WO2012039717A1 - Hepatitis c virus inhibitors - Google Patents

Hepatitis c virus inhibitors Download PDF

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Publication number
WO2012039717A1
WO2012039717A1 PCT/US2010/050138 US2010050138W WO2012039717A1 WO 2012039717 A1 WO2012039717 A1 WO 2012039717A1 US 2010050138 W US2010050138 W US 2010050138W WO 2012039717 A1 WO2012039717 A1 WO 2012039717A1
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mmol
solvent
nmr
mhz
calcd
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PCT/US2010/050138
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French (fr)
Inventor
Makonen Belema
Piyasena Hewawasam
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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 PCT/US2010/050138 priority Critical patent/WO2012039717A1/en
Priority to JP2013530121A priority patent/JP5619289B2/en
Priority to EA201370078A priority patent/EA201370078A1/en
Priority to EP10761101.4A priority patent/EP2619195A1/en
Priority to CA2812699A priority patent/CA2812699A1/en
Priority to CN2010800703399A priority patent/CN103249730A/en
Priority to MX2013002927A priority patent/MX2013002927A/en
Priority to BR112013008148A priority patent/BR112013008148A2/en
Publication of WO2012039717A1 publication Critical patent/WO2012039717A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero 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
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings

Definitions

  • the present disclosure is generally directed to antiviral compounds, and specifically directed to compounds which can inhibit the function of the NS5 A protein encoded by Hepatitis C virus (HCV), compositions comprising such compounds, and methods for inhibiting the function of the NS5A protein.
  • HCV Hepatitis C virus
  • HCV BACKGROUND OF THE DISCLOSURE HCV is a major human pathogen, infecting an estimated 170 million persons worldwide - roughly five times the number infected by human immunodeficiency virus type 1. A substantial fraction of these HCV infected individuals develop serious progressive liver disease, including cirrhosis and hepatocellular carcinoma.
  • HCV is a positive-stranded RNA virus. Based on a comparison of the deduced amino acid sequence and the extensive similarity in the 5' untranslated region, HCV has been classified as a separate genus in the Flaviviridae family. All members of the Flaviviridae family have enveloped virions that contain a positive stranded RNA genome encoding all known virus-specific proteins via translation of a single, uninterrupted, open reading frame.
  • this polyprotein In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the structural and non- structural (NS) proteins.
  • NS structural and non- structural
  • 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 be a metalloprotease and cleaves at the NS2- NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 (also referred to herein as NS3 protease) 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 by both acting as a cofactor for the NS3 protease and assisting in the membrane localization of NS3 and other viral replicase components.
  • NS3-NS4A complex The formation of a NS3-NS4A complex is necessary for proper protease activity resulting in increased proteolytic efficiency of the cleavage events.
  • the NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities.
  • NS5B (also referred to herein as HCV polymerase) is a RNA-dependent RNA polymerase that is involved in the replication of HCV with other HCV proteins, including NS5A, in a replicase complex.
  • HCV NS5A protein is described, for example, in the following references: S. L. Tan, et al., Virology, 284: 1-12 (2001); K.-J. Park, et al, J. Biol. Chem., 30711-30718 (2003); T. L. Tellinghuisen, et al, Nature, 435, 374 (2005); R. A. Love, et al, J. Virol, 83, 4395 (2009); N. Appel, et al, J. Biol. Chem., 281, 9833 (2006); L. Huang, J. Biol. Chem., 280, 36417 (2005); C. Rice, et al, WO2006093867.
  • X is hydrogen (H) or halogen and Z is hydrogen;
  • X and Z together with the carbon atoms to which they are attached, form a five- to eight-membered aromatic or non-aromatic fused ring optionally containing one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur; wherein the five- to eight-membered ring is optionally substituted with one, two, or three substitutents independently selected from alkoxy, alkoxyalkyl, alkoxycarbonyl, alkyl, alkylsulfonyl, aryl, arylalkyl, arylsulfonyl, carboxy, formyl, halo, haloalkoxy, haloalkyl, hydroxy, hydroxyalkyl, -NR a R b , ( R a R b )alkyl, ( R a R b )carbonyl, oxo, and spirocycle;
  • X' is hydrogen (H) or halogen and Z' is hydrogen;
  • X' and Z' together with the carbon atoms to which they are attached, form a five- to eight-membered aromatic or non-aromatic fused ring optionally containing one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur; wherein the five- to eight-membered ring is optionally substituted with one, two, or three substitutents independently selected from alkoxy, alkoxyalkyl, alkoxycarbonyl, alkyl, alkylsulfonyl, aryl, arylalkyl, arylsulfonyl, carboxy, formyl, halo, haloalkoxy, haloalkyl, hydroxy, hydroxyalkyl, -NR a R b , ( R a R b )alkyl, ( R a R b )carbonyl, oxo, and spirocycle;
  • Y and Y' are each independently -CH 2 -, -CH2CH2-, or -CH2O-, wherein the
  • R p is hydrogen or Ci to C 4 alkyl
  • R q is hydrogen, alkyl, or halo
  • R p and R q together with the carbon atoms to which they are attached, form a cycloalkyl ring
  • R v is selected from hydrogen, alkyl, halo, and hydroxy
  • R v and R q together with the carbon atom to which they are attached, form an ethylenyl group or a cycloalkyl ring;
  • R p ' is hydrogen or Ci to C 4 alkyl
  • R q ' is hydrogen, alkyl, or halo
  • R p ' and R q ' together with the carbon atoms to which they are attached, form a cycloalkyl ring
  • R v are independently selected from hydrogen, alkyl, halo, and hydroxy; or R v and R q , together with the carbon atom to which they are attached, form an ethylenyl group or a cycloalkyl ring;
  • R w and R w are independently selected from hydrogen and alkyl
  • R 1 is hydrogen or -C(0)R x ;
  • R 2 is hydrogen or -C(0)R y ;
  • R x and R y are independently selected from cycloalkyl, heteroaryl, heterocyclyl, alkoxy, and alkyl, said alkyl being substituted by one or more substituents independently selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, -OR 3 , -C(0)OR 4 , -NR a R b , and -C(0)NR c R d ,
  • any said aryl and heteroaryl may optionally be substituted with one or more substituents independently selected from alkenyl, alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, -C(0)OR 4 , -OR 5 , -NR a R b , ( R a R b )alkyl, and (MeO)(HO)P(0)0-, and
  • any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more substituents independently selected from alkyl, hydroxyl, halogen, aryl, -NR a R b , oxo, and -C(0)OR 4 ;
  • R 3 is hydrogen, alkyl, or arylalkyl
  • R 4 is alkyl or arylalkyl
  • R 5 is hydrogen, alkyl, or arylalkyl
  • R a and R b are independently selected from hydrogen, alkyl, cycloalkyl, arylalkyl, heteroaryl, -C(0)R 6 , -C(0)OR 7 , -C(0)NR c R d , and (NR c R d )alkyl, or alternatively, R a and R b , together with the nitrogen atom to which they are attached, form a five- or six-membered ring or bridged bicyclic ring structure, wherein said five- or six-membered ring or bridged bicyclic ring structure optionally may contain one or two additional heteroatoms independently selected from nitrogen, oxygen, and sulfur and may contain one, two, or three substituents independently selected from Ci to Ce alkyl, Ci to C 4 haloalkyl, aryl, hydroxyl, Ci to Ce alkoxy, Ci to C 4 haloalkoxy, and halogen;
  • R 6 is alkyl
  • R 7 is alkyl, arylalkyl, cycloalkyl, or haloalkyl
  • R c and R d are independently selected from hydrogen, alkyl, arylalkyl, and cycloalkyl.
  • X is hydrogen or chloro (CI) and Z is hydrogen;
  • X' is hydrogen or chloro (CI) and Z' is hydrogen; or
  • Y is -CH 2 -, -CH2CH2-, or -CH2O-, wherein the -CH 2 0- is drawn such that the oxygen atom is bound to the carbon atom substituted with R v and R q ;
  • R p is hydrogen or Ci to C 4 alkyl
  • R q is hydrogen, alkyl, or halo
  • R v is selected from hydrogen, alkyl, halo, and hydroxy
  • R v and R q together with the carbon atom to which they are attached, form an ethylenyl group or a cycloalkyl ring.
  • the present disclosure provides a compound of Formula (la), or a pharmaceutically acceptable salt thereof, wherein:
  • R 1 is -C(0)R x ;
  • R 2 is -C(0)R y ;
  • R x and R y are independently alkyl substituted by at least one -NR a R b , characterized by Formula (A):
  • n 0 or 1 ;
  • R 8 is hydrogen or alkyl
  • R 9 is selected from hydrogen, cycloalkyl, aryl, heteroaryl, heterocyclyl, and alkyl optionally substituted with a substituent selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, heterobicyclyl, -OR 3 , -C(0)OR 4 , -NR a R b , and -C(0)NR c R d , wherein any said aryl and heteroaryl may optionally be substituted with one or more substituents independently selected from alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, -C(0)OR 4 , -OR 5 , -NR a R b , ( R a R b )alkyl, and (MeO)(HO)P(0)0-, and
  • any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more substituents independently selected from alkyl, hydroxyl, halogen, aryl, -NR a R b , oxo, and -C(0)OR 4 ; and
  • R 3 , R 4 , R 5 , R a , R b , R c , and R d are defined as in Formula (I).
  • the present disclosure provides a compound of Formula (la) or a pharmaceutically acceptable salt thereof, wherein m is 0;
  • R 8 is hydrogen or Ci to C 4 alkyl
  • R 9 is selected from hydrogen, Ci to Ce alkyl optionally substituted with - OR 12 , C 3 to C 6 cycloalkyl, allyl, -CH 2 C(0)NR c R d , ( R c R d )alkyl,
  • j is 0 or 1 ;
  • k is 1, 2, or 3;
  • n is 0 or an integer selected from 1 through 4;
  • each R 10 is independently hydrogen, Ci to C 4 alkyl, Ci to C 4 haloalkyl, halogen, nitro, -OBn, or (MeO)(OH)P(0)0-;
  • R 11 is hydrogen, Ci to C 4 alkyl, or benzyl
  • R 12 is hydrogen, Ci to C 4 alkyl, or benzyl
  • R a is hydrogen or Ci to C 4 alkyl
  • R b is Ci to C 4 alkyl, C3 to Ce cycloalkyl, benzyl, 3-pyridyl, pyrimidin-5-yl, acetyl, -C(0)OR 7 , or -C(0)NR c R d ;
  • R 7 is Ci to C 4 alkyl or Ci to C 4 haloalkyl
  • R c is hydrogen or Ci to C 4 alkyl
  • R d is hydrogen, Ci to C 4 alkyl, or C3 to Ce cycloalkyl.
  • the present disclosure provides a compound of Formula (la) or a pharmaceutically acceptable salt thereof, wherein m is 0; R 8 is hydrogen;
  • R 9 is phenyl optionally substituted with one up to five substituents independently selected from Ci to Ce alkyl, Ci to C 4 haloalkyl, halogen, Ci to alkoxy, hydroxyl, cyano, and nitro; and
  • NR a R b is a heterocyclyl or heterobicyclyl group selected from:
  • n 0, 1
  • each R 13 is independently selected from Ci to Ce alkyl, phenyl,
  • R 14 is Ci to C 6 alkyl, phenyl, benzyl, or -C(0)OR 15 group, wherein R 15 is Ci to C 4 alkyl, phenyl, or benzyl.
  • the present disclosure provides a compound of Formula (la) or a pharmaceutically acceptable salt thereof, wherein m is 1 ;
  • R 8 is hydrogen
  • R 9 is Ci to e alkyl, arylalkyl, or heteroarylalkyl
  • R a is hydrogen
  • R b is -C(0)OR 7 , wherein R 7 is Ci to C 6 alkyl.
  • the present disclosure provides a compound of Formula (la) or a pharmaceutically acceptable salt thereof, wherein R 1 is -C(0)R x ;
  • R 2 is -C(0)R y ;
  • R x and R y are heteroaryl or heterocyclyl independently selected from:
  • n is 0 or an integer selected from 1 through 4;
  • each R 13 is independently selected from hydrogen, Ci to Ce alkyl, Ci to C 4 haloalkyl, phenyl, benzyl, Ci to Ce alkoxy, Ci to C 4 haloalkoxy, heterocyclyl, halogen, NR c R d , hydroxyl, cyano, and oxo, where R c and R d are independently hydrogen or Ci to C 4 alkyl; and
  • R 14 is hydrogen (H), Ci to C 6 alkyl, benzyl, or -C(0)OR 4 , wherein R 4 is Ci to C 6 alkyl.
  • the present disclosure provides a compound of Formula (la) or a pharmaceutically acceptable salt thereof, wherein R 1 is -C(0)R x ;
  • R 2 is -C(0)R y ;
  • R x and R y are cycloalkyl independently selected from: ( Rl3 )n _ NR a R b
  • j 0, 1, 2, or 3;
  • k 0, 1 , or 2;
  • n is 0 or an integer selected from 1 through 4;
  • each R 13 is independently selected from hydrogen, Ci to Ce alkyl, Ci to C 4 haloalkyl, Ci to Ce alkoxy, halogen, hydroxyl, cyano, and nitro;
  • R a and R b are each independently hydrogen, Ci to Ce alkyl, or -C(0)OR , wherein R 7 is Ci to Ce alkyl.
  • the present disclosure provides a compound of Formula (la) or a pharmaceutically acceptable salt thereof, wherein R 1 is -C(0)R x ;
  • R 2 is -C(0)R y ;
  • R x and R y are independently arylalkyl, wherein aryl part of said arylalkyl may optionally be substituted with (NR a R b )alkyl;
  • R a and R b are independently hydrogen, Ci to Ce alkyl, or benzyl, or alternatively, R a and R b , together with the nitrogen atom to which they are attached, form a five- or six-membered ring selected from
  • R 15 is hydrogen, Ci to C(, alkyl, or benzyl.
  • the present disclosure provides a compound of Formula (la) or a pharmaceutically acceptable salt thereof, wherein 1 and R 2 are the same and are selected from the group consisting of:
  • a squiggle bond ( ⁇ > ⁇ ) in the structure indicates that a stereogenic center to which the bond is attached can take either (R)- or (S)- configuration so long as chemical bonding principles are not violated.
  • the present disclosure provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein R 1 is -C(0)R x ;
  • R 2 is -C(0)R y ;
  • R x and R y are both t-butoxy.
  • the present disclosure provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein
  • R 1 and R 2 are both hydrogen.
  • X and X' are independently hydrogen (H) or halogen
  • R p is hydrogen or Ci to C 4 alkyl
  • R q is hydrogen, or alternatively, R p and R q , together with the carbon atoms to which they are attached, form a cyclopropyl ring
  • R p ' is hydrogen or Ci to C 4 alkyl
  • R q ' is hydrogen, or alternatively, R p ' and R q ', together with the carbon atoms to which they are attached, form a cyclopropyl ring
  • R 1 is hydrogen or -C(0)R x ;
  • R 2 is hydrogen or -C(0)R y ;
  • R x and R y are independently selected from cycloalkyl, heteroaryl, heterocyclyl, alkoxy, and alkyl, said alkyl being substituted by one or more substituents independently selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, -OR 3 , -C(0)OR 4 , -NR a R b , and -C(0)NR c R d ,
  • any said aryl and heteroaryl may optionally be substituted with one or more substituents independently selected from alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, -C(0)OR 4 , -OR 5 , -NR a R b , ( R a R b )alkyl, and (MeO)(HO)P(0)0-, and
  • any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more substituents independently selected from alkyl, hydroxyl, halogen, aryl, -NR a R b , oxo, and
  • R 3 is hydrogen, alkyl, or arylalkyl
  • R 4 is alkyl or arylalkyl
  • R 5 is hydrogen, alkyl, or arylalkyl
  • R a and R b are independently selected from hydrogen, alkyl, cycloalkyl, arylalkyl, heteroaryl, -C(0)R 6 , -C(0)OR 7 , -C(0)NR c R d , and (NR c R d )alkyl, or alternatively, R a and R b , together with the nitrogen atom to which they are attached, form a five- or six-membered ring or bridged bicyclic ring structure, wherein said five- or six-membered ring or bridged bicyclic ring structure optionally may contain one or two additional heteroatoms independently selected from nitrogen, oxygen, and sulfur and may contain one, two, or three substituents independently selected from Ci to Ce alkyl, Ci to C 4 haloalkyl, aryl, hydroxyl, Ci to Ce alkoxy, Ci to C 4 haloalkoxy, and halogen;
  • R 6 is alkyl
  • R 7 is alkyl, arylalkyl, or haloalkyl
  • R c and R d are independently selected from hydrogen, alkyl, arylalkyl, and cycloalkyl.
  • the present disclosure provides a composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, wherein Formula (I) is defined according to any of the embodiments described above in the first aspect of the present disclosure.
  • composition further comprises at least one additional compound having anti-HCV activity.
  • At least one of the additional compounds is an interferon or a ribavirin.
  • the interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, and lymphoblastiod interferon tau.
  • the present disclosure provides a composition
  • a composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and at least one additional compound having anti-HCV activity, wherein at least one of the additional compounds is selected from 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.
  • the present disclosure provides a composition
  • a composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and at least one additional compound having anti-HCV activity, wherein at least one of the additional compounds is effective to inhibit the function of a target selected from HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, and IMPDH for the treatment of an HCV infection.
  • a target selected from HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, and IMPDH for the treatment of an HCV infection.
  • the present disclosure provides a method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein Formula (I) is defined according to any of the embodiments described above in the first aspect of the present disclosure.
  • At least one of the additional compounds is an interferon or a ribavirin.
  • the interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, and lymphoblastiod interferon tau.
  • the present disclosure provides a method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one additional compound having anti-HCV activity prior to, after or simultaneously with the compound of
  • the present disclosure provides a method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one additional compound having anti-HCV activity prior to, after or simultaneously with the compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein at least one of the additional compounds is effective to inhibit the function of a target selected from HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, and IMPDH for the treatment of an HCV infection.
  • a target selected from HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, and IMPDH for the treatment of an HCV infection.
  • compositions comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; and (2) a method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • the "pyrrolidinyl-imidazole” moiety on the left side of the “linker” is independent from the “pyrrolidinyl-imidazole” moiety on the right side of the linker group in respect to, e.g., (1) tautomer form of imidazole ring, (2) absolute configuration of the stereogenic centers on the pyrrolidine ring, and (3) substituents on the pyrrolidine nitrogen, i.e., R 1 and R 2 are independent from each other, although in some circumstances they are preferably the same.
  • the stereogenic carbon center to which the imidazole group is attached can take either (R)- or (S)- configuration as depicted below:
  • this disclosure is intended to cover all possible stereoisomers even when a single stereoisomer, or no stereochemistry, is described in a structure.
  • the linkage between a benzene ring of the linker group and an imidazole ring of a pyrrolidinyl-imidazole moiety can take place in either the C-4 or the C-5 position (see below) of the imidazole ring.
  • a bonding of a benzene ring to the C-4 position may be equivalent to a bonding of the benzene ring to the C-5 osition as shown in the followin e uation:
  • this disclosure is intended to cover all possible tautomers even when a structure depicts only one of them.
  • a floating bond e.g., ⁇ ⁇
  • a floating substituent e.g.,
  • any substituent or variable at a particular location in a molecule be independent of its definitions elsewhere in that molecule.
  • substituent ( 10 ) n when n is 2, each of the two R 10 groups may be the same or different.
  • aryl, cycloalkyl, heteroaryl, and heterocyclyl groups of the present disclosure may be substituted as described in each of their respective definitions.
  • aryl part of an arylalkyl group may be substituted as described in the definition of the term "aryl.”
  • acetyl refers to -C(0)CH 3 .
  • alkenyl refers to a monovalent, straight or branched hydrocarbon chain having one or more, preferably one to two, double bonds therein.
  • the double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group.
  • Suitable alkenyl groups include, but are not limited to, C 2 to C 10 alkenyl groups, such as vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl, 4-(2- methyl-3-butene)-pentenyl.
  • An alkenyl group can be unsubstituted or substituted with one or two suitable substituents.
  • alkoxy refers to an alkyl group attached to the parent molecular moiety through an oxygen atom.
  • Representative examples of alkoxy group include, but are not limited to, methoxy (CH 3 O-), ethoxy (CH 3 CH 2 0-), and t-butoxy ((CH 3 ) 3 CO-).
  • alkoxy alkyl refers to an alkyl group substituted with one, two, or three alkoxy groups.
  • alkoxycarbonyl refers to an alkoxy group attached to the parent molecular moiety through a carbonyl group.
  • alkyl refers to a group derived from a straight or branched chain saturated hydrocarbon by removal of a hydrogen from one of the saturated carbons.
  • the alkyl group preferably contains from one to ten carbon atoms.
  • alkyl group examples include, but are not limited to, methyl, ethyl, isopropyl, and tert-butyl.
  • alkylcarbonyl refers to an alkyl group attached to the parent molecular moiety through a carbonyl group.
  • Representative examples of alkylcarbonyl group include, but are not limited to, acetyl (-C(0)CH 3 ), propanoyl (-C(0)CH 2 CH 3 ), n-butyryl (-C(0)CH 2 CH 2 CH 3 ), and 2,2-dimethylpropanoyl or pivaloyl (-C(0)C(CH 3 ) 3 ).
  • alkylsulfonyl refers to an alkyl group attached to the parent molecular moiety through a sulfonyl group.
  • aryl refers to a group derived from an aromatic carbocycle by removal of a hydrogen atom from an aromatic ring.
  • the aryl group can be monocyclic, bicyclic or polycyclic, wherein in bicyclic or polycyclic aryl group, the aromatic carbocycle can be fused onto another four- to six-membered aromatic or non-aromatic carbocycle.
  • Representative examples of aryl groups include, but are not limited to, phenyl, indanyl, indenyl, naphthyl, and 1,2,3,4- tetrahydronaphth-5 -yl.
  • arylalkyl refers to an alkyl group substituted with one, two, or three aryl groups, wherein aryl part of the arylalkyl group may optionally be substituted by one to five substituents independently selected from Ci to Ce alkyl, Ci to C4 haloalkyl, Ci to Ce alkoxy, halogen, cyano, and nitro groups.
  • substituents independently selected from Ci to Ce alkyl, Ci to C4 haloalkyl, Ci to Ce alkoxy, halogen, cyano, and nitro groups.
  • Represented examples of arylalkyl include, but are not limited to, benzyl, 2 -phenyl- 1 -ethyl (PhCH 2 CH 2 -), (naphth- l-yl)methyl, and (naphth-2-yl)methyl.
  • arylsulfonyl refers to an aryl group attached to the parent molecular moiety through a sulfonyl group.
  • benzyl refers to a methyl group on which one of the hydrogen atoms is replaced by a phenyl group, wherein said phenyl group may optionally be substituted by one to five substituents independently selected from methyl, trifluoromethyl (-CF 3 ), methoxy (-OCH 3 ), halogen, and nitro (-N0 2 ).
  • benzyl group include, but are not limited to, PhCH 2 -, 4-MeO-C 6 H 4 CH 2 -, and 2,4,6-tri-methyl-C 6 H 4 CH 2 -.
  • bridged bicyclic ring refers to a ring structure comprising a bridgehead between two of the ring members, wherein the ring and the bridgehead optionally may independently comprise one or more, preferably one to two, heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • bridged bicyclic ring structure examples include, but are not limited to:
  • Cap refers to the group which is placed on the nitrogen atom of the pyrrolidine ring in the compounds of formula (I). It should be understood that “Cap” or “cap” can also refer to the reagent which is a precursor to the final “cap” in compounds of formula (I) and is used as one of the starting materials in the reaction to append a group on the pyrrolidine nitrogen that results in the final product, a compound which contains the functionalized pyrrolidine that will be present in the compound of formula (I).
  • carbonyl refers to -C(O)-.
  • cyano refers to -CN.
  • cycloalkyl refers to a group derived from a saturated carbocycle, having preferably three to eight carbon atoms, by removal of a hydrogen atom from the saturated carbocycle, wherein the saturated carbocycle can optionally be fused onto one or two other aromatic or nonaromatic carbocycles.
  • Representative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, and 1,2,3,4-tetrahydronaphth-l-yl.
  • halo and halogen, as used herein, refer to F, CI, Br, or I.
  • haloalkoxy refers to a haloalkyl group attached to the parent molecular moiety through an oxygen atom.
  • haloalkyl refers to an alkyl group substituted by at least one halogen atom.
  • the haloalkyl group can be an alkyl group of which all hydrogen atoms are substituted by halogens.
  • Representative examples of haloalkyl include, but are not limited to, trifluoromethyl (CF 3 -), 1-chloroethyl (C1CH 2 CH 2 -), and 2,2,2-trifluoroethyl (CF 3 CH 2 -).
  • heteroaryl refers to group derived from a monocyclic, bicyclic, or polycyclic compound comprising at least one aromatic ring comprising one or more, preferably one to three, heteroatoms independently selected from nitrogen, oxygen, and sulfur, by removal of a hydrogen atom from an aromatic ring thereof.
  • heteroaryl rings have less aromatic character than their all-carbon counterparts.
  • a heteroaryl group need only have some degree of aromatic character.
  • heteroaryl groups include, but are not limited to, pyridyl, pyridazinyl, pyrimidyl, pyrazyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3,)- and (l,2,4)-triazolyl, pyrazinyl, pyrimidinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, isoxazolyl, oxazolyl, indolyl, quinolinyl, isoquinolinyl, benzisoxazolyl, benzothiazolyl, benzothienyl, and pyrrolopyridinyl.
  • heteroarylalkyl refers to an alkyl group substituted with one, two, or three heteroaryl groups.
  • heterocyclyl refers to a ring structure comprising two fused or bridged rings that include carbon and one or more, preferably one to three, heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • the heterobicyclic ring structure is a subset of heterocyclic ring and can be saturated or unsaturated. Examples of heterobicyclic ring structures include, but are not limited to, tropane, quinuclidine, and 7-azabicyclo[2.2.1]heptane.
  • heterocyclyl refers to a group derived from a monocyclic, bicyclic, or polycyclic compound comprising at least one nonaromatic ring comprising one or more, preferably one to three, heteroatoms independently selected from nitrogen, oxygen, and sulfur, by removal of a hydrogen atom from the nonaromatic ring.
  • the heterocyclyl group encompasses the heterobicyclyl group.
  • the heterocyclyl groups of the present disclosure can be attached to the parent molecular moiety through a carbon atom or a nitrogen atom in the group.
  • heterocyclyl groups include, but are not limited to, morpholinyl, oxazolidinyl, piperazinyl, piperidinyl, pyrrolidinyl, tetrahydrofuryl, thiomorpholinyl, and indolinyl.
  • heterocyclylalkyl refers to an alkyl group substituted with one, two, or three heterocyclyl groups.
  • hydroxy or "hydroxyl,” as used herein, refer to -OH.
  • hydroxyalkyl refers to an alkyl group substituted with one, two, or three hydroxy groups.
  • nitro refers to -N0 2 .
  • -NR a R b refers to two groups, R a and R b , which are attached to the parent molecular moiety through a nitrogen atom, or alternatively R a and R b , together with the nitrogen atom to which they are attached, form a 5- or 6- membered ring or a fused- or bridged-bicyclic ring structure optionally containing one, two, or three additional heteroatom independently selected from nitrogen, oxygen, and sulfur.
  • -NR c R d is defined similarly.
  • (NR a R b )alkyl refers to an alkyl group substituted with one, two, or three -NR a R b groups.
  • ( R c R d )alkyl is defined similarly.
  • sulfonyl refers to -SO 2 -.
  • trialkylsilyl refers to -S1R 3 , wherein each R is Ci to C 4 alkyl or phenyl.
  • the three R groups may be the same or different.
  • TMS trimethylsilyl
  • TDPS tert-butyldiphenylsilyl
  • TBDMS tert-butyldimethylsilyl
  • TIPS triisopropylsilyl
  • Certain compounds of the present disclosure 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 disclosure includes each conformational isomer of these compounds and mixtures thereof.
  • isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include deuterium and tritium.
  • isotopes of carbon include 13 C and 14 C.
  • Isotopically-labeled compounds of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein, using an appropriate isotopically- labeled reagent in place of the non-labeled reagent otherwise employed. Such compounds may have a variety of potential uses, for example as standards and reagents in determining biological activity. In the case of stable isotopes, such compounds may have the potential to favorably modify biological, pharmacological, or pharmacokinetic properties.
  • the compounds of the present disclosure can exist as pharmaceutically acceptable salts.
  • pharmaceutically acceptable salt represents salts or zwitterionic forms of the compounds of the present disclosure which are water or oil-soluble or dispersible, 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 benefit/risk ratio, and are effective for their intended use.
  • the salts can be prepared during the final isolation and purification of the compounds or separately by reacting a suitable nitrogen atom with a suitable acid.
  • Representative acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate; digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2 -hydroxy ethanesulfonate, lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate, persulfate, 3- phenylproprionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, gluta
  • Basic addition salts can be prepared during the final isolation and purification of the compounds by reacting a carboxy group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation or with ammonia or an organic primary, secondary, or tertiary amine.
  • a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation or with ammonia or an organic primary, secondary, or tertiary amine.
  • suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation or with ammonia or an organic primary, secondary, or tertiary amine.
  • the cations of pharmaceutically acceptable salts include lithium, sodium, potassium, calcium, magnesium, and aluminum, as well as nontoxic quaternary amine cations such as ammonium,
  • tetramethylammonium tetraethylammonium
  • methylamine dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, ⁇ , ⁇ -dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, ⁇ , ⁇ -dibenzylphenethylamine, and ⁇ , ⁇ '- dibenzylethylenediamine.
  • Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine,
  • compositions which include therapeutically effective amounts of compounds of Formula (I) or pharmaceutically acceptable salts thereof, and one or more, preferably one to three, pharmaceutically acceptable carriers, diluents, or excipients.
  • therapeutically effective amount refers to 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 term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously.
  • the compounds of Formula (I) and pharmaceutically acceptable salts thereof are as described above.
  • the carrier(s), diluent(s), or excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • a process for the preparation of a pharmaceutical formulation including admixing a compound of Formula (I), or a pharmaceutically acceptable salt thereof, with one or more, preferably one to three, pharmaceutically acceptable carriers, diluents, or excipients.
  • pharmaceutically acceptable refers 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 benefit/risk ratio, and are effective for their intended use.
  • compositions may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. Dosage levels of between about 0.01 and about 250 milligram per kilogram (“mg/kg”) body weight per day, preferably between about 0.05 and about 100 mg/kg body weight per day of the compounds of the present disclosure are typical in a monotherapy for the prevention and treatment of HCV mediated disease. Typically, the pharmaceutical compositions of this disclosure 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.
  • mg/kg milligram per kilogram
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending on the condition being treated, the severity of the condition, the time of administration, the route of administration, the rate of excretion of the compound employed, the duration of treatment, and the age, gender, weight, and condition of the patient.
  • Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.
  • treatment is initiated with small dosages substantially less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached.
  • 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.
  • compositions of this disclosure comprise a combination of a compound of the present disclosure 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 150%, and more preferably between about 10 and 80% of the dosage normally administered in a monotherapy regimen.
  • compositions may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intracutaneous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, intravenous, or intradermal injections or infusions) route.
  • Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). Oral administration or administration by injection are preferred.
  • compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in- water liquid emulsions or water-in-oil emulsions.
  • the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
  • an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
  • Powders are prepared by comminuting the compound to a suitable fine size and mixing with a similarly comminuted pharmaceutical carrier such as an edible carbohydrate, as, for example, starch or mannitol. Flavoring, preservative, dispersing, and coloring agent can also be present.
  • Capsules are made by preparing a powder mixture, as described above, and filling formed gelatin sheaths.
  • Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol can be added to the powder mixture before the filling operation.
  • a disintegrating or solubilizing agent such as agar-agar, calcium carbonate, or sodium carbonate can also be added to improve the availability of the medicament when the capsule is ingested.
  • suitable binders include starch, gelatin, natural sugars such as glucose or beta- lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, and the like.
  • Lubricants used in these dosage forms include sodium oleate, sodium chloride, and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, betonite, xanthan gum, and the like. Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant, and pressing into tablets.
  • a powder mixture is prepared by mixing the compound, suitable comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, an aliginate, gelating, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt and/or and absorption agent such as betonite, kaolin, or dicalcium phosphate.
  • the powder mixture can be granulated by wetting with a binder such as syrup, starch paste, acadia mucilage, or solutions of cellulosic or polymeric materials and forcing through a screen.
  • the powder mixture can be run through the tablet machine and the result is imperfectly formed slugs broken into granules.
  • the granules can be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc, or mineral oil.
  • the lubricated mixture is then compressed into tablets.
  • the compounds of the present disclosure can also be combined with a free flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps.
  • a clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material, and a polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different unit dosages.
  • Oral fluids such as solution, syrups, and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of the compound.
  • Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic vehicle.
  • Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and
  • polyoxyethylene sorbitol ethers preservatives, flavor additive such as peppermint oil or natural sweeteners, or saccharin or other artificial sweeteners, and the like can also be added.
  • dosage unit formulations for oral administration can be microencapsulated.
  • the formulation can also be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax, or the like.
  • the compounds of Formula (I), and pharmaceutically acceptable salts thereof can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.
  • Liposomes can be formed from a variety of phopholipids, such as cholesterol, stearylamine, or phophatidylcholines.
  • the compounds of Formula (I) and pharmaceutically acceptable salts thereof may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.
  • the compounds may also be coupled with soluble polymers as targetable drug carriers.
  • Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysine substituted with palitoyl residues.
  • the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels.
  • a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels.
  • compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharm. Res., 3(6):318 (1986).
  • compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils.
  • the formulations are preferably applied as a topical ointment or cream.
  • the active ingredient may be employed with either a paraffinic or a water-miscible ointment base.
  • the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in oil base.
  • Pharmaceutical formulations adapted for topical administrations to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.
  • compositions adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes.
  • Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or as enemas.
  • compositions adapted for nasal administration wherein the carrier is a solid include a course powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or nasal drops, include aqueous or oil solutions of the active ingredient.
  • Fine particle dusts or mists which may be generated by means of various types of metered, dose pressurized aerosols, nebulizers, or insufflators.
  • compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations.
  • compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, and soutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
  • patient includes both human and other mammals.
  • treating refers to: (i) preventing a disease, disorder or condition from occurring in a patient that may be predisposed to the disease, disorder, and/or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder, or condition, i.e., arresting its development; and (iii) relieving the disease, disorder, or condition, i.e., causing regression of the disease, disorder, and/or condition.
  • the compounds of the present disclosure can also be administered with a cyclosporin, for example, cyclosporin A.
  • Cyclosporin A has been shown to be active against HCV in clinical trials (Hepatology, 38: 1282 (2003); Biochem. Biophys. Res. Commun., 313 :42 (2004); J. Gastroenterol, 38:567 (2003)).
  • Table 1 lists some illustrative examples of compounds that can be administered with the compounds of this disclosure.
  • the compounds of the disclosure can be administered with other anti-HCV activity compounds in combination therapy, either jointly or separately, or by combining the compounds into a composition.
  • inhibitor Brand Name Physiological Class Type of Inhibitor or Source Company
  • the compounds of the present disclosure 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 structural biology studies to further enhance knowledge of the HCV disease mechanisms. Further, the compounds of the present disclosure are useful in establishing or determining the binding site of other antiviral compounds, for example, by competitive inhibition.
  • the compounds of this disclosure 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.
  • This disclosure is intended to encompass compounds having Formula (I) when prepared by synthetic processes or by metabolic processes including those occurring in the human or animal body (in vivo) or processes occurring in vitro.
  • TFA trifluoroacetic acid
  • MeCN or ACN for acetonitrile
  • LDA lithium diisopropylamide
  • DMSO dimethylsulfoxide
  • h or hr or hrs for hours
  • Boc or BOC for tert- butoxycarbonyl
  • HATU for 0-(7-azabenzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate
  • RT or Rt or rt for retention time or room temperature (context will dictate); Me for methyl; DMF for N,N-dimethylformamide; Pd(Ph 3 P) 4 for tetrakistriphenyl phosphine palladium; MeOH for methanol
  • Solvent A 0.1 % TFA in 10% methanol/90% water
  • Solvent B 0.1 % TFA in 90% methanol/10% water
  • Solvent A 0.2 % H 3 P0 4 in 10% methanol/90% water
  • Solvent B 0.2 % H 3 P0 4 in 90% methanol/10% water
  • Solvent A 0.1 % TFA in 10% methanol/90% water
  • Solvent B 0.1 % TFA in 90% methanol/10% water
  • Solvent A 0.2 % H 3 P0 4 in 10% methanol/90% water
  • Solvent B 0.2 % H 3 P0 4 in 90% methanol/10% water Condition 5
  • Solvent A 0.1 % TFA in 10% methanol/90% water
  • Solvent B 0.1 % TFA in 90% methanol/10% water Condition 6
  • Solvent A 0.1 % TFA in 10% methanol/90% water
  • Solvent B 0.1 % TFA in 90% methanol/10% water
  • Solvent A 0.1 % TFA in 10% methanol/90% water
  • Solvent B 0.1 % TFA in 90% methanol/10% water
  • Slovent A 0.1% TFA in 5% CH 3 CN/95%H 2 0
  • Solvent B 0.1% TFA in 95% CH 3 CN/5% H 2 0
  • Slovent A 0.1% TFA in 5% CH 3 CN/95%H 2 0
  • Solvent B 0.1% TFA in 95% CH 3 CN/5% H 2 0 Condition 10
  • Slovent A 0.1% TFA in 5% CH 3 CN/95%H 2 0
  • Slovent A 0.1% TFA in 5% CH 3 CN/95%H 2 0
  • Wavelength 1 220 nm
  • Wavelength 2 254 nm
  • Solvent A 0.1 % TFA in 5% MeCN/95% water
  • Solvent B 0.1 % TFA in 95% MeCN/5% water
  • Wavelength 1 220 nm
  • Wavelength 2 254 nm
  • Solvent A 0.1 % TFA in 5% MeCN/95% water
  • Solvent B 0.1 % TFA in 95% MeCN/5% water
  • Solvent A 0.1 % TFA in 10% methanol/90% water
  • Solvent B 0.1 % TFA in 90% methanol/10% water
  • Solvent A Buffer: CH 3 CN (95:5)
  • Solvent B Buffer: CH 3 CN (5:95)
  • Slovent A Buffer: CH 3 CN (95:5)
  • Solvent B Buffer: CH 3 CN (5:95)
  • Buffer 0.05% TFA in H 2 0 (pH 2.5, adjusted with dilute ammonia)
  • Wavelength 220 & 254 nm Condition 10e.2 (for a homogeneity index assessment on a Agilent 1200 series LC system)
  • Slovent A Buffer: CH 3 CN (95:5)
  • Solvent B Buffer: CH 3 CN (5:95)
  • Buffer 0.05% TFA in H 2 0 (pH 2.5, adjusted with dilute ammonia)
  • Solvent A 10 mM NH 4 HCO 3 in H 2 0 (pH 9.5, adjusted with dilute ammonia)
  • Solvent A Buffer: CH 3 CN (95:5)
  • Solvent B Buffer: CH 3 CN (5:95)
  • Buffer 0.05% TFA in H 2 0 (pH 2.5, adjusted with dilute ammonia)
  • Slovent A ACN (10%) 4 - 20 mM NH 4 OAc in H 2 0 (90%)
  • Solvent B ACN (90%) ⁇ + ⁇ 20 mM NH 4 OAc in H 2 0 (10%)
  • Solvent B CH 3 CN (98%) + 10 mM NH 4 COOH in H 2 0 (2%)
  • Solvent B CH 3 CN (98%) + 10 mM NH 4 COOH in H 2 0 (2%)
  • Solvent A 0.1 % TFA in 10% methanol/90% water
  • Solvent B 0.1 % TFA in 90% methanol/10% water
  • Solvent A 5% methanol/95% water : 10 mM ammonium acetate
  • Solvent B 95% methanol/5% water : 10 mM ammonium acetate
  • Mobile phase B 0.05% TFA in acetonitrile Solvent system: Hold 10%B: 0-lmin; 10-98%B: l-32min; Hold 98%B: 32-35 min; 98-10%B: 35-35.3 min; hold 10%B: 35.3-40 min
  • 4,4'-Oxybis-(benzoic acid) dimethyl ester (1.5 g, 5.24 mmol) was added to a solution of chloroiodomethane (3.05 mL, 41.92 mmol) in tetrahydrofuran (50 mL) and the resulting solution was cooled to -78 °C.
  • a 1.8M solution of LDA (29 mL, 52 mmol) in tetrahydrofuran was added dropwise and the resulting brown suspension was stirred at -78 °C for 15 min.
  • a solution of glacial acetic acid in tetrahydrofuran (10 mL in 50 mL) was then added slowly and the brown mixture was stirred at -78 °C for 10 min.
  • N,N-Diisopropylethylamine (1.13 mL, 6.47 mmol) was added dropwise, over 15 minutes, to a heterogeneous mixture of N-Boc-L-proline (0.8 g, 3.73 mmol),
  • HATU 52 mg, 0.137 mmol was added to a mixture of OL-le (35 mg, 0.06 mmol), diisopropylethylamine (58 ⁇ L, 0.33 mmol) and Cap-1 (22 mg, 0.12 mmol) in dimethylformamide (3 mL), and the resulting mixture was stirred at ambient for 3 h.
  • Examples OL-2 and OL-3 were prepared as TFA salts by substituting the respective acids for Cap-1 using the same method described for Example OL-1.
  • OL-4b was prepared from OL-4a, according to the same procedure used for the preparation of OL-la.
  • OL-4c was prepared from Example OL-4b, according to the same procedure used for the preparation of Example OL- lb.
  • LC Cond. 2): Anal. Calcd. for Ci 6 H 17 2 0 3 (M+H) + 285.12; found: 285.15.
  • Example OL-4d Anal. Calcd. for Ci 6 H 17 2 0 3 (M+H) + 285.12; found: 285.15.
  • Example OL-4d was prepared from Example OL-4c, according to the same procedure used for the preparation of Example OL- lc.
  • Example OL-4e was prepared from Example OL-4d, according to the same procedure used for the preparation of Example OL-ld.
  • LRMS Anal. Calcd. for C 36 H45 6 0 5 (M+H) + 641.3
  • Example OL-4f was prepared from Example OL-4e, according to the same procedure used for the preparation of Example OL-le.
  • X H NMR 500 MHz, DMSO- d 6 ) ⁇ ppm 1.94 - 2.06 (m, 2 H), 2.09 - 2.23 (m, 2 H), 2.28 - 2.38 (m, 1 H), 2.37 - 2.47 (m, 3 H), 3.27 - 3.50 (m, 4 H), 4.90 (br. s, 1 H), 4.99 (br.
  • Example OL-4 was prepared from Example OL-4f according to the same procedure used for the preparation of Example OL-1.
  • Examples OL-5 to OL-6 were prepared as TFA salts by substituting the respective acids for Cap-1 according to the same method described for Example OL-
  • Example OL-7a (Diazomethyl)trimethylsilane (15 mL, 30 mmol, 2 M in hexanes) was added dropwise to a suspension of Example OL-7a (2.04 g, 7.5 mmol) in methanol (30 mL) at 5 °C (ice-water bath). When the addition was complete, the cooling bath was removed and the mixture was stirred at ambient overnight. The solvent was removed under reduced pressure and the remaining residue was taken up in ethyl acetate, washed with water, sodium bicarbonate and brine, dried (MgSC ⁇ ), filtered and concentrated in vacuo.
  • Example OL-7b was purified by flash chromatography (silica gel, 10% ethyl acetate/hexanes) to provide Example OL-7b as white solid (1.5 g).
  • LC (Cond. 1): RT 2.56 min; LRMS: Anal. Calcd. for Ci 7 Hi 6 0 5 (M+H) + 301.11 ; found: 301.22.
  • Example OL-7c was prepared from Example OL-7b, according to the same procedure used for the preparation of Example OL-la.
  • LC (Cond. 1): RT 2.36 min;
  • LRMS Anal. Calcd. for Ci 7 H 15 Cl 2 0 3 (M+H) + 337.04 found: 337.09. Exam le OL-7d
  • Example OL-7d was prepared from Example OL-7c, according to the same procedure used for the preparation of Example OL- lb.
  • LRMS Anal. Calcd. for ⁇ 7 ⁇ 19 ⁇ 1 2 0 3 (M+H) + 299.14 found: 299.19.
  • Example OL-7e was prepared from Example OL-7d, according to the same procedure used for the preparation of Example OL- lc.
  • Example OL-7f was prepared from Example OL-7e, according to the same procedure used for the preparation of Example OL-ld.
  • LC Cond. 1): RT
  • Example OL-7g was prepared from Example OL-7fe, according to the same procedure used for the preparation of Example OL-le.
  • Example OL-7 was prepared from Example OL-7g according to the same procedure used for the preparation of Example OL-1.
  • Example OL-8 was prepared as TFA salt by substituting the respective acid for Cap-1 according to the same method described for Example OL-7.
  • Example OL-9b was prepared from Example OL-9a, according to the same procedure used for the preparation of Example OL-la.
  • Example OL-9c was prepared from Example OL-9b, according to the same procedure used for the preparation of Example OL-lb.
  • LC (Cond. 3): RT 1.90 min; LRMS: Anal. Calcd. for
  • Example OL-9d was prepared from Example OL-9c, according to the same procedure used for the preparation of Example OL- lc.
  • X H NMR 500 MHz, CDC1 3 ) ⁇ ppm 1.38 - 1.56 (m, 18 H), 1.83 - 2.00 (m, 4 H), 2.08 - 2.38 (m, 4 H), 3.01 (s, 4 H), 3.32 - 3.65 (m, 4 H), 4.28 (br. s, 1 H), 4.39 (br.
  • Example OL-9e was prepared from Example OL-9d, according to the same procedure used for the preparation of Example OL-ld.
  • Example OL-9f was prepared from Example OL-9e, according to the same procedure used for the preparation of Example OL-le.
  • Example OL-9 was prepared from Example OL-9f, according to the same procedure used for the preparation of Example OL-1.
  • Examples OL-10 to OL-13 were prepared as TFA salts by substituting the respective acids for Cap-1 according to the same method described for Example OL- 9.
  • Triethyl silane (5.84 mL, 36.55 mmol) was added dropwise to a solution of methyl 4-formylbenzoate (5 g, 30.46 mmol) and bismuth tribromide (0.273 g, 0.61 mmol) in acetonitrile (75 mL) and the opaque solution was stirred at ambient for 15 min. Volatiles were removed under reduced pressure and the remaining residue was taken up in ethyl acetate. The organic layer was then washed with water and brine, dried (MgSC ⁇ ), filtered and concentrated in vacuo.
  • Example OL-14b was prepared from Example OL-14a, according to the same procedure used for the preparation of Example OL-la.
  • LRMS Anal. Calcd. for CisHnC ⁇ Os (M+H) + 351.05, found: 351.06.
  • Example OL-14c was prepared from Example OL-14b, according to the same procedure used for the preparation of Example OL-lb.
  • Example OL-14d was prepared from Example OL-14c, according to the same procedure used for the preparation of Example OL- lc.
  • Example OL-14e was prepared from Example OL-14d, according to the same procedure used for the preparation of Example OL-ld.
  • Example OL-14f was prepared from Example OL-14e, according to the same procedure used for the preparation of Example OL-le.
  • Example OL-14 was prepared from Example OL-14f, according to the same procedure used for the preparation of Example OL- 1.
  • Examples OL-15 to OL-19 were prepared as TFA salts by substituting the respective acids for Cap-1 according to the same method described for Example OL- 14.
  • Example OL-20a was submitted to flash chromatography (silica gel; 20 % ethyl acetate/hexanes) to provide Example OL-20a as a clear oil that solidified upon standing (2.49 g).
  • LC (Cond. 1): RT 2.56 min;
  • Example OL-20b was prepared from Example OL-20a, according to the same procedure used for the preparation of Example OL-la.
  • LC (Cond. 1): RT 2.34 min;
  • LRMS Anal. Calcd. for C18H17CI2O3 (M+H) + 351.05 found: 351.12.
  • Example OL-20c was prepared from Example OL-20b, according to the same procedure used for the preparation of Example OL-lb.
  • LC (Cond. 1): RT 1.32 min;
  • LRMS Anal. Calcd. for Ci 8 H 21 N 2 03 (M+H) + 313.15 found: 313.24. Exam le OL-20d
  • Example OL-20d was prepared from Example OL-20c, according to the same procedure used for the preparation of Example OL- lc.
  • Example OL-20e was prepared from Example OL-20d, according to the same procedure used for the preparation of Example OL-ld.
  • Example OL-20f was prepared from Example OL-20e, according to the same procedure used for the preparation of Example OL-le.
  • Example OL-20 was prepared from Example OL-20f, according to the same procedure used for the preparation of Example OL- 1.
  • Examples OL-21 to OL-25 were prepared as TFA salts by substituting the respective acids for Cap-1 according to the same method described for Example OL- 20.
  • N,N-Diisopropylethylamine (18 mL, 103.3 mmol) was added dropwise, over 15 minutes, to a heterogeneous mixture of N-Boc-L-proline (7.14 g, 33.17 mmol), HATU (13.32 g, 35.04 mmol), the HCl salt of 2-amino-l-(4-bromophenyl)ethanone (8.13 g, 32.44 mmol), and DMF (105 mL), and stirred at ambient condition for 55 minutes. Most of the volatile component was removed in vacuo, and the resulting residue was partitioned between ethyl acetate (300 mL) and water (200 mL).
  • the resulting material was recrystallized from ethyl acetate/hexanes to provide two crops of imidazole D-lb as a light-yellow, dense solid, weighing 5.85 g.
  • the mother liquor was concentrated in vacuo and submitted to a flash chromatography (silica gel; 30% ethyl
  • Ci 8 H 23 Br 3 0 2 392.0974; found 392.0959.
  • Relative retention time 2.83 minutes (R), 5.34 minutes (5).
  • Pd(Ph 3 P) 4 (469 mg, 0.41 mmol) was added to a pressure tube containing a mixture of D-lb (4.01 g, 10.22 mmol), bis(pinacolato)diboron (5.42 g, 21.35 mmol), potassium acetate (2.57 g, 26.21 mmol) and 1,4-dioxane (80 mL).
  • the pressure tube was purged with nitrogen, capped and heated in an oil bath at 80 °C for 16.5 hours.
  • the reaction mixture was filtered and the filtrate was concentrated in vacuo.
  • Example D-lc contaminated with pinacol, as an off- white dense solid; the relative mole ratio of Example D-lc to pinacol was about 10: 1 ( X H NMR).
  • the sample weighed 3.925 g after -2.5 days exposure to high vacuum.
  • 3 ⁇ 4 NMR 500 MHz, DMSO-d 6 ) ⁇ ppm 1.45-1.10 (m, 21H), 2.27-1.77 (m, 4H), 3.36 (m, 1H), 3.52 (app br s, 1H), 4.86-4.70 (m, 1H), 7.79-7.50/ 7.34-7.27 (m, 5H), 12.22/11.94/ 11.87 (m, 1H).
  • LC (Cond. 7): RT 1.64 min;
  • LRMS Anal. Calcd. for [M+H] + C24H 3 5BN 3 O4: 440.27; found 440.23.
  • Tetrakistriphenylphosphine palladium (17.5 mg, 0.015 mmol) was added in one portion to a stirred suspension of D-lc (320 mg, 0.85 mmol), 1,4- diiodobenzene (100 mg, 0.30 mmol) and sodium bicarbonate (180 mg, 2.18 mmol) in
  • D-le was prepared from D-ld, according to the same procedure used for the preparation of OL-le except that methanol (1 mL) was used instead of
  • Example D-1 (TFA salt) was prepared from D-le, according to the same procedure used for the preparation of Example OL- 1. This gave Example D-1 as a tan solid (37.8 mg, 54 % yield).
  • Example D-2 (TFA salt) was prepared from pyrrolidine D-le, according to the same procedure used for the preparation of Example OL- 1. This gave Example D-2 as a light yellow solid (15.4 mg, 38 % yield).
  • X H NMR 500 MHz, DMSO-i3 ⁇ 4) ⁇ ppm 2.50-1.70 (m, 14 H), 2.98-2.62 (m, 6 H), 4.05-2.98 (m, 4 H), 5.79-5.12 (m, 4 H), 8.20-7.10 (m, 24 H), 10.25 (br s, 2 H). Note: The signal of the imidazole NH was too broad to assign a chemical shift.
  • step a
  • D-3bl and D-3b2 were prepared from D-3a and the appropriate Boc -protected prolines according to the same procedure used for the preparation of Example M3, step g.
  • D-3cl and D-3c2 were prepared from D-3bl and D-3b2 respectively according to the same procedure used for the preparation of Example M3, step h.
  • Ethynyltrimethylsilane (0.59 mL, 4.25 mmol) was added to a solution of D-lb (1.5 g, 3.82 mmol), triphenylphosphine (0.20 g, 0.77 mmol), diethylamine (4.25 mL, 40.70 mmol), copper (I) iodide (40 mg, 0.21 mmol) and /raws-dichloro(bis- triphenylphosphine)palladium (II) (149 mg, 0.21 mmol) in dry DMF (1.4 mL) at rt in a microwave vessel. The vessel was capped and irradiated for 25 min at 120 °C.
  • D-3d2 to D-3d4 were prepared according to the procedures described for D-
  • Tetrakis(triphenylphosphine)palladium (83 mg, 0.072 mmol) was added in one portion to a stirred, argon-degassed mixture of Example D-3cl (300 mg, 0.717 mmol), D-3el (315 mg, 0.932 mmol), triethylamine (0.40 mL, 2.87 mmol) and copper(I) iodide (13.7 mg, 0.072 mmol) in anhydrous DMF (6 mL) at rt in a thick- walled, screw-top vial.
  • D-3f2 to D-3f4 were prepared according to the procedure described for D-3fl .
  • Activated manganese dioxide (2.0 g, 23.12 mmol) was added in one portion to a stirred solution of D-3fl (260 mg, 0.39 mmol) in dry dichloromethane (5 mL). The suspension was stirred at rt for 6 h before additional activated manganese dioxide (1.0 g) was added. The suspension was stirred further for 14 h at rt before it suction-filtered through a pad of Celite with MeOH and the pad was washed with additional MeOH several times. The filtrate was then concentrated in vacuo to yield D-3gl (225.1 mg, 82% yield) as a yellow solid. A small amount (approx.
  • D-3hl to D-3h6 were prepared from D-3fl, D-3gl, D-3f2, D-3G, D-3g2 and D-3f4, respectively, according to the same procedure used for the preparation of OL- le except that methanol (1 mL) was used instead of dichloromethane. This gave D- 3hl to D-3h6 as hydrochloride salts (or TFA salts when purified further with preparative HPLC) upon concentration of the solvent(s) in vacuo.
  • Examples D-3 to D-11 were prepared from D-3hl to D-3h6 and the appropriate acids according to the same procedure used for the preparation of Example OL-1. This gave Examples D-3 to D-11 as TFA salts after HPLC purification.
  • HATU (0.069 g, 0.181 mmol) was added to the DMF solution of pyrrolodine Mlb/4HC1 (70.5 mg, 0.103 mmol), (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid (36.3 mg, 0.207 mmol) and DIEA (0.1 mL, 0.573 mmol) and stirred at room temperature for 70 min.
  • the volatile component was removed in vacuo and the residue was dissolved in MeOH and submitted to a reverse phase HPLC purification (MeOH/water/TFA) to afford the TFA salt of Example Ml as an off-white foam (68.9 mg).
  • LC (Cond. 9 and 10): >95% homogeneity index.
  • Example M2 to M2.1 were prepared as TFA salts from pyrrolidine Mlb and appropriate acids according to the procedure described for the preparation of Example Ml with a modified purification protocol noted in the table below.
  • Chloroiodomethane (stabilized over copper; 3.0 mL, 41.2 mmol) was added dropwise over 10 min, and stirred while maintaining the bath temperature at -25 °C for 1 h and between -25 °C and -21 °C for 18.5 h.
  • the reaction mixture was opened to the air and quenched by the slow addition of 50% saturated NaHCC solution (40 mL), and then removed from the cooling bath and stirred at ambient temperature for 20 min. It was filtered through a filter paper and the white cake was washed with 50 mL of toluene.
  • the organic phase of the filtrate was separated and washed with water (40 mL, 2x), dried (MgS0 4 ) and concentrated in vacuo.
  • the crude material was purified using a BIOTAGE® system (350 g silica gel; sample was loaded with 7% EtOAc/hexanes; eluted with 7-20% EtOAc/hexanes) to afford a mixture of methanopyrrolidines M3d-1 and M3d-2 as a colorless viscous oil (3.69 g, 90.7%). [Note: the exact cis/trans-isomer ratio was not determined at this stage].
  • TBAF (7.27 mL of 1.0 M in THF, 7.27 mmol) was added dropwise over 5 min to a THF (30 mL) solution of silyl ether M3d-l/-2 (3.13 g, 6.93 mmol) and the mixture stirred at ambient temperature for 4.75 h.
  • saturated NH 4 CI solution 5 mL
  • most of the volatile material was removed in vacuo and the residue partitioned between CH 2 CI 2 (70 mL) and 50% saturated NH 4 C1 solution (30 mL).
  • the aqueous phase was extracted with CH 2 CI 2 (30 mL), and the combined organic phase was dried (MgS0 4 ), filtered, concentrated in vacuo and then exposed to high vacuum overnight.
  • keto-ester M3g (3.07g, 7.24 mmol), ammonium acetate (5.48 g, 71.1 mmol) and Xylene (70 mL), capped and heated with an oil bath (140 °C) for 4.5 hr.
  • the reaction mixture was allowed to cool to room temperature and the volatile component was removed in vacuo.
  • CH 2 CI 2 100 mL
  • 50% saturated aHC03 solution (30 mL) were added to the residue, vigorously stirred until gas evolution ceased, and the phases were separated.
  • the organic layer was dried (MgSC ⁇ ), concentrated in vacuo, and purified with a BIOTAGE® (240 g silica gel; sample was loaded with (3 ⁇ 4(3 ⁇ 4; 40-80% EtOAc/hexanes) to afford imidazole M3h as a dark yellow foam (2.40 g).
  • BIOTAGE® 240 g silica gel; sample was loaded with (3 ⁇ 4(3 ⁇ 4; 40-80% EtOAc/hexanes) to afford imidazole M3h as a dark yellow foam (2.40 g).
  • Example M3 along with its analogs Examples M4-M7 highlighted in the table below, were prepared as TFA salts from pyrrolidine M3i (.4HC1) by employing the procedure described for the synthesis of Example Ml and appropriate acids.
  • Example M7 an equimolar mixture of (S)-2-(methoxycarbonylamino)-3- methylbutanoic acid and (S)-2-(methoxycarbonylamino)-2-(tetrahydro-2H-pyran-4- yl)acetic acid was employed for the coupling step and the resultant statistical mixture of products was separated by the HPLC technique described for Example Ml .
  • Example M3 LC (Cond. 9 and 10): >95% homogeneity index.
  • LC/MS (Cond. 3): R t 1.89 min.
  • NCS (0.0195 g, 0.143 mmol) was added to a DMF (2 mL) solution of the free base form of Example M3 (obtained from the TFA salt via a standard MCX free- basing protocol; 0.109 g, 0.139 mmol), and stirred at room temperature for 16 hr and at 50°C for 25 hr. Most of the solvent was removed in vacuo, and the residue was dissolved in MeOH and submitted to a reverse phase HPLC purification
  • Example M8 (MeOH/water/TFA) to afford the TFA salt of Example M8 (50 mg) and Example M9 (17.5 mg).
  • Example R t (Cond. 3); % homogeneity index (Cond. 9a and 10a);
  • NCS (0.021 g, 0.158 mmol) was added to a DMF (1.5 mL) solution of the free base form of Example M6 (obtained from the TFA salt via a standard MCX free- basing protocol; 0.1059 g, 0.122 mmol), and stirred at 50°C for 24 hr.
  • the reaction mixture was diluted with MeOH (2.5 mL) and submitted to a reverse phase HPLC purification condition (XTERRA, 30X100 mm, S5; MeOH/water/TFA).
  • Example M9.1 38.8 mg
  • Example M9.2 32.6 mg

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Abstract

This disclosure concerns novel compounds of Formula (I) as defined in the specification and compositions comprising such novel compounds. These compounds are useful antiviral agents, especially in inhibiting the function of the NS5A protein encoded by Hepatitis C virus (HCV). Thus, the disclosure also concerns a method of treating HCV related diseases or conditions by use of these novel compounds or a composition comprising such novel compounds.

Description

HEPATITIS C VIRUS INHIBITORS
FIELD OF THE DISCLOSURE
The present disclosure is generally directed to antiviral compounds, and specifically directed to compounds which can inhibit the function of the NS5 A protein encoded by Hepatitis C virus (HCV), compositions comprising such compounds, and methods for inhibiting the function of the NS5A protein.
BACKGROUND OF THE DISCLOSURE HCV is a major human pathogen, infecting an estimated 170 million persons worldwide - roughly five times the number infected by human immunodeficiency virus type 1. A substantial fraction of these HCV infected individuals develop serious progressive liver disease, including cirrhosis and hepatocellular carcinoma.
The current standard of care for HCV, which employs a combination of pegylated-interferon and ribavirin, has a non-optimal success rate in achieving sustained viral response and causes numerous side effects. Thus, there is a clear and long- felt need to develop effective therapies to address this undermet medical need.
HCV is a positive-stranded RNA virus. Based on a comparison of the deduced amino acid sequence and the extensive similarity in the 5' untranslated region, HCV has been classified as a separate genus in the Flaviviridae family. All members of the Flaviviridae family have enveloped virions that contain a positive stranded RNA genome encoding all known virus-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 due to the high error rate of the encoded RNA dependent RNA polymerase which lacks a proof-reading capability. At least six major genotypes have been characterized, and more than 50 subtypes have been described with distribution worldwide. The clinical significance of the genetic heterogeneity of HCV has demonstrated a propensity for mutations to arise during monotherapy treatment, thus additional treatment options for use are desired. The possible modulator effect of genotypes on pathogenesis and therapy remains elusive. 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 structural 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 be a metalloprotease and cleaves at the NS2- NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 (also referred to herein as NS3 protease) 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 by both acting as a cofactor for the NS3 protease and assisting in the membrane localization of NS3 and other viral replicase components. The formation of a NS3-NS4A complex is necessary for proper protease activity resulting in increased proteolytic efficiency of the cleavage events. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5B (also referred to herein as HCV polymerase) is a RNA-dependent RNA polymerase that is involved in the replication of HCV with other HCV proteins, including NS5A, in a replicase complex.
Compounds useful for treating HCV-infected patients are desired which selectively inhibit HCV viral replication. In particular, compounds which are effective to inhibit the function of the NS5A protein are desired. The HCV NS5A protein is described, for example, in the following references: S. L. Tan, et al., Virology, 284: 1-12 (2001); K.-J. Park, et al, J. Biol. Chem., 30711-30718 (2003); T. L. Tellinghuisen, et al, Nature, 435, 374 (2005); R. A. Love, et al, J. Virol, 83, 4395 (2009); N. Appel, et al, J. Biol. Chem., 281, 9833 (2006); L. Huang, J. Biol. Chem., 280, 36417 (2005); C. Rice, et al, WO2006093867.
The present disclosure provides compounds which selectively inhibit HCV viral replication, as characterized by Formula (I):
Figure imgf000004_0001
(I),
or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000004_0002
X is hydrogen (H) or halogen and Z is hydrogen; or
X and Z, together with the carbon atoms to which they are attached, form a five- to eight-membered aromatic or non-aromatic fused ring optionally containing one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur; wherein the five- to eight-membered ring is optionally substituted with one, two, or three substitutents independently selected from alkoxy, alkoxyalkyl, alkoxycarbonyl, alkyl, alkylsulfonyl, aryl, arylalkyl, arylsulfonyl, carboxy, formyl, halo, haloalkoxy, haloalkyl, hydroxy, hydroxyalkyl, -NRaRb, ( RaRb)alkyl, ( RaRb)carbonyl, oxo, and spirocycle;
X' is hydrogen (H) or halogen and Z' is hydrogen; or
X' and Z', together with the carbon atoms to which they are attached, form a five- to eight-membered aromatic or non-aromatic fused ring optionally containing one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur; wherein the five- to eight-membered ring is optionally substituted with one, two, or three substitutents independently selected from alkoxy, alkoxyalkyl, alkoxycarbonyl, alkyl, alkylsulfonyl, aryl, arylalkyl, arylsulfonyl, carboxy, formyl, halo, haloalkoxy, haloalkyl, hydroxy, hydroxyalkyl, -NRaRb, ( RaRb)alkyl, ( RaRb)carbonyl, oxo, and spirocycle;
Y and Y' are each independently -CH2-, -CH2CH2-, or -CH2O-, wherein the
-CH2O- is drawn such that the oxygen atom is bound to the carbon atom substituted with Rv and Rq or Rv' and Rq';
Rp is hydrogen or Ci to C4 alkyl;
Rq is hydrogen, alkyl, or halo; or
Rp and Rq, together with the carbon atoms to which they are attached, form a cycloalkyl ring;
Rv is selected from hydrogen, alkyl, halo, and hydroxy; or
Rv and Rq, together with the carbon atom to which they are attached, form an ethylenyl group or a cycloalkyl ring;
Rp' is hydrogen or Ci to C4 alkyl;
Rq' is hydrogen, alkyl, or halo; or
Rp' and Rq', together with the carbon atoms to which they are attached, form a cycloalkyl ring;
Rv are independently selected from hydrogen, alkyl, halo, and hydroxy; or Rv and Rq , together with the carbon atom to which they are attached, form an ethylenyl group or a cycloalkyl ring;
Rw and Rw are independently selected from hydrogen and alkyl;
R1 is hydrogen or -C(0)Rx;
R2 is hydrogen or -C(0)Ry;
Rx and Ry are independently selected from cycloalkyl, heteroaryl, heterocyclyl, alkoxy, and alkyl, said alkyl being substituted by one or more substituents independently selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, -OR3, -C(0)OR4, -NRaRb, and -C(0)NRcRd,
wherein any said aryl and heteroaryl may optionally be substituted with one or more substituents independently selected from alkenyl, alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, -C(0)OR4, -OR5, -NRaRb, ( RaRb)alkyl, and (MeO)(HO)P(0)0-, and
wherein any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more substituents independently selected from alkyl, hydroxyl, halogen, aryl, -NRaRb, oxo, and -C(0)OR4;
R3 is hydrogen, alkyl, or arylalkyl;
R4 is alkyl or arylalkyl; R5 is hydrogen, alkyl, or arylalkyl;
Ra and Rb are independently selected from hydrogen, alkyl, cycloalkyl, arylalkyl, heteroaryl, -C(0)R6, -C(0)OR7, -C(0)NRcRd, and (NRcRd)alkyl, or alternatively, Ra and Rb, together with the nitrogen atom to which they are attached, form a five- or six-membered ring or bridged bicyclic ring structure, wherein said five- or six-membered ring or bridged bicyclic ring structure optionally may contain one or two additional heteroatoms independently selected from nitrogen, oxygen, and sulfur and may contain one, two, or three substituents independently selected from Ci to Ce alkyl, Ci to C4 haloalkyl, aryl, hydroxyl, Ci to Ce alkoxy, Ci to C4 haloalkoxy, and halogen;
R6 is alkyl;
R7 is alkyl, arylalkyl, cycloalkyl, or haloalkyl; and
Rc and Rd are independently selected from hydrogen, alkyl, arylalkyl, and cycloalkyl.
In a first embodiment of the first aspect the present disclosure provides a compound of Formula (I) further characterized by Formula (la):
Figure imgf000006_0001
(la),
or a pharmaceutically acceptable salt or a tautomer thereof, wherein:
X is hydrogen or chloro (CI) and Z is hydrogen; or
X and Z, together with the carbon atoms to which they are attached, form a six-membered aromatic or non-aromatic fused ring;
X' is hydrogen or chloro (CI) and Z' is hydrogen; or
X' and Z', together with the carbon atoms to which they are attached, form a six-membered aromatic or non-aromatic fused ring;
Y is -CH2-, -CH2CH2-, or -CH2O-, wherein the -CH20- is drawn such that the oxygen atom is bound to the carbon atom substituted with Rv and Rq;
Rp is hydrogen or Ci to C4 alkyl; Rq is hydrogen, alkyl, or halo; or
Rp and Rq, together with the carbon atoms to which they are attached, form a cycloalkyll ring; and
Rv is selected from hydrogen, alkyl, halo, and hydroxy; or
Rv and Rq, together with the carbon atom to which they are attached, form an ethylenyl group or a cycloalkyl ring.
In a second embodiment of the first aspect the present disclosure provides a compound of Formula (I) further characterized by Formula (lb):
Figure imgf000007_0001
(lb),
or a pharmaceutically acceptable salt or a tautomer thereof.
In a third embodiment of the first aspect the present disclosure provides a compound of Formula (I) further characterized by Formula (Ic):
Figure imgf000007_0002
(Ic),
or a pharmaceutically acceptable salt or a tautomer thereof.
In a fourth embodiment of the first aspect the present disclosure provides a compound of Formula (I) further characterized by Formula (Id):
Figure imgf000007_0003
(Id),
or a pharmaceutically acceptable salt or a tautomer thereof. In a fifth embodiment of the first aspect the present disclosure provides a compound of Formula (la), or a pharmaceutically acceptable salt thereof, wherein:
R1 is -C(0)Rx;
R2 is -C(0)Ry;
Rx and Ry are independently alkyl substituted by at least one -NRaRb, characterized by Formula (A):
Figure imgf000008_0001
(A),
wherein:
m is 0 or 1 ;
R8 is hydrogen or alkyl;
R9 is selected from hydrogen, cycloalkyl, aryl, heteroaryl, heterocyclyl, and alkyl optionally substituted with a substituent selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, heterobicyclyl, -OR3, -C(0)OR4, -NRaRb, and -C(0)NRcRd, wherein any said aryl and heteroaryl may optionally be substituted with one or more substituents independently selected from alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, -C(0)OR4, -OR5, -NRaRb, ( RaRb)alkyl, and (MeO)(HO)P(0)0-, and
wherein any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more substituents independently selected from alkyl, hydroxyl, halogen, aryl, -NRaRb, oxo, and -C(0)OR4; and
R3, R4, R5, Ra, Rb, Rc, and Rd are defined as in Formula (I).
In a sixth embodiment of the first aspect the present disclosure provides a compound of Formula (la) or a pharmaceutically acceptable salt thereof, wherein m is 0;
R8 is hydrogen or Ci to C4 alkyl;
R9 is selected from hydrogen, Ci to Ce alkyl optionally substituted with - OR12, C3 to C6 cycloalkyl, allyl, -CH2C(0)NRcRd, ( RcRd)alkyl,
Figure imgf000009_0001
wherein j is 0 or 1 ;
k is 1, 2, or 3;
n is 0 or an integer selected from 1 through 4;
each R10 is independently hydrogen, Ci to C4 alkyl, Ci to C4 haloalkyl, halogen, nitro, -OBn, or (MeO)(OH)P(0)0-;
R11 is hydrogen, Ci to C4 alkyl, or benzyl;
R12 is hydrogen, Ci to C4 alkyl, or benzyl;
Ra is hydrogen or Ci to C4 alkyl;
Rb is Ci to C4 alkyl, C3 to Ce cycloalkyl, benzyl, 3-pyridyl, pyrimidin-5-yl, acetyl, -C(0)OR7, or -C(0)NRcRd;
R7 is Ci to C4 alkyl or Ci to C4 haloalkyl;
Rc is hydrogen or Ci to C4 alkyl; and
Rd is hydrogen, Ci to C4 alkyl, or C3 to Ce cycloalkyl.
In a seventh embodiment of the first aspect the present disclosure provides a compound of Formula (la) or a pharmaceutically acceptable salt thereof, wherein m is 0; R8 is hydrogen;
R9 is phenyl optionally substituted with one up to five substituents independently selected from Ci to Ce alkyl, Ci to C4 haloalkyl, halogen, Ci to alkoxy, hydroxyl, cyano, and nitro; and
NRaRb is a heterocyclyl or heterobicyclyl group selected from:
Figure imgf000010_0001
wherein n is 0, 1,
each R13 is independently selected from Ci to Ce alkyl, phenyl,
trifluoromethyl, halogen, hydroxyl, methoxy, and oxo; and
R14 is Ci to C6 alkyl, phenyl, benzyl, or -C(0)OR15 group, wherein R15 is Ci to C4 alkyl, phenyl, or benzyl.
In an eighth embodiment of the first aspect the present disclosure provides a compound of Formula (la) or a pharmaceutically acceptable salt thereof, wherein m is 1 ;
R8 is hydrogen;
R9 is Ci to e alkyl, arylalkyl, or heteroarylalkyl;
Ra is hydrogen; and
Rb is -C(0)OR7, wherein R7 is Ci to C6 alkyl.
In a ninth embodiment of the first aspect the present disclosure provides a compound of Formula (la) or a pharmaceutically acceptable salt thereof, wherein R1 is -C(0)Rx;
R2 is -C(0)Ry;
Rx and Ry are heteroaryl or heterocyclyl independently selected from:
Figure imgf000011_0001
wherein n is 0 or an integer selected from 1 through 4;
each R13 is independently selected from hydrogen, Ci to Ce alkyl, Ci to C4 haloalkyl, phenyl, benzyl, Ci to Ce alkoxy, Ci to C4 haloalkoxy, heterocyclyl, halogen, NRcRd, hydroxyl, cyano, and oxo, where Rc and Rd are independently hydrogen or Ci to C4 alkyl; and
R14 is hydrogen (H), Ci to C6 alkyl, benzyl, or -C(0)OR4, wherein R4 is Ci to C6 alkyl.
In a tenth embodiment of the first aspect the present disclosure provides a compound of Formula (la) or a pharmaceutically acceptable salt thereof, wherein R1 is -C(0)Rx;
R2 is -C(0)Ry;
Rx and Ry are cycloalkyl independently selected from: (Rl3)n _ NRaRb
Figure imgf000012_0001
wherein
j is 0, 1, 2, or 3;
k is 0, 1 , or 2;
n is 0 or an integer selected from 1 through 4;
each R13 is independently selected from hydrogen, Ci to Ce alkyl, Ci to C4 haloalkyl, Ci to Ce alkoxy, halogen, hydroxyl, cyano, and nitro; and
Ra and Rb are each independently hydrogen, Ci to Ce alkyl, or -C(0)OR , wherein R7 is Ci to Ce alkyl.
In an eleventh embodiment of the first aspect the present disclosure provides a compound of Formula (la) or a pharmaceutically acceptable salt thereof, wherein R1 is -C(0)Rx;
R2 is -C(0)Ry;
Rx and Ry are independently arylalkyl, wherein aryl part of said arylalkyl may optionally be substituted with (NRaRb)alkyl; and
Ra and Rb are independently hydrogen, Ci to Ce alkyl, or benzyl, or alternatively, Ra and Rb, together with the nitrogen atom to which they are attached, form a five- or six-membered ring selected from
Figure imgf000012_0002
^— / , and ^— / , wherein R15 is hydrogen, Ci to C(, alkyl, or benzyl.
In a twelfth embodiment of the first aspect the present disclosure provides a compound of Formula (la) or a pharmaceutically acceptable salt thereof, wherein 1 and R2 are the same and are selected from the group consisting of:
Figure imgf000012_0003
Figure imgf000013_0001
wherein a squiggle bond ( ·~>~ ) in the structure indicates that a stereogenic center to which the bond is attached can take either (R)- or (S)- configuration so long as chemical bonding principles are not violated.
In a thirteenth embodiment of the first aspect the present disclosure provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein R1 is -C(0)Rx;
R2 is -C(0)Ry; and
Rx and Ry are both t-butoxy.
In a fourteenth embodiment of the first aspect the present disclosure provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein
R1 and R2 are both hydrogen.
In a second aspect the present disclosure provides a compound of Formula
(II):
Figure imgf000014_0001
(I),
or a pharmaceutically acceptable salt thereof, wherein:
CH- -C≡C- -OCH2- -CH20-
Figure imgf000014_0002
X and X' are independently hydrogen (H) or halogen;
Rp is hydrogen or Ci to C4 alkyl, and Rq is hydrogen, or alternatively, Rp and Rq, together with the carbon atoms to which they are attached, form a cyclopropyl ring;
Rp' is hydrogen or Ci to C4 alkyl, and Rq' is hydrogen, or alternatively, Rp' and Rq', together with the carbon atoms to which they are attached, form a cyclopropyl ring;
R1 is hydrogen or -C(0)Rx;
R2 is hydrogen or -C(0)Ry;
Rx and Ry are independently selected from cycloalkyl, heteroaryl, heterocyclyl, alkoxy, and alkyl, said alkyl being substituted by one or more substituents independently selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, -OR3, -C(0)OR4, -NRaRb, and -C(0)NRcRd,
wherein any said aryl and heteroaryl may optionally be substituted with one or more substituents independently selected from alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, -C(0)OR4, -OR5, -NRaRb, ( RaRb)alkyl, and (MeO)(HO)P(0)0-, and
wherein any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more substituents independently selected from alkyl, hydroxyl, halogen, aryl, -NRaRb, oxo, and
-C(0)OR4; R3 is hydrogen, alkyl, or arylalkyl;
R4 is alkyl or arylalkyl;
R5 is hydrogen, alkyl, or arylalkyl;
Ra and Rb are independently selected from hydrogen, alkyl, cycloalkyl, arylalkyl, heteroaryl, -C(0)R6, -C(0)OR7, -C(0)NRcRd, and (NRcRd)alkyl, or alternatively, Ra and Rb, together with the nitrogen atom to which they are attached, form a five- or six-membered ring or bridged bicyclic ring structure, wherein said five- or six-membered ring or bridged bicyclic ring structure optionally may contain one or two additional heteroatoms independently selected from nitrogen, oxygen, and sulfur and may contain one, two, or three substituents independently selected from Ci to Ce alkyl, Ci to C4 haloalkyl, aryl, hydroxyl, Ci to Ce alkoxy, Ci to C4 haloalkoxy, and halogen;
R6 is alkyl;
R7 is alkyl, arylalkyl, or haloalkyl; and
Rc and Rd are independently selected from hydrogen, alkyl, arylalkyl, and cycloalkyl.
In a third aspect the present disclosure provides a composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, wherein Formula (I) is defined according to any of the embodiments described above in the first aspect of the present disclosure.
In a first embodiment of the third aspect the composition further comprises at least one additional compound having anti-HCV activity.
In a second embodiment of the third aspect at least one of the additional compounds is an interferon or a ribavirin.
In a third embodiment of the third aspect the interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, and lymphoblastiod interferon tau.
In a fourth embodiment of the third aspect the present disclosure provides a composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and at least one additional compound having anti-HCV activity, wherein at least one of the additional compounds is selected from 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.
In a fifth embodiment of the third aspect the present disclosure provides a composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and at least one additional compound having anti-HCV activity, wherein at least one of the additional compounds is effective to inhibit the function of a target selected from HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, and IMPDH for the treatment of an HCV infection.
In a fourth aspect the present disclosure provides a method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein Formula (I) is defined according to any of the embodiments described above in the first aspect of the present disclosure.
In a first embodiment of the fourth aspect the method further comprises
administering at least one additional compound having anti-HCV activity prior to, after or simultaneously with the compound of Formula (I), or a pharmaceutically acceptable salt thereof.
In a second embodiment of the fourth aspect at least one of the additional compounds is an interferon or a ribavirin.
In a third embodiment of the fourth aspect the interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, and lymphoblastiod interferon tau.
In a fourth embodiment of the fourth aspect the present disclosure provides a method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one additional compound having anti-HCV activity prior to, after or simultaneously with the compound of
Formula (I), or a pharmaceutically acceptable salt thereof, wherein at least one of the additional compounds is selected from 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.
In a fifth embodiment of the fourth aspect the present disclosure provides a method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one additional compound having anti-HCV activity prior to, after or simultaneously with the compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein at least one of the additional compounds is effective to inhibit the function of a target selected from HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, and IMPDH for the treatment of an HCV infection.
The compounds of the present disclosure can be effective to inhibit the function of the HCV NS5A protein. In particular, the compounds of the present disclosure can be effective to inhibit the HCV lb genotype or multiple genotypes of HCV. Therefore, this disclosure also encompasses: (1) compositions comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; and (2) a method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
Other aspects of the present disclosure may include suitable combinations of embodiments disclosed herein.
Yet other aspects and embodiments may be found in the description provided herein.
The description of the present disclosure herein should be construed in congruity with the laws and principals of chemical bonding. In some instances it may be necessary to remove a hydrogen atom in order to accommodate a substituent at any given location.
Certain features of the structure of Formula (I) are further illustrated below:
Figure imgf000018_0001
Pyrrolidinyl-imidazole
Pyrrolidinyl-imidazole
(Left)
(Right)
(I).
In Formula (I), as depicted above, the "pyrrolidinyl-imidazole" moiety on the left side of the "linker" is independent from the "pyrrolidinyl-imidazole" moiety on the right side of the linker group in respect to, e.g., (1) tautomer form of imidazole ring, (2) absolute configuration of the stereogenic centers on the pyrrolidine ring, and (3) substituents on the pyrrolidine nitrogen, i.e., R1 and R2 are independent from each other, although in some circumstances they are preferably the same.
It should be understood that the depiction of a pyrrolidine moiety on the "left" side or on the "right" side is for illustration purpose only, which does not in any way limit the scope of the disclosure.
In the linker group of Formula (I), the linkage between "L" and the two benzene rings encompasses all the following combinations:
Figure imgf000018_0002
wherein the "para-para," "para-meta," and "meta-meta" linkages are preferred. Likewise, in Formula (I), when L is a phenylene ( * l¾¾i/J ) group, it can link to the adjacent two benzene rings by the following manners:
Figure imgf000019_0001
wherein the "para" and "meta" arrangements are preferred, and the "para" arrangement is the more preferred.
In Formula (I), when L is a vinylene (-CH=CH-) group, it can take either trans- or cis- configuration, as depicted below:
Figure imgf000019_0002
(cis-), (trans-). In Formula (I), when L is a cyclopropylene (¾ ) group, the two benzene substituents can be either trans- or cis- to each other, forming one of the following four configurations:
Figure imgf000019_0003
)
In a pyrrolidine ring of a pyrrolidinyl-imidazole moiety, the stereogenic carbon center to which the imidazole group is attached can take either (R)- or (S)- configuration as depicted below:
Figure imgf000020_0001
(R-) (S-)
When a cyclopropyl ring is fused onto a pyrrolidine ring of a pyrrolidinyl- imidazole moiety, i.e., when (Rp, Rq) together is -CH2-, the CH2 group of the fused cyclopropyl ring can take either a- or β- position relative to the pyrrolidine ring, as depicted below:
Figure imgf000020_0002
Thus, this disclosure is intended to cover all possible stereoisomers even when a single stereoisomer, or no stereochemistry, is described in a structure.
In Formula (I), the linkage between a benzene ring of the linker group and an imidazole ring of a pyrrolidinyl-imidazole moiety can take place in either the C-4 or the C-5 position (see below) of the imidazole ring. As a person of ordinary skill in the art would understand, due to tautomerization of the imidazole ring, a bonding of a benzene ring to the C-4 position may be equivalent to a bonding of the benzene ring to the C-5 osition as shown in the followin e uation:
Figure imgf000020_0003
The sample principle also applies to substituent X or X'.
Thus, this disclosure is intended to cover all possible tautomers even when a structure depicts only one of them.
In this disclosure, a floating bond (e.g., \~ ) or a floating substituent (e.g.,
-R13) on a structure indicates that the bond or substituent can attach to any available position of the structure by removal of a hydrogen from the available position. It should be understood that in a bicyclic or polycyclic ring structure, unless specifically defined otherwise, the position of a floating bond or a floating substituent does not limit the position of such bond or substituent to a specific ring. Thus, the following two substituents should be construed to be equivalent:
Figure imgf000021_0001
It should be understood that the compounds encompassed by the present disclosure are those that are suitably stable for use as pharmaceutical agent.
It is intended that the definition of any substituent or variable at a particular location in a molecule be independent of its definitions elsewhere in that molecule. For example, for substituent ( 10)n, when n is 2, each of the two R10 groups may be the same or different.
All patents, patent applications, and literature references cited in the specification are herein incorporated by reference in their entirety. In the case of inconsistencies, the present disclosure, including definitions, will prevail.
DEFINITIONS
Definitions have been provided above for each of the groups defined. In addition, the following definitions shall be used.
As used herein, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise.
Unless stated otherwise, all aryl, cycloalkyl, heteroaryl, and heterocyclyl groups of the present disclosure may be substituted as described in each of their respective definitions. For example, the aryl part of an arylalkyl group may be substituted as described in the definition of the term "aryl."
The term "acetyl," as used herein, refers to -C(0)CH3.
The term "alkenyl," as used herein, refers to a monovalent, straight or branched hydrocarbon chain having one or more, preferably one to two, double bonds therein. The double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group. Suitable alkenyl groups include, but are not limited to, C2 to C10 alkenyl groups, such as vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl, 4-(2- methyl-3-butene)-pentenyl. An alkenyl group can be unsubstituted or substituted with one or two suitable substituents.
The term "alkoxy," as used herein, refers to an alkyl group attached to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy group include, but are not limited to, methoxy (CH3O-), ethoxy (CH3CH20-), and t-butoxy ((CH3)3CO-).
The term "alkoxy alkyl," as used herein, refers to an alkyl group substituted with one, two, or three alkoxy groups.
The term "alkoxycarbonyl," as used herein, refers to an alkoxy group attached to the parent molecular moiety through a carbonyl group.
The term "alkyl," as used herein, refers to a group derived from a straight or branched chain saturated hydrocarbon by removal of a hydrogen from one of the saturated carbons. The alkyl group preferably contains from one to ten carbon atoms.
Representative examples of alkyl group include, but are not limited to, methyl, ethyl, isopropyl, and tert-butyl.
The term "alkylcarbonyl," as used herein, refers to an alkyl group attached to the parent molecular moiety through a carbonyl group. Representative examples of alkylcarbonyl group include, but are not limited to, acetyl (-C(0)CH3), propanoyl (-C(0)CH2CH3), n-butyryl (-C(0)CH2CH2CH3), and 2,2-dimethylpropanoyl or pivaloyl (-C(0)C(CH3)3).
The term "alkylsulfonyl," as used herein, refers to an alkyl group attached to the parent molecular moiety through a sulfonyl group.
The term "allyl," as used herein, refers to the -CH2CH=CH2 group.
The term "aryl," as used herein, refers to a group derived from an aromatic carbocycle by removal of a hydrogen atom from an aromatic ring. The aryl group can be monocyclic, bicyclic or polycyclic, wherein in bicyclic or polycyclic aryl group, the aromatic carbocycle can be fused onto another four- to six-membered aromatic or non-aromatic carbocycle. Representative examples of aryl groups include, but are not limited to, phenyl, indanyl, indenyl, naphthyl, and 1,2,3,4- tetrahydronaphth-5 -yl. The term "arylalkyl," as used herein, refers to an alkyl group substituted with one, two, or three aryl groups, wherein aryl part of the arylalkyl group may optionally be substituted by one to five substituents independently selected from Ci to Ce alkyl, Ci to C4 haloalkyl, Ci to Ce alkoxy, halogen, cyano, and nitro groups. Represented examples of arylalkyl include, but are not limited to, benzyl, 2 -phenyl- 1 -ethyl (PhCH2CH2-), (naphth- l-yl)methyl, and (naphth-2-yl)methyl.
The term "arylsulfonyl," as used herein, refers to an aryl group attached to the parent molecular moiety through a sulfonyl group.
The term "benzyl," as used herein, refers to a methyl group on which one of the hydrogen atoms is replaced by a phenyl group, wherein said phenyl group may optionally be substituted by one to five substituents independently selected from methyl, trifluoromethyl (-CF3), methoxy (-OCH3), halogen, and nitro (-N02).
Representative examples of benzyl group include, but are not limited to, PhCH2-, 4-MeO-C6H4CH2-, and 2,4,6-tri-methyl-C6H4CH2-.
The term "bridged bicyclic ring," as used herein, refers to a ring structure comprising a bridgehead between two of the ring members, wherein the ring and the bridgehead optionally may independently comprise one or more, preferably one to two, heteroatoms independently selected from nitrogen, oxygen, and sulfur.
Illustrated examples of a bridged bicyclic ring structure include, but are not limited to:
Figure imgf000023_0001
The terms "Cap" and "cap," as used herein, refer to the group which is placed on the nitrogen atom of the pyrrolidine ring in the compounds of formula (I). It should be understood that "Cap" or "cap" can also refer to the reagent which is a precursor to the final "cap" in compounds of formula (I) and is used as one of the starting materials in the reaction to append a group on the pyrrolidine nitrogen that results in the final product, a compound which contains the functionalized pyrrolidine that will be present in the compound of formula (I). The term "carbonyl," as used herein, refers to -C(O)-.
The term "carboxyl," or "carboxy," as used herein, refers to -C02H.
The term "cyano," as used herein, refers to -CN.
The term "cycloalkyl," as used herein, refers to a group derived from a saturated carbocycle, having preferably three to eight carbon atoms, by removal of a hydrogen atom from the saturated carbocycle, wherein the saturated carbocycle can optionally be fused onto one or two other aromatic or nonaromatic carbocycles. Representative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, and 1,2,3,4-tetrahydronaphth-l-yl.
The term "formyl," as used herein, refers to -CHO.
The terms "halo" and "halogen," as used herein, refer to F, CI, Br, or I.
The term "haloalkoxy," as used herein, refers to a haloalkyl group attached to the parent molecular moiety through an oxygen atom.
The term "haloalkyl," as used herein, refers to an alkyl group substituted by at least one halogen atom. The haloalkyl group can be an alkyl group of which all hydrogen atoms are substituted by halogens. Representative examples of haloalkyl include, but are not limited to, trifluoromethyl (CF3-), 1-chloroethyl (C1CH2CH2-), and 2,2,2-trifluoroethyl (CF3CH2-).
The term "heteroaryl," as used herein, refers to group derived from a monocyclic, bicyclic, or polycyclic compound comprising at least one aromatic ring comprising one or more, preferably one to three, heteroatoms independently selected from nitrogen, oxygen, and sulfur, by removal of a hydrogen atom from an aromatic ring thereof. As is well known to those skilled in the art, heteroaryl rings have less aromatic character than their all-carbon counterparts. Thus, for the purposes of the disclosure, a heteroaryl group need only have some degree of aromatic character. Illustrative examples of heteroaryl groups include, but are not limited to, pyridyl, pyridazinyl, pyrimidyl, pyrazyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3,)- and (l,2,4)-triazolyl, pyrazinyl, pyrimidinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, isoxazolyl, oxazolyl, indolyl, quinolinyl, isoquinolinyl, benzisoxazolyl, benzothiazolyl, benzothienyl, and pyrrolopyridinyl.
The term "heteroarylalkyl," as used herein, refers to an alkyl group substituted with one, two, or three heteroaryl groups. The term "heterobicyclyl," as used herein, refers to a ring structure comprising two fused or bridged rings that include carbon and one or more, preferably one to three, heteroatoms independently selected from nitrogen, oxygen, and sulfur. The heterobicyclic ring structure is a subset of heterocyclic ring and can be saturated or unsaturated. Examples of heterobicyclic ring structures include, but are not limited to, tropane, quinuclidine, and 7-azabicyclo[2.2.1]heptane.
The term "heterocyclyl," as used herein, refers to a group derived from a monocyclic, bicyclic, or polycyclic compound comprising at least one nonaromatic ring comprising one or more, preferably one to three, heteroatoms independently selected from nitrogen, oxygen, and sulfur, by removal of a hydrogen atom from the nonaromatic ring. The heterocyclyl group encompasses the heterobicyclyl group. The heterocyclyl groups of the present disclosure can be attached to the parent molecular moiety through a carbon atom or a nitrogen atom in the group. Examples of heterocyclyl groups include, but are not limited to, morpholinyl, oxazolidinyl, piperazinyl, piperidinyl, pyrrolidinyl, tetrahydrofuryl, thiomorpholinyl, and indolinyl.
The term "heterocyclylalkyl," as used herein, refers to an alkyl group substituted with one, two, or three heterocyclyl groups.
The terms "hydroxy" or "hydroxyl," as used herein, refer to -OH.
The term "hydroxyalkyl," as used herein, refers to an alkyl group substituted with one, two, or three hydroxy groups.
The term "nitro," as used herein, refers to -N02.
The term "-NRaRb," as used herein, refers to two groups, Ra and Rb, which are attached to the parent molecular moiety through a nitrogen atom, or alternatively Ra and Rb, together with the nitrogen atom to which they are attached, form a 5- or 6- membered ring or a fused- or bridged-bicyclic ring structure optionally containing one, two, or three additional heteroatom independently selected from nitrogen, oxygen, and sulfur. The term "-NRcRd" is defined similarly.
The term "(NRaRb)alkyl," as used herein, refers to an alkyl group substituted with one, two, or three -NRaRb groups. The term "( RcRd)alkyl" is defined similarly.
The term "oxo," as used herein, refers to =0.
The term "sulfonyl," as used herein, refers to -SO2-. The term "trialkylsilyl," as used herein, refers to -S1R3, wherein each R is Ci to C4 alkyl or phenyl. The three R groups may be the same or different.
Representative examples of "trialkylsilyl" include, but are not limited to, trimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBS or TBDMS), and triisopropylsilyl (TIPS).
Asymmetric centers exist in the compounds of the present disclosure. These centers are designated by the symbols "R" or "S", depending on the configuration of substituents around the chiral carbon atom. It should be understood that the disclosure encompasses all stereochemical isomeric forms, or mixtures thereof, which possess the ability to inhibit NS5A. Individual stereoisomers of compounds can be prepared synthetically from commercially available starting materials which contain chiral centers or by preparation of mixtures of enantiomeric products followed by separation such as conversion to a mixture of diastereomers followed by separation or recrystallization, chromatographic techniques, or direct separation of enantiomers on chiral chromatographic columns. Starting compounds of particular stereochemistry are either commercially available or can be made and resolved by techniques known in the art.
Certain compounds of the present disclosure 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 disclosure includes each conformational isomer of these compounds and mixtures thereof.
The term "compounds of the present disclosure", and equivalent expressions, are meant to embrace compounds of Formula (I), and pharmaceutically acceptable enantiomers, diastereomers, and salts thereof. Similarly, references to intermediates are meant to embrace their salts where the context so permits.
The present disclosure is intended to include all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include deuterium and tritium. Isotopes of carbon include 13C and 14C. Isotopically-labeled compounds of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein, using an appropriate isotopically- labeled reagent in place of the non-labeled reagent otherwise employed. Such compounds may have a variety of potential uses, for example as standards and reagents in determining biological activity. In the case of stable isotopes, such compounds may have the potential to favorably modify biological, pharmacological, or pharmacokinetic properties.
The compounds of the present disclosure can exist as pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt," as used herein, represents salts or zwitterionic forms of the compounds of the present disclosure which are water or oil-soluble or dispersible, 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 benefit/risk ratio, and are effective for their intended use. The salts can be prepared during the final isolation and purification of the compounds or separately by reacting a suitable nitrogen atom with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate; digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2 -hydroxy ethanesulfonate, lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate, persulfate, 3- phenylproprionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para-toluenesulfonate, and undecanoate. Examples of acids which can be employed to form pharmaceutically acceptable addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric.
Basic addition salts can be prepared during the final isolation and purification of the compounds by reacting a carboxy group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation or with ammonia or an organic primary, secondary, or tertiary amine. The cations of pharmaceutically acceptable salts include lithium, sodium, potassium, calcium, magnesium, and aluminum, as well as nontoxic quaternary amine cations such as ammonium,
tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, Ν,Ν-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, Ν,Ν-dibenzylphenethylamine, and Ν,Ν'- dibenzylethylenediamine. Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine,
diethanolamine, piperidine, and piperazine.
When it is possible that, for use in therapy, therapeutically effective amounts of a compound of Formula (I), as well as pharmaceutically acceptable salts thereof, may be administered as the raw chemical, it is possible to present the active ingredient as a pharmaceutical composition. Accordingly, the disclosure further provides pharmaceutical compositions, which include therapeutically effective amounts of compounds of Formula (I) or pharmaceutically acceptable salts thereof, and one or more, preferably one to three, pharmaceutically acceptable carriers, diluents, or excipients. The term "therapeutically effective amount," as used herein, refers to 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 term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously. The compounds of Formula (I) and pharmaceutically acceptable salts thereof, are as described above. The carrier(s), diluent(s), or excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. In accordance with another aspect of the present disclosure there is also provided a process for the preparation of a pharmaceutical formulation including admixing a compound of Formula (I), or a pharmaceutically acceptable salt thereof, with one or more, preferably one to three, pharmaceutically acceptable carriers, diluents, or excipients. The term "pharmaceutically acceptable," as used herein, refers 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 benefit/risk ratio, and are effective for their intended use.
Pharmaceutical formulations may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. Dosage levels of between about 0.01 and about 250 milligram per kilogram ("mg/kg") body weight per day, preferably between about 0.05 and about 100 mg/kg body weight per day of the compounds of the present disclosure are typical in a monotherapy for the prevention and treatment of HCV mediated disease. Typically, the pharmaceutical compositions of this disclosure 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 carrier materials to produce a single dosage form will vary depending on the condition being treated, the severity of the condition, the time of administration, the route of administration, the rate of excretion of the compound employed, the duration of treatment, and the age, gender, weight, and condition of the patient. Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient. Generally, treatment is initiated with small dosages substantially less than the optimum dose of the compound. 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 disclosure comprise a combination of a compound of the present disclosure 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 150%, and more preferably between about 10 and 80% of the dosage normally administered in a monotherapy regimen.
Pharmaceutical formulations may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intracutaneous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, intravenous, or intradermal injections or infusions) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). Oral administration or administration by injection are preferred.
Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in- water liquid emulsions or water-in-oil emulsions.
For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Powders are prepared by comminuting the compound to a suitable fine size and mixing with a similarly comminuted pharmaceutical carrier such as an edible carbohydrate, as, for example, starch or mannitol. Flavoring, preservative, dispersing, and coloring agent can also be present.
Capsules are made by preparing a powder mixture, as described above, and filling formed gelatin sheaths. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol can be added to the powder mixture before the filling operation. A disintegrating or solubilizing agent such as agar-agar, calcium carbonate, or sodium carbonate can also be added to improve the availability of the medicament when the capsule is ingested.
Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta- lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, and the like.
Lubricants used in these dosage forms include sodium oleate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, betonite, xanthan gum, and the like. Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant, and pressing into tablets. A powder mixture is prepared by mixing the compound, suitable comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, an aliginate, gelating, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt and/or and absorption agent such as betonite, kaolin, or dicalcium phosphate. The powder mixture can be granulated by wetting with a binder such as syrup, starch paste, acadia mucilage, or solutions of cellulosic or polymeric materials and forcing through a screen. As an alternative to granulating, the powder mixture can be run through the tablet machine and the result is imperfectly formed slugs broken into granules. The granules can be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc, or mineral oil. The lubricated mixture is then compressed into tablets. The compounds of the present disclosure can also be combined with a free flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material, and a polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different unit dosages.
Oral fluids such as solution, syrups, and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of the compound. Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic vehicle.
Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and
polyoxyethylene sorbitol ethers, preservatives, flavor additive such as peppermint oil or natural sweeteners, or saccharin or other artificial sweeteners, and the like can also be added.
Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The formulation can also be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax, or the like.
The compounds of Formula (I), and pharmaceutically acceptable salts thereof, can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.
Liposomes can be formed from a variety of phopholipids, such as cholesterol, stearylamine, or phophatidylcholines.
The compounds of Formula (I) and pharmaceutically acceptable salts thereof may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysine substituted with palitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels.
Pharmaceutical formulations adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharm. Res., 3(6):318 (1986).
Pharmaceutical formulations adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils.
For treatments of the eye or other external tissues, for example mouth and skin, the formulations are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in oil base. Pharmaceutical formulations adapted for topical administrations to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.
Pharmaceutical formulations adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes. Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or as enemas.
Pharmaceutical formulations adapted for nasal administration wherein the carrier is a solid include a course powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or nasal drops, include aqueous or oil solutions of the active ingredient.
Pharmaceutical formulations adapted for administration by inhalation include fine particle dusts or mists, which may be generated by means of various types of metered, dose pressurized aerosols, nebulizers, or insufflators.
Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations.
Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, and soutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
It should be understood that in addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
The term "patient" includes both human and other mammals.
The term "treating" refers to: (i) preventing a disease, disorder or condition from occurring in a patient that may be predisposed to the disease, disorder, and/or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder, or condition, i.e., arresting its development; and (iii) relieving the disease, disorder, or condition, i.e., causing regression of the disease, disorder, and/or condition.
The compounds of the present disclosure can also be administered with a cyclosporin, for example, cyclosporin A. Cyclosporin A has been shown to be active against HCV in clinical trials (Hepatology, 38: 1282 (2003); Biochem. Biophys. Res. Commun., 313 :42 (2004); J. Gastroenterol, 38:567 (2003)).
Table 1 below lists some illustrative examples of compounds that can be administered with the compounds of this disclosure. The compounds of the disclosure 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 imgf000034_0001
Brand Name Physiological Class Type of Inhibitor or Source Company
Target
Pyrazolopyrimidine Antiviral HCV inhibitors Arrow Therapeutics compounds and salts Ltd.
From
WO 2005/047288
May 26, 2005
Levovirin Antiviral IMPDH inhibitor Ribapharm Inc., Costa
Mesa, CA
Merimepodib Antiviral IMPDH inhibitor Vertex
(VX-497) Pharmaceuticals Inc.,
Cambridge, MA
XTL-6865 Antiviral Monoclonal antibody XTL
(XTL-002) Biopharmaceuticals
Ltd., Rehovot, Israel
Telaprevir Antiviral NS3 serine protease Vertex
(VX-950, inhibitor Pharmaceuticals Inc.,
LY-570310) Cambridge, MA / Eli
Lilly and Co., Inc., Indianapolis, IN
HCV-796 Antiviral NS5B replicase Wyeth / Viropharma inhibitor
NM-283 Antiviral NS5B replicase Idenix / Novartis inhibitor
GL-59728 Antiviral NS5B replicase Gene Labs / Novartis inhibitor
GL-60667 Antiviral NS5B replicase Gene Labs / Novartis inhibitor
2'C MeA Antiviral NS5B replicase Gilead
inhibitor
PSI 6130 Antiviral NS5B replicase Roche
inhibitor
R1626 Antiviral NS5B replicase Roche
inhibitor Brand Name Physiological Class Type of Inhibitor or Source Company
Target
2'C Methyl Antiviral NS5B replicase Merck
adenosine inhibitor
JTK-003 Antiviral RdRp inhibitor Japan Tobacco Inc.,
Tokyo, Japan
Levovirin Antiviral Ribavirin ICN Pharmaceuticals,
Costa Mesa, CA
Ribavirin Antiviral Ribavirin Schering-Plough
Corporation, Kenilworth, NJ
Viramidine Antiviral Ribavirin prodrug Ribapharm Inc., Costa
Mesa, CA
Heptazyme Antiviral Ribozyme Ribozyme
Pharmaceuticals Inc., Boulder, CO
BILN-2061 Antiviral Serine protease Boehringer Ingelheim inhibitor Pharma KG,
Ingelheim, Germany
SCH 503034 Antiviral Serine protease Schering-Plough inhibitor
Zadazim Immune modulator Immune modulator SciClone
Pharmaceuticals Inc., San Mateo, CA
Ceplene Immunomodulator Immune modulator Maxim
Pharmaceuticals Inc., San Diego, CA
CELLCEPT® Immunosuppressant HCV IgG F. Hoffmann-La immunosuppressant Roche LTD, Basel,
Switzerland
Civacir Immunosuppressant HCV IgG Nabi
immunosuppressant Biopharmaceuticals
Inc., Boca Raton, FL Brand Name Physiological Class Type of Inhibitor or Source Company
Target
Albuferon - a Interferon Albumin IFN-a2b Human Genome
Sciences Inc., Rockville, MD
Infergen A Interferon IFN alfacon-1 InterMune
Pharmaceuticals Inc., Brisbane, CA
Omega IFN Interferon IFN-co Intarcia Therapeutics
IFN-P and EMZ701 Interferon IFN- and EMZ701 Transition
Therapeutics Inc., Ontario, Canada
REBIF® Interferon IFN- ia Serono, Geneva,
Switzerland
Roferon A Interferon IFN-a2a F. Hoffmann-La
Roche LTD, Basel, Switzerland
Intron A Interferon IFN-a2b Schering-Plough
Corporation, Kenilworth, NJ
Intron A and Zadaxin Interferon IFN-a2b/a 1 -thymosin RegeneRx Biopharma.
Inc., Bethesda, MD / SciClone
Pharmaceuticals Inc, San Mateo, CA
Rebetron Interferon IFN-a2b / ribavirin Schering-Plough
Corporation, Kenilworth, NJ
Actimmune Interferon INF-γ InterMune Inc.,
Brisbane, CA
Interferon-β Interferon Interferon- -la Serono
Multiferon Interferon Long lasting IFN Viragen / Valentis
Wellferon Interferon Lymphoblastoid IFN- GlaxoSmithKline pic, anl Uxbridge, UK Brand Name Physiological Class Type of Inhibitor or Source Company
Target
Omniferon Interferon natural IFN-a Viragen Inc.,
Plantation, FL
Pegasys Interferon PEGylated IFN-a2a F. Hoffmann-La
Roche LTD, Basel, Switzerland
Pegasys and Ceplene Interferon PEGylated IFN- Maxim
a2a/immune Pharmaceuticals Inc., modulator San Diego, CA
Pegasys and Interferon PEGylated IFN- F. Hoffmann-La Ribavirin a2a/ribavirin Roche LTD, Basel,
Switzerland
PEG-Intron Interferon PEGylated IFN-a2b Schering-Plough
Corporation, Kenilworth, NJ
PEG-Intron / Interferon PEGylated IFN- Schering-Plough Ribavirin a2b/ribavirin Corporation,
Kenilworth, NJ
IP-501 Liver protection Antifibrotic Indevus
Pharmaceuticals Inc., Lexington, MA
IDN-6556 Liver protection Caspase inhibitor Idun Pharmaceuticals
Inc., San Diego, CA
ITMN-191 Antiviral Serine protease InterMune
(R-7227) inhibitor Pharmaceuticals Inc.,
Brisbane, CA
GL-59728 Antiviral NS5B replicase Genelabs
inhibitor
ANA-971 Antiviral TLR-7 agonist Anadys
Boceprevir Antiviral Serine protease Schering-Plough inhibitor
TMS-435 Antiviral Serine protease Tibotec BVBA, inhibitor Mechelen, Belgium Brand Name Physiological Class Type of Inhibitor or Source Company
Target
BI-201335 Antiviral Serine protease Boehringer Ingelheim inhibitor Pharma KG,
Ingelheim, Germany
MK-7009 Antiviral Serine protease Merck
inhibitor
PF-00868554 Antiviral Replicase inhibitor Pfizer
ANA598 Antiviral Non-Nucleoside Anadys
NS5B polymerase Pharmaceuticals, Inc., inhibitor San Diego, CA, USA
IDX375 Antiviral Non-Nucleoside Idenix
replicase inhibitor Pharmaceuticals,
Cambridge, MA, USA
BILB 1941 Antiviral NS5B polymerase Boehringer Ingelheim inhibitor Canada Ltd R&D,
Laval, QC, Canada
PSI-7851 Antiviral Nucleoside Pharmasset,
polymerase inhibitor Princeton, NJ, USA
VCH-759 Antiviral NS5B polymerase ViroChem Pharma inhibitor
VCH-916 Antiviral NS5B polymerase ViroChem Pharma inhibitor
GS-9190 Antiviral NS5B polymerase Gilead
inhibitor
Peg-interferon lamda Antiviral Interferon ZymoGenetics /
Bristol-Myers Squibb
The compounds of the present disclosure 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 structural biology studies to further enhance knowledge of the HCV disease mechanisms. Further, the compounds of the present disclosure are useful in establishing or determining the binding site of other antiviral compounds, for example, by competitive inhibition. The compounds of this disclosure 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.
This disclosure is intended to encompass compounds having Formula (I) when prepared by synthetic processes or by metabolic processes including those occurring in the human or animal body (in vivo) or processes occurring in vitro.
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 of the abbreviations used are as follows: TFA for trifluoroacetic acid; min or min. or mins for minutes; MeCN or ACN for acetonitrile; LDA for lithium diisopropylamide; DMSO for dimethylsulfoxide; h or hr or hrs for hours; Boc or BOC for tert- butoxycarbonyl; HATU for 0-(7-azabenzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate; RT or Rt or rt for retention time or room temperature (context will dictate); Me for methyl; DMF for N,N-dimethylformamide; Pd(Ph3P)4 for tetrakistriphenyl phosphine palladium; MeOH for methanol; MeOD for CD4OD; TEA for triethylamine; Ph for phenyl; TBDPS for tert-butyldiphenylsilyl; Et3N or TEA for triethylamine; DMAP for Ν,Ν-dimethylaminopyridine; EtOAc for ethyl acetate; TBAF for tetrabutylammonium fluoride; THF for tetrahydrofuran; DIEA or DIPEA or iPr2NEt for diisopropylethylamine; NCS for N- chlorosuccinimide; NBS for N-bromosuccinimide; DCM for dichloromethane; SEM for 2-(trimethylsilyl)ethoxymethyl; DCE for 1,2-dichloroethane; EDCI for l-ethyl-3- (3-dimethylaminopropyl)carbodiimide; DBU for l,8-diazabicyclo[5.4.0]undec-7-ene; Pd(t-Bu3P)2 for palladium bis(tributylphosphine); HMDS for hexamethyldisilazide; TMSCHN2 for trimethylsilyldiazomethane; H-D-Ser-OBzl for D-serine benzyl ester; i-PrOH for isopropanol; LiHMDS for lithium hexamethyldisilazide; DIBAL or DIBALH for diisobutylaluminum hydride; TBDMS for tert-butyldimethylsilyl; CBz for carbobenzyloxy; Bn for benzyl; DEAD for diethyl azodicarboxylate; mCPBA for meta-chloroperoxybenzoic acid; TMSCN for trimethylsilyl cyanide; dpppe for 1,5-
Bis(diphenylphosphino) pentane; TMEDA for tetramethylethylenediamine; OAc for acetate; DMA for Ν,Ν-dimethylacetamide; and d for days. The present disclosure will now be described in connection with certain embodiments which are not intended to limit its scope. On the contrary, the present disclosure covers all alternatives, modifications, and equivalents as can be included within the scope of the claims. Thus, the following examples, which include specific embodiments, will illustrate one practice of the present disclosure, it being understood that the examples are for the purposes of illustration of certain embodiments and are presented to provide what is believed to be the most useful and readily understood description of its procedures and conceptual aspects.
Starting materials can be obtained from commercial sources or prepared by well- established literature methods known to those of ordinary skill in the art.
EXAMPLES
Unless noted otherwise, purity assessments were conducted on Shimadzu LC system, and retention time (Rt) determination and low resolution mass analysis were conducted on a Shimadzu LC system coupled with Waters MICROMASS® ZQ MS system. It should be noted that retention times may vary slightly between machines.
Condition 1
Column = PHENOMENEX®, CI 8, 3.0 X 50 mm, 10 μιη
Start %B = 0
Final %B = 100
Gradient time = 2 min
Stop time = 3 min
Flow Rate = 4 mL/min
Wavelength = 220 nm
Solvent A = 0.1 % TFA in 10% methanol/90% water
Solvent B = 0.1 % TFA in 90% methanol/10% water
Condition 2
Column = XTERRA®, CI 8, 3.0 x 50 mm, S7
Start %B = 0
Final %B = 100 Gradient time = 2 min
Stop time = 3 min
Flow Rate = 5 mL/min
Wavelength = 220 nm
Solvent A = 0.2 % H3P04 in 10% methanol/90% water Solvent B = 0.2 % H3P04 in 90% methanol/10% water
Condition 3
Column = PHENOMENEX®, CI 8, 3.0 X 50 mm, 10 μιη Start %B = 0
Final %B = 100
Gradient time = 3 min
Stop time = 4 min
Flow Rate = 4 mL/min
Wavelength = 220 nm
Solvent A = 0.1 % TFA in 10% methanol/90% water
Solvent B = 0.1 % TFA in 90% methanol/10% water
Condition 4
Column = XTERRA®, CI 8, 3.0 x 50 mm, S7
Start %B = 0
Final %B = 100
Gradient time = 10 min
Stop time = 11 min
Flow Rate = 5 mL/min
Wavelength = 220 nm
Solvent A = 0.2 % H3P04 in 10% methanol/90% water Solvent B = 0.2 % H3P04 in 90% methanol/10% water Condition 5
Column = PHENOMENEX®, CI 8, 3.0 X 50 mm, 10 μιη Start %B = 0
Final %B = 100
Gradient time = 4 min
Stop time = 5 min
Flow Rate = 4 mL/min
Wavelength = 220 nm
Solvent A = 0.1 % TFA in 10% methanol/90% water Solvent B = 0.1 % TFA in 90% methanol/10% water Condition 6
Column = Phenomenex-Luna, CI 8, 4.6 X 50 mm, S 10
Start %B = 0
Final %B = 100
Gradient time = 3 min
Stop time = 4 min
Flow Rate = 4 mL/min
Wavelength = 220 nm
Solvent A = 0.1 % TFA in 10% methanol/90% water Solvent B = 0.1 % TFA in 90% methanol/10% water
Condition 7
Column = Phenomenex-Luna, CI 8, 3. OX 50 mm, S 10 Start %B = 0
Final %B = 100
Gradient time = 2 min
Stop time = 3 min
Flow Rate = 4 mL/min
Wavelength = 220 nm
Solvent A = 0.1 % TFA in 10% methanol/90% water Solvent B = 0.1 % TFA in 90% methanol/10% water
Condition 9 Column = Waters Sunfire, C18, 4.6X150 mm, 3.5 μιη
Start %B = 10
Final %B = 50
Gradient time = 20 min
Stop time = 25 min
Flow Rate = 1 mL/min
Wavelength = 220 & 254 nm
Slovent A = 0.1% TFA in 5% CH3CN/95%H20
Solvent B = 0.1% TFA in 95% CH3CN/5% H20
Condition 9a
Same as Condition 9 with the exception that Stop time = 35 min Condition 9a.1
Column = Waters Sunfire, C18, 4.6X150 mm, 3.5 μιη
Start %B = 30
Final %B = 100
Gradient time = 20 min
Stop time = 25 min
Flow Rate = 1 mL/min
Wavelength = 220 & 254 nm
Slovent A = 0.1% TFA in 5% CH3CN/95%H20
Solvent B = 0.1% TFA in 95% CH3CN/5% H20 Condition 10
Column = Waters Xbridge phenyl, CI 8, 4.6X150 mm, 3 μιη
Start %B = 10
Final %B = 50
Gradient time = 20 min
Stop time = 25 min
Flow Rate = 1 mL/min Wavelength = 220 & 254 nm
Slovent A = 0.1% TFA in 5% CH3CN/95%H20
Solvent B = 0.1% TFA in 95% CH3CN/5% H20 Condition 10a
Same as Condition 10 with the exception that Stop time = 35 min
Condition 10a.1
Column = Waters Xbridge phenyl, CI 8, 4.6X150 mm, 3 μιη Start %B = 40
Final %B = 100
Gradient time = 20 min
Stop time = 25 min
Flow Rate = 1 mL/min
Wavelength = 220 & 254 nm
Slovent A = 0.1% TFA in 5% CH3CN/95%H20
Solvent B = 0.1% TFA in 95% CH3CN/5% Precondition 10b
Column = Sunfire, CI 8, 3.0 X 150 mm, 3.5 μιη
Start %B = 10
Final %B = 40
Gradient time = 15 min
Stop time = 18 min
Flow Rate = 1 mL/min
Wavelength 1 = 220 nm
Wavelength 2 = 254 nm
Solvent A = 0.1 % TFA in 5% MeCN/95% water
Solvent B = 0.1 % TFA in 95% MeCN/5% water
Condition 10c Column = Xbridge Phenyl, C18, 3.0 X 150 mm, 3.5 μιη
Start %B = 10
Final %B = 40
Gradient time = 15 min
Stop time = 18 min
Flow Rate = 1 mL/min
Wavelength 1 = 220 nm
Wavelength 2 = 254 nm
Solvent A = 0.1 % TFA in 5% MeCN/95% water
Solvent B = 0.1 % TFA in 95% MeCN/5% water
Condition lOd
Column = PHENOMENEX®-Luna, CI 8, 2.0 X 50 mm, 3 μιη
Start %B = 0
Final %B = 100
Gradient time = 4 min
Stop time = 5 min
Flow Rate = 0.8 mL/min
Wavelength = 220 nm
Solvent A = 0.1 % TFA in 10% methanol/90% water
Solvent B = 0.1 % TFA in 90% methanol/10% water
Oven temp. = 40 °C
Condition lOe (Agilent 1200 series LC system)
Column = Xbridge phenyl, C18, 4.6X150 mm, 3.5 μιη
Solvent A = Buffer: CH3CN (95:5)
Solvent B = Buffer: CH3CN (5:95)
Buffer = 0.05% TFA in H20 (pH 2.5, adjusted with dilute ammonia) Start %B = 10
Final %B = 100
Gradient time = 12 min Isocratic time = 3 min
Stop time = 23 min
Flow Rate = 1 mL/min
Wavelength = 220 & 254 nm
Condition lOe.l (for a homogeneity index assessment on a Agilent 1200 series LC system)
Column = Sunfire C 18 ((4.6x 150) mm, 3.5 μιη)
Slovent A = Buffer: CH3CN (95:5)
Solvent B = Buffer: CH3CN (5:95)
Buffer = 0.05% TFA in H20 (pH 2.5, adjusted with dilute ammonia)
Start %B = 10
Final %B = 100
Gradient time = 12 min
Isocratic time = 3 min
Stop time = 23 min
Flow Rate = 1 mL/min
Wavelength = 220 & 254 nm Condition 10e.2 (for a homogeneity index assessment on a Agilent 1200 series LC system)
Column = Sunfire C 18 ((4.6x 150) mm, 3.5 μιη)
Slovent A = Buffer: CH3CN (95:5)
Solvent B = Buffer: CH3CN (5:95)
Buffer = 0.05% TFA in H20 (pH 2.5, adjusted with dilute ammonia)
Start %B = 0
Final %B = 50
Gradient time- 1 = 15 min
Final %B = 100
Gradient time-2 = 3 min
Isocratic time = 5 min Stop time = 28 min
Flow Rate = 1 mL/min
Wavelength = 220 & 254 nm
Condition 10e.3 (for a homogeneity index assessment on a Agilent 1200 series LC system)
Column = Xbridge phenyl ((4.6x150) mm, 3.5 μιη)
Solvent A = 10 mM NH4HCO3 in H20 (pH 9.5, adjusted with dilute ammonia)
Solvent B = MeOH
Start %B = 10
Final %B = 100
Gradient time = 12 min
Isocratic time = 8 min
Stop time = 23 min
Flow Rate = 1 mL/min
Wavelength = 220 & 254 nm
Condition 10e.4 (for a homogeneity index assessment on a Agilent 1200 series LC system)
Column = Xbridge phenyl ((4.6x150) mm, 3.5 μιη)
Solvent A = Buffer: CH3CN (95:5)
Solvent B = Buffer: CH3CN (5:95)
Buffer = 0.05% TFA in H20 (pH 2.5, adjusted with dilute ammonia)
Start %B = 10
Final %B = 100
Gradient time = 25 min
Isocratic time = 5 min
Stop time = 36 min
Flow Rate = 1 mL/min
Wavelength = 220 & 254 nm Condition 10e.5 (LC-MS analysis on a Agilent LC-1200 series coupled with 6140 single quad, mass spectrometer, ESI +ve mode, MS range: 100-2000)
Column = Zorbax SB CI 8 1 ((4.6x50) mm, 5 μιη)
Slovent A = MeOH (10%) 4 - 0.1% TFA in H20 (90%)
Solvent B = MeOH (90%) - +■ 0.1% TFA in H20 (10%)
Start %B = 0
Final %B = 100
Gradient time = 2 min
Stop time = 3 min
Flow Rate = 5 mL/min
Wavelength = 220 nm
Condition 10e.6 (LC-MS analysis on a Agilent LC-1200 series coupled with 6140 single quad, mass spectrometer, ESI +ve mode & -ve mode, MS range: 100-2000)
Column = Purospher@star RP-18 ((4.0x55) mm, 3 μιη)
Slovent A = ACN (10%) 4 - 20 mM NH4OAc in H20 (90%)
Solvent B = ACN (90%)■ +■ 20 mM NH4OAc in H20 (10%)
Start %B = 0
Final %B = 100
Gradient time = 2.0 min
Isocratic time = 0.5 min
Stop time = 3 min
Flow Rate = 2.5 mL/min
Wavelength = 220 nm
Condition 10e.7 (LC-MS analysis on a Agilent LC-1200 series coupled with 6330 ion trap mass spectrometer, ESI +ve mode & -ve mode, MS range: 100-2000) Column = Ascentis Express C18 (2.1X50 mm, 2.7 μιη)
Slovent A = CH3CN (2%) + 10 mM NH4COOH in H20 (98%)
Solvent B = CH3CN (98%) + 10 mM NH4COOH in H20 (2%)
Start %B = 0 Final %B = 100
Gradient time = 1.5 min
Isocratic time = 1.7 min
Stop time = 4 min
Flow Rate = 1.0 mL/min
Wavelength = 220 nm
Condition lOf (Agilent LC-1200 series coupled with 6140 single quad, mass spectrometer, ESI +ve mode)
Column = Zorbax SB, C18, 4.6X50 mm, 5 μιη
Slovent A = MeOH (10%) + 0.1% TFA in H20 (90%)
Solvent B = MeOH (90%) + 0.1% TFA in H20 (10%)
Start %B = 0
Final %B = 100
Gradient time = 3 min
Isocratic time = 1 min
Stop time = 5 min
Flow Rate = 5 mL/min
Wavelength = 220 nm
Condition lOg (Agilent LC-1200 series coupled with 6140 single quad, mass spectrometer, ESI +ve mode)
Column = Ascentis Express, C-8, 2.1X5 mm, 2.7 μιη
Slovent A = CH3CN (2%) + 10 mM NH4COOH in H20 (98%)
Solvent B = CH3CN (98%) + 10 mM NH4COOH in H20 (2%)
Start %B = 0
Final %B = 100
Gradient time = 1.5 min
Isocratic time = 1.7 min
Stop time = 4 min
Flow Rate = 1 mL/min Wavelength = 220 nm Condition lOh
Column = PHENOMENEX®-Luna, CI 8, 4.6 X 30 mm, S10
Start %B = 0
Final %B = 100
Gradient time = 3 min
Stop time = 4 min
Flow Rate = 4 mL/min
Wavelength = 220 nm
Solvent A = 0.1 % TFA in 10% methanol/90% water
Solvent B = 0.1 % TFA in 90% methanol/10% water
Condition 11
Column = PHENOMENEX®-Luna, C 18, 50X2 mm, 3 μιη
Start %B = 0
Final %B = 100
Gradient time = 4 min
Stop time = 5 min
Flow Rate = 0.8 mL/min
Wavelength = 220 nm
Solvent A = 5% methanol/95% water : 10 mM ammonium acetate
Solvent B = 95% methanol/5% water : 10 mM ammonium acetate
Oven temperature = 40 °C
Condition 12 (Waters Acquity HPLC with Waters PDA UV-Vis detection and Waters SQ MS-ESCI probe)
Column = Waters Acquity BEH, C18, 150 X 2.1 mm ID, 1.7 μιη (at 35 °C) Mobile phase A = 0.05% TFA in water
Mobile phase B = 0.05% TFA in acetonitrile Solvent system: Hold 10%B: 0-lmin; 10-98%B: l-32min; Hold 98%B: 32-35 min; 98-10%B: 35-35.3 min; hold 10%B: 35.3-40 min
Flow rate = 0.35 ml/min
UV detection = 335 nm
EXAMPLES
Exam le OL- 1
Figure imgf000052_0001
Exam le OL-1, step a
Figure imgf000052_0002
4,4'-Oxybis-(benzoic acid) dimethyl ester (1.5 g, 5.24 mmol) was added to a solution of chloroiodomethane (3.05 mL, 41.92 mmol) in tetrahydrofuran (50 mL) and the resulting solution was cooled to -78 °C. A 1.8M solution of LDA (29 mL, 52 mmol) in tetrahydrofuran was added dropwise and the resulting brown suspension was stirred at -78 °C for 15 min. A solution of glacial acetic acid in tetrahydrofuran (10 mL in 50 mL) was then added slowly and the brown mixture was stirred at -78 °C for 10 min. before allowing it to reach room temperature. The mixture was taken up in ethyl acetate and brine (1 : 1, 50 mL) and the organic layer was then separated, washed with a sat. solution of sodium bicarbonate and brine, dried (MgSC^), filtered, and concentrated in vacuo. The residue was submitted to flash chromatography (silica gel; 15 % ethyl acetate/hexanes) to provide OL-1 a as a pale brown solid (0.6 g). XH NMR (500 MHz, DMSO-d6) δ ppm 5.17 (s, 4 H), 7.22 (d, J=8.85 Hz, 4 H), 8.06 (d, J=8.85 Hz, 4 H). LC (Cond. 2): RT = 1.53 minutes, 97% homogeneity index; LCMS: Anal. Calcd. For (M-H)+ C16HiiCl203: 321.01; found: 321.30.
Exam le OL-1, step b
Figure imgf000053_0001
A mixture of OL-la (0.6 g, 1.85 mmol) and sodium diformylamide (0.42 g, 4.45 mmol) in acetonitrile (20 mL) was heated to reflux for 4 h. The solvent was the removed under reduced pressure and the remaining residue was redissolved in a 5% HC1 solution in ethanol (30 mL) and heated to reflux temperature for 2 h. The mixture was cooled in an ice-water bath and the resulting precipitate was filtered, washed with ethanol and ether and dried in vacuo. The recovered pale brow solid was used without further purification (0.66 g). XH NMR (500 MHz, DMSO-d6) δ ppm 4.54 (s, 4 H), 7.25 (d, J=8.85 Hz, 4 H), 8.11 (d, J=8.55 Hz, 4 H), 8.52 (br. s, 6 H). LC (Cond. 1): RT = 1.24 min; LRMS: Anal. Calcd. For (M+H)+ C^Hn^Os 285.12; found: 285.19.
Exam le OL-1, step c
Figure imgf000053_0002
N,N-Diisopropylethylamine (1.13 mL, 6.47 mmol) was added dropwise, over 15 minutes, to a heterogeneous mixture of N-Boc-L-proline (0.8 g, 3.73 mmol),
HATU (1.48 g, 3.88 mmol), OL-lb (0.66 g, 1.85 mmol) and dimethylformamide (15 mL), and stirred at ambient condition for 4 h. Most of the volatile components were removed in vacuo, and the resulting residue was partitioned between ethyl acetate (30 mL) and water (20 mL). The organic layer was washed with water (20 mL) and brine, dried (MgS04), filtered, and concentrated in vacuo. A silica gel mesh was prepared from the residue and submitted to flash chromatography (silica gel; 65-85 % ethyl acetate/ hexanes) to provide OL-lc as a pale brown solid (0.7 g). XH NMR (500 MHz, DMSO-d6) δ ppm 1.39/1.34 (rotomers, s, 18 H), 1.70 - 1.91 (m, 6 H), 2.00 - 2.25 (m, J=8.55 Hz, 2 H), 3.22 - 3.33 (m, 2 H), 3.32 - 3.47 (m, J=10.68, 10.68 Hz, 2 H), 4.08 - 4.26 (m, 2 H), 4.56 (ddd, J=36.93, 18.16, 5.34 Hz, 4 H), 7.19 (d, J=8.85 Hz, 4 H), 8.06 (d, J=7.93 Hz, 4 H), 8.11 - 8.24 (m, J=5.49 Hz, 2 H). LC (Cond. 1): RT = 2.55 min; LRMS: Anal. Calcd. for (M+H)+ C36H47 4O9 679.33; found: 679.44.
Exam le OL-1, step d
Figure imgf000054_0001
A mixture of OL-lc (0.7 g, 1.025 mmol) and ammonium acetate (0.79 g, 10 mmol) in xylenes (5 mL) was heated in a sealed tube at 140 °C for 1.2 hours under microwave radiation. The volatile component was removed in vacuo, and the residue was partitioned carefully between ethyl acetate and water, whereby enough saturated sodium bicarbonate solution was added so as to make the pH of the aqueous phase slightly basic after the shaking of the biphasic system. The layers were separated, and the aqueous layer was extracted with additional ethyl acetate. The combined organic phase was washed with brine, dried (MgS04), filtered, and concentrated in vacuo. The resulting material submitted to a flash chromatography (silica gel; 15- 25% acetone/dichloromethane) to provide OL-ld (0.13 g). ¾ NMR (500 MHz, DMSO-d6) δ ppm 1.17/1.48 (rotomers, s, 18 H), 1.76 - 1.93 (m, 4 H), 1.93 - 2.06 (m, J=10.07 Hz, 2 H), 2.10 - 2.30 (m, 2 H), 3.32 - 3.39 (m, 2 H), 3.45 - 3.62 (m, 2 H), 4.69 - 4.80 (m, 1 H), 4.83 (d, J=7.02 Hz, 1 H), 6.97 (d, J=7.93 Hz, 4 H), 7.00 - 7.22 (m, 1 H), 7.40/7.62 (rotomers, m, 2 H), 7.73 (d, J=8.55 Hz, 3 H), 11.74/11.81/12.07 (rotomers, s, 2 H). LC (Cond. 2): RT = 1.53 minutes, 93% homogeneity index; LCMS: Anal. Calcd. for (M+H)+ C36H44N605: 641.34; found: 641.50. Exam le OL-1, step e
Figure imgf000055_0001
To a solution of OL-1 d (0.13 g, 0.2 mmol) in 30 mL dichloromethane was added 1 mL of a 4.0M solution of HQ in dioxane. The reaction was stirred for 2 hours at room temperature and concentrated under reduced pressure. The resulting residue was redissolved in a minimum amount of methanol and the desired product was triturated with ether, filtered and dried in vacuo. A pale tan solid (0.1 lg) was recovered and used without further purification. XH NMR (500 MHz, DMSO-d6) δ ppm 1.92 - 2.07 (m, 2 H), 2.12 - 2.25 (m, 2 H), 2.37 - 2.47 (m, 4 H), 3.30 - 3.47 (m, 4 H), 4.90 - 5.02 (m, 2 H), 7.15 (d, J=8.55 Hz, 4 H), 7.91 (d, J=8.24 Hz, 4 H), 7.97 (s, 2 H), 9.74 (br. s, 2 H), 10.25 (br. s, 2 H). Note: the signal of the imidazole NH was too broad to assign a chemical shift. LC (Cond. 1): RT = 1.68 min; LRMS: Anal. Calcd. for (M+H)+ C26H29N60 441.24; found: 441.30. Example OL-1
HATU (52 mg, 0.137 mmol) was added to a mixture of OL-le (35 mg, 0.06 mmol), diisopropylethylamine (58 \L, 0.33 mmol) and Cap-1 (22 mg, 0.12 mmol) in dimethylformamide (3 mL), and the resulting mixture was stirred at ambient for 3 h.
The volatile component was removed in vacuo, and the residue was purified by a reverse phase HPLC system (water/methanol/TFA) to provide the TFA salt of
Example OL-1 as an off-white solid (32 mg). XH NMR (500 MHz, DMSO-D6) δ ppm
1.82 - 1.93 (m, 2 H), 1.94 - 2.08 (m, 4 H), 2.14 - 2.24 (m, 2 H), 2.29 - 2.40 (m, 4 H),
2.83 (s, 4 H), 2.97 - 3.07 (m, 4 H), 3.97 (t, J=8.24 Hz, 2 H), 4.89 - 5.20 (m, 2 H), 5.34 - 5.73 (m, 2 H), 7.00 - 7.22 (m, 6 H), 7.49 - 7.64 (m, 9 H), 7.70 - 7.89 (m, 5 H), 10.20 (br.s, 2 H). LC (Cond. 1): RT = 2.48 min; LRMS: Anal. Calcd. for (M+H)+
C46H51N8O3 763.41; found: 763.56; HRMS: Anal. Calcd. for (M+H)+ C46H51N8O3 763.4084; found: 763.4109. Exam les OL-2 and OL-3
Figure imgf000056_0001
Examples OL-2 and OL-3 were prepared as TFA salts by substituting the respective acids for Cap-1 using the same method described for Example OL-1.
Figure imgf000056_0003
Exam le OL-4
Figure imgf000056_0002
Example OL-4, step a
Figure imgf000057_0001
Methyl 3-bromobenzoate (5 g, 23.2 mmol), methyl 4-hydroxybenzoate (5.3 g, 34.87 mmol), cesium carbonate (15.12 g, 46.4 mmol), copper (I) iodide (0.44 g, 2.32 mmol) and Ν,Ν-dimethyl hydrochloride (0.97 g, 6.96 mmol) were combined in dioxane (100 ml) in a sealed tube and the resulting mixture was heated to 90 °C for 15 h. The solvent was removed under reduced pressure and the residue was taken up in ethyl acetate/water (1 : 1, 200 mL). The organic layer was washed with saturated aq. sodium carbonate, water and brine, dried (MgSC^), filtered and concentrated in vacuo. The resulting material was purified by flash chromatography (silica gel, 5% ethyl acetate/hexanes) to provide OL-4a as a clear oil that solidified upon standing (5.8 g). ¾ NMR (500 MHz, DMSO-d6) δ ppm 3.83 (s, 6 H), 7.10 (d, J=8.55 Hz, 2 H), 7.43 (dd, J=8.09, 2.59 Hz, 1 H), 7.56 (s, 1 H), 7.61 (t, J=7.93 Hz, 1 H), 7.81 (d, J=7.63 Hz, 1 H), 7.98 (d, J=8.55 Hz, 2 H). LC (Cond. 2): RT = 1.53 minutes;
LCMS: Anal. Calcd. for (M+l)+ Ci6H1505: 287.01; found: 287.23.
Example OL-4, step b
Figure imgf000057_0002
OL-4b was prepared from OL-4a, according to the same procedure used for the preparation of OL-la. ¾ NMR (500 MHz, DMSO-d6) δ ppm 5.14 (s, 2 H), 5.20 (s, 2 H), 7.13 (d, J=8.85 Hz, 2 H), 7.43 - 7.49 (m, 1 H), 7.64 (t, J=8.09 Hz, 1 H), 7.66 - 7.68 (m, 1 H), 7.85 (d, J=7.93 Hz, 1 H), 8.02 (d, J=8.85 Hz, 2 H). LC (Cond. 1): RT = 1.51 min; LRMS: Anal. Calcd. for (M+H)+ Ci6H13Cl203 323.02; found: 323.07. Example OL-4, step c
Figure imgf000058_0001
OL-4c was prepared from Example OL-4b, according to the same procedure used for the preparation of Example OL- lb. XH NMR (500 MHz, DMSO-d6) δ ppm. 4.52 (s, 2 H), 4.56 (s, 2 H), 7.16 (d, J=8.85 Hz, 2 H), 7.51 (dd, J=8.24, 2.44 Hz, 1 H), 7.68 (t, J=7.93 Hz, 1 H), 7.71 - 7.75 (m, 1 H), 7.91 (d, J=7.63 Hz, 1 H), 8.07 (d, J=8.85 Hz, 2 H), 8.60 (s, 6 H). LC (Cond. 2): Anal. Calcd. for Ci6H17 203 (M+H)+ 285.12; found: 285.15. Example OL-4d
Boc
Figure imgf000058_0002
Example OL-4d was prepared from Example OL-4c, according to the same procedure used for the preparation of Example OL- lc. XH NMR (500 MHz, DMSO- d6) 5 ppm 1.38/1.33 (rotomers, s, 18 H), 1.69 - 1.91 (m, 6 H), 1.95 - 2.19 (m, 2 H), 3.20 - 3.31 (m, 2 H), 3.32 - 3.47 (m, 2 H), 4.08 - 4.23 (m, 2 H), 4.43 - 4.68 (m, 4 H), 7.10 (d, J=8.55 Hz, 2 H), 7.42 (dd, J=8.09, 1.98 Hz, 1 H), 7.57 - 7.70 (m, 2 H), 7.86 (d, J=7.02 Hz, 1 H), 8.03 (d, J=7.93 Hz, 2 H), 8.08 - 8.24 (m, 2 H). LC (Cond. 1): RT = 2.53 min; LRMS: Anal. Calcd. for C36H47 4O9 (M+H)+ 679.33; found: 679.42. Example OL-4e
Figure imgf000059_0001
Example OL-4e was prepared from Example OL-4d, according to the same procedure used for the preparation of Example OL-ld. XH NMR (500 MHz, DMSO- d6) 5 ppm 1.14/1.17 /1.38/1.40(rotomers, s, 18 H), 1.71 - 2.06 (m, 6 H), 2.09 - 2.28 (m, J=25.79, 13.89 Hz, 2 H), 3.31 - 3.40 (m, 2 H), 3.51 (s, 2 H), 4.65 - 4.89 (m, 2 H), 6.80 (t, J=8.39 Hz, 1 H), 6.91 - 7.10 (m, 2 H), 7.28 - 7.55 (m, 5 H), 7.58 - 7.80 (m, 2 H), 11.66 - 12.21 (m, 2 H). LC (Cond. 1): RT = 2.98 min; LRMS: Anal. Calcd. for C36H45 605 (M+H)+ 641.34; found: 641.39.
Exam le OL-4f
Figure imgf000059_0002
Example OL-4f was prepared from Example OL-4e, according to the same procedure used for the preparation of Example OL-le. XH NMR (500 MHz, DMSO- d6) δ ppm 1.94 - 2.06 (m, 2 H), 2.09 - 2.23 (m, 2 H), 2.28 - 2.38 (m, 1 H), 2.37 - 2.47 (m, 3 H), 3.27 - 3.50 (m, 4 H), 4.90 (br. s, 1 H), 4.99 (br. s, 1 H), 7.00 (d, J=7.93 Hz, 1 H), 7.13 (d, J=8.55 Hz, 2 H), 7.49 (t, J=7.93 Hz, 1 H), 7.62 (s, 1 H), 7.69 (d, J=7.63 Hz, 1 H), 7.92 (d, J=8.55 Hz, 2 H), 7.99 (s, 2 H), 9.58 (br. s, 1 H), 9.84 (br. s, 1 H), 10.25 (br. d, J=22.28 Hz, 2 H). Note: the signal of the imidazole NH was too broad to assign a chemical shift. LC (Cond. 1): RT = 1.72 min; LRMS: Anal. Calcd. for C26H29N60 (M+H)+ 441.24; found: 441.29. Example OL-4
Example OL-4 was prepared from Example OL-4f according to the same procedure used for the preparation of Example OL-1. XH NMR (500 MHz, DMSO- d6) δ ppm 1.83 - 1.92 (m, 4 H), 1.95 - 2.09 (m, 8 H), 2.1 1 - 2.23 (m, 4 H), 2.75 - 2.88 (m, 2 H), 2.92 (s, 1 H), 2.96 - 3.12 (m, 2 H), 3.97 (t, J=8.39 Hz, 2 H), 5.14 (dd, J=17.70, 7.02 Hz, 2 H), 5.41 (s, 2 H), 6.91 - 7.11 (m, J=49.75 Hz, 2 H), 7.14 (d, J=7.93 Hz, 2 H), 7.44 - 7.68 (m, 12 H), 7.79 (d, J=8.54 Hz, 2 H), 7.84 - 8.02 (m, J=9.16 Hz, 2 H), 10.21 (s, 1 H). Note: the signal of the imidazole NH was too broad to assign a chemical shift. LC (Cond. 1): RT = 1.78 min; LRMS: Anal. Calcd. for C46H51N8O3 (M+H)+ 763.41; found: 763.56. HRMS: Anal. Calcd. for C46H51N8O3 (M+H)+ 763.4084; found: 763.4067.
Exam les OL-5 and OL-6
Figure imgf000060_0001
Examples OL-5 to OL-6 were prepared as TFA salts by substituting the respective acids for Cap-1 according to the same method described for Example OL-
4.
Figure imgf000060_0002
Example 0 RT (LC-Cond.); MS data
Acid
OL-6 RT = 1.96 min. LC (Cond. 2); LCMS: Anal. Calcd.
for: C46H47 807 (M+H)+ 823.36; Found: 823.5 L HRMS: Anal. Calcd. for: C46H47 807 (M+H)+
Figure imgf000061_0001
823.3568; Found: 823.3588
Cap-4
Exam le OL-7
Figure imgf000061_0002
Exam le OL-7a
Figure imgf000061_0003
To a solution of 4-(chloromethyl)benzoic acid (8.53 g, 50 mmol) and sodium hydroxide (10 g, 0.25 mol) in water (40 mL) was added a solution of 4- hydroxybenzoic acid (6.9 g, 50 mmol) and sodium hydroxide (6 g, 0.15 mol) in water (50 mL). The resulting mixture was heated to reflux temperature for 15 h and, after cooling to room temperature, it was filtered and acidified with a solution of sulfuric acid in water (1 : 1, 200 mL). The resulting precipitate was filtered off, washed with water and dried in vacuo. The off-white solid (9.3 g) was used without further purification. XH NMR (500 MHz, DMSO-d6) δ ppm 5.27 (s, 1 H), 7.10 (d, J=8.85 Hz, 2 H), 7.56 (d, J=8.24 Hz, 2 H), 7.89 (d, J=8.85 Hz, 2 H), 7.96 (d, J=8.24 Hz, 2 H), 12.36 (br. s, 2 H). LC (Cond. 2), Note: The molecule did not ionize well in the LC/MS system and therefore an exact mass was not obtained. Exam le OL-7b
Figure imgf000062_0001
(Diazomethyl)trimethylsilane (15 mL, 30 mmol, 2 M in hexanes) was added dropwise to a suspension of Example OL-7a (2.04 g, 7.5 mmol) in methanol (30 mL) at 5 °C (ice-water bath). When the addition was complete, the cooling bath was removed and the mixture was stirred at ambient overnight. The solvent was removed under reduced pressure and the remaining residue was taken up in ethyl acetate, washed with water, sodium bicarbonate and brine, dried (MgSC^), filtered and concentrated in vacuo. The resulting material was purified by flash chromatography (silica gel, 10% ethyl acetate/hexanes) to provide Example OL-7b as white solid (1.5 g). XH NMR (500 MHz, DMSO-d6) δ ppm 3.80 (s, 3 H), 3.85 (s, 3 H), 5.29 (s, 2 H), 7.13 (d, J=8.85 Hz, 2 H), 7.59 (d, J=8.24 Hz, 2 H), 7.91 (d, J=9.16 Hz, 2 H), 7.98 (d, J=8.24 Hz, 2 H). LC (Cond. 1): RT = 2.56 min; LRMS: Anal. Calcd. for Ci7Hi605 (M+H)+ 301.11 ; found: 301.22.
Example OL-7c
Figure imgf000062_0002
Example OL-7c was prepared from Example OL-7b, according to the same procedure used for the preparation of Example OL-la. XH NMR (500 MHz, DMSO- d6) δ ppm 5.09 (s, 2 H), 5.18 (s, 2 H), 5.34 (s, 2 H), 7.15 (d, J=8.85 Hz, 2 H), 7.62 (d, J=8.55 Hz, 2 H), 7.95 (d, J=9.16 Hz, 2 H), 8.00 (d, J=8.55 Hz, 2 H). LC (Cond. 1): RT = 2.36 min; LRMS: Anal. Calcd. for Ci7H15Cl203 (M+H)+ 337.04 found: 337.09. Exam le OL-7d
Figure imgf000063_0001
Example OL-7d was prepared from Example OL-7c, according to the same procedure used for the preparation of Example OL- lb. XH NMR (500 MHz, DMSO- de) δ ppm 4.49 (s, 2 H), 4.57 (s, 2 H), 5.38 (s, 2 H), 7.19 (d, J=8.85 Hz, 2 H), 7.66 (d, J=8.24 Hz, 2 H), 8.00 (d, J=8.85 Hz, 2 H), 8.05 (d, J=8.24 Hz, 2 H), 8.45 (s, 6 H). LC (Cond. 1): RT = 1.26 min; LRMS: Anal. Calcd. for Οι7Η19Ν1203 (M+H)+ 299.14 found: 299.19.
Exam le OL-7e
Figure imgf000063_0002
Example OL-7e was prepared from Example OL-7d, according to the same procedure used for the preparation of Example OL- lc. XH NMR (500 MHz, DMSO- d6) δ ppm 1.30 - 1.47 (m, 18 H), 1.65 - 1.89 (m, 6 H), 2.01 - 2.20 (m, 2 H), 3.24 - 3.34 (m, 2 H), 3.34 - 3.43 (m, 2 H), 4.09 - 4.25 (m, 2 H), 4.44 - 4.68 (m, 4 H), 5.33 (s, 2 H), 7.13 (d, J=8.85 Hz, 2 H), 7.60 (d, J=8.24 Hz, 2 H), 7.99 (dd, J=20.29, 8.09 Hz, 4 H), 8.04 - 8.22 (m, J=31.74 Hz, 2 H). LC (Cond. 2): RT = 1.53 minutes, 97% homogeneity index; LCMS: Anal. Calcd. for
Figure imgf000063_0003
(M+l)+ 693.35; found: 693.32. Exam le OL-7f
Figure imgf000064_0001
Example OL-7f was prepared from Example OL-7e, according to the same procedure used for the preparation of Example OL-ld. XH NMR (500 MHz, DMSO- d6) δ ppm 1.06 - 1.52 (m, 18 H), 1.69 - 2.08 (m, 6 H), 2.08 - 2.30 (m, 2 H), 3.33 - 3.43 (m, 2 H), 3.52 (s, 2 H), 4.75 (s, 1 H), 4.83 (s, 1 H), 5.06 (s, 2 H), 6.92 - 7.10 (m, J=7.63 Hz, 2 H), 7.20 - 7.33 (m, 1 H), 7.40 (d, J=7.63 Hz, 2 H), 7.46 (d, J=8.55 Hz, 1 H), 7.64 (d, J=7.63 Hz, 2 H), 7.75 (d, J=7.93 Hz, 2 H), 11.47 - 12.18 (m, 2 H). LC (Cond. 1): RT = 2.94 min; LRMS: Anal. Calcd. for C37H47 6O3 (M+H)+ 655.36 found: 655.39.
Exam le OL-7g
Figure imgf000064_0002
Example OL-7g was prepared from Example OL-7fe, according to the same procedure used for the preparation of Example OL-le. XH NMR (500 MHz, DMSO- d6) δ ppm 1.93 - 2.07 (m, 2 H), 2.13 - 2.26 (m, 2 H), 2.40 - 2.48 (m, 3 H), 3.31 - 3.52 (m, 5 H), 4.91 - 5.09 (m, 2 H), 5.21 (s, 2 H), 7.17 (d, J=8.85 Hz, 2 H), 7.57 (d, J=8.24 Hz, 2 H), 7.87 (d, J=8.85 Hz, 2 H), 7.93 (d, J=8.24 Hz, 2 H), 8.03 (d, J=16.79 Hz, 2 H), 9.78 (s, 1 H), 9.95 (s, 1 H), 10.31 (s, 2 H). Note: the signal of the imidazole NH was too broad to assign a chemical shift. LC (Cond. 2): RT = 0.62 minutes; LCMS: Anal. Calcd. for C27H31N6O (M+l)+ 455.26; found: 455.34.
Example OL-7
Example OL-7 was prepared from Example OL-7g according to the same procedure used for the preparation of Example OL-1. XH NMR (500 MHz, DMSO- d6) δ ppm 1.90 (d, J=2.44 Hz, 2 H), 2.04 (d, J=3.66 Hz, 4 H), 2.21 (s, 2 H), 2.36 (d, J=1.83 Hz, 2 H), 2.42 - 2.46 (m, 4 H), 2.83 (br. s, 4 H), 2.99 - 3.11 (m, 2 H), 3.98 (d, J=7.02 Hz, 2 H), 5.18 (s, 4 H), 5.42 (d, J=8.55 Hz, 2 H), 7.08 - 7.28 (m, 3 H), 7.50 - 7.67 (m, 1 1 H), 7.73 (d, J=7.32 Hz, 2 H), 7.80 (d, J=7.93 Hz, 2 H), 7.85 - 7.98 (m, J=10.38 Hz, 1 H). Note: the signal of the imidazole NH was too broad to assign a chemical shift. LC (Cond. 3): RT = 2.94 min; LRMS: Anal. Calcd. for C47H52N8O3 (M+H)+ 777.42 found: 777.51 ; HRMS: Anal. Calcd. for C47H53N8O3 (M+H)+ 777.4241 found: 777.4265.
Exam le OL-8
Figure imgf000065_0001
Example OL-8 was prepared as TFA salt by substituting the respective acid for Cap-1 according to the same method described for Example OL-7.
Figure imgf000065_0003
Figure imgf000065_0002
Exam le OL-9a
Figure imgf000066_0001
Palladium on carbon (10%, 300 mg) was added to a solution of dimethyl 4,4'- (ethene-l,2-diyl)dibenzoate (2g, 6.76 mmol) in methanol (100 mL). The suspension was purged with N2; placed under 1 atm of ¾ (balloon) and stirred at ambient overnight. The mixture was then filtered through a plug of diatomaceous earth
(CELITE ) and the solvent was concentrated under reduced pressure. A white solid was recovered (1.42 g) and used without further purification. XH NMR (500 MHz, CDC13) δ ppm 2.98 (s, 4 H), 3.90 (s, 6 H), 7.18 (d, J=7.32 Hz, 4 H), 7.93 (d, J=7.32 Hz, 4 H). LC (Cond. 1): RT = 2.65 min; LRMS: Anal. Calcd. for Ci8H1904 (M+H)+ 299.13 found: 299.21.
Exam le OL-9b
Figure imgf000066_0002
Example OL-9b was prepared from Example OL-9a, according to the same procedure used for the preparation of Example OL-la. XH NMR (500 MHz, CDCI3) δ ppm 3.02 (s, 4 H), 4.67 (s, 4 H), 7.25 (d, J=8.24 Hz, 4 H), 7.86 (d, J=8.24 Hz, 4 H). LC (Cond. 2): RT = 0.62 min; LCMS: Anal. Calcd. for Ci8H17Cl202 (M+l)+ 335.06; found: 335.12.
Exam le OL-9c
Figure imgf000066_0003
Example OL-9c was prepared from Example OL-9b, according to the same procedure used for the preparation of Example OL-lb. XH NMR (500 MHz, DMSO- d6) δ ppm 3.03 (s, 4 H), 4.51 (s, 4 H), 7.43 (d, J=8.55 Hz, 4 H), 7.92 (d, J=8.24 Hz, 4 H), 8.50 (s, 6 H). LC (Cond. 3): RT = 1.90 min; LRMS: Anal. Calcd. for
Ci8H21N202 (M+H)+ 297.16 found: 297.24.
Example OL-9d
Figure imgf000067_0001
Example OL-9d was prepared from Example OL-9c, according to the same procedure used for the preparation of Example OL- lc. XH NMR (500 MHz, CDC13) δ ppm 1.38 - 1.56 (m, 18 H), 1.83 - 2.00 (m, 4 H), 2.08 - 2.38 (m, 4 H), 3.01 (s, 4 H), 3.32 - 3.65 (m, 4 H), 4.28 (br. s, 1 H), 4.39 (br. s, 1 H), 4.63 - 4.71 (m, 2 H), 4.72 - 4.84 (m, J=4.58 Hz, 2 H), 7.01 (s, 1 H), 7.18 - 7.33 (m, 4 H), 7.47 (s, 1 H), 7.87 (d, J=7.93 Hz, 4 H). LC (Cond. 1): RT = 2.56 min; LRMS: Anal. Calcd. for
C38H51 4O8 (M+H)+ 691.37, found: 691.48.
Exam le OL-9e
Figure imgf000067_0002
Example OL-9e was prepared from Example OL-9d, according to the same procedure used for the preparation of Example OL-ld. XH NMR (500 MHz, DMSO- d6) 5 ppm 1.15/1.39(rotomers, s, 18 H), 1.75 - 1.92 (m, 3 H), 1.96 (s, 3 H), 2.09 - 2.27 (m, J=32.04 Hz, 2 H), 2.81 - 2.90 (m, 4 H), 3.31 - 3.40 (m, 2 H), 3.52 (s, 2 H), 4.66 - 4.88 (m, J=35.40 Hz, 2 H), 7.14 (d, J=7.02 Hz, 4 H), 7.21 (s, 1 H), 7.31 - 7.41 (m, 1 H), 7.51 (d, J=6.41 Hz, 1 H), 7.61 (d, J=7.93 Hz, 3 H), 11.62 - 12.08 (m, 2 H). LC (Cond. 1): RT = 2.20 min; LRMS: Anal. Calcd. for
Figure imgf000067_0003
(M+H)+ 653.38, found: 653.47. Exam le OL-9f
Example OL-9f was prepared from Example OL-9e, according to the same procedure used for the preparation of Example OL-le. XH NMR (500 MHz, DMSO- d6) δ ppm 1.92 - 2.05 (m, 2 H), 2.1 1 - 2.25 (m, 2 H), 2.40 - 2.47 (m, 4 H), 2.89 - 3.03 (m, 4 H), 3.32 - 3.39 (m, 2 H), 3.40 - 3.49 (m, 2 H), 5.02 (t, J=7.78 Hz, 2 H), 7.32 (d, J=8.24 Hz, 4 H), 7.80 (d, J=8.24 Hz, 4 H), 9.91 (s, 2 H), 10.32 (s, 2 H). Note: the signal of the imidazole NH was too broad to assign a chemical shift. LC (Cond. 5): RT = 2.52 min; LRMS: Anal. Calcd. for C28H33N6 (M+H)+ 453.28, found: 453.31.
Example OL-9
Example OL-9 was prepared from Example OL-9f, according to the same procedure used for the preparation of Example OL-1. XH NMR (500 MHz, DMSO- d6) δ ppm 1.82 - 1.95 (m, 2 H), 1.97 - 2.10 (m, 4 H), 2.15 - 2.27 (m, J=4.58 Hz, 2 H), 2.28 - 2.45 (m, 4 H), 2.72 - 2.88 (m, 4 H), 2.94 (s, 5 H), 2.98 - 3.07 (m, 2 H), 3.97 (t, J=9.77 Hz, 2 H), 5.17 (d, J=6.10 Hz, 2 H), 5.41 (s, 2 H), 7.12 (s, 1 H), 7.38 (d, J=7.32 Hz, 4 H), 7.51 - 7.62 (m, 9 H), 7.69 (d, J=7.93 Hz, 4 H), 7.93 (s, 2 H). Note: the signal of the imidazole NH was too broad to assign a chemical shift. LC (Cond. 5): RT = 2.87 min; LRMS: Anal. Calcd. for C48H55N8O2 (M+H)+ 775.44 found: 775.51 ; HRMS: Anal. Calcd. for C48H55N8O2 (M+H)+ 775.4448 found: 775.4454.
Exam les OL-10 to OL-13
Figure imgf000068_0002
Examples OL-10 to OL-13 were prepared as TFA salts by substituting the respective acids for Cap-1 according to the same method described for Example OL- 9.
Figure imgf000069_0001
Exam le OL-14
Figure imgf000070_0001
Triethyl silane (5.84 mL, 36.55 mmol) was added dropwise to a solution of methyl 4-formylbenzoate (5 g, 30.46 mmol) and bismuth tribromide (0.273 g, 0.61 mmol) in acetonitrile (75 mL) and the opaque solution was stirred at ambient for 15 min. Volatiles were removed under reduced pressure and the remaining residue was taken up in ethyl acetate. The organic layer was then washed with water and brine, dried (MgSC^), filtered and concentrated in vacuo. The recovered solid was then submitted to flash chromatography (silica gel; 10-20% ethyl acetate/hexanes) to provide OL-14a (3.05 g). ¾ NMR (500 MHz, CDC13) δ ppm 3.84 (s, 6 H), 4.65 (s, 4 H), 7.51 (d, J=8.24 Hz, 4 H), 7.95 (d, J=8.24 Hz, 4 H). LC (Cond. 2): RT = 1.68 min; LCMS: Anal. Calcd. for Ci8H1905 (M+H)+ 315.12; found: 315.26.
Example OL-14b
Figure imgf000070_0002
Example OL-14b was prepared from Example OL-14a, according to the same procedure used for the preparation of Example OL-la. XH NMR (500 MHz, DMSO- d6) δ ppm 4.67 (s, 4 H), 5.19 (s, 4 H), 7.55 (d, J=8.24 Hz, 4 H), 7.98 (d, J=8.24 Hz, 4 H). LC (Cond. 5): RT = 3.85 min; LRMS: Anal. Calcd. for CisHnC^Os (M+H)+ 351.05, found: 351.06. Example OL-14c
Figure imgf000071_0001
2 HCI
Example OL-14c was prepared from Example OL-14b, according to the same procedure used for the preparation of Example OL-lb. XH NMR (500 MHz, DMSO- de) δ ppm 4.55 (s, 4 H), 4.69 (s, 4 H), 7.57 (d, J=8.24 Hz, 4 H), 8.02 (d, J=8.55 Hz, 4 H), 8.53 (s, 6 H). LC (Cond. 1): RT = 1.29 min; LRMS: Anal. Calcd. for
Ci8H21N203 (M+H)+ 313.15, found: 313.21.
Exam le OL-14d
Figure imgf000071_0002
Example OL-14d was prepared from Example OL-14c, according to the same procedure used for the preparation of Example OL- lc. XH NMR (500 MHz, DMSO- d6) δ ppm 1.34/1.40 (rotomers, s, 18 H), 1.70 - 1.92 (m, 6 H), 1.99 - 2.20 (m, 2 H), 3.24 - 3.33 (m, 2 H), 3.35 - 3.44 (m, 2 H), 4.11 - 4.25 (m, 2 H), 4.50 - 4.65 (m, 4 H), 4.66 (s, 4 H), 7.53 (d, J=8.24 Hz, 4 H), 7.99 (d, J=7.93 Hz, 4 H), 8.09 - 8.23 (m, 2 H). LC (Cond. 1): RT = 2.49 min; LRMS: Anal. Calcd. for C38H51 4O9 (M+H)+ 707.36 found: 707.50.
Exam le OL-14e
Figure imgf000071_0003
Example OL-14e was prepared from Example OL-14d, according to the same procedure used for the preparation of Example OL-ld. XH NMR (500 MHz, DMSO- d6) δ ppm 1.14/ 1.39 (rotomers, s, 18 H), 1.76 - 2.06 (m, 6 H), 2.08 - 2.30 (m, 2 H), 3.31 - 3.41 (m, 2 H), 3.52 (s, 2 H), 4.49 (s, 4 H), 4.70 - 4.78 (m, 1 H), 4.79 - 4.90 (m, 1 H), 7..29/7 '.35 (rotomers, d, J=7.93 Hz, 4 H), 7.40 - 7.54 (m, 2 H), 7.71/761
(rotomers, d, J=7.93 Hz, 4 H), 1 1.72 - 12.19 (m, 2 H). LC (Cond. 1): RT = 2.14 min; LRMS: Anal. Calcd. for
Figure imgf000072_0001
(M+H)+669.38, found: 669.53.
Exam le OL-14f
Figure imgf000072_0002
Example OL-14f was prepared from Example OL-14e, according to the same procedure used for the preparation of Example OL-le. XH NMR (500 MHz, DMSO- d6) δ ppm 1.94 - 2.06 (m, 2 H), 2.1 1 - 2.25 (m, 2 H), 2.38 - 2.48 (m, 4 H), 3.32 - 3.50 (m, 4 H), 4.60 (s, 4 H), 4.92 - 5.06 (m, 2 H), 7.48 (d, J=7.93 Hz, 4 H), 7.89 (d, J=7.93 Hz, 4 H), 8.04 (s, 2 H), 9.78 (s, 2 H), 10.28 (s, 2 H). Note: the signal of the imidazole NH was too broad to assign a chemical shift. LC (Cond. 1): RT =1.69 min; LRMS: Anal. Calcd. for C28H33N60 (M+H)+ 469.27 found: 469.34. Example OL- 14
Example OL-14 was prepared from Example OL-14f, according to the same procedure used for the preparation of Example OL- 1. XH NMR (500 MHz, DMSO- d6) δ ppm 1.82 - 2.1 1 (m, 6 H), 2.18 (d, J=6.10 Hz, 2 H), 2.31 - 2.47 (m, 4 H), 2.98 - 3.10 (m, 2 H), 3.92 - 4.07 (m, 2 H), 4.59 (s, 4 H), 5.10 - 5.22 (m, 2 H), 5.42 (s, 2 H), 7.07 - 7.13 (m, 2 H), 7.40 - 7.51 (m, 4 H), 7.54 - 7.67 (m, 10 H), 7.75 (d, J=7.32 Hz, 4 H). Note: the signal of the imidazole NH was too broad to assign a chemical shift. The signals for the Me groups of the cap fell underneath the solvent peak. LC (Cond, 4): RT = 1.83 min; LRMS: Anal. Calcd. for C48H55N8O3 (M+H)+ 791.44; found: 791.60. HRMS: Anal. Calcd. for C48H55N8O3 (M+H)+ 791.4397; found: 791.4406. Exam les OL-15 to OL-19
Figure imgf000073_0001
Examples OL-15 to OL-19 were prepared as TFA salts by substituting the respective acids for Cap-1 according to the same method described for Example OL- 14.
Figure imgf000073_0002
Example 0 RT (LC-Cond.); MS data
Acid
OL-19 RT = 1.43 min. LC (Cond. 2); LCMS: Anal. Calcd.
for: CseHes ioOs (M+H)+ 957.51 ; Found: 957.84 HRMS: Anal. Calcd. for: CseHgj ioOs (M+H)+
957.5139; Found: 957.5142
Figure imgf000074_0001
Exam le OL-20
Figure imgf000074_0002
Example OL-20a
Figure imgf000074_0003
Sodium hydride (0.76 g, 18.95 mmol, 60% wt) was slowly added to a solution of methyl 4-(hydroxymethyl)benzoate (3 g, 18.05 mmol) in dimethylformamide (25 mL). The resulting dark blue solution was stirred at ambient for 15 min and methyl 3-(bromomethyl)benzoate (3.76g, 16.41 mmol) was added in one portion. The resulting yellow solution was stirred at ambient for 1 h and then the volatiles were removed under reduced pressure. The residue was taken up in ethyl acetate and washed with water and brine, dried (MgS04), filtered and concentrated in vacuo.
The residue was submitted to flash chromatography (silica gel; 20 % ethyl acetate/hexanes) to provide Example OL-20a as a clear oil that solidified upon standing (2.49 g). ¾ NMR (300 MHz, CDC13) δ ppm 3.91 (d, J=1.83 Hz, 6 H), 4.61 (d, J=2.56 Hz, 4 H), 7.39 - 7.49 (m, 3 H), 7.54 - 7.61 (m, 1 H), 7.93 - 8.00 (m, 1 H), 7.99 - 8.07 (m, 3 H). LC (Cond. 1): RT =2.56 min; LRMS: Anal. Calcd. for Ci8H1905 (M+H)+ 315.12 found: 315.22.
Example OL-20b
Figure imgf000075_0001
Example OL-20b was prepared from Example OL-20a, according to the same procedure used for the preparation of Example OL-la. XH NMR (500 MHz, CDCI3) δ ppm 4.65 (d, J=4.58 Hz, 4 H), 4.69 (d, J=3.36 Hz, 4 H), 7.47 - 7.54 (m, J=7.63, 7.63 Hz, 3 H), 7.62 (d, J=7.63 Hz, 1 H), 7.89 (d, J=7.93 Hz, 1 H), 7.93 - 8.00 (m, 3 H). LC (Cond. 1): RT =2.34 min; LRMS: Anal. Calcd. for C18H17CI2O3 (M+H)+ 351.05 found: 351.12.
Example OL-20c
Figure imgf000075_0002
Example OL-20c was prepared from Example OL-20b, according to the same procedure used for the preparation of Example OL-lb. XH NMR (300 MHz, DMSO- d6) δ ppm 4.57 (d, J=2.93 Hz, 4 H), 4.68 (d, J=5.85 Hz, 4 H), 7.52 - 7.67 (m, J=8.60, 8.60 Hz, 3 H), 7.73 (d, J=7.68 Hz, 1 H), 7.90 - 8.10 (m, 4 H), 8.53 (s, 6 H). LC (Cond. 1): RT =1.32 min; LRMS: Anal. Calcd. for Ci8H21N203 (M+H)+ 313.15 found: 313.24. Exam le OL-20d
Figure imgf000076_0001
Example OL-20d was prepared from Example OL-20c, according to the same procedure used for the preparation of Example OL- lc. XH NMR (500 MHz, DMSO- de) δ ppm 1.39/1.34 (rotomers, s, 18 H), 1.69 - 1.95 (m, 6 H), 2.00 - 2.18 (m, J=23.80 Hz, 2 H), 3.23 - 3.33 (m, 2 H), 3.34 - 3.43 (m, 2 H), 4.11 - 4.23 (m, 2 H), 4.50 - 4.63 (m, 4 H), 4.65 (d, J=6.71 Hz, 4 H), 7.48 - 7.56 (m, 3 H), 7.66 (d, J=7.63 Hz, 1 H), 7.89 - 8.02 (m, 4 H), 8.06 - 8.25 (m, 2 H). LC (Cond. 1): RT =2.53 min; LRMS: Anal. Calcd. for C38H51 4O9 (M+H)+ 707.36 found: 707.44.
Exam le OL-20e
Figure imgf000076_0002
Example OL-20e was prepared from Example OL-20d, according to the same procedure used for the preparation of Example OL-ld. XH NMR (500 MHz, DMSO- d6) δ ppm 1.14/1.39 (rotomers, s, 18 H), 1.76 - 2.07 (m, 6 H), 2.08 - 2.29 (m, 2 H), 3.31 - 3.40 (m, 2 H), 3.52 (br. s, 2 H), 4.44 - 4.59 (m, J=8.55 Hz, 4 H), 4.75 (br. s, 1 H), 4.83 (br. s, 1 H), 7.08 - 7.23 (m, 2 H), 7.30 (d, J=7.63 Hz, 2 H), 7.34 - 7.50 (m, 2 H), 7.53 - 7.67 (m, 1 H), 7.68 - 7.79 (m, 3 H), 11.70 - 12.22 (m, 2 H). LC (Cond. 1): RT =2.19 min; LRMS: Anal. Calcd. for
Figure imgf000076_0003
(M+H)+ 669.39, found: 669.40. Exam le OL-20f
Figure imgf000077_0001
Example OL-20f was prepared from Example OL-20e, according to the same procedure used for the preparation of Example OL-le. XH NMR (500 MHz, DMSO- D6) δ ppm 1.94 - 2.08 (m, 2 H), 2.12 - 2.24 (m, 2 H), 2.39 - 2.48 (m, J=5.19 Hz, 4 H), 3.27 - 3.49 (m, 4 H), 4.61 (d, J=4.88 Hz, 4 H), 4.94 - 5.09 (m, J=7.93 Hz, 2 H), 7.38 (d, J=7.32 Hz, 1 H), 7.45 - 7.53 (m, 3 H), 7.83 (d, J=7.63 Hz, 1 H), 7.90 (d, J=7.02 Hz, 3 H), 8.07 (s, 2 H), 9.86 (br. s, 2 H), 10.33 (br. s, 2 H). Note: the signal of the imidazole NH was too broad to assign a chemical shift. LC (Cond. 1): RT = 1.75 min; LRMS: Anal. Calcd. for C28H32N60 (M+H)+ 469.27, found: 469.23.
Example OL-20
Example OL-20 was prepared from Example OL-20f, according to the same procedure used for the preparation of Example OL- 1. XH NMR (500 MHz, DMSO- d6) δ ppm 1.84 - 1.95 (m, J=5.19 Hz, 2 H), 1.95 - 2.09 (m, J=8.24 Hz, 4 H), 2.15 - 2.28 (m, 2 H), 3.02 (q, J=7.73 Hz, 2 H), 3.93 - 4.05 (m, 2 H), 4.61 (br. s, 4 H), 5.12 - 5.22 (m, 2 H), 5.42 (br.s, 2 H), 7.12 (br. s, 1 H), 7.39 (s, 1 H), 7.45 - 7.51 (m, J=7.32 Hz, 3 H), 7.53 - 7.64 (m, 9 H), 7.66 - 7.82 (m, 4 H), 7.96 (s, 2 H). Note: the signal of the imidazole NH was too broad to assign a chemical shift. The signals for the Me groups of the cap fell underneath the solvent peak. LC (Cond. 4): RT = 1.93 min; LRMS: Anal. Calcd. for C48H55N8O3 (M+H)+ 791.44; found: 791.57. HRMS: Anal. Calcd. for C48H55N8O3 (M+H)+. 791.4397; found: 791.4373. Exam les OL-21 to OL-25
Figure imgf000078_0001
Examples OL-21 to OL-25 were prepared as TFA salts by substituting the respective acids for Cap-1 according to the same method described for Example OL- 20.
Figure imgf000078_0002
Figure imgf000079_0001
Exam le D-l
Figure imgf000079_0002
Example D- 1 , step a
Figure imgf000080_0001
N,N-Diisopropylethylamine (18 mL, 103.3 mmol) was added dropwise, over 15 minutes, to a heterogeneous mixture of N-Boc-L-proline (7.14 g, 33.17 mmol), HATU (13.32 g, 35.04 mmol), the HCl salt of 2-amino-l-(4-bromophenyl)ethanone (8.13 g, 32.44 mmol), and DMF (105 mL), and stirred at ambient condition for 55 minutes. Most of the volatile component was removed in vacuo, and the resulting residue was partitioned between ethyl acetate (300 mL) and water (200 mL). The organic layer was washed with water (200 mL) and brine, dried (MgS04), filtered, and concentrated in vacuo. A silica gel mesh was prepared from the residue and submitted to flash chromatography (silica gel; 50-60 % ethyl acetate/hexanes) to provide D-la as a white solid (12.8 g). XH NMR (500 MHz, DMSO-d6) δ ppm 1.40/1.34 (two app br s, 9 H), 1.90-1.70 (m, 3 H), 2.18-2.20 (m, 1 H), 3.30-3.23 (m, 1 H), 3.43-3.35 (m, 1 H), 4.22-4.12 (m, 1 H), 4.53 (dd, J= 18.1, 5.6, 1 H), 4.61 (dd, J= 18.3, 5.7, 1 H), 7.75 (br d, J=8.6, 2 H), 7.92 (br d, J=8.0, 2 H), 8.25-8.14 (m, 1H). LC (Cond. 7): RT = 1.70 min; LRMS: Anal. Calcd. for (M+Na)+ Ci8H23BrN2Na04: 433.07 found 433.09.
Exam le D-l, step b
Figure imgf000080_0002
A mixture of D-la (12.8 g, 31.12 mmol) and H4OAc (12.0 g, 155.7 mmol) in xylenes (155 mL) was heated in a sealed tube at 140 °C for 2 hours. The volatile component was removed in vacuo, and the residue was partitioned carefully between ethyl acetate and water, whereby enough saturated NaHCCh solution was added so as to make the pH of the aqueous phase slightly basic after the shaking of the biphasic system. The layers were separated, and the aqueous layer was extracted with additional ethyl acetate. The combined organic phase was washed with brine, dried (MgS04), filtered, and concentrated in vacuo. The resulting material was recrystallized from ethyl acetate/hexanes to provide two crops of imidazole D-lb as a light-yellow, dense solid, weighing 5.85 g. The mother liquor was concentrated in vacuo and submitted to a flash chromatography (silica gel; 30% ethyl
acetate/hexanes) to provide an additional 2.23 g of Example D-lb. lH NMR (500 MHz, DMSO-d6) δ ppm 1.40/1.15 (app br s, 9 H), 2.30-1.75 (m, 4 H), 3.36 (m, 1 H), 3.52 (app br s, 1 H), 4.86-4.70 (m, 1 H), 7.72-7.46/7.28 (m, 5 H), 12.17/11.92/11.86 (m, 1 H). LC (Cond. 7): RT = 1.71 min; LRMS: Anal. Calcd. for [M+H]+
Ci8H23Br 302: 392.10; found 391.96; HRMS: Anal. Calcd. for [M+H]+
Ci8H23Br 302: 392.0974; found 392.0959.
The optical purity of the two samples of Example D-lb was assessed according to the chiral HPLC conditions noted below (ee > 99% for the combined crops; ee = 96.7% for the sample from flash chromatography):
Column: Chiralpak AD, 10 um, 4.6 x 50 mm
Solvent: 2% ethanol/heptane (isocratic)
Flow rate: 1 mL/min
Wavelength: either 220 or 254 nm
Relative retention time: 2.83 minutes (R), 5.34 minutes (5).
Exam le D-l, step c
Figure imgf000081_0001
Pd(Ph3P)4 (469 mg, 0.41 mmol) was added to a pressure tube containing a mixture of D-lb (4.01 g, 10.22 mmol), bis(pinacolato)diboron (5.42 g, 21.35 mmol), potassium acetate (2.57 g, 26.21 mmol) and 1,4-dioxane (80 mL). The pressure tube was purged with nitrogen, capped and heated in an oil bath at 80 °C for 16.5 hours. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The crude residue was partitioned carefully between CH2C12 (150 mL) and an aqueous medium (50 mL water and 10 mL saturated NaHCC^ solution). The aqueous layer was extracted with CH2C12, and the combined organic phase was dried (MgS04), filtered, and concentrated in vacuo. The resulting material was purified with flash chromatography (sample was loaded with eluting solvent; 20-35% ethyl
acetate/CH^Cy to provide Example D-lc, contaminated with pinacol, as an off- white dense solid; the relative mole ratio of Example D-lc to pinacol was about 10: 1 (XH NMR). The sample weighed 3.925 g after -2.5 days exposure to high vacuum. ¾ NMR (500 MHz, DMSO-d6) δ ppm 1.45-1.10 (m, 21H), 2.27-1.77 (m, 4H), 3.36 (m, 1H), 3.52 (app br s, 1H), 4.86-4.70 (m, 1H), 7.79-7.50/ 7.34-7.27 (m, 5H), 12.22/11.94/ 11.87 (m, 1H). LC (Cond. 7): RT = 1.64 min; LRMS: Anal. Calcd. for [M+H]+ C24H35BN3O4: 440.27; found 440.23.
Exam le D-l, step d
Figure imgf000082_0001
Tetrakistriphenylphosphine palladium (17.5 mg, 0.015 mmol) was added in one portion to a stirred suspension of D-lc (320 mg, 0.85 mmol), 1,4- diiodobenzene (100 mg, 0.30 mmol) and sodium bicarbonate (180 mg, 2.18 mmol) in
dimethoxyethane (2.4 mL) and water (7 mL) and heated to 90 °C for 2 h. The reaction was diluted with ethyl acetate and washed with saturated sodium
bicarbonate. The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The filtrate was taken up in methanol, filtered through a nylon syringe filter and then purified by preparative HPLC to provide D- 1 d as a white solid (101.1 mg, 20% yield). ¾ NMR (500 MHz, DMSO-i¾) δ ppm 1.17 (br s,
9 H), 1.41 (br s, 9 H), 1.80 - 2.06 (m, 4 H), 2.13 - 2.32 (m, 4 H), 3.35 - 3.43 (m, 2 H),
3.56 (br. s., 2 H), 4.80 (br s, 2 H), 7.54 (d, J=10.07 Hz, 2 H), 7.73 (d, J=7.63 Hz, 4 H), 7.79 (s, 4 H), 7.83 (d, J=8.24 Hz, 4 H). LC (Cond. 6): RT = 2.55 min; HRMS: Anal. Calcd. for (M+H)+ C42H49 604 701.3815; found: 701.3790.
Exam le D-1, step e
Figure imgf000083_0001
D-le was prepared from D-ld, according to the same procedure used for the preparation of OL-le except that methanol (1 mL) was used instead of
dichloromethane. This gave D-le as a tan solid and as a hydrochloride salt (101.5 mg, 96 % yield). XH NMR (500 MHz, DMSO-d6) δ ppm 1.93 - 2.30 (m, 9 H), 2.32 - 2.44 (m, 3 H), 4.69 - 4.86 (m, 2 H), 7.68 - 7.87 (m, 12 H), 7.90 (d, J= 8.24 Hz, 2 H), 9.48 - 9.75 (m, 2 H). LC (Cond. 6): RT = 1.99 min; HRMS: Anal. Calcd. for (M+H)+ C32H33N6 501.2767; found: 501.2753.
Example D-1
Example D-1 (TFA salt) was prepared from D-le, according to the same procedure used for the preparation of Example OL- 1. This gave Example D-1 as a tan solid (37.8 mg, 54 % yield). ¾ NMR (500 MHz, DMSO-i¾) δ ppm 2.05 (d, J=8.55 Hz, 8 H), 3.49 - 3.57 (m, 10 H), 3.94 (br s, 2 H), 5.52 (d, J=7.63 Hz, 2 H), 7.31 - 7.47 (m, 10 H), 7.72 (d, J=7.02 Hz, 2 H), 7.87 - 8.01 (m, 12 H). LC (Cond. 6): RT = 2.41 min; HRMS: Anal. Calcd. for (M+H)+ C52H5i 806 883.3932; found: 883.3947.
-2
Figure imgf000084_0001
Example D-2 (TFA salt) was prepared from pyrrolidine D-le, according to the same procedure used for the preparation of Example OL- 1. This gave Example D-2 as a light yellow solid (15.4 mg, 38 % yield). XH NMR (500 MHz, DMSO-i¾) δ ppm 2.50-1.70 (m, 14 H), 2.98-2.62 (m, 6 H), 4.05-2.98 (m, 4 H), 5.79-5.12 (m, 4 H), 8.20-7.10 (m, 24 H), 10.25 (br s, 2 H). Note: The signal of the imidazole NH was too broad to assign a chemical shift. LC (Cond. 6): RT = 2.08 min; HRMS: Anal. Calcd. For (M+H)+ C52H55N8O2 823.4448; found: 823.4467. -3
Figure imgf000084_0002
Example D-3, step a
Figure imgf000084_0003
A solution of bromine (683 μί, 13.33 mmol) in acetic acid (7 mL) was added dropwise to a cold (10 °C) solution of 6-bromo-3,4-dihydronaphthalen-l(2H)-one (purchased from J & W PharmLab, LLC) (3.00 g, 13.33 mmol) and 48% hydrogen bromide (20 μί, 13.33 mmol) in acetic acid (120 mL). The mixture was allowed to warm up to rt after the addition was complete and allowed to stir at rt for 1 h before it was diluted with dichloromethane and washed with water (3x), saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. There was isolated D-3a (4.19 g, 97% yield) as a colorless oil which solidified on standing under high vacuum to a white solid. This material was used without further purification. XH NMR (500 MHz, DMSO-d6) δ ppm 7.85 (1 H, d, J=8.5 Hz), 7.71 (1 H, s), 7.62 (1 H, dd, J=8.5, 1.8 Hz), 5.06 (1 H, dd, J=6.1, 3.7 Hz), 2.93 - 3.15 (2 H, m), 2.55 - 2.64 (1 H, m), 2.32 - 2.42 (1 H, m). RT = 2.67 min (Cond. 6); LC/MS: Anal. Calcd. for [M+H]+ Ci0H9 79Br2O: 304.90; found: 304.91.
Examples D-3bl and D-3b2 (step b)
D-3bl and D-3b2 were prepared from D-3a and the appropriate Boc -protected prolines according to the same procedure used for the preparation of Example M3, step g.
Figure imgf000085_0001
Examples D-3cl and D-3c2 (step c)
D-3cl and D-3c2 were prepared from D-3bl and D-3b2 respectively according to the same procedure used for the preparation of Example M3, step h.
Figure imgf000086_0001
Example D-3dl (step d)
Figure imgf000087_0001
Ethynyltrimethylsilane (0.59 mL, 4.25 mmol) was added to a solution of D-lb (1.5 g, 3.82 mmol), triphenylphosphine (0.20 g, 0.77 mmol), diethylamine (4.25 mL, 40.70 mmol), copper (I) iodide (40 mg, 0.21 mmol) and /raws-dichloro(bis- triphenylphosphine)palladium (II) (149 mg, 0.21 mmol) in dry DMF (1.4 mL) at rt in a microwave vessel. The vessel was capped and irradiated for 25 min at 120 °C. Two identical 1.5 g reactions were run in tandem. The reaction mixtures were diluted with ether and ethyl acetate, combined and shaken with 0. IN HQ. After standing for 20 min, the suspension was suction-filtered and the pad was washed with ether and ethyl acetate. The organic phase was then separated, washed with brine, dried over sodium sulfate, and concentrated. There was isolated the crude product (4.2 g) as a brownish-red foam which was taken up in dichloromethane and added directly to a Thompson 1 10 g silica gel column. Gradient elution of the residue with
20% ethyl acetate in dichloromethane to 100% ethyl acetate furnished D-3dl (2.8 g, 40% yield) as a yellow solid after evaporation of the eluant which was taken forward directly. RT = 2.37 min (Cond. 3); LC/MS: Anal. Calcd for C23H32 3O2S1 [M+H]+ 410.23; found: 410.12.
D-3d2 to D-3d4 were prepared according to the procedures described for D-
3
Figure imgf000087_0002
Figure imgf000088_0001
Example D-3el (step e)
Figure imgf000088_0002
Potassium carbonate (91 mg, 0.66 mmol) was added in one portion to a stirred solution of D-3dl (2.7 g, 6.6 mmol) in MeOH (60 mL) at rt. The mixture was stirred for 1 h before it was concentrated down in vacuo. The residue was taken up in dichloromethane and added directly to a 130 g Thompson silica gel column.
Gradient elution of the residue with 15% ethyl acetate in hexanes to 100% ethyl acetate furnished D-3el (2.04 g, 87% yield) as a yellow foam after evaporation of the eluant. A small amount (approx. 20 mg) of the product was then subjected to preparative HPLC to afford a purer sample of D-3el as an off-white solid. XH NMR (500 MHz, MeOD, imidazole N-H missing) δ ppm 7.68 (2 H, d, J=7.9 Hz), 7.46 (2 H, d, J=8.2 Hz), 7.34 - 7.40 (1 H, m), 4.89 (1 H, m), 3.64 - 3.73 (1 H, m), 3.49 (1 H, m), 3.49 (1 H, s), 3.31 - 3.34 (1 H, m), 2.25 - 2.44 (1 H, m), 1.99 - 2.1 1 (3 H, m), 1.25 and 1.47 (9 H, 2s). RT = 1.63 min (Cond. 3), LC/MS: Anal. Calcd for C2oH24 302 (M+H)+ 338.19; found: 338.12. D-3e2 to D-3e5 were prepared according to the procedures described for alkyne D-3el.
Figure imgf000089_0002
Example D-3fl (step f)
Figure imgf000089_0001
Tetrakis(triphenylphosphine)palladium (83 mg, 0.072 mmol) was added in one portion to a stirred, argon-degassed mixture of Example D-3cl (300 mg, 0.717 mmol), D-3el (315 mg, 0.932 mmol), triethylamine (0.40 mL, 2.87 mmol) and copper(I) iodide (13.7 mg, 0.072 mmol) in anhydrous DMF (6 mL) at rt in a thick- walled, screw-top vial. The mixture was stirred at rt for 16 h and at 40 °C for 16 h before additional Cul (10 mg), TEA (0.4 mL) and Pd(PPh3)4 catalyst (40 mg) were added since the reaction was not complete after 32 h as judged by LCMS. The mixture was stirred further at 60 °C for 10 h before it was cooled to rt, diluted with ethyl acetate, THF and water and suction- filtered. The organic phase of the filtrate was separated, washed with saturated sodium bicarbonate solution and brine prior to drying over anhydrous sodium sulfate and evaporation. The residue was taken up in dichloromethane and added directly to a Thompson 80 g silica gel column. Gradient elution of the residue with 30% ethyl acetate in hexanes to 100% ethyl acetate followed by 0% methanol in ethyl acetate to 20% methanol in ethyl acetate furnished D-3fl (366.7 mg, 68% yield) as a golden brown foam after evaporation of the eluant. A small amount (approx. 20 mg) of product was then subjected to preparative HPLC to afford a purer sample of Example D-3fl as a tan solid. lH NMR (500 MHz, MeOD, imidazole N-H's missing) δ ppm 7.73 (2 H, d, J=8.2 Hz), 7.47 - 7.57 (4 H, m), 7.41 (2 H, br s), 4.96 - 5.09 (1 H, m), 4.89 - 4.96 (1 H, m), 3.69 (2 H, br s), 3.54 (2 H, br s), 3.04 - 3.13 (2 H, m), 2.83 - 2.94 (2 H, m), 2.30 - 2.50 (2 H, m), 1.94 -
2.17 (6 H, m), 1.27 - 1.49 (18 H, 2s). RT = 2.06 min (Cond. 3); LC/MS: Anal. Calcd for C40H47N6O4 (M+H)+ 675.37; found: 675.26.
D-3f2 to D-3f4 were prepared according to the procedure described for D-3fl .
Figure imgf000090_0001
Figure imgf000091_0001
-3gl (step g)
Figure imgf000091_0002
Activated manganese dioxide (2.0 g, 23.12 mmol) was added in one portion to a stirred solution of D-3fl (260 mg, 0.39 mmol) in dry dichloromethane (5 mL). The suspension was stirred at rt for 6 h before additional activated manganese dioxide (1.0 g) was added. The suspension was stirred further for 14 h at rt before it suction-filtered through a pad of Celite with MeOH and the pad was washed with additional MeOH several times. The filtrate was then concentrated in vacuo to yield D-3gl (225.1 mg, 82% yield) as a yellow solid. A small amount (approx. 20 mg) of the product was then subjected to preparative HPLC to afford a purer sample of D- 3gl as a light tan solid. XH NMR (500 MHz, MeOD, imidazole N-H's missing) δ ppm 8.43 (1 H, d, J=8.5 Hz), 8.19 (1 H, br s), 7.68 - 7.78 (5 H, m), 7.62 (2 H, d, J=8.2 Hz), 7.54 (1 H, s), 4.90 - 5.27 (2 H, m), 3.76 - 3.85 (1 H, m), 3.66 - 3.74 (1 H, m), 3.51 - 3.66 (2 H, m), 2.35 - 2.60 (2 H, m), 1.94 - 2.21 (6 H, m), 1.49 (6 H, br s), 1.27 (6 H, br s), 1.14 (6 H, br s). RT = 2.12 min (Cond. 3); LC/MS: Anal. Calcd for C40H45N6O4 (M+H)+ 673.35; found: 673.30. D-3g2 was prepared according to the procedure described for D-3gl.
Figure imgf000092_0001
Example D-3hl (step h)
Figure imgf000092_0002
D-3hl to D-3h6 were prepared from D-3fl, D-3gl, D-3f2, D-3G, D-3g2 and D-3f4, respectively, according to the same procedure used for the preparation of OL- le except that methanol (1 mL) was used instead of dichloromethane. This gave D- 3hl to D-3h6 as hydrochloride salts (or TFA salts when purified further with preparative HPLC) upon concentration of the solvent(s) in vacuo.
Figure imgf000092_0003
Figure imgf000093_0001
Example D-3 to D- 11 (final step)
Figure imgf000094_0001
Examples D-3 to D-11 were prepared from D-3hl to D-3h6 and the appropriate acids according to the same procedure used for the preparation of Example OL-1. This gave Examples D-3 to D-11 as TFA salts after HPLC purification.
Figure imgf000094_0002
Figure imgf000095_0001
Figure imgf000096_0001
From D-3h6 and Cap 170 Example Ml
Figure imgf000097_0001
Example Ml, Step a
Figure imgf000097_0002
Pd(Ph3P)4 (0.078 g, 0.067 mmol) was added to a DMF (5 mL) solution of (S)- tert-butyl 2-(5-(4-bromophenyl)-lH-imidazol-2-yl)pyrrolidine-l-carboxylate(D-lb) (0.675g, 1.721 mmol) and 1 ,2-bis(trimethylstannyl)ethyne (0.3134 g, 0.891 mmol) in a pressure tube, the mixture was flushed with nitrogen for 1 min and then heated at 90 °C for 15 h. The volatile component was removed in vacuo and the residue was directly submitted to a BIOTAGE® purification (110 g; EtOAc) to afford alkyne Mia as a yellowish orange foam containing unidentified impurity and residual solvent (430 mg). ¾ NMR (DMSO, δ = 2.50 ppm, 400 MHz): 12.23/11.98/11.91 (three br s, 2H), 7.80-7.35 (m, 10H), 4.84-4.76 (m, 2H), 3.54 (m, 2H), 3.39-3.33 (m, 2H), 2.28-1.78 (m, 8H), 1.40 (br s, 7.54H), 1.16 (br s, 10.46H). LC/MS: Anal. Calcd. for [M+H]+ C38H45N604: 649.35; found 649.27.
Example Ml, Step b
Figure imgf000097_0003
HCl/dioxanes (4N; 8 mL, 32.0 mmol), CH2C12 (1 mL) and MeOH (1.0 mL) added to bis-carbamate Mia (0.427 g, 0.658 mmol), and the heterogeneous mixture was stirred for 5 hr. The volatile component was removed in vacuo, and the residue was exposed to high vacuum to afford Mlb (.4HC1) as an off-white solid (452 mg), which was submitted to the next step as such. XH NMR analysis indicates that the sample may contain residual dioxane (~1 mol equiv). XH NMR (DMSO, δ = 2.50 ppm, 400 MHz): 10.29 (br s, 2H), 9.73 (br s, 2H), 8.11 (s, 2H), 7.96 (d, J = 8.3, 4H), 7.67 (d, J = 8.6, 4H), 4.97 (br m, 2H), 3.47-3.31 (m, 4H), 2.50-2.36 ('m' partially overlapped with solvent signal, 4H), 2.23-2.14 (m, 2H), 2.07-1.95 (m, 2H). LC/MS: Anal. Calcd. for [M+H]+ C28H29N6: 449.25; found 49.23. Example Ml
HATU (0.069 g, 0.181 mmol) was added to the DMF solution of pyrrolodine Mlb/4HC1 (70.5 mg, 0.103 mmol), (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid (36.3 mg, 0.207 mmol) and DIEA (0.1 mL, 0.573 mmol) and stirred at room temperature for 70 min. The volatile component was removed in vacuo and the residue was dissolved in MeOH and submitted to a reverse phase HPLC purification (MeOH/water/TFA) to afford the TFA salt of Example Ml as an off-white foam (68.9 mg). LC (Cond. 9 and 10): >95% homogeneity index. LC/MS (Cond. 3): Rt = 1.78 min. 'H NMR (DMSO, δ = 2.50 ppm, 400 MHz): 8.10 (br s, 2H), 7.88-7.83 (m, 4H), 7.71 (d, J = 8.3, 3.91H), 7.34 (d, J = 8.5, 2H; NHC02), 6.92 (app br s, 0.09H), 5.52 (br m, 0.17H), 5.12 (app t, 1.93H), 4.11 (app t, 2H), 3.89-3.77 (m, 4H), 3.54 (s, 5.52H), 3.33 (s, 0.48H), 2.41-2.33 (m, 2H), 2.21-1.93 (m, 8H), 0.89 (app t, 0.91H), 0.83/0.79 (two overlapping d, J = 6.8/6.8, 11.09 H). LC/MS: Anal. Calcd. for
[M+H]+ C42H5i 806: 763.39; found 763.33.
Example M2 to M2.1
Example M2 to M2.1 were prepared as TFA salts from pyrrolidine Mlb and appropriate acids according to the procedure described for the preparation of Example Ml with a modified purification protocol noted in the table below.
Figure imgf000098_0001
Example R Analytical data
M2 Purified using two different reverse phase
HPLC conditions (Column: Phenomenex Luna,
0 NHC02Me
30x100 mm, S10; MeOH/water/TFA) and (Waters Sunfire, 30x100 mm, S5;
CH3CN/water/TFA). LC (Cond. 9 and 10): >95% homogeneity index. LC/MS (Cond. 3): Rt = 2.04 min. LC/MS: Anal. Calcd. for
[M+H]+ C48H47 806: 831.36; found 841.41.
M2.1 Purified using two different reverse phase
HPLC conditions (Column: Xbridge, 19x100 mm, S5; MeOH/water/TFA) and (Waters
0 NHC02Me
Sunfire, 30x100 mm, S5; CH3CN/water/TFA). LC (Cond. 9 and 10): 95% homogeneity index. LC/MS (Cond. lOd): Rt = 2.82 min. LC/MS: Anal. Calcd. for [M+H]+ eHssNgOg: 847.41 ; found 847.25.
Example M3
Figure imgf000099_0001
Example M3, Step a
TB
Figure imgf000099_0002
To a solution of (S)-5-(hydroxymethyl)pyrrolidin-2-one (10 g, 87 mmol) in CH2CI2 (50 mL) was added tert-butylchlorodiphenylsilane (25.6 g, 93 mmol), Et3 (12.1 mL, 87 mmol) and DMAP (1.06 g, 8.7 mmol). The mixture was stirred at room temperature until the starting pyrrolidinone was completely consumed, and then it was diluted with (¾(¾ (50 mL) and washed with water (50 mL). The organic layer was dried (Na2S04), filtered, and evaporated in vacuo, and the crude material was submitted to flash chromatography (silica gel; 30 to 100% of EtOAc/hexanes) to afford ether M3a as a colorless oil (22.7 g, 74% yield). XH-NMR (400 MHz, DMSO- d6, δ = 2.5 ppm) 7.69 (br s, 1H), 7.64-7.61 (m, 4H), 7.50-7.42 (m, 6H), 3.67-3.62 (m, 1H), 3.58-3.51 (m, 2H), 2.24-2.04 (m, 3H), 1.87-1.81 (m, 1H), 1.00 (s, 9H). LC/MS (M+H)+ = 354.58.
Example M3, Step b
Figure imgf000100_0001
Di-tert-butyl dicarbonate (38.5 g, 177 mmol) was added in portions as a solid over 10 min to a CH2C12 (200 mL) solution of silyl ether M3a (31.2 g, 88.3 mmol), Et3N (8.93 g, 88 mmol), and DMAP (1.08 g, 8.83 mmol) and stirred for 18 h at 24 °C. Most of the volatile material was removed in vacuo and the crude material taken up in 20% EtOAc/Hex and applied to a 2 L funnel containing 1.3 L of silica gel and then eluted with 3 L of 20% EtO Ac/hex and 2 L of 50% EtOAc). Upon
concentration of the desired fractions in a rotary evaporator, a white slurry of solid formed which was filtered, washed with hexanes and dried in vacuo to afford carbamate M3b as a white solid (32.65 g, 82% yield). XH-NMR (400 MHz, DMSO- d6, δ = 2.5 ppm) 7.61-7.59 (m, 2H), 7.56-7.54 (m, 2H), 7.50-7.38 (m, 6H), 4.18 (m, 1H), 3.90 (dd, J = 10.4, 3.6, 1H), 3.68 (dd, J = 10.4, 2.1, 1H), 2.68-2.58 (m, 1H), 2.40-2.33 (m, 1H), 2.22-2.12 (m, 1H), 2.01-1.96 (m, 1H), 1.35 (s, 9H), 0.97 (s, 9H). LC/MS (M-Boc+H)+ = 354.58.
Figure imgf000100_0002
A three-necked flask equipped with a thermometer and a nitrogen inlet was charged with carbamate M3b (10.05 g, 22.16 mmol) and toluene (36 mL), and lowered into -55 °C cooling bath. When the internal temperature of the mixture reached -50 °C, lithium triethylborohydride (23 mL of 1.0 M/THF, 23.00 mmol) was added dropwise over 30 min and the mixture stirred for 35 min while maintaining the internal temperature between -50 °C and -45 °C. Hunig's base (16.5 mL, 94 mmol) was added dropwise over 10 min. Then, DMAP (34 mg, 0.278 mmol) was added in one batch, followed by the addition of trifluoroacetic anhydride (3.6 mL, 25.5 mmol) over 15 min, while maintaining the internal temperature between -50 °C and -45 °C. The bath was removed 10 min later, and the reaction mixture was stirred for 14 h while allowing it to rise to ambient temperature. It was diluted with toluene (15 mL), cooled with an ice-water bath, and treated slowly with water (55 mL) over 5 min. The phases were separated and the organic layer washed with water (50 mL, 2x) and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel; 5% EtOAc/hexanes) to afford dihydropyrrole M3c as a colorless viscous oil (7.947 g, 82% yield). LC/MS (Cond. 7): Rt = 2.41 min. XH-NMR (400 MHz, DMSO-d6, δ = 2.5 ppm) 7.62-7.58 (m, 4H), 7.49-7.40 (m, 6H), 6.47 (br s, 1H), 5.07/5.01 (overlapping br d, 1H), 4.18 (br s, 1H), 3.89 (br s, 0.49H), 3.69 (br s, 1.51H), 2.90-2.58 (br m, 2H), 1.40/1.26 (overlapping br s, 9H), 0.98 (s, 9H). LC/MS: [M+Na]+ = 460.19.
Figure imgf000101_0001
M3d-1 : frans-isomer
M3d-2: c/s-isomer
Diethylzinc (19 mL of ~1.1 M in toluene, 20.9 mmol) was added dropwise over 15 min to a cooled (-30 °C) toluene (27 mL) solution of dihydropyrrole M3c
(3.94 g, 9.0 mmol). Chloroiodomethane (stabilized over copper; 3.0 mL, 41.2 mmol) was added dropwise over 10 min, and stirred while maintaining the bath temperature at -25 °C for 1 h and between -25 °C and -21 °C for 18.5 h. The reaction mixture was opened to the air and quenched by the slow addition of 50% saturated NaHCC solution (40 mL), and then removed from the cooling bath and stirred at ambient temperature for 20 min. It was filtered through a filter paper and the white cake was washed with 50 mL of toluene. The organic phase of the filtrate was separated and washed with water (40 mL, 2x), dried (MgS04) and concentrated in vacuo. The crude material was purified using a BIOTAGE® system (350 g silica gel; sample was loaded with 7% EtOAc/hexanes; eluted with 7-20% EtOAc/hexanes) to afford a mixture of methanopyrrolidines M3d-1 and M3d-2 as a colorless viscous oil (3.69 g, 90.7%). [Note: the exact cis/trans-isomer ratio was not determined at this stage].
LC/MS (Cond. 7): Rt = 2.39 min ¾-NMR (400 MHz, DMSO-d6, δ = 2.5 ppm) 7.62- 7.60 (m, 4H), 7.49-7.40 (m, 6H), 3.77/3.67 (overlapping br s, 3H), 3.11-3.07 (m, 1H), 2.23 (app br s, 1H), 2.05-2.00 (m, 1H), 1.56-1.50 (m, 1H), 1.33 (very broad s, 9H), 1.00 (s, 9H), 0.80 (m, 1H), 0.30 (m, 1H). LC/MS: [M+Na = 474.14. e
Figure imgf000102_0001
Boc
M3e-1 : frans-isomer
M3e-2: c/s-isomer
TBAF (7.27 mL of 1.0 M in THF, 7.27 mmol) was added dropwise over 5 min to a THF (30 mL) solution of silyl ether M3d-l/-2 (3.13 g, 6.93 mmol) and the mixture stirred at ambient temperature for 4.75 h. After the addition of saturated NH4CI solution (5 mL), most of the volatile material was removed in vacuo and the residue partitioned between CH2CI2 (70 mL) and 50% saturated NH4C1 solution (30 mL). The aqueous phase was extracted with CH2CI2 (30 mL), and the combined organic phase was dried (MgS04), filtered, concentrated in vacuo and then exposed to high vacuum overnight. The crude material was purified using a BIOTAGE® (silica gel; 40-50% EtOAc/hexanes) to afford a mixture of alcohols M3e-1 and M3e- 2, contaminated with traces of a lower Rf spot, as a colorless oil (1.39 g, -94% yield). [Note: the exact cis/trans isomer ratio was not determined at this stage.] XH-NMR
(400 MHz, DMSO-d6, δ = 2.5 ppm) 4.70 (t, J 3.44 (m, 1H), 3.33-3.27 (m, 1H), 3.08-3.04 (m, 1H), 2.07 (br m, 1H), 1.93-1.87 (m, 1H), 1.51-1.44 (m, 1H), 1.40 (s, 9H), 0.76-0.71 (m, 1H), 0.26 (m, 1H). LC/MS (M+Na)+ = 236.20. f
Figure imgf000103_0001
M3f-1 : trans isomer
M3f-2: cis isomer
A semi-solution of NaI04 (6.46 g, 30.2 mmol) in H20 (31 mL) was added to a solution of alcohol M3e-l/-2 (2.15 g, 10.08 mmol) in CH3CN (20 mL) and CC14 (20 mL). RuC¾ (0.044 g, 0.212 mmol) was added immediately and the heterogeneous reaction mixture was stirred vigorously for 75 min. The reaction mixture was diluted with H2O (60 mL) and extracted with CH2CI2 (50 mL, 3x). The combined organic phase was treated with 1 mL MeOH, allowed to stand for about 5 min, and then filtered through a pad of diatomaceous earth (CELITE®). The CELITE® was washed with CH2CI2 (50 mL), and the filtrate was concentrated in vacuo to afford a light charcoal-colored solid. ^- MR analysis of this crude material indicated a
1.00:0.04:0.18 mole ratio of trans acid M3f-l :cis acid M3f-2:presumed side product, tert-butyl 3-oxo-2-azabicyclo[3.1.0]hexane-2-carboxylate. The crude material was dissolved in EtOAc (~10 mL) with heating and allowed to stand at ambient temperature with seeding. About 15 min into the cooling phase, a rapid crystal formation was observed. About 1 h later, hexanes (~6 mL) was added and the mixture refrigerated overnight (it did not appear that additional material precipitated out). The mixture was filtered and washed with ice/water-cooled hexanes/EtOAc (2: 1 ratio; 20 mL) and dried under high vacuum to afford the first crop of acid M3f-1 (off- white crystals, 1.222 g). The mother liquor was concentrated in vacuo, and the residue dissolved in ~3 mL of EtOAc with heating, allowed to stand at ambient temperature for 1 h, and then 3 mL hexanes was added and stored in a refrigerator for ~15 h. A second crop of acid M3f-1 was retrieved similarly (grey crystals, 0.133 g), for a combined yield of 59%. Acid M3f-1 : Rt = 1.48 min under the following HPLC conditions: Solvent gradient from 100% A : 0% B to 0% A : 100% B (A = 0.1% TFA in 1 :9 MeOH/H20; B = 0.1% TFA in 9: 1 MeOH/H20) over 3 min; detection @ 220 nm; PHENOMENEX®-Luna 3.0X50 mm S10 column. MP (dec.) for the first crop = 147.5-149.5 °C. XH-NMR (400 MHz, DMSO-d6, δ = 2.5 ppm) 12.46 (s, 1H), 3.88 (app br s, 1H), 3.27 (app br s, 1H; overlapped with water signal), 2.28 (br m, 1H), 2.07 (app br s, 1H), 1.56 (app s, 1H), 1.40/1.34 (two overlapped s, 9H), 0.71 (m, 1H), 0.45 (m, 1H). 13C-NMR (100.6 MHz, DMSO-d6, δ = 39.21 ppm) 172.96, 172.60, 154.45, 153.68, 78.74, 59.88, 59.58, 36.91, 31.97, 31.17, 27.77, 27.52, 14.86, 14.53, 13.69. LC/MS [M+Na = 250.22. Anal. Calcd. for CnH17N04: C, 58.13; H, 7.54; N, 6.16. Found (for first crop): C, 58.24; H, 7.84; N, 6.07. Optical rotation (10 mg/mL in CHCI3): [α]ο = -216 and -212 for the first and second crop, respectively.
Example M3, Step g
Figure imgf000104_0001
DIEA (1.3 mL, 7.44 mmol) was added dropwise over 2 min to a semi- heterogeneous mixture of acid M3f-1 (1.697 g, 7.47 mmol) and 2-bromo-l-(4- bromophenyl)ethanone (2.01 g, 7.23 mmol) in CH3CN (30 mL) and stirred at room temperature for 8 hr. The volatile component was removed in vacuo and the residue was taken up in CH2CI2 (100 mL), washed with water (30 mL), dried (MgS04) and concentrated in vacuo to afford ketoester M3g as an off-white viscous semi-foamy oil
(3.076 g). ¾-NMR (400 MHz, DMSO-d6, δ = 2.5 ppm): 7.92 (d, J = 8.3, 2H), 7.78 (d, J = 8.5, 2H), 5.61-5.42 (m, 2H), 4.16 (m, 1H), 3.34 ('m' partially overlapped with water signal, 1H), 2.40 (m, 2H), 1.63 (m, 1H), 1.41/1.35 (two overlapped V, 9H), 0.74 (m, 1H), 0.53 (m, 1H). LC/MS: Anal. Calcd. for [M+Na]+ Ci9H22 79Br a05: 446.06; found 446.06. Example M3, Step h
Figure imgf000105_0001
A 350 ml pressure tube was charged with keto-ester M3g (3.07g, 7.24 mmol), ammonium acetate (5.48 g, 71.1 mmol) and Xylene (70 mL), capped and heated with an oil bath (140 °C) for 4.5 hr. The reaction mixture was allowed to cool to room temperature and the volatile component was removed in vacuo. CH2CI2 (100 mL) and 50% saturated aHC03 solution (30 mL) were added to the residue, vigorously stirred until gas evolution ceased, and the phases were separated. The organic layer was dried (MgSC^), concentrated in vacuo, and purified with a BIOTAGE® (240 g silica gel; sample was loaded with (¾(¾; 40-80% EtOAc/hexanes) to afford imidazole M3h as a dark yellow foam (2.40 g). XH-NMR (400 MHz, DMSO-d6, δ = 2.5 ppm): 12.21 (s, 0.11H), 1.93 (s, 0.89H), 7.69 (d, J = 8.8, 1.8H), 7.62-7.55 (m, 0.4H), 7.53 (br d, J = 2, 0.87H), 7.49 (d, J = 8.5, 1.8H), 7.29 (br d, J = 1.6, 0.13H), 4.59 (m, 1H), 3.41 (m, 1H), 2.37-2.17 (br m, 2H), 1.62 (m, 1H), 1.21 (very broad 's', 9H), 0.75 (m, 1H), 0.54 (m, 1H). LC/MS: Anal. Calcd. for [M+H]+ Ci9H2381Br 302: 406.10; found, 406.14.
Example M3, Step i
Figure imgf000105_0002
Pyrrolidine M3i (4HC1) was prepared from bromide M3h according to the procedure described for the synthesis of pyrrolidine Mlb (.4HC1) from bromide D- lb. XH-NMR (400 MHz, DMSO-d6, δ = 2.5 ppm): 10.51 (app br s, 4H), 8.08 (s, 2H), 7.92 (d, J= 7.8, 4H), 7.66 (d, J = 8.5, 4H), 4.78 (m, 2H), 3.42 (m, 2H), 2.65 (m, 2H), -2.53 ('m' partially overlapped with solvent signal, 2H), 1.94 (m, 2H), 1.10 (m, 2H), 0.86 (m, 2H). LC/MS: Anal. Calcd. for [M+H]+ C3oH29 6: 473.25; found 473.21. Example M3 (and Examples M4-M7)
Example M3, along with its analogs Examples M4-M7 highlighted in the table below, were prepared as TFA salts from pyrrolidine M3i (.4HC1) by employing the procedure described for the synthesis of Example Ml and appropriate acids. In the case of Example M7 an equimolar mixture of (S)-2-(methoxycarbonylamino)-3- methylbutanoic acid and (S)-2-(methoxycarbonylamino)-2-(tetrahydro-2H-pyran-4- yl)acetic acid was employed for the coupling step and the resultant statistical mixture of products was separated by the HPLC technique described for Example Ml .
Example M3 : LC (Cond. 9 and 10): >95% homogeneity index. LC/MS (Cond. 3): Rt = 1.89 min. LC/MS: Anal. Calcd. for [M+H]+ C44H5i 806: 787.39; found 787.40.
Figure imgf000106_0001
Figure imgf000106_0002
Figure imgf000107_0001
-M9
Figure imgf000107_0002
NCS (0.0195 g, 0.143 mmol) was added to a DMF (2 mL) solution of the free base form of Example M3 (obtained from the TFA salt via a standard MCX free- basing protocol; 0.109 g, 0.139 mmol), and stirred at room temperature for 16 hr and at 50°C for 25 hr. Most of the solvent was removed in vacuo, and the residue was dissolved in MeOH and submitted to a reverse phase HPLC purification
(MeOH/water/TFA) to afford the TFA salt of Example M8 (50 mg) and Example M9 (17.5 mg).
Figure imgf000107_0003
Example Rt (Cond. 3); % homogeneity index (Cond. 9a and 10a);
LC/MS data
M9 2.95 min; >95%; LC/MS: Anal. Calcd. for [M+H]+ C44H49Cl2 806:
855.32; found 855.24 -M9.2
Figure imgf000108_0001
NCS (0.021 g, 0.158 mmol) was added to a DMF (1.5 mL) solution of the free base form of Example M6 (obtained from the TFA salt via a standard MCX free- basing protocol; 0.1059 g, 0.122 mmol), and stirred at 50°C for 24 hr. The reaction mixture was diluted with MeOH (2.5 mL) and submitted to a reverse phase HPLC purification condition (XTERRA, 30X100 mm, S5; MeOH/water/TFA). The resultant sample was repurified with a different reverse phase HPLC condition (Waters-Sunfire, 30X100 mm, S5; acetonitrile/water/TFA) to afford Example M9.1 (38.8 mg) and Example M9.2 (32.6 mg) as TFA salts.
Figure imgf000108_0002
Example M9.3
Figure imgf000109_0001
a
Figure imgf000109_0002
To a cooled (0°C) CH2CI2 (22 mL) was added diethylzinc (1M in hexanes,
60.5 mL, 60.5 mmol) under nitrogen followed by the dropwise addition of trifluoroacetic acid (5.1 mL, 66.2 mmol) in 10 mL CH2CI2 over 15 min. The reaction was stirred for 15 min, and then diiodomethane (5.4 mL, 66.9 mmol) in 10 mL CH2CI2 was added dropwise to the reaction. The reaction was continued to stir at 0 °C for 1 hr, and then (E)-l,2-diphenylethene (2 g, 11.10 mmol) in 10 mL CH2CI2 was added. The mixture was removed from the cold bath and stirred at
~25 °C under nitrogen for 20 h. The reaction was quenched with 0. IN HCl (50 mL), the layers were separated, and the aqueous layer was extracted with hexanes (2 x 100 mL). The combined organic phase was washed with saturated NaHCC^ (aq) (50 mL) and brine (50 mL), dried over MgSC^, filtered, and concentrated in vacuo. The residue was submitted to a silica gel flash chromatography (hexanes) to afford biphenyl M9.3a as a colorless oil (1.6 g). XH NMR (400 MHz, CDCI3) δ ppm 7.34- 7.31 (m, 4H), 7.24-7.17 (m, 6H), 2.22-2.18 (m, 2H), 1.51-1.47 (m, 2H).
Example M9.3, step b
Figure imgf000110_0001
M9.3b1 M9.3b2
To a solution of biphenyl M9.3a (3.3 g, 16.99 mmol) in 1,2-dimethoxyethane (197 mL) and water (82 mL) was added NBS (12.09 g, 67.9 mmol). The reaction flask was covered with aluminum foil and stirred at room temperature for 51 hr. The reaction was partitioned between diethyl ether and water, and the organic layer was washed with brine, dried over MgSC^, filtered, and concentrated in vacuo. The residue was submitted to a flash chromatography (sample was loaded with chloroform; eluted with hexanes) to afford a stereoisomeric mixture of dibrominated product as a white solid (3.1 g). The stereoisomeric mixture was separated by chiral SFC (Chiral pak AD-H column, 30X250 mm, 5 μιη; 80% CO2-20% EtOH; 35 °C; 150 bar; 70 mL/min for 20 min; 220 nm) to isolate two enantiomers: M9.3M (white solid, 1.1 g); ¾ NMR (400 MHz, CDCI3) δ ppm 7.43 (d, J = 8.6 Hz, 4H), 7.02 (d, J
= 8.6 Hz, 4H), 2.12-2.09 (m, 2H), 1.47-1.43 (m, 2H); OR: +361.79, 3.15 mg in 1 mL CHCI3, λ = 589 nm, 50 mm cell. M9.3b2 (white solid, 1.2 g); XH NMR (400 MHz, CDCI3) δ ppm 7.43 (d, J = 8.6 Hz, 4H), 7.02 (d, J = 8.6 Hz, 4H), 2.12-2.09 (m, 2H),
1.47-1.43 (m, 2H); OR: -376.70, 3.03 mg in 1 mL CHCI3, λ = 589 nm, 50 mm cell.
Example M9.3, Step c
Figure imgf000110_0002
To a solution of dibromide M9.3M (0.8806 g, 2.501 mmol) and tributyl(l- ethoxyvinyl)stannane (2.71 g, 7.50 mmol) in 1,4-dioxane (17 mL) was added dichlorobis(triphenylphosphine)-palladium(II) (0.105 g, 0.150 mmol). The reaction was thoroughly flushed with nitrogen, sealed, and heated at 80 °C for 16 h. The reaction was removed from the heat, IN HC1 (aq) (17 mL) was added, and the mixture was stirred for 4 h. The mixture was diluted with water (20 mL) and extracted with EtOAc (3 x 50 mL). The combined organic layer was dried over MgS04, filtered, and concentrated in vacuo. The residue was purified with a flash chromatography (35% ethyl acetate/hexanes), and the retrieved sample was triturated with hexanes (3 x 50 mL) to afford ketone M9.3c as an off-white solid (412.5 mg). ¾ NMR (400 MHz, CDC13) δ ppm 7.92 (d, J = 8.5 Hz, 4H), 7.23 (d, J = 8.6 Hz, 4H), 2.61 (s, 6H), 2.32-2.28 (m, 2H), 1.67-1.63 (m, 2H). LC/MS: Anal. Calcd. for
[M+H]+ Ci9H1902: 279.14; found 279.13.
Example M9.3, Step d
Figure imgf000111_0001
To a solution of ketone M9.3c (0.4072 g, 1.463 mmol) in THF (7 mL) was added phenyltrimethylammonium tribromide (1.10 g, 2.93 mmol), and the reaction mixture was stirred at ~25 °C for 15 h. The volatile component was removed in vacuo, and the residue was partitioned between water (25 mL) and (¾(¾ (100 mL). The organic layer was dried over MgSO, filtered, and concentrated in vacuo to afford dibromide M9.3d, which was used without further purification. LC/MS: Anal. Calcd. for [M+H]+ Ci9H17 79Br202: 434.96; found 434.98.
Example M9.3, Step e
Figure imgf000111_0002
To a solution of dibromide M9.3d (-1.463 mmol) and acid M3fl (0.698 g, 3.07 mmol) in acetonitrile (10 mL) was added N,N-diisopropylethylamine (0.537 mL, 3.07 mmol), and the reaction was stirred at ~25 °C for 5h. The volatile component was removed in vacuo, and the residue was taken up in chloroform (4 mL) and loaded onto a silica gel column and eluted with 8% ethyl acetate/methylene chloride over 1296 mL solvents to afford diketoester M9.3e as a light yellow foam containing unidentified impurities (0.723 g). XH NMR (400 MHz, DMSO-d6) δ ppm 7.90 (d, J = 8.0 Hz, 4H), 7.37 (d, J = 8.3 Hz, 4H), 5.59-5.40 (m, 3H), 4.24-4.09 (m, 2H), 3.37-3.25 (m, 2H), 2.47-2.36 (m, 5H), 1.87-1.80 (m, 2H), 1.72-1.68 (m, 2H), 1.57-1.49 (m, 2H), 1.41 (s, 7H), 1.35 (s, 11H), 0.80-0.67 (m, 2H), 0.58-0.48 (m, 2H). LC/MS: Anal. Calcd. for [M+H]+ C4iH48 N2 Na O i0: 751.32; found 751.55.
Example M9.3, Step f
Figure imgf000112_0001
A mixture of diketoester M9.3e (0.723 g, 0.992 mmol) and ammonium acetate (1.529 g, 19.84 mmol) in xylene (10 mL) in a sealed reaction vessel was heated at 140 °C for 2.5 h. After the reaction was allowed to cool to ambient condition, the volatile component was removed in vacuo. The residue was taken up in 20% MeOH/CHCl3 (50 mL) and treated with saturated NaHC03 (aq) (20 mL), stirred and the layers were separated. The aqueous layer was extracted further with 20% MeOH/CHCl3 (2 x 50 mL), and the combined organic phase was dried over MgS04, filtered, and concentrated in vacuo. The residue was taken up in CHCI3 (4 mL), loaded onto a silica gel column and eluted with 45% ehtyl acetate/methylene chloride over 1296 mL to afford imidazole M9.3f as an orange solid (261.7 mg). XH
NMR (400 MHz, DMSO-d6) δ ppm 12.08 (s, 0.5H), 11.81 (s, 1.5H), 7.64 (d, J = 8.3 Hz, 3H), 7.55 (d, J = 8.3 Hz, 1H), 7.41 (d, J = 1.7 Hz, 1.7H), 7.19-1.17 (m, 1.3H), 7.11 (d, J = 8.3 Hz, 3H), 4.59 (app br s, 2H), 3.41 (app br s, 2H), 2.37-2.14 (m, 6H), 1.68-1.57 (m, 2H), 1.48-1.40 (m, 2H), 1.40-0.95 (br s, 18H), 0.79-0.69 (m, 2H), 0.59- 0.48 (m, 2H). LC/MS: Anal. Calcd. for [M+H]+ C4iH49 N6 04: 689.38; found
689.43.
Example M9.3, Step g
- I l l - H
A solution of carbamate M9.3f (0.2391 g, 0.347 mmol) in 25% TFA/CH2Q2
(1.7 mL) was stirred at ~25 °C for lh. The volatile component was removed in vacuo to afford the TFA salt of pyrrolidine M9.3g as a tan foam (328 mg). XH NMR (400 MHz, DMSO-d6) δ ppm 10.01 (br s, 2H), 7.70-7.68 (m, 6H), 7.20 (d, J = 8.6 Hz, 4H), 4.63-4.58 (m, 2H), 3.37-3.33 (m, 2H), 2.51-2.42 (m, 6H), 2.24-2.20 (m, 2H), 1.93-1.86 (m, 2H), 1.51-1.47 (m, 2H), 1.16-1.09 (m, 2H), 0.084-0.78 (m, 2H).
LC/MS: Anal. Calcd. for [M+H]+ C31H33 N6: 489.28; found 489.26. Example M9.3
To a solution of pyrrolidine M9.3g/TFA salt (0.0834 g, 0.088 mmol), (S)-2- (methoxycarbonylamino)-3-methylbutanoic acid (0.034 g, 0.194 mmol), and N,N- diisopropylethylamine (0.123 mL, 0.706 mmol) in DMF (1.5 mL) was added HATU (0.070 g, 0.185 mmol), and the mixture was stirred at 25 °C for 4h. The reaction mixture was diluted with MeOH (2.5 mL) and submitted to a reverse phase HPLC purification (XTERRA, 30X100 mm, S5; MeOH/H20/TFA) to afford Example M9.3 as an off-white foam (58 mg). ¾ NMR (400 MHz, DMSO-d6) δ ppm 14.90-14.20 (bs, 3H), 8.00 (br s, 2H), 7.68 (d, J = 8.3 Hz, 4H), 7.34 (d, J = 8.6 Hz, 4H), 7.25 (d, J = 8.6 Hz, 2H), 5.00-4.96 (m, 2H), 4.42-4.39 (m, 2H), 3.79-3.69 (m, 2H), 3.54 (s, 6H), 2.38-2.31 (m, 5H), 2.16-2.07 (m, 2H), 1.95-1.88 (m, 2H), 1.61-1.58 (m, 2H), 0.97- 0.90 (m, 8H), 0.80 (d, J = 6.5 Hz, 8H). Rt = 1.96 min (Cond. 3); >95% homogeneity index (Cond. 9 and 10); LC/MS: Anal. Calcd. for [M+H]+ C45H55 N8 06: 803.42; found 803.43. Example M9.4
Figure imgf000114_0001
Example M9.4 was prepared from pyrrolidine M9.3g/TFA salt and (S)-2- (methoxycarbonylamino)-2-(tetrahydro-2H-pyran-4-yl)acetic acid according to the same procedure used for the preparation of Example M9.3. XH NMR (400 MHz, DMSO-d6) δ ppm 15-14 (bs, 6H), 7.98 (br s, 2H), 7.66 (d, J = 8.3 Hz, 4H), 7.34-7.32 (m, 5H), 4.98-4.94 (m, 2H), 4.50-4.46 (m, 2H), 3.84-3.78 (m, 6H), 3.54 (s, 6H), 3.32- 3.18 (m, 4H), 2.39-2.29 (m, 4H), 2.1 1-1.97 (m, 2H), 1.97-1.88 (m, 2H), 1.60-1.57 (m, 2H), 1.51-1.26 (m, 8H), 0.99-0.87 (m, 2H), 0.84-0.72 (m, 2H). Rt = 1.77 min (Cond. 3); >95% homogeneity index (Cond. 9 and 10); LC/MS: Anal. Calcd. for [M+H]+ C49H59 8O8: 887.45; found 887.50.
Examples M9.5 and M9.6
Figure imgf000114_0002
Examples M9.5 and M9.6 were prepared as TFA salts starting from dibromide M9.3b2 according to the procedures described for the preparation of
Examples M9.3 and M9.4 from the corresponding stereoisomer dibromide M9.3M.
Figure imgf000114_0003
Figure imgf000115_0001
Example M9.7
Figure imgf000115_0002
Example M9.7 (TFA salt) was prepared from dibromide M9.3d and acid M12.2c according to the procedures described for the preparation of Examples M9.3. ¾ NMR (400 MHz, DMSO-d6) δ ppm 14.90-14.42 (br s, 4H), 8.02 (br s, 2H), 7.70 (d, J = 8.5 Hz, 4H), 7.36 (d, J = 8.6 Hz, 4H), 7.25 (d, J = 8.3 Hz, 2H), 5.07 (dd, J = 10.7 Hz/7.1 Hz, 2H), 4.18-4.13 (app t, 2H), 4.12-4.08 (app t, 2H), 3.55 (s, 6H), 3.42- 3.37 (app t, 2H), 2.49-2.39 (m, 2H), 2.37-2.31 (app t, 2H), 2.02-1.93 (m, 2H), 1.87- 1.78 (app q, 2H), 1.62-1.59 (app t, 2H), 1.14 (d, J = 6.3 Hz, 6H), 0.82 (d, J = 6.5 Hz, 6H), 0.77 (d, J = 6.8 Hz, 6H). Rt = 3.12 min (Cond. lOd); >95% homogeneity index (Cond. 9 and 10); LC/MS: Anal. Calcd. for [M+H]+ C45H59 N8 06: 807.46; found 807.45.
Example M9.8
Figure imgf000115_0003
Example M9.7 (TFA salt) was prepared from dibromide M9.3d and acid M12.2c according to the procedures described for the preparation of Examples M9.4. Rt = 3.01 min (Cond. lOd); >95% homogeneity index (Cond. 9 and 10); LC/MS: Anal. Calcd. for [M+H]+ C49H63 808: 891.48; found 891.52.
Example M10
Figure imgf000116_0001
Exam le M10, Step a
Figure imgf000116_0002
The title compound, containing a diastereomeric impurity, was prepared in 5 steps from (S)-methyl 5 -oxopyrrolidine-2-carboxylate by employing the procedure described J. Med. Chem., 49:3520-3535 (2006) for the synthesis of its ethyl ester analog. XH NMR (CDC13, 400 MHz): 4.35 (m, 0.5H), 4.25 (m, 0.5H), 4.05 (m, 0.5H), 3.90 (m, 0.5H), 3.73 (s, 3H), 2.20 (m, 1H), 2.00 (m, 2H), 1.65 (m, 1H), 1.50/1.40 (two overlapping br s, 9H), 1.31 (d, J = 6.0, 3H).
Exam le M10, Step b
Figure imgf000116_0003
A solution of lithium hydroxide (0.23g, 9.62 mmol) in water (5 mL) was added dropwise to a solution of ester MlOa (1.8g, 7.4 mmol) in ethanol (10 mL), and stirred at room temperature for 17 hr. Most of the solvent was evaporated, and the residue was diluted with water, 1 N HCl was added dropwise to bring it to pH 3. It was extracted with ethyl acetate (20 mL, 4x), dried (Na2S04) and evaporated in vacuo to afford a colorless oil, which yielded crystals when dissolved in
EtOAc/hexanes solvent system and allowed to stand at room temperature. The white solid was filtrated and dried in vacuo (1.42g). XH NMR (CDC13, 400 MHz, d = 7.24 ppm): 4.35 (m, 1H), 3.95 (m, 1H), 2.35 (m, 1H), 2.05 (m, 2H), 1.70 (m, 1H), 1.50 (br s, 9H), 1.25 (d, J = 7.1, 3H).
Example M10, Step c
Figure imgf000117_0001
To a solution of acid MlOb (2.16 g, 9.42 mmol) and 2-bromo-l-(4- bromophenyl)ethanone (2.62 g, 9.42 mmol) in acetonitrile (50 mL) was added slowly diisopropylethylamine (1.645 mL, 9.42 mmol), and the reaction mixture was stirred at room temperature for 5 hr. Solvent was removed in vacuo, and the residue was partitioned between ethyl acetate and water (1 : 1, 100 mL). The organic layer was washed with sat. aHC03, dried ( a2S04) and concentrated in vacuo to afford ketoester MlOc as white solid (3.9g), which was used in the next step without further purification. XH NMR (400 MHz , DMSO-d6, δ = 2.5 ppm): 7.92 (d, J = 8.3, 2H), 7.78 (d, J = 8.5, 2H), 5.6-5.4 (m, 2H), 4.35 (m, 1H), 3.85 (m, 1H), 2.25 (m, 1H), 2.05 (m, 2H), 1.60 (m, 1H), 1.5/1.4 (two overlapping br s, 9H), 1.18 (d, J = 6.6, 3H). LC/MS: Anal. Calcd. for [M+Na Ci9H2481BrNaN05: 450.07; found: 450.00.
Example M10, Step d
Figure imgf000117_0002
To a solution of ketoester MlOc (3.9 g, 9.15 mmol) in xylene (60 mL) in a
500 mL pressure tube, ammonium acetate (7.05 g, 91 mmol) was added. The reaction vessel was sealed and heated at 140 °C for 5 hr. The solvent was removed in vacuo, and the residue was partitioned between (¾(¾ (100 mL) and water (100 mL). The organic layer was washed (Sat. aHCOs), dried ( a2S04), and evaporated in vacuo. The resulting crude material was purified with flash chromatograph (30- 100% EtOAc/Hexane) to afford bromide MlOd as a brown foam (3.0 g, yield 81%). ¾ NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 11.77 (s, 1H), 7.70 (d, J = 8.5, 2H), 7.52 (br s, 1H), 7.50 (d, J = 8.5, 2H), 4.80 (m, 1H), 3.85 (m, 1H), 2.10 (m, 3H), 2.70 (m, 1H), 1.5/1.3 (overlapping br s, 9H), 1.20 (m, 3H). LC/MS: Anal. Calcd. for [M+H]+ Ci9H2579Br 302: 406.11; found: 406.18.
Example M10, Step e
Figure imgf000118_0001
Pyrrolidine MlOe (HCl salt) was prepared bromide MIOd according to the procedure described for the synthesis of pyrrolidine Mlb (HCl salt) from bromide
Example M10 (TFA salt) was prepared from bromide pyrrolidine MlOe (HCl salt) according to the procedure described for the synthesis of Example Ml from pyrroldine M3i (HCl salt). ¾ NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 8.05 (s, 2H), 7.85 (d, J = 8.1, 4H), 7.69 (d, J = 7.6, 4H), 7.56 (d, J = 8.0, 2H), 5.5 (m, 0.4H), 5.0 (m, 1.6H), 4.75 (1.6H), 4.10 (m, 0.4H), 3.95 (m, 2H), 3.50 (s, 6H), 2.50-2.30 (m, 4H), 2.10 (m, 2H), 1.85 (m, 4H), 1.46 (d, J = 6.6, 5H), 1.20 (d, J = 6.8, 1H), 0.9-0.9 (m, 7.2H), 0.74 (d, J = 6.8, 4.8H). LC (Cond. 9 and 10): >95% homogeneity index. LC/MS (Cond. 3): Rt = 2.03 min. LC/MS: Anal. Calcd. for [M+H]+ C44H55 806: 791.42; found: 791.39.
Examples M11-M12
The TFA salts Examples Ml 1-M12 were prepared according to the procedure described for Example M10 and by employing appropriate acids.
Figure imgf000119_0001
Example Ml 2.1
Figure imgf000119_0002
To a solution of pyrrolidine MIOe/HCl salt (119 mg, 0.191 mmol), (S)-2-
(methoxycarbonylamino)-2-(tetrahydro-2H-pyran-4-yl)acetic acid (49.0 mg, 0.226 mmol) and (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid (40.9 mg, 0.233 mmol) in DMF (5 mL) was added DIEA (0.200 mL, 1.147 mmol) and HATU (148 mg, 0.390 mmol), and the mixture was stirred at room temperature for 1 hr. Solvent was removed in vacuo and the residue was dissolved in methanol and purified with a reverse phase HPLC (MeOH/TFA/Water) to separate the statistical mixture of products, one of which was Example M12.1 (TFA salt; light yellow foam; 55 mg). ¾ NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 8.09-7.82 (m, 6H), 7.68-7.55 (m, 6H), 5.00 (m, 2H), 4.64 (app t, 2H), 4.21-3.75 (m, 6H), 3.53 (m, 6H), 3.17 (m, 2H), 2.33- 2.21 (m, 6H), 1.85 (m, 3H), 1.62-1.17 (overlay of 'd' and 'm', J for 'd' = 6.6 Hz, 9H), 0.88 (m, 3.6H), 0.74 (d, J = 6.8 Hz, 2.4H). LC (Cond. 9 and 10): >95% homogeneity index. LC/MS (Cond. lOh): Rt = 1.92 min. LC/MS: Anal. Calcd. for [M+H]+ C46H57 8O7: 833.44; found: 833.40.
Figure imgf000120_0001
Example M12.2, Step a
Figure imgf000120_0002
M12.2a-1 M12.2a-2 M12.2a-3
The above three esters were prepared from (S)-l-tert-butyl 2-methyl 5- oxopyrrolidine-l,2-dicarboxylate according to the procedure described in Tetrahedon Letters, 2003, 3203-3205.
Exa Step b
Figure imgf000120_0003
Borane-methyl sulfide complex (5.44 mL, 10.88 mmol) was added to a solution of ester M12.2a-2 (1.4 g, 5.44 mmol) in THF (25 mL), and the reaction mixture was heated at 40 °C for 7 hr. The volatile component was removed in vacuo and the residue was partitioned between EtOAc and water (50 mL each). The aqueous layer was extracted with EtOAc (30 mL), and the combined organic phase was dried with Na2S04, and concentrated in vacuo. The resultant colorless oil was purified with a flash chromatography (0-50% EtOAc/Hexane) to afford ester Ml 2.2b as a colorless oil (0.77 g). XH NMR (CDC13, δ = 7.24 ppm, 400 MHz): 4.29-4.18 (m, 1H), 3.78-3.66 (m, 4H), 2.99 (app t, J = 10.1, 1H), 2.43-2.97 (m, 1H), 2.43-2.37 (m, 1H), 2.30-2.18 (m, 1H), 1.60-1.52 (m, 1H), 1.47/1.42 (two 's', 9H), 1.08-1.05 (m, 3H).
Exam le Ml 2.2, Step c
Figure imgf000121_0001
To a solution of ester M12.2b (1.69 g, 6.95 mmol) in ethanol (10 mL) was added solution of LiOH (0.250 g, 10.42 mmol) in water (5.00 mL), and the reaction mixture was stirred at room temperature for 5 hr. The organic solvent was evaporated in vacuo and the residue was diluted with water (10 mL) and washed with ether (10 mL). It was chilled in ice-water bath, and acidified to a pH range of ~2 with IN HCl. It was then extracted with EtOAc (20 mL, 3x). The organic layer was dried with Na2S04 and concentrated in vacuo to afford acid M12.2c as a colorless oil, which became a white solid upon extended exposure to high vacuum (1.38g). XH NMR (CDC13, δ = 7.24 ppm, 400 MHz): 4.39-4.22 (m, 1H), 3.80-3.69 (m, 0.91H), 3.59-3.35 (m, 0.18H), 3.03-2.89 (m, 0.91H), 2.51-2.22 (m, 2H), 1.98-1.91 (m, 0.71H), 1.68-1.60 (0.29H), 1.50/1.44 (two 's', 9H), 1.09 (app m, 3H).
Figure imgf000121_0002
To a solution of M12.2c (1.38 g, 6.02 mmol) and 2-bromo-l-(4- bromophenyl)ethanone (1.673 g, 6.02 mmol) in acetonitrile (35 mL) was added DIEA (1.051 mL, 6.02 mmol). It was stirred at room temperature for 5 hrs. The solvent was evaporated in vacuo and water (50 mL) and EtOAc (70 mL) were added, organic layer was separated and washed by sat. aHC03 (30 mL), dried with Na2S04, evaporated in vacuo to give crude M12.2d as a red oil (2.71 g), which was used in the next step without further purification. XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 7.91 (m, 2H), 7.78 (d, J = 8.5 Hz, 2H), 5.60-4.90 (m, 2H), 4.29 (app t, 1H), 3.61 (m, 1H), 2.85 (m, 1H), 2.35-1.80 (m, 2H), 1.65 (m, 1H), 1.40-1.32 (two s, 9H), 1.02 (d, J = 6.5 Hz, 3H). LC/MS: Anal. Calcd. for [M+Na Ci9H24 81BrNNa05: 450.07; found: 450.11.
Exam le Ml 2.2, step e
Figure imgf000122_0001
To a pressure tube containing a solution of ketoester M12.2d (2.57 g, 6.03 mmol) in xylene (50 mL was added ammonium acetate (4.65 g, 60.3 mmol). The vessel was capped and heated at 140 °C for 5 hrs. The volatile component was removed in vacuo and the residue was partitioned between DCM (50 mL) and water (40 mL). The organic layer was dried with a2S04, and concentrated in vacuo. The resulting crude mixture was purified with flash chromatograph (30-100%
EtOAc/Hexane) to afford imidazole M12.2.e as a brown solid (1.24g). XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 7.69 (d, J = 8.5 Hz, 2H), 7.58-7.48 (m, 3H), 4.70 (m, 1H), 3.65 (m, 1H), 3.02(m, 1H), 2.37 (m, 1H), 2.22 (m, 1H), 1.74-1.54 (m, 1H), 1.37-1.08 (two s, 9H), 1.03 (d, J = 6.3 Hz, 3H). LC/MS: Anal. Calcd. for
[M+H]+ Ci9H25 79Br 302: 406.11; found: 406.18.
Example M12.2, step f
Figure imgf000122_0002
Pyrrolidine M12.2f (HC1 salt) was prepared bromide M12.2e according to the procedure described for the synthesis of pyrrolidine Mlb (HC1 salt) from bromide Dlb. Example Ml 2.2
To a mixture of pyrrolidine M12.2f /HC1 salt (200 mg, 0.321 mmol) and (S)- 2-(methoxycarbonylamino)-3-methylbutanoic acid (124 mg, 0.707 mmol) in DMF (5 mL) were added DIEA (0.337 mL, 1.928 mmol) and HATU (250 mg, 0.659 mmol), and the mixture was stirred at room temperature for 2 hrs. The volatile component was removed in vacuo and the residue was purified with a reverse phase HPLC (Phenomenex-Luna 3 OX 100mm, S10 Axia, MeOH/TFA/Water). The resultant sample was repurified with a different reverse phase HPLC (Water-Sunfire
30X100mm S5, ACN/TFA/Water) to afford the TFA salt of Example M12.2 as a light yellow foam (77.1 mg). XH NMR (Methanol-d4, δ = 3.29 ppm, 400 MHz): 7.90 (s, 2H), 7.74 (d, J = 8.6 Hz, 4H), 7.67 (d, J = 8.5 Hz, 4H), 5.18 (m, 2H), 4.30 (app t, 2H), 4.18 (d, J= 7.3 Hz, 2H), 3.63 (s, 6H), 3.38 (m, 2H), 2.63 (m, 2H), 2.51 (m, 2H), 1.98 (m, 2H), 1.81 (m, 2H), 1.21 (d, J = 6.3 Hz, 6H), 0.83-0.90 (m, 12H). LC (Cond. 9 and 10): >95% homogeneity index. LC/MS (Cond. lOh): Rt = 2.01 min. LC/MS: Anal. Calcd. for [M+H]+ C44H55 806: 791.42; found: 791.46.
Example M12.3
Figure imgf000123_0001
Example M12.3 (TFA salt) was prepared from pyrrolidine M12.2f /HC1 and (S)-2-(methoxycarbonylamino)-2-(tetrahydro-2H-pyran-4-yl)acetic acid according to the procedure described for the preparation of Example M12.2. RT: LC (Cond. 9 and 10): >95% homogeneity index. LC/MS (Cond. lOh): Rt = 1.89 min. LC/MS: Anal. Calcd. for [M+H]+ QsHsg sOs: 875.45; found 875.42. -M12.5
Figure imgf000124_0001
a solution of Example M12.2 (free-based using MCX column and 2N
NH3/MeOH) (80 mg, 0.101 mmol) in DMF (5 mL) was added NCS (17.56 mg, 0.131 mmol), and heated at 50 °C for 3 hrs. Additional NCS (5 mg, 0.037 mmol) was added to the mixture and heating was continued for 5 more hours. The volatile component was removed in vacuo and the residue was dissoled in MeOH and submitted to a reverse phase HPLC condition (MeOH/TF A/Water) to afford Example M12.4/TFA salt (light yellow foam, 24 mg) and Example M12.5/TFA salt (light yellow foam, 28 mg). Example M12.4/TFA salt: XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 8.17(s, 1H), 7.83-7.73 (m, 6H), 7.66 (d, J = 8.6 Hz, 2H), 7.30 (d, J = 8.3 Hz, 1H), 7.22 (d, J = 8.3 Hz, 1H), 5.05 (m, 1H), 4.85 (m, 1H), 4.16-4.02 (m, 4H), 3.52 (s, 6H), 3.37 (m, 1H), 3.23 (m, 1H), 2.54-2.24 (m, 4H), 1.87-1.63 (m, 4H), 1.13- 1.08 (m, 6H), 0.85-0.74 (m, 12H). LC (Cond. 9a.1 and 10a.1): >95% homogeneity index. LC/MS (Cond. 11): Rt = 4.26 min. LC/MS: Anal. Calcd. for [M+H]+
C44H54CI 8O8: 825.39; found: 825.50. Example M12.5/TFA salt: (DMSO-d6, δ = 2.5 ppm, 400 MHz): 7.79 (d, J = 8.4 Hz, 4H), 7.66 (d, J = 8.4 Hz, 4H), 7.22 (d, J = 8.3 Hz, 2H), 4.85 (m, 2H), 4.13 (app t, 2H), 4.04 (app t, 2H), 3.53 (s, 6H), 3.24 (app t, 2H), 2.39 (m, 2H), 2.26 (m, 2H), 1.90 (m, 2H), 1.66 (m, 2H), 1.09 (d, J = 6.5 Hz, 6H), 0.85 (d, J = 6.8 Hz, 6H), 0.80 (d, J = 6.5 Hz, 6H). LC (Cond. 9a and 10a.1): >95% homogeneity index. LC/MS (Cond. 11): Rt = 4.44 min. LC/MS: Anal. Calcd. for [M+H]+ C44H53Ci2 806: 859.35; found: 859.30. -M12.7
Figure imgf000125_0001
Example 12.6 - 12.7 were prepared as TFA salts from Example M12.3 according to the procedure described for the preparation of Example 12.4 - 12.5.
Figure imgf000125_0004
Example Ml 2.8
Figure imgf000125_0002
Example Ml 2.8, step a
Figure imgf000125_0003
To a mixture of pyrrolidine M12.2f/HCl salt (120 mg, 0.193 mmol) and (S)-2- (methoxycarbonylamino)-3-methylbutanoic acid (35.1 mg, 0.200 mmol) in DMF (2 mL), were added DIEA (0.168 mL, 0.964 mmol) followed by HATU (70.4 mg, 0.185 mmol), and the mixture was stirred at room temperature for 1 hr. The volatile component was removed in vacuo and the residue was dissoled in MeOH and submitted to a reverse phase HPLC condition (MeOH/TF A/Water) to isolate the TFA salt of pyrrolidine M12.8a as yellow solid (36mg). The sample was free based (MCX column; MeOH wash; 2 N NH3 /MeOH elution) to afford a yellow solid (22.3 mg). LC/MS: Anal. Calcd. for [M+H]+ C37H44N7O3: 634.35; found: 634.35.
Example Ml 2.8
To a solution of pyrrolidine M12.8a (22.3 mg, 0.035 mmol) and (S)-2- (methoxycarbonylamino)-2-(tetrahydro-2H-pyran-4-yl)acetic acid (12.99 mg, 0.060 mmol) in DMF (2 mL) were added DIEA (0.018 mL, 0.106 mmol) and HATU (13.65 mg, 0.036 mmol), and the mixture was stirred at room temperature for 2 hrs. The volatile component was removed in vacuo and the residue was dissoled in MeOH and submitted to a reverse phase HPLC condition (Column: Phenomenex-Luna
3 OX 100mm, S 10 Axia; MeOH/TF AJ Water). The resulting sample was repurified with a second reverse phase HPLC (Column: Water-Sunfire 30X100mm S5;
ACN/TF A/Water) to afford the TFA salt of Example Ml 2.8 as a light yellow foam (16 mg). XH NMR (Methanol-d4, δ = 3.29 ppm, 400 MHz): 7.92 (d, J = 5.0 Hz, 2H), 7.77 (d, J = 8.5 Hz, 4H), 7.72-7.69 (m, 4H), 5.21 (m, 2H), 4.39-4.22 (m, 4H), 3.92 (m, 2H), 3.67 (s, 6H), 3.47-3.28 (m, 4H), 2.68 (m, 2H), 2.54 (m, 2H), 2.06-1.85 (m, 4H), 1.25 (d, J = 6.5 Hz, 6H), 0.93 (d, J = 6.7 Hz, 3H), 0.91 (d, J = 7.3 Hz, 6H). LC (Cond. 9 and 10): >95% homogeneity index. LC/MS (Cond. lOd): Rt = 3.10 min. LC/MS: Anal. Calcd. for [M+H]+
Figure imgf000126_0001
833.44; found: 833.47.
Example M12.9
Figure imgf000126_0002
Figure imgf000127_0001
To a solution of mixture of M12.2a-1 and M12.2a-2 (4.75g, 18.46 mmol) was added Superhydride (19.20 mL, 19.20 mmol) dropwise at -50 °C in a dryice/acetone bath for 10 min. Hunig's base (13.58 mL, 78 mmol) was added, and stirred for 10 min and DMAP (0.122 g, 0.997 mmol) was added as a solid, stirred for 15 min, and trifluoroacetic anhydride (2.98 mL, 21.08 mmol) was added dropwise over 15 mins Then the dryice/acetone bath was removed, and the reaction mixture was stirred for 4 hr while allowing it to thaw to room temperature. The reaction mixture was washed with water (50 mL), sat. NaCl (30 mL), and concentrated in vacuo. The resulting crude material was purified with flash chromatography (8-60% EtOAc/Hexane) to afford ester M12.9a as a yellow oil (2.85 g). ¾ NMR (CDC13, 400 MHz): 6.36 (s, 0.5H), 6.25 (s, 0.5H), 4.70-4.57 (m, 1H), 3.78 (s, 3H), 2.96 (m, 1H), 2.54 (m, 1H), 1.70 (s, 3H), 1.50 (s, 4.5H), 1.44 (s, 4.5H).
Example Ml 2.9, Step b
Figure imgf000127_0002
M12.9b-1 M12.9b-2
Diethylzinc (1.1 M in toluene, 59.1 mL, 65.0 mmol) was added dropwise over 20 min to a cooled (-23 °C) toluene (60 mL) solution of ester M12.9a (5.23 g, 21.68 mmol), and stirred for 10 min. Chloroiodomethane (9.44 mL, 130 mmol) was added dropwise over 10 min, and the reaction mixture was stirred at -21 °C for 16 hr. Sat. aHC03 (60 mL) was added to the reaction mixture, the cooling bath was removed, and the mixture was stirred for 10 min. It was then filtered, and the filter cake was washed with toluene (50 mL). The filterate was partitioned, and the organic layer was dried with Na2S04, and concentrated in vacuo. The resulting crude material was purified with flash chromatography (2-10% EtOAc/Hexane) to afford ester ester M12.9b-1 (first elute; colorless oil; 2.88 g) and ester M12.9b-2 (second elute;
colorless oil; l.Olg). Relative stereochemical assignment was made based on NOE studies. Ester M12.9b-1 : ¾ NMR (CDC13, 400 MHz): 4.65-4.52 (m, 1H), 3.72 (s, 3H), 3.28-3.17 (m, 1H), 2.44-2.32 (m, 1H), 2.16-2.10 (m, 1H), 1.51-1.42 (two s, 9H), 1.24 (s, 3H), 1.07 (m, 1H), 0.69-0.60 (m, 1H).
Exam le M12.9, Step c
Figure imgf000128_0001
To a solution of M12.9b-1 (2.88 g, 1 1.28 mmol) in Ethanol (20 mL) was added a solution of LiOH (0.324 g, 13.54 mmol) in water (10.00 mL), and the mixture was stirred at room temperature for 6 hr. Most of the volatile component was removed in vacuo, and the residue was partitioned between water (20 mL) and ether (20 mL). The aqueous layer was chilled in an ice-water bath, acidified with a IN HC1 to a pH region of 2, and extracted with EtOAc (30 mL, 4x). The combined organic phase was dried with Na2S04, evaporated in vacuo to give acid M12.9c as a sticky solid (2.55g). XH NMR (CDC13, 400 MHz): 4.64 (m, 1H), 3.25 (appt s, 1H), 2.70-2.40 (m, 1H), 2.14 (m, 1H), 1.54-1.44 (m, 9H), 1.27 (s, 3H), 1.10-0.80 (m, 1H), 0.67 (m, 1H). Exam le Ml 2.9, Step d
Figure imgf000128_0002
To a solution of acid M12.9c (2.05 g, 8.50 mmol) in Acetonitrile (50 mL) was added 2-bromo-l-(4-bromophenyl)ethanone (2.361 g, 8.50 mmol) followed by DIEA (1.484 mL, 8.50 mmol), and the reaction mixture was stirred at room temperature for 16 hr. Most of the volatile component was removed in vacuo, and the residue was partitioned between EtOAc (50 mL) and water (50 mL). The organic layer was washed with sat. NaHC03 (30 mL) and sat. NaCl (20 mL), dried with Na2S04, and evaporated in vacuo to afford ketoester M12.9d as white foam (3.5g). LC/MS: Anal. Calcd. for [M+Na]+ C20H24 81Br NaO5: 462.07; found: 461.91.
Exam le M12.9, Step e
Figure imgf000129_0001
To a mixture of ketoester M12.9e (3.5 g, 7.99 mmol) and xylene (80 mL) in a pressure tube was added ammonium acetate (6.16 g, 80 mmol), and the reaction mixture was capped and heated at 140 °C for 4.5 hr. The volatile component was removed in vacuo and the residue was partitioned between DCM (70 mL) and water (70 mL). The organic layer was washed with sat. aHC03 (30 mL), and
concentrated in vacuo. The resulting crude material was purified with flash chromatograph (40-100% EtOAc/Hexane) to afford imidazole M12.9e as a brown solid (2.8 g). XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 7.68 (m, 2H), 7.57-7.49 (m, 3H), 5.08 (m, 1H), 3.20 (m, 1H), 2.45-2.09 (M, 2H), 1.69-1.52 (m, 1H), 1.42- 1.16 (m, 12H), 0.62 (m, 1H). LC/MS: Anal. Calcd. for [M+H]+ C2oH2581BrN302: 420.11 ; found: 420.02.
Exam le M12.9, Step f
Figure imgf000129_0002
To a pressure tube containing a solution of bromide M12.9e (0.354 g, 1.007 mmol) and l,2-bis(trimethylstannyl)ethyne (0.354 g, 1.007 mmol) in DMF (15 mL) was added Pd (Ph3P)4 (0.070 g, 0.060 mmol), and the reaction mixture was degassed for 10 min and the reaction vessel was capped and heated at 90 °C for 14 hr. Most of the volatile component was removed in vacuo, and the residue was partitioned between DCM (60 mL) and water (40 mL). The organic layer was dried with a2S04, and evaporated in vacuo. The resulting crude material was purified with flash chromatograph (40-100% EtOAc/hexanes) to afford alkyne M12.9f as a red solid (0.3 g). LC/MS: Anal. Calcd. for [M+H]+ C42H49 604: 701.38; found: 701.43.
Example Ml 2.9, Step g
Figure imgf000130_0001
4N HC1 in dioxane (3.90 mL, 128 mmol) was added to carbamate M12.9f (0.3 g, 0.428 mmol), and the mixture was stirred at room temperature for 5 hr. The volatile component was removed in vacuo and the residue was dried under high vacuum overnight to afford the HC1 salt of pyrrolidine M12.9g as a yellow solid (0.27 g). LC/MS: Anal. Calcd. for [M+H]+ C32H33 6: 501.28; found: 501.22.
Example M12.9
To a mixture of M12.9g/HCl salt (60 mg, 0.093 mmol) and (S)-2-
(methoxycarbonylamino)-3-methylbutanoic acid (35.8 mg, 0.204 mmol) in DMF (2 mL) were added DIEA (0.097 mL, 0.557 mmol) and HATU (72.7 mg, 0.191 mmol), and the reaction mixture was stirred at room temperature for 2 hr. The volatile component was removed in vacuo and the residue was dissoled in MeOH and submitted to a reverse phase HPLC purification (Phenomenex-Luna 3 OX 100mm, S10 Axia, MeOH/TF A/Water). The resulting sample was repurified with a second reverse phase HPLC (Water-Sunfire 30X150mm OBD, ACN/TFA/Water) to afford the TFA salt of Example M12.9 as light yellow foam (37 mg). LC/MS (Cond. lOd): Rt = 3.08 min. LC (Cond. 9 and 10): >95% homogeneity index. LC/MS: Anal. Calcd. for [M+H]+ C46H55 806: 815.42; found: 815.46. Example Ml 3
Figure imgf000131_0001
a
Figure imgf000131_0002
(SEM regiochemistry was not determined)
To a solution of bromide M3h (1.0 g, 2.47 mmol) in DMF (25 mL) was added sodium hydride (60%; 0.109 g, 2.72 mmol), and the reaction mixture was stirred at room temperature for 20 min. Then (2-(chloromethoxy)ethyl)trimethylsilane (0.482 mL, 2.72 mmol) was added dropwise, and the reaction was stirred for 21h. The volatile component was removed in vacuo, and the residue was partitioned between ethyl acetate (25 mL) and water (25 mL). The aqueous layer was extracted with ethyl acetate (2 x 25 mL), and the combined organic phase was washed with brine (25 mL), dried over MgS04, filtered, and concentrated in vacuo. The residue was taken up in CHCI3 (2 mL) and loaded on a Thomson's silica gel cartridge eluting with 25% ethyl acetate/hexanes to afford bromide M13a of unknown regio-chemical make up as light yellow foam (1.171 g). XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 7.71 (s, 1H), 7.69 (d, J = 8.6, 2H), 7.52 (d, J = 8.6, 2H), 5.62-5.31 (br s, 1H), 5.26 (d, J = 10.8, 1H), 4.84^1.66 (app br s, 1H), 3.57-3.36 (app br s, 1H), 3.50 (t, J = 8.1, 2H), 2.44-2.20 (app br s, 2H), 1.77-1.60 (app br s, 1H), 1.52-1.23 (br s, 4H), 1.23-0.96 (br s, 5H), 0.92-0.79 (m, 2H), 0.78-0.69 (m, 1H), 0.64- 0.55 (app br s, 1H), 0.00 (s, 9H). LC/MS: Anal. Calcd. for [M+H]+ C25H3779Br 303Si: 534.18; found: 533.99.
Figure imgf000132_0001
(SEM regiochemistry was not determined)
To a solution of bromide M13a (0.5542 g, 1.037 mmol) and triethylamine (0.434 mL, 3.11 mmol) in 2-propanol (2.000 mL) and water (1 mL) in a sealed reaction vessel was added potassium vinyltrifluoroborate (0.181 g, 1.348 mmol) followed by 1 , 1 '-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex (0.085 g, 0.104 mmol). The reaction mixture was flushed with nitrogen, capped and heated at 100 °C for 17h. The volatile component was removed in vacuo, and the residue was partitioned between ethyl acetate (25 mL) and water (25 mL), and the aqueous phase was extracted with ethyl acetate (2 x 25 mL). The combined organic phase was dried over MgS04, filtered, and concentrated in vacuo. The residue was taken up in CHCI3 (4 mL) and loaded onto a Thomson's silica gel cartridge eluting with 25% ethyl acetate/hexanes to afford alkene Ml 3b as a light yellow viscous oil (345.4 mg). ¾ NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 7.71 (d, J = 8.3, 2H), 7.67 (s, 1H), 7.45 (d, J = 8.3, 2H), 6.72 (dd, J = 17.6, 1 1.1, 1H), 5.8 (dd, J = 0.8, 17.6, 1H), 5.60-5.29 (app br s, 1H), 5.26 (d, J = 10.8, 1H), 5.22 (d, J = 11.8, 1H), 4.83^.67 (app br s, 1H), 3.59 - 3.35 (app br s, 1H), 3.51 (t, J = 8.1, 2H), 2.41-2.23 (app br s, 2H), 1.76-1.59 (app br s, 1H), 1.51-1.22 (app br s, 4H), 1.22- 0.97 (app br s, 5H), 0.93-0.80 (m, 2H), 0.80-0.71 (m, 1H), 0.65-0.56 (app br s, 1H), 0.00 (s, 9H). LC/ S: Anal. Calcd. for [M+H]+ C27H40N3O3S1: 482.28; found: 482.24.
Example M13, Step c
Figure imgf000133_0001
(SEM regiochemistry was not determined)
To a solution of alkene M13b (0.748 g, 1.552 mmol) in CH2C12 (2 mL) was added Grubbs 2nd Generation Catalyst (0.132 g, 0.155 mmol), and stirred at room temperature under nitrogen for 18 hr. The reaction was loaded onto a Thomson's silica gel cartridge eluting with 30% ethyl acetate/hexanes to afford M13c as a tan foam (0.50 g). XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 7.74 (d, J = 8.4, 4H), 7.68 (s, 2H), 7.58 (d, J = 8.3, 4H), 7.22 (s, 2H), 5.72-5.34 (bs, 3H), 5.34-5.21 (m, 2H), 4.94-4.63 (app br s, 3H), 3.52 (t, J = 7.8, 4H), 2.45-2.20 (app br s, 4H), 1.88- 1.57 (app br s, 2H), 1.57-1.26 (br s, 8H), 1.26-0.98 (br s, 10H), 0.93-0.80 (m, 4H), 0.80-0.69 (m, 2H), 0.69 -0.51 (app bs, 2H), 0.00 (s, 18H).
Example M13, Step d
Figure imgf000133_0002
4N HCl/Dioxane (13 mL) was carefully added to a flask containing Ml 3c
(0.4799 g, 0.513 mmol) that was cooled with an ice/water bath. A mixture of water (4 mL) and 12N HC1 (2 mL) was added to the above mixture and the cooling bath was removed and stirring of the reaction mixture continued for 24h. MeOH (2 mL) was added to the reaction, and stirring was continued for 17h. All solvents were removed in vacuo to afford pyrrolidine M13d (.4HC1) as a yellow/tan solid (317 mg). ¾ NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 10.78-9.87 (very br s, ~3H), 8.01 (s, 2H), 7.87 (d, J = 8.4, 4H), 7.71 (d, J = 8.6, 4H), 7.35 (s, 2H), 4.76 (app t, J = 8.8, 2H), 3.43 (app t, J = 5.1, 2H), 2.64 (m, 2H), 2.55-2.52 (m, 2H), 1.95 (m, 2H), 1.16- 1.05 (m, 2H), 0.86 (m, 2H). LC/MS: Anal. Calcd. for [M+H]+ C3oH3iN6: 475.26; found: 475.26. Example Ml 3
The TFA salt of Example Ml 3 was prepared from pyrrolidine M13d (.4HC1) and (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid according to the procedure described for the preparation of Example Ml. lH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 8.14-7.95 (br s, 2H), 7.80-7.74 (m, 8H), 7.40 (s, 2H), 7.25 (d, J = 8.3, 2H), 5.0 (app t, J = 7.3, 2H), 4.41 (app t, J = 7.6, 2H), 3.81-3.67 (m, 2H), 3.54 (s, 6H), 2.57-2.46 (overlapped with DMSO-d6) (m, 2H), 2.44-2.31 (m, 2H), 2.19-2.05 (m, 2H), 1.98-1.87 (m, 2H), 1.02-0.89 (m, 7.5H), 0.89-0.70 (m, 8.5H). LC (Cond. 3): ¾ = 1.92 mm; LC/ S: Anal. Calcd. for [M+H]+ C44H53 806: 789.41; found:
789.46.
Examples M14-M15
Example M14 (TFA salt) was prepared from pyrrolidine M13d and appropriate acid by employing the procedure described for the synthesis of Example Ml. In the case of Example M15 an equimolar mixture of (S)-2- (methoxycarbonylamino)-3-methylbutanoic acid and (S)-2-(methoxycarbonylamino)- 2-(tetrahydro-2H-pyran-4-yl)acetic acid was employed for the coupling step and the resultant statistical mixture of products was separated by the HPLC technique described for Example Ml.
Figure imgf000134_0001
Figure imgf000134_0002
Figure imgf000135_0001
Example l
Figure imgf000135_0002
Example Nl, Step a
Figure imgf000135_0003
To a suspension of 2-amino- 1 -(4-bromophenyl)ethanone, HC1 (4.0 g, 15.97 mmol) in DCM (50.0 mL) was added sodium bicarbonate (4.02 g, 47.9 mmol). Then Boc-anhydride (3.89 mL, 16.77 mmol) was added to the solution and the reaction mixture was warmed to rt and stirred for 18 hrs. Then DIEA (3 mL, 17.18 mmol) was added and the reaction mixture was stirred at rt for 2 hrs, LCMS showed that the reaction was complete. The reaction mixture was diluted with EtOAc and water, the organic phase was washed with 5% citric acid, water and sat. NaCl, dried over anhydrous Na2S04, filtered and concentrated to yield bromide N1A (5.0 g) as a yellow solid, which was used in the next step without further purification. XH NMR (400 MHz, MeOD) ppm 7.89 (2 H, m), 7.68 (2 H, m, J=8.53 Hz), 4.52 (2 H, s), 1.38 - 1.51 (9 H, m). LC/MS (Cond. lOd): Rt = 3.56 min. LC/MS: Anal. Calcd. For
[M+Naf Ci3H16BrNaN03: 336.03; found: 335.97.
Example Nl, Step b
Figure imgf000136_0001
To a solution of carbamate Nla (2.0 g, 6.37 mmol) in DMF (5 mL) was added l,2-bis(trimethylstannyl)ethyne (1.119 g, 3.18 mmol). The reaction mixture was degassed, tetrakis(triphenylphosphine)palladium(0) (0.184 g, 0.159 mmol) was added, and the mixture was heated at 90 °C for 4 hrs. The crude reaction mixture was charged to a 90 g silica gel cartridge which was eluted with a 20 min gradient of 0-60% EtOAc in hexane. Alkyne ib (0.83 g) was collected as a yellow solid.
LC/MS (Cond. lOd): Rt = 4.1 min. LC/MS: Anal. Calcd. For [M+Naf
C28H32NaN206: 515.23; found: 515.10.
Example Nl, Step c
Figure imgf000136_0002
To a solution of alkyne Nib (1.13 g, 2.294 mmol) in 1,4-dioxane (5 mL) was added 4 M HC1 in dioxane (4 mL, 16.00 mmol). The reaction mixture was stirred at rt for 2 hrs. The reaction mixture was concentrated to dryness to yield a yellow solid. The solid was washed with hexane and EtOAc, then dried to yield aminoketone Nlc, 2 HC1 (0.508 g). 'H NMR (400 MHz, MeOD) ppm 8.10 (4 H, d, J=8.53 Hz), 7.78 (4 H, d, J=8.53 Hz), 4.64 (4 H, s). LC/MS (Cond. lOd): Rt = 1.94 min. LC/MS: Anal. Calcd. For [M+H]+ Ci8H17N202: 293.12; found: 293.07. Example Nl, Step d
Figure imgf000137_0001
To a mixture of (S)-l-(tert-butoxycarbonyl)-4,4-difluoropyrrolidine-2- carboxylic acid (0.25 g, 0.995 mmol), aminoketone Nlc, 2 HC1 (0.182 g, 0.498 mmol), HATU (0.189 g, 0.498 mmol) in DMF (2 mL) was added DIEA (0.521 mL, 2.99 mmol). The reaction mixture was stirred at rt for 3 hrs. The crude mixture was charged to an 80 g silica gel cartridge which was eluted with a 20 min gradient of 0- 100% EtOAc in hexane. ketoamide Nld (0.12 g) was collected as a yellow solid. LC/MS (Cond. lOd): Rt = 4.08 min. LC/MS: Anal. Calcd. For [M+H]+
C38H43F4 4O8 : 759.29; found: 759.03.
Example Nl, Step e
Figure imgf000137_0002
In a sealed tube, a mixture of ketoamide Nld (0.12 g, 0.158 mmol) and ammonium acetate (0.122 g, 1.582 mmol) in xylene (2 mL) was heated at 140 °C for 4 hrs. The reaction mixture was partitioned between EtOAc and water, the organic layer was washed with sat. NaHC03 and sat. NaCl, dried over anhydrous Na2S04, filtered and concentrated. The residue was dissolved in a small amount of methylene chloride and charged to a 40 g silica gel cartridge which was eluted with a 20 min gradient of 0-100% EtOAc in hexane. Imidazole Nle (0.054 g) was collected as a yellow solid. LC/MS (Cond. lOd): Rt = 3.3 min. LC/MS: Anal. Calcd. For [M+H]+ C38H41F4N604: 721.3; found: 721.10. Example Nl, Step f
Figure imgf000138_0001
To a solution of imidazole Nle (0.054 g, 0.075 mmol) in 1,4-Dioxane was added 4 M HC1 in dioxane (0.5 mL, 2.0 mmol) plus a few drops of MeOH. The reaction mixture was stirred at rt for 4 hrs, and concentrated to yield pyrrolidine Nlf, 4 HC1 (0.055 g) as a pale yellow solid. LC/MS (Cond. lOd): Rt = 2.94 min. LC/MS: Anal. Calcd. For [M+H]+ C28H25F4N6: 520.2; found: 521.10.
Example Nl
To a mixture of (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid (0.014 g, 0.079 mmol), pyrrolidine Nlf, 4 HC1 (0.025 g, 0.038 mmol), HATU (0.030 g, 0.079 mmol) in DMF (2 mL) was added DIEA (0.039 mL, 0.225 mmol). The reaction mixture was stirred at rt for 18 hrs, diluted with MeOH, filtered and purified by reverse phase HPLC to yield TFA salt of Example Nl (0.026 g) as a white solid. LC (Cond. 10b and 10c): >95% homogeneity index. LC/MS (Cond. lOd): Rt = 3.11 min. LC/MS: Anal. Calcd. For [M+H]+ C42H47F4N806: 835.35; found: 835.16.
Example N2
Figure imgf000138_0002
Pyrrolidine Nlf was coupled with 2-(methoxycarbonylamino)-2-(tetrahydro- 2H-pyran-4-yl)acetic acid by employing the procedure described for the synthesis of Example Nl . The resultant three diastereomers (TFA salts) were separated by employing the following condition: Column = Waters-Sunfire 30 X 100 mm S5; Start %B = 0; Final %B = 32; Gradient time = 25 min; Stop time = 25 min; Flow Rate = 40 mL/min; Wavelength = 220 nm; Solvent A = 0.1 % TFA in 10% MeCN/90% water; Solvent B = 0.1 % TFA in 90% MeCN/10% water.
Figure imgf000139_0002
Example N5-N7.3
Example N5-N7.3 (TFA salt) were prepared starting from aminoketone Nlc and appropriate starting materials, obtained from commercial sources, by employing the procedures described for the synthesis of Example Nl.
Figure imgf000139_0001
Figure imgf000139_0003
Figure imgf000140_0001
Example N7.8 & N7.9
Figure imgf000141_0001
When the commercially available (S)-l-(tert-butoxycarbonyl)-2,5-dihydro- lH-pyrrole-2-carboxylic acid was used in place of (S)-l-(tert-butoxycarbonyl)-4,4- difluoropyrrolidine-2-carboxylic acid in the procedure described for the synthesis Example Nl, two diastereomers (Example N7.8 & N7.9) were isolated from the final step. The two diastereomers (TFA salts) were separated by employing the following condition: Column = Waters-Sunfire 30 X 100 mm S5; Start %B = 0; Final %B = 25; Gradient time = 30 min; Stop time = 30 min; Flow Rate = 40 mL/min; Wavelength = 220 nm; Solvent A = 0.1 % TFA in 10% MeCN/90% water; Solvent B = 0.1 % TFA in 90% MeCN/10% water. Example N7.8: XH NMR (400 MHz, MeOD) ppm 7.94 (2 H, s), 7.74 - 7.81 (4 H, m), 7.67 - 7.73 (4 H, m), 6.33 - 6.42 (2 H, m), 5.98 - 6.09 (4 H, m), 4.85 (2 H, d, J=1.76 Hz), 4.63 - 4.76 (2 H, m), 4.14 (2 H, d, J=8.03 Hz), 3.66 (6 H, s), 1.95 - 2.05 (2 H, m), 0.93 (6 H, d, J=6.53 Hz), 0.87 (6 H, d, J=6.78 Hz). LC (Cond. lOd): Rt = 2.933 min; LC/MS: Anal. Calcd. for [M+H]+ C42H46 806:
758.35; found: 759.41. Example N7.9: XH NMR (400 MHz, MeOD) ppm 7.92 (2 H, d, J=14.81 Hz), 7.75 - 7.83 (4 H, m), 7.69 (4 H, dd, J=8.66, 2.13 Hz), 6.37 (2 H, td, J=6.65, 2.26 Hz), 6.03 - 6.09 (1 H, m), 5.98 - 6.03 (3 H, m), 4.85 (2 H, d, J=1.76 Hz), 4.72 (1 H, d, J=5.02 Hz), 4.59 - 4.68 (1 H, m), 4.15 (2 H, dd, J=8.03, 4.77 Hz), 3.64 - 3.69 (3 H, m), 3.55 - 3.62 (3 H, m), 1.95 - 2.11 (2 H, m), 1.03 (6 H, d, J=6.53 Hz), 0.93 (3 H, d, J=6.78 Hz), 0.87 (3 H, d, J=6.78 Hz). LC (Cond. lOd): Rt = 2.920 min; LC/MS: Anal. Calcd. for [M+H]+ C42H46N806: 758.35; found: 759.34.
Example N8
Figure imgf000142_0001
Figure imgf000142_0002
Pyrrolidine N8a (HC1 salt) was prepared staring from aminoketone Nlc and
(lS,3S,5S)-2-(tert-butoxycarbonyl)-2-azabicyclo[3.1.0]hexane-3-carboxylic acid (for its preparative method, see WO2004/052850) according to the procedures described for the preparation of pyrrolidine Nlf. XH NMR (MeOD, δ = 3.33 ppm, 400 MHz): 7.98 (s, 2H ), 7.87 (d, J = 8.4, 4H), 7.69 (d, J = 8.4, 4H), 5.59 (dd, J = 10.2, 5.4, 2H), 3.61-3.56 (m, 2H), 3.05-2.98 (m, 2H), 2.67 (dd, J = 14.2, 5.4, 2H), 2.20-2.14 (m, 2H), 1.28-1.22 (m, 2H), 1.19-1.13 (m, 2H). LC/MS: Anal. Calcd. for [M+H]+ C3oH29 6: 473.24; found 473.2.
Example N8
Example N8 was prepared from pyrrolidine N8a according to the procedure described for the preparation of Example l with the exception that the purification was conducted under a modified reverse phase HPLC condition (ACN/water/ NH4OAc). Example N8 was retrieved as a pale-yellow solid. LC (Cond. lOe): >99% homogeneity index. LC/MS (Cond. lOf): Rt = 2.21 min. XH NMR (DMSO, δ = 2.50 ppm, 400 MHz): 12.33 (s, 0.28H), 11.86 (s, 1.72H), 7.74 (d, J = 8.4, 3.42H), 7.65 (d, J = 8.4, 0.58H), 7.55 (d, J = 8.4, 0.58H), 7.51 (s, 2H), 7.49 (d, J = 8.4, 3..42H), 7.31 (d, J = 8.8, 2H), 5.39 (app d, 0.15H), 3.34 (dd, J = 10.8, 2.4, 3.85H), 4.29 (app t, 2H), 3.90 (br m, 2H), 3.55 (s, 6H), 2.64-2.56 (m, 2H), 2.17 (app d, 2H), 2.08-1.98 (m, 2H), 1.79-1.74 (m, 4H), 0.92-0.83 (m, 14H). LC/MS: Anal. Calcd. for [M+H]+
C44H5iN806: 787.39; found 787.8. Example N9
Figure imgf000143_0001
Compound N9 (TFA salt) was prepared from pyrrolidine N8a and (5)-2- (methoxycarbonylamino)-2-(tetrahydro-2H-pyran-4-yl)acetic acid according to the procedure described for the preparation of Example l with the exception that ACN/water/TFA solvent system was employed for the purification step. LC (Cond. lOe): >97% homogeneity index. LC/MS (Cond. lOg): Rt = 1.82 min. LC/MS: Anal. Calcd. for [M+H]+ C48H55 8O8: 871.41 ; found 871.6.
Example 10
Figure imgf000143_0002
A solution of (S)-5-oxopyrrolidine-2-carboxylic acid (20 g, 154 mmol) in
MeOH (140 mL) was cooled to 0 °C. Then thionyl chloride (1 1.2 mL, 154 mmol) was added drop wise and stirred for 30 minutes. The reaction mixture was brought to RT and stirred for 3 h. Then the reaction mixture was concentrated under reduced pressure, the resulting residue was dissolved in EtOAc (200 mL) and the organic layer was washed with aHC03 solution. The aqueous phase was extracted with
EtOAc (2 x 100 mL), and the combined organic layer was washed with brine, dried over Na2S04 and concentrated in vacuo to obtain crude ester NlOa (8.0 g), which was used as such in the next step. 1H NMR (DMSO-d6, δ = 2.50 ppm, 400 MHz): δ 7.99 (s, 1H), 4.21-4.17 (m, 1H), 3.67 (s, 3H), 2.39-2.29 (m, 1H), 2.17-2.09 (m, 2H), 2.02- 1.95 (m, 1H).
E b
Figure imgf000144_0001
A solution of ester NlOa (6.5 g, 45.4 mmol) and DMAP (0.277 g, 2.2 mmol) in toluene (32 mL) was cooled to 0 °C. Then (Boc^O (10.69 mL, 46.6 mmol) was added drop wise, and the mixture was stirred for 15 minutes at 0 °C and at RT for 12 h. The reaction mixture was quenched with aHC03 solution and stirred for 45 minutes. The separated organic layer was washed with water and followed by brine, dried over Na2S04 and concentrated in vacuo. The resulting crude material was purified by flash chromatography (Silica gel, EtOAc: petroleum ether, 40:60) to afford carbamate NlOb (7 g). XH NMR (CDC13> δ = 7.26 ppm, 400 MHz): δ 4.62 (dd,
J = 9.4, 3.0, 1H), 3.78 (s, 3H), 2.68-2.59 (m, 1H), 2.53-2.45 (m, 1H), 2.37-2.27 (m, 1H), 2.06-1.98 (m, 1H), 1.48 (s, 9H). c
Figure imgf000144_0002
A solution of carbamate 10b (7 g, 28.8 mmol) in THF (25 mL) was cooled to -78 °C, and super hydride (1 M in THF, 34.5 mL, 34.5 mmol) was added drop wise and stirred for 2 h. The reaction mixture was quenched with saturated aHC03 solution and allowed to thaw to 0 °C. Then 30% aq. H202 (15 mL ) was added and stirred for 20 minutes. The reaction mixture was extracted with Et20 (3 x 100 mL), washed with brine, dried over Na2S04 and concentrated in vacuo. To the resulting crude aminol (28.8 mmol) in MeOH (25 mL) was added PTSA (656 mg, 3.45 mmol) and stirred for 12 h. The reaction mixture was quenched with NaHCCb solution and extracted with Et20 (3 x 100 mL). The organic phase was washed with brine, dried over a2S04 and concentrated in vacuo to afford a mixture of diastereomers of ether
NIOc (5.7 g), which was used as such in the next step. XH NMR (CDCI3, δ = 7.26 ppm, 400 MHz): δ 5.30-5.13 (m, 1H), 4.38-4.23 (m, 1H), 3.74/3.71/3.70 (s, 3H), 3.43/3.40/3.39/3.35 (s, 3H), 2.41-2.26 (m, 1H), 2.17-1.87 (m, 3H), 1.48/1.42/1.40 (s, 9H). d
Figure imgf000145_0001
A solution of CuBr:Me2S (10.15 g, 49.0 mmol) in Et20 (130 mL) was cooled to -40 °C, MeMgBr (3 M in Et20, 16.45 mL, 49.0 mmol) was added drop wise, and the reaction mixture was stirred for 45 minutes. Then the reaction mixture was cooled to -78 °C and BF3:Et20 (6.2 mL, 49.0 mmol) was added. After being stirred for 30 minutes, crude ether NIOc (3.2 g, 12.0 mmol) in Et20 (30 mL) was added drop wise, and it was stirred while allowing it to thaw from -78 °C to RT over 3 h, and stirring was continued for 1 h at RT. The reaction mixture was quenched with saturated NH4C1/ aq.N]¾ solution (1 : 1, 50 mL) and stirred for 1 h at RT. The organic layer was separated and the aqueous layer was extracted with Et20 (3 x 100 mL). The combined organic phase was washed with aHC03 solution, water, brine, dried over Na2S04 and concentrated in vacuo. The crude was submitted to a chiral purification (Column = Kromasil-(R,R) Whelk-01, 30 X 250 mm, 5 μ; Isocratic (n- hexane:EtOH, 95:5); Stop time = 30 min; Flow Rate = 20 mL/min; Wavelength =
220 nm) to afford the dominant stereoisomer, trans diastereomer NlOd (850 mg). XH NMR (CDCI3, δ = 7.26 ppm, 400 MHz): δ 4.33/4.26 (dd, J = 9.0, 1.6, 1H), 4.18-4.04 (m, 1H), 3.71/3.70 (s, 3H), 2.30-2.05 (m, 2H), 1.94-1.88 (m, 1H), 1.57-1.50 (m, 1H), 1.46/1.39 (s, 9H), 1.19/1.15 (d, J = 6.4, 3H).
E e
Figure imgf000145_0002
A solution of ester NIOd (700 mg, 2.88 mmol) in EtOH (4 mL) was cooled to 0 °C, and LiOH (103 mg, 4.32 mmol) in water (2 mL) was added, and the mixture was stirred for overnight. The reaction mixture was then concentrated under reduced pressure, the residue was dissolved in water and extracted with Et20 (20 mL). The aqueous phase was acidified to pH 4 with 1 N HC1 and extracted with EtOAc (100 mL). The organic layer was washed with brine, dried over Na2S04 and concentrated in vacuo to afford acid NlOe (630 mg). XH NMR (CDC13> δ = 7.26 ppm, 400 MHz): δ 4.39 (d, J = 6.8, 0.7H), 4.28 (d, J = 8.8, 0.3H), 4.18-4.09 (m, 0.3H), 3.98-3.93 (m, 0.7H), 2.38-2.29 (m, 1H), 2.17-2.08 (m, 2H), 1.62-1.54 (m, 1H), 1.49/1.41 (s, 9H), 1.22-1.17 (m, 3H). LC/MS: Anal. Calcd. for [M-H]" CuH18N04: 228.13; found 228.2.
Example 10, Step f
Figure imgf000146_0001
Bis-carbamate lOf.1 was prepared from acid IO.e according to the procedure described for the synthesis of bis-carbamate le. To a solution of bis- carbamate NlOf.1 (320 mg, 0.47 mmol) in MeOH (6 mL) was added HCl/dioxane (4N, 20 mL) at 0 °C and the mixture was stirred for 2 h while allowing it to thaw to room temperature. The volatile component was removed in vacuo, and the residue was co-evaporated with anhydrous DCM (3 x 5 mL) and dried under high vacuum to afford pyrrolidine 10f.2 (4HC1, 300 mg) as a pale yellow solid, which was submitted to the next step as such. XH NMR (DMSO-d6, δ = 2.50 ppm, 400 MHz): δ 9.76 (br s, 2H), 9.49 (br s, 2H), 7.90 (d, J = 8.4, 4H), 7.61 (d, J = 8.4, 4H), 4.97-4.91 (m, 2H), 3.94-3.81(m, 2H), 2.56-2.42 (obscured, 2H), 2.40-2.30 (m, 4H), 1.73-1.68 (m, 2H), 1.39 (d, J = 6.4, 6H). LC/MS: Anal. Calcd. for [M+H]+ C3oH33 6: 477.27; found 477.2.
Example 10 HATU (106 mg, 0.280 mmol) was added to the DMF (5 mL) solution of pyrrolidine N10f.2 (84 mg, 0.136 mmol), (S)-2-(methoxycarbonylamino)-3- methylbutanoic acid (50 mg, 0.286 mmol) and DIEA (0.1 mL, 0.546 mmol) and stirred at room temperature for 2 h. The volatile component was removed in vacuo and the residue was dissolved in EtOAc (50 mL). The organic layer was washed with saturated NaHCC solution, saturated NH4C1 and the aqueous phase was extracted with EtOAc (25 mL). The combined extracts were washed with water, brine, dried over Na2S04 and concentrated in vacuo. The crude was submitted to reverse phase HPLC purification (ACN/water/NH4OAc) to afford Example N10 as a free base (25 mg). LC (Condition We and lOe. l) >97% homogeneity index.
LC/MS (Condition lOe.5) Rt = 1.63 min. XH NMR (DMSO-d6, δ = 2.50 ppm, 400 MHz): δ 12.21/12.08/11.83 (s, 2H), 7.78-7.74 (m, 3H), 7.67-7.47 (m, 6H), 6.98-6.94 (m, 1H), 5.18 (d, J = 8.0, 0.2H), 5.04 (d, J = 8.8, 1.8H), 4.37-4.34 (m, 2H), 4.25-4.21 (m, 1.4H), 4.0-3.95 (m, 0.6H), 3.55 (s, 4H), 3.35-3.32 (obscured, 2H), 2.10-1.49 (m, 10H), 1.35 (d, J = 6.4, 4H), 1.17 (d, J = 6.4, 2H), 0.95 (d, J = 6.8, 4H), 0.91 (d, J = 6.4, 2H), 0.82/0.77 (d, J = 6.8, 6H). LC/MS: Anal. Calcd. for [M+H]+
Figure imgf000147_0001
791.42; found 791.4.
Example Ni l
Figure imgf000147_0002
Example Ni l (TFA salt) was prepared from pyrrolidine If (4HC1 salt) and (S)-2-(methoxycarbonylamino)-2-(tetrahydro-2H-pyran-4-yl)acetic acid according to the procedure described for Example N10 with the exception that ACN/water/TFA was used for the HPLC purification step. LC (Condition We and lOe. l): >98% homogeneity index. LC/MS (Condition 10e.5)\ Rt = 1.44 min. LC/MS: Anal. Calcd. for [M+H]+ C48H59N808: 875.44; found 875.4. Example Nl 1.1 & N11.2
Figure imgf000148_0001
Example 1 1.1, Step a
Figure imgf000148_0002
Pyrrolidine 1 1.1a (4HC1 salt) was prepared from (S)-5-(tert-butoxycarbonyl)- 5-azaspiro[2.4]heptane-6-carboxylic acid (see Patent application # US2009/0068140 for its preparation) according to the procedure described for pyrrolidine Nlf.2. XH NMR (DMSO-d6, δ = 2.50 ppm, 400 MHz): δ 10.26 (br s, 2H), 9.50 (br s, 2H), 7.94 (s, 2H), 7.91 (d, J = 8.4, 4H), 7.62 (d, J = 8.4, 4H), 5.09-4.99 (m, 2H), 3.45-3.38 (m, 2H), 3.19-3.16 (m, 2H), 2.57-2.48 (obscured, 2H), 2.25 (d, J = 7.6, 1.1H), 2.22 (d, J = 7.2, 0.9H), 0.86-0.69 (m, 8H). LC/MS: Anal. Calcd. for [M+H]+ C32H33 6: 501.27; found 501.2. Example Nl 1.1 & N11.2
Example Nl 1.1 (TFA salt) was prepared from pyrrolidine Nl 1. la (4HC1 salt) and (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid according to the procedure described for Example N 10. LC (Condition lOe and lOe. l) >95% homogeneity index. LC/MS (Condition lOe.5) Rt = 1.61 min. XH NMR (DMSO-d6, δ = 2.50 ppm, 400 MHz): δ 8.06 (br s, 2H), 7.84 (d, J = 8.4, 4H), 7.62 (app d, 4H), 7.4
(d, J = 7.6, 2H), 5.25 (t, J = 7.6, 2H), 4.03 (app t, 2H), 3.86 (d, J = 9.4, 2H), 3.74 (d, J
= 9.4, 2H), 3.55 (s, 6H), 2.23 (d, J = 7.6, 4H), 2.02-1.98 (m, 2H), 0.94-0.89 (m, 2H),
0.84 (d, J = 6.8, 6H), 0.81 (d, J = 6.4, 6H), 0.68-0.62 (m, 6H). LC/MS: Anal. Calcd. for [M+H C46H55 806: 815.42; found 815.4. The diastereomer Example Nl 1.2 (TFA salt; eluted second from column; ΧΗ NMR analysis suggested non-symmetric molecule) was also isolated. LC (Condition 10e) >92% homogeneity index. LC/MS (Condition lOe.5) Rt = 1.65 min. LC/MS: Anal. Calcd. for [M+H]+ Οκ¾5 806: 815.42; found 815.4.
Example N12
Figure imgf000149_0001
Example N12 (TFA salt) was prepared from Pyrrolidine 1 1.1a (4HC1 salt) and (S)-2-(methoxycarbonylamino)-2-(tetrahydro-2H-pyran-4-yl)acetic acid according to the procedure described for Example N10. LC (Condition lOe and lOe.l) >99% homogeneity index. LC/MS (Condition lOe.5): Rt = 1.51 min. LC/MS: Anal. Calcd. for [M+H CsoHsg sC 899.44; found 899.4.
Example N13
Figure imgf000149_0002
E a
Figure imgf000149_0003
A solution of ester NlOb (5 g, 20.57 mmol) in THF (50 mL) was cooled to - 78 °C, and LiHMDS (1M in THF, 20.57 mL, 20.57 mmol) was added drop wise and the mixture was stirred for 45 minutes at same temperature. Then Mel (3.18 mL, 51.44 mmol) was added drop wise and stirred for 2 h at same temperature, and stirring was continued under inert atmosphere while allowing the bath temperature to thaw to RT over 12 h. The reaction mixture was quenched with AcOH (2.35 mL, 41.15 mmol)) and water (8 mL). The volatile component was removed in vacuo and the residue was dissolved in EtOAc (250 mL). The organic layer was separated and washed with water, and the aqueous phase was back extracted with EtOAc (100 mL). The combined organic extract was washed with brine, dried over Na2S04 and concentrated in vacuo. The resulting crude material was purified by flash chromatography (Silica gel; EtOAc: petroleum ether, 30:70) to afford the methylated product N13a (1.45 g,). 'H NMR (CDC13, 5 = 7.26 ppm, 400 MHz): δ 4.58 (d, J = 9 1H), 3.78 (s, 3H), 2.73-2.66 (m, 1H), 2.31-2.26 (m, 1H), 1.98-1.89 (m, 1H), 1.51 (s 9H), 1.22 (d, J = 6.8, 3H).
Example N13, Step b
Figure imgf000150_0001
To a solution of ester Nl 3 a (1 g, 3.89 mmol) in THF (18 mL) was added BH3:Me2S (3.89 mL, 7.78 mmol) drop wise under nitrogen atmosphere, and the reaction mixture was heated at 40 °C for 7 h. The volatile component was removed in vacuo and the residue was dissolved in EtOAc (100 mL) and was washed with water and the aqueous phase was back extracted with EtOAc (50 mL). The combined extract was washed with brine, dried over Na2S04 and concentrated in vacuo. The crude was purified by flash chromatography (Silica gel, EtOAc:
petroleum ether, 25:75) to afford proline N13b (410 mg). XH NMR (CDC13> δ = 7.26 ppm, 400 MHz): δ 4.36 (dd, J = 8.8, 2.0, 0.4H), 4.26 (dd, J = 8.8, 3.2, 0.6H), 3.75- 3.64 (m, 1H), 3.71 (s, 3H), 2.99-2.87 (m, 1H), 2.42-2.36 (m, 1H), 2.09-2.03 (m, 1H), 1.88-1.80 (m, 1H), 1.46/1.40 (s, 9H), 1.033/1.027 (d, J = 6.8, 3H). Example N13, Step c
Figure imgf000151_0001
A solution of proline N13b (400 mg, 1.64 mmol) in EtOH (6 mL) was cooled to 0 °C, and LiOH (63 mg, 2.63 mmol) in water (3 mL) was added and the mixture was stirred at RT for 5 h. It was then concentrated under reduced pressure, and the residue was dissolved in water and extracted with Et20 (20 mL). The aqueous phase was acidified to pH 4 with 1 N HC1 and extracted with EtOAc (100 mL). The organic phase was washed with brine, dried over Na2S04 and concentrated in vacuo to afford acid N13c (630 mg). ¾ NMR (CDC13> δ = 7.26 ppm, 400 MHz): δ 4.31 (d, J = 7.2, 1H), 3.47 (t, J = 8.4 , 1H), 2.81 (app t, 1H), 2.53-2.44 (m, 1H), 2.38-1.80 (m, 2H), 1.47 (s, 9H), 1.02 (d, J = 6.4, 3H). LC/MS: Anal. Calcd. for [M-H]"
CiiH18N04: 228.13; found 227.7. Example 13, Step d
Figure imgf000151_0002
Pyrrolidine N13d (4HC1 salt) was prepared from acid 13c according to the procedure described for the preparartion of pyrrolidine N10f.2. lH NMR (MeOD δ = 3.34 ppm, 400 MHz): δ 7.87 (s, 2H), 7.86 (d, J = 8.0, 4H), 7.65 (d, J = 8.0, 4H), 5.21- 5.18 (m, 2H), 3.62-3.58 (m, 2H), 3.12-3.07 (m, 2H), 2.83-2.78 (m, 2H), 2.69-2.62 (m, 2H), 2.31-2.22 (m, 2H), 1.27 (d, J = 6.4, 6H). LC/MS: Anal. Calcd. for [M+H]+ C3oH33 6: 477.27; found 477.2.
Example N13 Example N13 (TFA salt) was prepared from pyrrolidine N13d (4HC1 salt) and (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid according to the procedure described for the preparation of Example N10. LC (Condition We and lOe. l): >98% homogeneity index. LC/MS (Condition 10e.5)\ Rt = 1.57 min. XH NMR (DMSO-d6, δ = 2.50 ppm, 400 MHz): δ 8.01-7.81 (m, 6H), 7.69-7.62 (m, 4H), 7.33 (d, J = 8.4, 2H), 5.22-5.16 (m, 2H), 4.09 (app t, 2H), 3.98 (t, J = 8.4, 2H), 3.56 (s, 6H), 3.39-3.35 (m, 2H), 2.69-2.65 (m, 2H), 2.20-2.11 (m, 2H), 2.05-1.92 (m, 4H), 1.07 (d, J = 6.4, 6H), 0.87 (d, J = 6.8, 6H), 0.83 (d, J = 6.4, 6H). LC/MS: Anal. Calcd. for [M+H]+ C44H55 806: 791.42; found 791.4.
Example 14
Figure imgf000152_0001
E a
Figure imgf000152_0002
A solution of ester NlOb (1 g, 4.1 1 mmol) in THF (20 mL) was cooled to -78 °C, and LiHMDS (1M in THF, 8.2 mL, 8.22 mmol) was added to it drop wise and stirred for 15 minutes. Then Mel (0.51 mL, 8.22 mmol) was added drop wise and stirred at -78 °C for 15 minutes and at RT for 2 h. The volatile component was removed in vacuo and the residue was dissolved in EtOAc (100 mL), and washed with NH4C1 solution. The aqueous phase was back extracted with EtOAc (50 mL), and the combined extract was washed with water, brine, dried over Na2S04 and concentrated in vacuo. The crude was purified by flash chromatography (Silica gel; EtOAc: petroleum ether, 30:70) to afford dimethylated product N14a (700 mg). XH NMR (CDCI3, δ = 7.26 ppm, 400 MHz): δ 4.53 (dd, J = 9.2, 5.2, 1H), 3.79 (s, 3H), 2.21 (dd, J = 13.2, 9.6, 1H), 1.97-1.88 (m, 1H), 1.50 (s, 9H), 1.21 (s, 6H).
Example 14, Step b
Figure imgf000153_0001
A solution of ester N14a (1.4 g, 5.16 mmol) in THF (25 mL) was cooled to - 78 °C and Superhydride (1M in THF, 6.19 mL, 6.19 mmol) was added to it drop wise under the N2. The reaction mixture was stirred for 30 minutes, quenched with saturated aHC03 solution and allowed to warm up to 0 °C. Then 30% aqueous H2O2 (3 mL) was added and stirred for 20 minutes at 0 °C. The reaction mixture was concentrated under reduced pressure and the residue was dissolved in DCM (100 mL) and washed with water. The aqueous phase was extracted with DCM (2 x 50 mL), and the combined organic extract was washed with brine, dried over Na2S04 and concentrated in vacuo to afford crude N 14b (1.25 g) which was used as such in the next step. c
Figure imgf000153_0002
To a solution of crude N14b (1.25 g, 5.16 mmol) in DCM (30 mL) was added EtsSiH (0.84 mL, 5.32 mmol), the reaction mixture was cooled to -78 °C and
BF3:Et20 (0.73 mL, 5.83 mmol) was added drop wise under the N2. After 30 minutes of stirring, additional Et3SiH (0.84 mL, 5.32 mmol) followed by BF3:Et20 (0.73 mL, 5.83 mmol) were added. After stirring for 2 h at -78 °C, the reaction mixture was quenched with NaHC03 solution and extracted with DCM (2 x 100 mL). The combined organic extract was dried over Na2S04 and concentrated in vacuo. The resultant crude material was purified by flash chromatography (Silica gel; EtOAc: petroleum ether, 25:75) to afford proline N14c (470 mg). ¾ NMR (CDC13> δ = 7.26 ppm, 400 MHz): δ 4.34/4.27 (t, J = 8.4, 1H), 3.74/3.73 (s, 3H), 3.38-3.18 (m, 2H), 2.02 (dd, J = 12.4, 8.0, 1H), 1.78-1.70 (m, 1H), 1.47/1.42 (s, 9H), 1.13/1.06 (s, 6H).
Example 14, Step d
Figure imgf000154_0001
A solution of proline N14c (500 mg, 1.94 mmol) in EtOH (8 mL) was cooled to 0 °C, LiOH (93 mg, 3.89 mmol) in water (4 mL) was added, and the reaction mixture was stirred at RT for 6 h. It was then concentrated under reduced pressure, and the resulting crude material was dissolved in water and extracted with Et20 (20 mL). The aqueous phase was acidified to pH 4 with 1 N HC1 and extracted with
EtOAc (100 mL). The organic phase was washed with brine, dried over Na2S04 and concentrated in vacuo to afford acid 14d (454 mg). XH NMR (CDC13, δ = 7.26 ppm, 400 MHz): δ 4.42-4.23 (m, 1H), 3.26 (d, J = 10.8, 1H), 3.10 (d, J = 10.8, 1H), 2.30- 2.20 (m, 1H), 1.96-1.82 (m, 1H), 1.50/1.45 (s, 9H), 1.11/1.07 (s, 6H).
Example 14, Step e
Figure imgf000154_0002
Pyrrolidine 14e (4HC1 salt) was prepared from acid 14d according to the procedure described for the preparation of pyrrolidine N10f.2. XH NMR (DMSO-d6, δ = 2.50 ppm, 400 MHz): δ 10.28 (br s, 2H), 9.50 (br s, 2H), 7.91-7.55 (m, 6H), 7.30- 7.05 (m, 4H), 5.05-4.96 (m, 2H), 3.19-3.01 (m, 4H), 2.33-2.26 (m, 4H), 1.24 (s, 6H), 1.22 (s, 6H). LC/MS: Anal. Calcd. for [M+H]+ C32H37 6: 505.3; found 505.2.
Example 14 Example N14 (free base) was prepared from pyrrolidine 14e (4HC1 salt) and (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid according to the procedure described for Example N10 with the exception that the crude was purified with an HPLC using CHsCN/Water/TFA solvent system and the resultant TFA salt product was partitioned between EtOAc and 10% aHC03, and the organic phase was dried ( a2S04) and evaporated in vacuo. LC (Condition lOe and lOe. l): >97%
homogeneity index. LC/MS (Condition lOe.5) Rt = 1.64 min. XH NMR (DMSO-d6, δ = 2.50 ppm, 400 MHz): δ 12.21/1 1.87 (s, 2H), 7.78-7.73/7.67-7.63 (m, 4H), 7.57- 7.53 (m, 2H), 7.51-7.44 (m, 4H), 7.21 (d, J = 8.0, 2H), 4.99 (t, J = 8.6, 2H), 4.01 (t, J = 8.0, 2H), 3.78 (d, J = 9.8, 2H), 3.54/3.52 (s, 6H), 3.45 (d, J = 9.8, 2H), 2.07-1.88 (m, 6H), 1.17 (s, 6H), 1.03 (s, 6H), 0.85 (d, J = 6.8, 6H), 0.82 (d, J = 6.8, 6H).
LC/MS: Anal. Calcd. for [M+H]+ C46H59 806: 819.45; found 819.4.
Example N15
Figure imgf000155_0001
Example N15 (TFA salt) was prepared from pyrrolidine 14e (4HC1 salt) and (S)-2-(methoxycarbonylamino)-2-(tetrahydro-2H-pyran-4-yl)acetic acid according to the procedure described for Example 10 with the exception that the reverse phase HPLC system used ACN/Water/TFA solvent . LC (Condition lOe and lOe. l): >96% homogeneity index. LC/MS (Condition 10e.5)\ Rt = 1.55 min. LC/MS: Anal. Calcd. for [M+H]+ CsoHes sC 903.47; found 903.4.
Example 16.1, N 16.2 & N16.3
Figure imgf000156_0001
E a
Figure imgf000156_0002
A solution of NaHMDS (1M in THF, 13.56 mL, 13.56 mmol) in THF (25 mL) was cooled to -78 °C, and a THF (15 mL) solution of (S)-l-tert-butyl 2-methyl 4-oxopyrrolidine-l,2-dicarboxylate (3 g, 12.33 mmol) was added drop wise. After 15 minutes of stirring, N-phenyl-bis(trifluoromethanesulfonimide) (4.4 g, 12.33 mmol) in THF (15 mL) was added, and the reaction mixture was stirred at -78 °C for 3 h. It was then quenched with aHC03 solution at -78 °C, and the mixture was then stirred at RT. The organic layer was separated and the aqueous phase was extracted with EtOAc (2 x 50 mL). The combined organic extract was washed with water and brine, dried over Na2S04 and concentrated in vacuo. The crude was purified by flash chromatography (Silica gel; EtOAc: petroleum ether, 15:85) to afford triflate N16.1a (4.3 g). 1H MR (CDCl3, 5 = 7.26 ppm, 400 MHz): 5 5.75-5.74/5.71-5.69 (m, 1H), 5.08-5.06/5.02-4.99 (m, 1H), 4.40-4.25 (m, 2H), 3.77/3.76 (s, 3H), 1.48/1.43 (s, 9H).
Example 16.1, Step b
Figure imgf000156_0003
A solution of Cul (1.46 g, 7.6 mmol) in THF (30 mL) was cooled to -30 UC, MeMgBr (3 M in Et20, 16.0 mmol) was added drop wise, and the reaction mixture was stirred for 30 minutes. It was then cooled to -78 °C, and triflate N16. la (2.4 g, 6.4 mmol) in THF (10 niL) was added drop wise. After being stirred for 2 h, the reaction mixture was quenched with NH4C1 solution. The organic layer was separated and the aqueous phase was extracted with EtOAc (2 x 100 mL). The combined organic phase was washed with water and brine, dried over Na2S04 and concentrated in vacuo. The resulting crude material was purified by flash chromatography (Silica gel; EtOAc: petroleum ether, 15:85) to afford N16. lb (560 mg). ¾ NMR (CDCI3, δ = 7.26 ppm, 400 MHz): δ 5.37/5.33 (br s, 1H), 4.99- 4.97/4.91-4.89 (m, 1H), 4.21-4.02 (m, 2H), 3.74/3.73 (s, 3H), 1.80/1.78 (s, 3H), 1.49/1.43 (s, 9H).
Example N 16.1, Step c
Figure imgf000157_0001
A solution N16.1b (560 mg, 2.32mmol) in EtOH (10 mL) was cooled to 0 UC, and LiOH (89 mg, 3.71 mmol) in water (5 mL) was added, and the mixture was stirred for overnight. It was then concentrated under reduced pressure, and the resiue was dissolved in water and extracted with Et20 (20 mL). The aqueous phase was acidified to pH 4 and extracted with EtOAc (100 mL). The organic layer was washed with brine, dried over Na2S04 and concentrated in vacuo to afford acid N 16.1c of unknown stereochemical integrity (490 mg). XH NMR (CDCI3, δ = 7.26 ppm, 400 MHz): δ 5.57/5.37 (br s, 1H), 5.07/4.94 (br s, 1H), 4.16-4.01 (m, 2H), 1.81(s, 3H), 1.52/1.46 (s, 9H).
Example N 16.1, Step d
Figure imgf000158_0001
Pyrrolidine N16. Id (4HC1 salt) was prepared from acid N16. lc according to the procedure described for the preparation of pyrrolidine N10f.2. XH NMR (MeOD δ = 3.34 ppm, 400 MHz): δ 8.09 (s, 2H), 7.88 (d, J = 8.6, 4H), 7.33 (d, J = 8.6, 4H), 6.01 (s, 2H), 5.79 (s, 2H), 4.29 (d, J = 15.2, 2H), 4.21 (d, J = 15.2, 2H), 2.07 (s, 6H). LC/MS: Anal. Calcd. for [M+H]+ C3oH29 6: 473.24; found 473.2.
Example 16.1, N 16.2 & N16.3
When pyrrolidine 16. Id (4HC1) was reacted with (S)-2- (methoxycarbonylamino)-3-methylbutanoic acid and purified according to the procedure described for Example 10, with the exception of using
CI¾CN/Water/TFA for the HPLC purification, the following three diastereomers were isolated. Example 16.1 (TFA salt; eluted first). LC (Condition lOe and lOe. l): >97% homogeneity index. LC/MS (Condition lOe.5) Rt = 1.54 min. LC/MS: Anal. Calcd. for [M+H]+ C44H5i 806: 787.39; found 787.4. Example N16.2 (TFA salt; eluted second): LC (Condition lOe and lOe. l): >97% homogeneity index.
LC/MS (Condition 10e.5): Rt = 1.56 min. XH NMR (MeOD, δ = 3.34 ppm, 400 MHz): δ 7.92 (s, 2H), 7.89-7.77 (m, 4H), 7.72-7.66 (m, 4H), 6.03-5.97 (m, 2H), 5.68-5.64 (m, 2H), 4.81-4.76 (m, 2H), 4.64-4.47 (m, 2H), 4.15 (d, J = 8.4, 2H), 3.68 (s, 6H), 2.01 (s, 6H), 2.00-1.92 (m, 2H), 0.94 (d, J = 6.8, 6H), 0.88 (d, J = 6.8, 6H). LC/MS: Anal. Calcd. for [M+H]+ C44H5iN806: 787.39; found 787.4. Example N 16.3 (TFA salt; eluted last): LC (Condition We and lOe. l): >92% homogeneity index. LC/MS (Condition 10e.6)\ Rt = 1.78 min. LC/MS: Anal. Calcd. for [M+H]+ C44H5iN806: 787.39; found 787.4. Example N17.1 & N17.2
Figure imgf000159_0001
The following two diastereomers were isolated when pyrrolidine 16. Id (4HC1) was reacted with (S)-2-(methoxycarbonylamino)-2-(tetrahydro-2H-pyran-4- yl)acetic acid and purified according to the procedure described for Example 10 with the exception that CH3CN/Water/TFA was used for the reverse phase HPLC purification. Example 17.1 (TFA salt; eluted first): LC (Condition lOe and lOe.l): >94% homogeneity index. LC/MS (Condition 10e.5): Rt = 1.46 min. LC/MS: Anal. Calcd. for
Figure imgf000159_0002
871.41 ; found 871.2. Example 17.2 (TFA salt; eluted second): LC (Condition lOe and lOe.l): >93% homogeneity index. LC/MS (Condition lOe.5) Rt = 1.48 min. LC/MS: Anal. Calcd. for [M+H]+ C4gH55NgOg: 871.41 ; found 871.4.
Example N22
Figure imgf000159_0003
Example N22, Step a
Figure imgf000159_0004
A solution of l-bromo-4-iodo-2-methylbenzene (5.0 g, 16.83 mmol) in dioxane (75 mL) was purged with 2 for 10 minutes. Then PdCl2(Pli3P)2 (428 mg 0.609 mmol) and tri-n-butyl(l-ethoxyvinyl)stanane (5.94 mL, 17.59 mmol) was added, and the reaction mixture was purged with 2 for 10 minutes and heated at 80 °C for overnight. Water (25 mL) was added to the reaction mixture and cooled to 0 °C, NBS (3 g, 17.1 mmol) was added and the mixture was stirred for 30 minutes. The volatile component was removed under reduced pressure, and water was added to the resulting residue and it was extracted with DCM (3 x 100 mL). The combined organic extract was dried over Na2S04 and concentrated in vacuo. The crude was purified by flash chromatography (ISCO; EtOAc: petroleum ether, 2:98) to afford ketone N22a (2.6 g). ¾ NMR (CDCI3, δ = 7.26 ppm, 400 MHz): δ 7.84 (d, J = 0.8, 1H), 7.65-7.62 (m, 2H), 4.32 (s, 2H), 2.48 (s, 3H).
Example N22, Step b
Figure imgf000160_0001
To a stirred solution of (lR,3S,5R)-2-(/er/-butoxycarbonyl)-2- azabicyclo[3.1.0]hexane-3-carboxylic acid (2.23 g, 9.89 mmol) and TEA (1.3 mL, 9.80 mmol) in ACN (30 mL) was added dibromide N22a (2.6 g, 8.99 mmol) in ACN (45 mL), and the reaction mixture was stirred for 90 minutes at room temperature. The volatile component was removed in vacuo, the residue was partitioned between water and DCM, and the organic layer was washed with brine, dried over Na2S04 and concentrated in vacuo. The resulting crude material was purified by flash chromatography (ISCO; EtOAc: petroleum ether, 40:80) to afford ketoester N22b
(1.6 g). *H NMR (DMSO-d6, δ = 2.50 ppm, 400 MHz): δ 7.98 (s, 1H), 7.79 (d, J = 8.2, 1H), 7.71 (d, J = 8.2, 1H), 5.59-5.42 (m, 2H), 4.18-4.12 (m, 1H), 3.40-3.32 (m, 1H), 2.43 (s, 3H), 2.41-2.37 (m, 2H), 1.68-1.60 (m, 1H), 1.42/1.32 (s, 9H), 0.78-0.73 (m, 1H), 0.56-0.51 (m, 1H). LC/MS: Anal. Calcd. for [M+H-Boc]+ Ci5H17BrN03: 338.03; found 338.0. Example N22, Step c
Figure imgf000161_0001
To a solution of N22b (1.6 g, 3.66 mmol) in xylenes (30 mL) was added NH4OAc (2.82 g, 36.6 mmol) and the reaction mixture was purged with 2 for 10 minutes. The reaction mixture was heated in a pressure tube at 130 °C for over night. The volatile component was evaporated under reduced pressure and the crude was treated with saturated aHC03 solution, stirred and then extracted with DCM (200 mL). The organic layer was washed with water and brine, and dried over Na2S04 and concentrated in vacuo. The resulting crude material was purified by flash chromatography (ISCO; MeOH:DCM: 0.3 :99.7) to afford imidazole N22c (1.1 g). XH NMR (CDCI3, δ = 7.26 ppm, 400 MHz): δ 10.8-10.2 (br s, 1H), 7.68 (br s, 1H), 7.52- 7.40 (m, 2H), 7.23 (br s, 1H), 4.86-4.78 (m, 1H), 3.60-3.30 (m, 1H), 3.25-3.20 (m, 1H), 2.42 (s, 3H), 2.40-2.32 (m, 1H), 1.80-1.70 (m, 1H), 1.48 (s, 9H), 0.90-0.81 (m, 1H), 0.49-0.42 (m, 1H). LC/MS: Anal. Calcd. for [M+H]+ CzoH^Br sCb: 418.1 1 ; found 420.2.
Example N22, Step d
Figure imgf000161_0002
A solution of imidazole N22c (940 mg, 2.25 mmol) in DMF (47 mL) was purged with N2 for 10 minutes, Pd(Ph3P)4 (98 mg, 0.084 mmol) and bis- trimethylstanylacetylene (395 mg, 1.12 mmol) were added, and the reaction mixture was purged with 2 for additional 10 minutes and heated at 90 °C for over night. The volatile component was removed under reduced pressure and the resulting crude material was purified by flash chromatography (ISCO; MeOH :DCM, 0.5:99.5) to afford alkyne N22d (230 mg). XH NMR (DMSO-d6, δ = 2.50 ppm, 400 MHz): δ 12.2/11.9 (br s, 2H), 7.73/7.65 (br s, 2H), 7.62 (app d, 2H), 7.55 (d, J = 2.0, 2H), 7.45 (d, J = 8.0, 2H), 4.65-4.53 (m, 2H), 3.49-3.39 (m, 2H), 2.57-2.44 (obscured, 6H), 2.38-2.19 (m, 4H), 1.68-1.59 (m, 2H), 1.38-1.15 (br s, 18H), 0.80-0.71 (m, 2H), 0.58- 0.52 (m, 2H). LC/MS: Anal. Calcd. for [M+H]+ C42H49 604: 701.37; found 701.4.
Example N22, Step e
Figure imgf000162_0001
To a solution of alkyne N22d (80 mg, 0.1 14 mmol) in MeOH (1.5 mL) was added HCl/dioxane (4M, 5 mL) at 0 °C, and the mixture was stirred at room temperature for 2 h. The volatile component was removed in vacuo, the residue was co-evaporated with anhydrous DCM (3 x 5 mL) and dried under high vacuum to afford pyrrolidine N22e (4HC1, 80 mg) as a pale yellow solid, which was submitted to the next step as such. 1H NMR (MeOD δ = 3.34 ppm, 400 MHz): δ 8.12 (s, 2H), 7.84 (br s, 2H), 7.74-7.69 (m, 4H), 5.03-4.96 (m, 2H), 3.71-3.62 (m, 2H), 2.90-2.67 (m, 4H), 2.65 (s, 6H), 2.21-2.14 (m, 2H), 1.12-1.05 (m, 2H), 0.93-0.84 (m, 2H). LC/MS: Anal. Calcd. for [M+H]+ C32H33 6: 501.27; found 501.2.
Example N22
HATU (88.5 mg, 0.233 mmol) was added to a solution of pyrrolidine N22e (80 mg, 0.114 mmol), (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid (41.8 mg, 0.239 mmol) and DIEA (0.15 mL, 0.91 mmol) in DMF (5 mL). The reaction mixture was stirred at RT for 2 h. The volatile component was removed in vacuo and the residue was dissolved in EtOAc (50 mL), and washed with saturated aHC03 solution and saturated NH4C1, and the combined aqueous phase was back extracted with EtOAc (25 mL). The combined organic extract was washed with water and brine, dried over Na2S04 and concentrated in vacuo. The resulting crude material was submitted to a reverse phase HPLC purification (ACN/water/TFA) to afford
Example N22 (40 mg, TFA salt) as an off white solid. LC (Condition lOe and lOe. l): >98% homogeneity index. LC/MS (Condition 10e.5)\ Rt = 1.64 min. XH NMR (MeOD δ = 3.34 ppm, 400 MHz): δ 7.86 (s, 2H), 7.69 (br s, 2H), 7.66 (d, J = 8.4, 2H), 7.58 (dd, J = 8.4, 1.4, 2H), 5.13/5.11 (d, J = 6.8, 2H), 4.56 (d, J = 6.8, 2H), 3.82- 3.79 (m, 2H), 3.68 (s, 6H), 2.70-2.65 (m, 2H), 2.63 (s, 6H), 2.51-2.43 (m, 2H), 2.20- 2.06 (m, 4H), 1.14-1.08 (m, 2H), 1.02 (d, J = 6.8, 6H), 0.93 (d, J = 6.8, 6H), 0.91- 0.85 (m, 2H). LC/MS: Anal. Calcd. for [M+H]+ C46H55 806: 815.42; found 815.4.
Example N23
Figure imgf000163_0001
Example N23 (TFA salt) was prepared from pyrrolidine N22e (4HC1 salt) and
(S)-2-(methoxycarbonylamino)-2-(tetrahydro-2H-pyran-4-yl)acetic acid according to the procedure described for Example N22. LC (Condition lOe. l and 10e.4)\ >94% homogeneity index. LC/MS (Condition 10e.5)\ Rt =1.53 min. LC/MS: Anal. Calcd. for [M+H CsoHjg sC 899.44; found 899.4.
Example N24
Figure imgf000163_0002
Example N24, Step a
Figure imgf000164_0001
To a solution of (lR,3S,5R)-2-(/er/-butoxycarbonyl)-2- azabicyclo[3.1.0]hexane-3-carboxylic acid (4.52 g, 20.0 mmol) in THF (60 mL), cooled to -25 °C, TEA (2.78 mL, 20.0 mmol) followed by isobutyl chloroformate (2.58 mL, 20.0 mmol) were added. After 30 minutes of stirring, the reaction mixture was warmed to -10 °C and CH2 2 in Et20 (3.0 eq ) was added and stirred at the same temperature for 3 h and at room temperature for 12 h. The volatile components were evaporated under reduced pressure and the crude was purified by flash
chromatography (ISCO, Silica gel; EtOAc: petroleum ether, 30:70) to afford N24a (3.9 g) as a yellow solid. XH NMR (CDCI3, δ = 7.26 ppm, 400 MHz): δ 5.50 (s, 1H), 4.01 (br s, 1H), 3.35 (br s, 1H), 2.27 (br s, 2H), 1.60-1.56 (m, 1H), 1.45 (s, 9H), 0.83- 0.80 (m, 1H), 0.44 (br s, 1H). b
Figure imgf000164_0002
Aqueous HBr (48%, 2.0 mL) was added to a stirred solution of N24a (2.0 g, 7.96 mmol) in Et20 (20 mL) at 0 °C. After 5 minutes of stirring, the reaction mixture was diluted with EtOAc (100 mL) and the organic phase was washed with water (50 mL) and brine (25 mL), dried over Na2S04 and concentrated in vacuo to afford crude N24b (2.02 g) as a pale yellow liquid which was used as such in the next step. H
NMR (CDC13, 5 = 7.26 ppm, 400 MHz): δ 4.21-4.14 (m, 1H), 4.10 (s, 2H), 3.52/3.40 (m, 1H), 2.41-2.17 (m, 2H), 1.68-1.61 (m, 1H), 1.47 (s, 9H), 0.85-0.78 (m, 1H), 0.52 (br s, 1H).
Example N24, Step c
Figure imgf000165_0001
To a stirred solution of 4-bromobenzimidamide (384 mg, 1.65 mmol) in THF:H20 (10 mL: 2.5 mL), K2C03 (456 mg, 3.30 mmol) was added followed by N24b (500 mg, 1.65 mmol) in THF (10 mL) and stirred at RT for 24 h. The volatile component was removed under reduced pressure and the crude was dissolved in EtOAc (100 mL) and the organic phase was washed with water (50 mL) and brine (25 mL), dried over Na2S04 and concentrated in vacuo to afford a mixture of the N- alkylated precursor and targeted product. To this mixture (1.65 mmol) in xylenes (8 mL) was added NH4OAc (1.27 g, 16.5 mmol), and the reaction mixture was transferred into a pressure tube, purged with N2 for 10 minutes and heated at 130 °C for overnight. The volatile component was removed under reduced pressure, the crude was treated with saturated aHC03 solution and extracted with EtOAc (100 mL). The organic layer was washed with water (50 mL) and brine (25 mL), dried over Na2S04 and concentrated in vacuo. The resulting crude material was purified by flash chromatography (Silica gel; MeOFLDCM: 2:98) to afford imidazole N24c (350 mg) as a pale yellow solid. 1H NMR (MeOD δ = 3.34 ppm, 400 MHz): δ 7.79- 7.76 (m, 2H), 7.63-7.61 (m, 2H), 6.96 (s, 1H), 4.76 (app t, 1H), 3.49 (app t, 1H), 2.44-2.32 (m, 2H), 1.71-1.64 (m, 1H), 1.38 (s, 9H), 0.92-0.87 (m, 1H), 0.58-0.52 (m, 1H). LC/MS: Anal. Calcd. for [M+H]+ Ci9H23BrN302: 404.09; found 405.0.
Example N24, Step d
Figure imgf000165_0002
A solution of N24c (135 mg, 0.33 mmol) in DMF (2 mL) was purged with N2 for 10 minutes, Pd(Ph3P)4 (14 mg, 0.012 mmol), bis-trimethylstanylacetylene (58 mg, 0.16 mmol) were added, and the reaction mixture was purged with N2 for additional 10 minutes and heated with a microwave at 90 C for 3 h. The reaction mixture was treated with EtOAc (50 mL) and washed with water (25 mL) and brine (10 mL), dried over Na2S04 and concentrated in vacuo. The resulting crude was purified by flash chromatography (ISCO, basic AI2O3; EtOAc: petroleum ether, 15:85) to afford alkyne N24d (14 mg) as a pale yellow solid. XH NMR (MeOD δ = 3.34 ppm, 400 MHz): δ 7.90 (d, J = 8.8, 4H), 7.63 (d, J = 8.8, 4H), 6.99 (s, 2H), 4.79 (br s, 2H), 3.50 (br s, 2H), 2.44-2.35 (m, 4H), 1.73-1.69 (m, 2H), 1.47 (s, 18H), 0.94-0.86 (m, 2H), 0.59 (br s, 2H). LC/MS: Anal. Calcd. for [M+H]+ C40H45N6O4: 673.34; found 673.4.
Example N24, Step e
Figure imgf000166_0001
To a solution of N24d (14 mg, 0.020 mmol) in MeOH (0.5 mL) was added HCl/dioxane (4M, 1 mL) at 0 °C, and the mixture was stirred at room temperature for 2 h. The volatile component was removed in vacuo, the residue was co-evaporated with anhydrous DCM (3 x 5 mL) and dried under high vacuum to afford pyrrolidine N24e (4HC1, 12.8 mg) as a pale yellow solid, which was submitted to the next step as such. XH NMR (MeOD, δ = 3.34 ppm, 400 MHz): δ 8.08 (d, J = 8.4, 4H), 7.88 (s, 2H), 7.86 (d, J = 8.4, 4H), 4.78 (obscured, 2H), 3.54 (app t, 2H), 2.73-2.66 (m, 4H), 2.12-2.09 (m, 2H), 1.08-1.02 (m, 2H), 0.92-0.81 (m, 2H). LC/MS: Anal. Calcd. for [M+H]+ C3oH29 6: 473.24; found 473.2.
Example N24
Figure imgf000167_0001
HATU (16 mg, 0.042 mmol) was added to a solution of N24e (12.8 mg, 0.020 mmol), (S)-2-(methoxycarbonylamino)-2-(tetrahydro-2H-pyran-4-yl)acetic acid (9 mg, 0.04 mmol) and DIEA (0.014 mL, 0.08 mmol) in DMF (2 mL). After stirring for 2 h at RT, the reaction mixture was dissolved in EtOAc (50 mL), and washed with saturated NH4C1 (25 mL), saturated NaHCCh solution (25 mL), brine (10 mL), dried over Na2S04 and concentrated in vacuo. The crude was submitted to a reverse phase HPLC purification (ACN/water/TFA) to afford Example N24 (8 mg, TFA salt) as white solid. LC (Condition 10e.2 and lOe.8) >92% homogeneity index. LC/MS (Condition 10e.6) Rt = 1.46 min. XH MR (MeOD δ = 3.34 ppm, 400 MHz): δ 7.94 (d, J = 8.4, 4H), 7.83 (d, J = 8.4, 4H), 7.52 (s, 2H), 5.25-5.20 (m, 2H), 4.64 (d, J = 7.6, 2H), 3.99-3.90 (m, 4H), 3.74-3.69 (m, 2H), 3.69 (s, 6H), 3.42-3.30 (m, 4H), 2.52-2.43 (m, 4H), 2.07-2.02 (m, 4H), 1.64-1.31 (m, 8H), 1.18-1.16 (m, 2H), 0.88- 0.83 (m, 2H). LC/MS: Anal. Calcd. for [M+H]+ QsHss sOs: 871.41 ; found 871.4.
Example N25
Figure imgf000167_0002
Example N25 (free base) was prepared similarly from pyrrolidine N24e and (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid according to the procedure described for Example N24, with the notable exception that CH3CN/Water/NH40Ac was used for the reverse phase HPLC purification step. LC (Condition We and 10e.9) >96% homogeneity index. LC/MS (Condition 10e.6) Rt =1.67 min. LC/MS: Anal. Calcd. for [M+H]+ C44H5iN806: 787.39; found 787.4. Example J 1 to J 1.1
Figure imgf000168_0001
DIPEA (2.8 mL, 16 mmol) was added to a slurry of acid M3f (2.00 g, 8.80 mmol) and 2-bromo-l-(3-bromophenyl)ethanone (2.22 g, 8.00 mmol) in acetonitrile (25 mL) and the reaction mixture was stirred overnight at rt. The reaction mixture was concentrated and purified with a Biotage Horizon (80g S1O2, 10-25%
EtOAc/hexanes) to yield ketoester Jla (3.37 g) as a viscous light yellow oil. LC-MS retention time 1.853 min; m/z 423 and 425.98 (1 : 1) (MH+). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Waters Xterra MS 7u C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220nM. The elution conditions employed a flow rate of 4 mL/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 2 min, a hold time of 1 min, and an analysis time of 3 min where solvent A was 5% MeOH / 95% ¾0 / 10 mM ammonium acetate and solvent B was 5% H2O / 95% MeOH / 10 mM ammonium acetate. MS data was determined using a
Micromass Platform for LC in electrospray mode. XH NMR (400 MHz, CDCI3) δ ppm 8.04 (t, J=1.8 Hz, 1H), 7.83 (d, J=7.8 Hz, 1H), 7.75 (d, J=8.0 Hz, 1H), 7.38 (t, J=7.9 Hz, 1H), 5.33 - 5.59 (m, 1H), 5.13 - 5.32 (m, 1H), 4.22 (br s, 1H), 3.40 - 3.63 (m, 1H), 2.52 - 2.63 (m, 1H), 2.46 (dd, J=13.1, 9.3 Hz, 1H), 1.63 - 1.73 (m, 1H), 1.47 (br s, 9H), 0.86 (br s, 1H), 0.51 (br s, 1H).
Example Jl, step b
Figure imgf000169_0001
A solution of ketoester Jla (3.33 g, 7.85 mmol) in xylene (75 mL) was added to ammonium acetate (5.94 g, 77 mmol) in a pressure vessel and the reaction mixture was stirred under nitrogen for 5 min. The reaction vessel was sealed and then placed into an oil bath which had been preheated to 140 °C and the reaction was held at that temperature for 6h. The reaction was cooled to rt, stirred overnight and reheated at 140 °C for 5 hr. Additional ammonium acetate (3.0g) was added and the reaction was stirred at 145 C for 8 hrs, cooled to rt and concentrated under high vacuum to a brown oil. The oil was partitioned between DCM (~ 200 mL) and 1/2 sat. sodium bicarbonate (-200 mL). The organic layer was dried (MgSC^), filtered and concentrated. The crude orange solidified foam was purified on a Biotage Horizon (20-50% EtOAc/hexanes, 160 g Si02) to yield imidazole Jib (2.03 g) as a yellow solidified foam. LC-MS retention time 2.450 min; m/z 404 and 406.06 (1 : 1) (MH+). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Phenomenex-Luna lOu C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220 nM. The elution conditions employed a flow rate of 4 mL/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% MeOH / 95% ¾0 / 10 mM ammonium acetate and solvent B was 5% ¾0 / 95% MeOH / 10 mM ammonium acetate. MS data was determined using a Micromass Platform for LC in electrospray mode. XH NMR (400 MHz, MeOD) δ ppm 7.89 (br s, 1H), 7.65 (d, J=6.5 Hz, 1H), 7.32 - 7.44 (m, 2H), 7.26 (t, J=7.8 Hz, 1H), 4.66 (br s, 1H), 3.52 - 3.63 (m, 1H), 2.51 (dd, J=13.1, 8.8 Hz, 1H), 2.25 - 2.37 (m, 1H), 1.66 - 1.75 (m, 1H), 1.29 (br s, 9H), 0.84 (ddd, J=8.2, 6.0, 5.8 Hz, 1H), 0.56 - 0.63 (m, 1H).
Example Jl, step c
Figure imgf000170_0001
Nitrogen was bubbled through a stirred solution of bromide Jib (854 mg, 2.18 mmol) and l,2-bis(trimethylstannyl)ethyne (403 mg, 1.15 mmol) in DMF (10 mL) for 10 minutes. Then Pd(PPh3)4 (79 mg, 0.069 mmol) was added, nitrogen was bubbled through the reaction for 1 min and then the reaction was heated at 90 °C under nitrogen for 17h. The reaction mixture was cooled to rt, concentrated to a viscous oil and purified on a Biotage Horizon (40 g S1O2, 70-100% EtOAc/hexanes, loaded with DCM)) to yield alkyne Jlc (500 mg) as a yellow solidified foam.
LC-MS retention time 2.876 min; m/z 649.51 (MH+). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Phenomenex-Luna lOu C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220 nM. The elution conditions employed a flow rate of 4 mL/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% MeOH / 95% ¾0 / 10 mM ammonium acetate and solvent B was 5% H2O / 95% MeOH / 10 mM ammonium acetate. MS data was determined using a Micromass Platform for LC in electrospray mode. XH NMR (400 MHz, MeOD) δ ppm 7.89 (s, 2H), 7.67 - 7.74 (m, 2H), 7.35 - 7.44 (m, 6H), 4.85 - 5.04 (m, 2H), 3.64 - 3.74 (m, 2H), 3.47 - 3.56 (m, 2H), 2.29 - 2.45 (m, 2H), 1.90 - 2.13 (m, 6H), 1.48 (br s, 6H), 1.26 (br s, 12H).
Example Jl, step d
Figure imgf000171_0001
TFA (3 mL, 38.9 mmol) was added to a stirred solution of carbamate Jlc (238 mg, 0.367 mmol) in DCE (7 mL) and the reaction was stirred at rt for lh. The reaction mixture was concentrated under vacuum to yield a TFA salt of pyrrolidine Jld (260 mg) as a yellow solid.
LC-MS retention time 2.505 min; m/z 449.22 (MH+). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Phenomenex-Luna lOu C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220 nM. The elution conditions employed a flow rate of 4 mL/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% MeOH / 95% FLO / 10 mM ammonium acetate and solvent B was 5% FLO / 95% MeOH / 10 mM ammonium acetate. MS data was determined using a Micromass Platform for LC in electrospray mode. XH NMR (400 MHz, MeOD) δ ppm 7.96 - 8.00 (m, 2H), 7.79 (dt, J=6.6, 2.1 Hz, 2H), 7.65 (s, 2H), 7.41 - 7.48 (m, 4H), 4.91 (t, J=7.8 Hz, 2H), 3.44 - 3.61 (m, 4H), 2.51 - 2.62 (m, 2H), 2.27 - 2.45 (m, 4H), 2.16 - 2.24 (m, 2H).
Example Jl to Jl . l
HATU (61.8 mg, 0.162 mmol) was added to a stirred solution of a TFA salt of pyrrolidine Jld (1 10 mg, 0.12 mmol) and (S)-2-(methoxycarbonylamino)-3- methylbutanoic acid (28.5 mg, 0.162 mmol) in DMF (0.7 mL) and Hunig'sBase (0.10 mL, 0.54 mmol). The reaction mixture was stirred at rt for 3 hours and then concentrated under a stream of nitrogen. The reaction was purified in two injections by preparative HPLC (MeOH/water /10 mM ammonium acetate) to yield Example Jl (31.5 mg) as a light yellow solid and Example Jl. l (41.1 mg) as a a light yellow solid. Example Jl: LC-MS retention time 2.605 min; m/z 763.36 (MH+). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Phenomenex-Luna lOu CI 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220 nM. The elution conditions employed a flow rate of 4 mL/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% MeOH / 95% ¾0 / 10 mM ammonium acetate and solvent B was 5% H20 / 95% MeOH / 10 mM ammonium acetate. MS data was determined using a Micromass Platform for LC in electrospray mode. The XH NMR presents as a 4 : 1 mixture of rotamers. The major rotamer is: XH NMR (400 MHz, MeOD) δ ppm 7.86 (s, 2H), 7.64 - 7.70 (m, 2H), 7.36 - 7.43 (m, 4H), 7.34 (s, 2H), 5.17 (dd, J=7.8, 5.3 Hz, 2H), 4.24 (d, J=7.3 Hz, 2H), 3.95 - 4.04 (m, 2H), 3.84 - 3.92 (m, 2H), 3.66 (s, 6H), 2.17 - 2.42 (m, 6H), 2.00 - 2.13 (m, 4H), 0.96 (d, J=6.8 Hz, 6H), 0.91 (d, J=6.8 Hz, 6H).
Example Jl. l: LC-MS retention time 2.620 min; m/z 606.23 (MH+). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Phenomenex-Luna lOu CI 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220 nM. The elution conditions employed a flow rate of 4 mL/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% MeOH / 95% H20 / 10 mM ammonium acetate and solvent B was 5% H20 / 95% MeOH / 10 mM ammonium acetate. MS data was determined using a Micromass Platform for LC in electrospray mode. The XH NMR presents as a 3 : 1 mixture of rotamers. XH NMR (400 MHz, MeOD) δ ppm 7.94 (s, 1H), 7.87 (s, 1H), 7.74 (ddd, J=5.6, 3.4, 1.8 Hz, 1H), 7.65 - 7.70 (m, 1H), 7.51 (s, 1H), 7.36 - 7.44 (m, 6H), 7.35 (s, 1H), 5.18 (dd, J=7.7, 5.4 Hz, 1H), 4.68 (t, J=7.8 Hz, 1H), 4.24 (d, J=7.5 Hz, 1H), 3.95 - 4.06 (m, 1H), 3.85 - 3.92 (m, 1H), 3.66 (s, 3H), 3.35 - 3.52 (m, 2H), 2.01 - 2.50 (m, 9H), 0.96 (d, J=6.8 Hz, 3H), 0.91 (d, J=6.8 Hz, 3H). Example J2
Figure imgf000173_0001
HATU (118 mg, 0.310 mmol) was added to a stirred solution of a TFA salt of pyrrolidine Jld (93.4 mg, 0.103 mmol) and (S)-2-(methoxycarbonylamino)-2- (tetrahydro-2H-pyran-4-yl)acetic acid (67.3 mg, 0.310 mmol) in DIPEA (0.18 mL, 1.0 mmol) and DMF (1 mL) and the reaction was stirred at rt for 2h. The reaction was concentrated under a stream of nitrogen, dissolved into MeOH and purified by preparative HPLC (MeOH/water 10 mM ammonium acetate) to yield Example J2 (18.1 mg) as a light pink solid
LC-MS retention time 2.428 min; m/z 847.39 (MH+). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Phenomenex-Luna lOu C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220 nM. The elution conditions employed a flow rate of 4 mL/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% MeOH / 95% ¾0 / 10 mM ammonium acetate and solvent B was 5% ¾0 / 95% MeOH / 10 mM ammonium acetate. MS data was determined using a Micromass Platform for LC in electrospray mode. The XH NMR exhibits as a 4: 1 mixture of rotamers. The major rotamer is : 1H NMR (400 MHz, MeOD) δ ppm 7.83 (s, 2H), 7.62 - 7.67 (m, 2H), 7.33 - 7.42 (m, 6H), 5.15 (dd, J=7.5, 5.5 Hz, 2H), 4.32 (d, J=8.3 Hz, 2H), 3.89 - 4.08 (m, 6H), 3.66 (s, 6H), 3.33 - 3.50 (m, 6H), 2.23 - 2.40 (m, 4H), 2.13 - 2.22 (m, 2H), 1.92 - 2.10 (m, 4H), 1.61 - 1.71 (m, 4H), 1.32 - 1.56 (m, 4H). Example J3
Figure imgf000174_0001
Example J3, step a
Figure imgf000174_0002
Bromide J3a was prepared from acid M3f according to the procedure described for bromide Jib.
Example J3, step b
Figure imgf000174_0003
Nitrogen was bubbled through a stirred solution of bromidie J3a (825 mg, 2.041 mmol) and l,2-bis(trimethylstannyl)ethyne (378 mg, 1.074 mmol) in DMF (10 mL) for 10 minutes. Then, Pd(PPh3)4 (74.5 mg, 0.064 mmol) was added to the reaction mixture, nitrogen was bubbled through the reaction for 1 min and then the reaction was heated at 90 °C under nitrogen for 17h. The reaction mixture was cooled to rt, concentrated to a thick black oil, diluted with DCM and purified on a Biotage Horizon (40 g Si02, 60-100% EtOAc/hexanes) to yield alkyne J3b (312 mg) as a yellow solid. LC-MS retention time 2.800 min; m/z 671.53 (MH-). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a
Phenomenex-Luna lOu CI 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220 nM. The elution conditions employed a flow rate of 4 mL/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% MeOH / 95% ¾0 / 10 mM ammonium acetate and solvent B was 5% H20 / 95% MeOH / 10 mM ammonium acetate. MS data was determined using a Micromass Platform for LC in electrospray mode. XH NMR (400
MHz, MeOD) δ ppm 7.89 (s, 2H), 7.70 (ddd, J=5.6, 3.3, 1.9 Hz, 2H), 7.38 - 7.43 (m, 6H), 4.69 (br s, 2H), 3.59 (br s, 2H), 2.53 (dd, J=13.2, 8.9 Hz, 2H), 2.28 - 2.39 (m, 2H), 1.68 - 1.77 (m, 2H), 1.29 - 1.31 (m, 18H), 0.85 (dt, J=8.5, 5.8 Hz, 2H), 0.58 - 0.64 (m, 2H).
Example J3, step c
Figure imgf000175_0001
TFA (3 mL, 38.9 mmol) was added to a stirred solution of carbamate J3b (316 mg, 0.470 mmol) in DCE (7 mL) and the reaction was stirred at rt for lh. The reaction mixture was concentrated under vacuum to yield a TFA salt of pyrrolidine J3c (360 mg) as a yellow solid. LC-MS retention time 2.71 lmin; m/z 473.24 (MH+). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Phenomenex-Luna lOu C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220 nM. The elution conditions employed a flow rate of 4 mL/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% MeOH / 95% ¾0 / 10 mM ammonium acetate and solvent B was 5% ¾0 / 95% MeOH / 10 mM ammonium acetate. MS data was determined using a Micromass Platform for LC in electrospray mode.
Example J3 HATU (69.6 mg, 0.183 mmol) was added to a stirred solution of a TFA salt of pyrrolidine J3c (56.7 mg, 0.061 mmol) and (S)-2-(methoxycarbonylamino)-3- methylbutanoic acid (32.1 mg, 0.183 mmol) in DMF (0.7 mL) and Hunig'sBase (0.1 1 mL, 0.61 mmol) and the reaction mixture was stirred at rt for 3 h. The reaction was concentrated under a stream of nitrogen, dissolved into MeOH and purified in two injections by preparative HPLC (MeOH/water 10 mM ammonium acetate) to yield Example J3 (31.8 mg) as a light yellow solid. LC-MS retention time 2.605 min; m/z 787.36 (MH+). LC data was recorded on a Shimadzu LC-10AS liquid
chromatograph equipped with a Phenomenex-Luna lOu CI 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220 nM. The elution conditions employed a flow rate of 4 mL/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% MeOH / 95% ¾0 / 10 mM ammonium acetate and solvent B was 5% ¾0 / 95% MeOH / 10 mM ammonium acetate. MS data was determined using a Micromass Platform for LC in electrospray mode. ¾ NMR (400 MHz, MeOD) δ ppm 7.87 (s, 2H), 7.65 - 7.71 (m, 2H), 7.37 - 7.43 (m, 4H), 7.36 (s, 2H), 5.16 (dd, J=8.8, 4.8 Hz, 2H), 4.59 (d, J=6.5 Hz, 2H), 3.67 (s, 6H), 3.63 - 3.68 (m, 2H), 2.49 - 2.58 (m, 2H), 2.37 - 2.45 (m, 2H), 2.10 - 2.21 (m, 2H), 1.98 - 2.07 (m, 2H), 1.12 (ddd, J=8.7, 5.6, 5.5 Hz, 2H), 1.01 (d, J=6.8 Hz, 6H), 0.93 (d, J=6.8 Hz, 6H), 0.78 (br s, 2H).
Example J4
Figure imgf000176_0001
HATU (120 mg, 0.317 mmol) was added to a stirred solution of a TFA salt of pyrrolidine J3c (98 mg, 0.106 mmol) and (S)-2-(methoxycarbonylamino)-2- (tetrahydro-2H-pyran-4-yl)acetic acid (68.8 mg, 0.317 mmol) in DIPEA (0.18 mL, 1.1 mmol) and DMF (1 mL) and the reaction was stirred at rt for 3h. The reaction was concentrated under a stream of nitrogen, dissolved into MeOH and purified in by preparative HPLC (MeOH/water 10 mM ammonium acetate) and then repurified by prep HPLC (MeOH/water 0.1% TFA) to yield a TFA salt of Example J4 (18 mg) as a white solid. LC-MS retention time 2.463 min; m/z 869.40 (MH-). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a
Phenomenex-Luna lOu CI 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220 nM. The elution conditions employed a flow rate of 4 mL/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% MeOH / 95% ¾0 / 10 mM ammonium acetate and solvent B was 5% H20 / 95% MeOH / 10 mM ammonium acetate. MS data was determined using a Micromass Platform for LC in electrospray mode. XH NMR (400 MHz, MeOD) δ ppm 7.89 - 7.92 (m, 2H), 7.87 (s, 2H), 7.74 (dt, J=7.8, 1.5 Hz, 2H), 7.61 - 7.66 (m, 2H), 7.54 - 7.60 (m, 2H), 5.11 (dd, J=9.0, 7.0 Hz, 2H), 4.59 (d, J=7.5 Hz, 2H), 3.91 - 3.99 (m, 4H), 3.83 (t, J=4.8 Hz, 2H), 3.67 (s, 6H), 3.33 - 3.45 (m, 4H), 2.67 (dd, J=13.7, 9.4 Hz, 2H), 2.43 - 2.52 (m, 2H), 2.02 - 2.14 (m, 4H), 1.39 - 1.63 (m, 8H), 1.03 - 1.11 (m, 2H), 0.83 - 0.90 (m, 2H).
Example J5
Figure imgf000177_0001
Example J5, Step a
Figure imgf000177_0002
Nitrogen was bubbled through a stirred solution of (S)-tert-butyl 2-(4-(4- bromophenyl)-lH-imidazol-2-yl)pyrrolidine-l-carboxylate (bromide D-lb) (400 mg, 1.02 mmol), 4-ethynylphenylboronic acid (149 mg, 1.02 mmol) and sodium bicarbonate (214 mg, 2.55 mmol) in DME (8 mL) and water (2 mL) for 15 min. Then Pd(PPh3)4 (58.9 mg, 0.051 mmol) was added and the nitrogen bubbling was continued for 5 min. before the reaction vessel was sealed and heated at 90 °C overnight. The reaction was cooled, diluted with THF, washed with brine and concentrated to dryness. The residue was purified by Biotage Horizon (60-90% EtOAc/hexanes) to yield carbamate J5a (85 mg) as a yellow solid. LC-MS retention time 2.91 1 min; m/z 723.64 (MH-). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Phenomenex-Luna lOu CI 8 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220nM. The elution conditions employed a flow rate of 4 mL/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% MeOH / 95% H20 / 10 mM ammonium acetate and solvent B was 5% H20 / 95% MeOH / 10 mM ammonium acetate. MS data was determined using a Micromass Platform for LC in electrospray mode. XH NMR (400 MHz, MeOD) δ ppm 7.78 (d, J=8.0 Hz, 2H), 7.72 (d, J=8.0 Hz, 2H), 7.64 - 7.69 (m, 4H), 7.58 (d, J=8.3 Hz, 2H), 7.52 (d, J=8.3 Hz, 2H), 7.31 - 7.41 (m, 2H), 4.86 - 5.03 (m, 2H), 3.68 (br s, 2H), 3.46 -
3.57 (m, 2H), 2.37 (br s, 2H), 1.89 - 2.13 (m, 6H), 1.47 (br s, 6H), 1.25 (br s, 12H).
Example J5, Step b
Figure imgf000178_0001
TFA (250 μΐ^, 3.24 mmol) was added to a solution of carbamate J5a in DCE
(1 mL) and the reaction was stirred at rt for 1.5 hr. The reaction was concentrated under a stream of nitrogen to provide a TFA salt of pyrrolidine J5b (45 mg). LC-MS retention time 2.928 min; m/z 525.32 (MH+). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Phenomenex-Luna lOu CI 8 3.0 x 50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220nM. The elution conditions employed a flow rate of 4 mL/min, a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 4 min, a hold time of 1 min, and an analysis time of 5 min where solvent A was 5% MeOH / 95% H20 / 10 mM ammonium acetate and solvent B was 5% H20 / 95% MeOH / 10 mM ammonium acetate. MS data was determined using a Micromass Platform for LC in electrospray mode.
Example J5
HATU (53 mg, 0.14 mmol) was added to a solution of a TFA salt of J5b (45 mg, 0.046 mmol) and (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid (24.2 mg, 0.138 mmol) in DMF (0.7 mL) and TEA (0.038 mL, 0.28 mmol) and the reaction was stirred for 30 min. The reaction was diluted with MeOH, filtered and purified by prep HPLC (MeOH/water with 10 mM ammonium acetate) to yield Example J5 (14.7 mg) as a light yellow solid. LC-MS retention time 2.728 min; m/z 839.57 (MH+). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Phenomenex-Luna lOu C18 3.0x50mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220nM. The elution conditions employed a flow rate of 4 mL/min , a gradient of 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B, a gradient time of 4 min, a hold time of 1 min, and an analysis time of 5 min where solvent A was 5% MeOH / 95% ¾0 / 10 mM ammonium acetate and solvent B was 5% ¾0 / 95% MeOH / 10 mM ammonium acetate. MS data was determined using a Micromass Platform for LC in electrospray mode. XH NMR (400 MHz, MeOD) δ ppm 7.73 - 7.78 (m, 2H), 7.65 - 7.71 (m, 6H), 7.57 - 7.62 (m, 2H), 7.52 (d, J=8.5 Hz, 2H), 7.33 - 7.36 (m, 2H), 5.18 (dt, J=7.6, 5.2 Hz, 2H), 4.21 -
4.27 (m, 2H), 3.96 - 4.05 (m, 2H), 3.84 - 3.92 (m, 2H), 3.66 (s, 6H), 2.17 - 2.42 (m, 6H), 2.00 - 2.13 (m, 4H), 0.95 (dd, J=6.8, 1.3 Hz, 6H), 0.91 (dd, J=6.8, 1.0 Hz, 6H). Synthesis of common caps
Compound analysis conditions: Purity assessment and low resolution mass analysis were conducted on a Shimadzu LC system coupled with Waters Micromass ZQ MS system. It should be noted that retention times may vary slightly between machines. Additional LC conditions applicable to the current section, unless noted otherwise.
Cond-MS-Wl Column = XTERRA 3.0 X 50 mm S7 Start %B = 0
Final %B = 100
Gradient time = 2 min
Stop time = 3 min
Flow Rate = 5 mL/min
Wavelength = 220 nm
Solvent A 0.1% TFA in 10% methanol/90%H2O Solvent B 0.1% TFA in 90% methanol/ 10% H20
Cond.-MS-W2
Column = XTERRA 3.0 X 50 mm S7 Start %B = 0
Final %B = 100
Gradient time = 3 min
Stop time = 4 min
Flow Rate = 4 mL/min
Wavelength = 220 nm
Solvent A : 0.1%) TFA in 10% methanol/90%H2O Solvent B 0.1% TFA in 90% methanol/ 10% H20
Cond.-MS-W5
Column = XTERRA 3.0 X 50 mm S7 Start %B = 0
Final %B = 30
Gradient time = 2 min
Stop time = 3 min
Flow Rate = 5 mL/min
Wavelength = 220 nm
Solvent A = 0.1% TFA in 10% methanol/90%H2O Solvent B 0.1% TFA in 90% methanol/ 10% H20 Cond.-Dl
Column = XTERRA C18 3.0 X 50 mm S7 Start %B = 0
Final %B = 100
Gradient time = 3 min
Stop time = 4 min
Flow Rate = 4 mL/min
Wavelength = 220 nm
Solvent A = 0.1% TFA in 10% methanol/90%H2O Solvent B 0.1% TFA in 90% methanol/ 10% H20
Cond.-D2
Column = Phenomenex-Luna 4.6 X 50 mm S10 Start %B = 0
Final %B = 100
Gradient time = 3 min
Stop time = 4 min
Flow Rate = 4 mL/min
Wavelength = 220 nm
Solvent A = 0.1% TFA in 10% methanol/90%H2O Solvent B 0.1% TFA in 90% methanol/10% H20
Cond.-MDl
Column = XTERRA 4.6 X 50 mm S5 Start %B = 0
Final %B = 100
Gradient time = 3 min
Stop time = 4 min
Flow Rate = 4 mL/min
Wavelength = 220 nm
Solvent A = 0.1% TFA in 10% methanol/90%H2O Solvent B : 0.1% TFA in 90% methanol/10% H20 Cond.-M3
Column = XTERRA C18 3.0 X 50 mm S7
Start %B = 0
Final %B = 40
Gradient time = 2 min
Stop time = 3 min
Flow Rate = 5 mL/min
Wavelength = 220 nm
Solvent A = 0.1% TFA in 10% methanol/90%H2O
Solvent B = 0.1% TFA in 90% methanol/10% H20
Condition OL1
Column = Phenomenex-Luna 3.0 X 50 mm S10
Start %B = 0
Final %B = 100
Gradient time = 4 min
Stop time = 5 min
Flow Rate = 4 mL/min
Wavelength = 220 nm
Solvent A = 0.1% TFA in 10% methanol/90%H2O
Solvent B = 0.1% TFA in 90% methanol/ 10% H20
Condition OL2
Column = Phenomenex-Luna 50X 2 mm 3 u
Start %B = 0
Final %B = 100
Gradient time = 4 min
Stop time = 5 min
Flow Rate = 0.8 mL/min
Oven Temp = 40 °C
Wavelength = 220 nm = 0.1% TFA in 10% Acetonitrile/90%H2O = 0.1% TFA in 90% Acetonitrile/10% H20
Condition I
Column Phenomenex-Luna 3.0 X 50 mm S10 Start %B 0
Final %B 100
Gradient time 2 min
Stop time 3 min
Flow Rate 4 mL/min
Wavelength 220 nm
Solvent A 0.1% TFA in 10% methanol/90%H2O Solvent B 0.1% TFA in 90% methanol/ 10% H20 Condition II
Column Phenomenex-Luna 4.6 X 50 mm S10 Start %B 0
Final %B 100
Gradient time 2 min
Stop time 3 min
Flow Rate 5 mL/min
Wavelength 220 nm
Solvent A 0.1% TFA in 10% methanol/90%H2O Solvent B 0.1% TFA in 90% methanol/ 10% H20 Condition III
Column XTERRA CI 8 3.0 x 50mm S7
Start %B 0
Final %B 100
Gradient time = 3 min
Stop time = 4 min
Flow Rate 4 mL/min
Wavelength 220 nm 0.1% TFA in 10% methanol/90%H2O
0.1% TFA in 90% methanol/ 10% H20 -1
Figure imgf000184_0001
A suspension of 10% Pd/C (2.0g) in methanol (10 mL) was added to a mixture of (R)-2-phenylglycine (lOg, 66.2 mmol), formaldehyde (33 mL of 37% wt. in water), IN HC1 (30 mL) and methanol (30 mL), and exposed to H2 (60 psi) for 3 hours. The reaction mixture was filtered through diatomaceous earth (Celite®), and the filtrate was concentrated in vacuo. The resulting crude material was
recrystallized from isopropanol to provide the HC1 salt of Cap-1 as a white needle (4.0 g). Optical rotation: -117.1° [c=9.95 mg/mL in H20; λ=589 nm]. ¾ NMR (DMSO-d6, δ=2.5 ppm, 500 MHz): δ 7.43-7.34 (m, 5H), 4.14 (s, 1H), 2.43 (s, 6H); LC (Cond. I): RT=0.25; LC/MS: Anal. Calcd. for [M+H]+ Ci0H14NO2 180.10; found 180.17; HRMS: Anal. Calcd. for [M+H]+ Ci0Hi4NO2 180.1025; found 180.1017. -2
Figure imgf000184_0002
NaBI¾CN (6.22g, 94 mmol) was added in portions over a few minutes to a cooled (ice/water) mixture of (R)-2-Phenylglycine (6.02 g, 39.8 mmol) and methanol (100 mL), and stirred for 5 minutes. Acetaldehyde (10 mL) was added dropwise over 10 minutes and stirring was continued at the same cooled temperature for 45 minutes and at ambient temperature for ~6.5 hours. The reaction mixture was cooled back with ice-water bath, treated with water (3 mL) and then quenched with a dropwise addition of concentrated HC1 over ~ 45 minutes until the pH of the mixture was - 1.5 - 2.0. The cooling bath was removed and the stirring was continued while adding concentrated HC1 in order to maintain the pH of the mixture around 1.5-2.0. The reaction mixture was stirred overnight, filtered to remove the white suspension, and the filtrate was concentrated in vacuo. The crude material was recrystallized from ethanol to afford the HC1 salt of Cap-2 as a shining white solid in two crops (crop-1 : 4.16 g; crop-2: 2.19 g). ¾ NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 10.44 (1.00, br s, 1H), 7.66 (m, 2H), 7.51 (m, 3H), 5.30 (s, 1H), 3.15 (br m, 2H), 2.98 (br m, 2H), 1.20 (app br s, 6H). Crop-1: [a]25 -102.21° (c=0.357, H20); crop-2: [a]25 -99.7° (c=0.357, H20). LC (Cond. I): RT=0.43 min; LC/MS: Anal. Calcd. for [M+H]+ Ci2H18N02: 208.13; found 208.26. -3
Figure imgf000185_0001
Acetaldehyde (5.0 mL, 89.1 mmol) and a suspension of 10% Pd/C (720 mg) in methanol/H20 (4mL/l mL) was sequentially added to a cooled (~ 15 °C) mixture of (R)-2-phenylglycine (3.096g, 20.48 mmol), IN HC1 (30 mL) and methanol (40 mL). The cooling bath was removed and the reaction mixture was stirred under a balloon of H2 for 17 hours. An additional acetaldehyde (10 mL, 178.2 mmol) was added and stirring continued under H2 atmosphere for 24 hours [Note: the supply of H2 was replenished as needed throughout the reaction]. The reaction mixture was filtered through diatomaceous earth (Celite®), and the filtrate was concentrated in vacuo. The resulting crude material was recrystallized from isopropanol to provide the HC1 salt of (R)-2-(ethylamino)-2-phenylacetic acid as a shining white solid (2.846g). ¾ NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): δ 14.15 (br s, 1H), 9.55 (br s, 2H), 7.55-7.48 (m, 5H), 2.88 (br m, 1H), 2.73 (br m, 1H), 1.20 (app t, J=7.2, 3H). LC (Cond. I): RT=0.39 min; >95 % homogeneity index; LC/MS: Anal. Calcd. for
[M+H]+ CioH14N02: 180.10; found 180.18.
A suspension of 10% Pd/C (536 mg) in methanol/H20 (3 mL/1 mL) was added to a mixture of (R)-2-(ethylamino)-2-phenylacetic acid/HCl (1.492g, 6.918 mmol), formaldehyde (20 mL of 37% wt. in water), IN HC1 (20 mL) and methanol (23 niL). The reaction mixture was stirred under a balloon of ¾ for ~72 hours, where the H2 supply was replenished as needed. The reaction mixture was filtered through diatomaceous earth (Celite®) and the filtrate was concentrated in vacuo. The resulting crude material was recrystallized from isopropanol (50 mL) to provide the HC1 salt of Cap-3 as a white solid (985 mg). XH NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): δ 10.48 (br s, 1H), 7.59-7.51 (m, 5H), 5.26 (s, 1H), 3.08 (app br s, 2H), 2.65 (br s, 3H), 1.24 (br m, 3H). LC (Cond. I): RT=0.39 min; >95 % homogeneity index; LC/MS: Anal. Calcd. for [M+H]+ CnH16N02: 194.12; found 194.18; HRMS: Anal. Calcd. for [M+H]+ CnH16N02: 194.1180; found 194.1181. -4
Figure imgf000186_0001
ClC02Me (3.2 mL, 41.4 mmol) was added dropwise to a cooled (ice/water) THF (410 mL) semi-solution of (R)-/er/-butyl 2-amino-2-phenylacetate/HCl (9.877 g, 40.52 mmol) and diisopropylethylamine (14.2 mL, 81.52 mmol) over 6 min, and stirred at similar temperature for 5.5 hours. The volatile component was removed in vacuo, and the residue was partitioned between water (100 mL) and ethyl acetate (200 mL). The organic layer was washed with IN HC1 (25 mL) and saturated aHC03 solution (30 mL), dried (MgS04), filtered, and concentrated in vacuo. The resultant colorless oil was triturated from hexanes, filtered and washed with hexanes (100 mL) to provide (R)-tert-butyl 2-(methoxycarbonylamino)-2-phenylacetate as a white solid (7.7 g). XH NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 7.98 (d, J=8.0, 1H), 7.37-7.29 (m, 5H), 5.09 (d, J=8, 1H), 3.56 (s, 3H), 1.33 (s, 9H). LC (Cond. I): RT=1.53 min; -90 % homogeneity index; LC/MS: Anal. Calcd. for [M+Na
Ci4H19NNa04: 288.12; found 288.15.
TFA (16 mL) was added dropwise to a cooled (ice/water) CH2CI2 (160 mL) solution of the above product over 7 minutes, and the cooling bath was removed and the reaction mixture was stirred for 20 hours. Since the deprotection was still not complete, an additional TFA (1.0 mL) was added and stirring continued for an additional 2 hours. The volatile component was removed in vacuo, and the resulting oil residue was treated with diethyl ether (15 mL) and hexanes (12 mL) to provide a precipitate. The precipitate was filtered and washed with diethyl ether/hexanes (~1 :3 ratio; 30 mL) and dried in vacuo to provide Cap-4 as a fluffy white solid (5.57 g). Optical rotation: -176.9° [c=3.7 mg/mL in H20; λ=589 nm]. XH NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): δ 12.84 (br s, 1H), 7.96 (d, J=8.3, 1H), 7.41-7.29 (m, 5H), 5.14 (d, J=8.3, 1H), 3.55 (s, 3H). LC (Cond. I): RT=1.01 min; >95 % homogeneity index; LC/MS: Anal. Calcd. for [M+H]+ Ci0H12NO4 210.08; found 210.17; HRMS: Anal. Calcd. for [M+H]+ Ci0H12NO4 210.0766; found 210.0756. -5
Figure imgf000187_0001
A mixture of (R)- 2-phenylglycine (1.0 g, 6.62 mmol), 1,4-dibromobutane (1.57 g, 7.27 mmol) and Na2C03 (2.10 g, 19.8 mmol) in ethanol (40 mL) was heated at 100 °C for 21 hours. The reaction mixture was cooled to ambient temperature and filtered, and the filtrate was concentrated in vacuo. The residue was dissolved in ethanol and acidified with IN HC1 to pH 3-4, and the volatile component was removed in vacuo. The resulting crude material was purified by a reverse phase HPLC (water/methanol/TFA) to provide the TFA salt of Cap-5 as a semi-viscous white foam (1.0 g). 'H NMR (DMSO-d6, 5=2.5, 500 MHz) δ 10.68 (br s, 1H), 7.51 (m, 5H), 5.23 (s, 1H), 3.34 (app br s, 2H), 3.05 (app br s, 2H), 1.95 (app br s, 4H); RT=0.30 minutes (Cond. I); >98% homogeneity index; LC/MS: Anal. Calcd. for [M+H]+ Ci2H16N02: 206.12; found 206.25.
Cap-6
Figure imgf000188_0001
The TFA salt of Cap-6 was synthesized from (R)-2-phenylglycine and 1 - bromo-2-(2-bromoethoxy)ethane by using the method of preparation of Cap-5. XH NMR (DMSO-d6, 5=2.5, 500 MHz) δ 12.20 (br s, 1H), 7.50 (m, 5H), 4.92 (s, 1H), 3.78 (app br s, 4H), 3.08 (app br s, 2H), 2.81 (app br s, 2H); RT=0.32 minutes (Cond. I); >98%; LC/MS: Anal. Calcd. for [M+H]+ Ci2H16N03: 222.11 ; found 222.20; HRMS: Anal. Calcd. for [M+H]+ Ci2H16N03: 222.1 130; found 222.1 121.
Cap-7
Cap-7a: enantiomer-1
Cap-7b: enantiomer-2
Figure imgf000188_0002
A CH2C12 (200 mL) solution of p-toluenesulfonyl chloride (8.65 g, 45.4 mmol) was added dropwise to a cooled (-5 °C) CH2CI2 (200 mL) solution of (S)- benzyl 2-hydroxy-2-phenylacetate (10.0 g, 41.3 mmol), triethylamine (5.75 mL, 41.3 mmol) and 4-dimethylaminopyridine (0.504 g, 4.13 mmol), while maintaining the temperature between -5 °C and 0 °C. The reaction was stirred at 0 °C for 9 hours, and then stored in a freezer (-25 °C) for 14 hours. It was allowed to thaw to ambient temperature and washed with water (200 mL), IN HC1 (100 mL) and brine (100 mL), dried (MgS04), filtered, and concentrated in vacuo to provide benzyl 2-phenyl-2- (tosyloxy)acetate as a viscous oil which solidified upon standing (16.5 g). The chiral integrity of the product was not checked and that product was used for the next step without further purification. XH NMR (DMSO-d6, 5=2.5, 500 MHz) 5 7.78 (d, J= 8.6, 2H), 7.43-7.29 (m, 10H), 7.20 (m, 2H), 6.12 (s, 1H), 5.16 (d, J=12.5, 1H), 5.10 (d, J= 12.5, 1H), 2.39 (s, 3H). RT=3.00 (Cond. Ill); >90% homogeneity index; LC/MS: Anal. Calcd. for [M+H]+ C22H2oNa05S: 419.09; found 419.04. A THF (75 mL) solution of benzyl 2-phenyl-2-(tosyloxy)acetate (6.0 g, 15.1 mmol), 1-methylpiperazine (3.36 mL, 30.3 mmol) and N,N-diisopropylethylamine (13.2 mL, 75.8 mmol) was heated at 65 °C for 7 hours. The reaction was allowed to cool to ambient temperature and the volatile component was removed in vacuo. The residue was partitioned between ethylacetate and water, and the organic layer was washed with water and brine, dried (MgSC^), filtered, and concentrated in vacuo. The resulting crude material was purified by flash chromatography (silica gel, ethyl acetate) to provide benzyl 2-(4-methylpiperazin-l-yl)-2-phenylacetate as an orangish-brown viscous oil (4.56 g). Chiral HPLC analysis (Chiralcel OD-H) indicated that the sample is a mixture of enantiomers in a 38.2 to 58.7 ratio. The separation of the enantiomers were effected as follow: the product was dissolved in 120 mL of ethanol/heptane (1 : 1) and injected (5 mL/injection) on chiral HPLC column (Chiracel OJ, 5 cm ID x 50 cm L, 20 μιη) eluting with 85: 15 Heptane/ethanol at 75 mL/min, and monitored at 220 nm. Enantiomer-1 (1.474 g ) and enantiomer-2 (2.2149 g) were retrieved as viscous oil. XH NMR (CDC13, δ=7.26, 500 MHz) 7.44- 7.40 (m, 2H), 7.33-7.24 (m, 6H), 7.21-7.16 (m, 2H), 5.13 (d, J= 12.5, 1H), 5.08 (d, J= 12.5, 1H), 4.02 (s, 1H), 2.65-2.38 (app br s, 8H), 2.25 (s, 3H). RT=2.10 (Cond. Ill); >98% homogeneity index; LC/MS: Anal. Calcd. for [M+H]+ C20H25N2O2: 325.19; found 325.20.
A methanol (10 mL) solution of either enantiomer of benzyl 2-(4- methylpiperazin-l-yl)-2-phenylacetate (1.0 g, 3.1 mmol) was added to a suspension of 10% Pd/C (120 mg) in methanol (5.0 mL). The reaction mixture was exposed to a balloon of hydrogen, under a careful monitoring, for <50 minutes. Immediately after the completion of the reaction, the catalyst was filtered through diatomaceous earth (Celite®) and the filtrate was concentrated in vacuo to provide Cap-7, contaminated with phenylacetic acid as a tan foam (867.6 mg; mass is above the theoretical yield). The product was used for the next step without further purification. XH NMR (DMSO-d6, 5=2.5, 500 MHz) δ 7.44-7.37 (m, 2H), 7.37-7.24 (m, 3H), 3.92 (s, 1H), 2.63-2.48 (app. br s, 2H), 2.48-2.32 (m, 6H), 2.19 (s, 3H); RT=0.31 (Cond. II); >90% homogeneity index; LC/MS: Anal. Calcd. for [M+H]+ Ci3H19 202: 235.14; found 235.15; HRMS: Anal. Calcd. for [M+H Ci3H19 202: 235.1447; found 235.1440.
The synthesis of Cap-8 and Cap-9 was conducted according to the synthesis of Cap-7 by using appropriate amines for the S 2 displacement step (i.e., 4- hydroxypiperidine for Cap-8 and (S)-3-fluoropyrrolidine for Cap-9) and modified conditions for the separation of the respective stereoisomeric intermedites, as described below. p-8
8a: enantiomer-1
8b: enantiomer-2
Figure imgf000190_0001
The enantiomeric separation of the intermediate benzyl 2-(4- hydroxypiperidin-l-yl)-2-phenyl acetate was effected by employing the following conditions: the compound (500 mg) was dissolved in ethanol/heptane (5 mL/45 mL). The resulting solution was injected (5 mL/injection) on a chiral HPLC column (Chiracel OJ, 2 cm ID x 25 cm L, 10 μιη) eluting with 80:20 heptane/ethanol at 10 mL/min, monitored at 220 nm, to provide 186.3 mg of enantiomer- 1 and 209.1 mg of enantiomer-2 as light-yellow viscous oils. These benzyl ester was hydrogenolysed according to the preparation of Cap-7 to provide Cap-8: XH NMR (DMSO-d6, δ=2.5, 500 MHz) 7.40 (d, J=7, 2H), 7.28-7.20 (m, 3H), 3.78 (s 1H), 3.46 (m, 1H), 2.93 (m,
1H), 2.62 (m, 1H), 2.20 (m, 2H), 1.70 (m, 2H), 1.42 (m, 2H). RT=0.28 (Cond. II); >98% homogeneity index; LC/MS: Anal. Calcd. for [M+H]+ Ci3H18N03: 236.13; found 236.07; HRMS: Calcd. for [M+H]+ Ci3H18N03: 236.1287; found 236.1283.
Cap-9 9a: diastereomer-1
9b: diastereomer-2
Figure imgf000191_0001
The diastereomeric separation of the intermediate benzyl 2-((S)-3- fluoropyrrolidin-l-yl)-2-phenylacetate was effected by employing the following conditions: the ester (220 mg) was separated on a chiral HPLC column (Chiracel OJ- H, 0.46 cm ID x 25 cm L, 5 μιη) eluting with 95% C02 / 5% methanol with 0.1% TFA, at 10 bar pressure, 70 mL/min flow rate, and a temperature of 35 °C. The HPLC elute for the respective stereoisomers was concentrated, and the residue was dissolved in CH2CI2 (20 mL) and washed with an aqueous medium (10 mL water + 1 mL saturated NaHCC solution). The organic phase was dried (MgSC^), filtered, and concentrated in vacuo to provide 92.5 mg of fraction- 1 and 59.6 mg of fraction-2. These benzyl esters were hydrogenolysed according to the preparation of Cap-7 to prepare Caps 9a and 9b. Cap-9a (diastereomer-1 ; the sample is a TFA salt as a result of purification on a reverse phase HPLC using H20/methanol/TFA solvent): XH NMR (DMSO-d6, δ=2.5, 400 MHz) 7.55-7.48 (m, 5H), 5.38 (d of m, J=53.7, 1H), 5.09 (br s, 1H), 3.84-2.82 (br m, 4H), 2.31-2.09 (m, 2H). RT=0.42 (Cond. I); >95% homogeneity index; LC/MS: Anal. Calcd. for [M+H]+ Ci2H15FN02: 224.11 ; found 224.14; Cap-9b (diastereomer-2): 'H NMR (DMSO-d6, δ=2.5, 400 MHz) 7.43-7.21 (m, 5H), 5.19 (d of m, J=55.9, 1H), 3.97 (s, 1H), 2.95-2.43 (m, 4H), 2.19-1.78 (m, 2H). RT=0.44 (Cond. I); LC/MS: Anal. Calcd. for [M+H]+ Ci2H15FN02: 224.1 1; found 224.14. - 10
Figure imgf000191_0002
To a solution of D-proline (2.0 g, 17 mmol) and formaldehyde (2.0 mL of 37% wt. in H20) in methanol (15 mL) was added a suspension of 10% Pd/C (500 mg) in methanol (5 mL). The mixture was stirred under a balloon of hydrogen for hours. The reaction mixture was filtered through diatomaceous earth (Celite R) and concentrated in vacuo to provide Cap- 10 as an off-white solid (2.15 g). XH NMR (DMSO-d6, 5=2.5, 500 MHz) 3.42 (m, 1H), 3.37 (dd, J=9.4, 6.1, 1H), 2.85-2.78 (m, 1H), 2.66 (s, 3H), 2.21-2.13 (m, 1H), 1.93-1.84 (m, 2H), 1.75-1.66 (m, 1H).
RT=0.28 (Cond. II); >98% homogeneity index; LC/MS: Anal. Calcd. for [M+H]+ C6H12N02: 130.09; found 129.96. - 11
Figure imgf000192_0001
A mixture of (2S,4R)-4-fluoropyrrolidine-2-carboxylic acid (0.50 g, 3.8 mmol), formaldehyde (0.5 mL of 37% wt. in H20), 12 N HC1 (0.25 mL) and 10% Pd/C (50 mg) in methanol (20 mL) was stirred under a balloon of hydrogen for 19 hours. The reaction mixture was filtered through diatomaceous earth (Celite®) and the filtrate was concentrated in vacuo. The residue was recrystallized from isopropanol to provide the HC1 salt of Cap-1 1 as a white solid (337.7 mg). ¾ NMR (DMSO-d6, 8=2.5, 500 MHz) 5.39 (d m, J=53.7, 1H), 4.30 (m, 1H), 3.90 (ddd, J=31.5, 13.5, 4.5, 1H), 3.33 (dd, J=25.6, 13.4, 1H), 2.85 (s, 3H), 2.60-2.51 (m, 1H), 2.39-2.26 (m, 1H). RT=0.28 (Cond. II); >98% homogeneity index; LC/MS: Anal. Calcd. for [M+H]+ C6HnFN02: 148.08; found 148.06.
Ca -12 (same as cap 52)
Figure imgf000192_0002
L-Alanine (2.0 g, 22.5 mmol) was dissolved in 10% aqueous sodium carbonate solution (50 mL), and a THF (50 mL) solution of methyl chloro formate (4.0 mL) was added to it. The reaction mixture was stirred under ambient conditions for 4.5 hours and concentrated in vacuo. The resulting white solid was dissolved in water and acidified with IN HC1 to a pH ~ 2-3. The resulting solutions was extracted with ethyl acetate (3 x 100 mL), and the combined organic phase was dried
( a2S04), filtered, and concentrated in vacuo to provide a colorless oil (2.58 g). 500 mg of this material was purified by a reverse phase HPLC (H^O/methanol/TFA) to provide 150 mg of Cap-12 as a colorless oil . XH NMR (DMSO-d6, 5=2.5, 500 MHz) 7.44 (d, J=7.3, 0.8H), 7.10 (br s, 0.2H), 3.97 (m, 1H), 3.53 (s, 3H), 1.25 (d, J=7.3, 3H).
Cap- 13
Figure imgf000193_0001
A mixture of L-alanine (2.5 g, 28 mmol), formaldehyde (8.4 g, 37 wt. %), IN
HCl (30 mL) and 10% Pd/C (500 mg) in methanol (30 mL) was stirred under a hydrogen atmosphere (50 psi) for 5 hours. The reaction mixture was filtered through diatomaceous earth (Celite®) and the filtrate was concentrated in vacuo to provide the HCl salt of Cap- 13 as an oil which solidified upon standing under vacuum (4.4 g; the mass is above theoretical yield). The product was used without further purification. ¾ NMR (DMSO-d6, 8=2.5, 500 MHz) δ 12.1 (br s, 1H), 4.06 (q, J=7.4, 1H), 2.76 (s, 6H), 1.46 (d, J=7.3, 3H). -14
Figure imgf000193_0002
Cap- 14
Step 1: A mixture of (R)-(-)-D-phenylglycine tert-butyl ester (3.00 g, 12.3 mmol), NaBH3CN (0.773 g, 12.3 mmol), KOH (0.690 g, 12.3 mmol) and acetic acid
(0.352 mL, 6.15 mmol) were stirred in methanol at 0 °C. To this mixture was added glutaric dialdehyde (2.23 mL, 12.3 mmol) dropwise over 5 minutes. The reaction mixture was stirred as it was allowed to warm to ambient temperature and stirring was continued at the same temperature for 16 hours. The solvent was subsequently removed and the residue was partitioned with 10% aqueous NaOH and ethyl acetate. The organic phase was separated, dried (MgSC^), filtered and concentrated to dryness to provide a clear oil. This material was purified by reverse-phase preparative HPLC (Primesphere C-18, 30 x 100mm; CH3CN-H2O-0.1% TFA) to give the intermediate ester (2.70 g, 56%) as a clear oil. XH NMR (400 MHz, CDC13) δ 7.53-7.44 (m, 3H), 7.40-7.37 (m, 2H), 3.87 (d, J= 10.9 Hz, 1H), 3.59 (d, J= 10.9 Hz, lH), 2.99 (t, J=1 1.2 Hz, 1H), 2.59 (t, J=l 1.4 Hz, 1H), 2.07-2.02 (m, 2H), 1.82 (d, J= 1.82 Hz, 3H), 1.40 (s, 9H). LC/MS: Anal. Calcd. for Ci7H25 02: 275; found: 276 (M+H)+.
Step 2: To a stirred solution of the intermediate ester (1.12g, 2.88mmol) in dichloromethane (10 mL) was added TFA (3 mL). The reaction mixture was stirred at ambient temperature for 4 hours and then it was concentrated to dryness to give a light yellow oil. The oil was purified using reverse-phase preparative HPLC
(Primesphere C-18, 30 x 100mm; CH3CN-H2O-0.1% TFA). The appropriate fractions were combined and concentrated to dryness in vacuo. The residue was then dissolved in a minimum amount of methanol and applied to applied to MCX LP extraction cartridges (2 x 6 g). The cartridges were rinsed with methanol (40 mL) and then the desired compound was eluted using 2M ammonia in methanol (50 mL). Product-containing fractions were combined and concentrated and the residue was taken up in water. Lyophilization of this solution provided the title compound (0.492 g, 78%) as a light yellow solid. XH NMR (DMSO-d6) δ 7.50 (s, 5H), 5.13 (s, 1H), 3.09 (br s, 2H), 2.92-2.89 (m, 2H), 1.74 (m, 4H), 1.48 (br s, 2H). LC/MS: Anal. Calcd. for Ci3H17N02: 219; found: 220 (M+H)+. - 15
Figure imgf000194_0001
(R)-Cap-15
Step 1: (S)-l-Phenylethyl 2-bromo-2-phenylacetate: To a mixture of a- bromophenylacetic acid (10.75 g, 0.050 mol), (S)-(-)-l-phenylethanol (7.94 g, 0.065 mol) and DMAP (0.61 g, 5.0 mmol) in dry dichloromethane (100 mL) was added solid EDCI (12.46 g, 0.065 mol) all at once. The resulting solution was stirred at room temperature under Ar for 18 hours and then it was diluted with ethyl acetate, washed (H20 x 2, brine), dried (Na2S04), filtered, and concentrated to give a pale yellow oil. Flash chromatography (Si02/ hexane-ethyl acetate, 4: 1) of this oil provided the title compound (11.64 g, 73%) as a white solid. XH NMR (400 MHz, CDC13) δ 7.53-7.17 (m, 10H), 5.95 (q, J=6.6 Hz, 0.5H), 5.94 (q, J=6.6 Hz, 0.5H), 5.41 (s, 0.5H), 5.39 (s, 0.5H), 1.58 (d, J=6.6 Hz, 1.5H), 1.51 (d, J=6.6 Hz, 1.5H).
Step 2: (S)-l-Phenylethyl (R)-2-(4-hydroxy-4-methylpiperidin-l-yl)- 2- phenylacetate: To a solution of (S)-l-phenylethyl 2-bromo-2-phenylacetate (0.464 g, 1.45 mmol) in THF (8 mL) was added triethylamine (0.61 mL, 4.35 mmol), followed by tetrabutylammonium iodide (0.215 g, 0.58 mmol). The reaction mixture was stirred at room temperature for 5 minutes and then a solution of 4-methyl-4- hydroxypiperidine (0.251 g, 2.18 mmol) in THF (2 mL) was added. The mixture was stirred for 1 hour at room temperature and then it was heated at 55-60 °C (oil bath temperature) for 4 hours. The cooled reaction mixture was then diluted with ethyl acetate (30 mL), washed (H20 x2, brine), dried (MgS04), filtered and concentrated. The residue was purified by silica gel chromatography (0-60% ethyl acetate-hexane) to provide first the (S,R)-isomer of the title compound (0.306 g, 60%) as a white solid and then the corresponding (S,S)-isomer (0.120 g, 23%), also as a white solid. (S,R)-isomer: ¾ NMR (CD3OD) δ 7.51-7.45 (m, 2H), 7.41-7.25 (m, 8H), 5.85 (q, J=6.6 Hz, IH), 4.05 (s, IH), 2.56-2.45 (m, 2H), 2.41-2.29 (m, 2H), 1.71-1.49 (m, 4H), 1.38 (d, J=6.6 Hz, 3H), 1.18 (s, 3H). LCMS: Anal. Calcd. for C22H27 03: 353; found: 354 (M+H)+. (S,S)-isomer: XH NMR (CD3OD) δ 7.41-7.30 (m, 5H), 7.20-7.14 (m, 3H), 7.06-7.00 (m, 2H), 5.85 (q, J=6.6 Hz, IH), 4.06 (s, IH), 2.70-2.60 (m, IH), 2.51 (dt, J=6.6, 3.3 Hz, IH), 2.44-2.31 (m, 2H), 1.75-1.65 (m, IH), 1.65- 1.54 (m, 3H), 1.50 (d, J=6.8 Hz, 3H), 1.20 (s, 3H). LCMS: Anal. Calcd. for C22H27N03: 353; found: 354 (M+H)+.
Step 3: (R)-2-(4-Hydroxy-4-methylpiperidin-l-yl)-2-phenylacetic acid: To a solution of (S)- 1 -phenylethyl (R)-2-(4-hydroxy-4-methylpiperidin- 1 -yl)-2- phenylacetate (0.185 g, 0.52 mmol) in dichloromethane (3 mL) was added trifluoroacetic acid (1 niL) and the mixture was stirred at room temperature for 2 hours. The volatiles were subsequently removed in vacuo and the residue was purified by reverse-phase preparative HPLC (Primesphere C-18, 20 x 100mm;
CH3CN-H2O-0.1% TFA) to give the title compound (as TFA salt) as a pale bluish solid (0,128 g, 98%). LCMS: Anal. Calcd. for Ci4H19N03: 249; found: 250 (M+H)+. - 16
Figure imgf000196_0001
(RJ-Cap-16
Step 1: (S)- 1 -Phenyl ethyl 2-(2-fluorophenyl)acetate: A mixture of 2- fluorophenylacetic acid (5.45 g, 35.4 mmol), (S)-l-phenylethanol (5.62 g, 46.0 mmol), EDCI (8.82 g, 46.0 mmol) and DMAP (0.561 g, 4.60 mmol) in CH2C12 (100 mL) was stirred at room temperature for 12 hours. The solvent was then
concentrated and the residue partitioned with H20-ethyl acetate. The phases were separated and the aqueous layer back-extracted with ethyl acetate (2x). The combined organic phases were washed (H20, brine), dried ( a2S04), filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (Biotage/ 0-20% ethyl acetate-hexane) to provide the title compound as a colorless oil (8.38 g, 92%). XH NMR (400 MHz, CD3OD) δ 7.32-7.23 (m, 7H), 7.10-7.04 (m, 2), 5.85 (q, J=6.5 Hz, 1H), 3.71 (s, 2H), 1.48 (d, J=6.5 Hz, 3H).
Step 2: (R)-((S)- 1 -Phenylethyl) 2-(2-fluorophenyl)-2-(piperidin- 1 -yl)acetate: To a solution of (S)-l -phenylethyl 2-(2-fluorophenyl)acetate (5.00 g, 19.4 mmol) in THF (1200 mL) at 0 °C was added DBU (6.19 g, 40.7 mmol) and the solution was allowed to warm to room temperature while stirring for 30 minutes. The solution was then cooled to -78 °C and a solution of CBr4 (13.5 g, 40.7 mmol) in THF (100 mL) was added and the mixture was allowed to warm to -10 °C and stirred at this temperature for 2 hours. The reaction mixture was quenched with saturated aq. NH4CI and the layers were separated. The aqueous layer was back-extracted with ethyl acetate (2x) and the combined organic phases were washed (H20, brine), dried ( a2S04), filtered, and concentrated in vacuo. To the residue was added piperidine (5.73 mL, 58.1 mmol) and the solution was stirred at room temperature for 24 hours. The volatiles were then concentrated in vacuo and the residue was purified by silica gel chromatography (Biotage/ 0-30% diethyl ether-hexane) to provide a pure mixture of diastereomers (2: 1 ratio by XH NMR) as a yellow oil (2.07 g, 31%), along with unreacted starting material (2.53 g, 51%). Further chromatography of the diastereomeric mixture (Biotage/ 0-10% diethyl ether-toluene) provided the title compound as a colorless oil (0.737 g, 11%). XH NMR (400 MHz, CD3OD) δ 7.52 (ddd, J=9.4, 7.6, 1.8 Hz, 1H), 7.33 - 7.40 (m, 1), 7.23 - 7.23 (m, 4H), 7.02 - 7.23 (m, 4H), 5.86 (q, J=6.6 Hz, 1H), 4.45 (s, 1H), 2.39 - 2.45 (m, 4H), 1.52 - 1.58 (m, 4H), 1.40 - 1.42 (m, 1H), 1.38 (d, J=6.6 Hz, 3H). LCMS: Anal. Calcd. for C21H24FNO2: 341; found: 342 (M+H)+.
Step 3: (R)-2-(2-fluorophenyl)-2-(piperidin-l-yl)acetic acid: A mixture of
(R)-((S)-l-phenylethyl) 2-(2-fluorophenyl)-2-(piperidin-l-yl)acetate (0.737 g, 2.16 mmol) and 20% Pd(OH)2/C (0.070 g) in ethanol (30 mL) was hydrogenated at room temperature and atmospheric pressure (H2 balloon) for 2 hours. The solution was then purged with Ar, filtered through diatomaceous earth (Celite®), and concentrated in vacuo. This provided the title compound as a colorless solid (0.503 g, 98%). ¾ NMR (400 MHz, CD3OD) δ 7.65 (ddd, J=9.1, 7.6, 1.5 Hz, 1H), 7.47-7.53 (m, 1H), 7.21-7.30 (m, 2H), 3.07-3.13 (m, 4H), 1.84 (br s, 4H), 1.62 (br s, 2H). LCMS: Anal. Calcd. for Ci3H16FN02: 237; found: 238 (M+H)+.
Figure imgf000197_0001
Step 1: (S)-l-Phenylethyl (R)-2-(4-hydroxy-4-phenylpiperidin-l-yl)- 2- phenylacetate: To a solution of (S)-l-phenylethyl 2-bromo-2-phenylacetate (1.50 g, 4.70 mmol) in THF (25 mL) was added triethylamine (1.31 mL, 9.42 mmol), followed by tetrabutylammonium iodide (0.347 g, 0.94 mmol). The reaction mixture was stirred at room temperature for 5 minutes and then a solution of 4-phenyl-4- hydroxypiperidine (1.00 g, 5.64 mmol) in THF (5 mL) was added. The mixture was stirred for 16 hours and then it was diluted with ethyl acetate (100 mL), washed (H20 x2, brine), dried (MgS04), filtered and concentrated. The residue was purified on a silica gel column (0-60% ethyl acetate-hexane) to provide an approximately 2: 1 mixture of diastereomers, as judged by 1H NMR. Separation of these isomers was performed using supercritical fluid chromatography (Chiralcel OJ-H, 30 x 250mm; 20% ethanol in CO2 at 35 °C), to give first the (R)-isomer of the title compound (0.534 g, 27%) as a yellow oil and then the corresponding (S)-isomer (0.271 g, 14%), also as a yellow oil. (S,R)-isomer: XH NMR (400 MHz, CD3OD) δ 7.55-7.47 (m, 4H), 7.44-7.25 (m, 10H), 7.25-7.17 (m, 1H), 5.88 (q, J=6.6 Hz, 1H), 4.12 (s, 1H), 2.82-2.72 (m, 1H), 2.64 (dt, J=l l.l, 2.5 Hz, 1H), 2.58-2.52 (m, 1H), 2.40 (dt, J=l l. l, 2.5 Hz, 1H), 2.20 (dt, J=12.1, 4.6 Hz, 1H), 2.10 (dt, J=12.1, 4.6 Hz, 1H), 1.72-1.57 (m, 2H), 1.53 (d, J=6.5 Hz, 3H). LCMS: Anal. Calcd. for C27H29 03: 415; found: 416 (M+H)+; (S,S)-isomer: HXNMR (400 MHz, CD3OD) δ 7.55-7.48 (m, 2H), 7.45-7.39 (m, 2H), 7.38-7.30 (m, 5H), 7.25-7.13 (m, 4H), 7.08-7.00 (m, 2H), 5.88 (q, J=6.6 Hz, 1H), 4.12 (s, 1H), 2.95-2.85 (m, 1H), 2.68 (dt, J= 11.1, 2.5 Hz, 1H), 2.57-2.52 (m, 1H), 2.42 (dt, J=l 1.1, 2.5 Hz, 1H), 2.25 (dt, J=12.1, 4.6 Hz, lH), 2.12 (dt, J=12.1, 4.6 Hz, 1H), 1.73 (dd, J=13.6, 3.0 Hz, 1H), 1.64 (dd, J=13.6, 3.0 Hz, 1H), 1.40 (d, J=6.6 Hz, 3H). LCMS: Anal. Calcd. for C27H29NO3: 415; found: 416 (M+H)+.
The following esters were prepared in similar fashion:
Figure imgf000199_0001
Figure imgf000200_0001
Chiral SFC Conditions for determining retention time
Condition I
Column: Chiralpak AD-H Column, 4.62x50 mm, 5μιη Solvents: 90% CO2-10% methanol with 0.1%DEA Temp: 35 °C
Pressure: 150 bar
Flow rate: 2.0 mL/min.
UV monitored @ 220 nm
Injection: 1.0 mg/3mL methanol
Condition II
Column: Chiralcel OD-H Column, 4.62x50 mm, 5μιη
Solvents: 90% CO2-10% methanol with 0.1%DEA
Temp: 35 °C
Pressure: 150 bar
Flow rate: 2.0 mL/min.
UV monitored @ 220 nm
Injection: 1.0 mg/mL methanol
Cap 17, Step 2; (R)-2-(4-Hydroxy-4-phenylpiperidin-l-yl)-2-phenylacetic acid: To a solution of (S)- 1 -phenylethyl (R)-2-(4-hydroxy-4-phenylpiperidin-l-yl)- 2-phenylacetate (0.350 g, 0.84 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (1 mL) and the mixture was stirred at room temperature for 2 hours. The volatiles were subsequently removed in vacuo and the residue was purified by reverse-phase preparative HPLC (Primesphere C-18, 20 x 100mm; CH3CN-H2O-0.1% TFA) to give the title compound (as TFA salt) as a white solid (0.230 g, 88%). LCMS: Anal. Calcd. for Ci9H21N03: 311.15; found: 312 (M+H)+. The following carboxylic acids were prepared in optically pure form in a similar fashion: Cap- 17a RT=2.21 (Cond'n II);
1H NMR (500 MHz, DMSO-d6) δ ppm 2.20- 2.35 (m, 2H) 2.34-2.47 (m, 2H) 3.37 (s, 4H) 3.71 (s, 1H) 5.06 (s, 2H) 7.06-7.53 (m, 10H). LCMS: Anal. Calcd. for: C2oH22N204 354.16; Found: 355.38 (M+H)+.
Cap- 17b RT=0.27 (Cond'n III);
LCMS: Anal. Calcd.
Figure imgf000202_0001
234.10; Found: 235.22 (M+H)+.
Cap- 17c RT=0.48 (Cond'n II);
LCMS: Anal. Calcd. for: C13H18N202 234.14; Found: 235.31 (M+H)+.
Cap- 17 d RT=2.21 (Cond'n I);
LCMS: Anal. Calcd.
Figure imgf000202_0002
Found: 296.33 (M+H)+.
LCMS Conditions for determining retention time
Condition I
Column: Phenomenex-Luna 4.6 X 50 mm S10 Start % B=0
Fianl % B=100
Gradient Time=4 min
Flow Rate=4 mL/min
Wavelength=220
Solvent A=10% methanol - 90% H20 - 0.1% TFA Solvent B=90% methanol - 10% H20 - 0.1% TFA
Condition II
Column: Waters-Sunfire 4.6 X 50 mm S5 Start % B=0
Fianl % B=100
Gradient Time=2 min
Flow Rate=4 mL/min
Wavelength=220
Solvent A=10% methanol - 90% H20 - 0.1% TFA
Solvent B=90% methanol - 10% H20 - 0.1% TFA
Condition III
Column: Phenomenex 10μ 3.0 X 50 mm
Start % B=0
Fianl % B=100
Gradient Time=2 min
Flow Rate=4 mL/min
Wavelength=220
Solvent A=10% methanol - 90% H20 - 0.1% TFA
Solvent B=90% methanol - 10% H20 - 0.1% TFA
Figure imgf000204_0001
Step 1; (R,S)-Ethyl 2-(4-pyridyl)-2-bromoacetate: To a solution of ethyl 4- pyridylacetate (1.00 g, 6.05 mmol) in dry THF (150 mL) at 0 °C under argon was added DBU (0.99 mL, 6.66 mmol). The reaction mixture was allowed to warm to room temperature over 30 minutes and then it was cooled to -78 °C. To this mixture was added CBr4 (2.21 g, 6.66 mmol) and stirring was continued at -78 °C for 2 hours. The reaction mixture was then quenched with sat. aq. NH4C1 and the phases were separated. The organic phase was washed (brine), dried (Na2S04), filtered, and concentrated in vacuo. The resulting yellow oil was immediately purified by flash chromatography (SiCV hexane-ethyl acetate, 1 : 1) to provide the title compound (1.40 g, 95%) as a somewhat unstable yellow oil. XH NMR (400 MHz, CDC13) δ 8.62 (dd, J=4.6, 1.8 Hz, 2H), 7.45 (dd, J=4.6, 1.8 Hz, 2H), 5.24 (s, 1H), 4.21-4.29 (m, 2H), 1.28 (t, J=7.1 Hz, 3H). LCMS: Anal. Calcd. for C9H10BrNO2: 242, 244; found: 243, 245 (M+H)+.
Step 2; (R,S)-Ethyl 2-(4-pyridyl)-2-(N,N-dimethylamino)acetate: To a solution of (R,S)-ethyl 2-(4-pyridyl)-2-bromoacetate (1.40 g, 8.48 mmol) in DMF (10 mL) at room temperature was added dimethylamine (2M in THF, 8.5 mL, 17.0 mmol). After completion of the reaction (as judged by thin layer chromatography) the volatiles were removed in vacuo and the residue was purified by flash
chromatography (Biotage, 40+M S1O2 column; 50%- 100% ethyl acetate-hexane) to provide the title compound (0.539 g, 31%) as a light yellow oil. XH NMR (400 MHz, CDCI3) δ 8.58 (d, J=6.0 Hz, 2H), 7.36 (d, J=6.0 Hz, 2H), 4.17 (m, 2H), 3.92 (s, 1H), 2.27 (s, 6H), 1.22 (t, J=7.0 Hz). LCMS: Anal. Calcd. for CiiH16N202: 208; found: 209 (M+H)+. Step 3; (R,S)-2-(4-Pyridyl)-2-(N,N-dimethylamino)acetic acid: To a solution of (R,S)-ethyl 2-(4-pyridyl)-2-(N,N-dimethylamino)acetate (0.200 g, 0.960 mmol) in a mixture of THF-methanol-H20 (1 : 1 : 1, 6 mL) was added powdered LiOH (0.120 g, 4.99 mmol) at room temperature. The solution was stirred for 3 hours and then it was acidified to pH 6 using IN HCl. The aqueous phase was washed with ethyl acetate and then it was lyophilized to give the dihydrochloride of the title compound as a yellow solid (containing LiCl). The product was used as such in subsequent steps. ¾ NMR (400 MHz, DMSO-d6) δ 8.49 (d, J=5.7 Hz, 2H), 7.34 (d, J=5.7 Hz, 2H), 3.56 (s, 1H), 2.21 (s, 6H).
The following examples were prepared in similar fashion using the method described above;
Figure imgf000205_0001
Figure imgf000206_0001
Figure imgf000207_0001
- 37
Figure imgf000207_0002
Step 1; (R,S)-Ethyl 2-(quinolin-3-yl)-2-(N,N-dimethylamino)-acetate: A mixture of ethyl N,N-dimethylaminoacetate (0.462 g, 3.54 mmol), K3P04 (1.90 g, 8.95 mmol), Pd(t-Bu3P)2 (0.090 g, 0.176 mmol) and toluene (10 mL) was degassed with a stream of Ar bubbles for 15 minutes. The reaction mixture was then heated at 100 °C for 12 hours, after which it was cooled to room temperature and poured into H20. The mixture was extracted with ethyl acetate (2x) and the combined organic phases were washed (H20, brine), dried (Na2S04), filtered, and concentrated in vacuo. The residue was purified first by reverse-phase preparative HPLC
(Primesphere C-18, 30 x 100mm; CH3CN-H20-5 mM NH4OAc) and then by flash chromatography (Si02/ hexane-ethyl acetate, 1 : 1) to provide the title compound (0.128 g, 17%) as an orange oil. XH NMR (400 MHz, CDC13) δ 8.90 (d, J=2.0 Hz, IH), 8.32 (d, J=2.0 Hz, IH), 8.03-8.01 (m, 2H), 7.77 (ddd, J=8.3, 6.8, 1.5 Hz, IH), 7.62 (ddd, J=8.3, 6.8, 1.5 Hz, IH), 4.35 (s, IH), 4.13 (m, 2H), 2.22 (s, 6H), 1.15 (t, J=7.0 Hz, 3H). LCMS: Anal. Calcd. for Ci5H18N202: 258; found: 259 (M+H)+. Step 2; (R,S) 2-(Quinolin-3-yl)-2-(N,N-dimethylamino)acetic acid: A mixture of (R,S)-ethyl 2-(quinolin-3-yl)-2-(N,N-dimethylamino)acetate (0.122 g, 0.472 mmol) and 6M HC1 (3 mL) was heated at 100 °C for 12 hours. The solvent was removed in vacuo to provide the dihydrochloride of the title compound (0.169 g, >100%) as a light yellow foam. The unpurified material was used in subsequent steps without further purification. LCMS: Anal. Calcd. for C 3H14 2O2: 230;
found: 231 (M+H)+. -38
Figure imgf000208_0001
Cap-38
Step 1; (R)-((S)-l-phenylethyl) 2-(dimethylamino)-2-(2-fluorophenyl)acetate and (S)-((S)-l-phenylethyl) 2-(dimethylamino)-2-(2-fluorophenyl)acetate: To a mixture of (RS)-2-(dimethylamino)-2-(2-fluorophenyl)acetic acid (2.60 g, 13.19 mmol), DMAP (0.209 g, 1.71 mmol) and (S)-l-phenylethanol (2.09 g, 17.15 mmol) in CH2CI2 (40 mL) was added EDCI (3.29 g, 17.15 mmol) and the mixture was allowed to stir at room temperature for 12 hours. The solvent was then removed in vacuo and the residue partitioned with ethyl acetate-H20. The layers were separated, the aqueous layer was back-extracted with ethyl acetate (2x) and the combined organic phases were washed (H20, brine), dried (Na2S04), filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (Biotage/ 0-50% diethyl ether-hexane). The resulting pure diastereomeric mixture was then separated by reverse-phase preparative HPLC (Primesphere C-18, 30 x 100mm; CH3CN-H20- 0.1% TFA) to give first (S)-l-phenethyl (R)-2-(dimethylamino)-2-(2- fluorophenyl)acetate (0.501 g, 13%) and then (S)-l-phenethyl (S)-2- (dimethylamino)-2-(2-fluorophenyl)-acetate (0.727 g. 18%), both as their TFA salts. (S,R)-isomer: ¾ NMR (400 MHz, CD3OD) δ 7.65-7.70 (m, 1H), 7.55-7.60 (ddd, J=9.4, 8.1, 1.5 Hz, 1H), 7.36-7.41 (m, 2H), 7.28-7.34 (m, 5H), 6.04 (q, J=6.5 Hz, 1H), 5.60 (s, 1H), 2.84 (s, 6H), 1.43 (d, J=6.5 Hz, 3H). LCMS: Anal. Calcd. for Ci8H2oFN02: 301 ; found: 302 (M+H)+; (S,S)-isomer: 'H NMR (400 MHz, CD3OD) δ 7.58-7.63 (m, 1H), 7.18-7.31 (m, 6H), 7.00 (dd, J=8.5, 1.5 Hz, 2H), 6.02 (q, J=6.5 Hz, 1H), 5.60 (s, 1H), 2.88 (s, 6H), 1.54 (d, J=6.5 Hz, 3H). LCMS: Anal. Calcd. for Ci8H2oFN02: 301; found: 302 (M+H)+.
Step 2; (R)-2-(dimethylamino)-2-(2-fluorophenyl)acetic acid: A mixture of (R)-((S)- 1 -phenylethyl) 2-(dimethylamino)-2-(2-fluorophenyl)acetate TFA salt (1.25 g, 3.01 mmol) and 20% Pd(OH)2/C (0.125 g) in ethanol (30 mL) was hydrogenated at room temperature and atmospheric pressure (H2 balloon) for 4 hours. The solution was then purged with Ar, filtered through diatomaceous earth (Celite®), and concentrated in vacuo. This gave the title compound as a colorless solid (0.503 g, 98%). 'H NMR (400 MHz, CD3OD) δ 7.53-7.63 (m, 2H), 7.33-7.38 (m, 2H), 5.36 (s, 1H), 2.86 (s, 6H). LCMS: Anal. Calcd. for Ci0Hi2FNO2: 197; found: 198 (M+H)+.
The S-isomer could be obtained from (S)-((S)-1 -phenylethyl) 2- (dimethylamino)-2-(2-fluorophenyl)acetate TFA salt in similar fashion. -39
Figure imgf000209_0001
cap-39
A mixture of (R)-(2-chlorophenyl)glycine (0.300 g, 1.62 mmol),
formaldehyde (35% aqueous solution, 0.80 mL, 3.23 mmol) and 20% Pd(OH)2/C (0.050 g) was hydrogenated at room temperature and atmospheric pressure (H2 balloon) for 4 hours. The solution was then purged with Ar, filtered through diatomaceous earth (Celite®) and concentrated in vacuo. The residue was purified by reverse-phase preparative HPLC (Primesphere C-18, 30 x 100mm; CH3CN-H20- 0.1% TFA) to give theTFA salt of the title compound (R)-2-(dimethylamino)-2-(2- chlorophenyl)acetic acid as a colorless oil (0.290 g, 55%). *H NMR (400 MHz, CD3OD) δ 7.59-7.65 (m, 2H), 7.45-7.53 (m, 2H), 5.40 (s, 1H), 2.87 (s, 6H). LCMS: Anal. Calcd. for Ci0H12ClNO2: 213; found: 214 (M+H)+.
Cap-40
Figure imgf000210_0001
cap-40
To an ice-cold solution of (R)-(2-chlorophenyl)glycine (1.00 g, 5.38 mmol) and NaOH (0.862 g, 21.6 mmol) in H2O (5.5 mL) was added methyl chloroformate (1.00 mL, 13.5 mmol) dropwise. The mixture was allowed to stir at 0 °C for 1 hour and then it was acidified by the addition of cone. HC1 (2.5 mL). The mixture was extracted with ethyl acetate (2x) and the combined organic phase was washed (H2O, brine), dried (Na2S04), filtered, and concentrated in vacuo to give the title compound (R)-2-(methoxycarbonylamino)-2-(2-chlorophenyl)acetic acid as a yellow-orange foam (1.31 g, 96%). 'H NMR (400 MHz, CD3OD) δ 7.39 - 7.43 (m, 2H), 7.29 - 7.31 (m, 2H), 5.69 (s, 1H), 3.65 (s, 3H). LCMS: Anal. Calcd. for Ci0H10ClNO4: 243; found: 244 (M+H)+. -41
Figure imgf000210_0002
cap-41
To a suspension of 2-(2-(chloromethyl)phenyl)acetic acid (2.00 g, 10.8 mmol) in THF (20 mL) was added morpholine (1.89 g, 21.7 mmol) and the solution was stirred at room temperature for 3 hours. The reaction mixture was then diluted with ethyl acetate and extracted with ¾0 (2x). The aqueous phase was lyophilized and the residue was purified by silica gel chromatography (Biotage/ 0-10% methanol- CH2CI2) to give the title compound 2-(2-(Morpholinomethyl)phenyl)acetic acid as a colorless solid (2.22 g, 87%). XH NMR (400 MHz, CD3OD) δ 7.37-7.44 (m, 3H), 7.29-7.33 (m, 1H), 4.24 (s, 2H), 3.83 (br s, 4H), 3.68 (s, 2H), 3.14 (br s, 4H). LCMS: Anal. Calcd. for C13H17NO3: 235; found: 236 (M+H)+.
The following examples were similarly prepared using the method described for Cap-41 :
Figure imgf000211_0002
Cap-45a
Figure imgf000211_0001
Cap-45a HMDS (1.85 niL, 8.77 mmol) was added to a suspension of (R)-2-amino-2- phenylacetic acid p-toluenesulfonate (2.83 g, 8.77 mmol) in (¾(¾ (10 mL) and the mixture was stirred at room temperature for 30 minutes. Methyl isocyanate (0.5 g, 8.77 mmol) was added in one portion stirring continued for 30 minutes. The reaction was quenched by addition of H20 (5 mL) and the resulting precipitate was filtered, washed with H20 and n-hexanes, and dried under vacuum. (R)-2-(3-methylureido)- 2-phenylacetic acid (1.5 g; 82 %).was recovered as a white solid and it was used without further purification. XH NMR (500 MHz, DMSO-d6) δ ppm 2.54 (d, J=4.88 Hz, 3H) 5.17 (d, J=7.93 Hz, 1H) 5.95 (q, J=4.48 Hz, 1H) 6.66 (d, J=7.93 Hz, 1H) 7.26-7.38 (m, 5H) 12.67 (s, 1H). LCMS: Anal. Calcd. for C10H12 2O3 208.08 found 209.121 (M+H)+; HPLC Phenomenex C- 18 3.0 x 46 mm, 0 to 100% B over 2 minutes, 1 minute hold time, A=90% water, 10% methanol, 0.1% TFA, B=10% water, 90% methanol, 0.1% TFA, RT=1.38 min, 90% homogeneity index. -46
Figure imgf000212_0001
cap-46
The desired product was prepared according to the method described for Cap- 45a. 'H NMR (500 MHz, DMSO-d6) δ ppm 0.96 (t, J=7.17 Hz, 3H) 2.94-3.05 (m, 2H) 5.17 (d, J=7.93 Hz, 1H) 6.05 (t, J=5.19 Hz, 1H) 6.60 (d, J=7.63 Hz, 1H) 7.26- 7.38 (m, 5H) 12.68 (s, 1H). LCMS: Anal. Calcd. for CiiH14N203 222.10 found 223.15 (M+H)+. HPLC XTERRA C-18 3.0 x 506 mm, 0 to 100% B over 2 minutes, 1 minute hold time, A=90% water, 10% methanol, 0.2% H3PO4, B=10% water, 90% methanol, 0.2% H3PO4, RT=0.87 min, 90% homogeneity index.
Figure imgf000213_0001
B Cap-47
Step 1; (R)-tert-butyl 2-(3,3-dimethylureido)-2-phenylacetate: To a stirred solution of (R)-tert-butyl-2-amino-2-phenylacetate (1.0 g, 4.10 mmol) and Hunig's base (1.79 mL, 10.25 mmol) in DMF (40 mL) was added dimethylcarbamoyl chloride (0.38 mL, 4.18 mmol) dropwise over 10 minutes. After stirring at room temperature for 3 hours, the reaction was concentrated under reduced pressure and the resulting residue was dissolved in ethyl acetate. The organic layer was washed with H20, IN aq. HC1 and brine, dried (MgS04), filtered and concentrated under reduced pressure. (R)-tert-butyl 2-(3,3-dimethylureido)-2-phenylacetate was obtained as a white solid ( 0.86 g; 75%) and used without further purification. XH NMR (500 MHz, DMSO-d6) δ ppm 1.33 (s, 9H) 2.82 (s, 6H) 5.17 (d, J=7.63 Hz, IH) 6.55 (d, J=7.32 Hz, IH) 7.24-7.41 (m, 5H). LCMS: Anal. Calcd. for Ci5H22N203 278.16 found 279.23 (M+H)+; HPLC Phenomenex LUNA C-18 4.6 x 50 mm, 0 to 100% B over 4 minutes, 1 minute hold time, A=90%> water, 10%> methanol, 0.1%> TFA, B=10% water, 90% methanol, 0.1% TFA, RT=2.26 min, 97% homogeneity index.
Step 2; (R)-2-(3,3-dimethylureido)-2-phenylacetic acid: To a stirred solution of ((R)-tert-butyl 2-(3,3-dimethylureido)-2-phenylacetate ( 0.86 g, 3.10 mmol) in CH2C12 (250 mL) was added TFA (15 mL) dropwise and the resulting solution was stirred at rt for 3 hours. The desired compound was then precipitated out of solution with a mixture of EtOAC:Hexanes (5:20), filtered off and dried under reduced pressure. (R)-2-(3,3-dimethylureido)-2-phenylacetic acid was isolated as a white solid (0.59g, 86%) and used without further purification. ¾ NMR (500 MHz, DMSO-d6) δ ppm 2.82 (s, 6H) 5.22 (d, J=7.32 Hz, IH) 6.58 (d, J=7.32 Hz, IH) 7.28 (t, J=7.17 Hz, IH) 7.33 (t, J=7.32 Hz, 2H) 7.38-7.43 (m, 2H) 12.65 (s, IH). LCMS: Anal. Calcd. for CnH14N203: 222.24; found: 223.21 (M+H)+. HPLC XTERRA C- 18 3.0 x 50 mm, 0 to 100% B over 2 minutes, 1 minute hold time, A=90% water, 10% methanol, 0.2% H3P04, B=10% water, 90% methanol, 0.2% H3P04, RT=0.75 min, 93% homogeneity index. -48
Figure imgf000214_0001
B Cap-48
Step 1; (R)-tert-butyl 2-(3-cyclopentylureido)-2-phenylacetate: To a stirred solution of (R)-2-amino-2-phenylacetic acid hydrochloride (1.0 g, 4.10 mmol) and Hunig's base (1.0 mL, 6.15 mmol) in DMF (15 mL) was added cyclopentyl isocyanate (0.46 mL, 4.10 mmol) dropwise and over 10 minutes. After stirring at room temperature for 3 hours, the reaction was concentrated under reduced pressure and the resulting residue was traken up in ethyl acetate. The organic layer was washed with H20 and brine, dried (MgS04), filtered, and concentrated under reduced pressure. (R)-tert-butyl 2-(3-cyclopentylureido)-2-phenylacetate was obtained as an opaque oil (1.32 g; 100 %) and used without further purification. XH NMR (500
MHz, CD3CI-D) δ ppm 1.50-1.57 (m, 2H) 1.58-1.66 (m, 2H) 1.87-1.97 (m, 2H) 3.89- 3.98 (m, 1H) 5.37 (s, 1H) 7.26-7.38 (m, 5H). LCMS: Anal. Calcd. for Ci8H26 203 318.19 found 319.21 (M+H)+; HPLC XTERRA C-18 3.0 x 50 mm, 0 to 100% B over 4 minutes, 1 minute hold time, A=90% water, 10% methanol, 0.1% TFA, B=10% water, 90% methanol, 0.1% TFA, RT=2.82 min, 96% homogeneity index.
Step 2; (R)-2-(3-cyclopentylureido)-2-phenylacetic acid: To a stirred solution of (R)-tert-butyl 2-(3-cyclopentylureido)-2-phenylacetate (1.31 g, 4.10 mmol) in CH2CI2 (25 mL) was added TFA (4 mL) and trietheylsilane (1.64 mL; 10.3 mmol) dropwise, and the resulting solution was stirred at room temperature for 6 hours. The volatile components were removed under reduced pressure and the crude product was recrystallized in ethyl acetate/pentanes to yield (R)-2-(3-cyclopentylureido)-2- phenylacetic acid as a white solid (0.69 g, 64%). XH NMR (500 MHz, DMSO-d6) δ ppm 1.17-1.35 (m, 2H) 1.42-1.52 (m, 2H) 1.53-1.64 (m, 2H) 1.67-1.80 (m, 2H) 3.75- 3.89 (m, IH) 5.17 (d, J=7.93 Hz, IH) 6.12 (d, J=7.32 Hz, IH) 6.48 (d, J=7.93 Hz, IH) 7.24-7.40 (m, 5H) 12.73 (s, IH). LCMS: Anal. Calcd. for Ci4H18 203: 262.31 ; found: 263.15 (M+H)+. HPLC XTERRA C- 18 3.0 x 50 mm, 0 to 100% B over 2 minutes, 1 minute hold time, A=90% water, 10% methanol, 0.2% H3P04, B=10% water, 90% methanol, 0.2% H3P04, RT=1.24 min, 100% homogeneity index. -49
Figure imgf000215_0001
cap-49
To a stirred solution of 2-(benzylamino)acetic acid (2.0 g, 12.1 mmol) in formic acid (91 mL) was added formaldehyde (6.94 mL, 93.2 mmol). After five hours at 70 °C, the reaction mixture was concentrated under reduced pressure to 20 mL and a white solid precipitated. Following filtration, the mother liquors were collected and further concentrated under reduced pressure providing the crude product. Purification by reverse-phase preparative HPLC (Xterra 30 X 100 mm, detection at 220 nm, flow rate 35 mL/min, 0 to 35% B over 8 min; A= 90% water, 10 % methanol, 0.1% TFA, B=10% water, 90 % methanol, 0.1% TFA) provided the title compound 2-(benzyl(methyl)-amino)acetic acid as its TFA salt (723 mg, 33%) as a colorless wax. ¾ NMR (300 MHz, DMSO-d6) δ ppm 2.75 (s, 3H) 4.04 (s, 2H) 4.34 (s, 2H) 7.29-7.68 (m, 5H). LCMS: Anal. Calcd. for: Ci0H13NO2 179.09; Found: 180.20 (M+H)+. -50
Figure imgf000215_0002
Cap-50 To a stirred solution of 3-methyl-2-(methylamino)butanoic acid (0.50 g, 3.81 mmol) in water ( 30 mL) was added K2CO3 (2.63 g, 19.1 mmol) and benzyl chloride (1.32 g, 11.4 mmol). The reaction mixture was stirred at ambient temperature for 18 hours. The reaction mixture was extracted with ethyl acetate (30 mL x 2) and the aqueous layer was concentrated under reduced pressure providing the crude product which was purified by reverse-phase preparative HPLC (Xterra 30 x 100mm, detection at 220 nm, flow rate 40 mL/min, 20 to 80% B over 6 min; A= 90% water, 10 % methanol, 0.1% TFA, B=10% water, 90 % methanol, 0.1% TFA) to provide 2- (benzyl(methyl)amino)-3-methylbutanoic acid, TFA salt (126 mg, 19%) as a colorless wax. ¾ NMR (500 MHz, DMSO-d6) δ ppm 0.98 (d, 3H) 1.07 (d, 3H) 2.33-2.48 (m, 1H) 2.54-2.78 (m, 3H) 3.69 (s, 1H) 4.24 (s, 2H) 7.29-7.65 (m, 5H). LCMS: Anal. Calcd. for: Ci3H19N02 221.14; Found: 222.28 (M+H)+.
Figure imgf000216_0001
Na2C03 (1.83g, 17.2 mmol) was added to NaOH (33 mL of 1M/H20, 33 mmol) solution of L-valine (3.9 g, 33.29 mmol) and the resulting solution was cooled with ice-water bath. Methyl chloro formate (2.8 mL, 36.1 mmol) was added dropwise over 15 min, the cooling bath was removed and the reaction mixture was stirred at ambient temperature for 3.25 hr. The reaction mixture was washed with ether (50 mL, 3x), and the aqueous phase was cooled with ice-water bath and acidified with concentrated HCl to a pH region of 1-2, and extracted with CH2CI2 (50 mL, 3x). The organic phase was dried (MgS04) and evaporated in vacuo to afford Cap-51 as a white solid (6 g). XH NMR for the dominant rotamer (DMSO-d6, ^2.5 ppm, 500
MHz): 12.54 (s, 1H), 7.33 (d, J=8.6, 1H), 3.84 (dd, J=8.4, 6.0, 1H), 3.54 (s, 3H), 2.03 (m, 1H), 0.87 (m, 6H). HRMS: Anal. Calcd. for [M+H]+ C7H14N04: 176.0923; found 176.0922. Cap 51 (alternate route)
Figure imgf000217_0001
DIEA (137.5 mL, 0.766 mol) was added to a suspension of (5)-tert-butyl 2- amino-3-methylbutanoate hydrochloride (75.0 g, 0.357 mol) in THF (900 mL), and the mixture was cooled to 0 °C (ice/water bath). Methyl chloroformate (29.0 mL, 0.375 mol) was added dropwise over 45 min, the cooling bath was removed and the heterogeneous mixture was stirred at ambient temperature for 3 h. The solvent was removed under diminished pressure and the residue partitioned between EtOAc and water (1 L each). The organic layer was washed with H20 (1 L) and brine (1 L), dried (MgS04), filtered and concentrated under diminished pressure. The crude material was passed through a plug of silica gel (1 kg), eluting with hexanes (4 L) and 15:85 EtOAc/hexanes (4 L) to afford (S)-tert-butyl 2-(methoxycarbonylamino)-3- methylbutanoate as a clear oil (82.0 g, 99% yield). XH-NMR (500 MHz, DMSO-i¾, δ = 2.5 ppm) 7.34 (d, J= 8.6, 1 H), 3.77 (dd, J= 8.6, 6.1, 1 H), 3.53 (s, 3 H), 1.94 - 2.05 (m, 1 H), 1.39 (s, 9 H), 0.83 - 0.92 (m, 6 H). 13C-NMR (126 MHz, DMSO-i¾, δ = 39.2 ppm) 170.92, 156.84, 80.38, 60.00, 51.34, 29.76, 27.62, 18.92, 17.95. LC/MS:
[M+Na]+ 254.17.
Trifluoroacetic acid (343 mL, 4.62 mol) and EtsSiH (142 mL, 0.887 mol) were added sequentially to a solution of (S)-tert-butyl 2-(methoxycarbonylamino)-3- methylbutanoate (82.0 g, 0.355 mol) in (¾(¾ (675 mL), and the mixture was stirred at ambient temperature for 4 h. The volatile component was removed under diminished pressure and the resultant oil triturated with petroleum ether (600 mL) to afford a white solid, which was filtered and washed with hexanes (500 mL) and petroleum ether (500 mL). Recrystallization from EtO Ac/petroleum ether afforded Cap-51 as white flaky crystals (54.8 g, 88 % yield). MP = 108.5-109.5 °C. XH NMR (500 MHz, DMSO-i¾, δ = 2.5 ppm) 12.52 (s, 1 H), 7.31 (d, J= 8.6, 1 H), 3.83 (dd, J = 8.6, 6.1, 1 H), 3.53 (s, 3 H), 1.94 - 2.07 (m, 1 H), 0.86 (dd, J= 8.9, 7.0, 6 H). 13C MR (126 MHz, DMSO-i¾, 8 = 39.2 ppm) 173.30, 156.94, 59.48, 51.37, 29.52, 19.15, 17.98. LC/MS: [M+H] = 176.11. Anal. Calcd. for C7H13N04: C, 47.99; H, 7.48; N, 7.99. Found: C, 48.17; H, 7.55; N, 7.99. Optical Rotation: [a] D = -4.16 (12.02 mg/mL; MeOH). Optical purity: >99.5 % ee. Note: the optical purity assessment was made on the methyl ester derivative of Cap-51, which was prepared under a standard TMSCHN2 (benzene/MeOH) esterification protocol. HPLC analytical conditions: column,
ChiralPak AD-H (4.6 x 250mm, 5μιη); solvent, 95% heptane / 5% IPA (isocratic); flow rate, 1 mL/min; temperature, 35 °C; UV monitored at 205 nm.
[Note: Cap 51 could also be purchased from Flamm.] Cap-5 Cap-12)
Figure imgf000218_0001
Cap-52 was synthesized from L-alanine according to the procedure described for the synthesis of Cap-51. For characterization purposes, a portion of the crude material was purified by a reverse phase HPLC (H20/methanol/TFA) to afford Cap-
52 as a colorless viscous oil. XH NMR (DMSO-d6, £=2.5 ppm, 500 MHz): 12.49 (br s, 1H), 7.43 (d, J=7.3, 0.88H), 7.09 (app br s, 0.12H), 3.97 (m, 1H), 3.53 (s, 3H), 1.25 (d, J=7.3, 3H).
Cap-53 to -64 were prepared from appropriate starting materials according to the procedure described for the synthesis of Cap-51, with noted modifications if any.
Figure imgf000218_0002
Figure imgf000219_0001
[M+H]+ C7H14N04 : 176.09; found
176.06.
Cap-58 XH NMR (DMSO-d6, δ = 2.5 ppm, 500
MHz): δ 12.63 (br s, 1H), 7.35 (s, lH), 7.31 ( d, J = 8.2, 1H), 6.92 (s, 1H), 4.33- 4.29 (m, 1H), 3.54 (s, 3H), 2.54(dd, J = 15.5, 5.4, 1H), 2.43 (dd, J = 15.6, 8.0,
Figure imgf000220_0001
1H). RT = 0.16 min (Cond. 2); LC/MS:
Anal. Calcd. for [M+H]+
Figure imgf000220_0002
: 191.07; found 191.14.
Cap- 59a: XH NMR (DMSO-d6, δ = 2.5 ppm, 400
(R) MHz): δ 12.49 (br s, 1H), 7.40 (d, J = 7.3,
Cap- 59b: 0.89H), 7.04 (br s, 0.1 1H), 4.00-3.95 (m, (S) 3H), 1.24 (d, J = 7.3, 3H), 1.15 (t, J =
Figure imgf000220_0003
7.2, 3H). HRMS: Anal. Calcd. for
[M+H]+ C6Hi2N04: 162.0766; found 162.0771.
Cap- 60 The crude material was purified with a reverse phase HPLC (H20/MeOH/TFA) to afford a colorless viscous oil that crystallized to a white solid upon exposure to high vacuum. XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): δ
Figure imgf000220_0004
12.38 (br s, 1H), 7.74 (s, 0.82H), 7.48 (s,
0.18H), 3.54/3.51 (two s, 3H), 1.30 (m, 2H), 0.98 (m, 2H). HRMS: Anal. Calcd. for [M+H]+ C6H10NO4: 160.0610; found 160.0604.
Cap- 61 XH NMR (DMSO-d6, δ = 2.5 ppm, 400
MHz): δ 12.27 (br s, 1H), 7.40 (br s, 1H),
Figure imgf000220_0005
3.50 (s, 3H), 1.32 (s, 6H). HRMS: Anal. Calcd. for [M+H]+ C6H12N04: 162.0766;
found 162.0765.
Cap- 62 XH NMR (DMSO-d6, δ = 2.5 ppm, 400
MHz): δ 12.74 (br s, 1H), 4.21 (d, J = 10.3, 0.6H), 4.05 (d, J = 10.0, 0.4H),
1 ° 3.62/3.60 (two singlets, 3H), 3.0 (s, 3H), O ^\ 2.14-2.05 (m, 1H), 0.95 (d, J = 6.3, 3H),
0.81 (d, J = 6.6, 3H). LC/MS: Anal. Calcd. for [M-H] C8H14N04: 188.09; found 188.05.
Cap- 63 [Note: the reaction was allowed to run for longer than what was noted for the general procedure.] XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 12.21 (br s, 1H), 7.42 (br s, 1H), 3.50 (s, 3H), 2.02-1.85 (m, 4H), 1.66-1.58 (m, 4H). LC/MS: Anal. Calcd. for [M+H]+ C8H14N04: 188.09; found 188.19.
Cap- 64 [Note: the reaction was allowed to run for longer than what was noted for the general procedure.] XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 12.35 (br s, 1H), 7.77 (s, 0.82H), 7.56/7.52 (overlapping br s, 0.18H), 3.50 (s, 3H), 2.47-2.40 (m, 2H), 2.14-2.07 (m, 2H), 1.93-1.82 (m, 2H).
Cap- 65
Figure imgf000222_0001
Methyl chloroformate (0.65 niL, 8.39 mmol) was added dropwise over 5 min to a cooled (ice-water) mixture of Na2C03 (0.449 g, 4.23 mmol), NaOH (8.2 mL of IM/H2O, 8.2 mmol) and (5)-2-amino-3-hydroxy-3-methylbutanoic acid (1.04 g, 7.81 mmol). The reaction mixture was stirred for 45 min, and then the cooling bath was removed and stirring was continued for an additional 3.75 hr. The reaction mixture was washed with CH2CI2, and the aqueous phase was cooled with ice-water bath and acidified with concentrated HC1 to a pH region of 1-2. The volatile component was removed in vacuo and the residue was taken up in a 2: 1 mixture of MeOH/CH^C^
(15 mL) and filtered, and the filterate was rotervaped to afford Cap-65 as a white semi-viscous foam (1.236 g). XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): δ 6.94 (d, J = 8.5, 0.9 H), 6.53 (br s, 0.1H), 3.89 (d, J = 8.8, 1H), 2.94 (s, 3H), 1.15 (s, 3H), 1.13 (s, 3H).
Cap-66 and -67 were prepared from appropriate commercially available starting materials by employing the procedure described for the synthesis of Cap-65.
Ca - 66
Figure imgf000222_0002
XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): δ 12.58 (br s, 1H), 7.07 (d, J = 8.3, 0.13H), 6.81 (d, J = 8.8, 0.67H), 4.10-4.02 (m, 1.15H), 3.91 (dd, J = 9.1, 3.5, 0.85H), 3.56 (s, 3H), 1.09 (d, J = 6.2, 3H). [Note: only the dominant signals of NH were noted].
Cap- 67
Figure imgf000223_0001
XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 12.51 (br s, 1H), 7.25 (d, J = 8.4, 0.75H), 7.12 (br d, J = 0.4, 0.05H), 6.86 (br s, 0.08H), 3.95-3.85 (m, 2H), 3.54 (s, 3H), 1.08 (d, J = 6.3, 3H). [Note: only the dominant signals of NH were noted].
Cap- 68
H 0
/Ογ Ν^ΟΗ
o
Methyl chloroformate (0.38 ml, 4.9 mmol) was added drop-wise to a mixture of IN NaOH (aq) (9.0 ml, 9.0 mmol), 1M NaHC03 (aq) (9.0 ml, 9.0 mol), L-aspartic acid β-benzyl ester (1.0 g, 4.5 mmol) and Dioxane (9 ml). The reaction mixture was stirred at ambient conditions for 3 hr, and then washed with Ethyl acetate (50 ml, 3x). The aqueous layer was acidified with 12N HC1 to a pH ~ 1-2, and extracted with ethyl acetate (3 x 50 ml). The combined organic layers were washed with brine, dried (Na2S04), filtered, and concentrated in vacuo to afford Cap-68 as a light yellow oil (1.37g; mass is above theoretical yield, and the product was used without further purification). ¾ NMR (DMSO-d6, δ = 2.5 ppm, 500 MHz): δ 12.88 (br s, 1H), 7.55 (d, J = 8.5, 1H), 7.40-7.32 (m, 5H), 5.13 (d, J = 12.8, 1H), 5.10 (d, J = 12.9, 1H), 4.42-4.38 (m, 1H), 3.55 (s, 3H), 2.87 (dd, J = 16.2, 5.5, 1H), 2.71 (dd, J =16.2, 8.3, 1H). LC (Cond. 2): RT = 1.90 min; LC/MS: Anal. Calcd. For [M+H]+ Ο3Η16Ν06: 282.10; found 282.12.
Cap-69a and -69b
Cap-69a: (R)-enantiomer
^N ^^\OH Cap-69b: (S)-enantiomer
NaCNBH3 (2.416 g, 36.5 mmol) was added in batches to a chilled (-15 °C) water (17 mL)/MeOH (10 mL) solution of alanine (1.338 g, 15.0 mmol). A few minutes later acetaldehyde (4.0 mL, 71.3 mmol) was added drop-wise over 4 min, the cooling bath was removed, and the reaction mixture was stirred at ambient condition for 6 hr. An additional acetaldehyde (4.0 mL) was added and the reaction was stirred for 2 hr. Concentrated HC1 was added slowly to the reaction mixture until the pH reached ~ 1.5, and the resulting mixture was heated for 1 hr at 40 °C. Most of the volatile component was removed in vacuo and the residue was purified with a Dowex® 50WX8-100 ion-exchange resin (column was washed with water, and the compound was eluted with dilute NH4OH, prepared by mixing 18 ml of NH4OH and 282 ml of water) to afford Cap-69 (2.0 g) as an off-white soft hygroscopic solid. XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): δ 3.44 (q, J = 7.1, 1H), 2.99-2.90 (m, 2H), 2.89-2.80 (m, 2H), 1.23 (d, J = 7.1, 3H), 1.13 (t, J = 7.3, 6H).
Cap-70 to -74x were prepared according to the procedure described for the synthesis of Cap-69 by employing appropriate starting materials.
Figure imgf000224_0001
[M+H]+ C9H2oN02: 174.15; found
174.15.
Cap- 73 XH NMR (DMSO-d6, δ = 2.5 ppm, 500
^ o MHz): δ 9.5 (br s, IH), 3.77 (dd, J =
10.8, 4.1, IH), 3.69-3.61 ( m, 2H), 3.26
S) (s, 3H), 2.99-2.88 (m, 4H), 1.13 (t, J =
1
7.2, 6H).
Cap- 74 XH NMR (DMSO-d6, δ = 2.5 ppm, 500
MHz): δ 7.54 (s, IH), 6.89 (s, IH), 3.81 (t, J = 6.6, k, lH), 2.82-2.71 (m, 4H), 2.63 (dd, J = 15.6, 7.0, IH), 2.36 (dd, J = 15.4, 6.3, IH), 1.09 (t, J = 7.2, 6H).
NH2
RT = 0.125 minutes (Cond. 2); LC/MS: Anal. Calcd. for [M+H]+ ϋ8Η17Ν203 : 189.12; found 189.13.
Cap-74x
LC/MS: Anal. Calcd. for [M+H] + CioH22N02 : 188.17; found 188.21 -75
Figure imgf000225_0001
Cap-75, step a
Figure imgf000226_0001
NaBH3CN (1.6 g, 25.5 mmol) was added to a cooled (ice/water bath) water (25 miymethanol (15 ml) solution of H-D-Ser-OBzl HC1 (2.0 g, 8.6 mmol).
Acetaldehyde (1.5 ml, 12.5 mmol) was added drop-wise over 5 min, the cooling bath was removed, and the reaction mixture was stirred at ambient condition for 2 hr. The reaction was carefully quenched with 12N HC1 and concentrated in vacuo. The residue was dissolved in water and purified with a reverse phase HPLC
(MeOH/H20/TFA) to afford the TFA salt of (R)-benzyl 2-(diethylamino)-3- hydroxypropanoate as a colorless viscous oil (1.9g). lH NMR (DMSO-d6, δ = 2.5 ppm, 500 MHz): δ 9.73 (br s, 1H), 7.52-7.36 (m, 5H), 5.32 (d, J = 12.2, 1H), 5.27 (d, J = 12.5, 1H), 4.54-4.32 (m, 1H), 4.05-3.97 (m, 2H), 3.43-3.21 (m, 4H), 1.23 (t, J = 7.2, 6H). LC/MS (Cond. 2): RT = 1.38 min; LC/MS: Anal. Calcd. for [M+H]+ C14H22NO3: 252.16; found 252.19.
Cap-75
NaH (0.0727 g, 1.82 mmol, 60%) was added to a cooled (ice-water) THF (3.0 mL) solution of the TFA salt (R)-benzyl 2-(diethylamino)-3-hydroxypropanoate (0.3019 g, 0.8264 mmol) prepared above, and the mixture was stirred for 15 min. Methyl iodide (56 μΐ,, 0.90 mmol) was added and stirring was continued for 18 hr while allowing the bath to thaw to ambient condition. The reaction was quenched with water and loaded onto a MeOH pre-conditioned MCX (6 g) cartridge, and washed with methanol followed by compound elution with 2N NH^/Methanol.
Removal of the volatile component in vacuo afforded Cap-15, contaminated with (R)-2-(diethylamino)-3-hydroxypropanoic acid, as a yellow semi-solid (100 mg). The product was used as is without further purification.
Cap-76
Figure imgf000227_0001
NaCNBH3 (1.60 g, 24.2 mmol) was added in batches to a chilled (-15 °C) water/MeOH (12 mL each) solution of (5)-4-amino-2-(tert-butoxycarbonylamino) butanoic acid (2.17 g, 9.94 mmol). A few minutes later acetaldehyde (2.7 mL, 48.1 mmol) was added drop-wise over 2 min, the cooling bath was removed, and the reaction mixture was stirred at ambient condition for 3.5 hr. An additional acetaldehyde (2.7 mL, 48.1 mmol) was added and the reaction was stirred for 20.5 hr. Most of the MeOH component was removed in vacuo, and the remaining mixture was treated with concentrated HC1 until its pH reached ~ 1.0 and then heated for 2 hr at 40 °C. The volatile component was removed in vacuo, and the residue was treated with 4 M HCl/dioxane (20 mL) and stirred at ambient condition for 7.5 hr. The volatile component was removed in vacuo and the residue was purified with Dowex ® 50WX8-100 ion-exchange resin (column was washed with water and the compound was eluted with dilute NH4OH, prepared from 18 ml of NH4OH and 282 ml of water) to afford intermediate (S)-2-amino-4-(diethylamino)butanoic acid as an off-white solid (1.73 g).
Methyl chloroformate (0.36 mL, 4.65 mmol) was added drop-wise over 1 1 min to a cooled (ice-water) mixture of a2C03 (0.243 g, 2.29 mmol), NaOH (4.6 mL of IM/H2O, 4.6 mmol) and the above product (802.4 mg). The reaction mixture was stirred for 55 min, and then the cooling bath was removed and stirring was continued for an additional 5.25 hr. The reaction mixture was diluted with equal volume of water and washed with CH2CI2 (30 mL, 2x), and the aqueous phase was cooled with ice-water bath and acidified with concentrated HC1 to a pH region of 2. The volatile component was then removed in vacuo and the crude material was free-based with MCX resin (6.0g; column was washed with water, and sample was eluted with 2.0 M NH3/MeOH) to afford impure Cap-76 as an off-white solid (704 mg). XH NMR (MeOH-d4, δ = 3.29 ppm, 400 MHz): δ 3.99 (dd, J = 7.5, 4.7, 1H), 3.62 (s, 3H), 3.25-3.06 (m, 6H), 2.18-2.09 (m, 1H), 2.04-1.96 (m, 1H), 1.28 (t, J = 7.3,
LC/MS: Anal. Calcd. for [M+H]+ Ci0H21 2O4: 233.15; found 233.24. -77a and -77b
Cap-77a: enantiomer-1
Cap-77b: enantiomer-2
Figure imgf000228_0001
The synthesis of Cap-11 was conducted according to the procedure described for Cap-1 by using 7-azabicyclo[2.2.1]heptane for the SN2 displacement step, and by effecting the enantiomeric separation of the intermediate benzyl 2-(7- azabicyclo[2.2.1]heptan-7-yl)-2-phenylacetate using the following condition: the intermediate (303.7 mg) was dissolved in ethanol, and the resulting solution was injected on a chiral HPLC column (Chiracel AD-H column, 30 x 250 mm, 5 um) eluting with 90% CO2-10% EtOH at 70 mL/min, and a temperature of 35 °C to provide 124.5 mg of enantiomer-1 and 133.8 mg of enantiomer-2. These benzyl esters were hydrogenolysed according to the preparation of Cap-1 to provide Cap-11:
¾ NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): δ 7.55 (m, 2H), 7.38-7.30 (m, 3H), 4.16 (s, 1H), 3.54 (app br s, 2H), 2.08-1.88 (m, 4 H), 1.57-1.46 (m, 4H). LC (Cond. 1): RT = 0.67 min; LC/MS: Anal. Calcd. for [M+H]+ Ci4H18N02: 232.13; found 232.18. HRMS: Anal. Calcd. for [M+H]+ CMH18N02: 232.1338; found 232.1340.
Ca -78
Figure imgf000228_0002
NaCNBH3 (0.5828 g, 9.27 mmol) was added to a mixture of the HC1 salt of (R)-2-(ethylamino)-2-phenylacetic acid (an intermediate in the synthesis of Cap-3; 0.9923 mg, 4.60 mmol) and (l-ethoxycyclopropoxy)trimethylsilane (1.640 g, 9.40 mmol) in MeOH (10 mL), and the semi-heterogeneous mixture was heated at 50 °C
- 221 - with an oil bath for 20 hr. More (l-ethoxycyclopropoxy)trimethylsilane (150 mg, 0.86 mmol) and NaCNB¾ (52 mg, 0.827 mmol) were added and the reaction mixture was heated for an additional 3.5 hr. It was then allowed to cool to ambient temperature and acidified to a ~ pH region of 2 with concentrated HCl, and the mixture was filtered and the filtrate was rotervaped. The resulting crude material was taken up in -PrOH (6 mL) and heated to effect dissolution, and the non-dissolved part was filtered off and the filtrate concentrated in vacuo. About 1/3 of the resultant crude material was purified with a reverse phase HPLC (E^O/MeOH/TFA) to afford the TFA salt of Cap-1% as a colorless viscous oil (353 mg). XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz; after D20 exchange): δ 7.56-7.49 (m, 5H), 5.35 (S, 1H), 3.35 (m, 1H), 3.06 (app br s, 1H), 2.66 (m, 1H), 1.26 (t, J = 7.3, 3H), 0.92 (m, 1H), 0.83-0.44 (m, 3H). LC (Cond. 1): RT = 0.64 mm; LC/ S: Anal. Calcd. for [M+H]+
Ci3H18N02: 220.13; found 220.21. HRMS: Anal. Calcd. for [M+H]+ Ci3H18N02: 220.1338; found 220.1343.
Ca -79
Figure imgf000229_0001
Ozone was bubbled through a cooled (-78 °C) CH2CI2 (5.0 mL) solution Cap- 55 (369 mg, 2.13 mmol) for about 50 min until the reaction mixture attained a tint of blue color. Me2S (10 pipet drops) was added, and the reaction mixture was stirred for 35 min. The -78 °C bath was replaced with a -10 °C bath and stirring continued for an additional 30 min, and then the volatile component was removed in vacuo to afford a colorless viscous oil.
NaBH3CN (149 mg, 2.25 mmol) was added to a MeOH (5.0 mL) solution of the above crude material and morpholine (500 iL, 5.72 mmol) and the mixture was stirred at ambient condition for 4 hr. It was cooled to ice-water temperature and treated with concentrated HCl to bring its pH to ~2.0, and then stirred for 2.5 hr. The volatile component was removed in vacuo, and the residue was purified with a combination of MCX resin (MeOH wash; 2.0 N HyMeOH elution) and a reverse phase HPLC (H^O/MeOH/TFA) to afford Cap-19 containing unknown amount of morpholine.
In order to consume the morpholine contaminant, the above material was dissolved in CH2C12 (1.5 mL) and treated with Et3N (0.27 mL, 1.94 mmol) followed by acetic anhydride (0.10 mL, 1.06 mmol) and stirred at ambient condition for 18 hr. THF (1.0 mL) and FLO (0.5 mL) were added and stirring continued for 1.5 hr. The volatile component was removed in vacuo, and the resultant residue was passed through MCX resin (MeOH wash; 2.0 N NH3/MeOH elution) to afford impure Cap- 19 as a brown viscous oil, which was used for the next step without further purification. Oa and -80b
Cap-80a: S/S-diastereomer
Cap-80b: S/R-diastereomer
Figure imgf000230_0001
SOCI2 (6.60 mL, 90.5 mmol) was added drop-wise over 15 min to a cooled (ice-water) mixture of (S)-3-amino-4-(benzyloxy)-4-oxobutanoic acid (10.04g, 44.98 mmol) and MeOH (300 mL), the cooling bath was removed and the reaction mixture was stirred at ambient condition for 29 hr. Most of the volatile component was removed in vacuo and the residue was carefully partitioned between EtOAc (150 mL) and saturated aHC03 solution. The aqueous phase was extracted with EtOAc (150 mL, 2x), and the combined organic phase was dried (MgS04), filtered, and concentrated in vacuo to afford (S)-l -benzyl 4-methyl 2-aminosuccinate as a colorless oil (9.706g). XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): δ 7.40-7.32 (m, 5H), 5.11 (s, 2H), 3.72 (app t, J = 6.6, 1H), 3.55 (s, 3H), 2.68 (dd, J = 15.9, 6.3, 1H), 2.58 (dd, J = 15.9, 6.8, 1H), 1.96 (s, 2H). LC (Cond. 1): RT = 0.90 min; LC/MS: Anal. Calcd. for [M+H]+ Ci2H16N04: 238.1 1; found 238.22. Pb(N03)2 (6.06 g, 18.3 mmol) was added over 1 min to a CH2C12 (80 mL) solution of (S)-l -benzyl 4-methyl 2-aminosuccinate (4.50 g, 19.0 mmol), 9-bromo-9- phenyl-9H-fluorene (6.44 g, 20.0 mmol) and Et3N (3.0 mL, 21.5 mmol), and the heterogeneous mixture was stirred at ambient condition for 48 hr. The mixture was filtered and the filtrate was treated with MgS04 and filtered again, and the final filtrate was concentrated. The resulting crude material was submitted to a Biotage purification (350 g silica gel, CH2C12 elution) to afford (S)-l -benzyl 4-methyl 2-(9- phenyl-9H-fluoren-9-ylamino)succinate as highly viscous colorless oil (7.93 g). XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): δ 7.82 (m, 2H), 7.39-7.13 (m, 16H), 4.71 (d, J = 12.4, 1H), 4.51 (d, J = 12.6, 1H), 3.78 (d, J = 9.1, NH), 3.50 (s, 3H), 2.99 (m, 1H), 2.50-2.41 (m, 2H, partially overlapped with solvent). LC (Cond. 1): RT = 2.16 min; LC/MS: Anal. Calcd. for [M+H]+ C3iH28N04: 478.20; found 478.19.
LiHMDS (9.2 mL of 1.0 M/THF, 9.2 mmol) was added drop-wise over 10 min to a cooled (-78 °C) THF (50 mL) solution of (S)-l -benzyl 4-methyl 2-(9- phenyl-9H-fluoren-9-ylamino)succinate (3.907 g, 8.18 mmol) and stirred for ~1 hr. Mel (0.57 mL, 9.2 mmol) was added drop-wise over 8 min to the mixture, and stirring was continued for 16.5 hr while allowing the cooling bath to thaw to room temperature. After quenching with saturated NH4C1 solution (5 mL), most of the organic component was removed in vacuo and the residue was partitioned between CH2C12 (100 mL) and water (40 mL). The organic layer was dried (MgS04), filtered, and concentrated in vacuo, and the resulting crude material was purified with a Biotage (350 g silica gel; 25% EtOAc/hexanes) to afford 3.65 g of a 2S/3S and 2S/3R diastereomeric mixtures of 1 -benzyl 4-methyl 3-methyl-2-(9-phenyl-9H-fluoren-9- ylamino)succinate in -1.0:0.65 ratio (XH NMR). The stereochemistry of the dominant isomer was not determined at this juncture, and the mixture was submitted to the next step without separation. Partial 1H NMR data (DMSO-d6, δ = 2.5 ppm, 400 MHz): major diastereomer, δ 4.39 (d, J = 12.3, 1H of CH2), 3.33 (s, 3H, overlapped with H20 signal), 3.50 (d, J = 10.9, NH), 1.13 (d, J = 7.1, 3H); minor diastereomer, δ 4.27 (d, J = 12.3, 1H of CH2), 3.76 (d, J = 10.9, NH), 3.64 (s, 3H), 0.77 (d, J = 7.0, 3H). LC (Cond. 1): RT = 2.19 min; LC/MS: Anal. Calcd. for [M+H]+ C32H30NO4: 492.22; found 492.15. Diisobutylaluminum hydride (20.57 ml of 1.0 M in hexanes, 20.57 mmol) was added drop-wise over 10 min to a cooled (-78 °C) THF (120 mL) solution of (2S)-l-benzyl 4-methyl 3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)succinate (3.37 g, 6.86 mmol) prepared above, and stirred at -78 °C for 20 hr. The reaction mixture was removed from the cooling bath and rapidly poured into ~1M H3PO4/H2O (250 mL) with stirring, and the mixture was extracted with ether (100 mL, 2x). The combined organic phase was washed with brine, dried (MgSC^), filtered and concentrated in vacuo. A silica gel mesh of the crude material was prepared and submitted to chromatography (25% EtOAc/hexanes; gravity elution) to afford l . lg of (2S,3S)-benzyl 4-hydroxy-3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate, contaminated with benzyl alcohol, as a colorless viscous oil and (2S,3R)-benzyl 4- hydroxy-3 -methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate containing the (2S,3R) stereoisomer as an impurity. The later sample was resubmitted to the same column chromatography purification conditions to afford 750 mg of purified material as a white foam. [Note: the (2S, 3S) isomer elutes before the (2S,3R) isomer under the above condition]. (2S, 3S) isomer: XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 7.81 (m, 2H), 7.39-7.08 (m, 16H), 4.67 (d, J = 12.3, 1H), 4.43 (d, J = 12.4, 1H), 4.21 (app t, J = 5.2, OH), 3.22 (d, J = 10.1, NH), 3.17 (m, 1H), 3.08 (m, 1H), -2.5 (m, 1H, overlapped with the solvent signal), 1.58 (m, 1H), 0.88 (d, J = 6.8, 3H). LC (Cond. 1): RT = 2.00 min; LC/MS: Anal. Calcd. for [M+H]+ C31H30NO3: 464.45; found 464.22. (2S, 3R) isomer: XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz): 7.81 (d, J = 7.5, 2H), 7.39-7.10 (m, 16H), 4.63 (d, J= 12.1, 1H), 4.50 (app t, J = 4.9, 1H), 4.32 (d, J = 12.1, 1H), 3.59-3.53 (m, 2H), 3.23 (m, 1H), 2.44 (dd, J = 9.0, 8.3, 1H), 1.70 (m, 1H), 0.57 (d, J = 6.8, 3H). LC (Cond. 1): RT = 1.92 min; LC/MS: Anal. Calcd. for [M+H]+ C31H30NO3: 464.45; found 464.52.
The relative stereochemical assignments of the DIBAL-reduction products were made based on NOE studies conducted on lactone derivatives prepared from each isomer by employing the following protocol: LiHMDS (50 μϊ^ of 1.0 M/THF, 0.05 mmol) was added to a cooled (ice-water) THF (2.0 mL) solution of (2S,3S)- benzyl 4-hydroxy-3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate (62.7 mg, 0.135 mmol), and the reaction mixture was stirred at similar temperature for ~2 hr. The volatile component was removed in vacuo and the residue was partitioned between (¾(¾ (30 mL), water (20 mL) and saturated aqueous NH4C1 solution (1 mL). The organic layer was dried (MgS04), filtered, and concentrated in vacuo, and the resulting crude material was submitted to a Biotage purification (40 g silica gel; 10-15% EtOAc/hexanes) to afford (3S,4S)-4-methyl-3-(9-phenyl-9H-fluoren-9- ylamino)dihydrofuran-2(3H)-one as a colorless film of solid (28.1 mg). (2S,3R)- benzyl 4-hydroxy-3 -methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate was elaborated similarly to (3S,4R)-4-methyl-3-(9-phenyl-9H-fluoren-9- ylamino)dihydrofuran-2(3H)-one. (3S,4S)-lactone isomer: XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz), 7.83 (d, J = 7.5, 2H), 7.46-7.17 (m, 11H), 4.14 (app t, J = 8.3, 1H), 3.60 (d, J = 5.8, NH), 3.45 (app t, J = 9.2, 1H), -2.47 (m, 1H, partially overlapped with solvent signal), 2.16 (m, 1H), 0.27 (d, J = 6.6, 3H). LC (Cond. 1): RT = 1.98 min; LC/MS: Anal. Calcd. for [M+Na C24H2i a02: 378.15; found 378.42. (3S,4R)-lactone isomer: XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz), 7.89 (d, J = 7.6, 1H), 7.85 (d, J = 7.3, 1H), 7.46-7.20 (m, 1 1H), 3.95 (dd, J = 9.1, 4.8,
1H), 3.76 (d, J = 8.8, 1H), 2.96 (d, J = 3.0, NH), 2.92 (dd, J = 6.8, 3, NCH), 1.55 (m, lH), 0.97 (d, J = 7.0, 3H). LC (Cond. 1): RT = 2.03 min; LC/MS: Anal. Calcd. for [M+Naf C24H2iNNa02: 378.15; found 378.49.
TBDMS-C1 (48 mg, 0.312 mmol) followed by imidazole (28.8 mg, 0.423 mmol) were added to a CH2CI2 (3 ml) solution of (2S,3S)-benzyl 4-hydroxy-3- methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate (119.5 mg, 0.258 mmol), and the mixture was stirred at ambient condition for 14.25 hr. The reaction mixture was then diluted with CH2CI2 (30 mL) and washed with water (15 mL), and the organic layer was dried (MgS04), filtered, and concentrated in vacuo. The resultant crude material was purified with a Biotage (40 g silica gel; 5% EtOAc/hexanes) to afford (2S,3S)-benzyl 4-(tert-butyldimethylsilyloxy)-3-methyl-2-(9-phenyl-9H-fluoren-9- ylamino)butanoate, contaminated with TBDMS based impurities, as a colorless viscous oil (124.4 mg). (2S,3R)-benzyl 4-hydroxy-3-methyl-2-(9-phenyl-9H- fluoren-9-ylamino)butanoate was elaborated similarly to (2S,3R)-benzyl 4-(tert- butyldimethylsilyloxy)-3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate.
(2S,3S)-silyl ether isomer: XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz), 7.82 (d, J = 4.1, 1H), 7.80 (d, J = 4.0, 1H), 7.38-7.07 (m, 16 H), 4.70 (d, J = 12.4, 1H), 4.42 (d, J = 12.3, 1H), 3.28-3.19 (m, 3H), 2.56 (dd, J = 10.1, 5.5, 1H), 1.61 (m, 1H), 0.90 (d, J = 6.8, 3H), 0.70 (s, 9H), -0.13 (s, 3H), -0.16 (s, 3H). LC (Cond. 1, where the run time was extended to 4 min): RT = 3.26 min; LC/MS: Anal. Calcd. for [M+H]+ C37H44NO3S1: 578.31 ; found 578.40. (2S,3R)-silyl ether isomer: XH NMR (DMSO- d6, δ = 2.5 ppm, 400 MHz), 7.82 (d, J = 3.0, 1H), 7.80 (d, J = 3.1, 1H), 7.39-7.10 (m, 16H), 4.66 (d, J = 12.4, 1H), 4.39 (d, J = 12.4, 1H), 3.61 (dd, J = 9.9, 5.6, 1H), 3.45 (d, J = 9.5, 1H), 3.41 (dd, J = 10, 6.2, 1H), 2.55 (dd, J = 9.5, 7.3, 1H), 1.74 (m, 1H), 0.77 (s, 9H), 0.61 (d, J = 7.1, 3H), -0.06 (s, 3H), -0.08 (s, 3H).
A balloon of hydrogen was attached to a mixture of (2S,3S)-benzyl 4-(tert- butyldimethylsilyloxy)-3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate (836 mg, 1.447 mmol) and 10% Pd/C (213 mg) in EtOAc (16 mL) and the mixture was stirred at room temperature for ~ 21 hr, where the balloon was recharged with ¾ as necessary. The reaction mixture was diluted with CH2CI2 and filtered through a pad of diatomaceous earth (Celite-545®), and the pad was washed with EtOAc (200 mL), EtOAc/MeOH (1 : 1 mixture, 200 mL) and MeOH (750 mL). The combined organic phase was concentrated, and a silica gel mesh was prepared from the resulting crude material and submitted to a flash chromatography (8:2: 1 mixture of EtOAc/i- PrOH/H20) to afford (2S,3S)-2-amino-4-(tert-butyldimethylsilyloxy)-3- methylbutanoic acid as a white fluffy solid (325 mg). (2S,3R)-benzyl 4-(tert- butyldimethylsilyloxy)-3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate was similarly elaborated to (2S,3R)-2-amino-4-(tert-butyldimethylsilyloxy)-3- methylbutanoic acid. (2S,3S)-amino acid isomer: XH NMR (Methanol-cU, δ = 3.29 ppm, 400 MHz), 3.76 (dd, J = 10.5, 5.2, 1H), 3.73 (d, J = 3.0, 1H), 3.67 (dd, J = 10.5, 7.0, 1H), 2.37 (m, 1H), 0.97 (d, J = 7.0, 3H), 0.92 (s, 9H), 0.10 (s, 6H).
LC/MS: Anal. Calcd. for [M+H]+ CiiH26 03Si: 248.17; found 248.44. (2S,3R)- amino acid isomer: XH NMR (Methanol-d4, δ = 3.29 ppm, 400 MHz), 3.76-3.75 (m, 2H), 3.60 (d, J = 4.1, lH), 2.16 (m, 1H), 1.06 (d, J = 7.3, 3H), 0.91 (s, 9H), 0.09 (s, 6H). Anal. Calcd. for [M+H]+ CiiH26N03Si: 248.17; found 248.44.
Water (1 mL) and NaOH (0.18 mL of 1.0 M/H20, 0.18 mmol) were added to a mixture of (2S,3S)-2-amino-4-(tert-butyldimethylsilyloxy)-3 -methylbutanoic acid (41.9 mg, 0.169 mmol) and a2C03 (1 1.9 mg, 0.1 12 mmol), and sonicated for about 1 min to effect dissolution of reactants. The mixture was then cooled with an ice- water bath, methyl chloroformate (0.02 mL, 0.259 mmol) was added over 30 s, and vigorous stirring was continued at similar temperature for 40 min and then at ambient temperature for 2.7 hr. The reaction mixture was diluted with water (5 mL), cooled with ice-water bath and treated drop-wise with 1.0 N HC1 aqueous solution (-0.23 mL). The mixture was further diluted with water (10 mL) and extracted with (¾(¾ (15 mL, 2x). The combined organic phase was dried (MgSC^), filtered, and concentrated in vacuo to afford Cop-80a as an off-white solid. (2S,3R)-2-amino-4- (tert-butyldimethylsilyloxy)-3-methylbutanoic acid was similarly elaborated to Cap- 80b. Cap-80a: XH NMR (DMSO-d6, δ = 2.5 ppm, 400 MHz), 12.57 (br s, 1H), 7.64 (d, J = 8.3, 0.3H), 7.19 (d, J = 8.8, 0.7H), 4.44 (dd, J = 8.1, 4.6, 0.3H), 4.23 (dd, J = 8.7, 4.4, 0.7H), 3.56/3.53 (two singlets, 3H), 3.48-3.40 (m, 2H), 2.22-2.10 (m, 1H), 0.85 (s, 9H), -0.84 (d, 0.9H, overlapped with t-Bu signal), 0.79 (d, J = 7, 2.1H), 0.02/0.01/0.00 (three overlapping singlets, 6H). LC/MS: Anal. Calcd. for [M+Na]+ Ci3H27 a05Si: 328.16; found 328.46. Cap-80b: XH NMR (CDC13, δ = 7.24 ppm, 400 MHz), 6.00 (br d, J = 6.8, 1H), 4.36 (dd, J = 7.1, 3.1, 1H), 3.87 (dd, J = 10.5, 3.0, 1H), 3.67 (s, 3H), 3.58 (dd, J = 10.6, 4.8, 1H), 2.35 (m, 1H), 1.03 (d, J = 7.1, 3H), 0.90 (s, 9H), 0.08 (s, 6H). LC/MS: Anal. Calcd. for [M+Na
Figure imgf000235_0001
328.16; found 328.53. The crude products were utilized without further purification.
Cap-81
Figure imgf000235_0002
Prepared according to the protocol described by Falb et al. Synthetic
Communications 1993, 23, 2839.
Cap-82 to Cap-85
Cap-%2 to Cap-85 were synthesized from appropriate starting materials according to the procedure described for Cap-51 or Cap- 13. The samples exhibited similar spectral profiles as that of their enantiomers (i.e., Cap-4, Cap- 13, Cap-51 and -52, respectively).
Figure imgf000236_0001
Cap-82 Cap-83 Cap-84 Cap-85
Cap-86
Me
Figure imgf000236_0002
To a mixture of O-methyl-L-threonine (3.0 g, 22.55 mmol), NaOH (0.902 g, 22.55 mmol) in H20 (15 mL) was added ClC02Me (1.74 mL, 22.55 mmol) dropwise at 0°C. The mixture was allowed to stir for 12 h and acidified to pH 1 using IN HCl. The aqueous phase was extracted with EtOAc and (2x250 mL) and 10% MeOH in
CH2CI2 (250 mL) and the combined organic phases were concentrated under in vacuo to afford a colorless oil (4.18 g, 97%) which was of sufficient purity for use in subsequent steps. 1HNMR (400 MHz, CDC13) δ 4.19 (s, 1H), 3.92-3.97 (m, 1H), 3.66 (s, 3H), 1.17 (d, J = 7.7 Hz, 3H). LCMS: Anal. Calcd. for C7H13NO5: 191; found: 190 (M-H)".
Cap-87
Figure imgf000236_0003
To a mixture of L-homoserine (2.0 g, 9.79 mmol), Na2C03 (2.08 g, 19.59 mmol) in H20 (15 mL) was added ClC02Me (0.76 mL, 9.79 mmol) dropwise at 0°C. The mixture was allowed to stir for 48 h and acidified to pH 1 using IN HCl. The aqueous phase was extracted with EtOAc and (2X250 mL) and the combined organic phases were concentrated in vacuo to afford a colorless solid (0.719 g, 28%) which was of sufficient purity for use in subsequent steps. 1HNMR (400 MHz, CDCI3) δ 4.23 (dd, J = 4.5, 9.1 Hz, 1H), 3.66 (s, 3H), 3.43-3.49 (m, 2H), 2.08 - 2.14 (m, 1H), 1.82 - 1.89 (m, 1H). LCMS: Anal. Calcd. for C7H13N05: 191; found: 192 (M+H)+.
Ca -88
Figure imgf000237_0001
A mixture of L-valine (1.0 g, 8.54 mmol), 3-bromopyridine (1.8 mL, 18.7 mmol), K2C03 (2.45 g, 17.7 mmol) and Cul (169 mg, 0.887 mmol) in DMSO (10 mL) was heated at 100°C for 12h. The reaction mixture was cooled to rt, poured into H2O (ca. 150 mL) and washed with EtOAc (x2). The organic layers were extracted with a small amount of H2O and the combined aq phases were acidified to ca. pH 2 with 6N HC1. The volume was reduced to about one-third and 20g of cation exchange resin (Strata) was added. The slurry was allowed to stand for 20 min and loaded onto a pad of cation exchange resin (Strata) (ca. 25g). The pad was washed with H20 (200 mL), MeOH (200 mL), and then NH3 (3M in MeOH, 2X200 mL). The appropriate fractions was concentrated in vacuo and the residue (ca. 1.1 g) was dissolved in H20, frozen and lyophyllized. The title compound was obtained as a foam (1.02 g, 62%). 1HNMR (400 MHz, DMSO-d6) δ 8.00 (s, br, 1H), 7.68 - 7.71 (m, 1H), 7.01 (s, br, 1H), 6.88 (d, J = 7.5 Hz, 1H), 5.75 (s, br, 1H), 3.54 (s, 1H), 2.04 - 2.06 (m, 1H), 0.95 (d, J = 6.0 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H). LCMS: Anal. Calcd. for CioH14 202: 194; found: 195 (M+H)+.
Ca -89
Figure imgf000237_0002
A mixture of L-valine (1.0 g, 8.54 mmol), 5-bromopyrimidine (4.03 g, 17.0 mmol), K2C03 (2.40 g, 17.4 mmol) and Cul (179 mg, 0.94 mmol) in DMSO (10 mL) was heated at 100°C for 12h. The reaction mixture was cooled to RT, poured into H2O (ca. 150 mL) and washed with EtOAc (x2). The organic layers were extracted with a small amount of H2O and the combined aq phases were acidified to ca. pH 2 with 6N HC1. The volume was reduced to about one-third and 20g of cation exchange resin (Strata) was added. The slurry was allowed to stand for 20 min and loaded onto a pad of cation exchange resin (Strata) (ca. 25g). The pad was washed with H20 (200 mL), MeOH (200 mL), and then NH3 (3M in MeOH, 2x200 mL). The appropriate fractions was concentrated in vacuo and the residue (ca. 1.1 g) was dissolved in H20, frozen and lyophyllized. The title compound was obtained as a foam (1.02 g, 62%). 1HNMR (400 MHz, CD3OD) showed the mixture to contain valine and the purity could not be estimated. The material was used as is in subsequent reactions. LCMS: Anal. Calcd. for C9H13N3O2: 195; found: 196 (M+H)+. -90
Figure imgf000238_0001
Cap-90 was prepared according to the method described for the preparation of Cap-l . The crude material was used as is in subsequent steps. LCMS: Anal. Calcd. for C11H15NO2: 193; found: 192 (M-H)".
The following caps were prepared according to the method used for preparation of cap 51 unless noted otherwise:
Figure imgf000238_0002
Figure imgf000239_0001
Figure imgf000240_0001
Figure imgf000241_0001
Figure imgf000242_0001
Figure imgf000243_0001
Cap-117 to Cap-123
For the preparation of Cap-I ll to Cap-123 the Boc amino acids were obtained from commercially sources and were deprotected by treatment with 25% TFA in (¾(¾. After complete reaction as judged by LCMS the solvents were removed in vacuo and the corresponding TFA salt of the amino acid was carbamoylated with methyl chloroformate according to the procedure described for Cap-51.
Figure imgf000244_0001
Figure imgf000245_0001
- 124
Figure imgf000245_0002
The hydrochloride salt of L-threonine tert-butyl ester was carbamoylated according to the procedure for Cap-51. The crude reaction mixture was acidified with IN HC1 to pH~l and the mixture was extracted with EtOAc (2X50 mL). The combined organic phases were concentrated in vacuo to give a colorless oil which solidified on standing. The aqueous layer was concentrated in vacuo and the resulting mixture of product and inorganic salts was triturated with EtOAc-CH2Cl2- MeOH (1 : 1 :0.1) and then the organic phase concentrated in vacuo to give a colorless oil which was shown by LCMS to be the desired product. Both crops were combined to give 0.52 g of a solid. 1HNMR (400 MHz, CD3OD) δ 4.60 (m, 1H), 4.04 (d, J = 5.0 Hz, 1H), 1.49 (d, J = 6.3 Hz, 3H). LCMS: Anal. Calcd. for C5H7N04: 145; found: 146 (M+H)+.
Cap- 125
/
-N O
BocHN OH
To a suspension of Pd(OH)2, (20%, 100 mg), aqueous formaldehyde (37% wt, 4 ml), acetic acid, (0.5 mL) in methanol (15 mL) was added (S)-4-amino-2-(tert- butoxycarbonylamino)butanoic acid (1 g, 4.48 mmol). The reaction was purged several times with hydrogen and was stirred overnight with an hydrogen balloon room temp. The reaction mixture was filtered through a pad of diatomaceous earth (Celite®), and the volatile component was removed in vacuo. The resulting crude material was used as is for the next step. LC/MS: Anal. Calcd. for C11H22N2O4: 246; found: 247 (M+H)+. - 126
Figure imgf000246_0001
cj-25 cap-126
This procedure is a modification of that used to prepare Cap-51. To a suspension of 3-methyl-L-histidine (0.80 g, 4.70 mmol) in THF (lOmL) and H2O (10 mL) at 0°C was added aHC03 (0.88 g, 10.5 mmol). The resulting mixture was treated with ClC02Me (0.40 mL, 5.20 mmol) and the mixture allowed to stir at 0°C. After stirring for ca. 2h LCMS showed no starting material remaining. The reaction was acidified to pH 2 with 6 N HC1.
The solvents were removed in vacuo and the residue was suspended in 20 mL of 20% MeOH in CH2CI2. The mixture was filtered and concentrated to give a light yellow foam (1.21 g,). LCMS and XH NMR showed the material to be a 9: l mixture of the methyl ester and the desired product. This material was taken up in THF (lOmL) and H20 (lOmL), cooled to 0°C and LiOH (249.1 mg, 10.4 mmol) was added. After stirring ca. lh LCMS showed no ester remaining. Therefore the mixture was acidified with 6N HC1 and the solvents removed in vacuo. LCMS and XH NMR confirm the absence of the ester. The title compound was obtained as its HC1 salt contaminated with inorganic salts (1.91 g, >100%). The compound was used as is in subsequent steps without further purification. 1HNMR (400 MHz, CD3OD) δ 8.84, (s, 1H), 7.35 (s, 1H), 4.52 (dd, J = 5.0, 9.1 Hz, 1H), 3.89 (s, 3H), 3.62 (s, 3H), 3.35 (dd, J = 4.5, 15.6 Hz, 1H, partially obscured by solvent), 3.12 (dd, J = 9.0, 15.6 Hz, 1H).LCMS: Anal. Calcd. for C9H13N3O4: 227.09; found: 228.09 (M+H)+. Cap- 127
Figure imgf000247_0001
cj-26 cap-127
Cap-127 was prepared according to the method for Cap-126 above starting from (S)-2-amino-3-(l-methyl-lH-imidazol-4-yl)propanoic acid (1.1 1 g, 6.56 mmol), NaHC03 (1.21 g, 14.4 mmol) and ClC02Me (0.56 mL, 7.28 mmol). The title compound was obtained as its HC1 salt (1.79 g, >100%) contaminated with inorganic salts. LCMS and XH NMR showed the presence of ca. 5% of the methyl ester. The crude mixture was used as is without further purification. 1HNMR (400 MHz, CD3OD) δ 8.90 (s, 1H), 7.35 (s, 1H), 4.48 (dd, J = 5.0, 8.6 Hz, 1H), 3.89 (s, 3H), 3.62 (s, 3H), 3.35 (m, 1H), 3.08 (m, 1H); LCMS: Anal. Calcd. for C9H13N3O4: 227.09; found: 228 (M+H)+.
Preparation of Cap
BocH
Figure imgf000247_0002
Figure imgf000247_0003
cj-29 cap-128
Step 1. Preparation of (S)-benzyl 2-(tert-butoxycarbonylamino)pent-4-ynoate (cj- 27b). BocHN C02Bn
cj-27b
To a solution oicj-27a (1.01 g, 4.74 mmol), DMAP (58 mg, 0.475 mmol) and iPr2NEt (1.7 mL, 9.8 mmol) in CH2C12 (100 mL) at 0°C was added Cbz-Cl (0.68 mL, 4.83 mmol). The solution was allowed to stir for 4 h at 0°C, washed (IN KHS04, brine), dried ( a2S04), filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (TLC 6: 1 hex:EtOAc) to give the title compound (1.30 g, 91%) as a colorless oil. 1HNMR (400 MHz, CDC13) δ 7.35 (s, 5H), 5.35 (d, br, J = 8.1 Hz, 1H), 5.23 (d, J = 12.2 Hz, 1H), 5.17 (d, J = 12.2 Hz, 1H), 4.48 - 4.53 (m, 1H), 2.68 - 2.81 (m, 2H), 2.00 (t, J = 2.5 Hz, 1H), 1.44 (s, 9H). LCMS: Anal. Calcd. for Ci7H21N04: 303; found: 304 (M+H)+.
Step 2. Preparation of (S)-benzyl 3-(l-benzyl-lH-l,2,3-triazol-4-yl)-2-(tert- butoxycarbonylamino)propanoat -28).
Figure imgf000248_0001
cj-28
To a mixture of (S)-benzyl 2-(tert-butoxycarbonylamino)pent-4-ynoate (0.50 g, 1.65 mmol), sodium ascorbate (0.036 g, 0.18 mmol), CuS04-5H20 (0.022 g, 0.09 mmol) and NaN3 (0.13 g, 2.1 mmol) in DMF-H20 (5 mL, 4: 1) at rt was added BnBr (0.24 mL, 2.02 mmol) and the mixture was warmed to 65°C. After 5h LCMS indicated low conversion. A further portion of NaN3 (100 mg) was added and heating was continued for 12h. The reaction was poured into EtOAc and H20 and shaken. The layers were separated and the aqueous layer extracted 3x with EtOAc and the combined organic phases washed (H20 x3, brine), dried (Na2S04), filtered, and concentrated. The residue was purified by flash (Biotage, 40+M 0-5% MeOH in CH2C12; TLC 3% MeOH in CH2C12) to afford a light yellow oil which solidified on standing (748.3 mg, 104%). The NMR was consistent with the desired product but suggests the presence of DMF. The material was used as is without further purification. 1HNMR (400 MHz, DMSO-d6) δ 7.84 (s, 1H), 7.27 - 7.32 (m, 10H), 5.54 (s, 2H), 5.07 (s, 2H), 4.25 (m, 1H), 3.16 (dd, J = 1.0, 5.3 Hz, 1H), 3.06 (dd, J = 5.3, 14.7 Hz), 2.96 (dd, J = 9.1, 14.7 Hz, 1H), 1.31 (s, 9H).
LCMS: Anal. Calcd. for C24H28 404: 436; found: 437 (M+H)+.
Step 3. Preparation of (S)-benzyl 3-(l-benzyl-lH-l,2,3-triazol-4-yl)-2- (methoxycarbonylamino)propanoate (cj-29).
Figure imgf000249_0001
cj-29
A solution of (S)-benzyl 3-(l-benzyl-lH-l,2,3-triazol-4-yl)-2-(tert- butoxycarbonylamino)propanoate (0.52 g, 1.15 mmol) in CH2CI2 was added TFA (4 mL). The mixture was allowed to stir at room temperature for 2h. The mixture was concentrated in vacuo to give a colorless oil which solidified on standing. This material was dissolved in THF-H20 and cooled to 0°C. Solid NaHC03 (0.25 g, 3.00 mmol) was added followed by ClCC^Me (0.25 mL, 3.25 mmol). After stirring for 1.5h the mixture was acidified to pH~2 with 6N HC1 and then poured into ¾0- EtOAc. The layers were separated and the aq phase extracted 2x with EtOAc. The combined org layers were washed (H20, brine), dried (Na2S04), filtered, and concentrated in vacuo to give a colorless oil (505.8 mg, 111%, NMR suggested the presence of an unidentified impurity) which solidified while standing on the pump. The material was used as is without further purification. 1HNMR (400 MHz, DMSO- d6) δ 7.87 (s, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.27 - 7.32 (m, 10H), 5.54 (s, 2H), 5.10 (d, J = 12.7 Hz, 1H), 5.06 (d, J = 12.7 Hz, 1H), 4.32 - 4.37 (m, 1H), 3.49 (s, 3H), 3.09 (dd, J = 5.6, 14.7 Hz, 1H), 2.98 (dd, J = 9.6, 14.7 Hz, 1H). LCMS: Anal.
Calcd. for C21H22N4O4: 394; found: 395 (M+H)+.
Step 4. Preparation of (S)-2-(methoxycarbonylamino)-3-(lH-l,2,3-triazol-4- yl)propanoic acid (Cap- 128).
Figure imgf000250_0001
Cap-128
(S)-benzyl 3 -(1 -benzyl- 1 H- 1 ,2,3-triazol-4-yl)-2- (methoxycarbonylamino)propanoate (502 mg, 1.1 1 mmol) was hydrogenated in the presence of Pd-C (82 mg) in MeOH (5 mL) at atmospheric pressure for 12h. The mixture was filtered through diatomaceous earth (Celite®) and concentrated in vacuo. (S)-2-(methoxycarbonylamino)-3-(lH-l,2,3-triazol-4-yl)propanoic acid was obtained as a colorless gum (266 mg, 11 1%) which was contaminated with ca. 10% of the methyl ester. The material was used as is without further purification. 1HNMR (400 MHz, DMSO-d6) δ 12.78 (s, br, 1H), 7.59 (s, 1H), 7.50 (d, J = 8.0 Hz, 1H), 4.19 - 4.24 (m, 1H), 3.49 (s, 3H), 3.12 (dd, J = 4.8 Hz, 14.9 Hz, 1H), 2.96 (dd, J = 9.9, 15.0 Hz, 1H). LCMS: Anal. Calcd. for C7H10N4O4: 214; found: 215 (M+H)+.
Preparation of Cap-129
CbzH
Figure imgf000250_0002
12g
Step 1. Preparation of (S)-2-(benzyloxycarbonylamino)-3-(lH-pyrazol-l- yl)propanoic acid (cj-31).
Figure imgf000250_0003
cj-31
A suspension of (S)-benzyl 2-oxooxetan-3-ylcarbamate (0.67 g, 3.03 mmol), and pyrazole (0.22 g, 3.29 mmol) in CH3CN (12 mL) was heated at 50 °C for 24h. The mixture was cooled to rt overnight and the solid filtered to afford (S)-2- (benzyloxycarbonylamino)-3-(lH-pyrazol-l-yl)propanoic acid (330.1 mg). The filtrate was concentrated in vacuo and then triturated with a small amount of C¾CN (ca. 4 mL) to afford a second crop (43.5 mg). Total yield 370.4 mg (44%). m.p. 165.5 - 168°C. lit m.p. 168.5 - 169.5 [Vederas et al. J. Am. Chem. Soc. 1985, 707, 7105]. 1HNMR (400 MHz, CD3OD) δ 7.51 (d, J = 2.0, 1H), 7.48 (s, J = 1.5 Hz, 1H), 7.24 - 7.34 (m, 5H), 6.23 m, 1H), 5.05 (d, 12.7 H, 1H), 5.03 (d, J = 12.7 Hz, 1H), 4.59 - 4.66 (m, 2H), 4.42 - 4.49 (m, 1H). LCMS: Anal. Calcd. for
Ci4H15N304: 289; found: 290 (M+H)+.
Step 2. Preparation of (S)-2-(methoxycarbonylamino)-3-(lH-pyrazol-l-yl)propanoic acid (Cap-129).
Figure imgf000251_0001
cap-129
(S)-2-(benzyloxycarbonylamino)-3-(lH-pyrazol-l-yl)propanoic acid (0.20 g, 0.70 mmol) was hydrogenated in the presence of Pd-C (45 mg) in MeOH (5 mL) at atmospheric pressure for 2h. The product appeared to be insoluble in MeOH, therefore the reaction mixture was diluted with 5mL ¾0 and a few drops of 6N HCl. The homogeneous solution was filtered through diatomaceous earth (Celite®), and the MeOH removed in vacuo. The remaining solution was frozen and lyophyllized to give a yellow foam (188.9 mg). This material was suspended in THF-H20 (1 : 1, lOmL) and then cooled to 0°C. To the cold mixture was added aHC03 (146.0 mg, 1.74 mmol) carefully (evolution of CO2). After gas evolution had ceased (ca. 15 min) ClC02Me (0.06 mL, 0.78 mmol) was added dropwise. The mixture was allowed to stir for 2h and was acidified to pH~2 with 6N HCl and poured into EtOAc. The layers were separated and the aqueous phase extracted with EtOAC (x5). The combined organic layers were washed (brine), dried (Na2S04), filtered, and concentrated to give the title compound as a colorless solid (1 17.8 mg, 79%).
1HNMR (400 MHz, DMSO-d6) δ 13.04 (s, 1H), 7.63 (d, J = 2.6 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.44 (d, J = 1.5 Hz, 1H), 6.19 (app t, J = 2.0 Hz, 1H), 4.47 (dd, J = 3.0, 12.9 Hz, 1H), 4.29 - 4.41 (m, 2H), 3.48 (s, 3H). LCMS: Anal. Calcd. for
C8Hn 304: 213; found: 214 (M+H)+. - 130
Figure imgf000252_0001
AcHN C02H
Cap- 130 was prepared by acylation of commercially available (R)- phenylglycine analgous to the procedure given in: Calmes, M.; Daunis, J.; Jacquier, R.; Verducci, J. Tetrahedron, 1987, 43(10), 2285. -131
Figure imgf000252_0002
Step a: Dimethylcarbamoyl chloride (0.92 mL, 10 mmol) was added slowly to a solution of (S)-benzyl 2-amino-3-methylbutanoate hydrochloride (2.44 g; 10 mmol) and Hunig's base (3.67 mL, 21 mmol) in THF (50 mL). The resulting white suspension was stirred at room temperature overnight (16 hours) and concentrated under reduced pressure. The residue was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried (MgSC^), filtered, and concentrated under reduced pressure. The resulting yellow oil was purified by flash chromatography, eluting with ethyl acetate:hexanes (1 : 1). Collected fractions were concentrated under vacuum providing 2.35 g (85%) of clear oil. XH NMR (300 MHz, DMSO-d6) δ ppm 0.84 (d, J=6.95 Hz, 3H), 0.89 (d, J=6.59 Hz, 3H), 1.98-2.15 (m, IH), 2.80 (s, 6H), 5.01-5.09 (m, J= 12.44 Hz, IH), 5.13 (d, J= 12.44 Hz, IH), 6.22 (d, J=8.05 Hz, IH), 7.26-7.42 (m, 5H). LC (Cond. 1): RT = 1.76 min; MS: Anal.
Calcd. for [M+H]+ Ci6H22 203: 279.17; found 279.03.
Step b: To a MeOH (50 mL) solution of the intermediate prepared above (2.35 g; 8.45 mmol) was added Pd/C (10%; 200 mg) and the resulting black suspension was flushed with N2 (3x) and placed under 1 atm of H2. The mixture was stirred at room temperature overnight and filtered though a microfiber filter to remove the catalyst. The resulting clear solution was then concentrated under reduced pressure to obtain 1.43 g (89%) of Cap-131 as a white foam, which was used without further purification. XH NMR (500 MHz, DMSO-d6) δ ppm 0.87 (d, J=4.27 Hz, 3H), 0.88 (d, J=3.97 Hz, 3H), 1.93-2.1 1 (m, IH), 2.80 (s, 6H), 3.90 (dd, J=8.39, 6.87 Hz, IH), 5.93 (d, J=8.54 Hz, IH), 12.36 (s, IH). LC (Cond. 1): RT = 0.33 min; MS: Anal. Calcd. for [M+H]+ CgHnNzOs: 189.12; found 189.04.
Cap-132
Figure imgf000253_0001
Cap-132 was prepared from (S)-benzyl 2-aminopropanoate hydrochloride according to the method described for Cap-131. ¾ NMR (500 MHz, DMSO-d6) δ ppm 1.27 (d, J=7.32 Hz, 3H), 2.80 (s, 6H), 4.06 (qt, IH), 6.36 (d, J=7.32 Hz, IH), 12.27 (s, IH). LC (Cond. 1): RT = 0.15 min; MS: Anal. Calcd. for [M+H]+ C6H13N203: 161.09; found 161.00.
Cap-133
Figure imgf000253_0002
Cap-133 was prepared from (S)-tert-butyl 2-amino-3-methylbutanoate hydrochloride and 2-fluoroethyl chloroformate according to the method described for Cap-47. XH NMR (500 MHz, DMSO-d6) δ ppm 0.87 (t, J = 6.71 Hz, 6H), 1.97-2.10 (m, IH), 3.83 (dd, J=8.39, 5.95 Hz, IH), 4.14-4.18 (m, IH), 4.20-4.25 (m, IH), 4.50- 4.54 (m, IH), 4.59-4.65 (m, IH), 7.51 (d, J = 8.54 Hz, IH), 12.54 (s, IH).
Cap-134
Figure imgf000254_0001
Cap-134 was prepared from (S)-diethyl alanine and methyl chloroformate according to the method described for Cap-51. XH NMR (500 MHz, DMSO-d6) δ ppm 0.72-0.89 (m, 6H), 1.15-1.38 (m, 4H), 1.54-1.66 (m, 1H), 3.46-3.63 (m, 3H), 4.09 (dd, J = 8.85, 5.19 Hz, 1H), 7.24 (d, J = 8.85 Hz, 1H), 12.55 (s, 1H). LC (Cond. 2): RT = 0.66 min; LC/MS: Anal. Calcd. for [M+H]+ C9H18N04: 204.12; found 204.02. -135
Figure imgf000254_0002
A solution of D-2-amino-(4-fluorophenyl)acetic acid (338 mg, 2.00 mmol), INHCl in diethylether (2.0 mL, 2.0 mmol) and formalin (37%, 1 mL) in methanol (5 mL) was subjected to balloon hydrogenation over 10% palladium on carbon (60 mg) for 16 h at 25 °C. The mixture was then filtered through Celite to afford the HC1 salt of Cap-135 as a white foam (316 mg, 80%). XH NMR (300 MHz, MeOH-d4) δ 7.59 (dd, J= 8.80, 5.10 Hz, 2H), 7.29 (t, J= 8.6 Hz, 2H), 5.17 (s, 1H), 3.05 (v br s, 3H), 2.63 (v br s, 3H); Rt = 0.19 min (Cond.-MS-W5); 95% homogenity index; LRMS: Anal. Calcd. for [M+H]+ Ci0H13FNO2: 198.09; found: 198.10. - 136
Figure imgf000254_0003
To a cooled (-50 °C) suspension of 1 -benzyl- lH-imidazole (1.58 g, 10.0 mmol) in anhydrous diethyl ether (50 mL) under nitrogen was added w-butyl lithium (2.5 M in hexanes, 4.0 niL, 10.0 mmol) dropwise. After being stirred for 20 min at - 50 °C, dry carbon dioxide (passed through Drierite) was bubbled into the reaction mixture for 10 min before it was allowed to warm up to 25 °C. The heavy precipitate which formed on addition of carbon dioxide to the reaction mixture was filtered to yield a hygroscopic, white solid which was taken up in water (7 mL), acidified to pH = 3, cooled, and induced to crystallize with scratching. Filtration of this precipitate gave a white solid which was suspended in methanol, treated with INHCl/diethyl ether (4 mL) and concentrated in vacuo. Lyophilization of the residue from water (5 mL) afforded the HC1 salt of Cap-136 as a white solid (817 mg, 40%). XH NMR (300 MHz, DMSO-d6) δ 7.94 (d, J= 1.5 Hz, 1H), 7.71 (d, J= 1.5 Hz, 1H), 7.50-7.31 (m, 5H), 5.77 (s, 2H); Rt = 0.51 min (Cond.-MS-W5); 95% homogenity index; LRMS: Anal. Calc. for [M+H]+ CnH12N202: 203.08; found: 203.11. - 137
Figure imgf000255_0001
- 137, step a
Figure imgf000255_0002
A suspension of l-chloro-3-cyanoisoquinoline (188 mg, 1.00 mmol; prepared according to the procedure in WO 2003/ 099274) (188 mg, 1.00 mmol), cesium fluoride (303.8 mg, 2.00 mmol), bis(tri-/er/-butylphosphine)palladium dichloride (10 mg, 0.02 mmol) and 2-(tributylstannyl)furan (378 μί, 1.20 mmol) in anhydrous dioxane (10 mL) under nitrogen was heated at 80 °C for 16 h before it was cooled to 25 °C and treated with saturated, aqueous potassium fluoride solution with vigorous stirring for 1 h. The mixture was partitioned between ethyl acetate and water and the organic phase was separated, washed with brine, dried over Na2S04, filtered and concentrated. Purification of the residue on silica gel (elution with 0% to 30% ethyl acetate/hexanes) afforded Cap-137, step a (230 mg, 105%) as a white solid which was carried forward directly. Rt = 1.95 min (Cond.-MS-W2); 90% homogeneity index; LRMS: Anal. Calc. for [M+H]+ Ci4H8N20: 221.07; found: 221.12. Cap-137
To a suspension of Cap 137, step a, (1 10 mg, 0.50 mmol) and sodium periodate (438 mg, 2.05 mmol) in carbon tetrachloride (1 mL), acetonitrile (1 mL) and water (1.5 mL) was added ruthenium trichloride hydrate (2 mg, 0.01 1 mmol). The mixture was stirred at 25 °C for 2 h and then partitioned between
dichloromethane and water. The aqueous layer was separated, extracted twice more with dichloromethane and the combined dichloromethane extracts were dried over a2S04; filtered and concentrated. Trituration of the residue with hexanes afforded Cap-137 (55 mg, 55%) as a grayish-colored solid. Rt = 1.10 min (Cond.-MS-W2); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]+ ΟιιΗ8Ν202: 200.08; found: 200.08.
Caps 138 to 158
Synthetic Strategy. Method A.
Figure imgf000256_0001
Figure imgf000256_0002
Cap-138, step a
Figure imgf000257_0001
To a stirred suspension of 5-hydroxyisoquinoline (prepared according to the procedure in WO 2003/ 099274) (2.0 g, 13.8 mmol) and triphenylphosphine (4.3 g, 16.5 mmol) in dry tetrahydrofuran (20 mL) was added dry methanol (0.8 mL) and diethyl azodicarboxylate (3.0 mL, 16.5 mmol) portionwise. The mixture was stirred at room temperature for 20 h before it was diluted with ethyl acetate and washed with brine, dried over Na2S04, filtered and concentrated. The residue was preabsorbed onto silica gel and chromatographed (elution with 40% ethyl acetate/hexanes) to afford Cap-138, step a (1.00 g, 45%) as a light yellow solid. XH NMR (CDC13, 500 MHz) δ 9.19 (s, 1H), 8.51 (d, J = 6.0 Hz, 1H), 7.99 (d, J = 6.0 Hz, 1H), 7.52-7.50 (m, 2H), 7.00-6.99 (m, 1H), 4.01 (s, 3H); Rt = 0.66 min (Cond.-D2); 95% homogeneity index; LCMS: Anal. Calc. for [M+H]+ Ci0H10NO: 160.08; found 160.1. -138, step b
Figure imgf000257_0002
To a stirred solution of Cap 138, step a (2.34 g, 14.7 mmol) in anhydrous dichloromethane (50 mL) at room temperature was added meto-chloroperbenzoic acid (77%, 3.42 g, 19.8 mmol) in one portion. After being stirred for 20 h, powdered potassium carbonate (2.0 g) was added and the mixture was stirred for 1 h at room temperature before it was filtered and concentrated in vacuo to afford Cap-138, step (2.15 g, 83%) as a pale, yellow solid which was sufficiently pure to carry forward directly. 'H NMR 400 MHz) δ 8.73 (d, J= 1.5 Hz, 1H), 8.1 1 (dd, J = 7.3, 1.7 Hz, 1H), 8.04 (d, J=7.1 Hz, 1H), 7.52 (t, J= 8.1 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 6.91 (d, J = 7.8 Hz, 1H), 4.00 (s, 3H); Rt = 0.92 min, (Cond.-D l); 90% homogenity index; LCMS: Anal. Calc. for [M+H]+ Ci0H10NO2: 176.07; found: 176.0.
Cap-138, step c
Figure imgf000258_0001
To a stirred solution of Cap 138, step b (0.70 g, 4.00 mmol) and triethylamine (1.1 mL, 8.00 mmol) in dry acetonitrile (20 mL) at room temperature under nitrogen was added trimethylsilylcyanide (1.60 mL, 12.00 mmol). The mixture was heated at 75 °C for 20 h before it was cooled to room temperature, diluted with ethyl acetate and washed with saturated sodium bicarbonate solution and brine prior to drying over a2S04 and solvent concentration. The residue was flash chromatographed on silica gel (gradient elution with 5% ethyl acetate in hexanes to 25% ethyl acetate in hexanes) to afford Cap-138, step c (498.7 mg, 68%) as a white, crystalline solid along with 223 mg (30%) of additional Cap-138, step c recovered from the filtrate. ¾ NMR (CDC13, 500 MHz) δ 8.63 (d, J= 5.5 Hz, 1H), 8.26 (d, J= 5.5 Hz, 1H), 7.88 (d, J= 8.5 Hz, 1H), 7.69 (t, J= 8.0 Hz, 1H), 7.08 (d, J= 7.5 Hz, 1H), 4.04 (s, 3H); Rt = 1.75 min, (Cond.-Dl); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]+ C11H9N2O: 185.07; found: 185.10.
Cap-138
Cap-138, step c (0.45 g, 2.44 mmol) was treated with 5N sodium hydroxide solution (10 mL) and the resulting suspension was heated at 85 °C for 4 h, cooled to 25 °C, diluted with dichloromethane and acidified with IN hydrochloric acid. The organic phase was separated, washed with brine, dried over Na2S04, concentrated to ¼ volume and filtered to afford Cap-138 (0.44g, 88.9%) as a yellow solid. XH NMR (DMSO-d6, 400 MHz) δ 13.6 (br s, 1H), 8.56 (d, J= 6.0 Hz, 1H), 8.16 (d, J = 6.0 Hz, 1H), 8.06 (d, J= 8.8 Hz, 1H), 7.71-7.67 (m, 1H), 7.30 (d, J= 8.0 Hz, 1H), 4.02 (s, 3H); Rt = 0.70 min (Cond.-Dl); 95% homogenity index; LCMS: Anal. Calc. for [M+H]+ CiiH10NO3: 204.07; found: 204.05.
Synthetic Strategy. Method B (derived from Tetrahedron Letters, 2001, 42, 6707).
Figure imgf000259_0001
-139
Figure imgf000259_0002
-139, step a
Figure imgf000259_0003
To a thick-walled, screw-top vial containing an argon-degassed suspension of l-chloro-6-methoxyisoquinoline (1.2 g, 6.2 mmol; prepared according to the procedure in WO 2003/ 099274), potassium cyanide (0.40 g, 6.2 mmol), 1,5- bis(diphenylphosphino)pentane (0.27 g, 0.62 mmol) and palladium (II) acetate (70 mg, 0.31 mmol) in anhydrous toluene (6 mL) was added Ν,Ν,Ν',Ν'- tetramethylethylenediamine (0.29 mL, 2.48 mmol). The vial was sealed, heated at 150 °C for 22 h and then allowed to cool to 25 °C. The reaction mixture was diluted with ethyl acetate, washed with water and brine, dried over Na2S04, filtered and concentrated. The residue was purified on silica gel (gradient elution with 5% ethyl acetate/hexanes to 25% ethyl acetate/hexanes) to afford Cap-139, step a (669.7 mg, 59%) as a white solid. ¾ NMR (CDC13, 500 MHz) δ 8.54 (d, J= 6.0 Hz, 1H), 8.22 (d, J= 9.0 Hz, 1H), 7.76 (d, J= 5.5 Hz, 1H), 7.41-7.39 (m, 1H), 7.13 (d, J= 2.0 Hz, 1H), 3.98 (s, 3H); Rt = 1.66 min (Cond.-Dl); 90% homogenity index; LCMS: Anal. Calc. for [M+H]+ C11H9N2O: 185.07; found: 185.2.
Cap-139
Cap-139 was prepared from the basic hydrolysis of Cap-139, step a with 5N NaOH according to the procedure described for Cap 138. XH NMR (400 MHz,
DMSO-d6) δ 13.63 (v br s, 1H), 8.60 (d, J= 9.3 Hz, 1H), 8.45 (d, J= 5.6 Hz, 1H), 7.95 (d, J= 5.9 Hz, 1H), 7.49 (d, J= 2.2 Hz, 1H), 7.44 (dd, J= 9.3, 2.5 Hz, 1H), 3.95 (s, 3H); Rt = 0.64 min (Cond.-Dl); 90% homogenity index; LCMS: Anal. Calc. for [M+H]+ CiiH10NO3: 204.07; found: 204.05.
Figure imgf000260_0001
-140, step a
Figure imgf000260_0002
To a vigorously-stirred mixture of l,3-dichloro-5-ethoxyisoquinoline (482 mg, 2.00 mmol; prepared according to the procedure in WO 2005/ 051410), palladium (II) acetate (9 mg, 0.04 mmol), sodium carbonate (223 mg, 2.10 mmol) and l,5-bis(diphenylphosphino)pentane (35 mg, 0.08 mmol) in dry
dimethylacetamide (2 mL) at 25 °C under nitrogen was added Ν,Ν,Ν',Ν'- tetramethylethylenediamine (60 mL, 0.40 mmol). After 10 min, the mixture was heated to 150 °C, and then a stock solution of acetone cyanohydrin (prepared from 457 μϊ^ of acetone cyanohydrin in 4.34 mL DMA) was added in 1 mL portions over 18 h using a syringe pump. The mixture was then partitioned between ethyl acetate and water and the organic layer was separated, washed with brine, dried over Na2S04, filtered and concentrated. The residue was purified on silica gel (gradient elution with 10% ethyl acetate in hexanes to 40% ethyl acetate in hexanes) to afford Cap-140, step a (160 mg, 34%) as a yellow solid. Rt = 2.46 min (Cond.-MS-W2); 90% homogenity index; LCMS: Anal. Calc. for [M+H]+ Ci2H9ClN20: 233.05; found: 233.08.
Cap-140 Cap-140 was prepared by the acid hydrolysis of Cap-140, step a with 12N HC1 as described in the procedure for the preparation of Cap 141, described below. Rt = 2.24 min (Cond.-MS-W2); 90% homogenity index; LCMS: Anal. Calc. for [M+H]+ C12HiiClN03: 252.04; found: 252.02. -141
Figure imgf000261_0001
-141, step a
Figure imgf000261_0002
Cap-141, step a was prepared from l-bromo-3-fluoroisoquinoline (prepared from 3-amino-l-bromoisoquinoline using the procedure outlined in J. Med. Chem. 1970, 13, 613) as described in the procedure for the preparation of Cap-140, step a (vide supra). ¾ NMR (500 MHz, CDC13) δ 8.35 (d, J= 8.5 Hz, 1H), 7.93 (d, J= 8.5 Hz, 1H), 7.83 (t, J= 7.63 Hz, 1H), 7.77-7.73 (m, 1H), 7.55 (s, 1H); Rt = 1.60 min (Cond.-Dl); 90% homogenity index; LCMS: Anal. Calc. for [M+H]+ Ci0H6FN2: 173.05; found: 172.99.
Cap-141
Cap-141, step a (83 mg, 0.48 mmol) was treated with 12NHC1 (3 mL) and the resulting slurry was heated at 80 °C for 16 h before it was cooled to room temperature and diluted with water (3 mL). The mixture was stirred for 10 min and then filtered to afford Cap-141 (44.1 mg, 48%) as an off-white solid. The filtrate was diluted with dichloromethane and washed with brine, dried over Na2S04, and concentrated to afford additional Cap-141 (29.30 mg, 32%) which was sufficiently pure to be carried forward directly. ¾ NMR (DMSO-d6, 500 MHz) δ 14.0 (br s, 1H), 8.59-8.57 (m, 1H), 8.10 (d, J= 8.5 Hz, 1H), 7.88-7.85 (m, 2H), 7.74-7.71 (m, 1H); Rt = 1.33 min (Cond.-Dl); 90% homogenity index; LCMS: Anal. Calc. for [M+H]+ CioH7FN02: 192.05; found: 191.97. -142
Figure imgf000262_0001
Cap-142, step a
Figure imgf000262_0002
Cap-142, step a was prepared from 4-bromoisoquinoline N-oxide as described in the two-step procedure for the preparation of Cap-138, steps b and = 1.45 min (Cond.-MS-Wl); 90% homogenity index; LCMS: Anal. Calc. for
[M+H]+ Ci0H6BrN2: 232.97; found: 233.00. -142, step b
Figure imgf000262_0003
To an argon-degassed suspension of Cap-142, step a (116 mg, 0.50 mmol), potassium phosphate tribasic (170 mg, 0.80 mmol), palladium (II) acetate (3.4 mg, 0.015 mmol) and 2-(dicyclohexylphosphino)biphenyl (11 mg, 0.03 mmol) in anhydrous toluene (1 mL) was added morpholine (61 μΐ,, 0.70 mmol). The mixture was heated at 100 °C for 16 h, cooled to 25 °C, filtered through diatomaceous earth (Celite®) and concentrated. Purification of the residue on silica gel (gradient elution with 10% to 70% ethyl acetate in hexanes) afforded Cap-142, step b (38 mg, 32%) as a yellow solid which was carried forward directly. Rt = 1.26 min (Cond.-MS-Wl); 90% homogenity index; LCMS: Anal. Calc. for [M+H Ci4H14N30: 240.11 ; found:
Cap-142
Cap-142 was prepared from Cap-142, step b with 5N sodium hydroxide as described in the procedure for Cap 138. Rt = 0.72 min (Cond.-MS-Wl); 90% homogenity index; LCMS: Anal. Calc. for [M+H]+ Ci4H15N203: 259.11 ; found: 259.08.
Figure imgf000263_0001
-143, step a
Figure imgf000263_0002
To a stirred solution of 3-amino-l-bromoisoquinoline (444 mg, 2.00 mmol) in anhydrous dimethylformamide (10 mL) was added sodium hydride (60%, unwashed, 96 mg, 2.4 mmol) in one portion. The mixture was stirred at 25 °C for 5 min before 2-bromoethyl ether (90%, 250 μί, 2.00 mmol) was added. This mixture was stirred further at 25 °C for 5 h and at 75 °C for 72 h before it was cooled to 25 °C, quenched with saturated ammonium chloride solution and diluted with ethyl acetate. The organic layer was separated, washed with water and brine, dried over Na2S04, filtered and concentrated. Purification of the residue on silica gel (gradient elution with 0% to 70% ethyl acetate in hexanes) afforded Cap-143, step a (180 mg, 31%) as a yellow solid. Rt = 1.75 min (Cond.-MS-Wl); 90% homogenity index; LCMS: Anal. Calc. for [M+H]+ Ci3H14BrN20: 293.03; found: 293.04.
Cap-143 To a cold (-60 °C) solution of Cap- 143, step a (154 mg, 0.527 mmol) in anhydrous tetrahydrofuran (5 mL) was added a solution of w-butyllithium in hexanes (2.5 M, 0.25 mL, 0.633 mmol). After 10 min, dry carbon dioxide was bubbled into the reaction mixture for 10 min before it was quenched with INHCl and allowed to warm to 25 °C. The mixture was then extracted with dichloromethane (3 x 30 mL) and the combined organic extracts were concentrated in vacuo. Purification of the residue by reverse phase HPLC (MeOH/water/TFA) afforded Cap-143 (16 mg, 12%). ¾ = 1.10 min (Cond.-MS-Wl); 90% homogenity index; LCMS: Anal. Calc. for [M+H]+ Ci4H15 203: 259.1 1; found: 259.08. -144
Figure imgf000264_0001
Cap-144, step a
Figure imgf000264_0002
1,3-Dichloroisoquinoline (2.75 g, 13.89 mmol) was added in small portions to a cold (0 °C) solution of fuming nitric acid (10 mL) and concentrated sulfuric acid (10 mL). The mixture was stirred at 0 °C for 0.5 h before it was gradually warmed to 25 °C where it stirred for 16 h. The mixture was then poured into a beaker containing chopped ice and water and the resulting suspension was stirred for 1 h at 0 °C before it was filtered to afford Cap-144, step a (2.73 g, 81%) as a yellow solid which was used directly. Rt = 2.01 min (Cond.-Dl); 95% homogenity index; LCMS: Anal. Calc. for [M+H]+ C9H5CI2 2O2: 242.97; found: 242.92.
Cap-144, step b
Figure imgf000265_0001
Cap- 144, step a (0.30 g, 1.23 mmol) was taken up in methanol (60 niL) and treated with platinum oxide (30 mg), and the suspension was subjected to Parr hydrogenation at 7 psi ¾ for 1.5 h before formalin (5 mL) and additional platinum oxide (30 mg) were added. The suspension was resubjected to Parr hydrogenation at 45 psi H2 for 13 h before it was suction- filtered through diatomaceous earth (Celite®) and concentrated down to ¼ volume. Suction-filtration of the ensuing precipitate afforded the title compound as a yellow solid which was flash chromatographed on silica gel (gradient elution with 5% ethyl acetate in hexanes to 25% ethyl acetate in hexanes) to afford Cap-144, step b (231 mg, 78%) as a pale, yellow solid. Rt = 2.36 min (Cond.-Dl); 95% homogenity index; XH NMR (400 MHz, CDC13) δ 8.02 (s, 1H), 7.95 (d, J= 8.6 Hz, 1H), 7.57-7.53 (m, 1H), 7.30 (d, J= 7.3 Hz, 1H), 2.88 (s, 6H); LCMS: Anal. Calc. for [M+H]+ C11H11CI2N2: 241.03; found: 241.02. HRMS: Anal. Calc. for [M+H]+ CnHnC^: 241.0299; found: 241.0296.
C -144, step c
Figure imgf000265_0002
Cap-144, step c was prepared from Cap-144, step b according to the procedure described for the preparation of Cap-139, step a. Rt = 2.19 min (Cond.- Dl); 95% homogenity index; LCMS: Anal. Calc. for [M+H]+ C12HnClN3: 232.06; found: 232.03. HRMS: Anal. Calc. for [M+H]+ C12H11CIN3: 232.0642; found: 232.0631.
Cap-144 Cap-144 was prepared according to the procedure described for Cap-141. Rt = 2.36 min (Cond.-Dl); 90%; LCMS: Anal. Calc. for [M+H]+ Ci2H12Cl 202: 238.01 ; found: 238.09.
Caps-145 to -162
Caps-145 to 162 were prepared from the appropriate 1 -chloroisoquinolines according to the procedure described for the preparation of Cap-138 (Method A) or Cap-139 (Method B) unless noted otherwise as outlined below.
Figure imgf000266_0001
1.40 min
(Cond.-
Dl);
95% ;
LCMS:
Cap- C02H Anal.
146 Prepared from A 5NNaOH Calc. for commercially available 3- [M+H]+ hydroxyisoquinoline CnHio
03: 204.07; found: 204.06.
0.87 min
(Cond.-
Dl);
95%;
LCMS:
Cap- C02H Anal.
147 Prepared from B 5NNaOH Calc. for commercially available 1- [M+H]+ chloro-4- CnHio hydroxyisoquinoline 03:
204.07; found: 204.05.
0.70 min
Cap- C02H (Cond.-
148 Prepared from A 5NNaOH Dl); commercially available 7- 95%; hydroxyisoquinoline LCMS:
Figure imgf000268_0001
204.07;
found:
204.04.
1.78 min
(Cond.-
Dl); 90%;
LCMS:
C02H
Cap- Anal.
Prepared from 5-methoxy- 151 B 12NHC1 Calc. for
1,3-dichloroisoquinoline,
[M+H]+ which can be synthesized
Figure imgf000269_0001
following the procedure in
03: WO 2005 / 051410.
238.03; found: 238.09.
1.65 min
(Cond.-
Dl);
95%;
LCMS:
C02H
Cap- Anal. 152 Prepared from B 12NHC1 Calc. for commercially available 6- [M+H]+ methoxy-1,3- C11H9CIN dichloroisoquinoline
03: 238.00; found: 238.09.
Figure imgf000270_0001
following the procedure in Anal. WO 2003/ 099274 Calc. for
[M+H]+
Figure imgf000271_0001
02: 208.02; found: 208.00.
0.60 min
(Cond.-
MS-W1);
90%;
C02H LCMS:
Cap- Prepared from 1,6- Anal.
156 dichloroisoquinoline, which B 5NNaOH Calc. for can be synthesized [M+H]+ following the procedure in C10H7CIN
WO 2003/ 099274 02:
208.02; found:
208.03.
1.49 min
CI (Cond.- Dl);
C02H 95%;
Cap-
Prepared from 1,4- LCMS: 157 B 12NHC1
dichloroisoquinoline, Anal. which can be synthesized Calc. for following the procedure in [M+H]+ WO 2003/ 062241 C10H17CI
NO:
Figure imgf000272_0001
Figure imgf000273_0001
953. [M+H]
CnHio
03: 204.07; found: 203.94. - 163
Figure imgf000274_0001
To a solution of 2-ketobutyric acid (1.0 g, 9.8 mmol) in diethylether (25 ml) was added phenylmagnesium bromide (22 ml, 1M in THF) dropwise. The reaction was stirred at ~25 °C under nitrogen for 17.5h. The reaction was acidified with IN HC1 and the product was extracted with ethyl acetate (3 x 100 ml). The combined organic layer was washed with water followed by brine and dried over MgS04. After concentration in vacuo, a white solid was obtained. The solid was recrystallized from hexanes/ethyl acetate to afford Cap-163 as white needles (883.5 mg). XH NMR (DMSO-d6, δ = 2.5 ppm, 500 MHz): 12.71 (br s, 1 H), 7.54-7.52 (m, 2H), 7.34-7.31 (m, 2H), 7.26-7.23 (m, 1H), 5.52-5.39 (br s, 1H), 2.11 (m, 1H), 1.88 (m, 1H), 0.79 (app t, J = 7.4 Hz, 3H). -164
Figure imgf000274_0002
A mixture of 2-amino-2-phenylbutyric acid (1.5 g, 8.4 mmol), formaldehyde (14 mL, 37% in water), IN HC1 (10 mL) and 10% Pd/C (0.5 mg) in MeOH (40 mL) was exposed to ¾ at 50 psi in a Parr bottle for 42 h. The reaction was filtered over Celite and concentrated in vacuo, the residue was taken up in MeOH (36 mL) and the product was purified with a reverse phase HPLC (MeOH/H20/TFA) to afford the TFA salt of Cap-164 as a white solid (1.7 g). 1H NMR (DMSO-d6, δ = 2.5 ppm, 500 MHz) 7.54-7.47 (m, 5H), 2.63 (m, 1H), 2.55 (s, 6H), 2.31 (m, 1H), 0.95 (app t, J = 7.3 Hz, 3H).
Cap- 165
Figure imgf000275_0001
To a mixture of 2-amino-2-indanecarboxylic acid (258.6 mg, 1.46 mmol) and formic acid (0.6 ml, 15.9 mmol) in 1,2-dichloroethane (7 ml) was added
formaldehyde (0.6 ml, 37% in water). The mixture was stirred at ~25 °C for 15 min then heated at 70 °C for 8h. The volatile component was removed in vacuo, and the residue was dissolved in DMF (14 mL) and purified by a reverse phase HPLC
(MeOH/H20/TFA) to afford the TFA salt of Cap-165 as a viscous oil (120.2 mg). XH
NMR (DMSO-d6, δ = 2.5 ppm, 500 MHz): 7.29-7.21 (m, 4 H), 3.61 (d, J = 17.4 Hz,
2H), 3.50 (d, J = 17.4 Hz, 2H), 2.75 (s, 6H). LC/MS: Anal. Calcd. for [M+H]+
Ci2H16N02: 206.12; found: 206.07.
Cap-166a and -166b
Cap-166a: Diastereomer-1
Cap-166b: Diastereomer-2
Figure imgf000275_0002
Cops-166a and -166b were prepared from (IS, 4S)-(+)-2-methyl-2,5- diazabicyclo[2.2.1]heptane (2HBr) according to the method described for the synthesis of Cap-7a and Cap-7b, with the exception that the benzyl ester intermediate was separated using a semi-prep Chrialcel OJ column, 20 x 250 mm, 10 μηι eluting with 85: 15 heptane/ethanol mixture at 10 mL/min elution rate for 25 min. Cap-166b: ¾ NMR (DMSO-d6, δ = 2.5 ppm, 500 MHz): 7.45 (d, J = 7.3 Hz, 2H), 7.27-7.19 (m, 3H), 4.09 (s, 1H), 3.34 (app br s, 1H), 3.16 (app br s, 1H), 2.83 (d, J= 10.1 Hz, 1H), 2.71 (m, 2H), 2.46 (m, 1H), 2.27 (s, 3H), 1.77 (d, J = 9.8 Hz, 1H), 1.63 (d, J = 9.8 Hz, 1H). LC/MS: Anal. Calcd. for [M+H]+ Ci4H19 202: 247.14; found: 247.1 1. -167
Figure imgf000276_0001
A solution of racemic Boc-l,3-dihydro-2H-isoindole carboxylic acid (l .Og,
3.8 mmol) in 20% TFA/CH2C12 was stirred at -25 °C for 4h. All the volatile component was removed in vacuo. A mixture of the resultant crude material, formaldehyde (15 mL, 37% in water), IN HC1 (10 mL) and 10% Pd/C (10 mg) in MeOH was exposed to H2 (40 PSI) in a Parr bottle for 23 h. The reaction mixture was filtered over Celite and concentrated in vacuo to afford Cap-167 as a yellow foam (873.5 mg). ¾ NMR (DMSO-d6, δ = 2.5 ppm, 500 MHz) 7.59-7.38 (m, 4H), 5.59 (s, 1H), 4.84 (d, J = 14 Hz, 1H), 4.50 (d, J = 14.1 Hz, 1H), 3.07 (s, 3H). LC/MS: Anal. Calcd. for [M+H]+ Ci0H12NO2: 178.09; found: 178.65. - 168
Figure imgf000276_0002
Racemic Cap-168 was prepared from racemic Boc-aminoindane-1 -carboxylic acid according to the procedure described for the preparation of Cap-167. The crude material was employed as such.
Cap-169
Figure imgf000277_0001
A mixture of 2-amino-2-phenylpropanoic acid hydrochloride (5.0 g, 2.5 mmol), formaldehyde (15 ml, 37% in water), IN HC1 (15 ml), and 10% Pd/C (1.32 g) in MeOH (60 mL) was placed in a Parr bottle and shaken under hydrogen (55 PSI) for 4 days. The reaction mixture was filtered through diatomaceous earth (Celite ) and concentrated in vacuo. The residue was taken up in MeOH and purified by reverse phase prep-HPLC (MeOH/water/TFA) to afford the TFA salt of Cap- 169 as a viscous semi-solid (2.1 g). XH NMR (CDC13, δ = 7.26 ppm, 500 MHz): 7.58-7.52 (m, 2 H), 7.39-7.33 (m, 3H), 2.86 (br s, 3H), 2.47 (br s, 3H), 1.93 (s, 3H). LC/MS: Anal. Calcd. for [M+H]+ CnHi6N02: 194.12; found: 194.12.
Ca - 170
Figure imgf000277_0002
To (5)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid (505mg; 3.18mmol; obtained from Astatech) in water (15ml) was added sodium carbonate (673mg;
6.35mmol), and the resultant mixture was cooled to 0 °C and then methyl chloroformate (0.26ml; 3.33mmol) was added dropwise over 5 minutes. The reaction was allowed to stir for 18 hours while allowing the bath to thaw to ambient temperature. The reaction mixture was then partitioned between IN HQ and ethyl acetate. The organic layer was removed and the aqueous layer was further extracted with 2 additional portions of ethyl acetate. The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated in vacuo to afford Cap-170 a colorless residue. ¾ NMR (500 MHz, DMSO-d6) δ ppm 12.65 (1 H, br s), 7.44 (1 H, d, J=8.24 Hz), 3.77 - 3.95 (3 H, m), 3.54 (3 H, s), 3.11 - 3.26 (2 H, m), 1.82 - 1.95 (1 H, m), 1.41 - 1.55 (2 H, m), 1.21 - 1.39 (2 H, m); LC/MS: Anal. Calcd. for [M+H]+ C9H16N05: 218.1 ; found 218.1. - 171
Figure imgf000278_0001
A solution of methyl 2-(benzyloxycarbonylamino)-2-(oxetan-3- ylidene)acetate (200 mg, 0.721 mmol; II Farmaco (2001), 56, 609-613) in ethyl acetate (7 ml) and CH2CI2 (4.00 ml) was degassed by bubbling nitrogen for lOmin. Dimethyl dicarbonate (0.1 16 ml, 1.082 mmol) and Pd/C (20 mg, 0.019 mmol) were then added, the reaction mixture was fitted with a hydrogen balloon and allowed to stir at ambient temperature overnight at which time TLC (95:5 CH2CI2 / MeOH: visulalized with stain made from lg Ce( H4)2S04, 6g ammonium molybdate, 6ml sulfuric acid, and 100ml water) indicated complete conversion. The reaction was filtered through celite and concentrated. The residue was purified via Biotage® (load with dichloromethane on 25 samplet; elute on 25S column with dichloromethane for 3CV then 0 to 5% MeOH / dichloromethane over 250ml then hold at 5% MeOH / dichloromethane for 250ml; 9ml fractions). Collected fractions containing desired material and concentrated to 120mg (81%) of methyl 2-(methoxycarbonylamino)-2- (oxetan-3-yl)acetate as a colorless oil. ¾ NMR (500 MHz, CHLOROFORM-D) δ ppm 3.29 - 3.40 (m, J=6.71 Hz, 1 H) 3.70 (s, 3 H) 3.74 (s, 3 H) 4.55 (t, J=6.41 Hz, 1 H) 4.58 - 4.68 (m, 2 H) 4.67 - 4.78 (m, 2 H) 5.31 (br s, 1 H). LC/MS: Anal. Calcd. for [M+H]+ C8Hi4N05: 204.2; found 204.0.
To methyl 2-(methoxycarbonylamino)-2-(oxetan-3-yl)acetate (50 mg, 0.246 mmol) in THF (2 mL) and water (0.5 mL) was added lithium hydroxide monohydrate (10.33 mg, 0.246 mmol). The resultant solution was allowed to stir overnite at ambient temperature. TLC (1 : 1 EA / Hex; Hanessian stain [lg Ce(NH4)2S04, 6g ammonium molybdate, 6ml sulfuric acid, and 100ml water]) indicated -10% starting material remaining. Added an additional 3mg LiOH and allowed to stir overnight at which time TLC showed no starting material remaining. Concentrated in vacuo and placed on high vac overnite providing 55mg lithium 2-(methoxycarbonylamino)-2-
(oxetan-3-yl)acetate as a colorless solid. XH NMR (500 MHz, MeOD) δ ppm 3.39 - 3.47 (m, 1 H) 3.67 (s, 3 H) 4.28 (d, J=7.93 Hz, 1 H) 4.64 (t, J=6.26 Hz, 1 H) 4.68 (t, J=7.02 Hz, 1 H) 4.73 (d, J=7.63 Hz, 2 H). - 172
Figure imgf000279_0001
-172, step a
Figure imgf000279_0002
The following diazotization step was adapted from Barton, A.; Breukelman, S. P.; Kaye, P. T.; Meakins, G. D.; Morgan, D. I J. C. 5". Perkin Trans I 1982, 159-164: A solution of NaN02 (166 mg, 2.4 mmol) in water (0.6 mL) was added slowly to a stirred, cold (0 °C) solution of methyl 2-amino-5-ethyl-l,3-thiazole-4- carboxylate (186 mg, 1.0 mmol), CuS04*5H20 (330 mg, 1.32 mmol), NaCl (260 mg, 4.45 mmol) and H2SO4 (5.5 mL) in water (7.5 mL). The mixture was stirred at 0 °C for 45 min and allowed to warm up to room temperature where it stirred further for 1 h before CuCl (1 18 mg) was added. This mixture was stirred further at room temperature for 16 h before it was diluted with brine and extracted with ether twice. The organic layers were combined, dried over MgS04 and concentrated to give methyl 2-chloro-5-ethylthiazole-4-carboxylate (i.e. Cap-172, step a) (175 mg, 85%) as an orange oil (80% pure) which was used directly in the next reaction. Rt = 1.99 min (Cond.-MDl); LC/MS: Anal. Calcd. for [M+H]+ C7H9C1N02S: 206.01; found: 206.05.
Cap-172
To a solution of methyl 2-chloro-5-ethylthiazole-4-carboxylate (175 mg) in THF/H20/MeOH (20 mL/ 3 mL/ 12 mL) was added LiOH (305 mg, 12.76 mmol). The mixture was stirred at room temperature overnight before it was concentrated down and neutralized with 1NHC1 in ether (25 mL). The residue was extracted twice with ethyl acetate and the organic layers were combined, dried over MgS04 and evaporated to yield Cap-172 (60 mg, 74%) as a red solid which was used without further purification. ¾ NMR (300 MHz, DMSO-d6) δ ppm 13.03-13.42 (1 H, m), 3.16 (2 H, q, J= 7.4 Hz), 1.23 (3 H, t, J= 7.5 Hz). Rt = 1.78 min (Cond.-MDl); LC/MS: Anal. Calcd. for [M+H]+ C6H7C1N02S: 191.99; found: 191.99. -173
Figure imgf000280_0001
C -173, step a
Figure imgf000280_0002
The following diazotization step was adapted from Barton, A.; Breukelman, S. P.; Kaye, P. T.; Meakins, G. D.; Morgan, D. J. J. C S. Perkin Trans I 1982, 159-164: A solution of NaN02 (150 mg, 2.17 mmol) in water (1.0 mL) was added dropwise to a stirred, cold (0 °C) solution of methyl 2-amino-5-ethyl-l,3-thiazole-4- carboxylate (186 mg, 1.0 mmol) in 50% H3PO2 (3.2 mL). The mixture was stirred at 0 °C for 1 h and allowed to warm up to room temperature where it stirred further for 2h. After recooling to 0 °C, the mixture was treated slowly with a solution of NaOH (85 mg) in water (10 mL). The mixture was then diluted with saturated aHC03 solution and extracted twice with ether. The organic layers were combined, dried over MgS04 and concentrated to give methyl 5-ethylthiazole-4-carboxylate (i.e. Cap- 173, step a) (134 mg, 78%) as an orange oil (85% pure) which was used directly in the next reaction. Rt = 1.58 min (Cond.-MDl); LC/MS: Anal. Calcd. for [M+H]+ C7H10NO2S: 172.05; found: 172.05.
Cap-173 To a solution of methyl 5-ethylthiazole-4-carboxylate (134 mg) in
THF/H20/MeOH (18 mL/ 2.7 mL/ 1 1 mL) was added LiOH (281 mg, 1 1.74 mmol). The mixture was stirred at room temperature overnight before it was concentrated down and neutralized with 1NHC1 in ether (25 mL). The residue was extracted twice with ethyl acetate and the organic layers were combined, dried over MgS04 and evaporated to yield Cap-173 (90 mg, 73%) as an orange solid which was used without further purification. XH NMR (300 MHz, DMSO-d6) δ ppm 12.74-13.04 (1 H, m), 3.20 (2 H, q, J= 7.3 Hz), 1.25 (3 H, t, J= 7.5 Hz). Rt = 1.27 min (Cond.- MD1); LC/ S: Anal. Calcd. for [M+H]+ C6H8N02S: 158.03; found: 158.04.
Figure imgf000281_0001
-174, step a
Figure imgf000281_0002
Triflic anhydride (5.0 g, 18.0 mmol) was added dropwise to a cold (0 °C) solution of methyl 3-hydroxypicolinate (2.5 g, 16.3 mmol) and TEA (2.5 mL, 18.0 mmol) in CH2CI2 (80 mL). The mixture was stirred at 0 °C for lh before it was allowed to warm up to room temperature where it stirred for an additional 1 h. The mixture was then quenched with saturated aHC03 solution (40 mL) and the organic layer was separated, washed with brine, dried over MgSC and concentrated to give methyl 3-(trifluoromethylsulfonyloxy)picolinate (i.e. Cap-174, step a) (3.38 g, 73%) as a dark brown oil (>95% pure) which was used directly without further purification. XH NMR (300 MHz, CDC13) δ ppm 8.72-8.79 (1 H, m), 7.71 (1 H, d, J = 1.5 Hz), 7.58-7.65 (1 H, m), 4.04 (3 H, s). Rt = 1.93 min (Cond.-MDl); LC/MS: Anal. Calcd. for [M+H]+ C8H7F3 O5S: 286.00; found: 286.08. Cap- 174
To a solution of methyl 3-(trifluoromethylsulfonyloxy)picolinate (570 mg, 2.0 mmol) in DMF (20 mL) was added LiCl (254 mg, 6.0 mmol), tributyl(vinyl)stannane (761 mg, 2.4 mmol) and bis(triphenylphosphine)palladium dichloride (42 mg, 0.06 mmol). The mixture was heated at 100 °C overnight before a saturated solution of KF (20 mL) was added to the reaction mixture at room temperature. This mixture was stirred for 4 h before it was filtered through diatomaceous earth (Celite ) and the pad was washed with ethyl acetate. The aqueous phase of the filtrate was then separated and concentrated down in vacuo. The residue was treated with 4NHC1 in dioxanes (5 mL) and the resulting mixture was extracted with methanol, filtered and evaporated to afford Cap-174 (260 mg) as a green solid which was slightly contaminated with inorganic salts but was used without further purification. XH NMR (300 MHz, DMSO-d6) δ ppm 8.21 (1 H, d, J= 3.7 Hz), 7.81-7.90 (1 H, m), 7.09 (1 H, dd, J= 7.7, 4.8 Hz), 6.98 (1 H, dd, J= 17.9, 1 1.3 Hz), 5.74 (1 H, dd, J= 17.9, 1.5 Hz), 5.20 (1 H, d, J= 11.0 Hz). Rt = 0.39 min (Cond.-MDl); LC/MS: Anal. Calcd. for [M+H]+ C8H8N02: 150.06; found: 150.07.
Figure imgf000282_0001
a
Figure imgf000282_0002
To a solution of methyl 3-(trifluoromethylsulfonyloxy)picolinate (i.e. Cap
173, step a) (570 mg, 2.0 mmol), an intermediate in the preparation of Cap-174, in
DMF (20 mL) was added LiCl (254 mg, 6.0 mmol), tributyl(vinyl)stannane (761 mg
2.4 mmol) and bis(triphenylphosphine)palladium dichloride (42 mg, 0.06 mmol).
The mixture was heated at 100 °C for 4 h before the solvent was removed in vacuo. The residue was taken up in acetonitrile (50 mL) and hexanes (50 mL) and the resulting mixture was washed twice with hexanes. The acetonitrile layer was then separated, filtered through Celite, and evaporated. Purification of the residue by flash chromatography on a Horizon instrument (gradient elution with 25% ethyl acetate in hexanes to 65% ethyl acetate in hexanes) afforded methyl 3-vinylpicolinate (i.e. Cap-175, step a) (130 mg, 40%) as a yellow oil. XH NMR (300 MHz, CDC13) δ ppm 8.60 (1 H, dd, J= 4.6, 1.7 Hz), 7.94 (1 H, d, J= 7.7 Hz), 7.33-7.51 (2 H, m), 5.72 (1 H, d, J= 17.2 Hz), 5.47 (1 H, d, J= 11.0 Hz), 3.99 (3 H, s). Rt = 1.29 min (Cond.-MDl); LC/MS: Anal. Calcd. for [M+H]+ C9H10NO2: 164.07; found: 164.06. b
Figure imgf000283_0001
Palladium on carbon (10%, 25 mg) was added to a solution of methyl 3- vinylpicolinate (120 mg, 0.74 mmol) in ethanol (10 mL). The suspension was stirred at room temperature under an atmosphere of hydrogen for 1 h before it was filtered
®
through Celite and the pad of diatomaceous earth (Celite ) was washed with methanol. The filtrate was concentrated down to dryness to yield methyl 3- ethylpicolinate (i.e. Cap-175, step b) which was taken directly into the next reaction. Rt = 1.15 min (Cond.-MDl); LC/MS: Anal. Calcd. for [M+H]+ C9H12N02: 166.09; found: 166.09.
Cap-175
To a solution of methyl 3-ethylpicolinate in THF/H20/MeOH (5 mL/ 0.75 mL/ 3 mL) was added LiOH (35 mg, 1.47 mmol). The mixture was stirred at room temperature for 2 d before additional LiOH (80 mg) was added. After an additional 24 h at room temperature, the mixture was filtered and the solvent was removed in vacuo. The residue was then treated with 4NHC1 in dioxanes (5 mL) and the resulting suspension was concentrated down to dryness to yield Cap-175 as a yellow solid which was used without further purification. XH NMR (300 MHz, DMSO-d6) δ ppm 8.47 (1 H, dd, J= 4.8, 1.5 Hz), 7.82-7.89 (1 H, m), 7.53 (1 H, dd, J= 7.7, 4.8 Hz), 2.82 (2 H, q, J= 7.3 Hz), 1.17 (3 H, t, J= 7.5 Hz). Rt = 0.36 min (Cond.-MDl); LC/MS: Anal. Calcd. for [M+H]+ C8H10NO2: 152.07; found: 152.10.
- 176
Figure imgf000284_0001
- 176, step a
Figure imgf000284_0002
A solution of l,4-dioxaspiro[4.5]decan-8-one (15 g, 96 mmol) in EtOAc (150 mL) was added to a solution of methyl 2-(benzyloxycarbonylamino)-2- (dimethoxyphosphoryl)acetate (21.21 g, 64.0 mmol) in 1,1,3,3-tetramethylguanidine (10.45 mL, 83 mmol) and EtOAc (150 mL). The resulting solution was the stirred at ambient temperature for 72 h and then it was diluted with EtOAc (25 mL). The organic layer was washed with IN HC1 (75 mL), H20 (100 mL) and brine (100 mL), dried (MgSC^), filtered and concentrated. The residue was purified via Biotage (5% to 25 % EtOAc/Hexanes; 300g column). The combined fractions containing the product were then concentrated under vacuum and the residue was re-crystallized from hexanes/EtOAc to give white crystals that corresponded to methyl 2- (benzyloxycarbonylamino)-2-(l,4-dioxaspiro[4.5]decan-8-ylidene)acetate (6.2 g) XH NMR (400 MHz, DC -d) δ ppm 7.30 - 7.44 (5 H, m), 6.02 (1 H, br. s.), 5.15 (2 H, s), 3.97 (4 H, s), 3.76 (3 H, br. s.), 2.84 - 2.92 (2 H, m), 2.47 (2 H, t, J=6.40 Hz), 1.74 - 1.83 (4 H, m). LC (Cond. OL1): Rt = 2.89 min. LC/MS: Anal. Calcd. For [M+Na]+ Ci9H23 a06: 745.21 ; found: 745.47.
Cap 176, step b
Figure imgf000285_0001
Ester Cap 176, step b was prepared from alkene Cap 176, step a according to the method of Burk, M. J.; Gross, M. F. and Martinez J. P. (J. Am. Chem. Soc., 1995, 117, 9375-9376 and references therein): A 500 mL high-pressure bottle was charged with alkene Cap 176, step a (3.5 g, 9.68 mmol) in degassed MeOH (200 mL) under a blanket of N2. The solution was then charged with (-)-l,2-Bis((2S,5S)-2,5- dimethylphospholano)ethane(cyclooctadiene)rhodium (I) tetrafluoroborate (0.108 g, 0.194 mmol) and the resulting mixture was flushed with 2 (3x) and charged with ¾ (3x). The solution was shaken vigorously under 70 psi of H2 at ambient temperature for 72 h. The solvent was removed under reduced pressure and the remaining residue was taken up in EtOAc. The brownish solution was then filtered through a plug of Silica Gel and eluted with EtOAc. The solvent was concentrated under vacuum to afford a clear oil corresponding to ester Cap 176, step b (3.4 g) .XH NMR (500 MHz, DC -d) δ ppm 7.28 - 7.43 (5 H, m), 5.32 (1 H, d, J=9.16 Hz), 5.06 - 5.16 (2 H, m), 4.37 (1 H, dd, J=9.00, 5.04 Hz), 3.92 (4 H, t, J=3.05 Hz), 3.75 (3 H, s), 1.64 - 1.92 (4 H, m), 1.37 - 1.60 (5 H, m). LC (Cond. OL1): Rt = 1.95 min. LC/MS: Anal. Calcd. For [M+H]+ Ci9H26N06: 364.18; found: 364.27.
Cap 176, step c
Figure imgf000285_0002
Ester Cap 176, step b (4.78 g, 13.15 mmol) was dissolved in THF (15 mL) followed by sequential addition of water (10 mL), glacial acetic acid (26.4 mL, 460 mmol) and dichloroacetic acid (5.44 niL, 65.8 mmol). The resulting mixture was stirred for 72 h at ambient temperature, and the reaction was quenched by slow addition of solid a2C03 with vigorous stirring until the release of gas was no longer visible. Crude product was extracted into 10% ethyl acetate-dichloromethane and the organic layers were combined, dried (MgS04) filtered and concentrated. The resulting residue was purified via Biotage (0 to 30% EtOAc/Hex; 25 g column) to afford ketone Cap 176, step c (3.86g) as a clear oil. ¾ NMR (400 MHz, DC -d) δ ppm 7.28 - 7.41 (5 H, m), 5.55 (1 H, d, J=8.28 Hz), 5.09 (2 H, s), 4.46 (1 H, dd, J=8.16, 5.14 Hz), 3.74 (3 H, s), 2.18 - 2.46 (5 H, m), 1.96 - 2.06 (1 H, m), 1.90 (1 H, ddd, J=12.99, 5.96, 2.89 Hz), 1.44 - 1.68 (2 H, m, J=12.36, 12.36, 12.36, 12.36, 4.77 Hz). LC (Cond. OL1): Rt = 1.66 min. LC/MS: Anal. Calcd. For [M+Na
Ci7H21 a05: 342.13; found: 342.10.
Cap 176, step d
Figure imgf000286_0001
Deoxo-Fluor® (3.13 mL, 16.97 mmol) was added to a solution of ketone Cap 176, step c (2.71 g, 8.49 mmol) in CH2C12 (50 mL) followed by addition of a catalytic ammount of EtOH (0.149 mL, 2.55 mmol). The resulting yellowish solution was stirred at rt overnight. The reaction was quenched by addition of sat. aq. aHC03 (25 mL) and the mixture was extracted with EtO Ac (3X75 mL)). The combined organic layers were dried (MgS04), filtered and dried to give a yellowish oil. The residue was purified via Biotage chromatography (2% to 15% EtO Ac/Hex; 90g column) and a white solid corresponding to the difluoro amino acid dilforide Cap 176, step d (1.5 g) was recovered. XH NMR (400 MHz, , DC -d) δ ppm 7.29 -
7.46 (5 H, m), 5.34 (1 H, d, J=8.28 Hz), 5.12 (2 H, s), 4.41 (1 H, dd, J=8.66, 4.89 Hz), 3.77 (3 H, s), 2.06 - 2.20 (2 H, m), 1.83 - 1.98 (1 H, m), 1.60 - 1.81 (4 H, m), 1.38 - 1.55 (2 H, m). 19F NMR (376 MHz, DC -d) δ ppm -92.15 (1 F, d, J=237.55 Hz), -102.44 (1 F, d, J=235.82 Hz). LC (Cond. OL1): Rt = 1.66 min. LC/MS: Anal. Calcd. For [2M+Na]+ C34H42F4N2Na08: 705.28; found: 705.18.
Figure imgf000287_0001
Difluoride Cap 176, step d (4 g, 11.72 mmol) was dissolved in MeOH (120 mL) and charged with Pd/C (1.247 g, 1.172 mmol). The suspension was flushed with 2 (3x) and the reaction mixture was placed under 1 atm of ¾ (balloon). The mixture was stirred at ambient temperature for 48 h. The suspension was then filtered though a plug of Celite and concentrated under vacuum to give an oil that corresponded to amino acid Cap 176, step e (2.04 g) and that was used without further purification. ¾ NMR (400 MHz, DMSO-de) δ ppm 3.62 (3 H, s), 3.20 (1 H, d, J=5.77 Hz), 1.91 - 2.09 (2 H, m), 1.50 - 1.88 (7 H, m), 1.20 - 1.45 (2 H, m). 19F NMR (376 MHz, DMSO-d6) δ ppm -89.39 (1 F, d, J=232.35 Hz), -100.07 (1 F, d, J=232.35 Hz). 13C MR (101 MHz, DMSO-de) δ ppm 175.51 (1 C, s), 124.10 (1 C, t, J=241.21, 238.90 Hz), 57.74 (1 C, s), 51.39 (1 C, s), 39.23 (1 C, br. s.), 32.02 - 33.83 (2 C, m), 25.36 (1 C, d, J=10.02 Hz), 23.74 (1 C, d, J=9.25 Hz). LC (Cond. OL2): Rt = 0.95 min. LC/MS: Anal. Calcd. For [2M+ttf Cntt^V AY\202 415.22; found: 415.40.
Figure imgf000287_0002
Methyl chloroformate (1.495 mL, 19.30 mmol) was added to a solution of amino acid Cap 176, step e (2 g, 9.65 mmol) and DIEA (6.74 mL, 38.6 mmol) in CH2CI2 (100 mL). The resulting solution was stirred at rt for 3 h and volatiles were removed under reduced pressure. The residue was purified via Biotage (0% to 20% EtO Ac/Hex; 90g column). A clear oil that solidified upon standing under vacuum and corresponding to carbamate Cap-176, step f (2.22 g) was recovered. XH NMR (500 MHz, CDC-3-ί δ ppm 5.27 (1 H, d, J=8.55 Hz), 4.39 (1 H, dd, J=8.85, 4.88 Hz), 3.77 (3 H, s), 3.70 (3 H, s), 2.07 - 2.20 (2 H, m), 1.84 - 1.96 (1 H, m), 1.64 - 1.82 (4 H, m), 1.39 - 1.51 (2 H, m). 19F NMR (471 MHz, CDC -d) δ ppm -92.55 (1 F, d, J=237.13 Hz), -102.93 (1 F, d, J=237.12 Hz). 13C NMR (126 MHz, COC -d) δ ppm 171.97 (1 C, s), 156.69 (1 C, s), 119.77 - 125.59 (1 C, m), 57.24 (1 C, br. s.), 52.48 (1 C, br. s.), 52.43 (1 C, s), 39.15 (1 C, s), 32.50 - 33.48 (2 C, m), 25.30 (1 C, d, J=9.60 Hz), 24.03 (1 C, d, J=9.60 Hz). LC (Cond. OL1): Rt = 1.49 min. LC/MS: Anal. Calcd. For [M+Na]+ CiiH17F2NNa04: 288.10; found: 288.03.
Cap- 176
A solution of LiOH (0.379 g, 15.83 mmol) in Water (25 mL) was added to a solution of carbamate Cap- 176, step f(2A g, 7.92 mmol) in THF (75 mL) and the resulting mixture was stirred at ambient temperature for 4 h. THF was removed under vacuum and the remaining aqueous phase was acidified with IN HC1 solution (2 mL) and then extracted with EtOAc (2 X 50 mL). The combined organic layers were dried (MgS04), filtered and concentrated to give a white foam corresponding to Cap-176 (1.92 g). XH NMR (400 MHz, DMSO-d6) δ ppm 12.73 (1 H, s), 7.50 (1 H, d, J=8.78 Hz), 3.97 (1 H, dd, J=8.53, 6.02 Hz), 3.54 (3 H, s), 1.92 - 2.08 (2 H, m), 1.57 - 1.90 (5 H, m), 1.34 - 1.48 (1 H, m), 1.27 (1 H, qd, J=12.72, 3.26 Hz). 19F NMR (376 MHz, DMSO-de) δ ppm -89.62 (1 F, d, J=232.35 Hz), -99.93 (1 F, d, J=232.35 Hz). LC (Cond. OL2): Rt = 0.76 min. LC/MS: Anal. Calcd. For [M-H]+ Ci0H14F2NO4: 250.09; found: 250.10.
BIOLOGICAL ACTIVITY
An HCV Replicon assay was utilized in the present disclosure, and was prepared, conducted and validated as described in commonly owned
PCT/US2006/022197 and in O'Boyle et. al. Antimicrob Agents Chemother. 2005 Apr;49(4): 1346-53. Assay methods incorporating luciferase reporters have also been used as described (Apath.com).
HCV-neo replicon cells and replicon cells containing resistance substitutions in the NS5A region were used to test the currently described family of compounds. The compounds were determined to have differing degrees of reduced inhibitory activity on cells containing mutations vs. the corresponding inhibitory potency against wild-type cells. Thus, the compounds of the present disclosure can be effective in inhibiting the function of the HCV NS5A protein and are understood to be as effective in combinations as previously described in application
PCT/US2006/022197 and commonly owned WO/04014852. It should be understood that the compounds of the present disclosure can inhibit multiple genotypes of HCV. Table 2 shows the EC50 (Effective 50% inhibitory concentration) values of representative compounds of the present disclosure against the HCV lb genotype. In one embodiment, compounds of the present disclosure are inhibitory versus la, lb, 2a, 2b, 3a, 4a, and 5a genotypes. EC50 values against HCV lb are as follows A (10- 350 nM); B (1-9.9 nM); C (0.1-0.99 nM); D (0.0006-0.099 nM).
Figure imgf000289_0001
Compound lb EC50 Range Name
No. (nM)
OL-4 0.07 D (lR)-2-((2S)-2-(4-(3-(4-(2-((2S)-l-((2R)-2- (dimethylamino)-2-phenylacetyl)-2- pyrrolidinyl)- 1 H-imidazol-4-yl)phenoxy) phenyl)- 1 H-imidazol-2-yl)- 1 -pyrrolidinyl)- N,N-dimethyl-2-oxo- 1 -phenylethanamine
OL-5 80 A (lR)-2-((2S)-2-(4-(3-(4-(2-((2S)-l-((2R)-2- hydroxy-2-phenylacetyl)-2-pyrrolidinyl)- 1 H- imidazol-4-yl)phenoxy)phenyl)- 1 H- imidazol-2-yl)- 1 -pyrrolidinyl)-2-oxo- 1 - phenylethanol
OL-6 D methyl (( 1 R)-2-((2S)-2-(4-(3 -(4-(2-((2 S)- 1 - ((2R)-2-((methoxycarbonyl)amino)-2- phenylacetyl)-2-pyrrolidinyl)- 1 H-imidazol- 4-yl)phenoxy)phenyl)- 1 H-imidazol-2-yl)- 1 - pyrrolidinyl)-2-oxo- 1 -phenylethyl)carbamate
OL-7 D (lR)-2-((2S)-2-(4-(4-((4-(2-((2S)-l-((2R)-2- (dimethylamino)-2-phenylacetyl)-2- pyrrolidinyl)- 1 H-imidazol-4-yl)benzyl)oxy) phenyl)- 1 H-imidazol-2-yl)- 1 -pyrrolidinyl)- N,N-dimethyl-2-oxo- 1 -phenylethanamine
OL-8 D methyl (( 1 R)-2-((2S)-2-(4-(4-((4-(2-((2 S)- 1 - ((2R)-2-((methoxycarbonyl)amino)-2- phenylacetyl)-2-pyrrolidinyl)- 1 H-imidazol- 4-yl)benzyl)oxy)phenyl)-lH-imidazol-2-yl)- 1 -pyrrolidinyl)-2-oxo- 1 - phenylethyl)carbamate Compound lb EC50 Range Name
No. (nM)
OL-9 D (lR)-2-((2S)-2-(4-(4-(2-(4-(2-((2S)-l-((2R)- 2-(dimethylamino)-2-phenylacetyl)-2- pyrrolidinyl)- 1 H-imidazol-5-yl)phenyl)ethyl) phenyl)- 1 H-imidazol-2-yl)- 1 -pyrrolidinyl)- N,N-dimethyl-2-oxo- 1 -phenylethanamine
OL-10 D (lR,l'R)-2,2'-(l,2-ethanediylbis(4,l- phenylene-lH-imidazole-4,2-diyl(2S)-2,l- pyrrolidinediyl))bis(2-oxo- 1 -phenylethanol)
OL-11 D dimethyl ( 1 ,2-ethanediylbis(4, 1 -phenylene- lH-imidazole-4,2-diyl(2S)-2,l- pyrrolidinediyl ((1 R)-2-oxo- 1 -phenyl-2, 1 - ethanediyl))) biscarbamate
OL-12 D N',N"'-( 1 ,2-ethanediylbis(4, 1 -phenylene- 1 H- imidazole-4,2-diyl(2S)-2,l-pyrrolidinediyl (( 1 R)-2-oxo- 1 -phenyl-2, 1 -ethanediyl)))bis( 1 - ethylurea)
OL-13 D 1 -cyclopentyl-3 -(( 1 R)-2-((2S)-2-(4-(4-(2-(4- (2-((2S)- 1 -((2R)-2-((cyclopentylcarbamoyl) amino)-2-phenylacetyl)-2-pyrrolidinyl)- 1 H- imidazol-5-yl)phenyl)ethyl)phenyl)- 1 H- imidazol-2-yl)- 1 -pyrrolidinyl)-2-oxo- 1 - phenylethyl)urea
OL-14 C ( 1 R)-2-((2S)-2-(4-(4-(((4-(2-((2S)- 1 -((2R)-2- (dimethylamino)-2-phenylacetyl)-2- pyrrolidinyl)-lH-imidazol-5-yl)benzyl)oxy) methyl)phenyl)- 1 H-imidazol-2-yl)- 1 - pyrrolidinyl)-N,N-dimethyl-2-oxo- 1 - phenylethanamine Compound lb EC50 Range Name
No. (nM)
OL-15 1.1 B (lR)-2-((2S)-2-(4-(4-(((4-(2-((2S)-l-((2R)-2- hydroxy-2-phenylacetyl)-2-pyrrolidinyl)- 1 H- imidazol-5-yl)benzyl)oxy)methyl)phenyl)- 1 H-imidazol-2-yl)- 1 -pyrrolidinyl)-2-oxo- 1 - phenylethanol
OL-16 D dimethyl (oxybis(methylene-4, 1-phenylene- 1 H-imidazole-4,2-diyl(2S)-2, 1 - pyrrolidinediyl ((1 R)-2-oxo- 1 -phenyl-2, 1 - ethanediyl))) biscarbamate
OL-17 D 1 -methyl-3 -(( 1 R)-2-((2 S)-2-(4-(4-(((4-(2- ((2S)- 1 -((2R)-2-((methylcarbamoyl)amino)- 2-phenylacetyl)-2-pyrrolidinyl)-lH- imidazol-5-yl)benzyl)oxy)methyl)phenyl)- 1 H-imidazol-2-yl)- 1 -pyrrolidinyl)-2-oxo- 1 - phenylethyl)urea
OL-18 D l-ethyl-3-((lR)-2-((2S)-2-(4-(4-(((4-(2- ((2S)-l-((2R)-2-((ethylcarbamoyl)amino)-2- phenylacetyl)-2-pyrrolidinyl)-lH-imidazol- 5 -yl)benzyl)oxy)methyl)phenyl)- 1 H- imidazol-2-yl)- 1 -pyrrolidinyl)-2-oxo- 1 - phenylethyl)urea
OL-19 B l-cyclopentyl-3-((lR)-2-((2S)-2-(4-(4-(((4- (2-((2S)- 1 -((2R)-2-((cyclopentylcarbamoyl) amino)-2-phenylacetyl)-2-pyrrolidinyl)- 1 H- imidazol-5-yl)benzyl)oxy)methyl)phenyl)- 1 H-imidazol-2-yl)- 1 -pyrrolidinyl)-2-oxo- 1 - phenylethyl)urea Compound lb EC50 Range Name
No. (nM)
OL-20 C ( 1 R)-2-((2S)-2-(4-(3 -(((4-(2-((2S)- 1 -((2R)-2- (dimethylamino)-2-phenylacetyl)-2- pyrrolidinyl)- 1 H-imidazol-4-yl)benzyl)oxy) methyl)phenyl)- 1 H-imidazol-2-yl)- 1 - pyrrolidinyl)-N,N-dimethyl-2-oxo- 1 - phenylethanamine
OL-21 C ( 1 R)-2-((2S)-2-(4-(3 -(((4-(2-((2S)- 1 -((2R)-2- hydroxy-2-phenylacetyl)-2-pyrrolidinyl)- 1 H- imidazol-4-yl)benzyl)oxy)methyl)phenyl)- 1 H-imidazol-2-yl)- 1 -pyrrolidinyl)-2-oxo- 1 - phenylethanol
OL-22 D (methyl ((lR)-2-((2S)-2-(4-(3-(((4-(2-((2S)- l-((2R)-2-((methoxycarbonyl)amino)-2- phenylacetyl)-2-pyrrolidinyl)- 1 H-imidazol- 4-yl)benzyl)oxy)methyl)phenyl)- 1 H- imidazol-2-yl)- 1 -pyrrolidinyl)-2-oxo- 1 - phenylethyl)carbamate
OL-23 C 1 -methyl-3 -(( 1 R)-2-((2 S)-2-(4-(3 - (((4-(2- ((2S)- 1 -((2R)-2-((methylcarbamoyl)amino)- 2-phenylacetyl)-2-pyrrolidinyl)-lH- imidazol-4-yl)benzyl)oxy)methyl)phenyl)- 1 H-imidazol-2-yl)- 1 -pyrrolidinyl)-2-oxo- 1 - phenylethyl)urea
OL-24 D l-ethyl-3-((lR)-2-((2S)-2-(4-(3-(((4-(2- ((2S)-l-((2R)-2-((ethylcarbamoyl)amino)-2- phenylacetyl)-2-pyrrolidinyl)-lH-imidazol- 4-yl)benzyl)oxy)methyl)phenyl)- 1 H- imidazol-2-yl)- 1 -pyrrolidinyl)-2-oxo- 1 - phenylethyl)urea Compound lb EC50 Range Name
No. (nM)
OL-25 D l-cyclopentyl-3-((lR)-2-((2S)-2-(4-(3-(((4- (2-((2S)- 1 -((2R)-2-((cyclopentylcarbamoyl) amino)-2-phenylacetyl)-2-pyrrolidinyl)- 1 H- imidazol-4-yl)benzyl)oxy)methyl)phenyl)- 1 H-imidazol-2-yl)- 1 -pyrrolidinyl)-2-oxo- 1 - phenylethyl)urea
D-l D dimethyl (1,1 ':4', 1 "-terphenyl-4,4"- diylbis(lH-imidazole-4,2-diyl(2S)-2,l- pyrrolidinediyl ((1 R)-2-oxo- 1 -phenyl-2, 1 - ethanediyl))) biscarbamate
D-2 C ( 1 R)-2-((2S)-2-(4-(4"-(2-((2S)- 1 -((2R)-2- (dimethylamino)-2-phenylacetyl)-2- pyrrolidinyl)- lH-imidazol-5-yl)- 1 , 1 ':4', 1 "- terphenyl-4-yl)- lH-imidazol-2-yl)- 1 - pyrrolidinyl)-N,N-dimethyl-2-oxo- 1 - phenylethanamine
0.006 D methyl ((1S)-1 -(((2S)-2-(4-(4-((2-((2S)- 1 -
((2S)-2-((methoxycarbonyl)amino)-3- methylbutanoyl)-2-pyrrolidinyl)-4,5-dihydro-
D-3 lH-naphtho[l,2-d]imidazol-7- yl)ethynyl)phenyl)- 1 H-imidazol-2-yl)- 1 - pyrrolidinyl)carbonyl)-2- methylpropyl)carbamate
D methyl (( 1 R)-2-((2S)-2-(4-(4-((2-((2 S)- 1 - ((2R)-2-((methoxycarbonyl)amino)-2- phenylacetyl)-2-pyrrolidinyl)-4,5-dihydro-
D-4
lH-naphtho[l,2-d]imidazol-7- yl)ethynyl)phenyl)- 1 H-imidazol-2-yl)- 1 - pyrrolidinyl)-2-oxo- 1 -phenylethyl)carbamate Compound lb EC50 Range Name
No. (nM)
D methyl (( 1 R)-2-((2S)-2-(7-((4-(2-((2 S) - 1 - ((2R)-2-((methoxycarbonyl)amino)-2- phenylacetyl)-2-pyrrolidinyl)- 1 H-imidazol-
D-5
4-yl)phenyl)ethynyl)- 1 H-naphtho [1,2- d]imidazol-2-yl)- 1 -pyrrolidinyl)-2-oxo- 1 - phenylethyl)carbamate
D methyl ((1S)-1 -(((2S)-2-(4-(4-((2-((2S)- 1 - ((2S)-2-((methoxycarbonyl)amino)-3- methylbutanoyl)-2-pyrrolidinyl)- 1 H-
D-6 naphtho[ 1 ,2-d]imidazol-7- yl)ethynyl)phenyl)- 1 H-imidazol-2-yl)- 1 - pyrrolidinyl)carbonyl)-2- methylpropyl)carbamate
D methyl (( 1 S)- 1 -((( 1 R,3 S,5R)-3 -(4-(4-((2- ((lR,3S,5R)-2-((2S)-2- ((methoxycarbonyl)amino)-3- methylbutanoyl)-2-azabicyclo[3.1.0]hex-3 -
D-7
yl)-lH-naphtho[l,2-d]imidazol-7- yl)ethynyl)phenyl)-lH-imidazol-2-yl)-2- azabicyclo[3.1.0]hex-2-yl)carbonyl)-2- methylpropyl)carbamate
methyl ((lR)-2-((lR,3S,5R)-3-(7-((4-(2-
D
((lR,3S,5R)-2-((2R)-2-
((methoxycarbonyl)amino)-2-phenylacetyl)-
D-8 0.005 2-azabicyclo[3.1.0]hex-3-yl)-lH-imidazol-4- yl)phenyl)ethynyl)- lH-naphtho[ 1 ,2- d]imidazol-2-yl)-2-azabicyclo[3.1.0]hex-2- yl)-2-oxo- 1 -phenylethyl)carbamate Compound lb EC50 Range Name
No. (nM)
D methyl (( 1 S)- 1 -(((1 R,3 S,5R)-3 -(7-((2- ((lR,3S,5R)-2-((2S)-2- ((methoxycarbonyl)amino)-3- methylbutanoyl)-2-azabicyclo[3.1.0]hex-3 -
D-9
yl)-4,5-dihydro-lH-naphtho[l,2-d]imidazol- 7-yl)ethynyl)-4,5-dihydro- lH-naphtho[ 1 ,2- d]imidazol-2-yl)-2-azabicyclo[3.1.0]hex-2- yl)carbonyl)-2-methylpropyl)carbamate
D methyl (( 1 S)- 1 -((( 1R,3 S,5R)-3 -(7-((2-((3 S)- 2-((2S)-2-((methoxycarbonyl)amino)-3- methylbutanoyl)-2-azabicyclo[3.1.0]hex-3 -
D-10 yl)-lH-naphtho[l,2-d]imidazol-7- yl)ethynyl)- 1 H-naphtho [1,2-d] imidazol-2- yl)-2-azabicyclo[3.1.0]hex-2-yl)carbonyl)-2- methylpropyl)carbamate
methyl ((lS)-2-((lR,3S,5R)-3-(7-((2-
D
((lR,3S,5R)-2-((2S)-2- ((methoxycarbonyl)amino)-3- methylbutanoyl)-2-azabicyclo[3.1.0]hex-3 -
D-l l
yl)-lH-naphtho[l,2-d]imidazol-7- yl)ethynyl)- 1 H-naphtho [1,2-d] imidazol-2- yl)-2-azabicyclo[3.1.0]hex-2-yl)-2-oxo- 1 - (tetrahydro-2H-pyran-4-yl)ethyl)carbamate
Ml D methyl ((1S)-1 -(((2S)-2-(4-(4-((4-(2-((2 S)- 1 - ((2S)-2-((methoxycarbonyl)amino)-3- methylbutanoyl)-2-pyrrolidinyl)- 1 H- imidazol-4-yl)phenyl)ethynyl)phenyl)- 1 H- imidazol-2-yl)-l-pyrrolidinyl)carbonyl)-2- methylpropyl) carbamate Compound lb EC50 Range Name
No. (nM)
M2 0.24 C dimethyl ( 1 ,2-ethynediylbis(4, 1 -phenylene- lH-imidazole-4,2-diyl(2S)-2,l- pyrrolidinediyl ((1 R)-2-oxo- 1 -phenyl-2, 1 - ethanediyl))) biscarbamate
M2.1 D dimethyl ( 1 ,2-ethynediylbis(4, 1 -phenylene- lH-imidazole-4,2-diyl(2S)-2,l- pyrrolidinediyl(( 1 S)-2-oxo- 1 -(tetrahydro-2H- pyran-4-yl)-2, 1 -ethanediyl)))biscarbamate
M3 D methyl (( 1 S)- 1 -((( 1 R,3 S,5R)-3 -(4-(4-((4-(2- ((lR,3S,5R)-2-((2S)-2-((methoxycarbonyl) amino)-3 -methylbutanoyl)-2- azabicyclo[3.1.0] hex-3 -yl)- lH-imidazol-4- yl)phenyl)ethynyl) phenyl)- 1 H-imidazol-2- yl)-2-azabicyclo[3.1.0] hex-2-yl)carbonyl)-2- methylpropyl)carbamate
M4 D dimethyl ( 1 ,2-ethynediylbis(4, 1 -phenylene- 1 H-imidazole-4,2-diyl(l R,3 S,5R)-2- azabicyclo [3.1.0]hexane-3 ,2-diyl((2 S)- 1 - oxo- 1 ,2-butanediyl)))biscarbamate
M5 C dimethyl ( 1 ,2-ethynediylbis(4, 1 -phenylene- 1 H-imidazole-4,2-diyl(l R,3 S,5R)-2- azabicyclo [3.1.0]hexane-3 ,2-diyl(( 1 S)-l- cyclobutyl-2-oxo-2, 1- ethanediyl)))biscarbamate
M6 0.0033 D dimethyl ( 1 ,2-ethynediylbis(4, 1 -phenylene- lH-imidazole-4,2-diyl(lR,3S,5R)-2- azabicyclo [3.1.0]hexane-3 ,2-diyl((l S)-2- oxo- 1 -(tetrahydro-2H-pyran-4-yl)-2, 1 - ethanediyl)))biscarbamate Compound lb EC50 Range Name
No. (nM)
M7 D methyl (( 1 S)- 1 -((( 1 R,3 S,5R)-3 -(4-(4-((4-(2-
((lR,3S,5R)-2-((2S)-2-((methoxycarbonyl) amino)-2-(tetrahydro-2H-pyran-4-yl)acetyl)-
2-azabicyclo[3.1.0]hex-3-yl)-lH-imidazol-4- yl)phenyl)ethynyl)phenyl)-lH-imidazol-2- yl)-2-azabicyclo[3.1.0]hex-2-yl)carbonyl)-2- methylpropyl)carbamate
M8 D methyl ((1S)-1 -((( 1 R,3 S,5R)-3 -(4-chloro-5 - (4-((4-(2-((lR,3S,5R)-2-((2S)-2- ((methoxycarbonyl)amino)-3- methylbutanoyl)-2-azabicyclo[3.1.0]hex-3 - yl)- 1 H-imidazol-4- yl)phenyl)ethynyl)phenyl)-lH-imidazol-2- yl)-2-azabicyclo[3.1.0]hex-2-yl)carbonyl)-2- methylpropyl)carbamate
M9 D methyl ((1S)-1 -((( 1 R,3 S,5R)-3 -(4-chloro-5 - (4-((4-(4-chloro-2-(( 1 R,3 S,5R)-2-((2S)-2- ((methoxycarbonyl)amino)-3- methylbutanoyl)-2-azabicyclo[3.1.0]hex-3 - yl)- 1 H-imidazol-5 - yl)phenyl)ethynyl)phenyl)-lH-imidazol-2- yl)-2-azabicyclo[3.1.0]hex-2-yl)carbonyl)-2- methylpropyl)carbamate
Compound lb EC50 Range Name
No. (nM)
D methyl ((lS)-2-((lR,3S,5R)-3-(4-(4-((4-(4- chloro-2-((lR,3S,5R)-2-((2S)-2-
((methoxycarbonyl)amino)-2-(tetrahydro-
2H-pyran-4-yl)acetyl)-2-
M9.1
azabicyclo [3.1.0]hex-3 -yl)- 1 H-imidazol-5 - yl)phenyl)ethynyl)phenyl)-lH-imidazol-2- yl)-2-azabicyclo[3.1.0]hex-2-yl)-2-oxo- 1 -
(tetrahydro-2H-pyran-4-yl)ethyl)carbamate dimethyl ( 1 ,2-ethynediylbis(4, 1 -
D
phenylene(4-chloro- 1 H-imidazole-5 ,2-
M9.2 diyl)(lR,3S,5R)-2-azabicyclo[3.1.0]hexane- 3 ,2-diyl(( 1 S)-2-oxo- 1 -(tetrahydro-2H-pyran- 4-yl)-2, 1 -ethanediyl)))biscarbamate
D methyl (( 1 S)- 1 -((( 1 R,3 S,5R)-3 -(4-(4- ((lS,2S)-2-(4-(2-((lR,3S,5R)-2-((2S)-2- ((methoxycarbonyl)amino)-3- methylbutanoyl)-2-azabicyclo[3.1.0]hex-3 -
M9.3
yl)- 1 H-imidazol-4- yl)phenyl)cyclopropyl)phenyl)- 1 H-imidazol- 2-yl)-2-azabicyclo[3.1.0]hex-2-yl)carbonyl)- 2-methylpropyl)carbamate
dimethyl ((1S,2S)-1,2-
D
cyclopropanediylbis(4, 1 -phenylene- 1 H- imidazole-4,2-diyl( 1 R,3 S,5R)-2-
M9.4
azabicyclo[3.1.0]hexane-3 ,2-diyl(( 1 S)-2- oxo- 1 -(tetrahydro-2H-pyran-4-yl)-2, 1 - ethanediyl)))biscarbamate Compound lb EC50 Range Name
No. (nM)
D methyl (( 1 S)- 1 -((( 1 R,3 S,5R)-3 -(4-(4- ((lR,2R)-2-(4-(2-((lR,3S,5R)-2-((2S)-2- ((methoxycarbonyl)amino)-3- methylbutanoyl)-2-azabicyclo[3.1.0]hex-3 -
M9.5
yl)- 1 H-imidazol-4- yl)phenyl)cyclopropyl)phenyl)- 1 H-imidazol- 2-yl)-2-azabicyclo[3.1.0]hex-2-yl)carbonyl)- 2-methylpropyl)carbamate
dimethyl ((1R,2R)-1,2-
D
cyclopropanediylbis(4, 1 -phenylene- 1 H- imidazole-4,2-diyl( 1 R,3 S,5R)-2-
M9.6
azabicyclo[3.1.0]hexane-3 ,2-diyl(( 1 S)-2- oxo- 1 -(tetrahydro-2H-pyran-4-yl)-2, 1 - ethanediyl)))biscarbamate
methyl ((lS)-l-(((2S,4S)-2-(4-(4-((lS,2S)-2-
(4-(2-((2S,4S)-l-((2S)-2-
((methoxycarbonyl)amino)-3- methylbutanoyl)-4-methyl-2-pyrrolidinyl)-
M9.7 D
lH-imidazol-4- yl)phenyl)cyclopropyl)phenyl)- 1 H-imidazol- 2-yl)-4-methyl- 1 -pyrrolidinyl)carbonyl)-2- methylpropyl)carbamate
M9.8 D dimethyl ((1S,2S)-1,2- cyclopropanediylbis(4, 1 -phenylene- 1 H- imidazole-4,2-diyl((2S,4S)-4-methyl-2,l- pyrrolidinediyl)(( 1 S)-2-oxo- 1 -(tetrahydro- 2H-pyran-4-yl)-2,l- ethanediyl)))biscarbamate Compound lb EC50 Range Name
No. (nM)
M10 D methyl ((lS)-l-(((2S,5S)-2-(4-(4-((4-(2-
((2S,5S)-l-((2S)-2-
((methoxycarbonyl)amino)-3- methylbutanoyl)-5-methyl-2-pyrrolidinyl)- lH-imidazol-4-yl)phenyl)ethynyl)phenyl)- lH-imidazol-2-yl)-5-methyl-l-pyrrolidinyl) carbonyl)-2-methylpropyl)carbamate
Mi l D dimethyl ( 1 ,2-ethynediylbis(4, 1 -phenylene- lH-imidazole-4,2-diyl((2S,5S)-5-methyl-2,l- pyrrolidinediyl)((2S)- 1 -oxo- 1 ,2-butanediyl))) biscarbamate
M12 D dimethyl ( 1 ,2-ethynediylbis(4, 1 -phenylene- lH-imidazole-4,2-diyl((2S,5S)-5-methyl-2,l- pyrrolidinediyl)(( 1 S)-2-oxo- 1 -(tetrahydro- 2H-pyran-4-yl)-2,l- ethanediyl)))biscarbamate
methyl ((lS)-l-(((2S,5S)-2-(4-(4-((4-(2-
D
((2S,5S)-l-((2S)-2-
((methoxycarbonyl)amino)-2-(tetrahydro- 2H-pyran-4-yl)acetyl)-5-methyl-2-
M12.1
pyrrolidinyl)- 1 H-imidazol-4- yl)phenyl)ethynyl)phenyl)-lH-imidazol-2- yl)-5-methyl- 1 -pyrrolidinyl)carbonyl)-2- methylpropyl)carbamate Compound lb EC50 Range Name
No. (nM)
D methyl ((lS)-l-(((2S,4S)-2-(4-(4-((4-(2-
((2S,4S)-l-((2S)-2-
((methoxycarbonyl)amino)-3- methylbutanoyl)-4-methyl-2-pyrrolidinyl)-
M12.2
lH-imidazol-4-yl)phenyl)ethynyl)phenyl)-
1 H-imidazol-2-yl)-4-methyl- 1 - pyrrolidinyl)carbonyl)-2- methylpropyl)carbamate
dimethyl ( 1 ,2-ethynediylbis(4, 1 -phenylene-
D
lH-imidazole-4,2-diyl((2S,4S)-4-methyl-2,l-
M12.3 0.014 pyrrolidinediyl)(( 1 S)-2-oxo- 1 -(tetrahydro- 2H-pyran-4-yl)-2,l- ethanediyl)))biscarbamate
D methyl ((lS)-l-(((2S,4S)-2-(4-(4-((4-(4- chloro-2-((2S,4S)-l-((2S)-2-
((methoxycarbonyl)amino)-3- methylbutanoyl)-4-methyl-2-pyrrolidinyl)-
M12.4
lH-imidazol-5-yl)phenyl)ethynyl)phenyl)-
1 H-imidazol-2-yl)-4-methyl- 1 - pyrrolidinyl)carbonyl)-2- methylpropyl)carbamate
methyl ((lS)-l-(((2S,4S)-2-(4-chloro-5-(4-
D
((4-(4-chloro-2-((2S,4S)-l-((2S)-2-
((methoxycarbonyl)amino)-3- methylbutanoyl)-4-methyl-2-pyrrolidinyl)-
M12.5
lH-imidazol-5-yl)phenyl)ethynyl)phenyl)-
1 H-imidazol-2-yl)-4-methyl- 1 - pyrrolidinyl)carbonyl)-2- methylpropyl)carbamate Compound lb EC50 Range Name
No. (nM)
D methyl ((lS)-2-((2S,4S)-2-(4-(4-((4-(4- chloro-2-((2S,4S)-l-((2S)-2-
((methoxycarbonyl)amino)-2-(tetrahydro-
2H-pyran-4-yl)acetyl)-4-methyl-2-
M12.6
pyrrolidinyl)-lH-imidazol-5- yl)phenyl)ethynyl)phenyl)-lH-imidazol-2- yl)-4-methyl- 1 -pyrrolidinyl)-2-oxo- 1 -
(tetrahydro-2H-pyran-4-yl)ethyl)carbamate dimethyl ( 1 ,2-ethynediylbis(4, 1 -
D
phenylene(4-chloro- 1 H-imidazole-5 ,2- diyl)((2S,4S)-4-methyl-2, 1 -
M12.7 0.061
pyrrolidinediyl)(( 1 S)-2-oxo- 1 -(tetrahydro- 2H-pyran-4-yl)-2,l- ethanediyl)))biscarbamate
methyl ((lS)-2-((2S,4S)-2-(4-(4-((4-(2-
D
((2S,4S)-l-((2S)-2- ((methoxycarbonyl)amino)-3 - methylbutanoyl)-4-methyl-2-pyrrolidinyl)-
M12.8
lH-imidazol-4-yl)phenyl)ethynyl)phenyl)- lH-imidazol-2-yl)-4-methyl-l-pyrrolidinyl)- 2-oxo- 1 -(tetrahydro-2H-pyran-4- yl)ethyl)carbamate
Compound lb EC50 Range Name
No. (nM)
D methyl (( 1 S)- 1 -((( 1 S,3 S,5 S)-3 -(4-(4-((4-(2-
((lS,3S,5S)-2-((2S)-2-
((methoxycarbonyl)amino)-3- methylbutanoyl)-5-methyl-2-
M12.9
azabicyclo [3.1.0]hex-3 -yl)- 1 H-imidazol-4- yl)phenyl)ethynyl)phenyl)-lH-imidazol-2- yl)-5-methyl-2-azabicyclo[3.1.0]hex-2- yl)carbonyl)-2-methylpropyl)carbamate
M13 D methyl (( 1 S)- 1 -(((1 R,3 S,5R)-3 -(4-(4-((E)-2- (4-(2-((lR,3S,5R)-2-((2S)-2- ((methoxycarbonyl) amino)-3- methylbutanoyl)-2-azabicyclo[3.1.0] hex-3- yl)- lH-imidazol-4-yl)phenyl)vinyl) phenyl)- 1 H-imidazol-2-yl)-2-azabicyclo [3.1.0] hex- 2-yl)carbonyl)-2-methylpropyl)carbamate
M14 0.017 D dimethyl ((E)- 1 ,2-ethenediylbis(4, 1 - phenylene-lH-imidazole-4,2- diyl(lR,3S,5R)-2-azabicyclo [3.1.0]hexane- 3 ,2-diyl(( 1 S)-2-oxo- 1 -(tetrahydro-2H-pyran- 4-yl)-2, 1 -ethanediyl))) biscarbamate
M15 D methyl ((lS)-2-((lR,3S,5R)-3-(4-(4-((E)-2- (4-(2-((lR,3S,5R)-2-((2S)-2- ((methoxycarbonyl) amino)-3- methylbutanoyl)-2-azabicyclo[3.1.0] hex-3- yl)- lH-imidazol-4-yl)phenyl)vinyl) phenyl)- 1 H-imidazol-2-yl)-2-azabicyclo [3.1.0] hex- 2-yl)-2-oxo-l-(tetrahydro-2H-pyran-4- yl)ethyl)carbamate Compound lb EC50 Range Name
No. (nM)
D methyl ((lS)-l-(((2S)-2-(4-(4-((4-(2-((2S)- 4,4-difluoro-l-((2S)-2- ((methoxycarbonyl)amino)-3- methylbutanoyl)-2-pyrrolidinyl)- 1 H-
Nl
imidazol-4-yl)phenyl)ethynyl)phenyl)- 1 H- imidazol-2-yl)-4,4-difluoro- 1 - pyrrolidinyl)carbonyl)-2- methylpropyl)carbamate
dimethyl (ethyne- 1 ,2-diylbis(4, 1 -phenylene-
D
lH-imidazole-5,2-diyl((2S)-4,4-
N2 difluoropyrrolidine-2, 1 -diyl)(( 1 S)-2-oxo- 1 - (tetrahydro-2H-pyran-4-yl)ethane-2, 1 - diyl)))biscarbamate
A methyl ((lR)-2-((2S)-2-(5-(4-((4-(2-((2S)- 4,4-difluoro-l-((2S)-2- ((methoxycarbonyl)amino)-2-(tetrahydro- 2H-pyran-4-yl)acetyl)pyrrolidin-2-yl)-lH-
N3 0.25
imidazol-5-yl)phenyl)ethynyl)phenyl)-lH- imidazol-2-yl)-4,4-difluoropyrrolidin- 1 -yl)- 2-oxo- 1 -(tetrahydro-2H-pyran-4- yl)ethyl)carbamate
A dimethyl (ethyne- 1 ,2-diylbis(4, 1 -phenylene- lH-imidazole-5,2-diyl((2S)-4,4-
N4 0.18 difluoropyrrolidine-2, 1 -diyl)(( 1 R)-2-oxo- 1 - (tetrahydro-2H-pyran-4-yl)ethane-2, 1 - diyl)))biscarbamate Compound lb EC50 Range Name
No. (nM)
D methyl ((1S)-1 -(((3 S)-3 -(4-(4-((4-(2-((3 S)-4-
((2S)-2-((methoxycarbonyl)amino)-3- methylbutanoyl)-3-morpholinyl)-lH-
N5
imidazol-4-yl)phenyl)ethynyl)phenyl)- 1 H- imidazol-2-yl)-4-morpholinyl)carbonyl)-2- methylpropyl)carbamate
A methyl ((1S)-1 -(((2S)-2-(4-(4-((4-(2-((2 S)- 1 - ((2S)-2-((methoxycarbonyl)amino)-3- methylbutanoyl)-2-piperidinyl)- 1 H-imidazol-
N6
4-yl)phenyl)ethynyl)phenyl)- 1 H-imidazol-2- yl)- 1 -piperidinyl)carbonyl)-2- methylpropyl)carbamate
methyl ((lS)-l-(((2S,4S)-4-hydroxy-2-(4-(4-
D
((4-(2-((2S,4S)-4-hydroxy-l-((2S)-2-
((methoxycarbonyl)amino)-3-
N7 methylbutanoyl)-2-pyrrolidinyl)- 1 H- imidazol-4-yl)phenyl)ethynyl)phenyl)- 1 H- imidazol-2-yl)-l-pyrrolidinyl)carbonyl)-2- methylpropyl)carbamate
methyl ((1S)-1 -(((2S)-2-(4-(4-((4-(2-((2 S)- 1 -
((2S)-2-((methoxycarbonyl)amino)-3- methylbutanoyl)-4-methylene-2-
N7.1 pyrrolidinyl)- 1 H-imidazol-4- yl)phenyl)ethynyl)phenyl)-lH-imidazol-2- yl)-4-methylene- 1 -pyrrolidinyl)carbonyl)-2- methylpropyl)carbamate Compound lb EC50 Range Name
No. (nM)
methyl ((lS)-l-(((2S,4R)-4-hydroxy-2-(4-(4-
((4-(2-((2S,4R)-4-hydroxy-l-((2S)-2-
((methoxycarbonyl)amino)-3-
N7.2 methylbutanoyl)-2-pyrrolidinyl)- 1 H- imidazol-4-yl)phenyl)ethynyl)phenyl)- 1 H- imidazol-2-yl)-l-pyrrolidinyl)carbonyl)-2- methylpropyl)carbamate
A methyl(( 1 S)- 1 -((( 1 S,3 S,5 S)-3 -(4-(4-((4-(2- ((lS,3S,5S)-2-((2S)-2- ((methoxycarbonyl)amino)-3- methylbutanoyl)-2-azabicyclo[3.1.0]hex-3 -
N8
yl)- 1 H-imidazol-4- yl)phenyl)ethynyl)phenyl)-lH-imidazol-2- yl)-2-azabicyclo[3.1.0]hex-2-yl)carbonyl)-2- methylpropyl)carbamate
dimethyl ( 1 ,2-ethynediylbis(4, 1 -phenylene-
D
1 H-imidazole-4,2-diyl(l S,3 S,5 S)-2-
N9 azabicyclo[3.1.0]hexane-3 ,2-diyl(( 1 S)-2- oxo- 1 -(tetrahydro-2H-pyran-4-yl)-2, 1 - ethanediyl)))biscarbamate
D methyl ((lS)-l-(((2S)-2-(4-(3-((3-(2-((2S)-l- ((2S)-2-((methoxycarbonyl)amino)-3- methylbutanoyl)-2-pyrrolidinyl)- 1 H-
Jl
imidazol-4-yl)phenyl)ethynyl)phenyl)- 1 H- imidazol-2-yl)-l-pyrrolidinyl)carbonyl)-2- methylpropyl)carbamate Compound lb EC50 Range Name
No. (nM)
A methyl ((lS)-2-methyl-l-(((2S)-2-(4-(3-((3- (2-((2S)-2-pyrrolidinyl)-lH-imidazol-4-
Jl. l 338.8 yl)phenyl)ethynyl)phenyl)-lH-imidazol-2- yl)-l- pyrrolidinyl)carbonyl)propyl)carbamate dimethyl ( 1 ,2-ethynediylbis(3 , 1 -phenylene-
C
lH-imidazole-4,2-diyl(2S)-2,l-
J2 0.12
pyrrolidinediyl(( 1 S)-2-oxo- 1 -(tetrahydro-2H- pyran-4-yl)-2, 1 -ethanediyl)))biscarbamate
D methyl (( 1 S) - 1 - ((( 1 R,3 S,5R)-3 - (4-(3 -((3 -(2- ((lR,3S,5R)-2-((2S)-2- ((methoxycarbonyl)amino)-3- methylbutanoyl)-2-azabicyclo[3.1.0]hex-3 -
J3
yl)- 1 H-imidazol-4- yl)phenyl)ethynyl)phenyl)-lH-imidazol-2- yl)-2-azabicyclo[3.1.0]hex-2-yl)carbonyl)-2- methylpropyl)carbamate
D dimethyl ( 1 ,2-ethynediylbis(3 , 1 -phenylene- 1 H-imidazole-4,2-diyl(l R,3 S,5R)-2-
J4 azabicyclo[3.1.0]hexane-3 ,2-diyl(( 1 S)-2- oxo- 1 -(tetrahydro-2H-pyran-4-yl)-2, 1 - ethanediyl)))biscarbamate
D methyl (( 1 S)- 1 -(((2S)-2-(4-(4'-((4-(2-((2S)- 1 -
((2S)-2-((methoxycarbonyl)amino)-3-
J5 methylbutanoyl)-2-pyrrolidinyl)- 1 H- imidazol-4-yl)phenyl)ethynyl)-4- biphenylyl)- 1 H-imidazol-2-yl)- 1 - pyrrolidinyl)carbonyl)-2- methylpropyl)carbamate Compound lb EC50 Range Name
No. (nM)
N7.3 D
N7.8 D
N7.9 1.10 B
N10 D
Ni l D
Nl l. l 0.00147 D
N1 1.2 D
N12 D
N13 D
N14 D
N15 D
N16.1 C
N16.2 D
N16.3 B
N17.1 D
N17.2 0.13 C
N22 D
N23 D
N24 C
N25
The compounds of the present disclosure may inhibit HCV by mechanisms in addition to or other than NS5A inhibition. In one embodiment the compounds of the present disclosure inhibit HCV replicon and in another embodiment the compounds of the present disclosure inhibit NS5A.
It will be evident to one skilled in the art that the present disclosure is not limited to the foregoing illustrative examples, and that it can be embodied in other specific forms without departing from the essential attributes thereof. It is therefore desired that the examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing examples, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:
Figure imgf000311_0001
Figure imgf000312_0001
Figure imgf000312_0002
-311 -
Figure imgf000313_0001
Figure imgf000313_0002
Figure imgf000314_0001
Figure imgf000315_0001
or a pharmaceutically acceptable salt thereof.
2. A composition comprising a compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
3. A method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of a compound of claim 1 , or a pharmaceutically acceptable salt thereof.
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