 C 18 3.0x50 mm
(Method G) - YMC C18 S5 4.6x50 mm] Gradient: 100% Solvent A 0% Solvent B to 0% Solvent A/100% Solvent B Gradient time: 2 min. (A, B, D, F, G); 8 min. (C, E) Hold time: 1 min. (A, B, D, F, G); 2 min. (C, E) Flow rate: 5 mL/min Detector Wavelength: 220 nm Solvent A: 10% MeOH / 90% H2O / 0.1 % TFA Solvent B: 10% H2O / 90% MeOH / 0.1% TFA.
The abbreviations used in the present application, including particularly in the illustrative examples which follow, are well-known to those skilled in the art. Some ofthe abbreviations used are as follows:
rt room temperature
Boc tert-butyloxycarbonyl
DMSO dimethylsulfoxide
EtOAc ethyl acetate t-BuOK potassium t-butoxide
Et2O diethyl ether
TBME tert-butylmethyl ether
THF tetrahydrofuran
GDI carbonyldiimidazole
DBU l,8-diazabicyclo[5.4.0]undec-7-ene
TFA trifluoroacetic acid
NMM N-methylmoφholine
HATU O-7-azabenzotriazol- 1 -yl
HBTU O-{lH-benzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate
HOBT N-hydroxybenzotriazole PyBrop bromo-bis-pynolidine-phosphonium hexafluorophosphate
DMF dimethylformamide MeOH methanol EDTA ethylenediaminetetraacetic acid HRMS high resolution mass spectrometry DMAP 4-dimethylaminopyridine
DIPEA diisopropylethylamine
DCM dichloromethane
DCE dichloroethane
The compounds and chemical intermediates ofthe present invention, described in the following examples, were prepared according to the following methods. It should be noted that the following exemplification section is presented in sections. Example numbers and compound numbers are not contiguous throughout the entire Examples portion ofthe application and hence, each section indicates a "break" in the numbering. The numbering within each section is generally contiguous.
Section A:
Preparation of Intennediates: Preparation of PI Intermediates: The PI intennediates described in this section can be used to prepare compounds of Formula I by the methods described herein.
I PI elements:
1. Preparation of racemic (lR,2S)/(lS,2R)-l-amino-2-vinylcyclopropane carboxylic acid ethyl ester
Figure imgf000062_0001
Method A Step 1
Figure imgf000063_0001
Glycine ethyl ester hydrochloride (303.8 g, 2.16 mole) was suspended in tert- butylmethyl ether (1.6 L). Benzaldehyde (231 g, 2.16 mole) and anhydrous sodium sulfate (154.6 g, 1.09 mole) were added and the mixture cooled to 0 °C using an ice- water bath. Triethylamine (455 mL, 3.26 mole) was added dropwise over 30 min and the mixture stined for 48 h at rt. The reaction was then quenched by addition of ice- cold water (1 L) and the organic layer was separated. The aqueous phase was extracted with tert-butyhnethyl ether (0.5 L) and the combined organic phases washed with a mixture of saturated aqueous NaHCO3 (1 L) and brine (1 L). The solution was dried over MgSO4, concentrated in vacuo to afford 392.4 g ofthe N- benzyl imine product as a thick yellow oil that was used directly in the next step. 1H ΝMR (CDC13, 300 MHz) 6 1.32 (t, J=7.1 Hz, 3H), 4.24 (q, J=7.1 Hz, 2H), 4.41 (d, J=l.l Hz, 2H), 7.39-7.47 (m, 3H), 7.78-7.81 (m, 2H), 8.31 (s, IH).
Step 2
Figure imgf000063_0002
toluene/RT
2 ) H30+
3 ) aOH
4 ) (BOC) 20
To a suspension of lithium tert-butoxide (84.06 g, 1.05 mol) in dry toluene (1.2 L), was added dropwise a mixture ofthe N-benzyl imine of glycine ethyl ester (100.4 g, 0.526 mol) and trø7M-l,4-dibromo-2-butene (107.0 g, 0.500 mol) in dry toluene (0.6 L) over 60 min. After completion ofthe addition, the deep red mixture was quenched by addition of water (1 L) and tert-butyhnethyl ether (TBME, 1 L). The aqueous phase was separated and extracted a second time with TBME (1 L). The organic phases were combined, 1 Ν HCI (1 L) was added and the mixture stined at room temperature for 2 h. The organic phase was separated and extracted with water (0.8 L). The aqueous phases were then combined, saturated with salt (700 g), TBME (1 L) was added and the mixture cooled to 0 °C. The stined mixture was then basifϊed to pH 14 by the dropwise addition of 10 N NaOH, the organic layer separated, and the aqueous phase extracted with TBME (2 x 500 mL). The combined organic extracts were dried (MgSO ) and concentrated to a volume of IL. To this solution of free amine, was added BOC2O or di-tert-butyldicarbonate (131.0 g, 0.6 mol) and the mixture stirred 4 days at rt. Additional di-tβrt-butyldicarbonate (50 g, 0.23 mol) was added to the reaction, the mixture refluxed for 3 h, and was then allowed cool to room temperature overnight. The reaction mixture was dried over MgSO4 and concentrated in vacuo to afford 80 g of crude material. This residue was purified by flash chromatography (2.5 Kg of SiO , eluted with 1% to 2% MeOH/CH2Cl2) to afford 57 g (53%) of racemic N-Boc-(lR,2S)/(lS,2R)-l-amino-2- vinylcyclopropane carboxylic acid ethyl ester as a yellow oil which solidified while sitting in the refrigerator: 1H ΝMR (CDC13, 300 MHz) δ 1.26 (t, J=7.1 Hz, 3H), 1.46 (s, 9H), 1.43-1.49 (m, IH), 1.76-1.82 (br m, IH), 2.14 (q, J=8.6 Hz, IH), 4.18 (q, J=7.2 Hz, 2H), 5.12 (ddJ=10.3, 1.7 Hz, IH), 5.25 (br s, IH), 5.29 (dd, J=17.6, 1.7 Hz, IH), 5.77 (ddd, J=17.6, 10.3, 8.9 Hz, IH); MS m/z 254.16 (M-l)
Step 3 Preparation of Racemic (1R,2S)/(1S,2R) l-amino-2-vinylcyclopropane carboxylic acid ethyl ester hydrochloride
Figure imgf000064_0001
N-Boc-(lR,2S)/(lS,2R)-l -amino-2-vinylcyclopropane carboxylic acid ethyl ester (9.39 g, 36.8 mmol) was dissolved in 4 Ν HCl dioxane (90 ml, 360 mmol) and was stined for 2 h at rt. The reaction mixture was concentrated to supply (lR,2S)/(lS,2R)-l-amino-2-vinylcyclopropane carboxylic acid ethyl ester hydrochloride in quantitative yield (7 g, 100%). 1H ΝMR (methanol-dø δ 1.32 (t, J=7.1, 3H), 1.72 (dd, J=10.2, 6.6 Hz, IH), 1.81 (dd, J=8.3, 6.6 Hz, IH), 2.38 (q, J=8.3 Hz, IH), 4.26-4.34 (m, 2H), 5.24 (dd, 10.3, 1.3 Hz, IH) 5.40 (d, J=17.2, IH), 5.69-5.81 (m, IH).
Alternate route for the preparation of Racemic N-Boc-l-amino-2- vinylcyclopropane carboxylic acid ethyl ester hydrochloride
Figure imgf000065_0001
To a solution of potassium tert-butoxide (11.55 g, 102.9 mmol) in THF (450 mL) at — 78 °C was added the commercially available NN-dibenzyl imine of glycine ethyl ester (25.0 g, 93.53 mmol) in THF (112 mL). The reaction mixture was warmed to 0 °C, stined for 40 min, and was then cooled back to -78 °C. To this solution was added trα7M-l,4-dibromo-2-butene (20.0 g, 93.50 mmol), the mixture stined for 1 h at 0°C and was cooled back to -78°C. Potassium tert-butoxide (11.55 g, 102.9 mmol) was added, the mixture immediately warmed to 0°C, and was stined one more hour before concentrating in vacuo. The crade product was taken up in Et2O (530 mL), IN aq. HCI solution (106 mL, 106 mmol) added and the resulting biphasic mixture stined for 3.5 h at rt. The layers were separated and the aqueous layer was washed with Et O (2x) and basified with a saturated aq. ΝaHCO3 solution. The desired amine was extracted with Et2O (3x) and the combined organic extract was washed with brine, dried (MgSO4), and concentrated in vacuo to obtain the free amine. This material was treated with a 4N HCI solution in dioxane (100 mL, 400 mmol) and concentrated to afford (lR,2S)/(lS,2R)-l-amino-2-vinylcyclopropane carboxylic acid ethyl ester hydrochloride as a brown semisolid (5.3 g, 34% yield) identical to the material obtained from procedure A, except for the presence of a small unidentified aromatic hnpurity (8%).
Resolution of N-Boc-(lR,2S)/(lS,2R)-l-amino-2-vinylcyclopropane carboxylic acid ethyl ester
Figure imgf000066_0001
Resolution A
To an aqueous solution of sodium phosphate buffer (0.1 M, 4.25 liter ("L"), pH 8) housed in a 12 Liter jacked reactor, maintained at 39°C, and stined at 300 φm was added 511 grams of Acalase 2.4L (about 425 mL) (Novozymes North America Inc.). When the temperature ofthe mixture reached 39°C, the pH was adjusted to 8.0 by the addition of a 50% NaOH in water. A solution ofthe racemic N-Boc- (lR,2S)/(lS,2R)-l-amino-2-vinylcyclopropane carboxylic acid ethyl ester (85g) in
850 mL of DMSO was then added over a period of 40 min. The reaction temperature was then maintained at 40°C for 24.5h during which time the pH ofthe mixture was adjusted to 8.0 at the 1.5h and 19.5h time points using 50% ΝaOH in water. After 24.5h, the enantio-excess ofthe ester was determined to be 97.2%, and the reaction was cooled to room temperature (26°C) and stined overnight (16h) after which the enantio-excess ofthe ester was determhied to be 100%. The pH ofthe reaction mixture was then adjusted to 8.5 with 50% NaOH and the resulting mixture was extracted with MTBE (2 x 2 L). The combined MTBE extract was then washed with 5% NaHCO3 (3 x 100 mL), water (3 x 100 mL), and evaporated in vacuo to give the enantiomerically pure N-Boc-(lR,2S)/-l-amino-2-vinylcyclopropane carboxylic acid ethyl ester as light yellow solid (42.55 g; purity: 97% @ 210 nm, containing no acid; 100% enantiomeric excess ("ee").
The aqueous layer from the extraction process was then acidified to pH 2 with 50% H2SO4 and extracted with MTBE (2 x 2 L). The MTBE extract was washed with water (3 x 100 mL) and evaporated to give the acid as light yellow solid (42.74 g; purity: 99% @ 210 nm, containing no ester).
Figure imgf000067_0001
1 R, 2S-ester 1S,2R-acid
Figure imgf000067_0002
Figure imgf000068_0001
Resolution B
To 0.5 mL 100 mM Heps*Na buffer (pH 8.5) in a well of a 24 well plate (capacity: 10 ml/well), 0.1 mL of Savinase 16.0L (protease from Bacillus clausii) (Novozymes North America Inc.) and a solution ofthe racemic N-Boc- (lR,2S)/(lS,2R)-l-amino-2-vinylcyclopropane carboxylic acid ethyl ester (10 mg) in 0.1 mL of DMSO were added. The plate was sealed and incubated at 250 rpm at 40°C. After 18h, enantio-excess ofthe ester was determined to be 44.3% as following: 0.1 mL ofthe reaction mixture was removed and mixed well with 1 mL ethanol; after centrifugation, 10 microliter ("μl") ofthe supernatant was analyzed with the chiral HPLC. To the remaining reaction mixture, 0.1 mL of DMSO was added, and the plate was incubated for additional 3 days at 250 φm at 40°C, after which four L of ethanol was added to the well. After centrifugation, 10 μl ofthe supernatant was analyzed with the chiral HPLC and enantio-excess ofthe ester was determined to be 100%.
Resolution C To 0.5 ml 100 mM Heps*Na buffer (pH 8.5) in a well of a 24 well plate (capacity: 10 mL/well), 0.1 ml of Esperase 8.0L, (protease from Bacillus halodurans) (Novozymes North America Inc.) and a solution ofthe racemic N-Boc- (lR,2S)/(lS,2R)-l-amino-2-vinylcyclopropane carboxylic acid ethyl ester (10 mg) in 0.1 mL of DMSO were added. The plate was sealed and incubated at 250 φm at 40°C. After 18 hour, enantio-excess ofthe ester was determined to be 39.6% as following: 0.1 mL ofthe reaction mixture was removed and mixed well with 1 mL ethanol; after cemifugation, 10 μl ofthe supernatant was analyzed with the chiral HPLC. To the remaining reaction mixture, 0.1 mL of DMSO was added, and the plate was incubated for additional 3 days at 250 φm at 40°C, after which four mL of ethanol was added to the well. After centrifugation, 10 μl ofthe supernatant was analyzed with the chiral HPLC and enantio-excess ofthe ester was determined to be 100%.
Samples analysis was canied out in the following manner:
1) Sample preparation: About 0.5 ml ofthe reaction mixture was mixed well with 10 volume of EtOH. After centrifugation, 10 μl ofthe supernatant was injected onto HPLC column.
2) Conversion determination:
Column: YMC ODS A, 4.6 x 50 mm, S-5 μm
Solvent: A, 1 mM HCI in water; B, MeCΝ
Gradient: 30% B for 1 min; 30% to 45% B over 0.5 min; 45% B for 1.5 min; 45% to 30% B over 0.5 min.
Flow rate: 2 ml/min
UV Detection: 210 nm
Retention time: acid, 1.2 min; ester, 2.8 min.
3) Enantio-excess determination for the ester:
Column: CHIRACEL OD-RH, 4.6 x 150 mm, S-5 μm Mobile phase: MeCΝ/50 mM HClO4 in water (67/33) Flow rate: 0.75 ml/min. UV Detection: 210 nm. Retention time:
(IS, 2R) isomer as acid: 5.2 min; Rcaemate: 18.5 min and 20.0 min; (IR, 2S) isomer as ester: 18.5 min.
2. Preparation of Λ?-Boc-(lR,25)-l-amino-2-cyclopropylcycIopropane carboxylic acid ethyl ester
Figure imgf000070_0001
A solution of N-Boc-(lR,2S)-l-amino-2-vinylcyclopropane carboxylic acid (255 mg, 1.0 rnmol) in ether (10 mL) was treated with palladium acetate (5 mg, 0.022 mmol). The orange/red solution was placed under an atmosphere of Ν2. An excess of diazomethane in ether was added dropwise over the course of 1 h. The resulting solution was stined at rt for 18 h. The excess diazomethane was removed using a stream of nitrogen. The resulting solution was concentrated by rotary evaporation to give the crade product. Flash chromatography (10% EtOAc/hexane) provided 210 mg C78%) of N-Boc-(lR,2S)-l-amino-2-cyclopropylcyclopropane carboxylic acid ethyl ester as a colorless oil. LC-MS (retention time: 2.13, similar to method A except: gradient time 3 min, Xtena MS C18 S7 3.0 x 50mm column), MS m/e 270 (M++l).
3. 1-tert-butoxycarbonylamino-cyclopropane-carboxylic acid is commercially available
Figure imgf000070_0002
4. Preparation of 1-aminocyclobutanecarboxylic acid methyl ester-hydrochloride
Figure imgf000071_0001
1-aminocyclobutanecarboxylic acid (100 mg, 0.869 mmol)(Tocris) was dissolved in 10 mL of MeOH, HCI gas was bubbled in for 2h. The reaction mixture was stined for 18 h, and then concentrated in vacuo to give 144 mg of a yellow oil. Trituration with 10 mL of ether provided 100 mg ofthe titled product as a white solid. !H NMR (CDCI3) δ 2.10-2.25 (m, IH), 2.28-2.42 (m, IH), 2.64-2.82 (m, 4H), 3.87 (s, 3H), 9.21 (br s, 3H).
5. Preparation of racemic (1R,2R)/(1S,2S) l-Amino-2- ethylcyclopropanecarboxylic acid tert-butyl ester, shown below.
Figure imgf000071_0002
ethyl syn to carboxy
Step 1 : Preparation of 2-Ethylcyclopropane- 1 , 1 -dicarboxylic acid di-tert-butyl ester, shown below.
Figure imgf000071_0003
To a suspension of benzyltriethylammonium chloride (21.0 g, 92.2 mmol) in a 50% aqueous NaOH solution (92.4 g in 185 mL H2O) was added 1,2-dibromobutane (30.0 g, 138.9 mmol) and di-tert-butylmalonate (20.0 g, 92.5 mmol). The reaction mixture was vigorously stined 18 h at rt, a mixture of ice and water was then added. The crude product was extracted with CH C12 (3x) and sequentially washed with water (3x), brine and the organic extracts combined. The organic layer was dried (MgSO4), filtered and concentrated in vacuo. The resulting residue was flash chromatographed (100 g SiO2, 3% Et2O in hexane) to afford the titled product (18.3 g, 67.8 mmol, 73% yield) which was used directly in the next reaction.
Step 2: Preparation of racemic 2-Ethylcyclopropane- 1,1 -dicarboxylic acid tert-butyl ester, shown below.
Figure imgf000072_0001
The product of Step 1 (18.3 g, 67.8 mmol) was added to a suspension of potassium tert-butoxide (33.55 g, 299.0 mmol) in dry ether (500 mL) at 0 °C, followed by H20 (1.35 mL, 75.0 mmol) and was vigorously stined overnight at rt. The reaction rnixture was poured in a mixture of ice and water and washed with ether (3x). The aqueous layer was acidified with a 10% aq. citric acid solution at 0°C and extracted with EtOAc (3x). The combined organic layers were washed with water (2x), brine, dried (MgSO4) and concentrated in vacuo to afford the titled product as a pale yellow oil (10 g, 46.8 mmol, 69% yield). Step 3: Preparation of (1R,2R)/(1S,2S) 2-Ethyl-l-(2- trimethylsilanylethoxycarbonylamino)cyclopropane-carboxylic acid tert-butyl ester, shown below.
Figure imgf000072_0002
To a suspension, ofthe product of Step 2 (10 g, 46.8 mmol) and 3 g of freshly activated 4A molecular sieves in dry benzene (160 mL), was added Et3N (7.50 mL, 53.8 mmol) and DPPA (11 mL, 10.21 mmol). The reaction mixture was refluxed for 3.5 h, 2-trimethylsilyl-ethanol (13.5 mL, 94.2 mmol) was then added, and the reaction mixture was refluxed ovemite. The reaction mixture was filtered, diluted with Et2O, washed with a 10% aqueous citric acid solution, water, saturated aqueous NaHCO3, water (2x), brine (2X), dried (MgSO4) and concentrated in vacuo. The residue was suspended with lOg of Aldrich polyisocyanate scavenger resin in 120 mL of CH C12, stirred at rt ovemite and filtered to afford the titled product (8 g, 24.3 mmol; 52%) as a pale yellow oil: 1H NMR (CDC13) δ 0.03 (s, 9H), 0.97 (m, 5H), 1.20 (bm, IH), 1.45 (s, 9H), 1.40-1.70 (m, 4H), 4.16 (m, 2H), 5.30 (bs, IH). Step 4: Preparation of racemic (IR,2R)/(1S,2S) l-Amino-2- ethylcyclopropanecarboxylic acid tert-butyl ester, shown below.
Figure imgf000073_0001
ethyl syn to carboxy To the product of Step 3 (3 g, 9 mmol) was added a 1.0 M TBAF solution in THF (9.3 mL, 9.3 mrnol) and the mixture heated to reflux for 1.5 h, cooled to rt and then diluted with 50O ml of EtOAc. The solution was successively washed with water (2x100 mL), brine (2x100 mL), dried (MgSO4), concentrated in vacuo to provide the title intermediate
6. Preparation of l-Amino-spiro[2.3]hexane-l-carboxylic acid methyl ester hydrochloride salt
Figure imgf000073_0002
Step 1 Preparation of [2,3]hexane- 1 , 1 -dicarboxylic acid dimethyl ester, shown below.
Figure imgf000073_0003
To a mixture of methylene-cyclobutane (1.5 g, 22 mmol) and Rh2(OAc)4 (125 mg,
0.27 mmol) in anhydrous CH2C12 (15 mL) was added 3.2 g (20 mmol) of dimethyl diazomalonate (prepared according to J. Lee et al. Synth. Comm., 1995, 25, 1511- 1515) at 0°C over a period of 6 h. The reaction mixture was then warmed to rt and stined for another 2 h. The mixture was concentrated and purified by flash chromatography (eluting with 10:1 hexane/Et2O to 5:1 hexane/Et O) to give 3.2 g
(72%) of [2,3 ]hexane- 1,1 -dicarboxylic acid dhnethyl ester as a yellow oil. 1H NMR (300 MHz, CDCI3) δ 3.78 (s, 6 H), 2.36 (m, 2 H), 2.09 (m, 3 H), 1.90 (m, 1 H), 1.67 (s, 2 H). LC-MS: MS m/z 199 (M++l). Step 2: Preparation of spiro[2,3]hexane-l,l -dicarboxylic acid methyl ester, shown below.
Figure imgf000074_0001
To the mixture of spiro [2,3]hexane- 1 , 1 -dicarboxylic acid dimethyl ester (200 mg, 1.0 rrrmol) in 2 L of MeOH and 0.5 mL of water was added KOH (78 mg, 1.4 mmol). This solution was stined at rt for 2 days. It was then acidified with dilute HCI and extracted two times with ether. The combined organic phases were dried
(MgS04) and concentrated to yield 135 mg (73%) of 2 as a white solid. *H NMR (300 MHz, CDCI3) δ 3.78 (s, 3 H), 2.36-1.90 (m, 8 H). LC-MS: MS m/z 185 (M÷+l)
Step 3: Preparation ofthe titled product, l-amino-spiro[2.3]hexane-l -carboxylic acid methyl ester hydrochloride salt.
To a rnixture of spiro[2,3]hexane- 1,1 -dicarboxylic acid methyl ester (660 mg, 3.58 mmol) in 3 mL of anhydrous t-BuOH was added 1.08 g (3.92 mmol) of DPP A and 440 mg (4.35 mmol) of Et3N. The mixture was heated at reflux for 21 h and then partitioned between H O and ether. The ether phase was dried over magnesium sulfate, filtered and concentrated in vacuo to yield an oil. To this oil was added 3 mL of a 4 M HCl/dioxane solution. This acidic solution was stined at rt for 2 h and then concentrated in vacuo. The residue was triturated with ether to give 400 mg (58 %) of desried prodict as a white solid. *H NMR (300 MHz, d6-DMSO) δ 8.96 (br s, 3 H), 3.71 (s, 3 H), 2.41 ( , 1 H), 2.12 (m, 4 H), 1.93 (m, 1 H), 1.56 (q, 2 H, J=8 Hz). LCMS of free amine: MS m/z 156 (M÷+l).
7. Preparation of l-Amino-spiro[2.4]heptane-l-carboxylic acid methyl ester hydrochloride salt, shown below, was prepared as follows.
Figure imgf000075_0001
Step 1: Spiro[2.4]heptane- 1,1 -dicarboxylic acid dimethyl ester, shown below, was prepared as follows.
Figure imgf000075_0002
Using the same procedure described in the preparation of l-Amino-spiro[2.3]hexane- 1 -carboxylic acid methyl ester hydrochloride salt 1.14g (13.9 mmol) of methylenecyclopentane and 2.0 g (12.6 mmol) of dimethyl diazomalonate were reacted to yield 1.8 g (67%) ofthe dimethyl ester. XH NMR (300 MHz, CDCI3) δ 3.73 (s, 6 H), 1.80 (m, 2 H), 1.70 (m, 4 H), 1.60 (m, 4 H). LC-MS: MS m/z 213 (M++l).
Step 2: Preparation of Spiro[2.4]heptane-l,l-dicarboxylic acid methyl ester, shown below, was prepared as follows.
Figure imgf000075_0003
Using the same procedure described in the preparation of 1 -Amino-spiro[2.3]hexane- 1 -carboxylic acid methyl ester hydrochloride salt 1.7 g (8.0 mmol) ofthe produc of Step 1 and 493 mg (8.8 mmol) of KOH gave 1.5 g
(94%) of spiro[2.4]heptane- 1,1 -dicarboxylic acid methyl ester. lH NMR (300 MHz, CDCI3) δ 3.80 (s, 3 H), 2.06 (d, 1 H, J=5 Hz), 1.99 (d, 1 H, J=5 Hz), 1.80-1.66 (m, 8 H). LC-MS: MS m/z 199 (M++l).
Step 3: Preparation of l-Arnino-spiro[2.4]heptane-l -carboxylic acid methyl ester hydrochloride salt, shown below, was prepared as follows.
Figure imgf000076_0001
Using the same procedure described above in preparation of 1-Amino- spiro[2.3]hexane-l -carboxylic acid methyl ester hydrochloride salt, 500 mg (2.5 mmol) ofthe product of Step 2, 705 mg (2.5 mmol) of DPP A and 255 mg (2.5 mmol) of Et3N gave 180 mg (35%) of this hydrochloride salt. 1H NMR (300 MHz, d6- DMSO) δ 8.90 (br s, 3 H), 3.74 (s, 3 H), 1.84 (m, 1 H), 1.69 (m, 4 H), 1.58 (m, 4 H), 1.46 (d, 1 H, J=6 Hz). LC-MS of free amine: MS m z 170 (M++l).
8. Preparation of l-Amino-spiro[2.2]pentane-l-carboxylic acid methyl ester hydrochloride salt, shown below, was prepared as follows.
Figure imgf000076_0002
Step 1: Spiro[2.2]pentane-l,l-dicarboxylic acid dimethyl ester, shown below, was prepared as follows.
Figure imgf000076_0003
To a mixture of methylenecyclopropane (1.0 g, 18.5 mmol)(prepared according to P. Binger US Patent Serial No. 5,723,714) and Rh2(OAc)4 (82mg, 0.185 mmol) in anhydrous CH2C1 (10 mL), was added dimethyl diazomalonate (2.9 g, 18.3 mmol) at 0°C. At the top ofthe flask was installed a cold finger, the temperature of which was kept at -10°C. The reaction mixture was warmed to rt and stined for another 2 h. The mixture was concentrated in vacuo and purified by flash chromatography (eluting with 10:1 hexane/Et20 to 5:1 hexane/Et2O) to give 0.85 g (25%) ofthe dimethyl ester as a yellow oil. ^HNMR (300 MHz, CDCI3) δ 3.73 (s, 6 H), 1.92 (s, 2 H), 1.04 (d, 4 H, J=3 Hz). Step 2: Sρiro[2.2]pentane-l,l-dicarboxylic acid methyl ester, shown below, was prepared as follows.
Figure imgf000077_0001
Using the same procedure described above in preparation of 1-Amino- spiro[2.3]hexane-l -carboxylic acid methyl ester hydrochloride salt, 800 mg (4.3 mmol) ofthe product of step 1 and 240 mg (4.3 mmol) of KOH gave 600 mg (82%) of Spiro[2.2]pentane- 1 , 1 -dicarboxylic acid methyl ester. 1 H NMR (300 MHz, CDCI3) δ 3.82 (s, 6 H), 2.35 (d, 1 H, J=3 Hz), 2.26 (d, 1 H, J=3 Hz), 1.20 (m, 1 H),
1.15 (m, 1 H), 1.11 (m, 1 H), 1.05 (m, 1 H). LRMS: MS m/z 169 (M+-l) (Method D).
Step 3: l-Amino-spiro[2.2]pentane-l -carboxylic acid methyl ester hydrochloride salt, shown below, was prepared as follows.
Figure imgf000077_0002
Using the same procedure described above for the preparation of 1 -Amino- spiro[2.3]hexane-l -carboxylic acid methyl ester hydrochloride salt, 400 mg (2.3 mmol) ofthe product of step 2, 700 mg (2.5 mmol) of DPPA and 278 mg (2.7 mmol) of Et3N gave 82 mg (20%) ofthe hydrochloride salt. 1H NMR (300 MHz, CDCI3) δ
9.19 (br s, 3 H), 3.81 (s, 3 H), 2.16, (d, J=5.5 Hz, 1 H), 2.01 (d, J=5.5 Hz, 1 H), 1.49 (m, 1 H), 1.24, (m, 1 H), 1.12 (m, 2 H). LRMS of free amine: MS m/z 142 (M++l).
9. Preparation of 5-Amino-spiro[2.3]hexane-5-carboxylic acid ethyl ester, shown below, was prepared as follows.
V -NH2 °" "OEt
Spiro[2.3]hexan-4-one (500 mg, 5 mmol), which was prepared from bicyclopropylidene (A. Meijere et al. Org. Syn. 2000, 78, 142-151) according to A. Meijere et al. J. Org. Chem. 1988, 53, 152-161, was combined with ammonium carbamate (1.17 g, 15 mmol) and potassium cyanide (812 mg, 12.5 mmol) in 50 mL of EtOH and 50 mL of water. The mixture was heated at 55 °C for 2 days. Then NaOH (7 g, 175 mmol) was added and the solution was heated under reflux overnight. The mixture was then chilled to 0 °C, acidified to pH 1 with concentrated HCI, and concentrated in vacuo. EtOH was added to the crude amino acid mixture and then concentrated to dryness (5x) so as to remove residual water. The residue dissolved in 100 mL of EtOH was cooled to 0 °C. It was then treated with 1 mL of SOCl2 and refluxed for 3 days. The solids were removed by filtration, and the filtrate was concentrated in vacuo to give the crade product. The crade product was partitioned between 3 N NaOH, NaCl and EtOAc. The organic phase was dried over potassium carbonate and concentrated. The residue was purified using column chromatography on C18 silica gel (eluting with MeOH/H2O) to yield 180 mg (21%) of 15 as an oil. H NMR (300 MHz, CDCI3) δ 8.20 (br s, 2 H), 4.27 (s, 2 H), 2.80 (s, 1 H), 2.54 (s, 1 H), 2.34 (m, 2 H), 1.31 (s, 3 H), 1.02 (s, 1 H), 0.66 (m, 3 H). 13C NMR (300 MHz, CDCI3) δ 170.2(s), 63.0(s), 62.8 (s), 26.1 (s), 26.0 (s), 24.9 (s),
13.9 (s), 11.4 (s), 10.9 (s). LC-MS: MS m/z 170 (M++l).
II Heterocycles to be used as starting material in the construction of P2 elements for subsequent incorporation into compounds of Formula I.
1. Isoquinolines
Figure imgf000079_0001
Commercial material using the chemsitry described Method 1 \ in the f0||0Wjng sections HO alkoxide promoted Method 2 coupling
Mitsunobu H coupling BroVc °
Figure imgf000079_0002
Compounds of Formula
Figure imgf000079_0003
Isoquinoline (1) and substituted analogues thereof, can be incoφorated into P2 elements using the two methods outline above and described in detail herein. Said P2 elements (3) can then be converted into compounds of Formula I using procedures analogous to those described herein for similar isoquinoline analogues.
2. Isoxazolepyridine and Oxazolepyridiιιe(l)
Compounds of Formula I
Compounds of Formula I
Figure imgf000079_0004
Isoxazole and oxazole heterocycle (1) and analogues thereof can be prepared using know chemistry and incoφorated into compounds of Formula I using the chemistry described herein for similar isoxazolepyridine intermediates as shown in section B.
III PI prime elements:
The P 1 prime elements prepared below can be used to prepare compounds of Formula I by using the methods described herein.
1. Preparation of cyclopropylsulfonamide:
O H2N-S-<] O
Step 1 : Preparation of N-tert-Butyl-(3-chloro)propylsulfonamide
Figure imgf000080_0001
tert-Bixtylamine (3.0 mol, 315.3 mL) was dissolved in THF (2.5 L). The solution was cooled to — 20°C. 3-Chloropropanesulfonyl chloride (1.5 mol, 182.4 mL) was added slowly. The reaction mixture was allowed to warm to rt and stined for 24 h. The mixture was filtered, and the filtrate was concentrated in vacuo. The residue was dissolved in CH C12 (2.0 L). The resulting solution was washed with 1 N HCI (1.0 L), water (1.0 L), brine (1.0 L) and dried over Na SC»4. It was filtered and concentrated in vacuo to give a slightly yellow solid, which was crystallized from hexane to afford the product as a white solid (316.0 g, 99%).
1H NM R (CDC13) δ 1.38 (s, 9H), 2.30-2.27 (m, 2H), 3.22 (t, J- 7.35 Hz, 2H), 3.68
(t, J- 6.2 Hz, 2H), 4.35 (b, IH).
Step 2: preparation of Cyclopropanesulfonic acid tert-butylamide
Figure imgf000080_0002
To a solution of N-tert-butyl-(3-chloro)propylsulfonamide (2.14 g, 10.0 mmol) in THF (100 mL) was added n-BuLi (2.5 M in hexane, 8.0 mL, 20.0 mmol) at -78°C. The reation mixture was allowed to warm up to room temperature over period of 1 h. The volatiles were removed in vacuo. The residue was partitioned between EtOAC and water (200 mL, 200 mL). The separated organic phase was washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was recrystallized from hexane to yield the desired product as a white solid (1.0 g, 56%). 1H NMR (CDC13) δ 0.98-1.00 (m, 2H), 1.18-1.19 (m, 2H), 1.39 (s, 9H), 2.48-2.51 (m, lH), 4.19 (b, IH).
Step 3: preparation of cyclopropylsulfonamide
O
11 ^i H2N-S-<|
O A solution of cyclopropanesulfonic acid tert-butylamide (110.0 g, 0.62 mol) in TFA (500 mL) was stined at room temperature for 16 h. The volatile was removed in vacuo. The residue was recrystallized from EtOAC/hexane (60 mL/240 mL) to yield the desired product as a white solid (68.5 g, 91%).
1H NMR (DMSO-d6) δ 0.84-0.88 (m, 2H), 0.95-0.98 (m, 2H), 2.41-2.58 (m, IH), 6.56 (b, 2H).
2. Alternate procedure for the preparation of cyclopropyl sulfonamide ft NH3 ( sat ) THF 0 l>- O,rcι — 0 °C to rt *" ^- β,rNH2
To a solution of 100 mL of THF cooled to 0 °C was bubbled in gaseous ammonia until saturation was reached. To this solution was added a solution of 5 g (28.45 mmol) of cyclopropylsulfonyl chloride (purchased from Anay Biopharma) in 50 mL of THF, the solution warmed to rt ovemite and stined one additional day. The mixture was concentrated until 1-2 mL of solvent remained, applied onto 30 g plug of SiO2 (eluted with 30% to 60% EtOAc/Hexanes) to afford 3.45g (100%) of cyclopropyl sulfonamide as a white solid. 1H NMR (Methanol-cL,) δ 0.94-1.07 (m, 4H), 2.52-2.60 (m, IH); 13C NMR (methanol-ch) δ 5.92, 33.01.
3. Preparation of cyclobutyl sulfonamide o
S-NH,
I I Λ
O To a solution of 5.0 g (37.0 mmol) of cyclobutyl bromide in 30 mL of anhydrous diethyl ether (Et2O) cooled to -78 °C was added 44 mL (74.8 mmol) of 1.7M tert- butyl lithium in pentanes and the solution slowly warmed to -35 °C over 1.5 h. This mixture was cannulated slowly into a solution of 5.0 g (37.0 mmol) freshly distilled sulfuryl chloride in 100 mL of hexanes cooled to -40 °C, warmed to 0 °C over 1 h and carefully concentrated in vacuo. This mixture was redissolved in Et2O, washed once with some ice-cold water, dried (MgSO4) and concentrated carefully. This mixture was redissolved in 20 mL of THF, added dropwise to 500 mL of saturated NH3 in THF and was allowed to stir oveniite. The mixture was concentrated in vacuo to a crude yellow solid and was recrystallized from the minimum amount of CH2C12 in hexanes with 1-2 drops of MeOH to afford 1.90 g (38%) of cyclobutylsulfonamide as a white solid. 1HNMR (CDC13) δ 1.95-2.06 (m, 2H), 2.30- 2.54 (m, 4H), 3.86 (p, J=8 Hz, IH), 4.75 (brs, 2H); 13C NTV1R (CDC13) δ 16.43, 23.93, 56.29. HRMS m z (M-H)" calcd for C4H8NSO2: 134.0276, found 134.0282.
4 Preparation of cyclopentyl sulfonamide
Figure imgf000082_0001
A solution of 18.5 mL (37.0 mmol) of 2M cyclopentyl-magnesium chloride in ether was added dropwise to a solution of 3.0 mL (37.0 mmol) freshly distilled sulfuryl chloride (obtained from Aldrich) in 100 mL of hexanes cooled to — 78 °C. The mixture was warmed to 0 °C over 1 h and was then carefully concentrated in vacuo. This mixture was redissolved in Et O (200 mL), washed once with some ice-cold water (200 mL), dried (MgSO4) and concentrated carefully. This mixture was redissolved in 35 mL of THF, added dropwise to 500 mL of saturated NH3 in THF and was allowed to stir overnite. The mixture was concentrated in vacuo to a crude yellow solid, the residue filtered through 50g of silica gel using 70% EtOAc-hexanes as the eluent and the solution was then concentrated. The residue was recrystallized from the minimum amount of CH2C12 in hexanes with 1-2 drops of MeOH to afford 2.49 g (41%) of cyclopentylsulfonamide as a white solid. XH NMR (CDC13) δ 1.58- 1.72 (m, 2H), 1.74-1.88 (m, 2H), 1.94-2.14 (m, 4H), 3.48-3.59 (m, IH), 4.80 (bs, 2H); 13C NMR (CDC13) δ 25.90, 28.33, 63.54; MS m/e 148 (M-H)". 5. Preparation of cyclohexyl sulfonamide
Figure imgf000083_0001
A solution of 18.5 mL (37.0 mmol) of 2M cyclohexylmagnesium chloride (TCI Americas) in ether was added dropwise to a solution of 3.0 mL (37.0 mmol) freshly distilled sulfuryl chloride in 100 mL of hexanes cooled to —78 °C. The mixture was warmed to 0 °C over 1 h and was then carefully concentrated in vacuo. This mixture was redissolved in Et O (200 mL), washed once with some ice-cold water (200 mL), dried (MgSO4) and concentrated carefullyThis mixture was redissolved in 35 mL of THF, added dropwise to 500 mL of saturated NH3 in THF and was allowed to stir overnite. The mixture was concentrated in vacuo to a crude yellow solid, the residue filtered through 50g of silica gel using 70% EtOAc-hexanes as the eluent and was concentrated. The residue was recrystallized from the minimum amount of CH2C12 in hexanes with 1-2 drops of MeOH to afford 1.66 g (30%) of cyclohexyl- sulfonamide as a white solid: 1H NMR (CDC13) δ 1.11-1.37 (m, 3H), 1.43- 1.56 (m, 2H), 1.67-1.76 (m, IH), 1.86-1.96 (m, 2H), 2.18-2.28 (m, 2H), 2.91 (tt, J=12, 3.5 Hz, IH), 4.70 (bs, 2H); 13CH NMR (CDC13) δ 25.04, 25.04, 26.56, 62.74; MS m/e 162 (M-IV.
6. Preparation of Substituted Cycloalkyllsulfonamides for use in the construction of compounds of Formula 1. la) tBuNH2
"1-pot Ib-lc") n-BuLi (2 eq.);
Figure imgf000083_0002
Preparation of N-ter^-Butyl-(3-chloro)propylsulfonamide. As described above.
Figure imgf000084_0001
Preparation of N-tert-Butyl-(l-methyl)cyclopropyI-sulf onamide. A solution of N-tert-Butyl-(3-chloro)propylsulfonamide (4.3 g, 20 mmol) was dissolved in dry THF (100 mL) and cooled to - 78 °C. To this solution was added n- BuLi (17.6 mL, 44 mmol, 2.5 M in hexane) slowly. The dry ice bath was removed and the reaction mixture was allowed to warm to rt over a period of 1.5 h. This mixture was then cooled to - 78°C, and a solution of n- uLi (20 mmol, 8 mL, 2.5 M in hexane) was added. The reaction mixture was warmed to rt, recooled to -78 °C over a period of 2 h and a neat solution of methyliodide (5.68 g, 40 mmol) added. The reaction mixture was allowed to warm to rt overnight, quenched with saturated ΝH4C1 (100 mL) at rt. It was extracted with EtOAc (100 mL). The organic phase was washed with brine (100 mL), dried (MgSO4), and concentrated in vacuo to give a yellow oil which was crystallized from hexane to afford the product as a slightly yellow solid (3.1 g, 81%): 1HNMR (CDC13) δ 0.79 (m, 2H), 1.36 (s, 9H), 1.52 (m, 2H), 1.62 (s, 3H), 4.10 (bs, IH)
Figure imgf000084_0002
Preparation of Example 3, l-methylcyclopropylsulfonamide. A solution of N-tert-Butyl-(l-methyl)cyclopropylsulfonamide (1.91 g, 10 mmol) was dissolved in TFA (30 mL), and the reaction mixture stined at rt for 16 h. The solvent was removed in vacuo to give a yellow oil which was crystallized from EtOAc / hexane (1 : 4, 40 mL) to yield Example 3, l-methylcyclopropylsulfonamide, as a white solid (1.25 g, 96%): 1H ΝMR (CDC13) δ 0.84 (m, 2H), 1.41 (m, 2H), 1.58 (s, 3H), 4.65 (bs, 2H). Anal. Calcd. For C4H9ΝO2S: C, 35.54; H, 6.71; N, 10.36. Found: C, 35.67; H, 6.80; N, 10.40.
Figure imgf000085_0001
Preparation of N-terr-Butyl-(l-allyl)cyclo ropylsulfonamide.
This compound, N-ter -Butyl-(l-allyl)cyclopropylsulfonamide, was obtained in 97% yield according to the procedure described in the synthesis o N-tert-Butyl-(l- methyl)cyclopropylsul-fonamide except 1.25 equivalents of allyl "bromide were used as electrophile. The compound was taken directly into the next reaction without purification: 1H ΝMR (CDC13) δ 0.83 (m, 2H), 1.34 (s, 9H), 1.37 (m, 2H), 2.64 (d, J = 7.3 Hz, 2H), 4.25 (bs, IH), 5.07-5.10 (m, 2H), 6.70-6.85 (m, IH).
Figure imgf000085_0002
Preparation of Example 4, 1-allylcyclopro-pylsulfonamide.
Example 4, 1-allylcyclopropylsulfonamide, was obtained in 40% yield from N-tert- butyl-(l-allyl)cyclopropylsulfonamide according to the procedure described in the synthesis of 1-Methylcyclopropylsulfonamide. The compound was purified by column chromotography over SiO using 2% MeOH in CH2C12 as the eluent: !H NMR (CDC13) δ 0.88 (rn, 2 H), 1.37 (m, 2 H), 2.66 (d, J=7.0 Hz, 2 H), 4.80 (s, 2 H), 5.16 (m, 2 H), 5.82 (m, 1 H); 13C NMR (CDC13) δ 11.2, 35.6, 40.7, 119.0, 133.6.
Figure imgf000085_0003
Preparation of N-to*t-Butyl-[l-(l-hydroxy)cyclohexyl]-cyclopropylsulfonamide.
This compound was obtained in 84% yield using to the procedure described for the synthesis ofN-tert-Butyl-(l-methyl)cyclopropylsul-fonamide except 1.30 equivalents of cyclohexanone were -used, followed by recrystallization from the minimum amount of 20% EtOAc in hexane: 1H ΝMR (CDC13) δ 1.05 (m, 4H), 1.26 (m, 2H), 1.37 (s, 9H), 1.57-1.59 (m, 6H), 1.97 (m, 2H), 2.87 (bs, IH), 4.55 (bs, IH).
Figure imgf000086_0001
Preparation of Example 5, l-(l-cycIohexenyl)cyclopropyl-sulfonamide.
This compound, l-(l-cyclohexenyl)-cyclopropylsulfonamide, Example 5, was obtamed in 85% yield from N-tert-butyl-[ 1 -(1 -hydroxy)cyclohexyl]- cyclopropylsulfonamide using the procedure described for the synthesis of 1- methylcyclopropylsulfonamide, followed by recrystallization from the minimum amount of EtOAc and hexane: 1H ΝMR (DMSO-d6) δ 0.82 (m, 2 H), 1.28 (m, 2 H), 1.51 (m, 2 H), 1.55 (m, 2 H), 2.01 (s, 2 H), 2.16 (s, 2 H), 5.89 (s, 1 H), 6.46 (s, 2 H); 13C ΝMR (DMSO-d6) δ 11.6, 21.5, 22.3, 25.0, 27.2, 46.9, 131.6, 132.2; LR-MS (ESI): 200 (M+-l).
Figure imgf000086_0002
Preparation of iV-tert-Butyl-(l-benzoyl)cyclopropyl-sulfonamide.
This compound was obtained in 66% yield using the procedure described for the synthesis ofN-tert-Butyl-(l-methyl)cyclopropylsulfonamide except 1.2 equivalents of methyl benzoate was used as the electrophile. The compound was purified by column chromatography over SiO2 using 30% to 100% CH2C12 in hexane: 1H ΝMR (CDC13) δ 1.31 (s, 9H), 1.52 (m, 2H), 1.81 (m, 2H), 4.16 (bs, IH), 7.46 (EQ, 2H), 7.57 (m, IH), 8.05 (d, J- 8.5 Hz, 2H).
Figure imgf000086_0003
Preparation of Example 6, 1-benzoylcyclo-propylsulfonamide.
This compound, Example 6, 1-benzoylcyclopropyl-sulfonamide, was obtained in 87%> yield fromN-tert-butyl(l-benzoyl)cyclopropylsul-fonamide using the procedure described for the synthesis of 1-Methylcyclopropylsulfonamide, followed by recrystallization from the minimum amount of EtOAc in hexane: 1H NMR (DMSO- d6) δ 1.39 (m, 2 H), 1.61 (m, 2 H), 7.22 (s, 2 H), 7.53 (t, J=7.6 Hz, 2 H), 7.65 (t, J=7.6 Hz, 1 H), 8.06 (d, J=8.2 Hz, 2 H); 13C NMR (DMSO-d6) δ 12.3, 48.4, 128.1, 130.0, 133.4, 135.3, 192.0.
Figure imgf000087_0001
Preparation of N-t'ert-Butyl-(l-benzyI)cyclβpropyl-sulfonamide.
This compound was obtained in 60% yield using the procedure described for the synthesis ofN-tert-Butyl-(l-methyl)cyclopro-jylsulfonamide except 1.05 equivalents of benzyl bromide were used, followed by trituration with 10% EtOAc in hexane : 1H ΝMR (CDC13) δ 0.92 (m, 2H), 1.36 (m, 2H), 1.43 (s, 9H), 3.25 (s, 2H), 4.62 (bs, IH), 7.29-7.36 ( , 5H).
pOT*>
Preparation of Example 7, l-Benzylcyclo-p>ropylsulfonamide.
This compound, Example 7, l-Benzylcyclopxopylsulfonamide, was obtained in 6>6% yield fromN-tert-butyl(l-benzyl)cyclopropylsul-fonamide using the procedure described for the synthesis of l-Methylcyclop>ropylsulfonamide, followed by recrystallization from the minimum amount o_f 10% EtOAc in hexane: 1H ΝMR (CDCI3) δ 0.90 (m, 2H), 1.42 (m, 2 H), 3.25 (s, 2 H), 4.05 (s, 2 H), 7.29 (m, 3 H), 7.34 (m, 2 H); 13C ΝMR (CDC13) δ 11.1, 36.8 , 41.9, 127.4, 128.8, 129.9, 136.5.
Figure imgf000087_0002
Preparation of N-tert-Butyl-(l-phenylamino>carboxy)-cyclopropylsulfonamidei-
This compound was obtained in 42% yield using the procedure described for the synthesis of N-tert-Butyl-(l-methyl)cycloprop3^1sulfonamide using 1 equivalent of phenylisocyanate, followed by recrystallization from the minimum amount of EtOAc in hexane'H NMR (CDC13) δ 1.38 (s, 9H), 1.67-1.71 (m, 4H), 4.30 (bs, IH), 7.10 (t, J = 7.5 Hz, IH), 7.34 (t, J- 7.5 Hz, 2H), 7.53 (t, J- 7.5 Hz, 2H).
Figure imgf000088_0001
Sections B through I : Preparation of Compounds Section B: Preparation of Compounds 100-113
Preparation of 2(S)-[l(R)-(l-Ethyl-cyclopropanesulfonyIaminocarbonyl)-2(S)- vinyl-cyclopropy arbamoyl]-N-[N-Boc-amino-(ιS)-t-butyl-acetyl]-4(R)-hydro3y- pyrrolidine
Figure imgf000088_0002
Step 1:
A mixture of l(R)-N-Boc-amino-2(S)-vinyl-cyclopropanecarboxylic acid (3.41 g, 15 mmol) and GDI (2.46 g, 16.5 mmol) in THF (40 ml) was refluxed for 1 h. The solution was cooled to room temperature and transfened into a solution of 1 -ethyl- cyclopropylsulfonamide (2.46 g, 16.5 mmol) in THF (10 ml). DBU (2.7 ml, 18 mol) was added and the resulting mixture was stined at room temperature overnight. The reaction was quenched with IN HCI to pH 1 and the solvent was evaporated in vacuo. The residue was extracted with EtOAc (3 x 100 ml). The combined organic extracts were washed with brine, dried over MgSO4 and concentrated in vacuo. The crade product was purified by flash column chromatograph (Biotage Flash 40M) eluted with EtOAc to give the desired product (1.72 g, 32%). 1H NMR (400 MHz, CD3OD) δ ppm 0.92 (m, 2 H), 0.98 (t, J=7.46 Hz, 3 H), 1.27 (dd, J=9.54, 5.38 Hz, 1 H), 1.47 (m, 11 H), 1.81 (dd, J=7.83, 5.62 Hz, 1 H), 1.91 (m, 2 H), 2.18 (m, 1 H), 5.09 (dd, J=10.27, 1.47 Hz, 1 H), 5.27 (dd, J=17.24, 1.59 Hz, 1 H), 5.53 (m, 1 H); MS 359 (M+H)+.
Step 2:
A solution of 1-ethyl-cyclopropanesulfonic acid [l(R)-N-Boc-amino-2(S)-vinyl- cyclopropanecarbonyl]-amide (Product of Step 1, 1.72 g, 4.8 mmol) in TFA (15 ml) and CH2C12 (15 ml) was stirred at room temperature for 2 h. The reaction mixture was concentrated in vacuo. The residue was suspended in IN HCI in Et2O (10 ml) and concentrated in vacuo. This procedure was repeated once. The precipitate was washed with Et O, collected by filteration and dried in desiccator to give the desired product (1.40 g, 99%); MS 259 (M+H)+.
Step 3:
To a mixture ofBoc-Hyp-OH (1.00 g, 4.3 mmol) and 1-ethyl-cyclopropane-sulfonic acid [l(R)-amino-2(S)-vinyl-cyclopropanecarbonyl] -amide hydrochloride (Product of Step 2, 1.27 g, 4.3 mmol) in CH2C12 (10 ml) was added DIPEA (2.2 ml, 13 mmol) and PyBOP (2.36 g, 4.5 mmol). The mixture was stirred at room temperature for 1 h and concentrated in vacuo. Purification by flash column chromatograph (Biotage Flash 40M) eluted with EtOAc gave the desired product (2.00 g, 99%). MS 494 (M+Na)+.
Step 4:
A solution of 2(S)-[l(R)-(l-ethyl-cyclopropanesulfonylammocarbonyl)-2(S)-vinyl- cyclopropylcarbamoyl]-4(R)-hydroxy-N-Boc-pynolidine (Product of Step 3, 2.00g, 4.2 mmol) in TFA (5 ml) and CH2C12 (5 ml) was stined at room temperature for 1 h and concentrated in vacuo. The residue was dissolved in CH2C12 (10 ml) and Boc-L- tert-leucine (0.98 g, 4.2 mmol), DIPEA (2 ml) and PyBOP (2.32 g, 4.5 mmol) were added. The resulting mixture was stirred at room temperature overnight and the solvent was removed in vacuo. The crade product was purified by flash column chromatograph (Biotage Flash 40M) eluted with EtOAc to provide the title compound (2.01 g, 81%). *H NMR (400 MHz, CDC13 ) δ ppm 0.96 (m, 5 H), 1.01 (s, 9 H), 1.39 (m, 12 H), 1.83 (m, 2 H), 1.96 (m, 2 H), 2.17 (m, 2 BL), 3.81 (m, 2 H), 4.29 (d, J=9.54 Hz, 1 H), 4.40 (dd, J=9.78, 7.34 Hz, 1 H), 4.48 (s, 1 H), 5.11 (d, J=l 1.49 Hz, 1 H), 5.28 (d, J=17.36 Hz, 1 H), 5.73 (m, 1 H), 6.66 (d, J=9 .29 Hz, 1 H); MS 607 (M+Na)+.
Preparation of ( 1 (S)- {2(S)-[ 1 (R)-( 1 -Ethyl-cyclopropanesulfonylaminocarbonyl)-2(S)- vmyl-cyclopropylcarbamoyl]-4-hydroxy-pynolidine-l-carbonyl}-2,2-dimethyl- propyl)-carbamic acid isopropyl ester
π H O
Figure imgf000090_0001
A solution of 2(S)-[l(R)-(l-ethyl-cyclopropanesulfonylaminocaxbonyl)-2(S)-vinyl- cyclopropylcarbamoyl]-N-[N-Boc-amino-(S)-t-butyl-acetyl]-4(/?)-hydroxy- pynolidine (585 mg, 1.0 mmol) in TFA (2 ml) and CH C12 (2 ml) was stined at room temperature for 1 h and concentrated in vacuo. The residue was dissolved in CH C1 (5 ml) and isopropyl chloroformate (1.0M in toluene, 1.0 ml, d.O mmol), DIPEA (0.5 ml) were added. The resulting mixture was stined at room temperature overnight and the solvent was removed in vacuo. The crade product was purified by flash column chromatograph (Biotage Flash 40M) eluted with EtOAc to provide the title compound (180 mg, 32%). MS 571 (M+H)+.
Example 100: Preparation of compound 100
Figure imgf000091_0001
Step l:
To a solution of 2-methoxy cinnamic acid (10.0 g) in CH2C12 (100 ml) was added thionyl chloride (40 ml). After refluxing for 4 h, the reaction mixture was concentrated in vacuo. The crude product was used for next step without purification.
Step 2:
To a solution of 3-(2-methoxy-phenyl)-acryloyl chloride (Product of Step 1, 11.0 g, 56 mmol)) in acetone (100 ml) was added sodium azide (9.1 g, 240 mmol) in water (30 ml). The mixture was stirred at room temperature for 3 h and the solvent was evaporated in vacuo. The residue was extracted with diphenylmethane (50 ml) and the organic layer was washed with water and dried over MgSO4. This solution was used for next step without purification.
Step 3:
Diphenylmethane (50 ml) was heated to 200°C, and the solution of 3-(2-nxethoxy- phenyl)-acryloyl azide in diphenylmethane (Product of Step 2) was added dropwise.
After completion of addition, the mixture was refluxed for 4 h and then cooled to room temperature. The precipitate was collected by filtration, washed with benzene and dried. The filtrates were concentrated in vacuo and purified by flash c olumn chromatograph (Biotage Flash 40M) eluted with EtOAc : hexane (1:1) to give the second batch of product (total 5.3 g, 54%).
Figure imgf000092_0001
Η NMR (400 MHz, CD3OD) δ ppm 3.95 (s, 3 H), 6.94 (d, J=7.3 Hz, 1 H), 7.08 (d, J=8.1 Hz, 1 H), 7.14 (d, J=7.3 Hz, 1 H), 7.43 (t, J=8.1 Hz, 1 H), 7.99 (d, J=8.1 Hz, 1 H), 10.92 (s, 1 H); MS 176 (M+H)+.
Step 4:
A solution of 5-methoxy-2H-isoquinolin-l-one (Product of Step 3, 5.3 g, 30 mmol) in POCl3 (50 ml) was refluxed for 4 h. After cooling and concentration, the residue was extracted with CHC13 (50 ml) and neutralized with IN NaOH. The organic layer was washed with brine, dried over MgSO4 and concentrated in vacuo. The crude product was purified by flash column chromatograph (Biotage Flash 40M) eluted wάth 5% EtOAc in hexane to give the desired product (5.4 g, 92%).
Figure imgf000092_0002
1H NMR (400 MHz, CDC13) δ ppm 4.01 (s, 3 H), 7.04 (d, J=7.8 Hz, 1 H), 7.57 (t, J=8.1 Hz, 1 H), 7.88 (d, J=8.6 Hz, 1 H), 7.97 (d, J=5.9 Hz, 1 H), 8.25 (d, J=5.9 Hz, 1 H); MS 194 (M+H)+.
Step 5:
A solution of l-chloro-5-methoxy-isoquinoline (Product of Step 4, 19 mg, 0.1 mmol) and 2(S)-[l(R)-(l-ethyl-cyclopropanesulfonylaminocarbonyl)~2(S)-vinyl- cyclopropylcarbamoyl]-N-[N-Boc-amino-(S)-t-butyl-acetyl]-4(R)-hydroxy- ι pynolidine (58 mg, 0.1 mmol) in DMF (1 ml) was cooled to -78°C and t-BuOK (1.0 M in THF, 0.75 ml) was added. The resulting mixture was warmed to room temperature and stirred overnight. The reaction was quenched with aqueous ΝH4C1 and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4 and concentrated in vacuo. The crade product was purified by Prep HPLC to give compound 100 (18 mg, 24%). 1H ΝMR (400 MHz, CD3OD) δ ppm 0.96 (m, 5 H), 1.04 (s, 9 H), 1.26 (s, 9 H), 1.42 (m, 1 H), 1.51 (m, 2 H), 1.85 (m, 2 H), 1.97 (m, 1 H), 2.25 (m, 2 H), 2.63 (dd, J=13.69, 7.58 Hz, 1 H), 3.99 (s, 3 H), 4.07 (dd, J=11.49, 3.42 Hz, 1 H), 4.26 (s, 1 H), 4.46 (d, J=l 1.98 Hz, 1 H), 4.55 (dd, J=9.29, 7.83 Hz, 1 H), 5.11 (d, J=10.27 Hz, 1 H), 5.28 (d, J=17.36 Hz, 1 H), 5.71 (m, 1 H), 5.85 (s, 1 H), 7.14 (d, J=8.07 Hz, 1 H), 7.43 (t, J=7.83 Hz, 1 H), 7.60 (d, J=5.62 Hz, 1 H), 7.74 (d, J=8.07 Hz, 1 H), 7.94 (d, J=6.11 Hz, 1 H); MS 742 (M+H)+.
Example 101: Preparation of compound 101
Figure imgf000093_0001
Compound 101 Compound 101 was prepared by following Steps 1 through 5 described in Example 100 except that the following modifications were made:
Step 1-3:
2-Chlorocinnamic acid (25.0 g) was used as starting material and 14.6 g (59%) of product was obtained.
Figure imgf000094_0001
1H NMR (400 MHz, CD3OD) δ ppm 7.22 (d, J=7.3 Hz, 1 H), 7.42 (t, J=7.8 Hz, 1 H), 7.73 (d, J=7.8 Hz, 1 H), 8.34 (d, J=8.1 Hz, 1 H), 10.61 (s, 1 H); MS 180 (M+H)+.
Step 4:
5-Chloro-2H-isoquinolin-l-one (Product of Step 3, 14.6 g) was used as starting material and 8.28 g (53%) of product was obtained.
Figure imgf000094_0002
1H NMR (400 MHz, CDC13) δ ppm 7.60 (dd, J=8. 6, 7.6 Hz, 1 H), 7.83 (m, 1 H), 8.00 (d, J=5.9 Hz, 1 H), 8.29 (dt, J=8.9, 1.0 Hz, 1 H), 8.38 (d, J=5.9 Hz, 1 H); MS 198 (M+H)+.
Step 5:
1,5-Dichloro-isoquinoline (Product of Step 4, 20 mg) was used as starting material and 24 mg (32%) of compound 101 was obtained. 1H NMR (400 MHz, CD3OD) δ ppm 0.96 ( , 5 H), 1.03 (s, 9 H), 1.21 (s, 9 H), 1.42 (m, 1 H), 1.52 (m, 2 H), 1.85 (m, 2 H), 1.97 (m, 1 H), 2.27 (m, 2 H), 2.65 (dd, J=13.57, 6.97 Hz, 1 H), 4.07 (d, J=11.49 Hz, 1 H), 4.22 (s, 1 H), 4.54 (m, 2 H), 5.12 (d, J=10.03 Hz, 1 H), 5.28 (d, J=17.12 Hz, 1 H), 5.71 (m, 1 H), 5.88 (s, 1 H), 7.48 (t, J=7.83 Hz, 1 H), 7.63 (d, J=5.38 Hz, 1 H), 7.81 (d, J=7.34 Hz, 1 H), 8.11 (d, J=6.11 Hz, 1 H), 8.19 (d, J=8.31 Hz, 1 H); MS 746 (M+H)+.
Example 102: Preparation of compound 102
Figure imgf000095_0001
Step 1: A solution of β-chlorocinnamic acid (11.0 g, 60 mmol), diphenylphosphoryl azide (15.7 g, 57 mmol) ), and triethylamine (10 ml) in benzene (80 ml) was stined for 1 h. The reaction mixture was concentrated in vacuo at < 50°C and purified by flash column chromatograph (Biotage Flash 40M) eluted with 10% EtOAc in hexane to give the desired product (4.1 g, 34%).
Step 2:
A solution of 3-chloro-3-phenyl-acryloyl azide (Product of Step 1, 4.1 g, 19.7 mmol) in diphenylmethane (40 ml) was heated up slowly and refluxed for 4 h. After cooling to room temperature, the precipitate was collected by filtration, washed with hexane and dried. The filtrates were concentrated in vacuo and purified by flash column chromatograph (Biotage Flash 40M) eluted with EtOAc : hexane (1:1) to give the second batch of product (total 3.1 g, 89%). !H NMR (400 MHz, CD3OD) δ ppm 7.34 (s, 1 H), 7.52 (t, J=7.58 Hz, 1 H), 7.77 (t, J=7.46 Hz, 1 H), 7.90 (d, J=8.07 Hz, 1 H), 8.39 (d, J=8.07 Hz, 1 H), 11.37 (s, 1 H); MS 180 (M+H)+. Step 3:
A solution of 4-chloro-2H-isoquinolin-l-one (Product of Step 2, 3.1 g, 17 mmol) in POCl (20 mL) was refluxed for 4 h. After cooling and concentration, the residue was extracted with CHC13 (50 ml) and neutralized with IN NaOH. The organic layer was washed with brine, dried over MgSO and concentrated in vacuo. The crade product was purified by flash column chromatograph (Biotage Flash 40M) eluted with 5% EtOAc in hexane to give the product (2.3 g, 66%). 1H NMR (400 MHz, CDC13) δ ppm 7.77 (ddd, J=8.31, 7.09, 1.22 Hz, 1 H), 7.88 (ddd, J=8.31, 7.09, 1.22 Hz, 1 H), 8.23 (d, J=8.31 Hz, 1 H), 8.34 (s, 1 H), 8.36 (d, J=8.56 Hz, 1 H); MS 198 (M+H)+.
Step 4:
A solution of 1,4-dichloro-isoquinoline (Product of Step 3, 20 mg, 0.1 mmol) and
2(S)-[l(R)-(l-ethyl-cyclopropanesulfonylaminocarbonyl)-2(S)-vinyl- cyclopropylcarbamoyl]-N-[N-Boc-amino-(S)-t-butyl-acetyl]-4(R)-hydroxy- pynolidine (58 mg, 0.1 mmol) in DMF (1 ml) was cooled to -78°C and t-BuOK (1.0 M in THF, 0.75 ml) was added. The resulting mixture was warmed to room temperature and stined overnight. The reaction was quenched with aqueous ΝH C1 and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4 and concentrated in vacuo. The crude product was purified by Prep HPLC to give compound 102 (29 mg, 39%). 1H NMR (400 MHz, CD3OD) δ ppm 0.96 (m, 5 H), 1.03 (s, 9 H), 1.21 (s, 9 H), 1.42 (m, 1 H), 1.53 (m, 2 H), 1.85 (m, 2 H), 1.98 (m, 1 H), 2.27 (m, 2 H), 2.64 (dd, J=13.45, 6.85 Hz, 1 H), 4.08 (m, 1 H), 4.22 (s, 1 H), 4.54 (m, 2 H), 5.12 (d, J=10.27 Hz, 1 H), 5.29 (d, J=17.12 Hz, 1 H), 5.71 (m, 1 H), 5.85 (s, 1 H), 7.64 (t, J=7.34 Hz, 1 H), 7.86 (m, 1 H), 8.10 (m, 2 H), 8.26 (d, J=8.07 Hz, 1 H); MS 768 (M+Na)+.
Example 103: preparation of compound 103
Figure imgf000097_0001
Compound 103
Compound 103 was prepared by following Steps 1 through 4 described in Example 102 except that the following modifications were made:
Step 1-2:
2-Trifluormethoxycinnamic acid (11.6 g) was used as starting material and 5.1 g
(44%>) of product was obtained.
Figure imgf000097_0002
1H NMR (400 MHz, CD3OD) δ ppm 6.79 (d, J=7.3 Hz, 1 H), 7.29 (d, J=7.3 Hz, 1 H), 7.57 (t, J=8.1 Hz, 1 H), 7.70 (d, J=7.8 Hz, 1 H), 8.30 (d, J=8.1 Hz, 1 H)., MS 230 (M+H)+.
Step 3: 5-trifluoromethoxy-2H-isoquinolin-l -one (Product of Step 2, 4.58 g) was used as starting material and 4.35 g (88%) of product was obtained.
Figure imgf000097_0003
!H NMR (400 MHz, CDC13) δ ppm 7.66 (m, 2 H), 7.87 (d, J=5.9 Hz, 1 H), 8.31 (m, 1 H), 8.37 (d, J=5.9 Hz, 1 H); MS 248 (M+H)+. Step 4: l-Chloro-5-trifluoromethoxy-isoquinoline (Product of Step 3, 25 mg) was used as starting material and 28 mg (35%) of compound 103 was obtained. 1H NMR (400 MHz, CD3OD) δ ppm 0.96 (m, 5 H), 1.04 (s, 9 H), 1.19 (s, 9 H), 1.42 (m, 1 H), 1.52 (m, 2 H), 1.85 (m, 2 H), 1.97 (m, 1 H), 2.28 (m, 2 H), 2.65 (dd, J=13.69, 7.09 Hz, 1 H), 4.07 (dd, J=11.98, 2.69 Hz, 1 H), 4.21 (s, 1 H), 4.56 (m, 2 H), 5.12 (d, J=10.27 Hz, 1 H), 5.29 (d, J=17.12 Hz, 1 H), 5.71 (m, 1 H), 5.89 (s, 1 H), 7.49 (d, J=5.87 Hz, 1 H), 7.58 (t, J=8.07 Hz, 1 H), 7.69 (d, J=7.34 Hz, 1 H), 8.12 (d, J=6.11 Hz, 1 H), 8.22 (d, J=8.31 Hz, 1 H); MS 818 (M+Na)+.
Example 104: Preparation of compound 104
Figure imgf000098_0001
Compound 104
Compound 104 was prepared by followmg Steps 1 through 4 described in Example 102 except that the following modifications were made:
Step 1-2: 2-Trifluormethylcinnamic acid (10.0 g) was used as starting material and 5.0 g (50%) of product was obtained.
Figure imgf000098_0002
1H NMR (400 MHz, CD3OD) δ ppm 6.83 (m, 1 H), 7.33 (d, J=7.58 Hz, 1 H), 7.63 (t, J=7.83 Hz, 1 H), 8.09 (d, J=7.58 Hz, 1 H), 8.57 (d, J=8.07 Hz, 1 H).
Step 3:
5-Trifluoromethyl-2H-isoquinolin-l-one (Product of Step 2, 4.4 g) was used and 3.5 g (73%) of product was obtained.
Figure imgf000099_0001
1H NMR (400 MHz, CDC13) δ ppm 7.75 (t, J=7.95 Hz, 1 H), 7.90 (m, 1 H), 8.12 (d, J=7.34 Hz, 1 H), 8.41 (d, J=6.11 Hz, 1 H), 8.60 (d, J=8.56 Hz, 1 H).
Step 4: l-Chloro-5-trifluoromethyl-isoquinoline (Product of Step 3, 23 mg) was used as starting material and 24 mg (31%) of compound 104 was obtained. !H NMR (400 MHz, CD3OD) δ ppm 0.96 (m, 5 H), 1.03 (s, 9 H), 1.17 (s, 9 H), 1.47 (m, 3 H), 1.92 (m, 3 H), 2.29 (m, 2 H), 2.66 (dd, J=13.08, 7.21 Hz, 1 H), 4.07 (d, J=l 1.00 Hz, 1 H), 4.20 (s, 1 H), 4.57 (m, 2 H), 5.12 (d, J=10.27 Hz, 1 H), 5.28 (d, J=16.87 Hz, 1 H), 5.71 (m, 1 H), 5.90 (s, 1 H), 7.54 (s, 1 H), 7.65 (m, 1 H), 8.15 (m, 2 H), 8.51 (d, J=7.83 Hz, 1 H); MS 802 (M+Na)+.
Example 105: Preparation of compound 105
7/
Figure imgf000099_0002
Compound 105 Compound 105 was prepared by following Steps 1 through 4 described in Example 102 except that the following modifications were made:
Step 1-2:
2-Chloro-3-methoxycinnamic acid (658 mg) was used as starting material and 360 mg (54%o) of product was obtained.
Figure imgf000100_0001
1H NMR (400 MHz, CD3OD) δ ppm 4.02 (s, 3 H), 6.91 (d, J=7.34 Hz, 1 H), 7.23 (d, J=7.58 Hz, 1 H), 7.35 (d, J=9.05 Hz, 1 H), 8.27 (d, J=9.05 Hz, 1 H).
Step 3:
5-Chloro-6-methoxy-2H-isoquinolin-l-one (Product of Step 2, 350 mg) was used and 300 mg (80%)) of product was obtained.
Figure imgf000100_0002
1H NMR (400 Hz, CDC13) δ ppm 4.09 (s, 3 H), 7.43 (d, J=9.29 Hz, 1 H), 7.93 (d, J=6.11 Hz, 1 H), 8.30 (m, 2 H); MS 229 (M+H)+.
Step 4: l,5-Dichloro-6-methoxy-isoquinoline (Product of Step 3, 23 mg) was used as starting material and 19 mg (25%>) of compound 105 was obtained. 1H NMR (400 MHz, CD3OD) δ ppm 0.97 (m, 5 H), 1.03 (s, 9 H), 1.21 (s, 9 H), 1.43 (m, 1 H), 1.52 (m, 2 H), 1.85 (m, 2 H), 1.96 (m, 1 H), 2.27 (m, 2 H), 2.63 (dd, J=13.21, 6.85 Hz, 1 H), 4.06 (m, 4 H), 4.21 (s, 1 H), 4.47 (d, J=11.25 Hz, 1 H), 4.57 (m, 1 H), 5.12 (d, J=10.52 Hz, 1 H), 5.29 (d, J=16.87 Hz, 1 H), 5.71 (m, 1 H), 5.85 (s, 1 H), 7.40 (d, J=8.80 Hz, 1 H), 7.55 (d, J=5.62 Hz, 1 H), 8.01 (d, J=6.11 Hz, 1 H), 8.21 (d, J=8.80 Hz, 1 H); MS 776 (M+H)+. - o-
Example 106: Preparation of compound 106
Figure imgf000101_0001
Compound 106
Compound 106 was prepared by following Steps 1 through 4 described in Example 102 except that the following modifications were made:
Step 1-2:
3-Chloro-2-methoxycinnamic acid (4.24 g) was used as starting material and 2.4 g (57%) of product was obtained.
Figure imgf000101_0002
1H NMR (400 MHz, CD3OD) δ ppm 3.93 (s, 1 H), 6.85 (d, J=7.34 Hz, 1 H), 7.24 (d, J=7.34 Hz, 1 H), 7.52 (d, J=8.80 Hz, 1 H), 8.03 (d, J=8.80 Hz, 1 H); MS 210 (M+H)+.
Step 3:
6-Chloro-5-methoxy-2H-isoquinolin-l-one (Product of Step 2, 2.1 g) was used and 1.9 g (83%) of product was obtained.
Figure imgf000102_0001
Η NMR (400 Hz, CDC13) δ ppm 4.03 (s, 2 H), 7.63 (d, J=9.05 Hz, 1 H), 7.86 (d, J=5.14 Hz, 1 H), 8.06 (d, J=9.05 Hz, 1 H), 8.32 (d, J=5.62 Hz, 1 H); MS 229 (M+H)+.
Step 4: l,6-Dichloro-5-methoxy-isoquinoline (Product of Step 3, 23 mg) was used as starting material and 28 mg (36%) of compound 106 was obtained. !H NMR (400 MHz, CD3OD) δ ppm 0.96 (m, 5 H), 1.03 (s, 9 H), 1.21 (s, 9 H), 1.42 ( , 1 H), 1.52 (m, 2 H), 1.85 (m, 2 H), 1.96 (m, 1 H), 2.27 (m, 2 H), 2.63 (dd, J=13.69, 7.09 Hz, 1 H), 3.98 (s, 3 H), 4.05 (dd, J=12.47, 2.20 Hz, 1 H), 4.20 (s, 1 H), 4.47 (d, J=11.74 Hz, 1 H), 4.56 (dd, J=9.90, 7.46 Hz, 1 H), 5.12 (d, J=10.27 Hz, 1 H), 5.29 (d, J=16.87 Hz, 1 H), 5.71 (m, 1 H), 5.86 (s, 1 H), 7.48 (d, J=8.80 Hz, 1 H), 7.52 (d, J=6.11 Hz, 1 H), 7.96 (d, J=9.05 Hz, 1 H), 8.06 (d, J=6.11 Hz, 1 H); MS 776 (M+H)+.
Example 107: Preparation of compound 107
Figure imgf000102_0002
Compound 107
Compound 107 was prepared by following Steps 1 through 4 described in Example 102 except that the following modifications were made: Step 1-2:
A nnxture of 6-methoxy-2H-isoquinolin-l-one (700 mg) andNCS (532 mg) in
MeCN (10 ml) was refluxed for 3 h. Filtration gave 600 mg (72%) ofthe desired product.
Figure imgf000103_0001
1H NMR (400 MHz, CD3OD) δ ppm 3.96 (s, 1 H), 7.19 (dd, J=8.80, 2.45 Hz, 1 H), 7.28 (d, J=2.45 Hz, 1 H), 7.34 (s, 1 H), 8.25 (d, J=9.05 Hz, 1 H); MS 210 (M+H)+.
Step 3: 4-Chloro-6-methoxy-2H-isoquinolin-l-one (Product of Step 2, 500 mg) was used as starting material and 400 mg of product was obtained.
Figure imgf000103_0002
1H NMR (400 Hz, CDCh) δ ppm 4.01 (s, 3 H), 7.35 (d, J=2.45 Hz, 1 H), 7.41 (d, J=2.45 Hz, 1 H), 8.24 (d, J=9.29 Hz, 1 H), 8.27 (s, 1 H); MS 229 (M+H)+.
Step 4: l,4-Dichloro-6-methoxy- isoquinoline (Product of Step 3, 42 mg) was used as starting material and 70 mg (45%) of compound 107 was obtained. 1H NMR (400 MHz, CD3OD) δ ppm 0.95 (m, 14 H), 1.23 (s, 9 H), 1.47 (m, 3 H), 1.88 (m, 3 H), 2.24 (m, 2 H), 2.62 (m, J=13.69, 7.09 Hz, 1 H), 3.97 (s, 3 H), 4.04 (m, J=8.80 Hz, 1 H), 4.22 (m, 1 H), 4.52 (m, 2 H), 5.12 (d, J=10.27 Hz, 1 H), 5.28 (d, J=16.87 Hz, 1 H), 5.71 (m, 1 H), 5.81 (s, 1 H), 7.19 (dd, J=9.05, 2.20 Hz, 1 H), 7.38 (d, J=1.96 Hz, 1 H), 8.00 (s, 1 H), 8.15 (d, J=9.05 Hz, 1 H); MS 776 (M+H)+.
Example 108: Preparation of compound 108
Figure imgf000104_0001
Compound 108
Compound 108 was prepared by followmg Steps 1 through 4 described in Example 102 except that the following modifications were made:
Step 1-2:
3-(2,3-Dihydro-benzo[l,4]dioxin-5-yl)-acrylic acid (4.12 g) was used as starting material and 2.2 g (53%) of product was obtained.
Figure imgf000104_0002
1H NMR (400 MHz, CD3OD) δ ppm 4.37 (m, 4 H), 6.83 (d, J=7.09 Hz, 1 H), 7.02 (d, J=8.80 Hz, 1 H), 7.12 (d, J=7.34 Hz, 1 H), 7.79 (d, J=8.80 Hz, 1 H); MS 204 (M+H)+.
Step 3: 2,3-Dihydro-7H-l,4-dioxa-7-aza-phenanthren-8-one (Product of Step 2, 2.05 g) was used as starting material and 1.5 g (68%) of product was obtained.
Figure imgf000104_0003
1H NMR (400 Hz, CDC13) δ ppm 4.42 (m, 4 H), 7.24 (d, J=9.05 Hz, 1 H), 7.77 (d, J=5.87 Hz, 1 H), 7.84 (d, J=9.05 Hz, 1 H), 8.18 (d, J=5.87 Hz, 1 H); MS 222 (M+H)+. Step 4:
8-Chloro-2,3-dihydro-l,4-dioxa-7-aza-phenanthrene (Product of Step 3, 22 mg) was used as starting material and 29 mg (38%) of compound 108 was obtained. 1H NMR (400 MHz, CD3OD) δ ppm 0.96 (m, 5 H) 1.04 (s, 9 H) 1.25 (s, 9 H) 1.48 (m, 3 H) 1.84 (m, 2 H) 1.96 (m, 1 H) 2.25 (m, 2 H) 2.63 (dd, J=13.69, 7.83 Hz, 1 H) 4.06 (m, 1 H) 4.22 (s, 1 H) 4.43 (m, 5 H) 4.54 (dd, J=10.03, 7.83 Hz, 1 H) 5.12 (d, J=10.03 Hz, 1 H) 5.28 (d, J=17.12 Hz, 1 H) 5.71 (m, 1 H) 5.82 (s, 1 H) 7.07 (d, J=8.80 Hz, 1 H) 7.48 (d, J=5.87 Hz, 1 H) 7.74 (d, J=9.29 Hz, 1 H) 7.89 (d, J=6.36 Hz, 1 H); MS 770 (M+H)+.
Example 109 and 110: Preparation of compound 109 and 110
Figure imgf000105_0001
Compound 109 Compound 110
Compound 109 and compound 110 were prepared by following Steps 1 through 4 described in Example 102 except that the following modifications were made:
Step 1-2:
4-Fluoro-3-methoxycinnamic acid (19.6 g) was used as starting material and 9.5 g
(48%) of product was obtained.
Figure imgf000105_0002
1H NMR (400 MHz, CD3COCD3) δ ppm 4.00 (s, 1 H), 6.49 (d, J=7.34 Hz, 1 H), 7.19 (d, J=7.09 Hz, 1 H), 7.29 (d, J=8.07 Hz, 1 H), 7.86 (d, J=l 1.74 Hz, 1 H). Step 3:
7-Fluoro-6-methoxy-2H-isoquinolin-l-one (Product of Step 2, 9.0 g) was used as starting material and 7.0 g (70%) of product was obtained.
Figure imgf000106_0001
5 1H NMR (400 MHz, CDC13) δ ppm 4.04 (s, 3 H), 7.17 (d, J=8.07 Hz, 1 H), 7.48 (d, J=5.62 Hz, 1 H), 7.94 (d, J=l 1.49 Hz, 1 H), 8.20 (d, J=5.62 Hz, 1 H).
Step 4: l-Chloro-7-fluoro-6-methoxy-isoquinoline (Product of step 3, 21 mg) was used as 0 starting material and 9 mg (12%) of compound 109 and 24 mg (32%) of compound 110 were obtained.
Compound 109 1H NMR (400 MHz, CD3OD) δ ppm 1.00 (m, 14 H), 1.25 (s, 9 H), 1.47 (m, 3 H), 1.84 (m, 2 H), 1.97 (m, 1 H), 2.25 (m, 2 H), 2.61 (dd, J=13.82, 6.97 Hz, 1 H), 4.00 (s, 3 H), 4.04 (dd, J=11.37, 3.55 Hz, 1 H), 4.22 (s, 1 H), 4.44 (d,5 J=11.98 Hz, 1 H), 4.55 (m, 1 H), 5.12 (d, J=10.03 Hz, 1 H), 5.28 (d, J=17.36 Hz, 1 H), 5.71 (m, 1 H), 5.83 (s, 1 H), 7.28 (d, J=5.62 Hz, 1 H), 7.37 (d, J=8.07 Hz, 1 H), 7.76 (d, J=11.49 Hz, 1 H), 7.91 (d, J=5.87 Hz, 1 H); MS 760 (M+H)+. Compound 110 1H NMR (400 MHz, Methanol-D4) δ ppm 0.95 (m, 5 H), 1.02 (s, 9 H), 1.30 (s, 9 H), 1.51 (m, 3 H), 1.84 (m, 2 H), 1.97 (td, J=14.92, 7.34 Hz, 1 H), 2.26O (m, 2 H), 2.58 (dd, J=13.45, 6.60 Hz, 1 H), 3.99 (s, 3 H), 4.09 (dd, J=11.86, 2.81 Hz, 1 H), 4.23 (s, 1 H), 4.32 (d, J=11.74 Hz, 1 H), 4.53 (dd, J=10.15, 6.72 Hz, 1 H), 5.12 (dd, J=10.39, 1.34 Hz, 1 H), 5.29 (dd, J=17.12, 1.22 Hz, 1 H), 5.39 (s, 1 H), 5.72 (m, 1 H), 7.43 (s, 1 H), 7.64 (s, 1 H), 7.72 (d, J=5.62 Hz, 1 H), 8.10 (d, J=5.87 Hz, 1 H); MS 776 (M+H)+. 5
Example 111: Preparation of compound 111
Figure imgf000107_0001
Compound 111
Compound 111 was prepared by following Steps 1 through 5 described in Example 100 except that, in step 5, 1,5-dichloro-isoquinoline (20 mg) and (1(S)-{2(S)-[1(R)- (l-ethyl-cyclopropanesulfonylaminocarbonyl)-2(S)-vinyl-cyclopropylcarbamoyl]-4- hydroxy-pynolidine-1 -carbonyl} -2,2 -dimethyl-propyl)-carbamic acid isopropyl ester (57 mg) were used as starting materials and 20 mg (27%) of compound 111 was obtained. 1H NMR (400 MHz, CD3OD) δ ppm 0.97 (m, 5 H), 1.04 (s, 9 H), 1.11 (d, J=6.11 Hz, 3 H), 1.28 (s, 3 H), 1.41 (m, 1 H), 1.50 (m, 2 H), 1.84 (m, 2 H), 1.96 (m, 1 H), 2.26 ( , 2 H), 2.65 (dd, J=13.69, 7.09 Hz, 1 H), 4.08 (dd, J=11.86, 3.55 Hz, 1 H), 4.27 (s, 1 H), 4.45 (m, 2 H), 4.57 (dd, J=10.03, 7.34 Hz, 1 H), 5.11 (d, J=10.27 Hz, 1 H), 5.28 (d, J=17.12 Hz, 1 H), 5.72 (m, 1 H), 5.88 (s, 1 H), 7.49 (t, J=7.95 Hz, 1 H), 7.63 (d, J=6.11 Hz, 1 H), 7.82 (d, J=7.34 Hz, 1 H), 8.11 (d, J=6.11 Hz, 1 H), 8.19 (d, J=8.56 Hz, 1 H); MS 732 (M+H)+
Example 112: Preparation of compound 112
Figure imgf000107_0002
Compound 112 Compound 112 was prepared by followmg Steps 1 through 4 described in Example 102 except that, in step 4, (l(S)-{2(S)-[l(R)-(l-ethyl-cyclopropanesulfonylamino- carbonyl)-2(S)-vinyl-cyclopropylcarbamoyl]-4-hydroxy-pynolidine-l-carbonyl}-2,2- dimethyl-propyl)-carbamic acid isopropyl ester (57 mg) was used as starting material and 14 mg (19%) of compound 112 was obtained. 1H NMR (400 MHz, CD3OD) δ ppm 0.96 (m, 5 H), 1.04 (s, 9 H), 1.09 (d, J=6.36 Hz, 3 H), 1.27 (s, 3 H), 1.39 (dd, J=9.54, 5.38 Hz, 1 H), 1.50 (m, 2 H), 1.83 (m, 2 H), 1.95 (m, 1 H), 2.26 (m, 2 H), 2.63 (dd, J=13.82, 6.97 Hz, 1 H), 4.05 (dd, J=l 1.74, 3.67 Hz, 1 H), 4.26 (m, 1 H), 4.43 (m, 2 H), 4.56 (dd, J=10.03, 7.09 Hz, 1 H), 5.10 (d, J=10.27 Hz, 1 H), 5.27 (d, J=17.12 Hz, 1 H), 5.69 (m, 1 H), 5.84 (s, 1 H), 6.87 (d, J=9.05 Hz, 1 H), 7.63 (t, J=7.70 Hz, 1 H), 7.85 (t, J=7.70 Hz, 1 H), 8.05 (s, 1 H), 8.10 (d, J=8.31 Hz, 1 H), 8.25 (d, J=8.31 Hz, 1 H); MS 732 (M+H)+.
Example 113: Preparation of compound 113
Figure imgf000108_0001
Step 1-4: l-Clxloro-5-methoxy-isoquinoline was prepared by the same Steps 1 through 4 described in Example 100.
Step 5:
To a cold solution of l-chloro-5-methoxy-isoquinoline (Product of Step 4, 1.94 g, 10 mmol) in CH2C12 (30 ml) was added MCBPA (77%, 5.6 g, 25 mmol) and the resulting mixture was stined at room temperature overnight. The reaction was quenched with 5N NaOH (5 ml). The organic layer was separated, washed with brine, dried over MgSO4 and concentrated in vacuo. Purification by flash column chromatograph (Biotage Flash 40M) eluted with 5% EtOAc in hexane provided the desired product (0.5 g, 24%). MS 210 (M+H)+.
Step 6:
A solution of l-chloro-5-methoxy-isoquinoline 2-oxide (Product of Step 5, 0.5 g, 2.4 mmol) in POCl3 (5 ml) was refluxed for 3 h. After cooling and concentration, the residue was extracted with CHCI3 (50 ml) and neutralized with IN NaOH. The organic layer was washed with brine, dried over MgSO4 and concentrated in vacuo. The crude product was purified by flash column chromatograph (Biotage Flash 40M) eluted with 5% EtOAc in hexane to give the desired product (0.43 g, 80%). MS 228 (M+H)+.
Step 7: A soltxtion of l,3-dichloro-5-methoxy-isoquinoline (Product of Step 6, 23 mg, 0.1 mmol) and 2(S)-[ 1 (R)-(l -ethyl-cyclopropanesulfonylaminocarbonyl)-2(S)-vinyl- cyclopropylcarbamoyi] -N- [N-Boc-amino-(S)-t-butyl-acetyl] -4(R)-hydroxy- pynolidine (58 mg, 0.1 mmol) in DMF (1 ml) was cooled to -78°C and t-BuOK (1.0 M in THF, 0.75 ml) was added. The resulting mixture was stined at room temperature overnight. The reaction was quenched with aqueous ΝH4C1 and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4 and concentrated in vacuo. The crade product was purified by Prep HPLC to give compound 113 (27 mg, 35%). 1H NMR (400 MHz, CD3OD) δ ppm 0.96 (m, 5 H), 1.04 (s, 9 H), 1.26 (s, 9 H), 1.48 (m, 3 H), 1.84 (m, 2 H), 1.95 (m, 1 H), 2.26 (m, 2 H), 2.63 (dd, J=13.82, 6.97 Hz, 1 H), 3.99 (s, 3 H), 4.07 (m, 1 H), 4.24 (s, 1 H), 4.46 (d, J=11.98 Hz, 1 H), 4.54 (dd, J=10.15, 7.21 Hz, 1 H), 5.11 (d, =10.52 Hz, 1 H), 5.28 (d, =17.12 Hz, 1 H), 5.71 (m, 1 H), 5.84 (s, 1 H), 7.16 (d, =7.58 Hz, 1 H), 7.42 (t, =8.07 Hz, 1 H), 7.59 (s, 1 H), 7.72 (d, .7=8.31 Hz, 1 H); MS 798 (M+Na)+.
Section C
Preparation of Compounds 200-220
Example 200: Preparation of Compound 200
Figure imgf000110_0001
Compound 200
Scheme 1
Figure imgf000111_0001
Step l:
A solution of 3-chloro-propane-l -sulfonic acid tert-butylamide (9.34 g, 43.7 mmol) was chilled to -78 °C and treated with 2.5M n-butyllithium in hexanes (36.7 mL, 92 mmol) dropwise over 20 min. The mixture was stined for an additional 10 min and was then allowed to warm to rt and stined for 1.5 h. The mixture was then recooled to -78 °C and was again treated with 2.5M w-butyllithium in hexanes (19.2 mL, 48 mmol) dropwise over 15 min. The mixture was allowed to warm to rt and stined for 2 h. The solution was again cooled to -78 °C and was treated with a solution of benzyl bromide (9.71 g, 56.8 mmol) in dry THF (10 mL) dropwise over 15 min. The mixture was then stined at rt for 18 h. Saturated aqueous ammonium chloride (50 mL) was added, and the mixture was concentrated in vacuo. To the residue was added ethyl acetate (300 mL) and water (100 mL). The resulting bilayer was shaken and the two phases were separated. The organic phase was washed with water (2 x 100 mL) and brine (50 mL). The combined aqueous washes were back-extracted with ethyl acetate (150 mL), and the combined organics were dried over magnesium sulfate, filtered and concentrated in vacuo to give a waxy brown oil. Purification of the crude product by silica gel chromatography (10% ethyl acetate in hexanes) gave the desired product as a white powder (7.75 g, 66.4% yield): 1H NMR (CDC13) δ 0.72-0.74 (m, 2 H), 1.26 (s, 9 H), 1.34-1.36 (m, 2 H), 3.26 (s, 2 H), 3.69 (s, 1 H), 7.19 (d, J=6.71 Hz, 2 H), 7.23-7.26 (m, 1 H), 7.29-7.31 (m, 2 H); MS m/z 290 ((M+Na)+).
Step 2:
The product from Example 200, Step 1 (7.70 g, 28.8 mmol) was dissolved in TFA (75 mL) and the mixture was stirred for 1 h. The mixture was then concentrated in vacuo to give 6.19 g (quantitative yield) ofthe desired product as a white solid. This material was used in the next step without further purification: 1H NMR (CD3OD) δ 0.58-0.66 (m, 2 H), 1.16-1.25 (m, 2 H), 3.33 (s, 2 H), 7.14-7.34 (m, 5 H); MS m/z 212 (MH+).
Step 3: A solution of 1 (R^-tert-butoxycarbonylamino-2(S -vinyl-cyclopropanecarboxylic acid (4.45 g, 19.6 mmol) and 1,1 '-carbonyldiimidazole (3.97 g, 24.5 mmol) in dry THF (60 mL) was heated to boiling under reflux for 90 min. Upon cooling to rt, the mixture was treated sequentially with the product from Example 200, Step 2 (5.17 g, 24.5 mmol) and l,8-diazabicyclo[5.4.0]undec-7-ene (6.26 g, 41.1 mmol). The resulting mixture was stined at rt for 72h, and was then concentrated in vacuo to a viscous brown oil. The residue was dissolved in ethyl acetate (300 mL) and was washed with IN HCI (3 x 75 mL) and then with brine (75 mL). The organic was dried over anhydrous magnesium sulfate, filtered, and concentrated. Purification by flash silica gel chromatography (DCM, then 1% MeOH in DCM) gave 8.4 g (quantitative yield) ofthe desired product as an off-white solid: MS m/z 443
((M+Na)+).
Step 4:
The product from Example 200, Step 3 (8.4 g, 19.6 mmol) was dissolved in a mixture of TFA (75 mL) and DCM (75 mL) and the resulting solution was stined for 2.5 h at rt. Concentration in vacuo to an oily residue, followed by addition of IN HCI in Et20 (35 mL) gave a white solid which was isolated by filtration and dried in vacuo to give 6.30 g (90.2% yield) ofthe desired product as an off-white powder: 1H NMR (CD3OD) δ 0.66-O.83 (m, 2 H), 1.41-1.50 (m, 1 H), 1.60 (ddd, J=10.89, 6.31, 4.76 Hz, 1 H), 1.71 (dd, J=10.06, 7.87 Hz, 1 H), 2.17 (t, J=7.87 Hz, 1 H), 2.35-2.47 (m, 1 H), 3.33 (s, 2 H), 5.37 (d, J=10.25 Hz, 1 H), 5.48 (d, J=17.20 Hz, 1 H), 5.78 (ddd, J=17.11, 10.15, 7.50 Hz, 1 H), 7.13-7.20 (m, 2 H), 7.24-7.35 (m, 3 H); MS m/z 321 (MH+), 343 ((M-HNa)+).
Scheme 2
Figure imgf000113_0001
Step 5:
A stined solution of Boc-L-4-hydroxyproline (12.3g, 53.3 mol) in DMSO (200 mL) was treated with solid potassium tert-butoxide (15.7g, 133 mmol), and the resulting mixture was stined for 100 min at rt. The reaction flask was lowered into a cool water bath and to the mixture was added solid 1-chloroisoquinoline (lO.Og, 58.1 mmol) in two batches over 30 min. The now deep red mixture was stined for 3h. The mixture was concentrated in vacuo to a brown residue and to it was added water (700 mL). The aqueous solution was washed successively with diethyl ether (3 x 250 mL) and then with ethyl acetate (2 x 200 mL). The aqueous solution was acidified with IN aqueous HCI to pH = 4, then the mixture was extracted with DCM (3 150 mL). The combined DCM extracts were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo to give 16.48 g (86% yield) of a slightly orange solid which was approximately 94% pure; the major impurity appeared to be 1- hydroxyisoquinoline: 1H NMR (CD3OD) δ 1.38-1.50 (m, 9 H), 2.01 (s, 0.3 H), 2.41- 2.47 (m, 1 H), 2.65 (s, 0.7 H), 2.70-2.75 (m, 1 H), 3.80-3.84 (m, 1 H), 3.87 (d, J=4.58 Hz, 0.7 H), 3.89 (d, J=4.58 Hz, 0.3 H), 4.48 (t, J=7.93 Hz, 0.7 H), 4.52 (t, J=7.93 Hz, 0.3 H), 5.77-5.79 (m, 1 H), 7.32-7.34 (m, 1 H), 7.58 (t, J=7.48 Hz, 1 H), 7.71 (t, J=7.63 Hz, 1 H), 7.81 (d, J=7.94 Hz, 1 H), 7.95 (d, J=6.10 Hz, 1 H), 8.17-8.20 (m, 1 H); MS m/z 359 (MH+), 739 ((2M+Na)+).
Step 6:
The product of Example 200, Step 4 (3.00 g, 8.41 mmol) was combined with the product of Example 2O0, Step 5 (3.21 g, 8.41 mmol), HATU (3.84 g, 10.1 mmol), DIPEA (3.26 g, 25.2 mmol) and DMF (75 mL) and the resulting solution was stined at rt for 4.5 h. The mixture was concentrated in vacuo to a residue and was then redissolved in ethyl acetate (250 mL) and washed successively with pH = 4 buffer (4 x 150 mL), water (100 mL) and brine (150 mL). The organic was dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. Purification by flash silica gel chromatography (40:1 DCM : MeOH), gave the product as 4.84 g (87.1% yield) of a beige foam solid: 1H NMR (CD3OD) δ 0.58-0.65 (m, 2 H), 1.41 (s, 9 H), 1.43-1.46 (m, 2 H), 1.50-1.53 (m, 1 H), 1.91 (dd, J=8.09, 5.34 Hz, 1 H), 2.26-2.35 (m, 2 H), 2.58 (dd, J=13.73, 6.71 Hz, 1 H), 3.33 (d, J=7.02 Hz, 2 H), 3.82 (d, J=12.51 Hz, 1 H), 3.87-3.90 ( , 1 H), 4.45 (dd, J=9.61, 7.17 Hz, 1 H), 5.17 (d, J=10.38 Hz, 1 H), 5.36 (d, J=17.09 Hz, 1 H), 5.77-5.84 (m, 2 H), 7.16 (d, J=7.02 Hz, 2 H), 7.22-7.25 (m, 1 H), 7.27-7.30 (m, 2 H), 7.32 (d, J=5.80 Hz, 1 H), 7.58 (t, J=7.48 Hz, 1 H), 7.71 (t, J=7.48 Hz, 1 H), 7.81 (d, J=8.24 Hz, 1 H), 7.95 (d, J=6.10 Hz, 1 H), 8.18 (d, J=8.55 Hz, 1 H); MS m z 661 (MH+).
Step 7:
The product of Example 200, Step 6 (4.00 g, 6.05 mmol) was dissolved in 1,4- dioxane (50 mL) and the solution was treated with 4.0M HCI in 1,4-dioxane (15 mL). The mixture was stirred overnight at rt. The mixture was concentrated in vacuo and the resulting reddish-brown powder and placed under high vacuum. The yield was quantitative: 1H NMR (CD3OD) δ 0.62-0.65 (m, 2 H), 1.40-1.44 (m, 2 H), 1.48-1.53 (m, 1 H), 2.00 (dd, J=8.05, 5.49 Hz, 1 H), 2.37-2.51 (m, 2 H), 3.00 (dd, J=14.09, 7.50 Hz, 1 H), 3.30 (d, J=1.83 Hz, 2 H), 3.86-3.89 ( , 2 H), 4.80 (dd, J=10.61, 7.32 Hz, 1 H), 5.22 (dd, J=10.25, 1.46 Hz, 1 H), 5.40 (dd, J=17.20, 1.46 Hz, 1 H), 5.71 (ddd, J=17.11, 10.15, 8.60 Hz, 1 H), 6.00 (t, J=3.29 Hz, 1 H), 7.14 (d, J=1.46 Hz, 1 H), 7.16 (d, J=1.83 Hz, 1 H), 7.24-7.33 (m, 3 H), 7.49 (d, J=6.22 Hz, 1 H), 7.66-7.72 (m, 1 H), 7.80-7.85 (m, 1 H), 7.90-7.92 ( , 1 H), 7.98 (d, J=6.22 Hz, 1 H), 8.42 (d, J=8.42 Hz, 1 H); MS m/z 561 (MH+).
Step 8:
The product of Example 200, Step 7 (1.90 g, 3.00 mmol) was combined with N-Boc- L-tβrt-leucine (0.763 g, 3.30 mmol), HATU (1.48 g, 3.90 mmol), DIPEA (1.55 g, 12.0 mmol) and DMF (50 mL) and the resulting solution was stined at rt for 18 h. The mixture was concentrated in vacuo to a residue and was then redissolved in ethyl acetate (150 mL) and washed successively with pH = 4 buffer (3 x 75 mL) and brine (50 mL). The organic was dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. Purification by flash silica gel chromatography (gradient: DCM, to 3% MeOH in DCM) gave 1.99 g (85.5% yield) ofthe desired product as an off-white foamy solid: 1H NMR (CD3OD) δ 0.61-0.67 (m, 2 H), 0.97 (s, 9 H), 1.25
(s, 9 H), 1.41-1.52 (m, 3 H), 1.91-1.93 (m, 1 H), 2.28-2.32 (m, 2 H), 2.65 (dd, J=13.73, 7.32 Hz, 1 H), 2.81 (s, 2 H), 4.07 (dd, J=l 1.29, 3.36 Hz, 1 H), 4.25 (d, J=9.16 Hz, 1 H), 4.48 (d, J=11.29 Hz, 1 H), 4.58 (dd, J=9.77, 7.63 Hz, 1 H), 5.18 (d, J=10.07 Hz, 1 H), 5.35 (d, J=17.40 Hz, 1 H), 5.78 (ddd, J=17.01, 9.61, 9.38 Hz, 1 H), 5.86 (s, 1 H), 7.14-7.16 (m, 2 H), 7.24-7.26 (m, 1 H), 7.28-7.33 (m, 3 H), 7.52 (t, J=7.63 Hz, 1 H), 7.70 (t, J=7.48 Hz, 1 H), 7.80 (d, J=7.93 Hz, 1 H), 7.95-7.97 (m, 1 H), 8.21 (d, J=8.55 Hz, 1 H); MS m z 774 (MH+).
Scheme 3
Figure imgf000116_0001
Figure imgf000116_0002
Compound 200
Step 9: A solution ofthe product from Example 200, Step 8 (1.50g, 1.94 mmol) in DCM (50 mL) and trifluoroacetic acid (50 mL) was stined for 3 h at rt. The mixture was concentrated in vacuo to a viscous residue, and was then dissolved in 1,2- dichloroethane and again concentrated in vacuo to give the desired bis-trifluoroacetic acid salt product as an off-white glassy solid (quantitative). The material was used directly in the next step without purification: MS m/z 674 (MH+).
Step 10: To a solution ofthe product from Example 200, Step 9 (125 mg, 0.138 mmol) in 1,2- dichloroethane (3 mL) was added -tolyl chloroformate (30.5 mg, 0.180 mmol) and NN-diisopropylethylamine (89.0 mg, 0.692 mmol). The mixture was agitated at rt for 72 h. The reaction mixture was washed with pH = 4 buffer solution (3 x 3 mL), and the washes were back-extracted with 1,2-dichloroethane (3 mL). The organic phases were combined and concentrated in vacuo. The crude product was then dissolved in MeOH and purified by reverse phase preparative HPLC to give Compound 200 as a glassy yellow solid (81.0 mg, 72.6% yield): 1H ΝMR (CD3OD) δ 0.64 (s, 2 H), 1.04 (s, 9 H), 1.08-1.12 (m, 1 H), 1.41-1.48 (m, 3 H), 1.93 (dd, J=8.05, 5.49 Hz, 1 H), 2.27-2.34 (m, 5 H), 2.63-2.69 (m, 1 H), 3.78 (s, 1 H), 4.09 (dd, J=11.71, 3.66 Hz, 1 H), 4.37 (s, 1 H), 4.43 (d, J=11.71 Hz, 1 H), 4.60 (dd, J=10.06, 7.50 Hz, 1 H), 5.18 (d, J=11.34 Hz, 1 H), 5.34 (d, J=15.74 Hz, 1 H), 5.72-5.81 (m, 1 H), 5.85 (s, 1 H), 6.78 (d, J=8.42 Hz, 2 H), 7.07 (d, J=8.05 Hz, 2 H), 7.14-7.17 (m, 2 H), 7.22-7.32 (m, 4 H), 7.37 (t, J=7.68 Hz, 1 H), 7.68 (t, J=7.32 Hz, 1 H), 7.78-7.81 (m, 1 H), 7.92 (d, J=5.86 Hz, 1 H), 8.17 (d, J=8.42 Hz, 1 H); MS m/z 808 (MH+), m/z 806 (M-l).
Example 201: Preparation of Compound 201
Figure imgf000117_0001
Compound 201
Compound 201 was prepared by following Step 10 of Example 200 except that phenyl chloroformate was used in place of />-tolyl chloroformate.
Step 10: Modifications: 28 mg (0.18 mmol) phenyl chloroformate used, 69.7 mg product obtained as a yellow glassy solid (63.4% yield): 1H NMR (CD3OD) δ 0.64 (s, 2 H), 1.04 (s, 9 H), 1.08-1.13 (m, 1 H), 1.41-1 .48 (m, 3 H), 1.93 (dd, J=8.05, 5.49 Hz, 1 H), 2.24-2.34 (m, 2 H), 2.64-2.70 (m, 1 H), 3.78 (s, 1 H), 4.10 (dd, J=l 1.89, 3.84 Hz, 1 H), 4.37 (s, 1 H), 4.44 (d, J=11.71 Hz, 1 H), 4.61 (dd, J=10.06, 7.50 Hz, 1 H), 5.16- 5.20 (m, 1 H), 5.34 (d, J=17.20 Hz, 1 H), 5.72-5.81 (m, 1 H), 5.85 (s, 1 H), 6.91 (d, J=7.68 Hz, 2 H), 7.14-7.19 (m, 3 H), 7.25-7.32 (m, 6 H), 7.37 (t, J=7.68 Hz, 1 H), 7.67 (t, J=7.50 Hz, 1 H), 7.77-7.80 (m, 1 H), 7.92 (d, J=5.86 Hz, 1 H), 8.18 (d, J=8.05 Hz, 1 H); MS m/z 794 (MH+), m/z 792 (M-l).
Example 202: Preparation of Compound 202
Figure imgf000118_0001
Compound 202
Compound 202 was prepared by following Step 10 of Example 200 except that 4- fluorophenyl chloroformate was used in place of /?-tolyl chloroformate.
Step 10:
Modifications: 32 mg (0.18 mmol) 4-fluorophenyl chloroformate used, 71.6 mg product obtained as a glassy yellow solid (63.7% yield): 1H NMR (CD3OD) δ 0.64 (s, 2 H), 1.03 (s, 9 H), 1.08-1.12 (m, 1 H), 1.41-1.48 (m, 3 H), 1.93 (dd, J=8.05, 5.49 Hz, 1 H), 2.25-2.35 (m, 2 H), 2.62-2.70 (m, 1 H), 3.78 (s, 1 H), 4.09 (dd, J=l 1.71, 3.66 Hz, 1 H), 4.35 (s, 1 H), 4.43 (d, J=11.71 Hz, 1 H), 4.62 (dd, J=9.88, 7.32 Hz, 1 H), 5.18 (d, J=11.34 Hz, 1 H), 5.35 (d, J=17.20 Hz, 1 H), 5.72-5.81 (m, 1 H), 5.85 (s, 1 H), 6.85-6.90 (m, 2 H), 6.98 (t, J=8.60 Hz, 2 H), 7.14-7.17 (m, 2 H), 7.24-7.32 (m, 4 H), 7.40 (t, J=7.50 Hz, 1 H), 7.69 (t, J=7.14 Hz, 1 H), 7.78-7.81 (m, 1 H), 7.92 (d, J=6.22 Hz, 1 H), 8.17 (d, J=8.05 Hz, 1 H); MS m/z 812 (MH+), m/z 810 (M-l).
Example 203: Preparation of Compound 203
Figure imgf000119_0001
Compound 203
Compound 203 was prepared by following Step 10 of Example 200 except that 4- methoxyphenyl chloroformate was used in place ofp-tolyl chloroformate.
Step 10:
Modifications: 33 mg (0.18 mmol) 4-methoxyphenyl chloroformate used, 80.2 mg product obtained as a yellow solid (70.3% yield): 1H NMR (CD3OD) δ 0.64 (s, 2 H), 1.03 (s, 9 H), 1.07-1.12 (m, 1 H), 1.41-1.48 (m, 3 H), 1.93 (dd, J=8.05, 5.49 Hz, 1 H), 2.25-2.35 (m, 2 H), 2.63-2.70 ( , 1 H), 3.77 (s, 3 H), 3.78 (s, 1 H), 4.09 (dd, J=11.53, 3.48 Hz, 1 H), 4.36 (s, 1 H), 4.43 (d, J=11.71 Hz, 1 H), 4.61 (dd, J=9.88, 7.32 Hz, 1 H), 5.18 (d, J=11.71 Hz, 1 H), 5.35 (d, J=16.47 Hz, 1 H), 5.72-5.81 (m, 1 H), 5.85 (s, 1 H), 6.80 (s, 4 H), 7.14-7.17 (m, 2 H), 7.24-7.32 (m, 4 H), 7.40 (t, J=7.50 Hz, 1 H), 7.68 (t, J=7.14 Hz, 1 H), 7.78-7.81 (m, 1 H), 7.92 (d, J=5.86 Hz, 1 H), 8.18 (d, J=8.05 Hz, 1 H); MS m/z 824 (MH+), m z 822 (M-l).
Example 204: Preparation of Compound 204
Figure imgf000120_0001
Compound 204
Compound 204 was prepared by following Step 10 of Example 200 except that chloroformic acid 2-methoxyethyl ester was used in place ofp-tolyl chloroformate.
Step 10:
Modifications: 25 mg (0.18 mmol) chloroformic acid 2-methoxyethyl ester used, 73.3 mg product obtained as a yellow glassy solid (68.3% yield): 1H NMR (CD3OD) δ 0.63 (s, 2 H), 0.97 (s, 9 H), 1.00-1.05 (m, 1 H), 1.41-1.47 (m, 3 H), 1.92 (dd, J=8.05, 5.49 Hz, 1 H), 2.24-2.35 (m, 2 H), 2.62-2.69 (m, 1 H), 3.27 (s, 3 H), 3.40- 3.44 (m, 2 H), 3.78 (s, 1 H), 3.82-3.89 (m, 1 H), 3.92-3.99 (m, 1 H), 4.08 (dd, J=11.71, 3.66 Hz, 1 H), 4.28-4.31 (m, 1 H), 4.44 (d, J=l 1.71 Hz, 1 H), 4.59 (dd, J=9.88, 7.32 Hz, 1 H), 5.18 (dd, J=10.25, 1.46 Hz, 1 H), 5.34 (dd, J=17.02, 1.65 Hz, 1 H), 5.72-5.84 (m, 1 H), 5.86 (s, 1 H), 7.13-7.16 (m, 2 H), 7.24-7.34 (m, 4 H), 7.55 (t, J=7.50 Hz, 1 H), 7.71 (t, J=7.14 Hz, 1 H), 7.80-7.83 (m, 1 H), 7.95-7.97 (m, 1 H), 8.21 (d, J=8.42 Hz, 1 H); MS m z 776 (MH+), m/z 774 (M-l).
Example 205: Preparation of Compound 205
Figure imgf000121_0001
Compound 205
Compound 205 was prepared by following Step 10 of Example 200 except that neopentyl chloroformate was used in place of z tolyl chloroformate.
Step 10:
Modifications: 27 mg (0.18 mmol) neopentyl chloroformate used, 67.1 mg product obtained as an off-white solid (61.5% yield): 1H NMR. (CD3OD) δ 0.64 (s, 2 H), 0.83 (s, 9 H), 0.98 (s, 9 H), 1.01-1.05 (m, 1 H), 1.42-1.48 (m, 3 H), 1.92 (dd, J=8.05, 5.49 Hz, 1 H), 2.24-2.35 (m, 2 H), 2.61-2.68 (m, 1 H), 3.39 (d, J=10.61 Hz, 1 H), 3.53- 3.56 (m, 1 H), 3.78 (s, 1 H), 4.08 (dd, J=11.53, 3.48 Hz, 1 H), 4.29-4.33 ( , 1 H), 4.45 (d, J=11.71 Hz, 1 H), 4.59 (dd, J=10.06, 7.14 Hz, 1 H), 5.18 (d, J=11.71 Hz, 1 H), 5.35 (d, J=16.83 Hz, 1 H), 5.73-5.85 (m, 1 H), 5.86 (d, J=2.20 Hz, 1 H), 7.14- 7.16 (m, 2 H), 7.24-7.34 (m, 4 H), 7.53 (t, J=7.50 Hz, 1 H), 7.70 (t, J=7.14 Hz, 1 H), 7.80-7.83 (m, 1 H), 7.95-7.97 (m, 1 H), 8.19 (d, J=8.05 Hz, 1 H); MS m/z 788 (MH+), m/z 786 (M-l).
Example 206: Preparation of Compound 206
Figure imgf000122_0001
Compound 206
Compound 206 was prepared by following Step 10 of Example 200 except that 2- fluoroethyl chloroformate was used in place ofp-tolyl chloroformate.
Step 10:
Modifications: 23 mg (0.18 mmol) 2-fluoroethyl chloroformate used, 75.1 mg product obtained as a yellow glassy solid (71.0% yield): 1H NMR (CD3OD) δ 0.63 (s, 2 H), 0.98 (s, 9 H), 1.00-1.06 (m, 1 H), 1.41-1.47 (m, 3 H), 1.92 (dd, J=8.05, 5.49 Hz, 1 H), 2.24-2.35 (m, 2 H), 2.62-2.69 (m, 1 H), 3.78 (s, 1 H), 3.91-4.10 (m, 3 H), 4.28-4.35 (m, 2 H), 4.42-4.51 (m, 2 H), 4.59 (dd, J=10.06, 7.14 Hz, 1 H), 5.18 (dd, J=10.25, 1.46 Hz, 1 H), 5.34 (dd, J=17.20, 1.46 Hz, 1 H), 5.72-5.84 (m, 1 H), 5.86 (s, 1 H), 7.13-7.16 (m, 2 H), 7.24-7.34 (m, 4 H), 7.52-7.57 (m, 1 H), 7.68-7.73 (m, 1 H), 7.80-7.82 (m, 1 H), 7.95-7.97 (m, 1 H), 8.20 (d, J=8.42 Hz, 1 H); MS m/z 764 (MH+), m z 762 (M-l).
Example 207: Preparation of Compound 207
Figure imgf000123_0001
Compound 207
Compound 207 was prepared by following Step 10 of Example 2O0 except that 2- methoxyphenyl chloroformate was used in place ofp-tolyl chloroformate.
Step 10:
Modifications: 33 mg (0.18 mmol) 2-methoxyphenyl chloroformate used, 77.7 mg product obtained as a yellow glassy solid (68.1% yield): 1H NMR (CD3OD) δ 0.64 (s, 2 H), 1.05 (s, 9 H), 1.13-1.16 (m, 1 H), 1.41-1.48 (m, 3 H), 1.92 (dd, J=8.05, 5.12 Hz, 1 H), 2.25-2.37 (m, 2 H), 2.63-2.70 (m, 1 H), 3.65 (s, 3 H), 3.78 (s, 2 H), 4.13
(dd, J=11.53, 3.48 Hz, 1 H), 4.35-4.39 (m, 2 H), 4.61 (dd, J=9.33, 7.50 Hz, 1 H), 5.18 (d, J=11.34 Hz, 1 H), 5.35 (d, J=16.83 Hz, 1 H), 5.73-5.82 (m, 1 H), 5.85 (s, 1 H), 6.87 (d, J=3.66 Hz, 2 H), 6.98 (d, J=8.42 Hz, 1 H), 7.14-7.17 (m, 3 H), 7.24-7.32 (m, 4 H), 7.42 (t, J=7.50 Hz, 1 H), 7.68 (t, J=7.32 Hz, 1 H), 7.77-7.80 (m, 1 H), 7.92 (d, J=5.49 Hz, 1 H), 8.18 (d, J=8.05 Hz, 1 H); MS m/z 824 (MH+), m/z 822 (M-l).
Example 208: Preparation of Compound 208
Figure imgf000124_0001
Compound 208
Compound 208 was prepared by following Step 10 of Example 200 except that 3- trifluoromethylphenyl chloroformate was used in place of z tolyl chloroformate.
Step 10:
Modifications: 40 mg (0.18 mmol) 3-trifluoromethylphenyl chloroformate used, 45.2 mg product obtained as a slightly yellow glassy solid (37.9% yield): MS m/z 862 (MH+), m/z 860 (M-l).
Example 209: Preparation of Compound 209
Figure imgf000124_0002
Compound 209
Compound 209 was prepared by following Step 10 of Example 200 except that 2-(- )- 7R -menthyl chloroformate was used in place ofp-tolyl chloroformate. Step 10:
Modifications: 40 mg (0.18 mmol) (-)-(7R)-menthyl chloroformate used, 74.9 rng product obtained as an off-white solid (63.2% yield): MS m/z 856 (MH+), m/z 854
(M-l).
Example 210: Preparation of Compound 210
Figure imgf000125_0001
Compound 210
Scheme 1
Figure imgf000125_0002
Product from Example 200, Step 9 Compound 210
Step 1:
A mixture ofthe product from Example 100, Step 9 (125 mg, 0.138 mmol), tert-"butyl acetic acid (20.7 mg, 0.18 mmol), HATU (68 mg, 0.18 mmol) andN- methylmorpholine (55 mg, 0.55 mmol) in 1,2-dichloroethane was stined for 24 h. at rt. The reaction mixture was washed with pH = 4 buffer solution (3 x 3 mL), and the washes were back-extracted with 1,2-dichloroethane (3 mL). The organic phases were combined and concentrated in vacuo. The crude product was then dissolved in MeOH and purified by reverse phase preparative HPLC to give the title compound (Compound 210) as a yellow glassy solid (60.7 mg, 56.8 % yield): 1H NMR (CD3OD) δ 0.59-0.69 (m, 2 H), 0.82 (s, 9 H), 0.98 (s, 9 H), 1.02-1.06 (m, 2 H), 1.42- 1.52 (m, 3 H), 1.92 (dd, J=8.23, 5.31 Hz, 1 H), 1.99 (s, 2 H), 2.23-2.35 ( , 2 H), 2.60-2.67 (m, 1 H), 4.11 (dd, J=11.89, 3.84 Hz, 1 H), 4.43 (d, J=12.08 Hz, 1 H), 4.57 (dd, J=10.25, 6.95 Hz, 1 H), 4.63-4.67 (m, 1 H), 5.18 <dd, J=10.25, 1.83 Hz, 1 H), 5.34 (dd, J=17.20, 1.46 Hz, 1 H), 5.73-5.82 (m, 1 H), 5.85-5.88 (m, 1 H), 7.14-7.16 (m, 2 H), 7.24-7.33 (m, 4 H), 7.53 (dt, J=7.50, 1.10 Hz, 1 H), 7.71 (dt, J=7.50, 1.10 Hz, 1 H), 7.78-7.82 (m, 1 H), 7.95 (d, J=5.86 Hz, 1 H>, 8.17 (d, J=8.05 Hz, 1 H); MS m z 772 (MH+), m/z 770 (M-l).
Example 211: Preparation of Compound 211
Figure imgf000126_0001
Compound 211
Compound 211 was prepared by following Step 1 of Example 210 except that methoxyacetic acid was used in place of tert-butyl acetic acid.
Step 1:
Modifications: 16 mg (0.18 mmol) methoxyacetic acid used, 65.8 mg product obtained as a yellow glassy solid (63.7% yield): *H NMR (CD3OD) δ 0.61-0.66 (m, 2 H), 0.99 (s, 9 H), 1.01-1.06 (m, 1 H), 1.42-1.52 (m, 3 H), 1.92 (dd, J=8.23, 5.31 Hz, 1 H), 2.24-2.37 (m, 2 H), 2.61-2.69 (m, 1 H), 3.35 (s, 3 H), 3.68 (d, J=15.00 Hz, 1 H), 3.78 (s, 1 H), 3.83 (d, J=15.00 Hz, 1 H), 4.13 (dd, J=ll .89, 3.84 Hz, 1 H), 4.36 (d, J=12.08 Hz, 1 H), 4.59 (dd, J=10.06, 7.14 Hz, 1 H), 4.65 (s, 1 H), 5.18 (dd, J=10.25, 1.83 Hz, 1 H), 5.35 (dd, J=17.20, 1.46 Hz, 1 H), 5.80 (ddd, J=17.20, 10.25, 9.15 Hz, 1 H), 5.86-5.89 (m, 1 H), 7.15 (dd, J=7.68, 1.46 Hz, 2 H), 7.24-7.34 (m, 4 H), 7.56 (ddd, J=8.23, 6.95, 1.28 Hz, 1 H), 7.71 (ddd, J=8.23, 6.95, 1.28 Hz, 1 H), 7.80-7.83 (m, 1 H), 7.96 (d, J=6.22 Hz, 1 H), 8.16 (d, J=8.42 Hz, 1 H); MS m/z 746 (MH+), m z 744 (M-l).
Example 212: Preparation of Compound 212
Figure imgf000127_0001
Compound 212
Compound 212 was prepared by following Step 1 of Example 210 except that methoxypropionic acid was used in place of tert-butyl acetic acid.
Step 1:
Modifications: 20 mg (0.18 mmol) methoxypropionic acid used, 73.9 mg product obtained as a yellow glassy solid (70.3 % yield): 1H NMR (CD3OD) δ 0.59-0.68 ( , 2 H), 0.98 (s, 9 H), 1.02-1.06 (m, 1 H), 1.46 (m, 3 H), 1.92 (dd, J=8.23, 5.31 Hz, 1 H), 2.24-2.47 (m, 4 H), 2.60-2.69 (m, 1 H), 3.25 (s, 3 H), 3.43-3.55 (m, 2 H), 3.78 (s, 1 H), 4.13 (dd, J=11.71, 4.03 Hz, 1 H), 4.38 (d, J=11.71 Hz, 1 H), 4.57 (dd, J=9.88, 7.32 Hz, 1 H), 4.62 (s, 1 H), 5.18 (dd, J=10.43, 1.65 Hz, 1 H), 5.35 (dd, J=17.20, 1.46 Hz, 1 H), 5.73-5.82 (m, 1 H), 5.85-5.89 (m, 1 H), 7.15 (dd, J=7.87, 1.28 Hz, 2 H), 7.22-7.34 (m, 4 H), 7.56 (ddd, J=8.23, 6.95, 1.28 Hz, 1 H), 7.71 (ddd, J=8.23, 6.95, 1.28 Hz, 1 H), 7.80-7.83 (m, 1 H), 7.96 (d, J=5.86 Hz, 1 H), 8.18 (d, J=8.42 Hz, 1 H); MS m/z 760 (MH+), m/z 758 (M-l).
Example 213: Preparation of Compound 213
Figure imgf000128_0001
Compound 213
Compound 213 was prepared by following Step 1 of Example 210 except that (S)- 1 ,4-benzodioxane-2-carboxylic acid was used in place of tert-but^yl acetic acid.
Step l:
Modifications: 33 mg (0.18 mmol) (S)-l,4-benzodioxane-2-carboxylic acid used,
72.4 mg product obtained as a yellow glassy solid (62.6% yield): 1H NMR (CD3OD) δ 0.61-0.67 (m, 2 H), 0.76 (s, 9 H), 0.78-0.82 (m, 1 H), 1.44-1.50 (m, 3 H), 1.92 (dd, J=8.42, 5.49 Hz, 1 H), 2.23-2.37 (m, 2 H), 2.61-2.69 (m, 1 H), 3.78 (s, 1 H), 4.09- 4.17 (m, 2 H), 4.32-4.37 (m, 2 H), 4.56-4.64 (m, 3 H), 5.18 (dd, J=10.25, 1.83 Hz, 1 H), 5.35 (dd, J=17.02, 1.28 Hz, 1 H), 5.75-5.84 (m, 1 H), 5.87-5.90 (m, 1 H), 6.79- 6.91 (m, 3 H), 7.04-7.07 (m, 1 H), 7.13-7.15 (m, 2 H), 7.23-7.35 C , 4 H), 7.53-7.59 (m, 1 H), 7.67-7.72 (m, 1 H), 7.80-7.83 (m, 1 H), 7.97 (d, J=5.86 Ηz, 1 H), 8.17 (d, J=8.42 Hz, 1 H); MS m/z 836 (MH+), m z 834 (M-l).
Example 215: Preparation of Compound 216
Figure imgf000129_0001
Scheme 1.
Figure imgf000129_0002
IN HCI , Et20, DCM
Stepl:
To a solution of lR-tert-butoxycarbonylamino-2S-vinyl-cyclopropanecarboxylic acid (2.3 g, 10.1 mmol) in THF (40 mL) was added GDI (1.80 g, 11.6 mmol) and was heated to 85 °C for 30 min. After let cool to rt, the reaction mixture was treated with 1-methyl-cyclopropanesulfonam (1.64 g, 12.1 mmol) and DBU (3.08 g, 20.2 mmol). After stining at rt for 16 h, the reaction was diluted with EtOAc (160 mL) and washed with 2x25 mL IN HCI. The combined aqueous layer was extracted with 1x50 mL EtOAc. The combined organic layer was washed with H2O (50 mL) , brine, dried over MgSO4 and concentrated to a light yellow solid product (2.75 g, 79%). The product was used as crade. MS m/z 367 (M+Na).
Step 2:
To a solution ofthe product from step 1 of Example 216 (2.66 g, 7.72 mmol) in DCM (15 mL) was added TFA (15 mL) and was stining rt for 15 min. Solvent was concentrated. The resulting brown oil was redissolved in DCE (30 mL) and reconcentrated. It was then1 redissolved in DCM (15 mL) and treated with a solution of IN HCI in Et2O (30 mL) dropwise. The light yellow precipitation product was obtained by cacuum filtration and washed with Et2O (2.2 g, quantatative yield). The product was used as crude: MS m z 245 (MH1").
Figure imgf000130_0001
Step 3:
To a solution mixture ofthe product from Step 5 of Example 200 (1.70 g, 4.38 mmol), DIEA (1.42 g, 10.95 mmol) and the product from Step 2 of Example 216 (1.23g, 4.38 mmol) in DCM (44 mL) was added HATU (2.16 g, 5.69 mmol). After stining the reaction mixture at rt for 5h, it was diluted with DCM (50 mL) and washed with 5% aqueous NaHCO3 (25 mL). The aqueous layer was extracted with 1x25 mL DCM. The combined organic layer was washed with 5% aqueous citric acid (50 mL), H2O (25 mL), brine , dried over MgSO4 and concentrated. Crude product was purified by reverse phase prep-HPLC to give a white foarm (1.78 mg, 66% yield): MS /z 615 (MH4).
Step 4:
To a solution ofthe product from step 3 of Example 216 (1.78 g, 2.90 mmol) in DCM (6 mL) was added TFA (6 mL) and was stining rt for 20 min. Solvent was concentrated. The resulting brown oil was redissolved in DCE (50 mL) and reconcentrated. It was then redissolved in DCM (5 mL) and treated with a solution of IN HCI in Et2O (30 mL) dropwise. The brown precipitation product was obtained by cacuum filtration and washed with Et2O (1.7 g, quantatative yield). The product was used as crude: MS m/z 515 (MH+). Step 5:
To a solution mixture ofthe product from Step 4 of Example 216 (1.25 g, 2.13 mmol), DIEA (1.10 g, 8.52 mmol) and Boc-L-Tle-OH (0.640, 2.76 xnmol) in DCM (21 mL) was added HATU (1.21 g, 3.20 mmol). After stining the reaction mixture at rt for 5h, it was diluted with DCM (30 mL) and washed with 5% aqueous N HCO3 (15 mL). The aqueous layer was extracted with 1x25 mL DCM. Tire combined organic layer was washed with 5% aqueous citric acid (30 mL), H^O (15 mL), brine , dried over MgSO4 and concentrated. Crade product was purified by flash column chromatography to give Compound 301 as a white foarm (1.24- g, 80% yield): MS m/z 728 ( H ).
Figure imgf000131_0001
Step 6:
To a solution of Compound 301 (1.1 g, 1.51 mmol) in DCM (5 mL) was added TFA (5 mL) and was stirring rt for 20 min. Solvent was concentrated. The resulting brown oil was redissolved in DCE (15 mL) and reconcentrated. It was then redissolved in DCM (3 mL) and treated with a solution of IN HCI in Et O (15 mL) dropwise. The brown precipitation product was obtained by cacuum filtration and washed with Et2O (1.04 g, quantatative yield). The product was used as crade: MS m/z 628(MH+).
Example 217: Preparation of Compound 217
Figure imgf000132_0001
Figure imgf000132_0002
Step l:
To a solution mixture of Compound 216 (0.102 g, 0.146 mmol) and DIEA (47.3 m g, 0.365 mmol) in THF (2 mL) was added DSC (55.9 mg, 0.218 mmol) . The reaction mixture was irradiated in a microwave to 80 °C for 15 min. After let cooled to rt, the reaction was treated with tert-butylamine (0.107 g, 1.46 mmol). It was then stirred at rt for lh, concentrated and purified by reversed-phase prep-HPLC to give a white solid product (62.2 mg, 60% yield). MS m/z 727 (MH+).
Example 218: Preparation of Compound 218
Figure imgf000132_0003
Compound 218 was prepared by the same methods as Compound 217 with the following modifications: Modifications: cyclopentylamine was used as a starting material to give Compound 218 (57.5 mg, 53% yield): MS m/z 739 (MH+).
Example 219: Preparation of Compound 219
Figure imgf000133_0001
Compound 219 was prepared by the same methods as Compound 217 with the following modifications:
Modifications: Tert-amylamine was used as a starting material to give Compound 219 (58.3 mg, 54% yield): MS m/z 741 (MH+).
Example 220: Preparation of Compound 220
Figure imgf000133_0002
Compound 220 was prepared by the same methods as Compound 217 with the following modifications:
Modifications: Tert-amylamine was used as a starting material to give Compound 220 (10.0 mg, 20% yield): MS m/z 711 (MH ). Section D
Preparation of Compounds 300-304
LC-MS condition:
Columns: (Method A) - Xtena MS CI 8 S7 3.0x50 mm
(Method B) - Xtena S7 3.0x50 mm (Method C) - Xtena S7 C18 3.0x50 mm
Solvent A: 10% MeOH - 90% H2O - 0.1% TFA Solvent B: 90% MeOH - 10% H2O - 0.1% TFA
Gradient time: 2 min. (A, B, C) Hold time: 1 min. (A, B, C)
Flow rate: 5 mL/min (A, B, C))
Example 300: Preparation of Compound 300 {l-[2-[l-(l-Ethyl- cyclopropanesulfonylaminocarbonyl)-2-vinyl-cyclopropylcarbamoyl]-4-(6-methoxy- isoquinolin-l-yloxy)-pynolidine-l-carbonyl]-2,2-dimethyl-propyl}-carbamic acid tert-butyl ester
Figure imgf000134_0001
compound 300
Figure imgf000135_0001
Figure imgf000135_0002
compound 300
step 1:
A sluny of P2 Boc-(4R)-(6-methoxy-isoquinoline-l-oxo)-S-proline]-Pl(lR,2S Vinyl Acca)-COOEt (7.88 g, 14.99 mmol) in 4M HCl dioxane (120 mL, 480 mmol) was stined for 2 h, removed the solvent in vacuo and azeotroped with dry dioxane. To the residue was added DMF (75 L), N-mehtylmorpholine (6.27 mL, 57.07 mmol), Boc- X-tert-leucine (5.20 g, 22.49 mmol), and HATU (8.53 g, 22.49 mmol). The reaction mixture was stined at rt ovemite and worked up by pouring the reaction mixmre into ice water and adjusted to pH 5 with aqueous 1.0 N HCI and extracted with EtOAc. The extract was washed with NaHCO3 (aq.), brine, dried (MgSO4) and concentrated. The residue was purified over Biotage 65M column (EtOAc-hexanes: 5-100%) to provide the product (8.07 g, 84%): Retention time: 1.88 method C) MS m/z 639 (M++l). Step 2:
To a suspension ofthe product (4.0 g, 6.26 mmol) of Step 1 of Example 384 {Boc - NH-P3(£-tert-BuGly)-P2[(4R)-(6-methoxyl-isoquinoline- 1 -oxo)-S-proline]-P 1 (1R,2S Vinyl Acca)-COOEt} in THF(250 mL), CH3OH (31 mL), and H2O (125 mL) was added LiOH (2.4 g, 100.2 mmol). The reaction mixture was stined for ovemite and then adjusted to pH 7 with aqueous 1.0 N HCI. The organic solvents were removed in vacuo. The aqueous residue was acidified to pH 4 and extracted with EtOAc (2x). The combined organic solvent was dried (Na2SO4/MgSO ), and concentrated in vacuo to supply the product (3.79 g, 99%): 1H NMR (methanol-cLi) δ ppm 1.05 (s, 9 H), 1.25 (m, 1 H), 1.29 (s, 9 H), 1.46 (m, 1 H), 1.72 (dd, J=8.24, 5.19 Hz, 1 H), 2.23 (q, J=8.55 Hz, 1 H), 2.68 (dd, J=13.89, 7.78 Hz, 1 H), 3.94 (s, 3 H), 4.05 (dd, J=l 1.60, 3.05 Hz, 1 H), 4.23 (d, J=8.85 Hz, 1 H), 4.46 (d, J=l 1.60 Hz, 1 H), 4.63 (t, J=8.39 Hz, 1 H), 5.10 (d, J=10.38 Hz, 1 H), 5.29 (d, J=17.40 Hz, 1 H), 5.85 (m, 2 H), 7.10 (d, J=9.16 Hz, 1 H), 7.19 (s, 1 H), 7.26 (d, J=5.49 Hz, 1 H), 7.91 (d, J=5.80 Hz, 1 H), 8.12 (d, J=9.16 Hz, 1 H). LC-MS (Retention time: 1.81 method C) MS m/z 611 (M++l).
Step 3:
A solution of GDI (0.142g, 0.87 mmol) and the product (0.400 g, 0.58 mmol) of Step 2 of Example 300 {BOCNH-P3(I-t-BuGly)-P2[(4R)-6-methoxy-sioquinoline-l-oxo)- S-proline]-Pl (1R,2S Vinyl Acca)-CO2H} in THF (8 mL) was heated at 70 °C for 60 min and allowed to cool down to rt. The reaction solution was evenly divided by syringe into four flask. To one ofthe flask was added 1-ethyl- cyclopropanesulfonamide (0.039 g, 0.26 mmol) and followed by the addition of a solution of neat DBU (0.048 mL, 0.32 mmol). The reaction was stined for ovemite, then filtered through syringe filter and purified by preparative HPLC (solvent B: 50% to 100%) and to provide the Comound 300 (0.0638 mg); ]H NMR (500 MHz, Solvent: methanol-th) δ ppm 0.94 (m, 2 H), 0.98 (t, J=7.48 Hz, 3 H), 1.03 (d, J=3.66 Hz, 9 H), 1.27 (s, 9 H), 1.41 (dd, J=9.16, 5.49 Hz, 1 H), 1.52 (m, 2 H), 1.85 (m, 2 H), 1.97 (m, 1 H), 2.25 (m, 2 H), 2.61 (dd, J=13.89, 7.17 Hz, 1 H), 3.92 (s, 3 H), 4.05 (m, 1 H), 4.25 (s, 1 H), 4.43 (d, J=12.21 Hz, 1 H), 4.54 (m, 1 H), 5.11 (d, J=10.38 Hz, 1 H), 5.28 (d, J=17.09 Hz, 1 H), 5.71 (m, 1 H), 5.83 (s, 1 H), 7.09 (d, J=8.85 Hz, 1 H), 7.18 (s, 1 H), 7.25 (d, J=5.80 Hz, 1 H), 7.88 (d, J=5.80 Hz, 1 H), 8.09 (d, J=9.46 Hz, 1 H); LC-MS (Retention time: 1.92 method A), MS m/z 742 (M++l).
Example 301: Preparation of Compound 301; (l-{4-(6-Methoxy-isoquinolin-l- yloxy)-2-[l-(l-methyl-cyclopropanesulfonylaminocarbonyl)-2-vinyl- cyclopropylcarbamoyl] -pynolidine- 1 -carbonyl } -2,2-dimethyl-propyl)-carbamic acid tert-butyl ester
Figure imgf000137_0001
Step 1:
Compound 301 was prepared in the same procedure as described in Step 3 of Example 300 in preparation of Compound 300 instead of 1-methyl- cyclopropanesulfonamide (0.039 g, 0.26 mmol), was used in the place of 1-ethyl- cyclopropanesulfonamide, but purified by combination of Prep-HPLC (solvent B: 40% to 100%) and P-TLC (MeOH/CH2Cl2: 0% to 5%) as a white foam (0.0721g ). 1H NMR (500 MHz, Solvent: methanol-dj) δ ppm 0.93 (m, 2 H), 1.06 (s, 9 H), 1.30 (s, 9 H), 1.43 (dd, J=9.31, 5.34 Hz, 1 H), 1.52 (s, 3 H), 1.59 (m, 2 H), 1.89 ( , 1 H), 2.28 (m, 2 H), 2.65 (dd, J=14.04, 7.32 Hz, 1 H), 3.95 (s, 3 H), 4.09 (m, 1 H), 4.29 (d, J=9.46 Hz, 1 H), 4.47 (d, J=11.29 Hz, 1 H), 4.57 (m, 1 H), 5.15 (d, J=10.68 Hz, 1 H), 5.32 (d, J=16.79 Hz, 1 H), 5.74 (m, 1 H), 5.86 (s, 1 H), 6.62 (d, J=9.16 Hz, 1 H), 7.12 (d, J=8.85 Hz, 1 H), 7.21 (s, 1 H), 7.27 (d, J=5.49 Hz, 1 H), 7.91 (d, J=6.10 Hz, 1 H), 8.12 (d, J=9.16 Hz, 1 H), LC-MS (Retention time: 1.86 method A), MS m/z 728 (iVlAl). Example 302: Preparation of Compound 302; (l-{4-(6-Methoxy-isoquinolin-l- yloxy)-2- [ 1 -( 1 -propyl-cyclopropanesulfonylaminocarbonyl)-2-vinyl- cyclopropylcarbamoyl]-pynolidine-l-carbonyl}-2,2-dimethyl-propyl)-carbamic acid tert-butyl ester
Figure imgf000138_0001
Step 1:
Compound 302 was prepared in the same procedure as described in Step 3 of Example 300 in preparation of Compound 300 instead of 1-methyl- cyclopropanesulfonamide (0.039 g, 0.26 mmol), was used in the place of 1-ethyl- cyclopropanesulfonamide, and purified by Prep-HPLC (solvent B: 50% to 100%) as a white foam (0.0628g ). *H NMR (500 MHz, Solvent: methanol-ch) δ ppm 0.91 (t, J=7.32 Hz, 4 H), 0.94 (m, 2 H), 1.04 (s, 9 H), 1.28 (s, 9 H), 1.45 (m, 3 H), 1.59 (m, 1 H), 1.73 (m, 1 H), 1.88 (m, 2 H), 2.26 (m, 2 H), 2.61 (dd, J=13.73, 7.32 Hz, 1 H), 3.92 (s, 3 H), 4.06 (m, 1 H), 4.25 (s, 1 H), 4.43 (d, J=12.21 Hz, 1 H), 4.54 (m, 1 H), 5.11 (d, J=10.07 Hz, 1 H), 5.29 (d, J=18.01 Hz, 1 H), 5.72 (m, 1 H), 5.84 (s, 1 H), 7.09 (d, J=8.85 Hz, 1 H), 7.18 (s, 1 H), 7.25 (d, J=6.10 Hz, 1 H), 7.88 (d, J=6.10 Hz, 1 H), 8.09 (d, J=8.85 Hz, 1 H); LC-MS (Retention time: 1.97 method A), MS m/z 756 (M++l).
Example 303: Preparation of Compound 303; {l-[2-[l-(l-Benzyl- cyclopropanesulfonylaminocarbonyl)-2-vinyl-cyclopropylcarbamoyl]-4-(6-methoxy- iso qumolm-l-yloxy)-pynolidine-l-carbonyl]-2,2-dimethyl-propyl}-carbamic acid tert- butyl ester
Figure imgf000139_0001
compound 303
Step 1:
Compound 303 was prepared in the same procedure as described in Step 3 of Example 300 in preparation of Compound 300 instead of 1-benzyl- cyclopropanesulfonamide (0.055 g, 0.26 mmol), was used in the place of 1-ethyl- cyclopropanesulfonamide, and purified by Prep-HPLC (solvent B: 50% to 100%) as a white foam (0.070g ). 1H NMR (500 MHz, Solvent: methanol-ch δ ppm 0.63 (m, 2 H), 0.96 (s, 9 H), 1.27 (s, 9 H), 1.44 (m, 3 H), 1.91 (m, 1 H), 2.25 (m, 2 H), 2.62 (dd, J=13.43, 7.02 Hz, 1 H), 3.28 (d, J=13.73 Hz, 1 H), 3.34 (m, 1 H), 3.91 (s, 3 H), 4.04 (m, 1 H), 4.23 (s, 1 H), 4.43 (d, J=11.90 Hz, 1 H), 4.56 (m, 1 H), 5.17 (d, J=10.07 Hz, 1 H), 5.34 (d, J=17.09 Hz, 1 H), 5.77 (m, 2 H), 7.08 (d, J=8.85 Hz, 1 H), 7.15 (m, 3 H), 7.26 (m, 4 H), 7.87 (d, J=6.10 Hz, 1 H), 8.09 (d, J=8.85 Hz, 1 H); LC-MS (Retention time: 2.03 method A), MS m/z 804 (M++l).
Example 304: Preparation of Compound 304; {l-[2-[l-(l-Chloro- cyclopropanesulfonylaminocarbonyl)-2-vinyl-cyclopropylcarbamoyl]-4-(6-methoxy- isoquinolm-l-yloxy)-pynolidine-l-carbonyl]-2,2-dimethyl-propyl}-carbamic acid tert-butyl ester
Figure imgf000140_0001
compound 304
Scheme 1 step 1 step 2 BuLi, NCS O O TFA ° °
SS ,Boc CI "s" ,Boc
NH2
Step 1:
To a solution of cyclopropylsulfonylamine tert-butyl carbamate (1.0 g, 4.52 mmol) dissolved in THF (10 mL) cooled to -78 °C, was added n-BuLi (6.4 mL, 10.2 mmol, 1.6 M in hexane) and the reaction mixture was stined for 1 h. To this solution was added a THF (10 mL) solution of NCS (0.86 g, 6.34 mmol). After stirred for 5 min, the bath was changed to ice bath and the mixture was stined for 3 hrs at the temperature. The reaction mixture was diluted with ice water, the pH was adjusted to <4. The aqueous mixtire was extracted with EtOAc. The combined extracts were dried.(MgSO ), concentrated and purified by flash chromatography over SiO using 5 0% to 60% EtOAc in hexanes as the eluent to afford 0.98 g (67%) of 1 - chloro- cyclopropanesulfonamide- tert-butylcarbarnate as a white solid: : !H NMR (500 MHz, CDC13) δ ppm 1.51 (m, 11 H), 2.01 (m, 2 H), 7.60 (s, 1 H). Step 2: 0 A mixture of cyclopropylsulfonylamhie tert-butyl carbamate 0.148g 0.58 mmol) and TFA (lmL) was stirred for 2.5 h at rt. Removed the solvent in vacuo to provide the product yield (0.09 g, 100%) as a light brown solid: 1H NMR (500 MHz, Methanol- dt) δ ppm 1.38 (m, 2 H), 1.70 (m, 2 H).
Scheme 2
Figure imgf000141_0001
_ 5 compound 304
Step 3:
A solution ofthe product (0.10 g, 0.16 mmol) of Step 2 of Example 300 in 0 preparation of Compound 300 and GDI (0.037 mg, 0.229 mmol) in THF (2 mL) was heated at 70 °C for 60 min and allowed to cool down to rt. 1- Chlorocyclopropylsulfonamide (0.027g, 0.14 mmol) and neat DBU (0.024 mL, 0.16 mmol) were added. The reaction mixture was stirred for ovemite and directly purified by prep-HPLC to provided the product, Compound 304, (34.1 mg, %) as a light 5 brown solid; 1H NMR (500 MHz, Solvent: methanol-ch) δ ppm 1.03 (s, 9 H), 1.27 (s, 9 H), 1.46 (m, 3 H), 1.86 (m, 1 H), 1.98 (m, 2 H), 2.29 (m, 2 H), 2.61 (dd, J=13.73, 7.32 Hz, 1 H), 3.92 (s, 3 H), 4.05 (m, 1 H), 4.24 (s, 1 H), 4.55 (m, 1 H), 5.13 (d, J=10.07 Hz, 1 H), 5.29 (d, J=16.79 Hz, 1 H), 5.70 (m, 1 H), 5.82 (s, 1 H), 7.08 (d, J=8.85 Hz, 1 H), 7.17 (s, 1 H), 7.24 (d, J=5.80 Hz, 1 H), 7.88 (d, J=5.80 Hz, 1 H), 8.09 (d, J=9.16 Hz, 1 H); LC-MS (Retention time: 1.83 method B), MS m/z 748 (M++l).
Section E
Preparation of Compounds 400-401
Example 400: Preparation of Compound 400.
Figure imgf000142_0001
Compound 400
Scheme 1
Figure imgf000142_0002
Compound 400
Step l:
To a solution of N-BOC-3-(R)-hydroxy-L-proline (231 mg, 1.0 mmol) in DMSO (10 mL) at the> ambient temperature was added potassium tert-butoxide (336 mg, 3.0 mmol) in one portion. The formed suspension was stirred at this temperature for 30 min before being cooled to 10°C. 1-Chloro-isoquinoline (180 mg, 1.1 mmol) was added as solid in one portion and the final mixture was stined at the ambient temperature for 12 h. Quenched with iced 5% citric acid (aq), extracted with EtO C (10O L). The aqueous phase was extracted with EtOAC again. The combined organic layers were washed with 5% citric acid (aq) and brine respectively, dried over MgSO , filtered. The filtrate was evaporated in vacuo to dryness to yield 329 mg (92%) ofthe desired product as an off-white foam. This material was used in the next step reaction as crude without further purification. 1H NMR (CD3OD) δ 1.42, 1.44 (rotamers, 9H), 2.39-2.44 (m, IH), 2.68-2.72 (m, IH), 3.80-3.87 (m, 2H), 4.44-4.52 (m, IH), 5.78 (b, IH), 7.32-7.33 (m, IH), 7.58 (t, J=7.8 Hz, IH), ), 7.71 (t, J=7.5 Hz, IH), 7.81 (d, J=8.0 Hz, IH), 7.95 (d, J=6.0 Hz, IH), 8J9 (d, J=8.0 Hz, IH); LC-MS (retention time: 1.61 min, method B), MS m/z 359 (MAH). Step 2:
To a mixture ofthe product of Example 400, Step 1 (114 mg, 0.32 mmol), HATU (253 mg, 0.67 mmol), and the product of Example XX, Step XX (107 mg, 0.33 mmol) in CH2C12 (5 mL) was added DIPEA (129 mg, 1.0 mmol) at 0°C. After stining at the ambient temperature for 12 h, the formed solution was diluted with CH C1 (5 mL), washed with iced 5% citric acid (aq). The organic layer was washed with 5% citric acid (aq) and brine respectively, dried over MgSO4, and filtered. The filtrate was evaporated in vacuo to dryness. The residue was purified by prep-HPLC to yield 86 mg (43%) ofthe desired product as a foam. 1H NMR (CD3OD) δ 0.09-0.10 ( 2H), 0.46-0.47 (m, 2H), 0.69-0.70 (m, IH), 1.13- 1.17 ( , 2H), 1.46 (s, 9H), 1.52-1.54 (m, 2H), 1.83-1.89 (m, 2H), 2.21-2.33 (m, 2H),
2.54-2.58 (m, IH), 3.83-3.89 (m, 2H), 4.41-4.43 (m, IH), 5.12 (d, J=10.5 Hz, IH), 5.31 (d, J=15.0 Hz, IH), 5.73-5.78 (m, IH), 5.81 (b, IH), 7.33-7.34 (m, IH), 7.59 (t, J=7.8 Hz, IH), 7.73 (t, J=7.5 Hz, IH), 7.83 (d, J=8.0 Hz, IH), 7.96 (d, J=6.0 Hz, IH), 8.19 (d, J=8.0 Hz, IH), 9.16 (b, IH); LC-MS (retention time: 1.86 min, method B), MS m/z 625 (MAH).
Step 3: A solution ofthe product of Example 400, Step 2 (77 mg, 0.12 mmol) in DCM (1 mL) and TFA (1 mL) was stined at room temperature for 1.5 h. The volatiles were removed in vacuo and the residue suspended in IN HCI in diethyl ether (5 mL) and concentrated in vacuo. This procedure was repeated once. The resulting mixture was triturated from pentane and filtered to give the desired compound as a hygroscopic, off-white solid (65 mg, 91%).
LC-MS (retention time: 1.35 min, method B), MS m/z 525 (MAH). Step 4: To a mixture ofthe product of Example 400, Step 3 (65 mg, 0.12 mmol), HATU (66 mg, 0.17 mmol), and j -Boc-t-Butyl-L-glycine (32 mg, 0.14 mmol) in CH2C12 (2 mL) was added DIPEA (39 mg, 0.35 mmol) at 0°C. After stining at the ambient temperature for 12 h, the formed solution was diluted with CH2C12 (5 mL), washed with iced 5% citric acid (aq). The organic layer was washed with 5% citric acid (aq) and brine respectively, dried over MgSO4, and filtered. The filtrate was evaporated in vacuo to dryness. The residue was purified by prep-HPLC to yield 45 mg (53%>) of Compound 400 as a white solid.
1H NMR (CD3OD) δ O.04-0.12 (m 2H), 0.44-0.48 (m, 2H), 0.67-0.70 (m, IH), 1.04 (s, 9H), 1.13-1.18 (m, 2H), 1.24 (s, 9H), 1.49-1.58 ( , 3H), 1.76-1.95 (m, 3H), 2.21- 2.31 (m, 2H), 2.61-2.65 (m, IH), 4.07-4.10 (m, IH), 4.26-4.27 (in, IH), 4.46-4.48 (m, IH), 4.54-4.56 (m, IH), 5.11 (d, J=10 Hz, IH), 5.26 (d, J=20 Hz, IH), 5.69-5.78 (m, IH), 5.88 (b, IH), 6.66-6.6 (b, IH), 7.32-7.33 (m, IH), 7.52 (t, J=7.8 Hz, IH), 7.70 (t, J=7.5 Hz, IH), 7.8O (d, J=8.0 Hz, IH), 7.96 (d, J=6.0 Hz, IH), 8.20 (d, J=8.0 Hz, IH); LC-MS (retention time: 1.92 min, method B), MS m/z 738 (MAH).
Example 401: Preparation of Compound 401.
Figure imgf000145_0001
Compound 401
Scheme 2
Figure imgf000145_0002
Compound 401
Step 1:
Figure imgf000145_0003
This product was prepared by the same procedure as described in Example 400, Step 2, except using the product of Example 200, Step 4 instead. LC-MS (retention time: 1.66 min, method B), MS m/z 661 (MAH). Step 2:
Figure imgf000146_0001
This product was prepared by the same procedure as described in Example 400, Step
3, except using the product of Example 401, Step 1 instead.
LC-MS (retention time: 1.21 min, method B), MS m/z 561 (MAH).
Step 3:
Compound 401 was prepared by the same procedure as described in Example 400,
Step 4, except using the product of Example 401, Step 2 instead.
1H NMR (CD3OD) δ 0.61-0.67 (m, 2H), 0.96 (s, 9H), 0.99-1.02 (m, 2H), 1.25 (s,
9H), 1.44-1.48 (m, 3H), 1.90-1.94 (m, IH), 2.25-2.30 (m, 2H), 2.62-2.69 (m, IH),
4.04-4.10 (m, IH), 4.24 (b, IH), 4.46-4.50 (m, IH), 4.54-4.58 (m, IH), 5.18 (d, J=10
Hz, IH), 5.34 (d, J=15 Hz, IH), 5.76-5.81 (m, IH), 5.86 (b, IH), 7.13-7.26 (m, 2H),
7.25-7.30 (m, 4H), 7.32 (t, J=7.8 Hz, IH), 7.79 (t, J=7.5 Hz, IH), 7.80 (d, J=8.0 Hz,
IH), 7.96 (d, J=6.0 Hz, IH), 8.21 (d, J=8.0 Hz, IH), 9.08 (b, IH);
LC-MS (retention time: 2.03 min, method B), MS m/z 774 (M++H).
Section F
Preparation of Compounds 500-502
Example 500: Preparation of Compound 500.
Figure imgf000146_0002
Compound 500 Scheme 1
Figure imgf000147_0001
Step 1 :
A mixture of 3,5-dimethyl-4-nitro-isoxazole (1.42 g, 10.0 mmol), phenylacetaldehyde (1.32 g, 11.0 mmol) in piperidine (1 mL) and ethanol (10 mL) was heated to reflux for 16 h. After cooling down to the ambient temperature, the product precipitated out was collected by filtration. The cake was washed with cold ethanol thoroughly to afford 1.20 g (53%) ofthe desired product as a white solid. 'HNMR (CDC13) δ 2.87 (s, 3H), 7.46-7.50 (nx, 3H), 7.56 (d, J=8.5 Hz, IH), 7.7-7.80 (m, 2FΪ); LC-M S (retention time: 1.19 min, method B), MS m/z 227 (M++H). Step 2 :
A solution of 3-methyl-5-phenyl-isoxazolo[4,5-b]pyridine 4-oxide (1.00 g, 4.40 mmol) and POCl3 (2.71 g, 17.7 mmol) in chloroform (10 mL) was heated to reflux for 1 h.. After cooling down to the ambient temperature, the final solution was diluted with chloroform (50 mL) and washed with NaHCO3 (aq.) (two 50 L portions) and brine, dried over MgSO4, filtered, evaporated. The residue was purified by flash chromatography (4:1 hexane-EtOAc) to afford 790 mg (73%) ofthe desired product as a white solid. XH NMR (CDCI3) δ 2.72 (s, 3H), 7.46-7.54 (m, 3H), 7.91 (s, IH), 8.00-8.03 (m, 2H); LC-MS (retention time: 1.76 min, method B), MS m/z 245, 247 (M^+H).
Scheme 2
Figure imgf000148_0001
Step 3:
To a mixture of 4-hydroxy-pynolidine-2-carboxylic acid methyl ester (H-Hyp-OMe HCI) (1.81 g, 10.0 mmol), HATU (5.70 g, 15.0 mmol), and N-BOC-t-butyl-L-glycine (2.42 g, 10.5 mmol) in CH2C12 (100 mL) was added DIPEA (3.47 g, 31.0 mmol) at 0°C. After stining at the ambient temperature for 12 h, the formed solution was diluted with CH C12 (100 mL), washed with iced 5% citric acid (aq). The organic layer was washed with 5% citric acid, IM NaOH, brine respectively, dried over MgSO , and filtered. The filtrate was evaporated in vacuo to provide 3.55 g (99%>) of the desired product as an off-white foam. This product was used for the next reaction as crude without further purification.
1H NMR (CD3OD) δ 1.04 (s, 9H), 1.43 (s, 9H), 1.99-2.03 (m, IH), 2.20-2.30 (m, IH), 3.69 (s, 3H), 3.70-3.79 (m, 2H), 4.28 (b, IH), 4.46 (b, IH), 4.74-4.80 ( , IH); LC-MS (retention time: 1.28 min, method B), MS m/z 359 (MAH). Step 4:
A mixture ofthe product of Example 500, Step 3 (3.55 g, 9.9 mmol) in THF ( 50 mL), MeOH ( 50 mL) and LiOH monohydrate ( 0.83 g, 19.9 mmol in 50 mL H2O) was stined at the ambient temperature over night. After removal ofthe volatiles in vacuo, the residue was dissolved in 0.1 M NaOH (100 mL). This aqueous solution was washed with ether (50 mL), acidified by IM HCI to pH4. Extracted with EtOAc (100 mL). The organic layer was washed with 5% citric acid and brine, dried over MgSO4, evaporated to dryness to give 3.20g (95%) ofthe desired product as a white foam. This product was used as crude without further purification. 1H NMR (CD3OD) δ 1.02 (s, 9H), 1.43 (s, 9H), 2.01-2.09 (rn, IH), 2.25-2.32 (m, IH), 3.70-3.85 (m, 2H), 4.26-4.30 (m, IH), 4.46-4.51 (m, 2H), 6.37-6.41 (m, IH); LC-MS (retention time: 1.14 min, method B), MS m/z 345 C AH). Step 5:
To a solution ofthe product of Example 500, Step 4 (1.01 g, 2.93 mmol) in DMSO (30 mL) was added potassium tert-butoxide (1.02 g, 9.08 mmol). The formed solution was stirred at the ambient temperature for 1 h before addition of 7-chloro-3- methyl-5-phenyl-isoxazolo[4,5-b]pyridine (0.75 g, 3.08 mrrxol). The final solution was stined for 12 h. Then was quenched with iced water, acidified with IM HCI to pH 4, extracted with EtOAc (two 200 mL portions). The organic layers were washed with brine, dried over MgSO4, filtered, evaporated. The residue was purified by prep- HPLC (60%B— 100%B, 15 min gradient) to afford 305 mg (19%) ofthe desired product as a pale yellow solid.
1H NMR (CD3OD) δ 1.02 (s, 9H), 1.17 (s, 9H), 2.37-2.47 (m, IH), 2.64 (s, 3H), 2.85- 2.93 (m, IH), 4.00-4.08 (m, IH), 4.14 (b, IH), 4.49-4.55 (m, IH), 4.62-4.71 (m, IH), 5.70 (m, IH), 7.45-7.53 (m, 3H), 7.56 (s, IH), 8.03-8.06 ( , 2H); LC-MS (retention time: 1.89 min, method B), MS m/z 553 (JVJMH).
Figure imgf000149_0001
Step 6:
To a mixture ofthe product of Example 500, Step 5 (82 mg, 0.15 mmol), HATU (84 mg, 0.22 mmol), and the product of Example 200, Step 4 (5S mg, 0.16 mmol) in CH2C12 (5 mL) was added DIPEA (50 mg, 0.44 mmol) at 0°C. After stining at the ambient temperature for 12 h, the formed solution was diluted with CH2C12 (15 mL), washed with iced 5% citric acid (aq). The organic layer was washed with 5% citric acid (aq) and brine respectively, dried over MgSO4, and filtered. The filtrate was evaporated in vacuo to dryness. The residue was purified by prep-HPLC to yield 42 mg (33%>) of Compound 500 as an off-white solid.
1H NMR (CD3OD) δ 0.62-0.67 (m, 2H), 0.95 (s, 9H), 0.99-1.02 (m, 2H), 1.19 (s, 9H), 1.44-1.47 (m, 3H), 1.91-1.94 (m, IH), 2.27-2.37 (m, 2H), 2.62-2.68 (m, 4H), 4.08-4.10 (m, IH), 4.17 (b, IH), 4.49-4.51 (m, IH), 4.57-4.60 (m, IH), 5.19 (d, J=10 Hz, IH), 5.36 (d, J=20 Hz, IH), 5.70 (b, IH), 5.77-5.83 (m, IH), 7.14-7.15 (m, 2H), 7.24-7.31 (m, 3H), 7.47-7.50 (m, 3H), 7.57 (s, IH), 8.04-8.06 (m, 2H), 9.16 (b, IH); LC-MS (retention time: 2.06 min, method B), MS m/z 855 (M++H).
Example 501: Preparation of Compound 501.
Figure imgf000150_0001
Compound 501
Scheme 1
Figure imgf000150_0002
Figure imgf000150_0003
Compound 501
Compound 501 was prepared by the same procedure as described in Example 500, Step 6.
1H NMR (CD3OD) δ 0.90-0.92 (m, 5H), 1.02 (s, 9H), 1.21 (s, 9H), 1.43-1.47 (m, 4H), 1.58-1.61 (m, IH), 1.70-1.76 (m, IH), 1.84-1.92 (m, 2H), 2.22-2.27 (m, IH), 2.32-2.38 (m, IH), 2.62-2.68 (m, 4H), 4.10-4.12 (m, IH), 4.19 (b, IH), 4.47-4.50 (m, IH), 4.55-4.58 (m, IH), 5.12 (d, J=15 Hz, IH), 5.30 (d, J=20 Hz, IH), 5.68-5.76 (m, 2H), 7.46-7.52 (m, 3H), 7.58 (s, IH), 8.05-8.06 (m, 2H), 9.24 (b, IH); LC-MS (retention time: 2.02 min, method B), MS m/z 807 (MAH).
Example 502: Preparation of Compound 502.
Figure imgf000151_0001
Compound 502
Scheme 1
Figure imgf000151_0002
Compound 502
Figure imgf000151_0003
Compound 502 was prepared by the same procedure as described in Example 500, Step 6,..
1H NMR (CD3OD) δ 0.90-1.00 (m, 9H), 1.02 (s, 9H), 1.20 (s, 9H), 1.41-1.58 (m, 6H), 1.71-1.78 (m, IH), 1.91-2.00 (m, IH), 2.34-2.39 (m, IH), 2.62-2.64 (m, 4H), 4.11-4.16 (m, IH), 4.19 (b, IH), 4.47-4.59 (m, 2H), 5.71 (b, IH), 7.46-7.51 (m, 3H), 7.57 (s, IH), 8.04-8.06 ( , 2H); LC-MS (retention time: 1.96 min, method B), MS m/z 809 (MAH).
Section G
Preparation of Compounds 600-605 Example 600: Preparation of Compound 600
Figure imgf000152_0001
Compound 600
Scheme 1.
Figure imgf000152_0002
Stepl.
To a solution of lR-tert-butoxycarbonylamino-2S-virιyl-cyclopropanecarboxylic acid (2.1 g, 9.24 mmol) in THF (26 mL) was added GDI I -87 g, 11.6 mmol) and was heated to 78 °C for 45 min. After let cool to rt, the reaction mixture was treated wit . 1 -cyclopropylmethyl-cyclopropanesulfonic acid amide
(2.11 g, 12.01 mmol) and DBU (2.95 g, 19.4 mmol). After stining at rt for 14 h, the reaction was diluted with EtOAc (50 mL) and washed with 4x50 mL IN HCI. The combined aqueous layer was extracted with 3x50 mL EtOAc. The combined organic layer was with brine, dried over MgSO4 and concentrated to a light brown solid product (3.48 g, 98%). The product was used as crade. 1H NMR (500 MHz, CD3OD) δ 0.07 (q, J=4.88 Hz, 2 H) 0.44-0.48 (m, 2 H) 0.68-0.72 (m, 1 H) 1.14 (s, 2 H) 1.28 (dd, J=9.46, 5.19 Hz, 1 H) 1.43 (d, J=7.02 Hz, 1 H) 1-46 (s, 9 H) 1.49-1.53 (m, 2 H) 1.81 (dd, J=7.78, 5.34 Hz, 1 H) 1.86 (s, 2 H) 2.16-2.20 (m, 1 H) 5.08 (dd, J=10.38, 1.22 Hz, 1 H) 5.27 (dd, J=17.24, 1.37 Hz, 1 H) 5.51-5.55 (m, 1 H).
Step 2.
To a solution ofthe product from step 1 of Example 600 (3.75 g, 9.75 mmol) in DCM (15 mL) was added TFA (15 mL) and was stining rt for 20 min. Solvent was concentrated under vacuum to give viscous brown oil in quantitative yield. The product was used as crude: MS m/z 285 (MH+).
Scheme 2
+
Figure imgf000153_0002
Figure imgf000153_0001
Figure imgf000153_0003
Step 3:
To a solution of 2-bromobenzoic acid (10 g, 49.75 mmol) in DMF (150mL) in a medium pressure flask (Chemglass) was added benzamidine (8.6 g, 54.73 mmol), K CO3 (20.6 g, 149.3 mmol), and copper powder (632 mg, 9.95 mmol). The reaction mixture was heated to 180C° for 4h. Copper and excess K2CO3 were removed by hot vacuum filtration through a celite pad and washed with hot MeOH. The filtrate was let cool to rt and the white pricipatation was obtained by vacuum fitration (7.5 g, 68%> yield): Step 4:
To a 0 °C sluny of Boc-cw-Hydroxyproline-OMe (5.0 g, 20.39 mmol) and the product from step 3 of Example 600 (4.53 g, 20.39 mmol) in THF (200 mL) was added Ph3P (6.42 g, 24.47 mmol) and diisopropyl azocarboxyljate (4.95 g, 24.47 mmol) dropwise. After stirring at rt for 24h, the reaction mixture was diluted with EtOAc (lOOmL) washed with 2x50 mL of 10% aqueous Na2CO3,'2x50 mL H2O. The aqueous layer was separated and back-extracted with 1x10*0 mL EtOAc. The combined organic layer was washed with brine, dried over MgS04 and concentrated to give a yellow viscous oil which was redissolved in minimal amount of EtOAc and hexanes to effect the precipitation of most ofthe PI13PO by-product at 4 °C. Ph3PO was removed by vacuum filtration and the liquid filtrate was concentrated. The resulting viscous oil was purified by a flash column chromatography (SiO2, 4:1 hex: EtOAc) to give a white foam product (8.86 g, 97% yield):
Step 5: a). The product from step 4 of Example 600 (4.05 g, 9.01 mmol) was dissolved in 50% TFA in DCM (45 mL) and stined at rt for 20 min. The solvent was concentrated and the resulting brown viscous oil was dried in vctcuo overnight. The product was used directly for the next reaction.
b) To a solution ofthe resulting brown viscous oil from step 5a of Example 600 (4.90 g, 7.39 mmol) and DIEA (3.83 g, 29.56 mmol) in DCM (50 mL) were added N-BOC X-tBuGly (1.88 g, 8.13 rnmole), HATU (3.37 g, 8.87 mmol). After stining at rt for 35 h, the reaction mixture was wahed with I Ν HCI (26 mL) and adjusted to pH=5 with saturated ΝaHCO3. The aqueous layer was extracted with 3x50 mL DCM. The combined orgo layer was washed with saturated NaHCO3 (50 mL), dried over MgSO4 and concentrated to give a viscous oil product. Pale yell ow precipatation product ( 2.7g, 65% yield) was obtamed from a solution of 1:3 Et2O:pentane.
Step 6: To a solution ofthe product from step 5a of Example 600 (1.51g, 2.68 rnmol) in THF (20 mL) was added IN NaOH (6.7 mL, 6.7 mmol). After stining at rt for 24 h, the rection mixture was extracted with 1x25 mL Et2O. The Et2O layer was washed 2x5 L H O. The combined aqueous layer was acidified with IN HCI to pH=5 and extracted with 3x50 mL DCM. The combined DCM layer was dried over MgSO4, concentrated and and dried under vacuum to give a white solid product (1.2 g, 82% yield):
Scheme 3
Figure imgf000155_0001
Compound 600
Figure imgf000155_0002
Step 7: To a solution ofthe product from step 6 of Example 600 (0.250 g, 0.456 mmol) and DIEA (0.177 g, 1.37 mmol) in DCM (5 mL) were added the product from step 2 of Example 600 (0.182 g, 0.456 mmol), HATU (0.225 g, 0.592 mmol). After stining at rt for 14 h, the reaction mixture was wahed with 5% aqueous NaHCO3 (5 mL), and 5% aqueous citric acid (5mL). DCM (25 mL) was used to extrated the two aqueous layer, started with the NaHCO3 layer. The combined orgo layer was dried over MgSO4 and concentrated to give a brown viscous oil which was purified by flash column chromatography to give a pale yellow solid nproduct ( 0.299 g, 80% yield): MS m/z 815 (MH+).
Example 601: Preparation of Compound 601
Figure imgf000156_0001
Compound 601
Scheme 1
Figure imgf000156_0002
Compound 601
Figure imgf000156_0003
Compound 601 was prepared by the same methods as Compound 600 with the following modifications:
Modifications: The product from step 4 of Example 200 was used as a starting material to give Compound 601 (0.309 g, 80% yield): MS m/z 851 (MH+).
Example 602: Preparation of Compound 602
Figure imgf000156_0004
Scheme 1
Figure imgf000157_0001
Stepl: To a solution of Example 600 (0.245 g, 0.301 mmol) in DCM (1.5 mL) was a-dded TFA (1.5 L). After stirring rt for 15 min, reaction mixture was concentrated, and dried under vacuum to give a light brow solid product (0.281 g, 99% yield). The product was used as crade: MS m/z 715 (MH*).
Example 603: Preparation of Example 603
Figure imgf000157_0002
Scheme 1
Figure imgf000157_0003
Compound 603 was prepared by the same methods as Compound 602 with the following modifications:
Modifications: Compound 601 was used as a starting material to give Compound 603 (0.290 g, quantatative yield): MS m/z 751 (MH+).
Example 604: Preparation of Compound 604
Figure imgf000158_0001
Scheme 1
Figure imgf000158_0002
Step 1:
To a solution n ixture of Compound 602 (50.0 mg, 0.053 mmol) and DIEA (28.0 mg, 0.212 mmol) in DCM (2 mL) was added acetic anhydride (21.7 mg, 0.212 mmol). After stining at rt for 14 h, reaction was concentrated and purified by reversed phased prep-HPLC to give a white solid product (32.3 mg, 81% yield): MS m/z 757 (MH+).
Example 605: Preparation of Compound 605
Figure imgf000159_0001
Scheme 1
Figure imgf000159_0002
Compound 605 was prepared by the same methods as Compound 600 with the following modifications:
Modifications: Compound 602 and pyrazine-2-carboxylic acid were used as starting materials to give Compound 605 (38.2 m g, 88% yield): MS m/z 821 (MH4).
Section H
Compounds 700-710
Compounds 700 ans 701 were prepared using the processes described herein. The preparation ofthe functionalized P2 Proline intermediate employed in the constraction of Compound 700 and Compound 701 is described in CT-2723
Figure imgf000160_0001
Figure imgf000160_0002
Section I
Preparation of Compound 800 Example 800: Preparation of Compound 800.
Figure imgf000160_0003
Figure imgf000161_0001
Step 1:
A mixture of 2-amino-6-methylpyridine (1.08 g, 10.0 mmol), ethyl benzoylacetate
(2.30 g, 12.0 mmol) and polyphosphoric acid (6.00 g, 61.2 mmol) was heated to 110°C for 5 h. After cooling to the ambient temperature, the mixture was poured into iced water (20 mL) and neutralized to pH 7 with 10 M NaOH. Extracted with CHCI3.
The organic layer was washed with brine, dried over MgSO4, filtered, evaporated.
The residue was purified by flash chromatography (1:1 hexane-EtOAc) to afford 510 mg (22%) ofthe desired product as a pale yellow solid. 1H NMR (CDCI3) δ 3.08 (s, 3H), 6.64 (d, J=7.0 Hz, IH), 6.71 (s, IH), 7.42-7.52 (m,
5H), 8.04-8.06 (m, 2H);
LC-MS (retention time: 1.21 min, method B), MS m/z 237 (M++H).
Step 2:
A solution of 6-methyl-2-phenyl-pyrido[l,2a]pyrimidin-4-one (489 mg, 2.07 mmol) in melted diphenyl ether (5 mL) was heated to gentle reflux for 5 h. After cooling to the ambient temperature, the formed suspension was diluted with diethyl ether (10 mL), filtered. The cake was washed with diethyl ether thoroughly to afford 450 mg
(92%) ofthe desired product as a brownish solid.
LC-MS (retention time: 1.25 min, method B), MS m/z 231 (MAH). Step 3:
A suspension of 7-methyl-2-phenyl-lH-[l,8]naphthyridin-4-one (450 mg, 1.91 mmol) in POCI3 (10 mL) was heated to gentle reflux for 3 h. Evaporated in vacuo.
The residue was was poured into iced water (20 mL) and neutralized to pH 10 with
10 M NaOH. Extracted with CHCI3. The organic layer was washed with brine, dried over MgSO4, filtered, evaporated. The residue was purified by flash chromatography (2:1 hexane-EtOAc) to afford 450 mg (92%>) ofthe desired product as a pink solid. 1H NMR (CD3OD) δ 2.80 (s, 3H), 7.54-7.56 (m, 3H), 7.61 (d, J=8.4 Hz, IH), 8.25- 8.30 (m, 3H), 8.58 (d, J=8.4 Hz, IH); LC-MS (retention time: 1.39 min, method B), MS m/z 255, 257 (M H).
Figure imgf000162_0001
Compound 800 Step 4:
This product was prepared by the same procedure as described in Example 500. LC-MS (retention time: 1.55 min, method B), MS m/z 563 (MAH). Step 5:
Compound 800 was prepared by the same procedure as described in Example 500, Step 6, except using the product of Example 800, Step 4 instead. 1H NMR (CD3OD) δ 0.64 (b, 2H), 0.98 (s, 9H), 1.26 (s, 9H), 1.45-1.47 (m, 3H), 1.91-1.94 (m, IH), 2.28-2.32 (m, 2H), 2.71-2.77 (m, 4H), 4.08-4.10 (m, IH), 4.21 (b, IH), 4.56-4.61 (m, 2H), 5.17 (d, J=10 Hz, IH), 5.32 (d, J=15 Hz, IH), 5.63 (b, IH), 5.73-5.79 (m, IH), 7.14-7.15 (m, 2H), 7.24-7.30 (m, 3H), 7.41-7.43 (m, IH), 7.53- 7.55 (m, 4H), 8.22-8.24 (m, 2H), 8.56 (d, J=10 Hz, IH); LC-MS (retention time: 1.87 min, method B), MS m/z 865 (MAH).
The following conditions were used for LC/MS analysis. Columns: Method A: YMC ODS-A CI 8 S7 (4.6 x 33 mm) Method B: YMC Xtena ODS S7 (3.0 x 50mm) Method C: Xtena ms C18 (4.6 x 33mm) Method D: YMC ODS-A C18 S3 (4.6 x 33 mm) Gradient: 100% solvent A/ 0% solvent B to 0% solvent A/ 100% solvent B
Gradient time: 3 min.
Hold Time: 1 min.
Flow Rate: 5 mL/min.
Detector Wavelength: 220 nm. Solvents: Solvent A: 10% MeOH/ 90% water/ 0.1% TFA. Solvent B: 90% MeOH/
10% water/ 0.1% TFA.
The following conditions were used for prep-HPLC separation.
Columns: Phenomenex-Luna 30X100 mm, S5
Gradient: 60% solvent A/ 40% solvent B to 0% solvent A/ 100% solvent B Gradient time: 15 min.
Stop Time: 20 min.
Flow Rate: 30 mL/min.
Detector Wavelength: 220 nm.
Solvents: Solvent A: 10% MeOH/ 90% water/ 0.1% TFA. Solvent B: 90% MeOH/ 10% water/ 0.1% TFA.
Section J Biological Studies
Recombinant HCV NS3/4A protease complex FRET peptide assay
The purpose of this in vitro assay was to measure the inhibition of HCV NS3 protease complexes, derived from the BMS strain, H77 strain or J4L6S strain, as described below, by compounds ofthe present invention. This assay provides an indication of how effective compounds ofthe present invention would be in inhibiting HCV NS3 proteolytic activity. - o -
Serum from an HCV-infected patient was obtained from Dr. T. Wright, San Francisco Hospital. An engineered full-length cDNA (compliment deoxyribonucleic acid) template ofthe HCV genome (BMS strain) was constructed from DNA fragments obtained by reverse transcription-PCR (RT-PCR) of serum RNA (ribonucleic acid) and using primers selected on the basis of homology between other genotype la strains. From the determination ofthe entire genome sequence, a genotype la was assigned to the HCV isolate according to the classification of Simmonds et al. (See P Sirnmonds, KA Rose, S Graham, SW Chan, F McOmish, BC Dow, EA Follett, PL Yap and H Marsden, J. Clin. Microbiol., 31 (6), 1493-1503 (1993)). The amino acid sequence ofthe nonstractural region, NS2-5B, was shown to be >97% identical to HCV genotype la (H77) and 87% identical to genotype lb (J4L6S). The infectious clones, H77 (la genotype) and J4L6S (lb genotype) were obtained from R. Purcell (NIH) and the sequences are published in Genbank (AAB67036, see Yanagi,M., Purcell,R.H., EmersomS.U. and Bukh,J. Proc. Natl. Acad. Sci. U.S.A. 94(16),8738-8743 (1997); AF054247, see Yanagi,M., St Claire,M., Shapiro,M., Emerson,S.U., PurcelLRH. and Bukh,J, Virology 244 (1), 161-172. (1998)).
The H77 and J4L6S strains were used for production of recombinant NS3/4A protease complexes. DNA encoding the recombinant HCV NS3/4A protease complex (amino acids 1027 to 1711) for these strains were manipulated as described by P. Gallinari et al. (see Gallinari P, Paolini C, Brennan D, Nardi C, Steinkuhler C, De Francesco R. Biochemistry. 38(17):5620-32, (1999)). Briefly, a three-lysine solubilizing tail was added at the 3 '-end ofthe NS4A coding region. The cysteine in the PI position ofthe NS4A-NS4B cleavage site (amino acid 1711) was changed to a glycine to avoid the proteolytic cleavage ofthe lysine tag. Furthermore, a cysteine to serine mutation was introduced by PCR at amino acid position 1454 to prevent the autolytic cleavage in the NS3 helicase domain. The variant DNA fragment was cloned in the pET21b bacterial expression vector (Novagen) and the NS3/4A complex was expressed in Escherichia. coli strain BL21 (DE3) (Invitrogen) following the protocol described by P. Gallinari et al. (see Gallinari P, Brennan D, Nardi C, Brunetti M, Tomei L, Steinkuhler C, De Francesco R., J Virol. 72(8):6758- 69 (1998)) with modifications. Briefly, the NS3/4A protease complex expression was induced with 0.5 millimolar (mM) Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 22 hours (h) at 20°C. A typical fermentation (1 Liter (L)) yielded approximately 10 grams (g) of wet cell paste. The cells were resuspended in lysis buffer (10 mL/g) consisting of 25 mM N-(2-Hydroxyethyl)Piperazine-N'-(2-Ethane Sulfonic acid) (HEPES), pH 7.5, 20% glycerol, 500 mM Sodium Chloride (NaCl), 0.5%) Triton X-100, 1 microgram/milliliter ("μg/mL") lysozyme, 5 M Magnesium Chloride (MgCl2), 1 μg/ml Dnasel, 5mM β-Mercaptoethanol (βME), Protease inhibitor - Ethylenediamine Tetraacetic acid (EDTA) free (Roche), homogenized and incubated for 20 minutes (min) at 4°C. The homogenate was sonicated and clarified by ultra-centrifugation at 235000. g for 1 h at 4°C. hnidazole was added to the supernatant to a final concentration of 15 mM and the pH adjusted to 8.0. The crude protein extract was loaded on a Nickel - Nitrilotriacetic acid (Ni-NTA) column pre- equilibrated with buffer B (25 mM HEPES, pH 8.0, 20% glycerol, 500 mM NaCl, 0.5% Triton X-100, 15 mM imidazole, 5 mM βME). The sample was loaded at a flow rate of 1 mL/min. The column was washed with 15 column volumes of buffer C (same as buffer B except with 0.2%> Triton X-100). The protein was eluted with 5 column volumes of buffer D (same as buffer C except with 200 mM Imidazole).
NS3/4A protease complex-containing fractions were pooled and loaded on a desalting column Superdex-S200 pre-equilibrated with buffer D (25mM HEPES, pH 7.5, 20% glycerol, 300 mM NaCl, 0.2% Triton X-100, 10 mM βME). Sample was loaded at a flow rate of 1 mL/min. NS3/4A protease complex-containing fractions were pooled and concentrated to approximately 0.5 mg/ml. The purity ofthe NS3/4A protease complexes, derived from the BMS, H77 and J4L6S strains, were judged to be greater than 90% by SDS-PAGE and mass spectrometry analyses.
The enzyme was stored at -80°C, thawed on ice and diluted prior to use in assay buffer. The substrate used for the NS3/4A protease assay was RET SI (Resonance Energy Transfer Depsipeptide Substrate; AnaSpec, Inc. cat # 22991)(FRET peptide), described by Taliani et al. in Anal. Biochem. 240(2):60-67
(1996). The sequence of this peptide is loosely based on the NS4A/NS4B natural cleavage site for the HCV NS3 protease except there is an ester linkage rather than an amide bond at the cleavage site. The peptide substrate was incubated with one ofthe three recombinant NS3/4A protease complexes, in the absence or presence of a compound ofthe present invention, and the formation of fluorescent reaction product was followed in real time using a Cytofluor Series 4000.
The reagents were as follow: HEPES and Glycerol (Ultrapure) were obtained from GIBCO-BRL. Dimethyl Sulfoxide (DMSO) was obtained from Sigma, β- Mercaptoethanol was obtained from Bio Rad.
Assay buffer: 50 mM HEPES, pH 7.5; 0.15 M NaCl; 0.1 % Triton; -15% Glycerol; 10 mM βME. Substrate: 2 μM final concentration (from a 2 mM stock solution in DMSO stored at -20°C). HCV NS3/4A protease type la (lb), 2-3 nM final concentration (from a 5 μM stock solution in 25 mM HEPES, pH 7.5, 20% glycerol, 300 mM NaCl, 0.2% Triton-XlOO, 10 mM βME). For compounds with potencies approaching the assay limit, the assay was made more sensitive by adding 50 μg/ml Bovine Serum Albumin (Sigma) to the assay buffer and reducing the end protease concentration to 300 pM.
The assay was performed in a 96-well polystyrene black plate from Falcon. Each well contained 25 μl NS3/4A protease complex in assay buffer, 50 μl of a compound ofthe present invention in 10% DMSO/assay buffer and 25 μl substrate in assay buffer. A control (no compound) was also prepared on the same assay plate. The enzyme complex was mixed with compound or control solution for 1 min before initiating the enzymatic reaction by the addition of substrate. The assay plate was read immediately using the Cytofluor Series 4000 (Perspective Biosystems). The instrument was set to read an emission of 340 nm and excitation of 490 mn at 25°C. Reactions were generally followed for approximately 15 min.
The percent inhibition was calculated with the following equation:
100 - [(δFinh/δFCOn)xl00] where δF is the change in fluorescence over the linear range ofthe curve. A nonlinear curve fit was applied to the inhibition-concentration data, and the 50% effective concentration (IC50) was calculated by the use of Excel Xl-fit software using the equation, y=A+((B-A)/(l+((C/x)ΛD))).
All ofthe compounds tested were found to inhibit the activity ofthe NS3/4A protease complex with IC50's of 1.2 μM or less. Further, compounds ofthe present invention, which were tested against more than one type of NS 3/4 A complex, were found to have similar inhibitory properties though the compounds uniformly demonstrated greater potency against the lb strains as compared to the la strains.
Specificity Assays The specificity assays were performed to demonstrate the in vitro selectivity ofthe compounds ofthe present invention in inhibiting HCV NS3/4A protease complex as compared to other serine or cysteine proteases.
The specificities of compounds ofthe present invention were determined against a variety of serine proteases: human neutrophil elastase (HNE), porcine pancreatic elastase (PPE) and human pancreatic chymotrypsin and one cysteine protease: human liver cathepsin B. In all cases a 96-well plate format protocol using colorhnetric p-nitroaniline (pNA) substrate specific for each enzyme was used as described previously (PCT Patent Application No. WO 00/09543) with some modifications to the serine protease assays. All enzymes were purchased from Sigma while the substrates were from Bachem.
Each assay included a 2 h enzyme-inhibitor pre-incubation at room temperature followed by addition of substrate and hydrolysis to ~30% conversion as measured on a Spectramax Pro microplate reader. Compound concentrations varied from 100 to 0.4 μM depending on their potency.
The final conditions for each assay were as follows: 50 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) pH 8, 0.5 M Sodium Sulfate (Na2SO4), 50 mM NaCl, 0.1 mM EDTA, 3% DMSO, 0.01% Tween-20 with:
133 μM succ-AAA-pNA and 20 nM HNE or 8 nM PPE; 100 μM succ-AAPF- pNA and 250 pM Chymotrypsin.
100 mM NaHPO4 (Sodium Hydrogen Phosphate) pH 6, 0.1 mM EDTA, 3% DMSO, 1 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride), 0.01% Tween-20, 30 μM Z-FR-pNA and 5 nM Cathepsin B (enzyme stock activated in buffer containing 20 mM TCEP before use).
The percentage of inhibition was calculated using the formula:
[l-((Winh-UVblank)/(UVotl-UVbiank))] x 100
A non-linear curve fit was applied to the inhibition-concentration data, and the 50% effective concentration (IC 0) was calculated by the use of Excel Xl-fit software.
Generation of HCV Replicon
An HCV replicon whole cell system was established as described by Lohmann V, Komer F, Koch J, Herian U, Theihnarm L, Bartenschlager R., Science 285(5424): 110-3 (1999). This system enabled us to evaluate the effects of our HCV
Protease compounds on HCV RNA replication. Briefly, using the HCV strain lb sequence described in the Lohmann paper (Assession number: AJ238799), an HCV cDNA was generated encoding the 5' internal ribosome entry site (IRES), the neomycin resistance gene, the EMCV (encephalomyocarditis viurs)-IRES and the HCV nonstractural proteins, NS3-NS5B, and 3' non-translated region (NTR). In vitro transcripts ofthe cDNA were transfected into the human hepatoma cell line, Huh7. Selection for cells constitutively expressing the HCV replicon was achieved in the presence ofthe selectable marker, neomycin (G418). Resulting cell lines were characterized for positive and negative strand RNA production and protein production over tune.
FRET Assay
Huh7 cells, constitutively expressing the HCV replicon, were grown in Dulbecco's Modified Eagle Media (DMEM) containing 10% Fetal calf serum (FCS) and 1 mg/ml G418 (Gibco-BRL). Cells were seeded the night before (1.5 x 104 cells/well) in 96-well tissue-culture sterile plates. Compound and no compound controls were prepared in DMEM containing 4% FCS, 1:100 Penicillin / Streptomysin, 1:100 L-glutamine and 5% DMSO in the dilution plate (0.5% DMSO final concentration in the assay). Compound / DMSO mixes were added to the cells and incubated for 4 days at 37°C. After 4 days, plates were rinsed thoroughly with Phosphate-Buffered Saline (PBS) (3 times 150 μl). The cells were lysed with 25 μl of a lysis assay reagent containing the FRET peptide (RET SI, as described for the in vitro enzyme assay). The lysis assay reagent was made from 5X cell Lucif erase cell culture lysis reagent (Promega #E153A) diluted to IX with distilled water, NaCl added to 150 mM final, the FRET peptide diluted to 10 μM final from a 2 mM stock in 100% DMSO. The plate was then placed into the Cytofluor 4000 instrument which had been set to 340 nm excitation / 490 nm emission, automatic mode for 21 cycles and the plate read in a kinetic mode. EC50 determinations were canied out as described for the IC50 determinations.
Luciferase Assay
As a secondary assay, EC50 determinations from the replicon FRET assay were confirmed in a luciferase reporter assay. Utilization of a replicon luciferase reporter assay was first described by Krieger et al (Krieger N, Lohmann V, and Bartenschlager R, J. Virol. 75(10):4614-4624 (2001)). The replicon construct described for our FRET assay was modified by replacing the resistance gene neomycin with the Blasticidin-resistance gene fused to the N-tenninus ofthe humanized form of Renilla luciferase (restriction sites Ascl / Pmel used for the subcloning). The adaptive mutation at position 1179 (serine to isoleucine) was also introduced (Blight KJ, Kolykhalov, AA, Rice, CM, Science 290(5498): 1972- 1974). The luciferase reporter assay was set up by seeding huh7 cells the night before at a density of 2 x 10^ cells per T75 flask. Cells were washed the next day with 7.5 ml Opti-MEM. Following the Invitrogen protocol, 40 μl DMRIE-C was vortexed with 5 ml Opti-MEM before adding 5 μg HCV reporter replicon RNA. The mix was added to the washed huh7 cells and left for 4 h at 37°C. In the mean time, serial compound dilutions and no compound controls were prepared in DMEM containing 10%> FCS and 5% DMSO in the dilution plate (0.5%. DMSO final concentration in the assay). Compound / DMSO mixes were added to each well of a 24-well plate. After 4 h, the transfection mix was aspirated, and cells washed with 5 ml of Opti-MEM before trypsinization. Trypsinized cells were resuspended in 10% DMEM and seeded at 2 x 10 cells/well in the 24-well plates containing compound or no compound controls. Plates were incubated for 4 days. After 4 days, media was removed and cells washed with PBS. 100 μl lx Renilla Luciferase Lysis Buffer (Promega) was immediately added to each well and the plates either frozen at -80°C for later analysis, or assayed after 15 min of lysis. Lysate (40 μl) from each well was transfened to a 96-well black plate (clear bottom) followed by 200 μl lx Renilla Luciferase assay substrate. Plates were read immediately on a Packard TopCount NXT using a luminescence program.
The percentage inhibition was calculated using the formula below:
% control = average luciferase signal in experimental wells (+ compound) average luciferase signal in DMSO control wells (- compound)
The values were graphed and analyzed using XLFit to obtain the EC50 value. Compounds in accordance with the present invention were tested for biological activity as described above and found to have activities in the ranges as follow:
IC50 Activity Ranges (NS3/4A BMS Strain): A is 10 - 100 micromolar (μM); B is 1 - 10 μM; C is 0.1 - 1 μM; D is O.lμM
EC50 Activity Range (for compounds tested): A is 10 - 100 μM; B is 1 - 10 μM; C is 0.1 - l μM; D is O.lμM
Note that by using the Patent example number and the Patent compound number shown in Table 2, the structures of compounds can be found herein.
In accordance with the present invention, preferably the compounds have a biological activity (EC50) of 10 μM or less, more preferably 1 μM or less and most preferably 100 nM or less.
Table 2 Biological activity
Figure imgf000171_0001
Ex 103, Cpd D C
103
Ex 104, Cpd D C
104
Ex 105, Cpd D D
105
Ex 106, Cpd D D
106
Ex 107, Cpd D D
107
Ex 108, Cpd D D
108
Ex 109, Cpd D D
109
Ex 110, Cpd D D
110
Ex 111, Cpd D D
111
Ex 112, Cpd D C,
112 Ex 1 13, Cpd D C 113
Ex 217, D D Cmpd 217
Ex 218, D D Cmpd 218
Ex 219, D D Cmpd 219
Ex 220, D C Cmpd 220
Ex 201 , B Cmpd 201
Ex 200, B Cmpd 200
Ex 202, B Cmpd 202
Ex 203, B Cmpd 203
Figure imgf000174_0001
Ex. 300. D D Cmpd 3O0
Ex. 301 . D D Cmpd 301
Ex. 302 D D Cmpd 302
Ex. 303 C C Cmpd 303
Ex. 304 D D Cmpd 304
Ex 400, D C Cmpd 400
Ex 401, C C Cmpd 401 Ex 50O, D C Cmpd 5O0
Ex 501 , D C Cmpd 501
Ex 502, C C Cmpd 502
Ex 600, D D Cmpd 600
Ex 601 , D C Cmpd 601
Ex 602, C B Cmpd 602
Ex 603, B Cmpd 603
Ex 604, D C Cmpd 604
Ex 605, C B Cmpd 605 Ex 700, D D Cmpd 700
Ex 701 , D C Cmpd 701
Ex 800, D C Cmpd 800
Those skilled in the art will recognize that although the invention has been described above with respect to specific aspects, other aspects are intended to be within the scope ofthe claims which follow. All documents referenced herein are hereby incoφorated by reference as if set out in full.

Claims

A compound having the formula
Figure imgf000178_0001
wherein:
Figure imgf000178_0002
O O o
Z I •S c c — -*- C — o — -\- c - or -^- C — NR6R7. p is 1, 2 or 3; q is O or 1 ;
Rt is C3-7 cycloalkyl, C4-7 cycloalkenyl; C6-ι0 aryl; C7-14 alkylaryl; C6- 10 aryloxy; C7-ι4 alkylaryloxy; C8-15 alkylarylester; Het; or C1-8 alkyl optionally substituted with C1-6 alkoxy, hydroxy, halo, C2-10 alkenyl, C2_ιo alkynyl, C3_7 cycloalkyl, C4-7 cycloalkenyl, C6-10 aryl, C7-14 alkylaryl, C6-10 aryloxy, C7-14 alkylaryloxy, C8-1 alkylarylester or Het; or Ri is trialkylsilane or halogen, provided q is 0;
(b) m is 1 or 2; (c) n is 1 or 2; (d) R2 is H, Cι-6 alkyl, C2-6 alkenyl or C3-7 cycloalkyl, each optionally substituted with halogen;
(e) R3 is Cι-8 alkyl optionally substituted with halo, cyano, amino, Cι-6 dialkylamino, C6-ιo aryl, C7_14 alkylaryl, C1-6 alkoxy, carboxy, hydroxy, aryloxy, C7-14 alkylaryloxy, C -6 alkylester or C8-ι5 alkylarylester; C3-ι2 alkenyl; C3-7 cycloalkyl or C4-ιo alkylcycloalkyl, wherein the cycloalkyl or alkylcycloalkyl are optionally substituted with hydroxy, C1-6 alkyl, C -6 alkenyl or C1-6 alkoxy; or R3 together with the carbon atom to which it is attached forms a C3-7 cycloalkyl group optionally substituted with C2-6 alkenyl;
(f) Y is H, phenyl substituted with nitro, pyridyl substituted with nitro, or C1-6 alkyl optionally substituted with cyano, OH or C3_7 cycloalkyl; provided that if i or R5 is H then Yis H;
(g) B is H, C1-6 alkyl, R4-(C=O)-, ^CA))-, R4-N(R5)-C(=O)-, R4-N(R5)-C(=S)-, R4SO2-, or R4-N(R5)-SO2-;
(h) R4 is (i) C1-10 alkyl optionally substituted with phenyl, carboxyl, Cι-6 alkanoyl, 1-3 halogen, hydroxy, -OC(O)C1-6 alkyl, Cι-6 alkoxy, amino optionally substituted with Cι-6 alkyl, amido, or (lower alkyl) amido; (ii) C3.-7 cycloalkyl, C3- cycloalkoxy, or C4-10 alkylcycloalklyl, each optionally substituted with hydroxy, carboxyl, (C1-6 alkoxy)carbonyl, amino optionally substituted with Cι- alkyl, amido, or (lower alkyl) amido; (iii) C6-ιo aryl or C7-16 arylalkyl, each optionally substituted with C1-6 alkyl, halogen, nitro, hydroxy, amido, (lower alkyl) amido, or amino optionally substituted with C1-6 alkyl; (iv) Het; (v) bicyclo(l .1. l)pentane; or (vi) -C(O)OCι-6 alkyl, C2-6alkenyl or C -6 alkynyl; (i) R5 is H; Cι-6 alkyl optionally substituted with 1-3 halogens; or Cι_6 alkoxy provided R. is C O alkyl; (j) X is O, S, SO, SO2, OCH2, CH2O or NH; (k) R1 is Het, C6-ιo aryl or C7-14 alkylaryl, each optionally substituted with
Ra; and (1) Ra is C1-6 alkyl, C3- cycloalkyl, C1-6 alkoxy, C3-7 cycloalkoxy, halo-Ci- β alkyl, CF3, mono-or di- halo-C1-6 alkoxy, cyano, halo, thioalkyl, hydroxy, alkanoyl, NO2, SH, , amino, Cι-6 alkylamino, di (Ci-6) alkylamino, di (Cι-6) alkylamide, carboxyl, (Cι-6) carboxyester, C1-6 alkylsulfone, C1-6 alkylsulfonamide, di (Ci-6) alkyl(alkoxy)amine, C6- 10 aryl, C7-14 alkylaryl, or a 5-7 membered monocyclic heterocycle; and
(m) R6 and R7 are each independently H; or Cι-6 alkyl, C2-ι0 alkenyl or C6- 10 aryl, each of which may be optionally substituted with halo, cyano, nitro, C1-6 alkoxy, amido, amino or phenyl;
or a pharmaceutically acceptable enantiomer, diastereomer, salt, solvate or prodrug thereof.
The compound of Claim 1 wherein
Figure imgf000180_0001
z is -^- C — 5 -^- C — O — or — C — NR6R7. p is 1, 2 or 3; q is 0 or 1 ;
Ri is C3-7 cycloalkyl, C4-7 cycloalkenyl; C -14 alkylaryl; C7-ι4 alkylaryloxy; C8-ι5 alkylarylester; or Cι-8 alkyl optionally substituted with Cι-6 alkoxy, hydroxy, halo, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C4-7 cycloalkenyl, C6-ιo aryl, C6-10 aryloxy, C8-15 alkylarylester or Het; or Ri is trialkylsilane or halogen, provided q is
O; and R6 and R7 are each independently H; or Cι-6 alkyl, C2-ιo alkenyl or C6- ιo aryl, each of which may be optionally substituted with halo, cyano, nitro, C1-6 alkoxy, amido, amino or phenyl.
3. The compound of Claim 1 wherin R is C1-6 alkyl, C2-6 alkenyl or C3-7 cycloalkyl.
4. The compoxmd of Claim 3 wherein R2 is C2-6 alkenyl.
5. The compound of Claim 1 wherein R3 is C1-8 alkyl optionally substituted with
Cβaryl, C1-6 alkoxy, carboxy, hydroxy, aryloxy, C7-j alkylaryloxy, C2-6 alkylester or Cg-15 alkylarylester; C -ι2 alkenyl; C3-7 cycloalkyl; or C4-ι0 alkylcycloalkyl.
6. The compound of Claim 5 wherein R3 is Cι-8 alkyl optionally substituted with Cι_6 alkoxy; or C3-7 cycloalkyl.
7. The compound of Claim 1 wherein Y is H.
8. The compound of Claim 1 wherein B is H, C1-6 alkyl, R4-(C=O)-, R4O(C=O)-, R4-N(R5)-C(=O)-, R4-N(R5)-C(=S)-, R4SO2-, or R4-N(R5)-SO2-.
9. The compound of Claim 8 wherein B is R4-(C=O)-, RtO(C=O)-, or R4-ΪN(R5)-C(=O)-.
10. The compound of Claim 9 wherein B is R4O(C=O)- and Rt is C1-6 alkyl.
11. The compound of Claim 1 wherein R is (i) C1-10 alkyl optionally substituted with phenyl, carboxyl, C1-6 alkanoyl, 1-3 halogen, hydroxy, Cι-6 alkoxy; (ii) C3- cycloalkyl, C3-7 cycloalkoxy, or C4-ι0 alkylcycloalklyl; or (iii) C6-10 aryl or C7-16 arylalkyl, each optionally substituted with C1-6 alkyl or halogen.
12. The compound of Claim 11 wherein R4 is (i) C1-10 alkyl optionally substituted with 1-3 halogen or C1-6 alkoxy; or (ii) C3-7 cycloalkyl or C -ι0 alkylcycloalkyl.
13. The compound of Claim 1 wherein R5 is H or Cι-6 alkyl optionally substituted with 1-3 halogens.
14. The compoxmd of Claim 13 wherein R5 is H.
15. The compound of Claim 1 wherein X is O or NH.
16. The compound of Claim 16 wherein R' is Het; or C6-10 aryl optionally substituted with Ra.
17. The compound of Claim 16 wherein R' is Het.
18. The compound of Claim 17 wherein the heterocycle contains 1 or 2 nitrogen atoms and optionally a sulfur atom or an oxygen atom in the ring.
19. The compound of Claim 18 wherein the heterocycle is substituted with at least one of C1-6 alkyl, C1-6 alkoxy, halo, C6-10 aryl, C7-ι4 alkylaryl, or a 5-7 membered monocyclic heterocycle.
20. The compound of Claim 1 wherein Ra is Cι-6 alkyl, C3-7 cycloalkyl, C1-6 alkoxy, halo-Cι-6 alkyl, halo, amino, C aryl, or a 5-7 membered monocyclic heterocycle.
21. A compound having the formula
Figure imgf000182_0001
wherein:
Figure imgf000183_0001
O O n
II II ° z is A- C — ^ -^ C — O — or — C — R6R7. p is 1, 2 or 3; q is 0 or 1 ;and
Ri is C3- cycloalkyl, C -7 cycloalkenyl; C -ι alkylaryl; C -ι4 alkylaryloxy; C8-ι5 alkylarylester; or Cι-8 alkyl optionally substituted with Ci-6 alkoxy, hydroxy, halo, C2-ιo alkenyl, C2-ιo alkynyl, C3- cycloalkyl, C -7 cycloalkenyl, C6-ι0 aryl, C6-ιo aryloxy, C8-ι5 alkylarylester or Het; or Ri is trialkylsilane or halogen, provided q is
0;
(b) R2 is Cι-6 alkyl, C2-6 alkenyl or C3- cycloalkyl;
(c) R3 is Cι-8 alkyl optionally substituted with C6aryl, C1-6 alkoxy, carboxy, hydroxy, aryloxy, C -ι alkylaryloxy, C2-6 alkylester, C8-i5 alkylarylester; C3-1 alkenyl, C3-7 cycloalkyl, or C4-10 alkylcycloalkyl;
(d) Y is H;
(e) B is H, C1-6 alkyl, R4-(C=O)-, R4θ(C=O)-, R4-N(R5)-C(=O)-, R4-N(R5)-C(=S)-, R4SO2-, or R4-N(R5)-SO2-;
(f) R4 is (i) Cϊ-io alkyl optionally substituted with phenyl, carboxyl, Cι-6 alkanoyl, 1-3 halogen, hydroxy, Cι-6 alkoxy; (ii) C3-7 cycloalkyl, C3-7 cycloalkoxy, or C4-ιo alkylcycloalklyl; or (iii) C6-ιo aryl or C7-16 arylalkyl, each optionally substituted with Cι_6 alkyl or halogen;
(g) R5 is H or Cι-6 alkyl optionally substituted with 1-3 halogens; (h) X is O or NH; (i) R' is Het; or C6.10 aryl optionally substituted with Ra;
(j) Ra is Cι-6 alkyl, C3-7 cycloalkyl, Cι-6 alkoxy, halo-Cι-6 alkyl, halo, amino, C6 aryl, or a 5-7 membered monocyclic heterocycle; and (k) R6 and R7 are each independently H; or Cι-6 alkyl, C2-10 alkenyl or C6- 10 aryl, each of which may be optionally substituted with halo, cyano, nitro, Cj-6 alkoxy, amido, amino or phenyl;
or a pharmaceutically acceptable enantiomer, diastereomer salt, solvate or prodrag thereof.
22. The compound of Claim 21 wherein R' is a bicyclic heterocycle.
23. The compound of Claim 22 wherein the heterocycle contains 1 or 2 nitrogen atoms and optionally a sulfur atom or an oxygen atom in the ring.
24. The compound of Claim 22 wherein the heterocycle is substituted with at least one of C1-6 alkyl, C1-6 alkoxy, halo, C6 aryl, and a 5-7 membered monocyclic heterocycle.
25. The compound of Claim 21 wherein R' is a bicyclic heterocycle containing 1 nitrogen atom and substituted with methoxy and at least one of a C6 aryl and a 5-7 membered monocyclic heterocycle.
26. The compound of Claim 21 wherein R' is a monocyclic heterocycle.
27. The compound of Claim 26 wherein the heterocycle contains 1 or 2 nitrogen atoms and optionally a sulfur atom or an oxygen atom in the ring.
28. The compoxmd of Claim 26 wherein the heterocycle is substituted with at least one of Cι-6 alkyl, C1-6 alkoxy, halo, C6-ιo aryl, C -ι4 alkylaryl, or a 5-7 membered monocyclic heterocycle.
29. The compound of Claim 21 wherein R' is a monoyclic heterocycle containing 1 or 2 nitrogen atoms and substituted with methoxy and at least one of a C6 aryl and a 5-7 membered monocyclic heterocycle.
30. A compoxmd having the formula
Figure imgf000185_0001
(III)
wherein:
Figure imgf000185_0002
CH
(a) A is 2 / P
p is 1, 2 or 3;
Ri is C7-ι4 alkylaryl; C1-8 alkyl optionally substituted with Cι-6 alkoxy,
C2-ιo alkenyl or C4-ιo alkylcycloalkyl; or Ri is xrialkylsilane or halogen;
(b) R2 is C2-6 alkenyl;
(c) R3 is Cι-8 alkyl;
(e) B is R4θ(C=O)-, or R4-N(H)-C(=O)-;
(f) Ri is Cno alkyl;
(g) R' is a bicyclic heterocycle optionally substituted with Ra; and (h) Ra is C1-6 alkyl, C1-6 alkoxy, halo, C6 aryl, or a 5-7 membered monocyclic heterocycle;
or a pharmaceutically acceptable enantiomer, diastereomer salt, solvate or prodrug thereof.
31. The compound of Claim 30 wherein Ri is cyclopropyl or cyclobutyl.
32. The compound of Claim 30 wherein R2 is vinyl.
33. The compound of Claim 30 wherein R is t-butyl.
34. The compound of Claim 30 wherein t is t-butyl.
35. The compound of Claim 30 wherein R' is quinoline or isoquinoline optionally substituted with Ra.
36. The compound of Claim 30 wherein Ri is cyclopropyl, R is vinyl, R3 is t- butyl, t is t-butyl, and R' is isoquinoline substituted with Ra.
37. The compound of Claim 36 wherein Ra is Cι-6 alkoxy.
38. The compound of Claim 37 wherein Ra further includes at least one of C6 aryl or a 5-7 membered monocyclic heterocycle.
50. A composition comprising the compound of Claim 1 and a pharmaceutically acceptable canier.
51. The composition according to Claim 39 further comprising a compound having anti-HCV activity.
52. The composition according to Claim 40 wherein the compound having anti-
HCV activity is an interferon.
53. The composition according to Claim 41 wherein the interferon is selected from the group consisting of interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, and lymphoblastiod interferon tau.
54. The composition according to Claim 40 wherein the compound having anti- HCV activity is selected from the group consisting of interleukin 2, interleukin 6, mterleukin 12, a compound that enhances the development of a type 1 helper T cell response, interfering RNA, anti-sense RNA, Imiqimod, ribavirin, an inosine 5'- monophospate dehydrogenase inhibitor, amantadine, and rimantadine.
55. The composition according to the Claim 39 further comprising an interferon and ribavirin.
56. The composition according to Claim 40 wherein the compound having anti- HCV activity is a small molecule compound.
57. The composition according to Claim 40 wherein the compound having anti- HCV activity is effective to inhibit the function of a target selected from the group consisting of HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH and a nucleoside analog for the treatment of an HCV infection.
58. A method of inhibiting the function ofthe HCV serine protease comprising contacting the HCV serine protease with the compound of Claim 1.
59. A method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount ofthe compound of Claim 1, or a pharmaceutically acceptable enantiomer, diastereomer, solvate, prodrag or salt thereof.
60. The method according to Claim 48 wherein the compound is effective to inhibit the function ofthe HCV NS5B protein.
61. The method according to Claim 48 further comprising administering another compoxmd having anti-HCV activity prior to, after or simultaneously with the compound of Claim 1.
62. The method according to Claim 50 wherein the other compound having anti- HCV activity is an interferon.
63. The method according to Claim 51 wherein the interferon is selected from the group consisting of interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, lymphoblastiod interferon tau.
64. The method according to Claim 50 wherein the other compound having anti- HCV activity is selected from the group consisting of interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, interfering RNA, anti-sense RNA, Imiqimod, ribavirin, an inosine 5'- monophospate dehydrogenase inhibitor, amantadine, and rimantadine.
65. The method according to Claim 53 wherein the compound having anti-HCV activity is a small molecule.
66. The method according to Claim 54 wherein the compound having anti-HCV activity is effective to hihibit the function of a target selected from the group consisting of HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH and a nucleoside analog for the treatment of an HCV infection.
67. The method according to Claim 50 wherein the other compound having anti- HCV activity is effective to inhibit the function of target in the HCV life cycle other than the HCV NS5B protein.
68. Use ofthe compound of Claim 1 for the manufacture of a medicament for treating HCV infection in a patient.
69. Use ofthe composition of Claim 39 for the manufacture of a medicament for treating HCV infection in a patient.
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JP2007535491A (en) 2007-12-06
WO2005051410A9 (en) 2005-07-28
US7135462B2 (en) 2006-11-14
JP4688815B2 (en) 2011-05-25
EP1684787B1 (en) 2014-04-16
EP1684787A1 (en) 2006-08-02
WO2005051410B1 (en) 2005-09-09
IS8476A (en) 2006-05-18
NO20062339L (en) 2006-08-10

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