Novel peptides as NS3-serine protease inhibitors of hepatitis C virus
The present invention discloses novel compounds which have HCV protease inhibitory activity as well as methods for preparing such compounds. In another embodiment, the invention discloses pharmaceutical compositions comprising such compounds as well as methods of using them to treat disorders associated with the HCV protease.
[0001] The present invention relates to novel hepatitis C virus (“HCV”) protease inhibitors, pharmaceutical compositions containing one or more such inhibitors, methods of preparing such inhibitors and methods of using such inhibitors to treat hepatitis C and related disorders. This invention specifically discloses novel peptide compounds as inhibitors of the HCV NS3/NS4a serine protease.
BACKGROUND OF THE INVENTION[0002] Hepatitis C virus (HCV) is a (+)-sense single-stranded RNA virus that has been implicated as the major causative agent in non-A, non-B hepatitis (NANBH), particularly in blood-associated NANBH (BB-NANBH)(see, International Patent Application Publication No. WO 89/04669 and European Patent Application Publication No. EP 381 216). NANBH is to be distinguished from other types of viral-induced liver disease, such as hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV), as well as from other forms of liver disease such as alcoholism and primary biliar cirrhosis.
[0003] Recently, an HCV protease necessary for polypeptide processing and viral replication has been identified, cloned and expressed; (see, e.g., U.S. Pat. No. 5,712,145). This approximately 3000 amino acid polyprotein contains, from the amino terminus to the carboxy terminus, a nucleocapsid protein (C), envelope proteins (E1 and E2) and several non-structural proteins (NS1, 2, 3, 4a, 5a and 5b). NS3 is an approximately 68 kda protein, encoded by approximately 1893 nucleotides of the HCV genome, and has two distinct domains: (a) a serine protease domain consisting of approximately 200 of the N-terminal amino acids; and (b) an RNA-dependent ATPase domain at the C-terminus of the protein. The NS3 protease is considered a member of the chymotrypsin family because of similarities in protein sequence, overall three-dimensional structure and mechanism of catalysis. Other chymotrypsin-like enzymes are elastase, factor Xa, thrombin, trypsin, plasmin, urokinase, tPA and PSA. The HCV NS3 serine protease is responsible for proteolysis of the polypeptide (polyprotein) at the NS3/NS4a, NS4a/NS4b, NS4b/NS5a and NS5a/NS5b junctions and is thus responsible for generating four viral proteins during viral replication. This has made the HCV NS3 serine protease an attractive target for antiviral chemotherapy.
[0004] It has been determined that the NS4a protein, an approximately 6 kda polypeptide, is a co-factor for the serine protease activity of NS3. Autocleavage of the NS3/NS4a junction by the NS3/NS4a serine protease occurs intramolecularly (i.e., cis) while the other cleavage sites are processed intermolecularly (i.e., trans).
[0005] Analysis of the natural cleavage sites for HCV protease revealed the presence of cysteine at P1 and serine at P1′ and that these residues are strictly conserved in the NS4a/NS4b, NS4b/NS5a and NS5a/NS5b junctions. The NS3/NS4a junction contains a threonine at P1 and a serine at P1′. The Cys→Thr substitution at NS3/NS4a is postulated to account for the requirement of cis rather than trans processing at this junction. See, e.g., Pizzi et al. (1994) Proc. Natl. Acad. Sci (USA) 91:888-892, Failla et al. (1996) Folding & Design 1:35-42. The NS3/NS4a cleavage site is also more tolerant of mutagenesis than the other sites. See, e.g., Kollykhalov et al. (1994) J. Virol. 68:7525-7533. It has also been found that acidic residues in the region upstream of the cleavage site are required for efficient cleavage. See, e.g., Komoda et al. (1994) J. Virol. 68:7351-7357.
[0006] Inhibitors of HCV protease that have been reported include antioxidants (see, International Patent Application Publication No. WO 98/14181), certain peptides and peptide analogs (see, International Patent Application Publication No. WO 98/17679, Landro et al. (1997) Biochem. 36:9340-9348, Ingallinella et al. (1998) Biochem. 37:8906-8914, Llinàs-Brunet et al. (1998) Bioorg. Med. Chem. Lett. 8:1713-1718), inhibitors based on the 70-amino acid polypeptide eglin c (Martin et al. (1998) Biochem. 37:11459-11468, inhibitors affinity selected from human pancreatic secretory trypsin inhibitor (hPSTI-C3) and minibody repertoires (MBip) (Dimasi et al. (1997) J. Virol. 71:7461-7469), cVHE2 (a “camelized” variable domain antibody fragment) (Martin et al.(1997) Protein Eng. 10:607-614), and &agr;1-antichymotrypsin (ACT) (Elzouki et al.) (1997) J. Hepat. 27:42-28). A ribozyme designed to selectively destroy hepatitis C virus RNA has recently been disclosed (see, BioWorld Today 9(217): 4 (Nov. 10, 1998)).
[0007] Reference is also made to the PCT Publications, No. WO 98/17679, published Apr. 30, 1998 (Vertex Pharmaceuticals Incorporated); WO 98/22496, published May 28, 1998 (F. Hoffmann-La Roche AG); and WO 99/07734, published Feb. 18, 1999 (Boehringer Ingelheim Canada Ltd.).
[0008] HCV has been implicated in cirrhosis of the liver and in induction of hepatocellular carcinoma. The prognosis for patients suffering from HCV infection is currently poor. HCV infection is more difficult to treat than other forms of hepatitis due to the lack of immunity or remission associated with HCV infection. Current data indicates a less than 50% survival rate at four years post cirrhosis diagnosis. Patients diagnosed with localized resectable hepatocellular carcinoma have a five-year survival rate of 10-30%, whereas those with localized unresectable hepatocellular carcinoma have a five-year survival rate of less than 1%.
[0009] Reference is made to A. Marchetti et al., Synlett, S1, 1000-1002 (1999) describing the synthesis of bicylic analogs of an inhibitor of HCV NS3 protease. A compound disclosed therein has the formula: 1
[0010] Reference is also made to W. Han et al, Bioorganic & Medicinal Chem. Left, (2000) 10, 711-713, which describes the preparation of certain &agr;-ketoamides, &agr;-ketoesters and &agr;-diketones containing allyl and ethyl functionalities.
[0011] Reference is also made to WO 00/09558 (Assignee: Boehringer Ingelheim Limited; Published Feb. 24, 2000) which discloses peptide derivatives of the formula: 2
[0012] where the various elements are defined therein. An illustrative compound of that series is: 3
[0013] Reference is also made to WO 00/09543 (Assignee: Boehringer Ingelheim Limited; Published Feb. 24, 2000) which discloses peptide derivatives of the formula: 4
[0014] where the various elements are defined therein. An illustrative compound of that series is: 5
[0015] Current therapies for hepatitis C include interferon-&agr; (INF&agr;) and combination therapy with ribavirin and interferon. See, e.g., Beremguer et al. (1998) Proc. Assoc. Am. Physicians 110(2):98-112. These therapies suffer from a low sustained response rate and frequent side effects. See, e.g., Hoofnagle et al. (1997) N. Engl. J. Med. 336:347. Currently, no vaccine is available for HCV infection.
[0016] Pending and copending U.S. patent applications, Serial No. 60/194,607, filed Apr. 5, 2000, and Serial No. 60/198,204, filed Apr. 19, 2000, Serial No. 60/220,110, filed Jul. 21, 2000, Serial No. 60/220,109, filed Jul. 21, 2000, Serial No. 60/220,107, filed Jul. 21, 2000, Serial No. 60/254,869, filed Dec. 12, 2000, and Serial No. 60/220,101, filed Jul. 21, 2000, disclose various types of peptides and/or other compounds as NS-3 serine protease inhibitors of hepatitis C virus.
[0017] There is a need for new treatments and therapies for HCV infection. It is, therefore, an object of this invention to provide compounds useful in the treatment or prevention or amelioration of one or more symptoms of hepatitis C.
[0018] It is a further object herein to provide methods of treatment or prevention or amelioration of one or more symptoms of hepatitis C.
[0019] A still further object of the present invention is to provide methods for modulating the activity of serine proteases, particularly the HCV NS3/NS4a serine protease, using the compounds provided herein.
[0020] Another object herein is to provide methods of modulating the processing of the HCV polypeptide using the compounds provided herein.
SUMMARY OF THE INVENTION[0021] In its many embodiments, the present invention provides a novel class of inhibitors of the HCV protease, pharmaceutical compositions containing one or more of the compounds, methods of preparing pharmaceutical formulations comprising one or more such compounds, and methods of treatment, prevention or amelioration or one or more of the symptoms of hepatitis C. Also provided are methods of modulating the interaction of an HCV polypeptide with HCV protease. Among the compounds provided herein, compounds that inhibit HCV NS3/NS4a serine protease activity are preferred. The present application discloses a compound, including enantiomers, stereoisomers, rotamers, tautomers, racemates and prodrug of said compound, and pharmaceutically acceptable salts or solvates of said compound, or of said prodrug, said compound having the general structure shown in Formula I: 6
[0022] wherein:
[0023] Y is selected from the group consisting of the following moieties: alkyl, alkyl-aryl, heteroalkyl, heteroaryl, aryl-heteroaryl, alkyl-heteroaryl, cycloalkyl, alkyloxy, alkyl-aryloxy, aryloxy, heteroaryloxy, heterocycloalkyloxy, cycloalkyloxy, alkylamino, arylamino, alkyl-arylamino, arylamino, heteroarylamino, cycloalkylamino and heterocycloalkylamino, with the proviso that Y maybe optionally substituted with X11 or X12;
[0024] X11 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocyclyl, heterocyclylalkyl, aryl, alkylaryl, arylalkyl, heteroaryl, alkylheteroaryl, or heteroarylalkyl, with the proviso that X11 may be additionally optionally substituted with X12;
[0025] X12 is hydroxy, alkoxy, aryloxy, thio, alkylthio, arylthio, amino, alkylamino, arylamino, alkylsulfonyl, arylsulfonyl, alkylsulfonamido, arylsulfonamido, carboxy, carbalkoxy, carboxamido, alkoxycarbonylamino, alkoxycarbonyloxy, alkylureido, arylureido, halogen, cyano, or nitro, with the proviso that said alkyl, alkoxy, and aryl may be additionally optionally substituted with moieties independently selected from X12;
[0026] R1 is COR5 or B(OR)2, wherein R5 is H, OH, OR8, NR9R10, CF3, C2F5, C3F7, CF2R6, R6, or COR7 wherein R7 is H, OH, OR8, CHR9R10, or NR9R10, wherein R6, R8, R9 and R10 are independently selected from the group consisting of H, alkyl, aryl, heteroalkyl, heteroaryl, cycloalkyl, cycloalkyl, arylalkyl, heteroarylalkyl, [CH(R1′)]pCOOR11, [CH(R1′)] pCONR12R13, [CH(R1′)]pSO2R11, [CH(R1′)]pCOR11, [CH(R1′)]pCH(OH)R11, CH(R1′)CONHCH(R2′)COO R11, CH(R1′)CONHCH(R2′)CONR12R13, CH(R1′)CONHCH(R2′)R11, CH(R1′)CONHCH(R2′)CONHCH(R3′)COO R11, CH(R1′)CONHCH(R2′)CONHCH(R3′)CONR12R13, CH(R1′)CONHCH(R′2)CONHCH(R3′)CONHCH(R4′)COO R11, CH(R1′)CONHCH(R2′)CONHCH(R3′)CONHCH(R4′)CONR12R13, CH(R1′)CONHCH(R2′)CONHCH(R3′)CONHCH(R4′)CONHCH(R5′)COO R11 and CH(R1′)CONHCH(R2′)CONHCH(R3′)CONHCH(R4′)CONHCH(R5′) CONR12R13, wherein R1′, R2′, R3′, R4′, R5′, R11, R12, R13, and R′ are independently selected from the group consisting of H, alkyl, aryl, heteroalkyl, heteroaryl, cycloalkyl, alkyl-aryl, alkyl-heteroaryl, aryl-alkyl and heteroaralkyl;
[0027] Z is selected from O, N, CH or CR;
[0028] W may be present or absent, and if W is present, W is selected from C═O, C═S, C(═N—CN), or SO2;
[0029] Q may be present or absent, and when Q is present, Q is CH, N, P, (CH2)p, (CHR)p, (CRR′)p, O, NR, S, or SO2; and when Q is absent, M may be present or absent; when Q and M are absent, A is directly linked to L;
[0030] A is O, CH2, (CHR)p, (CHR—CHR′)p, (CRR′)p, NR, S, SO2 or a bond;
[0031] E is CH, N, CR, or a double bond towards A, L or G;
[0032] G may be present or absent, and when G is present, G is (CH2)p, (CHR)p, or (CRR′)p; and when G is absent, J is present and E is directly connected to the carbon atom in Formula I as G is linked to;
[0033] J maybe present or absent, and when J is present, J is (CH2)p, (CHR)p, or (CRR′)p, SO2, NH, NR or O; and when J is absent, G is present and E is directly linked to N shown in Formula I as linked to J;
[0034] L may be present or absent, and when L is present, L is CH, CR, O, S or NR; and when L is absent, then M may be present or absent; and if M is present with L being absent, then M is directly and independently linked to E, and J is directly and independently linked to E;
[0035] M may be present or absent, and when M is present, M is O, NR, S, SO2, (CH2)p, (CHR)p(CHR—CHR′)p, or (CRR′)p;
[0036] p is a number from 0 to 6; and
[0037] R, R′, R2, R3 and R4 are independently selected from the group consisting of H; C1-C10 alkyl; C2-C10 alkenyl; C3-C8 cycloalkyl; C3-C8 heterocycloalkyl, alkoxy, aryloxy, alkylthio, arylthio, amino, amido, ester, carboxylic acid, carbamate, urea, ketone, aldehyde, cyano, nitro, halogen; (cycloalkyl)alkyl and (heterocycloalkyl)alkyl, wherein said cycloalkyl is made of three to eight carbon atoms, and zero to six oxygen, nitrogen, sulfur, or phosphorus atoms, and said alkyl is of one to six carbon atoms; aryl; heteroaryl; alkyl-aryl; and alkyl-heteroaryl;
[0038] wherein said alkyl, heteroalkyl, alkenyl, heteroalkenyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl moieties may be optionally and chemically-suitably substituted, with said term “substituted” referring to optional and chemically-suitable substitution with one or more moieties selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heterocyclic, halogen, hydroxy, thio, alkoxy, aryloxy, alkylthio, arylthio, amino, amido, ester, carboxylic acid, carbamate, urea, ketone, aldehyde, cyano, nitro, sulfonamido, sulfoxide, sulfone, sulfonyl urea, hydrazide, and hydroxamate;
[0039] further wherein said unit N—C—G—E—L—J—N represents a five-membered or six-membered cyclic ring structure with the proviso that when said unit N—C—G—E—L—J—N represents a five-membered cyclic ring structure, or when the bicyclic ring structure in Formula I comprising N, C, G, E, L, J, N, A, Q, and M represents a five-membered cyclic ring structure, then said five-membered cyclic ring structure lacks a carbonyl group as part of the cyclic ring.
[0040] Among the above-stated definitions for the various moieties of Formula I, the preferred groups for the various moieties are as follows:
[0041] Preferred definition for R1 is COR5 with R5 being H, OH, COOR8 or CONR9R10, where R8, R9 and R10 are defined above. Still preferred moiety for R1 is COCONR9R10, where R9 is H; and R10 is H, R14, [CH(R1′)]pCOOR11, [CH(R1′)]pCONR12R13, [CH(R1′)]pSO2R11, [CH(R1′)]pSO2N R12R13, [CH(R1′)]pCOR11, CH(R1′)CONHCH(R2′)COOR11, CH(R1′)CONHCH(R2′) CONR12R13, or CH(R1′)CONHCH(R2′)(R′), wherein R14 is H, alkyl, aryl, heteroalkyl, heteroaryl, cycloalkyl, alkyl-aryl, alkyl-heteroaryl, aryl-alkyl, alkenyl, alkynyl or heteroaralkyl. Among the above for R10, preferred moieties for R10 are: H, R14, CH(R1′)COOR11, CH(R1′)CH(R1′)COOR11, CH(R1′)CONR12R13, CH(R1′)CH(R1′)CONR12R13, CH(R1′)CH(R1′)SO2R11, CH(R1′)CH(R1′)SO2N R12R13, CH(R1′)CH(R1′)COR11, CH(R1′)CONHCH(R2′)COOR11, CH(R1′)CONHCH(R2′) CONR12R13, or CH(R1′)CONHCH(R2′)(R′), wherein R1′ is H or alkyl, and R2′ is phenyl, substituted phenyl, hetero atom-substituted phenyl, thiophenyl, cycloalkyl, piperidyl or pyridyl.
[0042] More preferred moieties are: for R1′ is H, for R11 is H, methyl, ethyl, allyl, tert-butyl, benzyl, &agr;-methylbenzyl, &agr;,&agr;-dimethylbenzyl, 1-methylcyclopropyl or 1-methylcyclopentyl; for
[0043] R′ is hydroxymethyl or CH2CONR12R13 where
[0044] NR12R13 is selected from the group consisting of: 7
[0045] wherein U6 is H, OH, or CH2OH;
[0046] R14 is preferably selected from the group consisting of: H, Me, Et, n-propyl, methoxy, cyclopropyl, n-butyl, 1-but-3-ynyl, benzyl, &agr;-methylbenzyl, phenethyl, allyl, 1-but-3-enyl, OMe, cyclopropylmethyl;
[0047] and R2′ is preferably independently selected from the group consisting of: 8
[0048] wherein:
[0049] U1 and U2 maybe same or different and are selected from H, F, CH2COOH, CH2COOMe, CH2CONH2, CH2CONHMe, CH2CONMe2, azido, amino, hydroxyl, substituted amino, substituted hydroxyl;
[0050] U3 and U4 maybe same or different and are selected from O and S;
[0051] U5 is selected from the moieties consisting of alkyl sulfonyl, aryl sulfonyl, heteroalkyl sulfonyl, heteroaryl sulfonyl, alkyl carbonyl, aryl carbonyl, heteroalkyl carbonyl, heteroaryl carbonyl, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl or a combination thereof.
[0052] Preferred moieties for R2 are: 9 10
[0053] Preferred moieties for R3 are: 11 12
[0054] wherein R31═OH or O-alkyl;
[0055] Y19 is selected from the following moieties: 13
[0056] and Y20 is selected from the following moieties: 14
[0057] Most preferred moieties for R3 are: 15
[0058] Some other preferred moieties are: for Z it is N, for R4 it is H, and for W it is C═O. Additionally, the moiety Z—C—R3 in Formula I, with R4 being absent, may be represented by the following structures: 16
[0059] Preferred moieties for Y are: 17 18 19 20 21 22 23
[0060] wherein:
[0061] Y11 is selected from H, COOH, COOEt, OMe, Ph, OPh, NHMe, NHAc, NHPh, CH(Me)2, 1-triazolyl, 1-imidazolyl, and NHCH2COOH;
[0062] Y12 is selected from H, COOH, COOMe, OMe, F, Cl, or Br;
[0063] Y13 is selected from the following moieties: 24
[0064] Y14 is selected from MeSO2, Ac, Boc, iBoc, Cbz, or Alloc;
[0065] Y15 and Y16 are independently selected from alkyl, aryl, heteroalkyl, and heteroaryl;
[0066] Y17 is CF3, NO2, CONH2, OH, COOCH3, OCH3, OC6H5, C6H5, COC6H5, NH2, or COOH; and
[0067] Y18 is COOCH3, NO2, N(CH3)2, F, OCH3, CH2COOH, COOH, SO2NH2, or NHCOCH3.
[0068] Y may be more preferably represented by: 25 26 27
[0069] wherein:
[0070] Y17═CF3, NO2, CONH2, OH, NH2, or COOH;
[0071] Y18═F, COOH,
[0072] Still more preferred moieties for Y are: 28 29
[0073] As shown in Formula I, the unit: 30
[0074] represents a cyclic ring structure, which may be a five-membered or six-membered ring structure. When that cyclic ring represents a five-membered ring, it is a requirement of this invention that that five-membered cyclic ring does not contain a carbonyl group as part of the cyclic ring structure. Preferably, that five-membered ring is of the structure: 31
[0075] wherein R and R′ are defined above. Preferred representations for that five-membered cyclic ring structure is: 32
[0076] where R20 is selected form the following moieties: 33
[0077] Furthermore, that five-membered ring, along with its adjacent two exocyclic carbonyls, may be represented as follows: 34
[0078] in which case, R21 and R22 may be the same or different and are independently selected from the following moieties: 35 36
[0079] Some preferred illustrations for the five-membered ring structure: 37
[0080] are as follows: 38
[0081] Additionally, the unit: 39
[0082] in Formula I may be represented by the following structures b and c: 40
[0083] Preferred definitions for b are: 41
[0084] In c, G and J are independently selected from the group consisting of (CH2)p, (CHR)p, (CHR—CHR′)p, and (CRR′)p; A and M are independently selected from the group consisting of O, S, SO2, NR, (CH2)p, (CHR)p, (CHR—CHR′)p, and (CRR′)p; and Q is CH2, CHR, CRR′, NH, NR, O, S, SO2, NR, (CH2)p, (CHR)p, and (CRR′)p. Preferred definitions for c are: 42 43
[0085] When the cyclic ring structure is depicted as: 44
[0086] its most preferred illustrations are as follows: 45 46 47
[0087] Some of the still preferred moieties for the unit: 48
[0088] shown above, are: 49 50
[0089] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. Thus, for example, the term alkyl (including the alkyl portions of alkoxy) refers to a monovalent group derived from a straight or branched chain saturated hydrocarbon by the removal of a single atom having from 1 to 8 carbon atoms, preferably from 1 to 6;
[0090] aryl—represents a carbocyclic group having from 6 to 14 carbon atoms and having at least one benzenoid ring, with all available substitutable aromatic carbon atoms of the carbocyclic group being intended as possible points of attachment. Preferred aryl groups include phenyl, 1-naphthyl, 2-naphthyl and indanyl, and especially phenyl and substituted phenyl;
[0091] aralkyl—represents a moiety containing an aryl group linked vial a lower alkyl;
[0092] alkylaryl—represents a moiety containing a lower alkyl linked via an aryl group;
[0093] cycloalkyl—represents a saturated carbocyclic ring having from 3 to 8 carbon atoms, preferably 5 or 6, optionally substituted.
[0094] heterocyclic—represents, in addition to the heteroaryl groups defined below, saturated and unsaturated cyclic organic groups having at least one O, S and/or N atom interrupting a carbocyclic ring structure that consists of one ring or two fused rings, wherein each ring is 5-, 6- or 7-membered and may or may not have double bonds that lack delocalized pi electrons, which ring structure has from 2 to 8, preferably from 3 to 6 carbon atoms, e.g., 2- or 3-piperidinyl, 2- or 3-piperazinyl, 2- or 3-morpholinyl, or 2- or 3-thiomorpholinyl;
[0095] halogen—represents fluorine, chlorine, bromine and iodine;
[0096] heteroaryl—represents a cyclic organic group having at least one O, S and/or N atom interrupting a carbocyclic ring structure and having a sufficient number of delocalized pi electrons to provide aromatic character, with the aromatic heterocyclyl group having from 2 to 14, preferably 4 or 5 carbon atoms, e.g., 2-, 3- or 4-pyridyl, 2- or 3-furyl, 2- or 3-thienyl, 2-, 4- or 5-thiazolyl, 2- or 4-imidazolyl, 2-, 4- or 5-pyrimidinyl, 2-pyrazinyl, or 3- or 4-pyridazinyl, etc. Preferred heteroaryl groups are 2-, 3- and 4-pyridyl; such heteroaryl groups may also be optionally substituted. Additionally, unless otherwise specifically defined, as stated above, the term “substituted or unsubstituted” or “optionally substituted” refers to the subject moiety being optionally and chemically-suitably substituted with a moiety belonging to R12 or R13. As used herein, “prodrug” means compounds that are drug precursors which, following administration to a patient, release the drug in vivo via some chemical or physiological process (e.g., a prodrug on being brought to the physiological pH or through enzyme action is converted to the desired drug form).
[0097] Also included in the invention are tautomers, rotamers, enantiomers and other optical isomers, as well as prodrugs, of compounds of Formula I, as well as pharmaceutically acceptable salts, solvates and derivatives thereof.
[0098] A further feature of the invention is pharmaceutical compositions containing as active ingredient a compound of Formula I (or its salt, solvate or isomers) together with a pharmaceutically acceptable carrier or excipient.
[0099] The invention also provides methods for preparing compounds of Formula I, as well as methods for treating diseases such as, for example, HCV, AIDS (Acquired Immune Deficiency Syndrome), and related disorders. The methods for treating comprise administering to a patient suffering from said disease or diseases a therapeutically effective amount of a compound of Formula I, or pharmaceutical compositions comprising a compound of Formula I.
[0100] Also disclosed is the use of a compound of Formula I for the manufacture of a medicament for treating HCV, AIDS, and related disorders.
[0101] Also disclosed is a method of treatment of a hepatitis C virus associated disorder, comprising administering an effective amount of one or more of the inventive compounds.
[0102] Also disclosed is a method of modulating the activity of hepatitis C virus (HCV) protease, comprising contacting HCV protease with one or more inventive compounds.
[0103] Also disclosed is a method of treating, preventing, or ameliorating one or more symptoms of hepatitis C, comprising administering an effective amount of one or more of the inventive compounds. The HCV protease is the NS3 or NS4a protease. The inventive compounds inhibit such protease. They also modulate the processing of hepatitis C virus (HCV) polypeptide.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS[0104] In one embodiment, the present invention discloses compounds of Formula I as inhibitors of HCV protease, especially the HCV NS3/NS4a serine protease, or a pharmaceutically acceptable derivative thereof, where the various definitions are given above.
[0105] Representative compounds of the invention which exhibit excellent HCV protease inhibitory activity are listed below in Tables 1 to 5 along with their activity (ranges of Ki* values in nanomolar, nM). Several compounds as well as addiitonal compounds are additionally disclosed in the claims. 1 TABLE 1 Compounds and HCV protease continuous assay results Compound from Example No. Ki* Range 1 C 2 C 3 C 4 C 5 C 6 C 7 C 8 C 9 C 10 C 11 C 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 C 20 C 21 C 22 C 23 C 24 C 25 C 26 C 27 C 28 C 29 C 30 C 31 C 32 C 33 C 34 C 35 C 36 C 37 C 38 C 39 C 40 C 41 C 42 C 43 C 44 C 45 C 46 C 47 C 48 C 49 C 50 C 51 C 52 C 53 C 54 C 55 C 56 C 57 C 58 C 59 C 60 C 61 C 62 C 63 C 64 C 65 C 66 C 67 C 68 B 69 C 70 C 71 B 72 C 73 B 74 C 75 C 76 A 77 B 78 A 79 C 80 A 81 C 82 A 83 B 84 C 85 C 86 B 87 B 88 A 89 B 90 C 91 C 92 C 93 C 94 C 95 C 96 C 97 C 98 B 99 B 100 A 101 A 102 C 103 C 104 C 105 C 106 C 107 B 108 A 109 A 110 A 111 A 112 A 113 B 114 A 115 B 116 A 117 A 118 A 119 A 120 A 121 B 122 B 123 A 124 B 125 B 126 B 127 A 128 A 129 A 130 B 131 A 132 A 133 A 134 B 135 A 136 A 137 A 138 A 139 A 140 B 141 A 142 A 143 B 144 B 145 C 146 A 147 A 148 B 149 A 150 A 151 A 152 A 153 A 154 A 155 B 156 B 157 B 158 C 159 B 160 A 161 A 162 A 163 C 164 A 165 C 166 B 167 A 168 C 169 B 170 B 171 A 172 A 173 A 174 A 175 A 176 B 177 B 178 A 179 A 180 B 181 A 182 B 183 A 184 A 185 A 186 A 187 A 188 A 189 B 190 B 191 B 192 A 193 A 194 B 195 A 196 B 197 A 198 A 199 A 200 A 201 B 202 A 203 B 204 B 205 B 206 B 207 B 208 A 209 A 210 A 211 A 212 A 213 B 214 B 215 B 216 B 217 C 218 A 219 A 220 A 221 A 222 A 223 B 224 C 225 C 226 A 227 A 228 C 229 A 230 A 231 A 232 C 233 C 234 C 235 C 236 B 237 C 238 A 239 C 240 A 241 C 242 B 243 C 244 B 245 C 246 B 247 A 248 A 249 C 250 C 251 B 252 C 253 C 254 B 255 B 256 A 257 C 258 A 259 A 260 C 261 C 262 A 263 B 264 B 265 C 266 B 267 A 268 C 269 A 270 C 271 A 272 C 273 C 274 C 275 C 276 A 277 B 278 A 279 B 280 A 281 C 282 C 283 C 284 C 285 C 286 C 287 C 288 B 289 B 290 C 291 C 292 C 293 C 294 C 295 C 296 B 297 C 298 C 299 B 300 B 301 C 302 C 303 B 304 C 305 C 306 C 307 B 308 B 309 C 310 C 311 C 312 C 313 B 314 A 315 B 316 B 317 A 318 A 319 A 320 A 321 C 322 C 323 C 324 C 325 A 326 A 327 C 328 B 329 B 330 A 331 A 332 A 333 B 334 B 335 B 336 A 337 A 338 C 339 A 340 C 341 C 342 C 343 A 344 C 345 C 346 C 347 B 348 B 349 C 350 C 351 C 352 C 353 C 354 C 355 C 356 A 357 A 358 C 359 A 360 B 361 B 362 C HCV continuous assay Ki* range: Category A = 1-100 nM; Category B = 101-1,000 nM; Category C > 1000 nM.
[0106] Some types of the inventive compounds and methods of synthesizing the various types of the inventive compounds of Formula I are listed below, then schematically described, followed by the illustrative Examples. 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108
[0107] Depending upon their structure, the compounds of the invention may form pharmaceutically acceptable salts with organic or inorganic acids, or organic or inorganic bases. Examples of suitable acids for such salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic, methanesulfonic and other mineral and carboxylic acids well known to those skilled in the art. For formation of salts with bases, suitable bases are, for example, NaOH, KOH, NH4OH, tetraalkylammonium hydroxide, and the like.
[0108] In another embodiment, this invention provides pharmaceutical compositions comprising the inventive peptides as an active ingredient. The pharmaceutical compositions generally additionally comprise a pharmaceutically acceptable carrier diluent, excipient or carrier (collectively referred to herein as carrier materials). Because of their HCV inhibitory activity, such pharmaceutical compositions possess utility in treating hepatitis C and related disorders.
[0109] In yet another embodiment, the present invention discloses methods for preparing pharmaceutical compositions comprising the inventive compounds as an active ingredient. In the pharmaceutical compositions and methods of the present invention, the active ingredients will typically be administered in admixture with suitable carrier materials suitably selected with respect to the intended form of administration, i.e. oral tablets, capsules (either solid-filled, semi-solid filled or liquid filled), powders for constitution, oral gels, elixirs, dispersible granules, syrups, suspensions, and the like, and consistent with conventional pharmaceutical practices. For example, for oral administration in the form of tablets or capsules, the active drug component may be combined with any oral non-toxic pharmaceutically acceptable inert carrier, such as lactose, starch, sucrose, cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, talc, mannitol, ethyl alcohol (liquid forms) and the like. Moreover, when desired or needed, suitable binders, lubricants, disintegrating agents and coloring agents may also be incorporated in the mixture. Powders and tablets may be comprised of from about 5 to about 95 percent inventive composition. Suitable binders include starch, gelatin, natural sugars, corn sweeteners, natural and synthetic gums such as acacia, sodium alginate, carboxymethylcellulose, polyethylene glycol and waxes. Among the lubricants there may be mentioned for use in these dosage forms, boric acid, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include starch, methylcellulose, guar gum and the like.
[0110] Sweetening and flavoring agents and preservatives may also be included where appropriate. Some of the terms noted above, namely disintegrants, diluents, lubricants, binders and the like, are discussed in more detail below.
[0111] Additionally, the compositions of the present invention may be formulated in sustained release form to provide the rate controlled release of any one or more of the components or active ingredients to optimize the therapeutic effects, i.e. HCV inhibitory activity and the like. Suitable dosage forms for sustained release include layered tablets containing layers of varying disintegration rates or controlled release polymeric matrices impregnated with the active components and shaped in tablet form or capsules containing such impregnated or encapsulated porous polymeric matrices.
[0112] Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for parenteral injections or addition of sweeteners and pacifiers for oral solutions, suspensions and emulsions. Liquid form preparations may also include solutions for intranasal administration.
[0113] Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier such as inert compressed gas, e.g. nitrogen.
[0114] For preparing suppositories, a low melting wax such as a mixture of fatty acid glycerides such as cocoa butter is first melted, and the active ingredient is dispersed homogeneously therein by stirring or similar mixing. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby solidify.
[0115] Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions and emulsions.
[0116] The compounds of the invention may also be deliverable transdermally. The transdermal compositions may take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.
[0117] Preferably the compound is administered orally, intravenously or subcutaneously.
[0118] Preferably, the pharmaceutical preparation is in a unit dosage form. In such form, the preparation is subdivided into suitably sized unit doses containing appropriate quantities of the active components, e.g., an effective amount to achieve the desired purpose.
[0119] The quantity of the inventive active composition in a unit dose of preparation may be generally varied or adjusted from about 1.0 milligram to about 1,000 milligrams, preferably from about 1.0 to about 950 milligrams, more preferably from about 1.0 to about 500 milligrams, and typically from about 1 to about 250 milligrams, according to the particular application. The actual dosage employed may be varied depending upon the patient's age, sex, weight and severity of the condition being treated. Such techniques are well known to those skilled in the art.
[0120] Generally, the human oral dosage form containing the active ingredients can be administered 1 or 2 times per day. The amount and frequency of the administration will be regulated according to the judgment of the attending clinician. A generally recommended daily dosage regimen for oral administration may range from about 1.0 milligram to about 1,000 milligrams per day, in single or divided doses.
[0121] Some useful terms are described below:
[0122] Capsule—refers to a special container or enclosure made of methyl cellulose, polyvinyl alcohols, or denatured gelatins or starch for holding or containing compositions comprising the active ingredients. Hard shell capsules are typically made of blends of relatively high gel strength bone and pork skin gelatins. The capsule itself may contain small amounts of dyes, opaquing agents, plasticizers and preservatives.
[0123] Tablet—refers to a compressed or molded solid dosage form containing the active ingredients with suitable diluents. The tablet can be prepared by compression of mixtures or granulations obtained by wet granulation, dry granulation or by compaction.
[0124] Oral gel—refers to the active ingredients dispersed or solubilized in a hydrophillic semi-solid matrix.
[0125] Powder for constitution refers to powder blends containing the active ingredients and suitable diluents which can be suspended in water or juices.
[0126] Diluent—refers to substances that usually make up the major portion of the composition or dosage form. Suitable diluents include sugars such as lactose, sucrose, mannitol and sorbitol; starches derived from wheat, corn, rice and potato; and celluloses such as microcrystalline cellulose. The amount of diluent in the composition can range from about 10 to about 90% by weight of the total composition, preferably from about 25 to about 75%, more preferably from about 30 to about 60% by weight, even more preferably from about 12 to about 60%.
[0127] Disintegrant—refers to materials added to the composition to help it break apart (disintegrate) and release the medicaments. Suitable disintegrants include starches; “cold water soluble” modified starches such as sodium carboxymethyl starch; natural and synthetic gums such as locust bean, karaya, guar, tragacanth and agar; cellulose derivatives such as methylcellulose and sodium carboxymethylcellulose; microcrystalline celluloses and cross-linked microcrystalline celluloses such as sodium croscarmellose; alginates such as alginic acid and sodium alginate; clays such as bentonites; and effervescent mixtures. The amount of disintegrant in the composition can range from about 2 to about 15% by weight of the composition, more preferably from about 4 to about 10% by weight.
[0128] Binder—refers to substances that bind or “glue” powders together and make them cohesive by forming granules, thus serving as the “adhesive” in the formulation. Binders add cohesive strength already available in the diluent or bulking agent. Suitable binders include sugars such as sucrose; starches derived from wheat, corn rice and potato; natural gums such as acacia, gelatin and tragacanth; derivatives of seaweed such as alginic acid, sodium alginate and ammonium calcium alginate; cellulosic materials such as methylcellulose and sodium carboxymethylcellulose and hydroxypropylmethylcellulose; polyvinylpyrrolidone; and inorganics such as magnesium aluminum silicate. The amount of binder in the composition can range from about 2 to about 20% by weight of the composition, more preferably from about 3 to about 10% by weight, even more preferably from about 3 to about 6% by weight.
[0129] Lubricant—refers to a substance added to the dosage form to enable the tablet, granules, etc. after it has been compressed, to release from the mold or die by reducing friction or wear. Suitable lubricants include metallic stearates such as magnesium stearate, calcium stearate or potassium stearate; stearic acid; high melting point waxes; and water soluble lubricants such as sodium chloride, sodium benzoate, sodium acetate, sodium oleate, polyethylene glycols and d'l-leucine. Lubricants are usually added at the very last step before compression, since they must be present on the surfaces of the granules and in between them and the parts of the tablet press. The amount of lubricant in the composition can range from about 0.2 to about 5% by weight of the composition, preferably from about 0.5 to about 2%, more preferably from about 0.3 to about 1.5% by weight.
[0130] Glident—material that prevents caking and improve the flow characteristics of granulations, so that flow is smooth and uniform. Suitable glidents include silicon dioxide and talc. The amount of glident in the composition can range from about 0.1% to about 5% by weight of the total composition, preferably from about 0.5 to about 2% by weight.
[0131] Coloring agents—excipients that provide coloration to the composition or the dosage form. Such excipients can include food grade dyes and food grade dyes adsorbed onto a suitable adsorbent such as clay or aluminum oxide. The amount of the coloring agent can vary from about 0.1 to about 5% by weight of the composition, preferably from about 0.1 to about 1%.
[0132] Bioavailability—refers to the rate and extent to which the active drug ingredient or therapeutic moiety is absorbed into the systemic circulation from an administered dosage form as compared to a standard or control.
[0133] Conventional methods for preparing tablets are known. Such methods include dry methods such as direct compression and compression of granulation produced by compaction, or wet methods or other special procedures. Conventional methods for making other forms for administration such as, for example, capsules, suppositories and the like are also well known.
[0134] Another embodiment of the invention discloses the use of the pharmaceutical compositions disclosed above for treatment of diseases such as, for example, hepatitis C and the like. The method comprises administering a therapeutically effective amount of the inventive pharmaceutical composition to a patient having such a disease or diseases and in need of such a treatment.
[0135] In yet another embodiment, the compounds of the invention may be used for the treatment of HCV in humans in monotherapy mode or in a combination therapy (e.g., dual combination, triple combination etc.) mode such as, for example, in combination with antiviral and/or immunomodulatory agents. Examples of such antiviral and/or immunomodulatory agents include Ribavirin (from Schering-Plough Corporation, Madison, New Jersey) and Levovirin™ (from ICN Pharmaceuticals, Costa Mesa, California), VP 50406™ (from Viropharma, Incorporated, Exton, Pa.), ISIS 14803™ (from ISIS Pharmaceuticals, Carlsbad, Calif.), Heptazyme™ (from Ribozyme Pharmaceuticals, Boulder, Colo.), VX 497™ (from Vertex Pharmaceuticals, Cambridge, Mass.), Thymosin™ (from SciClone Pharmaceuticals, San Mateo, Calif.), Maxamine™ (Maxim Pharmaceuticals, San Diego, Calif.), mycophenolate mofetil (from Hoffman-LaRoche, Nutley, N.J.), interferon (such as, for example, interferon-alpha, PEG-interferon alpha conjugates) and the like. “PEG-interferon alpha conjugates” are interferon alpha molecules covalently attached to a PEG molecule. Illustrative PEG-interferon alpha conjugates include interferon alpha-2a (Roferon™, from Hoffman La-Roche, Nutley, N.J.) in the form of pegylated interferon alpha-2a (e.g., as sold under the trade name Pegasys™), interferon alpha-2b (Intron™, from Schering-Plough Corporation) in the form of pegylated interferon alpha-2b (e.g., as sold under the trade name PEG-Intron™), interferon alpha-2c (Berofor Alpha™, from Boehringer Ingelheim, Ingelheim, Germany) or consensus interferon as defined by determination of a consensus sequence of naturally occurring interferon alphas (Infergen™, from Amgen, Thousand Oaks, Calif.).
[0136] As stated earlier, the invention includes tautomers, rotamers, enantiomers and other stereoisomers of the inventive compounds also. Thus, as one skilled in the art appreciates, some of the inventive compounds may exist in suitable isomeric forms. Such variations are contemplated to be within the scope of the invention.
[0137] Another embodiment of the invention discloses a method of making the compounds disclosed herein. The compounds may be prepared by several techniques known in the art. Representative illustrative procedures are outlined in the following reaction schemes. It is to be understood that while the following illustrative schemes describe the preparation of a few representative inventive compounds, suitable substitution of any of both the natural and unnatural amino acids will result in the formation of the desired compounds based on such substitution. Such variations are contemplated to be within the scope of the invention.
[0138] Abbreviations which are used in the descriptions of the schemes, preparations and the examples that follow are:
[0139] THF: Tetrahydrofuran
[0140] DMF: N,N-Dimethylformamide
[0141] EtOAc: Ethyl acetate
[0142] AcOH: Acetic acid
[0143] HOOBt: 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one
[0144] EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
[0145] NMM: N-Methylmorpholine
[0146] ADDP: 1,1′-(Azodicarbobyl)dipiperidine
[0147] DEAD: Diethylazodicarboxylate
[0148] MeOH: Methanol
[0149] EtOH: Ethanol
[0150] Et2O: Diethyl ether
[0151] DMSO: Dimethylsulfoxide
[0152] HOBt: N-Hydroxybenzotriazole
[0153] PyBrOP: Bromo-tris-pyrrolidinophosphonium hexafluorophosphate
[0154] DCM: Dichloromethane
[0155] DCC: 1,3-Dicyclohexylcarbodiimide
[0156] TEMPO: 2,2,6,6-Tetramethyl-1-piperidinyloxy
[0157] Phg: Phenylglycine
[0158] Chg: Cyclohexylglycine
[0159] Bn: Benzyl
[0160] Bzl: Benzyl
[0161] Et: Ethyl
[0162] Ph: Phenyl
[0163] iBoc: isobutoxycarbonyl
[0164] iPr: isopropyl
[0165] tBu or But: tert-Butyl
[0166] Boc: tert-Butyloxycarbonyl
[0167] Cbz: Benzyloxycarbonyl
[0168] Cp: Cylcopentyidienyl
[0169] Ts: p-toluenesulfonyl
[0170] Me: Methyl
[0171] HATU: O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
[0172] DMAP: 4-N,N-Dimethylaminopyridine
[0173] Bop: Benzotriazol-1-yl-oxy-tris(dimethylamino)hexafluorophosphate
[0174] General Preparative Schemes:
[0175] The following schemes describe the methods of synthesis of intermediate building blocks: 109
[0176] 110
[0177] 111
[0178] 112
[0179] 113
[0180] 114
[0181] 115
[0182] 116
[0183] 117 118
[0184] 119 120
[0185] 121 122
[0186] 123 124 125
[0187] 126
[0188] 127 128
[0189] Preparation of Intermediates:
PREPARATIVE EXAMPLE 1[0190] Step A: Compound (1.1) 129
[0191] To a stirred solution of Compound (1.08)(3.00 g, 12.0 mmol (S. L. Harbeson et al. J. Med. Chem. 37 No. 18 (1994) 2918-2929) in DMF (15 mL) and CH2Cl2 (15 mL) at −20° C. was added HOOBt (1.97 g, 12.0 mmol), N-methyl morpholine (4.0 mL, 36.0 mmol) and EDCl (2.79 g, 14.5 mmol) and stirred for 10 minutes, followed by addition of HCl.H2N—Gly—OBn (2.56 g, 13.0 mmol). The resulting solution was stirred at −20° C. for 2 hrs, kept refrigerated overnight and then concentrated to dryness, followed by dilution with EtOAc (150 mL). The EtOAc solution was then washed twice with saturated NaHCO3, H2O, 5% H3PO4, brine, dried over Na2SO4, filtered and concentrated to dryness to give the Compound (1.09) (4.5 g, 94%). LRMS m/z MH+=395.1.
[0192] Step B: Compound (1.1) 130
[0193] A solution of Compound (1.09) (7.00 g, 17.8 mmol) in absolute ethanol (300 mL) was stirred at room temperature under a hydrogen atmosphere in the presence of Pd—C (300 mg, 10%). The reaction progress was monitored by tic. After 2 h, the mixture was filtered through a celite pad and the resulting solution was concentrated in vacuo to give Compound (1.1) (5.40 g, quantitative). LRMS m/z MH+=305.1.
PREPARATIVE EXAMPLE 2[0194] Step A Compound (1.3) 131
[0195] A mixture of Compound (1.1) from Preparative Example 1, Step B above (1 eq.), Compound (1.2) (from Novabiochem, Catalog No. 04-12-5147) (1.03 eq.), HOOBt (1.03 eq.), N-methylmorpholine (2.2 eq.), and dimethylformamide (70 mL/g) was stirred at −20° C. EDCl (1.04 eq.) was added and the reaction stirred for 48 hr. The reaction mixture was poured into 5% aqueous KH2PO4 and extracted with ethyl acetate (2×). The combined organics were washed with cold 5% aqueous K2CO3, then 5% aqueous KH2PO4, then brine, and the organic layer was dried over anhydrous MgSO4. The mixture was filtered, then evaporated and the filtrate dried under vacuum, the residue was triturated with Et2O-hexane, and filtered to leave the title compound (1.3)(86% yield), C25H39N3O7 (493.60), mass spec. (FAB) M+1=494.3.
[0196] Step B Compound (1.4) 132
[0197] Compound (1.3) from Preparative Example 2, Step A (3.0 g) was treated with 4 N HCl/dioxane (36 mL) and stirred at room temperature for 7 min. The mixture was poured into 1.5 L cold (5° C.) hexane and stirred, then allowed to set cold for 0.5 hr. The mixture was suction-filtered in a dry atmosphere, and the collected solid was further dried to afford the title compound (1.4) (2.3 g, 88% yield), C20H31N3O5.HCl, H1 NMR (DMSO-d6/NaOD) &dgr; 7.38 (m, 5H), 5.25 (m, 1H), 4.3-4.1 (m, 1H), 3.8 (m, 2H), 3.4-3.3 (m, obscured by D2O), 1.7-1.1 (m, 4H), 1.35 (s, 9H), 0.83 (m, 3H).
PREPARATIVE EXAMPLE 3[0198] Compound (1.5) 133
[0199] Compound (1.3) from Preparative Example 2, Step A, was treated in essentially the same manner as in Preparative Example 7, Step A below to afford Compound (1.5).
PREPARATIVE EXAMPLE 4[0200] Compound (1.6) 134
[0201] Compound (1.5) from Preparative Example 3, was treated in essentially the same manner as in Preparative Example 2, Step B, to afford Compound (1.6).
PREPARATIVE EXAMPLE 5[0202] Step A Compound (2.09) 135
[0203] To a solution of dimethylamine hydrochloride (1.61 g, 19.7 mmol), N-Boc-phenylglycine, Compound (2.08)(4.50 g, 17.9 mmol, Bachem Co. # A-2225), HOOBt (3.07 g, 18.8 mmol) and EDCl (4.12 g, 21.5 mmol) in anhydrous DMF (200 mL) and CH2Cl2 (150 mL) at −20° C. was added NMM (5.90 mL, 53.7 mmol). After being stirred at this temperature for 30 min, the reaction mixture was kept in a freezer overnight (18 h). It was then allowed to warm to rt, and EtOAc (450 mL), brine (100 mL) and 5% H3PO4 (100 mL) were added. After the layers were separated, the organic layer was washed with 5% H3PO4 (100 mL), saturated aqueous sodium bicarbonate solution (2×150 mL), water (150 mL), and brine (150 mL), dried (MgSO4), filtered and concentrated in vacuo to afford Compound (2.09) (4.86 g) as a white solid, which was used without further purification.
[0204] Step B Compound (2.1) 136
[0205] Compound (2.09) from Preparative Example 5, Step A (4.70 g, crude) was dissolved in 4 N HCl (60 mL, 240 mmol) and the resulting solution was stirred at room temperature. The progress of the reaction was monitored by TLC. After 4 h, the solution was concentrated in vacuo to yield Compound (2.1) as a white solid which was used in the next reaction without further purification. LRMS m/z MH+=179.0.
PREPARATIVE EXAMPLE 6[0206] Step A Compound (2.2) 137
[0207] In essentially the same manner as Preparative Example 2, Step A. substituting phenylglycine N,N-dimethylamide hydrochloride in place of phenylglycine t-butyl ester hydrochloride, Compound (2.2) was prepared mass spec. (FAB) M+1=465.3.
[0208] Step B Compound (2.3) 138
[0209] Compound (2.2) from Step A (1.85 g) was reacted with 4 N HCl/dioxane (50 mL) at room temperature for 1 hr. The mixture was evaporated under vacuum in a 20° C. water bath, triturated under isopropyl ether, filtered, and dried to afford Compound (2.3) (1.57 g, 98% yield), C18H28N4O4.HCl, mass spec. (FAB) M+1=365.3
PREPARATIVE EXAMPLE 7[0210] Step A Compound (2.4) 139
[0211] A solution of Compound (2.2) from Preparative Example 5, Step A (2.0 g) in dichloromethane (60 mL) was treated with dimethylsulfoxide (3.0 mL) and 2,2-dichloroacetic acid (0.70 mL). The stirred mixture was cooled to 5° C. and then added 1 M dicyclohexylcarbodiimide/dichloromethane solution (8.5 mL). The cold bath was removed and the mixture stirred for 22 hr. Then added 2-propanol (0.5 mL), and stirred for an additional 1 hr. The mixture was filtered then washed with ice-cold 0.1 N NaOH (50 mL), then ice-cold 0.1 N HCl (50 mL), then 5% aqueous KH2PO4, then saturated brine. The organic solution was dried over anhydrous magnesium sulfate, then filtered. The filtrate was evaporated, and chromatographed on silica gel, eluting with ethyl acetate to afford Compound (2.3) (1.87 g, 94% yield), C23H34N4O6, mass spec. (FAB) M+1=463.3.
[0212] Step B Compound (2.5) 140
[0213] In essentially the same manner as Preparative Example 2, Step B, Compound (2.5) was prepared.
PREPARATIVE EXAMPLE 8[0214] Step A Compound (3.1) 141
[0215] In a flask were combined N-Cbz-hydroxyproline methyl ester (available from Bachem Biosciences, Incorporated, King of Prussia, Pa.), compound (3.01) (3.0 g), toluene (30 mL), and ethyl acetate (30 mL). The mixture was stirred vigorously, and then a solution of NaBr/water (1.28 g /5 mL) was added. To this was added 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO, 17 mg, from Aldrich Chemicals, Milwaukee, Wis.). The stirred mixture was cooled to 5° C. and then was added a prepared solution of oxidant [commercially available bleach, Clorox® (18 mL), NaHCO3 (2.75 g) and water to make up 40 mL] dropwise over 0.5 hr. To this was added 2-propanol (0.2 mL). The organic layer was separated, and the aqueous layer extracted with ethyl acetate. The organic extracts were combined, washed with 2% sodium thiosulfate, then saturated brine. The organic solution was dried over anhydrous MgSO4, filtered, and evaporated the filtrate under vacuum to leave a pale yellow gum suitable for subsequent reactions (2.9 g, 97% yield), C14H15NO5 (277.28), mass spec. (FAB) M+1=278.1.
[0216] Step B Compound (3.2). 142
[0217] Compound (3.1) from Step A above (7.8 g) was dissolved in dichloromethane (100 mL), and cooled to 15° C. To this mixture was first added 1,3-propanedithiol (3.1 mL), followed by freshly distilled boron trifluoride etherate (3.7 mL). The mixture was stirred at room temperature for 18 h. While stirring vigorously, a solution of K2CO3/water (2 g/30 mL)was carefully added, followed by saturated NaHCO3 (10 mL). The organic layer was separated from the aqueous layer (pH ˜7.4), washed with water (10 mL), then brine. The organic solution was dried over anhydrous MgSO4, filtered, and evaporated under vacuum. The residue was chromatographed on silica gel, eluting with toluene, then a with a gradient of hexane-Et2O (2:3 to 0:1) to afford a brown oil (7.0 g, 68% yield), C17H21NO4S2 (367.48), mass spec. (FAB) M+1=368.1.
[0218] Step C Compound (3.3) 143
[0219] A solution of compound (3.2) from Step B above (45 g) in acetonitrile (800 mL) at 20° C. was treated with freshly distilled iodotrimethylsilane (53 mL) at once. The reaction was stirred for 30 min., then poured into a freshly prepared solution of di-t-butyldicarbonate (107 g), ethyl ether (150 mL), and diisopropylethylamine (66.5 mL). The mixture stirred for 30 min. more then was washed with hexane (2×500 mL). Ethyl acetate (1000 mL) was added to the lower acetonitrile layer, and then the layer was washed with 10% aqueous KH2PO4 (2×700 mL), and brine. The filtrate was evaporated under vacuum in a 25° C. water bath, taken up in fresh ethyl acetate (1000 mL), and washed successively with 0.1 N HCl, 0.1 N NaOH, 10% aqueous KH2PO4, and brine. The organic solution was dried over anhydrous MgSO4, filtered, and evaporated under vacuum. The residue (66 g) was chromatographed on silica gel (2 kg), eluting with hexane (2 L), then Et2O/hexane (55:45, 2 L), then Et2O (2 L) to afford an orange gum which slowly crystallized on standing (28 g, 69% yield), C14H23NO4S2 (333.46), mass spec. (FAB) M+1=334.1.
[0220] Step D Compound (3.4) 144
[0221] A solution of compound (3.3) from Step C above (11 g) in dioxane (150 mL) at 20° C. was treated with 1 N aqueous LiOH (47 mL) and stirred for 30 h. The mixture was concentrated under vacuum in a 30° C. water bath to half volume. The remainder was diluted with water (300 mL), extracted with Et2O (2×200 mL). The aqueous layer was acidified to pH ˜4 with 12 N HCl (34 mL), extracted with ethyl acetate, and washed with brine. The organic solution was dried over anhydrous MgSO4, filtered, and evaporated under vacuum to leave Compound (3.4) (8.1 g, 78%), C13H21NO4S2 (319.44), mass spec. (FAB) M+1=320.1.
[0222] Step E Compound (3.5). 145
[0223] To a solution of compound (3.3) from Step C above (1 g) in dioxane (5 mL), was added 4 N HCl-dioxane solution (50 mL). The mixture was stirred vigorously for 1 hr. The mixture was evaporated under vacuum in a 25° C. water bath. The residue was triturated with Et2O, and filtered to leave the title compound (0.76 g, 93% yield), C9H15NO2S2.HCl (269.81), mass spec. (FAB) M+1=234.0.
PREPARATIVE EXAMPLE 9[0224] Step A Compound (3.6) 146
[0225] Following essentially the same procedure of Preparative Example 8, Step B, substituting ethane dithiol for propane dithiol, compound (3.6) was obtained.
[0226] Step B Compound (3.7). 147
[0227] Following essentially the same procedure of Preparative Example 8, Step C, substituting compound (3.6) for compound (3.2), the product compound (3.7) was obtained.
[0228] Step C Compound (3.8) 148
[0229] Following essentially the same procedure of Preparative Example 8, Step D, substituting compound (3.7) for compound (3.3) the product compound (3.8) was obtained.
[0230] Step D Compound (3.9) 149
[0231] Following essentially the same procedure of Preparative Example 8, Step E, substituting compound (3.7) for compound (3.3) the product compound (3.9) was obtained.
PREPARATIVE EXAMPLE 10[0232] Step A Compound (4.1) 150
[0233] In essentially the same manner as Preparative Example 2, Step A, Compound (4.1) was prepared C33H48N409S2 (708.89).
[0234] Step B Compound (4.2) 151
[0235] In essentially the same manner as Preparative Example 2, Step B, Compound (4.2) was prepared mass spec. (FAB) M+1=609.3.
[0236] Step C Compound (4.3) 152
[0237] In essentially the same manner as Preparative Example 2, Step A, Compound (4.3) was prepared, C41H61N5O10S2 (708.89), mass spec. (FAB) M+1=709.3.
[0238] Step D Compound (4.4) 153
[0239] In essentially the same manner as Preparative Example 7, Step A, Compound (4.4) was prepared.
PREPARATIVE EXAMPLE 11[0240] Step A Compound (4.5) 154
[0241] In essentially the same manner as Preparative Example 2, Step A, Compound (4.5) was prepared.
[0242] Step B, Compound (4.6) 155
[0243] In essentially the same manner as Preparative Example 2, Step B, Compound (4.6) was prepared.
[0244] Step C, Compound (4.7) 156
[0245] Compound (4.9) from Preparative Example 12, was reacted with Compound (4.6) from Step B above, in essentially the same manner as Preparative Example 2, Step A, to afford Compound (4.7).
[0246] Step D, Compound (4.8) 157
[0247] In essentially the same manner as Preparative Example 7, Step A, Compound (4.8) was prepared.
PREPARATIVE EXAMPLE 12[0248] Compound (4.9) 158
[0249] A solution of L-cyclohexylglycine (4.02) (1.0 eq.), dimethylformamide (20 mL/g), and diisopropylethylamine (1.1 eq.) at 5° C. is treated with isobutyl chloroformate (4.01) (1.1 eq.). The cold bath is removed and it is stirred for 6 hr. The reaction mixture is poured into 5% aqueous KH2PO4 and extracted with ethyl acetate (2×). The combined organics are washed with cold 5% aqueous K2CO3, then 5% aqueous KH2PO4, then brine, and the organics are dried over anhydrous MgSO4. The mixture is filtered, the filtrate evaporated under vacuum, the residue chromatographed if necessary or else the residue triturated with Et2O-hexane, and filtered to leave the title compound (4.9), C13H23NO4 (257.33).
PREPARATIVE EXAMPLE 13[0250] Compound (13.1) 159
[0251] In essentially the same manner as Preparative Example 12, substituting L-O-benzylthreonine (13.02) (Wang et al, J. Chem. Soc., Perkin Trans. 1, (1997) No. 5, 621-624.) for L-cyclohexylglycine (4.02) Compound (13.1) is prepared C16H23NO5 (309.36), mass spec. (FAB) M+1=310.2.
PREPARATIVE EXAMPLE 14[0252] 160
[0253] Compound (4.8) from Preparative Example 11, Step D (1.0 g) was reacted with a solution of anhydrous trifluoroacetic acid-dichloromethane (1:1, 50 mL) for 2 hr. The solution was diluted with xylene (100 mL) and evaporated under vacuum. The residue was triturated with Et2O, and filtered to leave the title compound (5.1) (0.9 g), C37H53N5O9S2 (775.98), mass spec. (FAB) M+1=776.5.
[0254] Step B Compound (5.2) 161
[0255] In essentially the same manner as Preparative Example 2, Step A, Compound (5.1) was reacted with ammonia (0.5 M 1,4-dioxane solution), to obtain the title compound (5.2) C37H54N6O8S2 (774.99), mass spec. (FAB) M+1=775.4.
PREPARATIVE EXAMPLE 15[0256] 162
[0257] A mixture of Compound (5.1) from Preparative Example 14, Step A (0.15 g), N,N-dimethylamine (0.12 mL of 2 M THF solution), dimethylformamide (10 mL), and PyBrOP coupling reagent (0.11 g) was cooled to 5° C., then diisopropylethylamine (DIEA or DIPEA, 0.12 mL) was added. The mixture was stirred cold for 1 min., then stirred at room temperature for 6 hr. The reaction mixture was poured into cold 5% aqueous H3PO4 (50 mL) and extracted with ethyl acetate (2×). The combined organics were washed with cold 5% aqueous K2CO3, then 5% aqueous KH2PO4, then brine. The organic solution was dried over anhydrous MgSO4, filtered, and evaporated under vacuum. The residue was chromatographed on silica gel, eluting with MeOH—CH2Cl2 to afford the title compound (5.3), C39H58N6O8S2 (803.05), mass spec. (FAB) M+1=803.5.
PREPARATIVE EXAMPLE 16[0258] Step A Compound (6.2) 163
[0259] In essentially the same manner as Preparative Example 2, Step A, Compound (6.1) hydroxyproline benzyl ester hydrochloride was reacted with Compound (4.9) from Preparative Example 12, to obtain the title compound (6.2), C25H36N2O6 (460.56), mass spec. (FAB) M+1=461.2.
[0260] Step B Compound (6.3) 164
[0261] In essentially the same manner as Preparative Example 8, Compound (6.3) was prepared, C25H34N2O6 (458.55), mass spec. (FAB) M+1=459.2.
[0262] Step C Compound (6.4) 165
[0263] A mixture of Compound (6.3) from Step B (1 g), 10% Pd/C (0.05 g), and EtOH (100 mL) was stirred under 1 atm. H2 for 6 hr. The mixture was filtered, and evaporated to dryness under vacuum to leave the title compound (6.4) (0.77 g), C18H28N2O6 (368.42) mass spec. (FAB) M+1=369.2.
PREPARATIVE EXAMPLE 17[0264] Step A Compound (7.1) 166
[0265] Compound (6.4) from Preparative Example 16, Step C, was reacted with Compound (2.3) from Preparative Example 6, Step B, in essentially the same manner as Preparative Example 2, Step A, to afford Compound (7.1), C36H54N6O9 (714.85), mass spec. (FAB) M+1=715.9.
[0266] Step B Compound (7.2) 167
[0267] Compound (7.1) was reacted in essentially the same manner as Preparative Example 7, Step A, to afford Compound (7.2), C36H52N6O9 (712.83), mass spec. (FAB) M+1=713.5.
[0268] Step C Compound (7.3) 168
[0269] Compound (7.2) from Step B above, was reacted in essentially the same manner as Preparative Example 8, Step B, with 1,4-butanedithiol, to obtain the title compound (7.3), C40H60N6O8S2 (817.07), mass spec. (FAB) M+1=817.5.
[0270] Using the above-noted procedures, the compounds in the attached Table 2 were prepared. As a general note to all the Tables that are attached hereto as well as to the Examples and Schemes in this specification, any open-ended nitrogen atom with unfulfilled valence in the chemical structures in the Examples and Tables refers to NH, or in the case of a terminal nitrogen, —NH2. Similarly, any open-ended oxygen atom with unfulfilled valence in the chemical structures in the Examples and Tables refers to —OH.
[0271] Solid Phase Synthesis:
[0272] General Procedure for Solid-Phase Coupling Reactions.
[0273] The synthesis was done in a reaction vessel which was constructed from a polypropylene syringe cartridge fitted with a polypropylene frit at the bottom. The Fmoc-protected amino acids were coupled under standard solid-phase techniques. Each reaction vessel was loaded with 100 mg of the starting Fmoc-Sieber resin (approximately 0.03 mmol). The resin was washed with 2 mL portions of DMF (2 times). The Fmoc protecting group was removed by treatment with 2 mL of a 20% v/v solution of piperidine in DMF for 20 min. The resin was washed with 2 mL portions of DMF (4 times). The coupling was done in DMF (2 mL), using 0.1 mmol of Fmoc-amino acid, 0.1 mmol of HATU [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate] and 0.2 mmol of DIPEA (N,N-diisopropylethylamine). After shaking for 2 h, the reaction vessel was drained and the resin was washed with 2 mL portions of DMF (4 times). The coupling cycle was repeated with the next Fmoc-amino acid or capping group.
[0274] General Procedure for Solid-Phase Dess-Martin Oxidation.
[0275] The synthesis was conducted in a reaction vessel which was constructed from a polypropylene syringe cartridge fitted with a polypropylene frit at the bottom. Resin-bound hydroxy compound (approximately 0.03 mmol) was treated with a solution of 0.12 mmol of Dess-Martin periodinane and 0.12 mmol of t-BuOH in 2 mL of DCM for 4 h. The resin was washed with 2 mL portions of a 20% v/v solution of iPrOH in DCM, THF, a 50% v/v solution of THF in water (4 times), THF (4 times) and DCM (4 times).
PREPARATIVE EXAMPLE 18[0276] Preparation of N-Fmoc-2′,3′-Dimethoxyphenylglycine Compound (901) 169
[0277] To a solution of potassium cyanide (1.465 g, 22.5 mmol) and ammonium carbonate (5.045 g, 52.5 mmol) in water (15 mL) was added a solution of 2,3-dimethoxybenzaldehye 901A (2.5 g, 15 mmol) in ethanol (15 mL). The reaction mixture was heated at 40° C. for 24 h. The volume of the solution was reduced to 10 mL by evaporating under reduced pressure. Concentrated hydrochloric acid (15 mL) was added and compound 901 B was obtained as a white precipitate. Compound 901 B was isolated by filtration (2.2 g, 9.3 mmol). Compound 901 B was dissolved in 10% w/w aqueous sodium hydroxide solution (15 mL) and the resulting solution was heated under reflux for 24 h. Concentrated hydrochloric acid was added and the pH was adjusted to neutral (pH 7). The resulting solution containing compound 901C was evaporated under reduced pressure. The residue was dissolved in 5% w/w aqueous sodium bicarbonate solution (150 mL). The solution was cooled to 0° C. in an ice bath and 1,4-dioxane (30 mL) and a solution of 9-fluorenylmethyl succinimidyl carbonate (2.7 g, 8 mmol) in 1,4-dioxane (30 mL) was added at 0° C. The reaction mixture was allowed to warm to room temperature and was stirred at room temperature for 24 h. 1,4-dioxane was evaporated under reduced pressure. The aqueous solution was washed with diethyl ether. Concentrated hydrochloric acid was added and the pH was adjusted to acidic (pH 1). Ethyl acetate was added the organic layer was washed with water and brine. The organic layer was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to afford the desired compound 901 as a white foamy solid (3.44 g, 7.9 mmol). MS (LCMS-Electrospray) 434.1 MH+.
PREPARATIVE EXAMPLE 19[0278] Compound (801) 170
[0279] To a solution of N-Fmoc-phenylalanine 801A (5 g, 12.9 mmol) in anhydrous DCM (22 mL) cooled to −30° C. in a dry ice-acetone bath was added N-methylpyrrolidine (1.96 mL, 16.1 mmol) and methyl chloroformate (1.2 mL, 15.5 mmol) sequentially. The reaction mixture was stirred at −30° C. for 1 h and a solution of N,O-dimethylhydroxylamine hydrochloride (1.51 g, 15.5 mol) and N-methylpyrrolidine (1.96 mL, 16.1 mmol) in anhydrous DCM (8 mL) was added. The reaction mixture was allowed to warm to room temperature and was stirred at room temperature overnight. Toluene was added and the organic layer was washed with dilute hydrochloric acid, aqueous sodium bicarbonate solution and brine. The organic layer was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to afforded compound 801 B (4 g, 9.29 mmol).
[0280] To a solution of Red-Al (6.28 mL, 21.4 mmol) in anhydrous toluene (8 mL) cooled to −20° C. in a dry ice-acetone bath was added a solution of compound 801B (4 g, 9.29 mmol) in anhydrous toluene (12 mL). The reaction mixture was stirred at −20° C. for 1.5 h. The organic layer was washed with dilute hydrochloric acid, aqueous sodium bicarbonate solution and brine. The organic layer was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the crude product 801 C was used in the next reaction without further purification.
[0281] To a solution of compound 801C (approx. 9.29 mmol) in hexane (15 mL) was added a solution of potassium cyanide (24 mg, 0.37 mmol) and tetrabutylammonium iodide (34 mg, 0.092 mmol) in water (4 mL) and acetone cyanohydrin (1.27 mL, 13.9 mmol) sequentially. The reaction mixture was stirred at room temperature for 24 h. Ethyl acetate was added and the organic layer was washed with water and brine. The organic layer was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to afford compound 801D (2.4 g, 6.03 mmol).
[0282] To a solution of compound 801 D (2.4 g, 6.03 mmol) in 1,4-dioxane (11 mL) was added concentrated hydrochloric acid (11 mL). The reaction mixture was heated at 80° C. for 3 h. Ethyl acetate (25 mL) and water (25 mL) was added. The organic layer was washed with brine and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to afford the desired compound 801 as a white foamy solid (2 g, 4.8 mmol). MS (LCMS-Electrospray) 418.1 MH+. 171 172
Example (301J):[0283] Scheme 8 Compound (301J) 173
[0284] Resin-bound compound 301B, 301C, 301D, 301E, 301F and 301G were prepared according to the general procedure for solid-phase coupling reactions started with 100 mg of Fmoc-Sieber resin (0.03 mmol). Resin-bound compound 301G was oxidized to resin-bound compound 301H according to the general procedure for solid-phase Dess-Martin oxidation. The resin-bound compound 301H was treated with 4 mL of a 2% v/v solution of TFA in DCM for 5 min. The filtrate was added to 1 mL of AcOH and the solution was concentrated by vacuum centrifugation to provide compound 301J (0.0069 g, 29% yield). MS (LCMS-Electrospray) 771.2 MH+.
[0285] Using the solid phase synthesis techniques detailed above, and the following moieties for the various functionalities in the compound of Formula 1, the compounds in Table 3 were prepared: 174 175 176 177 178 179 180 181 182 183 184 185 186 2 TABLE 3 Compounds prepared by Solid Phase Synthesis Ki* STRUCTURE CLASS 187 C 188 C 189 C 190 C 191 C 192 C 193 C 194 C 195 C 196 B 197 B 198 C 199 B 200 C 201 B 202 B 203 C 204 C 205 C 206 C 207 C 208 C 209 C 210 C 211 C 212 C 213 C 214 C 215 C 216 C 217 C 218 C 219 C 220 C 221 C 222 C 223 C 224 C 225 C 226 C 227 C 228 B 229 C 230 B 231 C 232 C 233 C 234 C 235 C 236 C 237 C 238 C 239 C 240 C 241 C 242 C 243 C 244 C 245 C 246 C 247 C 248 C 249 C 250 B 251 B 252 B 253 C 254 C 255 C 256 C 257 C 258 C 259 C 260 C 261 C 262 B 263 C 264 B 265 C 266 B 267 B 268 C 269 A 270 A 271 A 272 B 273 A 274 B 275 B 276 B 277 C 278 B 279 B 280 B 281 B 282 C 283 B 284 B 285 B 286 B 287 B 288 B 289 C 290 C 291 A 292 B 293 B 294 B 295 B 296 B 297 B 298 B 299 C 300 B 301 B 302 B 303 B 304 B 305 A 306 B 307 B 308 B 309 B 310 C 311 B 312 B 313 B 314 B 315 B 316 B 317 C 318 C 319 C 320 A 321 B 322 A 323 A 324 B 325 A 326 A 327 A 328 A 329 B 330 B 331 A 332 A 333 B 334 B 335 C 336 B 337 B 338 B 339 B 340 C 341 C 342 C 343 C 344 B 345 B 346 B 347 B 348 A 349 B 350 C 351 C 352 B 353 B 354 B 355 B 356 A 357 A 358 A 359 A 360 B 361 C 362 B 363 A 364 C 365 A 366 C 367 C 368 C 369 C 370 C 371 C 372 C 373 C 374 C 375 C 376 C 377 C 378 B 379 B 380 B 381 B 382 C 383 B 384 A 385 B 386 B 387 B 388 B 389 B 390 B 391 B 392 B 393 B 394 B 395 B 396 A 397 B 398 C 399 C 400 C 401 C 402 C 403 B 404 C 405 C 406 B 407 C 408 B 409 B 410 B 411 B 412 B 413 B 414 B 415 B 416 B 417 B 418 B 419 B 420 B 421 B 422 B 423 B 424 B 425 B 426 B 427 B 428 B 429 B 430 B 431 B 432 B 433 C 434 B 435 B 436 C 437 C 438 C 439 C 440 B 441 C 442 C 443 C 444 C 445 C 446 C 447 C 448 C 449 C 450 C 451 C 452 C 453 C 454 B 455 B 456 B 457 B 458 B 459 B 460 B 461 B 462 B 463 B 464 C 465 B 466 B 467 C 468 B 469 C 470 B 471 B 472 B 473 B 474 C 475 C 476 C 477 C 478 C 479 C 480 B 481 C 482 C 483 C 484 B 485 C 486 B 487 C 488 C 489 C 490 C 491 B 492 B 493 C 494 C 495 C 496 C
[0286] Additional compounds that were prepared and their activity (Ki*) ranges are given in the attached Tables 4 and 5. The procedure used to prepare the compounds in Tables 4 and 5 is outlined below.
[0287] I) Synthesis of Intermediates for the Compounds in Tables 4 and 5:
Example I Synthesis of 4,4-Dimethyl Proline Methyl Ester (H-Pro(4,4-diMe)—OMe)[0288] 497
[0289] Step 1. Synthesis of Tert-Butyl N-Tert-Butoxycarbonyl-4-Methyl-L-Pyroglutamate (Boc-PyroGlu(4-Methyl)-OtBu): 498
[0290] To a solution of tert-butyl N-tert-butoxycarbonyl-pyroglutamate (11.5 g, 40 mmol) in THF (200 mL) stirring at −78° C., was added a 1 M solution of lithium hexamethyldisilazide in THF (42 mL, 42 mmol) dropwise over 5 minutes. After 30 minutes, methyliodide (3.11 mL, 50 mmol) was added. After an additional 2 hours at −78° C., the cooling bath was removed and 50% saturated aqueous ammonium chloride (200 mL) was added. The solution was stirred for 20 minutes, then extracted with ether (3×200 mL). The combined organic layers were washed with brine (200 mL), dried (Na2SO4), filtered and concentrated. The residue was chromatographed with 1:1 ethylacetate/hexanes to give Boc-PyroGlu(4-methyl)-OtBu (10.6 grams, 35.4 mmol, 88%) as a mixture of isomers (2:1 cis to trans).
[0291] Step 2. Synthesis of Tert-Butyl N-Tert-Butoxycarbonyl-4,4-Dimethyl-L-Pyroglutamate (Boc-PyroGlu(4,4-Dimethyl)-OtBu): 499
[0292] To a solution of tert-butyl N-tert-butoxycarbonyl-4-methyl-L-pyroglutamate (1.2 g, 4.0 mmol) in tetrahydrofuran (20 mL) stirring at −78° C., was added a 1 M solution of lithium hexamethyldisilazide in tetrahydrofuran (4.4 mL, 4.4 mmol) dropwise over 5 minutes. After 30 minutes, methyliodide (0.33 mL, 5.2 mmol) was added. After an additional 3 hours at −78° C., the cooling bath was removed and 50% saturated aqueous ammonium chloride (40 mL) was added. The solution was stirred for 20 minutes, then extracted with ether (2×50 mL). The combined organic layers were washed with water(2×25 mL), saturated sodium bicarbonate (2×25 mL), brine (50 mL), dried (Na2SO4), filtered and concentrated to give Boc-PyroGlu(4,4-dimethyl)-OtBu (0.673 g, 54%).
[0293] Step 3. Synthesis of Tert-Butyl N-Tert-Butoxycarbonyl-4,4-Dimethylproline (Boc-Pro(4,4-Dimethyl)-OtBu) 500
[0294] Modification of known procedure: Pedregal, C.; Ezquerra, J.; Escribano, A.; Carreno, M. C.; Garcia Ruano, J. L. Tetrahedron Letters 1994, 35(13), 2053-2056).
[0295] To a solution of tert-butyl N-tert-butoxycarbonyl-4,4-dimethylpyroglutamate (2.0 mmol) in tetrahydrofuran (5 mL) stirring at −78° C., was added a 1M solution of lithium triethylborohydride in tetrahydrofuran (2.4 mL, 2.4 mmol) dropwise over 5 minutes. After 30 minutes, the cooling bath was removed and saturated aqueous sodium bicarbonate (5 mL) was added. The reaction mixture was immersed in an ice/water bath and 30% aqueous hydrogen peroxide (10 drops) was added. The solution was stirred for 20 minutes at 0° C., then the reaction mixture was concentrated in vacuo to remove the tetrahydrofuran. The aqueous solution was diluted with water (10 mL) and extracted with dichloromethane (3×40 mL). The organic layers were dried (Na2SO4), filtered and concentrated. The residue was dissolved in dichloromethane (20 mL) and triethylsilane (310 &mgr;L, 2.0 mmol), then cooled to −78° C. and boron trifluoride diethyletherate (270 &mgr;L, 2.13 mmol) was added dropwise. Stirring was continued for 30 minutes, at which time additional triethylsilane (310 &mgr;L, 2.0 mmol) and boron trifluoride diethyletherate (270 &mgr;L, 2.13 mmol) were added. After stirring at −78° C. for an additional two hours, the cooling bath was removed and saturated aqueous sodium bicarbonate (4 mL) was added. After 5 minutes the mixture was extracted with dichloromethane (3×40 mL). The organic layers were dried (Na2SO4), filtered and concentrated to give Boc-Pro(4,4-dimethyl)-OtBu.
[0296] Step 4. Synthesis of 4,4-Dimethylproline (H-Pro(4,4-Dimethyl)—OH): 501
[0297] A solution of tert-butyl N-tert-butoxycarbonyl-4,4-dimethylproline in dichloromethane (5 mL) and trifluoroacetic (5 mL) was stirred at room temperature for five hours. The solution was concentrated, dried under high vacuum and taken to the next step without further purification.
[0298] Step 5. Synthesis of N-Tert-Butoxycarbonyl 4,4-Dimethylproline (Boc-Pro(4,4-Dimethyl)—OH): 502
[0299] To a solution of 4,4-dimethylproline trifluoroacetic salt (1.5 mmol) in dioxane (7 mL), acetonitrile (12 mL) and diisopropylethylamine (700 &mgr;L, 4 mmol) was added a solution of di-tert-butyl-dicarbonate (475 mg, 2.18 mmol) in acetonitrile (5 mL). After stirring for 12 hours at room temperature the solution was concentrated in vacuo, dissolved in saturated aqueous sodium bicarbonate (50 mL) and washed with diethyl ether (3×40 mL). The aqueous layer was acidified to pH=3 with citric acid, then extracted with dichloromethane (3×40 mL). The combined organic layers were dried over sodium sulfate filtered and concentrated.
[0300] Step 6. Synthesis of 4,4-Dimethylproline Methylester Hydrochloride Salt (HCl.H-Pro(4,4-Dimethyl)—OMe): 503
[0301] To a solution of Boc-Pro(4,4-diMe)—OH (0.5 g, 2.06 mmol) in anhydrous methanol (8 ml) was added dropwise thionylchloride (448 I, 6.18 mmol) and the reaction was stirred for six hours at room temperature. The reaction mixture was concentrated to an amorphous solid (377 mg, 95%).
Example II. General Procedure for the Synthesis of N-Tertbutoxycarbonyl-4-alkyl-4-Methyl Proline[0302] 504
[0303] Compounds where R group is allyl and benzyl were synthesized following steps 1-4 below:
[0304] Step 1. Synthesis of Tert-Butyl N-Tert-Butoxycarbonyl-4-Alkyl-4-Methyl-L-Pyroglutamate: 505
[0305] To a solution of tert-butyl N-tert-butoxycarbonyl-4-methyl-L-pyroglutamate (10.2 g, mmol) (see Example I, step 1) in tetrahydrofuran (170 mL) stirring at −78° C., was added a 1 M solution of lithium hexamethyldisilazide in tetrahydrofuran (37.5 mL, 37.5 mmol) dropwise over 5 minutes. After 40 minutes, alkyl halide (61.4 mmol) was added. After an additional 3 hours at −78° C., the cooling bath was removed and 50% saturated aqueous ammonium chloride (200 mL) was added. The solution was stirred for 20 minutes, then extracted with ether (2×200 mL). The combined organic layers were diluted with hexanes (150 mL) and washed with saturated sodium bicarbonate (100 mL), water (2×100 mL) and brine (100 mL), dried (Na2SO4), filtered and concentrated. The residue was flash chromatographed using 20% ethylacetate in hexanes to give the pure tert-Butyl N-tert-butoxycarbonyl-4-alkyl-4-methyl-L-pyroglutamate.
[0306] Step 2. Synthesis of Tert-Butyl N-Tert-Butoxycarbonyl-4-Alkyl-4-Methylproline: 506
[0307] Modification of known procedure: Pedregal, C.; Ezquerra, J.; Escribano, A.; Carreno, M. C.; Garcia Ruano, J. L. Tetrahedron Letters (1994) 35(13), 2053-2056).
[0308] To a solution of tert-butyl N-tert-butoxycarbonyl-4-alkyl-4-methylpyroglutamate (16.6 mmol) in tetrahydrofuran (40 mL) stirring at −78° C., was added a 1 M solution of lithium triethylborohydride in tetrahydrofuran (20 mL, mmol) dropwise over 10 minutes. After 120 minutes, the cooling bath was allowed to warm to −25° C. at which point saturated aqueous sodium bicarbonate (40 mL) was added. The reaction mixture was immersed in an ice/water bath and 30% aqueous hydrogen peroxide (4 mL) was added. The solution was stirred for 10 minutes at 0° C., then the reaction mixture was concentrated in vacuo to remove the tetrahydrofuran. The aqueous solution was diluted with water (300 mL) and extracted with dichloromethane (3×200 mL). The organic layers were dried (sodium sulfate), filtered and concentrated. The residue was dissolved in dichloromethane (100 mL) and triethylsilane (2.6 mL, mmol), then cooled to −78° C. and boron trifluoride diethyletherate (2.2 mL, mmol) was added dropwise. Stirring was continued for 1 hour, at which time additional triethylsilane (2.6 mL, mmol) and boron trifluoride diethyletherate (2.2 mL, mmol) were added. After stirring at −78° C. for an additional 4 hours, the cooling bath was removed and saturated aqueous sodium bicarbonate (30 mL) and water (150 mL) were added. After 5 minutes the mixture was extracted with dichloromethane (3×200 mL). The organic layers were dried (Na2SO4), filtered and concentrated.
[0309] Step 3. Synthesis 4-Alkyl-4-Methylproline: 507
[0310] A solution of tert-butyl N-tert-butoxycarbonyl-4-alkyl-4-methylproline in dichloromethane (5 mL) and trifluoroacetic (5 mL) was stirred at room temperature for 5 hours. Toluene was added and the solution was concentrated and then dried under high vacuum.
[0311] Step 4. Synthesis of N-Tert-Butoxycarbonyl 4-Alkyl-4-Methylproline: 508
[0312] To a solution of 4-alkyl-4-methylproline trifluoroacetic salt (1.5 mmol) in dioxane (7 mL), acetonitrile (12 mL) and diisopropylethylamine (700 &mgr;L, 4 mmol) was added a solution of di-tert-butyl-dicarbonate (475 mg, 2.18 mmol) in acetonitrile(5 mL). After stirring for 12 hours at room temperature the solution was concentrated in vacuo, dissolved in saturated aqueous sodium bicarbonate (50 mL) and washed with diethyl ether (3×40 mL). The aqueous layer was acidified to pH=3 with 1N hydrochloric acid, then extracted with dichloromethane (3×40 mL). The combined organic layers were dried (Na2SO4), filtered and concentrated. The residue was purified by flash chromatography using 1:1 ethylacetate/hexanes with 1% acetic acid.
Example II. Synthesis of N-Tert-Butoxycarbonyl 4-Propyl-4-Methylproline[0313] 509
[0314] A solution of N-tertbutoxycarbonyl-4-allyl-4-methylproline (400 mg, 1.48 mmol) (see Example II Step 4) and 10% Pd on carbon (400 mg) in methanol (20 mL) was hydrogenated at 50 psi for 4 hours. The mixture was filtered and concentrated.
Example IV. Synthesis of Boc-4-Cyclohexylproline[0315] 510
[0316] A solution of the commercially available Boc-4-phenylproline (750 mg) and 5% Rh on carbon (750 mg) in methanol (15 mL) was hydrogenated at 50 psi for 24 hours. The mixture was filtered and concentrated to give 730 mg of product.
Example V Preparation of Fluorenylmethoxycarbonyl-Pro(4-Spirocyclopentane)-Carboxylic Acid[0317] 511
[0318] Step 1. Synthesis of Boc-Pyroglutamic(4-Allyl)-Tert-Butylester: 512
[0319] To a cooled (−78° C.) solution of the commercially available N-Bo c-tert-butyl pyroglutamate (10 g, 35.1 mmol) in THF (175 ml) was added lithium hexamethyldisilazide (36.8 mL, 36.8 mmol) over five minutes. Stirring continued for thirty minutes. A solution of allyl bromide (6.1 ml, 70.2 mmol) in THF (39 mL) was added dropwise to the first solution. After two hours at −78° C., the reaction was quenched by the slow addition of saturated ammonium chloride (50 mL) solution. The reaction mixture was then diluted with ethylacetate and the layers were separated. The organic layer dried over sodium sulfate and concentrated. Flash column chromatography carried out in 2:8 ethylacetate: hexanes afforded the product (6 g, 53%). NMR &dgr; ppm (CDCl3): 5.7 (m, 1H), 5.1 (dd, 2H), 4.4 (m, 1H), 2.6 (m, 2H), 2.4 (m, 1H), 1.8-2.2 (m, 1H), 1.45 (s, 9H), 1.4 (s, 9H).
[0320] Step 2. Synthesis of N-Boc-Pyroglutamic(4,4-Diallyl)-Tert-Butylester: 513
[0321] N-Boc-pyroglutamic(4-allyl)-tert-butylester obtained in the Step 1 above (2.68 g, 8.24 mmol) was subjected to a second alkylation with allyl bromide under similar conditions. Flash chromatography in 15:85 ethylacetate: hexanes provided 2.13 g product (71%) as a clear oil.
[0322] Step 3. Synthesis of Boc-Pro(4,4-Diallyl)-Tert-Butylester: 514
[0323] Part a: To a cooled (−78° C.) solution of Boc-PyroGlu(4,4-diallyl)-tert-butylester (2.13 g, 5.83 mmol) in tetrahydrofuran (14 ml) was added lithium triethylborohydride (1M in tetrahydrofuran, 7.29 ml, 7.29 mmol) over five minutes. After two hours at −78° C., the reaction was warmed-up to 0° C. and quenched by the slow addition of saturated sodium bicarbonate solution (20 ml) and 30% hydrogen peroxide (20 drops). Stirring continued for 20 minutes. The tetrahydrofuran was removed under reduced pressure and the remaining thick white residue was diluted with water (80 ml) and extracted three times with dichloromethane. The organic layer was dried, filtered and concentrated and taken to the next step without further purification.
[0324] Part b): To the product obtained in part (a) in dichloromethane (14 ml) was added triethylsilane (931 &mgr;l, 5.83 mmol) followed by boron trifluoride diethyl etherate (776 &mgr;l, 6.12 mmol). After thirty minutes more triethylsilane (931 &mgr;l, 5.83 mmol) and boron trifluoride diethyl etherate etherate (776 &mgr;l, 6.12 mmol) were added and the reaction was stirred at −78° C. for three hours at which time the reaction was quenched by the slow addition of saturated sodium bicarbonate solution and water. The reaction mixture was extracted with dichloromethane and the organic layer was dried, filtered and concentrated. Flash column chromatography in 15% ethylacetate in hexanes afforded 1.07 colorless oil (57%). NMR &dgr; ppm (CDCl3): 5.7-5.8 (m, 2H), 5.1 (m, 4H), 4.1-4.2 (2 dd's, 1H rotamers), 3.5-3.3 (dd, 1H) and 3.2 (dd, 1H) rotamers, 2.2-2.0 (m, 5H), 1.7(m, 1H), 1.46 (s, 9H), 1.43 (s, 9H).
[0325] Step 4. Synthesis of Boc-Pro(4-Spirocyclopentene)-Tert-Butylester: 515
[0326] To Boc-Pro(4,4-diallyl)-tert-butylester (1.07 g, 3.31 mmol) in dichloromethane (66 ml) was added 5% Bis(tricyclohexylphosphin)benzylidene ruthenium IV dichloride (Grubbs catalyst) and the mixture was heated at reflux for 1.5 hours. The reaction mixture was concentrated and the remaining residue was purified by flash column chromatography in 15% ethylacetate in hexanes. A yellow oil was obtained (0.57 g, 53%). NMR &dgr; ppm (CDCl3): 5.56 (bs, 2H), 4.2 and 4.1 (t, 1H, rotamers), 3.2-3.5 (m, 2H), 2.2-2.5 (m, 5H), 1.9 (dd, 1H) 1.47 and 1.46 (2 s's, 9H, rotamers), 1.45 and 1.44 (2 s's, 9H, rotamers).
[0327] Step 5. Synthesis of Boc-Pro(4-Spirocyclopentane)-Tert-Butylester: 516
[0328] A solution of Boc-Pro(4-spirocyclopentene)-tert-butylester (1.12 g) in methanol (18 ml), water (4 ml) and acetic acid (4 ml) was placed in the Parr shaker and was hydrogenated for three hours at 35 psi in the presence of 10% palladium on carbon (300 mg). The catalyst was filtered off and the filtrate was concentrated to a colorless oil (1.26 g). NMR &dgr; ppm (CDCl3): 4.1 and 4.2 (t, 1H, rotamers, 3.4 (d, 1H), 3.2 (d, 1H), 2.1 (m, 1H), 1.9 (m, 1H), 1.6-1.7 (m, 10H), 1.5 (3 s's, 18H, rotamers).
[0329] Step 6. Synthesis of Fmoc-Pro(4-Spirocyclopentane)-Carboxylic Acid: 517
[0330] The Boc-Pro(4-spirocyclopentane)-tert-butylester (1.26, 3.9 mmol) was treated with dichloromethane (10 ml) and trifluoroacetic acid (15 ml) for three hours. The reaction mixture was concentrated and the yellow oil obtained was dissolved in water (6 ml). Fluorenylmethyl succinyl carbonate (1.45 g, 4.3 mmol) dissolved in dioxane (6 ml) was added portionwise followed by the addition of potassium carbonate (2.16 g, 15.6 mmol). The reaction was stirred for 18 hours and concentrated. The remaining residue was diluted with the saturated sodium bicarbonate solution (10 mL) and washed with diethylether (3×10 ml). The aqueous layer was then acidified to pH ˜1 with 1N sodium bisulfate solution and extracted with ethylacetate. The organic layer was dried over sodium sulfate, filtered and concentrated to a beige foam (1.3 g, 100%).
Example VI Synthesis of Boc-Pro(4T-NH(Fmoc))—OH[0331] 518
[0332] Step 1. Synthesis of N&agr;-Tert-Butoxycarbonyl-Cis-4-Chloro-L-Proline Benzyl Ester: 519
[0333] A mixture of the commercially available N-tert-butoxycarbonyl-trans-4-hydroxy-proline (8.79 g, 38 mmol), potassium carbonate (13.0 g, 94 mmol), benzyl bromide (4.5 ml, 38 mmol) and dimethylformamide (150 mL) was stirred for 18 h. Addition of ethyl acetate (100 mL) was followed by filtration. The white cloudy filtrate was clarified by the addition of 1M HCl (100 mL). The layers were separated and the aqueous layer was extracted with additional ethyl acetate (2×100 mL). The combined organic layers were washed with water (2×50 mL), dried (sodium sulfate), filtered and concentrated. Toluene was added to the crude benzyl ester, and the solution was filtered and reconcentrated. Dichloromethane (70 mL) and carbon tetrachloride (70 mL) was added, followed by triphenylphosphine (21.11 g, 80 mmol). The reaction mixture was stirred for 10 h, quenched with ethanol (7 mL) and stirred for 5 more h. The solution was concentrated to approx. 100 ml, then dichloromethane (40 mL) was added, followed by the addition of ether (200 mL) while stirring. The solution was cooled for 4 h, filtered and concentrated to give a yellow-brown oil which was purified by flash chromatography using ether/hexane/dichloromethane 2:2:1 to give the title compound (9.13 g, 26.9 mmol, 71%) as a white solid.
[0334] Step 2. Synthesis of N&agr;-Tert-Butoxycarbonyl-Trans-4-Azido-L-Proline Benzyl Ester: 520
[0335] A solution of N&agr;-tert-butoxycarbonyl-cis-4-chloro-L-proline benzyl ester (9.0 g, 26.5 mmol) and sodium azide (7.36 g, 113 mmol) in dimethylformamide (270 mL) was heated at 75° C. for 2 days. Water (100 mL) was added and the reaction mixture was extracted with ethyl acetate (3×100 mL). The combined organic layers were washed with water (3×50 mL), dried (sodium sulfate), filtered and concentrated. The oil was purified by flash chromatography using ethyl acetate/hexanes 1:1 to give the title compound (8.59 g, 24.8 mmol, 94%).
[0336] Step 3. Synthesis of Boc-Pro(4T-NH(Fmoc))—OH: 521
[0337] A mixture of N-&agr;-t-butoxycarbonyl-trans-4-azido-L-proline benzyl ester (8.59 g, 24.8 mmol) and 10% palladium on carbon (900 mg) in ethanol (500 mL) was hydrogenated at 50 psi for 14 h using a Parr hydrogenation apparatus. The mixture was filtered, concentrated, dissolved in methanol (60 mL), refiltered and concentrated to give a colorless oil. The oil was dissolved in water (53 mL) containing sodium carbonate (5.31 g, 50.1 mmol) and a solution of fluorenylmethyl succinyl carbonate (8.37 g, 29.8 mmol) in dioxane (60 mL) was added over 40 min. The reaction mixture was stirred at room temperature for 17 h, then concentrated to remove the dioxane and diluted with water (200 mL). The solution was washed with ether (3×100 mL). The pH of the aqueous solution was adjusted to 2 by the addition of citric acid (caution! foaming!) and water (100 mL). The mixture was extracted with dichloromethane (400 mL, 100 mL, 100 mL) and the combined organic layers were dried (sodium sulfate), filtered and concentrated to give the title compound.
Example VII Synthesis of N-T-Butoxycarbonyl-4-Trans-(N-Fluorenylmethyloxycarbonyl Aminomethyl)-L-Proline (Boc-Pro(4T-MeNHFmoc)—OH)[0338] 522
[0339] Step 1. Synthesis Tert-Butoxycarbonyl Cis4-Hydroxy-L-Proline Benzyl Ester (Boc-Pro(4-Cis-OH)—OBn): 523
[0340] To a mixture of cis-hydroxy-L-proline (5 g, 38.1 mmol) in benzene (45 mL) and benzyl alcohol (45 mL) was added p-toluenesulfonic acid monohydrate (7.6 g, 40.0 mmol). The reaction mixture was heated at 125° C. for 20 h while water (2 ml) was removed using a Dean-Stark trap. The solution was filtered while still hot, and then ether (150 ml) was added. The solution was allowed to cool for three h at room temperature, then three h at 4° C. The resulting solid was collected, washed with ether (100 mL) and dried in vacuo for 1 h to give 13.5 grams of white solid. The solid was dissolved in dioxane (40 mL) and diisopropylethylamine (7.6 mL), and then di-tert-butyl-dicarbonate (10 g, 45.8 mmol) was added over 5 min while using an ice bath to maintain a constant reaction temperature. After 10 h at room temperature the reaction mixture was poured into cold water (200 mL) and extracted with ethyl acetate (3×200 mL). The combined organic layers were washed with water (3×100 mL) and saturated aqueous sodium chloride (50 mL), dried (sodium sulfate), filtered and concentrated. The crude product was purified by flash chromatography using 40-60% ethyl acetate in hexanes to give the title compound (10.04 g, 31.24 mmol, 82%).
[0341] Step 2. Synthesis of N-T-Butoxycarbonyl Cis-4-Mesyloxy-L-Proline Benzyl Ester (Boc-Pro(4-Cis-OMs)—OBn): 524
[0342] To a solution of Boc-Pro(4-cis-OH)—OBn (8.45 g, 26.3 mmol) in pyridine (65 mL) at 0° C., was added methanesulfonyl chloride (3.4 mL, 44 mmol) dropwise over 7 min. The reaction mixture was allowed to warm to room temperature over 2 h, then stirred overnight. A solution of 10% water in pyridine (20 mL) was added over 15 min and the reaction mixture was concentrated. The residue was dissolved in water and extracted with ethyl acetate (2×200 mL). The combined organic layers were washed with water (2×50 mL) saturated aqueous sodium bicarbonate (50 mL) and saturated aqueous sodium chloride (50 mL), dried (sodium sulfate), filtered and concentrated. The resulting residue was dissolved in toluene (100 mL) and concentrated to remove traces of pyridine. The residue was dried in vacuo for 30 min to afford the title compound (10.7 g, 102%), then used in the next step without purification.
[0343] Step 3. N-T-Butoxycarbonyl-Trans-4R-Cyano-L-Proline Benzylester (Boc-Pro(4-trans-CN)—OBn): 525
[0344] A solution of Boc-Pro(4-cis-OMs)—OBn (10.7 g, 26.3 mmol) and tetrabutylammonium cyanide (15.0 g, 56 mmol) in dimethylformamide (100 mL) was heated in an oil bath at 55° C. for 28 h. After cooling, water (150 mL) was added and the mixture was extracted with ethyl acetate (3×200 mL). The combined organic layers were washed with water (3×100 mL) and saturated aqueous sodium chloride (100 mL), dried (sodium sulfate), filtered and concentrated. The resulting residue was purified by flash chromatography (1:1 ether/hexanes) and then recrystallized from ethyl acetate/hexanes to provide the title compound (2.40 g, 7.26 mmol, 28%).
[0345] Step 4. N-T-Butoxycarbonyl-4-Trans-(N-Fluorenylmethyloxycarbonyl Aminomethyl)-L-Proline (Boc-Pro(4T-MeNHFmoc)—OH): 526
[0346] A mixture of the compound of Step 3 above (2.31 g, 7 mmol), water (10 mL), methanol (85 mL) and 10% palladium on carbon (700 mg) was hydrogenated at 50 psi for 11 h using a Parr hydrogenation apparatus. The mixture was filtered and concentrated. Water (15 mL) and sodium carbonate (1.5 g, 14.2 mmol) was added to the residue. A solution of fluorenylmethyl succinyl carbonate (2.36 g, 7.0 mmol) in dioxane (17 mL) was added over 5 min and stirring was continued for 28 h at room temperature. The reaction was concentrated in vacuo to a 15 mL volume, and water (100 mL) was added. The solution was washed with ether (3×75 mL). The pH of the aqueous solution was adjusted to 2 by the addition of citric acid (approx. 20 g, caution! foaming!) and water (100 mL). The mixture was extracted with dichloromethane (4×100 mL), and the combined organic layers were dried (sodium sulfate), filtered and concentrated. The crude product contained a major impurity which necessitated a three step purification. The crude product was dissolved in dichloromethane (50 mL) and trifluoroacetic acid (50 mL) and stirred for 5 h before being concentrated. The residue was purified by preparatory reverse-phase HPLC. The pure 4-(N-fluorenylmethyloxycarbonyl aminomethyl)proline trifluoroacetate salt (1.887 g, 3.93 mmol) was dissolved in dioxane (10 mL), acetonitrile (20 mL) and diisopropylethylamine (1.4 mL, 8 mmol). To the reaction mixture was added a solution of di-tert-butyldicarbonate (1.1 g, 5 mmol) in dioxane (5 mL). After stirring for 18 h, the pH of the solution was adjusted to 2 by the addition of citric acid (caution: foaming!) and water (100 mL). The mixture was extracted with ethyl acetate (3×150 mL) and the combined organic layers were washed with saturated aqueous sodium chloride (100 mL), dried (sodium sulfate), filtered and concentrated. The crude product was dissolved in saturated aqueous sodium bicarbonate(100 mL) and washed with ether (3×75 mL). The aqueous layer was adjusted to pH=3 by the addition of citric acid, then extracted with dichloromethane (4×100 mL). The combined organic layers were dried (sodium sulfate), filtered and concentrated to the title compound (1.373 g, 2.94 mmol, 42%).
Example VIII Synthesis of 3,4-Isopropylideneprolinol[0347] 527
[0348] Step I. Cyclopropanation Reaction (Tetrahedron Lett. 1993. 34(16). 2691 and 2695): 528
[0349] To a stirring solution of isopropyltriphenyl-phosphonium iodide (4.14 g, 9.58 mmol) in tetrahydrofuran (60 mL) at 0° C., was added n-butyllithium (1.6 M in hexanes, 5.64 mL, 9.02 mmol) over 5 min. After 30 min, a solution of enamide ((5R, 7S)-5-phenyl-5,6,7,7a-tetrahydro-6-oxapyrrolizin-3-one) (1.206 grams, 6.0 mmol) (see J. Org. Chem. 1999, 64(2), 547 for the synthesis of the enamide starting material) in tetrahydrofuran (40 mL) was added over 10 min. After an additional 10 min, the cooling bath was removed and the reaction mixture was stirred at room temperature for 4 hours. The reaction was poured into water (400 mL) and extracted with diethyl ether (400 mL) and ethylacetate (2×400 mL). The combined organic extracts were dried with sodium sulfate, filtered and concentrated to give the desired crude product. The residue was purified by flash chromatography eluting with 3:5:2 ethylacetate/hexanes/methylene chloride to give pure cyclopropanated product (750 mg, 3.08 mmol, 51%).
[0350] Step 2. Synthesis of 3,4-Isopropylideneprolinol P[3,4-(diMe-Cyclopropyl)]-Alcohol) (J. Org. Chem. (1999) 64(2). 330: 529
[0351] A mixture of the product obtained in step 1 above (1.23 grams, 5.06 mmol) and lithium aluminum hydride (1.0 M in THF, 15 mL, 15 mmol) was heated at reflux for 5 hours. After cooling to 0° C., the remaining aluminum hydride was carefully quenched by the dropwise addition of saturated aqueous sodium sulfate (1.5 mL) over 15 min. The mixture was diluted with ethylacetate (40 mL) and then filtered through celite. The filtrate was dried with sodium sulfate, filtered and concentrated to give crude N-benzyl aminoalcohol (1.25 grams), which was carried on to the next step without further purification. A solution of crude N-benzyl aminoalcohol (1.25 grams, 5.06 mmol) in 1:1 acetic acid/ethylacetate (30 mL) with 10% Pd/C (1 gram) was hydrogenated at 50 psi for 16 hours using a Parr hydrogenation apparatus. The reaction mixture was filtered to remove the carbon-based catalyst and the filtrate was concentrated. The residue was dissolved in water (30 mL) and the pH was adjusted to 13 with 50% NaOH. The mixture was extracted with ether (3×60 mL). The combined extract was dried with sodium sulfate, filtered and concentrated to give crude aminoalcohol (485 mg, 3.43 mmol). This material was taken to the next step without further purification.
Example IX Synthesis of iBoc-G(Chx)-Pro(3,4-Isopropylidene)-Carboxylic Acid:[0352] 530
[0353] Step 1. Synthesis of Isobutyloxycarbonyl-Cyclohexylglycine (iBoc-G(Chx)—OH 531
[0354] To a solution of the commercially available cyclohexylglycine hydrochloride (15 g, 77.4 mmol) in acetonitrile (320 ml) and water (320 ml) was added potassium carbonate. Isobutylchloroformate (11.1 ml, 85.1 mmol) was added to the clear solution over 15 minutes and the reaction was stirred for 17 hours. The acetonitrile was removed under reduced pressure and the remaining aqueous layer was extracted twice with ether (100 ml). The aqueous layer was then acidified to pH 1 with 6N hydrochloric acid and extracted with dichloromethane (3×300 ml). The organic layer was dried over sodium sulfate, filtered and concentrated to yield 18.64 g (94%) product as a white solid.
[0355] Step 2. Synthesis of Isobutyloxycarbonyl-cyclohexyiglycyl-3.4-Isopropylideneproline (iBoc-G(Chx)-P[3,4-(diMe-Cyclopropyl)]—OH): 532
[0356] a) Coupling Step
[0357] To a solution of iBoc-G(Chx)—OH (890 mg, 3.45 mmol) in acetonitrile (20 mL) was added HATU (1.33 g, 3.5 mmol), HOAt (476 mg, 3.5 grams) and then diisopropylethylamine (2.5 mL, 14 mmol). After a 2 minutes, 3,4-isopropylideneprolinol (485 mg, 3.43 mmol) was added and the reaction mixture was stirred overnight. Addition of saturated aqueous sodium bicarbonate was followed by extraction with ether and ethylacetate. The combined organic layers were dried, filtered and concentrated. The residue was purified by flash chromatography eluting with 1:1 ethylacetate/hexanes to give pure dipeptide alcohol iBoc-G(Chx)-3,4-isopropylideneprolinol (870 mg, 2.3 mmol, 67%)
[0358] b) Jones Oxidation Step
[0359] To a solution of dipeptide alcohol iBoc-G(Chx)-3,4-isopropylideneprolinol (100 mg, 0.26 mmol) in acetone (2 mL) stirring at 0° C. was added Jones reagent (300 1L) dropwise over 5 min. [Jones Reagent: Prepared from chromium trioxide (13.4 g) and concentrated sulfuric acid (11.5 mL) diluted with water to a total volume of 50 mL.] After stirring at 0° C. for 3 hours, isopropanol (500 &mgr;L) was added and stirring continued for an additional 10 minutes. The reaction mixture was diluted with water (20 mL) and extracted with ethylacetate (3×70 mL). The combined organic layers were dried, filtered and concentrated to give the dipeptide iBoc-G(Chx)-3,4-isopropylideneproline (100 mg, 0.25 mmol, 96%).
Example X Synthesis of N-Cbz-3,4-Methanoproline[0360] 533
[0361] Step 1. Synthesis of N-benzyl-3,4-Methanoprolinol: 534
[0362] A mixture of the benzylidene starting material (J. Org. Chem. 1999, 64(2), 547) (4.6 grams, 21.4 mmol) and lithium aluminum hydride (1.0 M in THF, 64 mL, 64 mmol) was heated at reflux for 5 hours. After cooling to 0° C., the remaining aluminum hydride was carefully quenched by the dropwise addition of saturated aqueous sodium sulfate (5 mL) over 15 min. The mixture was diluted with ethylacetate (200 mL) and then filtered through celite. The filtrate was dried with sodium sulfate, filtered and concentrated to give crude N-benzyl aminoalcohol (3.45 grams), which was carried on to the next step without further purification.
[0363] Step 2. Synthesis of N-Benzyloxycarbonyl-3,4-Methanoprolinol (CBz-P(3,4-CH2)-ol): 535
[0364] A solution of crude N-benzyl aminoalcohol (3 grams, 14.76 mmol) in methanol (120 mL) and concentrated HCl (1.5 mL) with 10% Pd/C (300 mg) was hydrogenated at 50 psi for 16 hours. The reaction mixture was filtered to remove the carbon-based catalyst and the filtrate was concentrated. The residue was dissolved in water/dioxane (100 mL) and diisopropylethylamine (3.2 mL) was added. Benzyl chloroformate (2.76 mL, 16.2 mmol) was added and the reaction was stirred overnight. The reaction mixture was concentrated, dissolved in 1 M HCl (100 mL) and extracted with ethylacetate (3×200 mL). The combined organic layers were dried with magnesium sulfate, filtered and concentrated. The residue was purified by flash chromatography using 1:3 ethylacetate/hexanes to give the N-Cbz-3,4-methanoprolinol (2.4 g)
[0365] Step 3. Synthesis of N-Benzyloxycarbonyl-3,4-Methanoproline (CBz-P(3,4-CH2)—OH): 536
[0366] To a solution of N-Cbz-3,4-methanoprolinol (2.2 g, 8.90 mmol) in acetone (68 mL) stirring at 0° C., was added Jones reagent (6.6 mL) dropwise over 5 min. [Jones Reagent: Prepared from chromium trioxide (13.4 g) and concentrated sulfuric acid (11.5 mL) diluted with water to a total volume of 50 mL.] After stirring at 0° C. for 3 hours, isopropanol (11 mL) was added and stirring continued for an additional 10 minutes. The reaction mixture was diluted with water (400 mL) and extracted with ethylacetate (3×500 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated to give N-Cbz-3,4-methanoproline (2.25 g, 96%)
Example XI Synthesis of Boc-(6S-Carboethoxymethano) Proline[0367] 537
[0368] The synthesis of the title compound was carried out according to the published procedure: Marinozzi, M.; Nataini, B.; Ni, M. H.; Costantino, G.; Pellicciari R. IL Farmaco (1995) 50 (5), 327-331.
Example XII Synthesis of Boc-3-Morpholine Carboxylic Acid[0369] 538
[0370] The synthesis of the title compound was carried out according to the published procedure: Kogami Y., Okawa, K. Bull. Chem. Soc. Jpn. (1987) 60, 2963-2965.
Example XIII Synthesis of N-Tert-Butoxycarbonyl 2-Aza-3S-Hydroxycarbonyl-[2,2,2]-Bicyclooctane[0371] 539
[0372] A solution of crude 2-aza-2-(1-phenylethyl)-3S-methoxycarbonyl-[2,2,2]-bicyclooct-5-ene (10 mmol) (Tetrahedron (1992) 48(44) 9707-9718) and 10% Pd on carbon (1 g) in methanol (30 mL) was acidified with 12N HCl then hydrogenated at 50 psi for 16 hours using a Parr hydrogenation apparatus. The reaction mixture was filtered to remove the carbon-based catalyst and the filtrate was concentrated. The residue was dissolved in concentrated HCl and stirred overnight. The solution was concentrated and redissolved in acetonitrile (50 mL). Diisopropylethylamine (3.5 mL) and di-tert-butyldicarbonate (1 g) were added. The reaction mixture was stirred for 24 hours and then concentrated. The residue was dissolved in CH2Cl2 and 5% aqueous sulfuric acid. The reaction mixture was extracted with CH2Cl2 and the combined organic layers were concentrated. The residue was dissolved in 10% saturated sodium bicarbonate, washed with diethyl ether (2×) and acidified with 5% aqueous sulfuric acid. The aqueous layer was extracted with ethylacetate (2×). The combined ethylacetate layers were dried filtered and concentrated to give N-tert-butoxycarbonyl 2-aza-3S-hydroxycarbonyl-[2,2,2]-bicyclooctane (650 mg).
Example XIV Synthesis of Isobutyloxycarbonyl-Cyclohexylglyoyl-4,4-Dimethyl Proline (iBoc-G(Chx)-P(4,4-Dimethyl)—OH)[0373] 540
[0374] Step I. Synthesis of iBoc-G(Chx)-P(4,4-Dimethyl)—OMe: 541
[0375] To a solution of iBoc-G(Chx)—OH (Example IX, Step 1.)(377 mg, 1.95 mmol) in acetonitrile (7 mL) was added successively HCl.HN-Pro(4,4-dimethyl)—OMe (Example I, step 6)(377 mg, 1.95 mmol), N-hydroxybenzotriazole (239 mg, 1.75 mmol), TBTU (845 mg, 2.63 mmol) and diisopropylethylamine (1.35 mL, 7.8 mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated and the remaining residue was dissolved in ethylacetate. The organic layer was washed twice with 10 ml portions of saturated sodium bicarbonate solution, 1 N hydrochloric solution, and brine. The organic layer was dried over sodium sulfate, filtered and concentrated to a white solid (612 mg, 79%).
[0376] Step 2. Synthesis of iBoc-G(Chx)—P(4,4-Dimethyl)—OH: 542
[0377] The methyl ester obtained in Step 1 above (612 mg, 1.54 mmol) in methanol (6 ml) was saponified in the presence of 2M lithium hydroxide (1.16 ml) for three hours. The methanol was removed under reduced pressure and the remaining residue was diluted with ethylacetate and acidified to pH=2 with 1N hydrochloric acid. The layers were separated and the organic layer was washed with water and brine, dried over sodium sulfate, filtered and concentrated.
Example XV Synthesis of L-phenylglycine Dimethylamide[0378] 543
[0379] Step 1. Synthesis of N-Benzyloxycabonyl-L-Phenylglycine Dimethylamide (CBz-Phg-NMe2): 544
[0380] N-benzyloxycarbonyl-L-phenylglycine (25 g, 88 mmols) was dissolved in THF (800 mL) and cooled to −10° C. N-methylmorpholine (9.7 mL, 88 mmols) and isobutylchloroformate (11.4 mL, 88.0 mmols) were added and the mixture allowed to stir for 1 minute. Dimethylamine (100 mL, 2M in THF) was added and the reaction was allowed to warm to room temperature. The mixture was filtered and the filtrate concentrated in vacuo to afford N-benzyloxycabonyl-L-phenylglycine 110 dimethylamide (32.5 g) as a yellow oil. 545
[0381] Step 2. Synthesis of L-Phenylglycine Dimethylamide (H-Phg-NMe2):
[0382] The N-benzyloxycarbonyl-L-phenylglycine dimethylamide (32.5 g) obtained above was dissolved in methanol (750 ml) and 10% palladium on activated carbon (3.3 g) was added. This mixture was hydrogenated on a Parr apparatus under 35 psi hydrogen for 2 hours. The reaction mixture was filtered and the solvent removed in vacuo and the residue recrystallized from methanol-hexanes to afford phenylglycine dimethylamide (26 g) as an off white solid. The ee of this material was determined to be >99% by HPLC analysis of the 2,3,4,6-tetra-O-acetylglucopyranosylthioisocyanate derivative.
Example XVI Synthesis of (1-Methylcyclohexyl) Glycine[0383] 546
[0384] Step 1. 1-Methyl-1-Hydroxymethylcyclohexane 547
[0385] To a solution of 1-methyl-1-hydroxycarbonylcyclohexane (10 g, 70 mmol) in tetrahydrofuran(300 mL) at 0° C. was added 1M diborane in tetrahydrofuran (200 mL, 200 mmol) over 90 minutes. The cooling bath was removed and the reaction mixture was stirred at room temperature for two days. The remaining borane was quenched by the slow addition of saturated sodium bisulfate (10 mL) over 90 min with cooling. Additional saturated sodium bisulfate (200 mL) was added and after 20 min of stirring the aqueous layer was removed. The organic layer was washed with water and saturated sodium chloride, dried, filtered and concentrated. The residue was purified by flash chromatography using 20% diethylether in hexanes to give 1-methyl-1-hydroxymethylcyclohexane (6.17 g, 48 mmol, 69%).
[0386] Step 2. 1-Methylcyclohexylcarboxaldehyde: 548
[0387] To a solution of 1-methyl-1-hydroxymethylcyclohexane (6.17 g, 48 mmol) and triethylamine (20.1 mL, 144 mmol) in dichloromethane (150 mL) at 0° C., was added a solution of pyridine sulfur trioxide complex (22.9 g, 144 mmol) in dimethylsulfoxide (150 mL) over 15 min. The cooling bath was allowed to warm to room temperature over two hours, at which time the reaction mixture was poured into brine with ice (400 mL). The layers were separated and the aqueous layer was extracted with dichloromethane (200 mL). The combined organic layers were diluted with hexanes (600 mL) and washed with 1M HCl (2×150 mL), saturated sodium chloride (2×100 mL), dried, filtered and concentrated. The residue was purified by flash chromatography to give 1-methylcyclohexylcarboxaldehyde (1.77 g, 13.8 mmol, 29%).
[0388] Step 3. Synthesis of N-formyl-N-Glycosyl-1-Methylcyclohexyl-Tert-Butylamide: 549
[0389] The synthesis of the 2,3,4-tri-O-pivaloyl- -D-arabinosylamine was carried out according to the published procedure (Kunz. H.; Pfrengle, W.; Ruck, K.; Wilfried, S. Synthesis (1991) 1039-1042).
[0390] To a solution of 1-methylcyclohexylcarboxaldehyde (1.17 g, 8.34 mmol), 2,3,4-tri-O-pivaloyl- -D-arabinosylamine (8.3 g, 20.7 mmol), formic acid (850 &mgr;L, 22.2 mmol) and tert-butylisocyanide (2.4 mL, 21.2 mmol) in tetrahydrofuran (170 mL) at −30° C. was added 0.5M zinc chloride in tetrahydrofuran (41 mL, 20.57 mmol). The solution was stirred at −20° C. for 3 days, then concentrated. The residue was diluted with CH2Cl2 (500 mL), washed with saturated sodium bicarbonate (2×500 mL), water (500 mL). The organic layer was dried, filtered and concentrated to give a clear oil. Flash chromatography (20% ethylacetate in hexanes) provided pure product (4.3 g, 6.6 mmol, 33%)
[0391] Step 4. Synthesis of (1-Methylcyclohexyl)Glycine: 550
[0392] A solution of the product obtained in step 3 above (4.3 g, 6.6 mmol) in dichloromethane (30 mL) and saturated anhydrous methanolic HCl (30 mL) was stirred overnight. The solution was concentrated and the residue was dissolved in water (100 mL) and washed with pentane (2×100 mL). The aqueous layer was concentrated and the residue was dissolved in 6N HCl (50 mL) and heated at reflux for 30 hours. The solution was concentrated to give the crude (1-methylcyclohexyl)glycine hydrochloride (790 mg, 3.82 mmol, 58%).
Example XVII Synthesis of (4,4-Dimethylcyclohexyl)Glycine:[0393] 551
[0394] Step 1. Synthesis of 4,4-Dimethylcyclohexanone: 552
[0395] A mixture of 4,4-dimethylcyclohex-2-en-1-one (12 mL, 91.2 mmol) and Degussa type 10% Pd on carbon (2 g) was hydrogenated at 40 psi for 18 hours. The mixture was filtered and concentrated (1H NMR showed a mixture of ketone and alcohol in a 5:3 ratio). The mixture was dissolved in acetone (400 mL) and cooled to 0° C. Jones reagent (40 mL) was added over 30 min and the cooling bath was removed. After 2 days the excess acetone was evaporated and the resulting residue was dissolved in water and diethylether. The ether layer was washed with water until colorless, dried, filtered and concentrated to give 4,4-dimethylcyclohexanone (7.4 g, 58.6 mmol, 64%).
[0396] Step 2. Synthesis of the Methyl Enol Ether of 4,4-Dimethylcyclohexylcarboxaldehyde: 553
[0397] To a solution of methoxymethyl triphenylphosphonium chloride (8.6 g) in tetrahydrofuran (125 mL) at 0° C. was added n-butyllithium (1.6M in hexanes, 14.3 mL) over 10 min. After 30 min the reaction mixture was cooled to −78° C. and a solution of 4,4-dimethylcyclohexanone (2.45 g, 19.1 mmol) in tetrahydrofuran (50 mL) was added over 20 min. After 1 hour the cooling bath was remove and the reaction was warmed slowly to 0° C. The reaction was diluted with saturated ammonium chloride (50 mL), ethylacetate (100 mL) and hexanes (100 mL). The organic layer was washed with water and brine, dried filtered and concentrated. The residue was stirred with hexanes (70 mL) for 10 min and filtered. The filtrate was concentrated and chromatographed using 25% ethylacetate in hexanes to give the title compound (1.925 g, 12.5 mmol, 65%).
[0398] Step 3: 4,4-Dimethylcyclohexylcarboxaldehyde: 554
[0399] A solution of the methyl enol ether of 4,4-dimethylcyclohexylcarboxaldehyde (1.925 g, 12.5 mmol) (Step II above), tetrahydrofuran (100 mL) and 6M HCl (20 mL) was stirred at room temperature for 4 hours. The reaction mixture was diluted with hexanes, diethylether, brine and water. The organic layer was dried, filtered and concentrated to give 4,4-dimethylcyclohexylcarboxaldehyde (1.0 g, 7.1 mmol, 57%).
[0400] Step 4. Synthesis of N-formyl-N-Glycosyl-4,4-Dimethylcyclohexyl-Tert-Butylamide: 555
[0401] To a solution of 4,4-dimethylcyclohexylcarboxaldehyde (1.17 g, 8.34 mmol), 2,3,4-tri-O-pivaloyl-&agr;-D-arabinosylamine (3.43 g, 8.55 mmol), formic acid (350 &mgr;L, 9.17 mmol) and tert-butylisocyanide (990 &mgr;L, 8.76 mmol) in THF (70 mL) at −30° C. was added 0.5M zinc chloride in tetrahydrofuran (17 mL, 8.5 mmol). The solution was stirred at −20° C. for 2 days, then concentrated. The residue was diluted with dichloromethane (200 mL), washed with saturated sodium bicarbonate (2×200 mL), water (200 mL). The organic layer was dried, filtered and concentrated to give a clear oil. Flash chromatography (20% ethylacetate in hexanes) provided pure product (2.1 g, 3.3 mmol, 39%)
[0402] Step 5. Synthesis of (4,4-Dimethylcyclohexyl)Glycine: 556
[0403] A solution of the Ugi product obtained in step 4 above (2.1 g, 3.3 mmol) in dichloromethane (20 mL) and saturated anhydrous methanolic HCl (20 mL) was stirred overnight. The solution was concentrated and the residue was dissolved in water (100 mL) and washed with pentane (2×100 mL). The aqueous layer was concentrated and the residue was dissolved in 6N HCl (40 mL) and heated at reflux for 30 hours. The solution was concentrated to give the crude (1-methylcyclohexyl)glycine hydrochloride (300 mg, 1.36 mmol, 41%).
Example XVIII Synthesis of Boc—nVal—(CHOH)—Gly—OH:[0404] 557
[0405] Step 1. Preparation of Boc-Norvalinol: 558
[0406] To a solution of Boc-norvaline (25.0 g, 0.115 mol) in tetrahydrofuran (461 mL), cooled to 0° C., was added borane/tetrahydrofuran complex (461 mL of a 1.0M solution in tetrahydrofuran) dropwise. After 1 h at 0° C., the solution was warmed to room temperature over a period of 1.5 h. TLC indicated that the reaction was complete. Methanol was added to quench the reaction. The solution was concentrated to yield the title compound (22.56 g, 96%) as a foamy syrup. TLC of the products indicated satisfactory purity. Rf=0.34 (40% ethyl acetate/hexanes).
[0407] Step 2. Preparation Boc-Norvalinal: 559
[0408] To Boc-norvalinol (7.77 g, 38 mmol), in anhydrous dimethylsulfoxide (153 mL) and toluene (153 mL) was added EDC (73.32 g, 382 mmol). After the solution was cooled to 0° C., dichloroacetic acid (15.8 mL, 191 mmol) was added dropwise. After addition was complete, the reaction was stirred for 15 min. The solution was allowed to warm to room temperature over a period of 2 h. The reaction mixture was concentrated to remove the toluene, then dissolved in ethyl acetate. The solution was washed successively with 1 N sodium bisulfate, saturated sodium bicarbonate and brine, dried over sodium sulfate, filtered and concentrated to afford crude Boc-norvalinal which was used directly in the next step. TLC Rf=0.84 (40% ethyl acetate/hexanes).
[0409] Step 3. Synthesis of Boc—nVal—(CHOH)—Gly—OEt: 560
[0410] To a solution of the crude Boc-norvalinal (4.18 g, 20.77 mmol) in dichloromethane (83 mL) was added ethylisocyanoacetate (2.72 ml, 24.93 mmol) and pyridine (6.72 ml, 83.09 mmol). After the solution was cooled to 0° C., trifluoroacetic acid (4.15 ml, 41.54 mmol) was added dropwise. After stirring for 1 h, the solution was stirred at room temperature for 18 hours while allowing the solvent from the reaction mixture in an uncovered vessel to evaporate under ambient conditions. The reaction mixture was concentrated, then dissolved in ethyl acetate. The solution was washed successively with 1N sodium bisulfate, saturated sodium bicarbonate and brine, dried over sodium sulfate, filtered and then concentrated. The residue was purified by flash chromatography eluting with 20% to 40% ethylacetate/hexanes to afford 2.8 g of the title compound as a yellow syrup. Low resolution mass spectroscopy confirmed the presence of the desired product (MH+333).
[0411] Step 4. Synthesis of Boc—nVal—(CHOH)—Gly—OH: 561
[0412] The product obtained (Boc—nVal—(CHOH)—Gly—OEt) (1.52 g, 4.70 mmol) dissolved in ethanol (23 ml) was saponified with 1N lithium hydroxide (18.81 ml) for two hours at room temperature. The reaction mixture was acidified to pH≅2 with Dowex® 50 WX8 ion exchange resin, stirred for 20 minutes and then filtered. The resin was washed well with ethanol and water and the combined filtrates were concentrated to a white foam (0.48 g, 33%).
Example XVIV Synthesis of (2R,3S,4S,5S)-Tert-Butyl N-CBz-3-Amino-2-Hydroxy-4,5 Methylene-Hexanoate[0413] 562
[0414] Step 1: 563
[0415] To a solution of tert-Butyl diethylphosphonoacetate (4.7 mL, 20 mmol) dissolved in THF (50 mL) at −78° C. was added 1.6M n-butyl lithium in hexanes (12.4 mL). After 30 minutes (1S, 2S)-2-methylcyclopropylcarboxaldehyde (1 g, 12 mmol) (Barrett, A. G. M.; Doubleday, W. W.; Kasdorf, K.; Tustin, G. J., J. Org. Chem. (1996) 61, 3280) in diethyl ether (100 mL) was added over 10 min. The reaction was warmed to 0° C. for 2 hours and to 6° C. for 12 hours. The reaction was quenched with saturated ammonium chloride (20 mL) and the organic layer was separated, washed with 50 mL brine and dried over sodium sulfate, filtered and concentrated to afford 3.5 g of a clear oil. Flash chromatography (20% ethylacetate in hexanes) afforded pure unsaturated tert-butylester (1.4 g).
[0416] Step 2: 564
[0417] To a solution of benzyl carbamate (3.55 g, 23.5 mmols) in n-propanol (24 mL) was added a solution of sodium hydroxide (900 mg,22.7 mmol)in water (48 mL), followed by tert-butylhypochlorite (2.57 mL, 22.7 mmol). After 15 minutes the reaction was cooled to 0° C. and (DHQ)2PHAL (350 mg, 0.45 mmol) was added in n-propanol (24 mL), followed by unsaturated tert-butyl ester (1.4 g) from above in n-propanol (48 mL). Finally potassium osmate (110 mg, 0.30 mmol) in water (2 mL) was added and the solution very rapidly developed a dark green color which persisted for 4 hours. After 6 hours saturated sodium sulfate (50 mL) was added and the mixture extracted with ethyl acetate (2×50 mL). The combined organic layers were washed with brine (30 mL), dried over sodium sulfate, filtered and concentrated. Flash chromatography with 20% ethylacetate in hexanes afforded the desired cBz protected amino tert-butylester as a white solid (316 mg).
[0418] Step 3: 565
[0419] A mixture of CBz protected amino tert-butylester (316 mg, 0.9 mmol) and 32 mg 10% palladium on carbon in 9 mL methanol was hydrogenated for 8 hours. The mixture was filtered and concentrated to afford the free amine as a clear oil (195 mg).
Example XX Synthesis of 1R,2-DimethylPropyl Chloroformate:[0420] 566
[0421] To the commercially available 2R-hydroxy-3-methylbutane (410 mg, 4.65 mmol) was added a solution of 20% phosgene in toluene (1 mL, 2 mmol). The solution was stirred for 6 hours to generate the chloroformate (2 mmol) which was reacted directly and immediately with the desired amine. The S-isomer was synthesized by the same procedure.
[0422] II) Representative Solution Phase Cynthesis of HCV Inhibitors
Example XXI Solution Phase Synthesis of iBoc-G(Chx)-Pro(4,4-dimethyl)-Leu-(CO)-Gly-Phg-Dimethylamide:[0423] 567
[0424] Step 1. Synthesis of Tert-Butyloxycarbonyl-Leucinal (Boc-Leu-CHO): 568
[0425] To a solution of the commercially available (Advanced Chem Tech) Boc-L-leucinol (0.78 g, 3.6 mmol) in anhydrous dichloromethane (17.5 ml) was added triethyl amine (2 ml, 14.36 mmol) and the mixture was cooled to 0° C. Dimethyl sulfoxide (17.5 ml) was added followed by sulfur trioxide pyridine complex (2.3 g, 14.36 mmol) and the reaction was stirred for two hours. TLC in 1:1 ethylacetate: hexanes confirmed the completion of the reaction. The reaction mixture was concentrated and the remaining residue diluted with ethylacetate. The ethylacetate layer was washed with 1M hydrochloric acid (2×75 ml) followed by saturated sodium bicarbonate solution (2×75 ml) and brine (75 ml). The organic layer was dried (sodium sulfate), filtered and concentrated to yield 775 mg of product.
[0426] Step 2. Synthesis of Boc-2-Hydroxy-3-Amino-5-Methyl Hexanoyl-Glycine Ethyl Ester (Boc—Leu—(CHOH)—Gly—OEt): 569
[0427] To a solution of Boc-Leucine aldehyde (0.77 g, 3.59 mmol) in anhydrous dichloromethane (24 ml) was added anhydrous pyridine (1.16 ml, 14.36 mmol) and ethylisocyanoacetate (0.4 ml, 4.66 mmol). The reaction mixture was cooled to 0° C. and trifluoroacetic acid (0.55 ml, {fraction (7/18)} mmol) was added over two minutes. The reaction mixture was capped and stirred at 4° C. for four days, and at room temperature for one day. The reaction mixture was diluted with dichloromethane (350 ml) and washed twice each with 75 ml portions of 1M hydrochloric acid, saturated sodium bicarbonate and brine. The organic layer was dried, filtered and concentrated. The residue obtained was subjected to flash chromatography in a 2″×6″ silica gel column using 10% ethylacetate in hexanes (800 ml) followed by 1:1 ethylacetate in hexanes (800 ml). The fractions corresponding to the product were pooled and concentrated to yield 980 mg (79%) product.
[0428] Step 3. Synthesis of Boc—Leu—(CHOH)—Gly—OH: 570
[0429] To a solution of Boc—Leu—(CHOH)—Gly—Oet (0.98 g, 2.83 mmol) in ethanol (11.3 ml) was added 2M lithium hydroxide (4.25 ml) and the reaction was stirred for five hours at room temperature. The ethanol was removed under reduced pressure and the aqueous layer was diluted with ethylacetate. The organic layer was washed with 1M hydrochloric acid followed by brine, dried, filtered and concentrated to yield 775 mg (86%) product as a white solid.
[0430] Step 4. Synthesis of Boc-Leu-(CHOH)-Gly-Pha-Dimethylamide: 571
[0431] To a solution of Boc—Leu—(CHOH)—Gly—OH (0.37 g, 1.18 mmol) in acetonitrile (23 ml) was added successively phenylglycine dimethylamide (obtained in Example XV, Step 2), EDC (0.34 g, 1.76 mmol), N-hydroxybenzotriazole (HOBt)(0.18 g, 1.18 mmol) and diisopropylethylamine (DIEA) (0.82 ml, 4.7 mmol) and the reaction was stirred for 18 hours at room temperature. The reaction mixture was concentrated and the remaining residue was diluted with ethylacetate and washed successively with two 75 ml portions of 1M hydrochloric acid, saturated sodium bicarbonate and brine. The organic layer was then dried filtered and concentrated. The crude product was subjected to flash chromatography in a 2″×6″ silica gel column using 4:1 ethylacetate: hexanes (700 ml) followed by ethylacetate (1000 ml) and 10% methanol in dichloromethane (600 ml). The fractions corresponding to the product were pooled and concentrated to yield 445 mg (80%) white solid.
[0432] Step 5. Synthesis of H-Leu-(CHOH)-Gly-Phg-Dimethylamide Trifluoroacetate Salt: 572
[0433] To a solution Boc-Leu-(CHOH)-Gly-Phg-dimethylamide (70 mg, 0.146 mmol) in dichloromethane (1 ml) was added trifluoroacetic acid (1 ml) and the reaction was stirred at room temperature for 1 hour. The reaction mixture was concentrated and taken to the next step without further purification.
[0434] Step 6. Synthesis of iBoc-G(Chx)-Pro(4,4-Dimethyl)-Leu-(CHOH)-Gly-Phg-Dimethylamide: 573
[0435] To a solution of iBoc-G(Chx)-P(4,4-diMe)—OH (Example XIV, step 2)(53 mg, 0.148 mmol) in acetonitrile (3 ml) was added successively TFA.2HN-Leu(CHOH)-Gly-Phg-NMe2 (61 mg, 0.148 mmol), N-Hydroxybenzotriazole (HOBt) (23 mg, 0.148 mmol), TBTU (71.5 mg, 0.222 mmol and diisopropylethyl amine (103I,0.593 mmol). The reaction was stirred at room temperature for 18 hours and concentrated. The remaining residue was dissolved in ethylacetate and washed with 1M hydrochloric acid (2×5 ml), saturated sodium bicarbonate solution (2×5 ml), and brine (2×5 ml). The organic layer was dried, filtered and concentrated. The product (100 mg) was taken to the next step without further purification.
[0436] Step 7. Synthesis of iBoc-G(Chx)-Pro(4,4-Dimethyl)-Leu-(CO)-Gly-Phg-Dimethylamide: 574
[0437] To a solution of iBoc-G(Chx)-Pro(4,4-dimethyl)-Leu-(CHOH)-Gly-Phg-dimethylamide (30 mg, 0.04 mmol) in dichloromethane (1 ml) was added the commercially available Dess-Martin reagent (Omega Chemical Company Inc.)(67.8 mg, 0.16 mmol) and the reaction was stirred at room temperature for 90 minutes. The reaction mixture was concentrated and the remaining residue was stirred in 5% sodium thiosulfate. It was then diluted with dichloromethane and the layers were separated. The organic layer was washed with sodium thiosulfate (4×3 ml), followed by water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated. The crude product was dissolved in hexanes and isopropyl alcohol and was subjected to HPLC purification using a normal phase Kromasil 5 silica column (Phenomenex, 250×21.20 mm, 100 angstrom pore size, 5 &mgr;m gel particles) eluting with a 30 minutes gradient consisting of 0 to 25% isopropyl alcohol in hexanes (25 ml/minutes). The fractions corresponding to the product were pooled and concentrated. Lyophilization from water yielded 6.7 mg white powder. Low resolution mass spectra confirmed the desired mass (MH+=741.4).
[0438] III) Solid Phase Synthesis:
[0439] Solid-phase synthesis is useful for the production of small amounts of certain compounds of the present invention. As with the conventional solid-phase synthesis of peptides, reactors for the solid-phase synthesis of peptidyl ketoamides are comprised of a reactor vessel with at least one surface permeable to solvent and dissolved reagents, but not permeable to synthesis resin of the selected mesh size. Such reactors include glass solid phase reaction vessels with a sintered glass frit, polypropylene tubes or columns with frits, or reactor Kans™ made by Irori Inc., San Diego Calif. The type of reactor chosen depends on volume of solid-phase resin needed, and different reactor types might be used at different stages of a synthesis. The following procedures will be referenced in the subsequent examples:
[0440] Procedure A: Coupling reaction: To the resin suspended in N-methylpyrrolidine (NMP) (10-15 mL/gram resin) was added Fmoc-amino acid (2 eq), HOAt (2 eq), HATU (2 eq) and diisopropylethylamine (4 eq). The mixture was let to react for 4-48 hours. The reactants were drained and the resin was washed successively with dimethylformamide, dichloromethane, methanol, dichloromethane and diethylether (use 10-15 mL solvent/gram resin). The resin was then dried in vacuo.
[0441] Procedure B: Fmoc deprotection: The Fmoc-protected resin was treated with 20% piperidine in dimethylformamide (10 mL reagent/g resin) for 30 minutes. The reagents were drained and the resin was washed successively with dimethylformamide, dichloromethane, methanol, dichloromethane and diethyl ether (10 mL solvent/gram resin).
[0442] Procedure C: Boc deprotection: The Boc-protected resin was treated with a 1:1 mixture of dichloromethane and trifluoroacetic acid for 20-60 minutes (10 mL solvent/gram resin). The reagents were drained and the resin was washed successively with dichloromethane, dimethylformamide, 5% diisopropylethylamine in dimethylformamide, dimethylformamide, dichloromethane and dimethylformamide (10 mL solvent/gram resin).
[0443] Procedure D: Semicarbazone hydrolysis: The resin was suspended in the cleavage cocktail (10 mL/g resin) consisting of trifluoroacetic acid: pyruvic acid: dichloromethane: water 9:2:2:1 for 2 hours. The reactants were drained and the procedure was repeated three more times. The resin was washed successively with dichloromethane, water and dichloromethane and dried under vacuum.
[0444] Procedure E: HF cleavage: The dried peptide-nVal(CO)—G—O—PAM resin (50 mg) was placed in an HF vessel containing a small stir bar. Anisole (10% of total volume) was added as a scavenger. In the presence of glutamic acid and cysteine amino acids, thioanisole (10%) and 1,2-ethanedithiol (0.2%) were also added. The HF vessel was then hooked up to the HF apparatus (Immuno Dynamics) and the system was flushed with nitrogen for five minutes. It was then cooled down to −70° C. with a dry ice/isopropanol bath. After 20 minutes, HF was distilled to the desired volume (10 mL HF/g resin). The reaction was let to proceed for one and a half hour at 0° C. Work up consisted of removing all the HF using nitrogen. Dichloromethane was then added to the resin and the mixture was stirred for five minutes. This was followed by the addition of 20% acetic acid in water (4 mL). After stirring for 20 minutes, the resin was filtered using a fritted funnel and the dichloromethane was removed under reduced pressure. The remaining residue and the mixture was washed with hexanes (2×) to remove scavengers. Meanwhile, the resin was soaked in 1 mL methanol. The aqueous layer (20% HOAC) was added back to the resin and the mixture was agitated for five minutes and then filtered. The methanol was removed under reduced pressure and the aqueous layer was lyophilized. The peptide was then dissolved in 10-25% methanol (containing 0.1% trifluoroacetic acid) and purified by reverse phase HPLC.
Example XXII Representative Solid Phase Synthesis of Hep C Inhibitors: (iBoc-G(Chx)-P(4T-NHSO2Ph)-nV-(CO)-G-G(Ph)—NH2)[0445] 575
[0446] Step 1. Synthesis of Fmoc—nV—(dpsc)—Gly—OH:
[0447] A) Synthesis of Allyl Isocyanoacetate (Steps a-b Below):
[0448] a) Synthesis of Isocyanoacetic Acid Potassium Salt: 576
[0449] Ethyl isocyanoacetate (96.6 ml, 0.88 mol) was added dropwise to a chilled solution of ethanol (1.5 L) and potassium hydroxide (59.52 g, 1.0 mol). The reaction was slowly warmed to room temperature. After two hours the precipitated product was filtered on a glass funnel and washed with several portions of chilled ethanol. The potassium salt of isocyanoacetic acid thus obtained was dried in vacuo to a golden-brown solid (99.92 g, 91.8%).
[0450] b) Synthesis of Allyl Isocyanoacetate: 577
[0451] To the product of part a (99.92 g, 0.81 mol) dissolved in acetonitrile (810 ml) was added allyl bromide (92 ml, 1.05 mol). After heating at reflux for four hours a dark brown solution was obtained. The reaction mixture was concentrated and the remaining residue was dissolved in ether (1.5 L) and washed three times with water (500 ml). The organic layer was dried, filtered and concentrated to a dark brown syrup. The crude was purified by vacuum distillation at 7 mm Hg (98 C) to a clear oil (78.92 g, 78%). NMR &dgr; ppm (CDCl3): 5.9 (m, 1H), 5.3 (m, 2H), 4.7 (d, 2H), 4.25 (s, 2H).
[0452] B) Synthesis of 9-Fluorenylmethoxycarbonyl-Norvalinal (Steps a-c Below):
[0453] a) Synthesis of 9-Fluorenylmethoxycarbonyl-L-Norvaline Methyl Ester (Fmoc—nVal—OMe): 578
[0454] To a chilled solution of the commercially available Fmoc-norvaline (25 g, 73.75 mmol) in anhydrous methanol (469 ml) was added thionyl chloride (53.76 ml, 737.5 mmol) over one hour. TLC in ethylacetate taken an hour later confirmed the completion of the reaction (Rf=0.85). The reaction mixture was concentrated and the remaining residue was dissolved in ethylacetate. The organic layer was washed with several 200 ml portions of saturated sodium bicarbonate followed by brine. The organic layer was dried, filtered and concentrated to afford Fmoc—norVal—OMe) as a white solid (26.03 g) in quantitative yield. NMR &dgr; ppm (CD3OD): 7.7 (m, 2H), 7.6 (m, 2H), 7.4 (m, 2H), 7.3 (m, 2H), 4.3 (m, 2H), 4.1 (m, 2H), 3.7 (s, 3H), 1.7 (m, 1H), 1.6 (m, 1H), 1.4 (m, 2H), 0.95 (t, 3H).
[0455] b) Synthesis of 9-Fluorenylmethoxycarbonyl-Norvalinol (Fmoc-nValinol): 579
[0456] To Fmoc—nVal—OMe (26.03 g, 73.75 mmol) in tetrahydrofuran (123 ml) and methanol (246 ml) was added calcium chloride (16.37 g, 147.49 mmol). The reaction mixture was cooled to 0° C. and sodium borohydride (11.16 g, 294.98 mmol) was added in several batches. To the thick paste obtained, methanol (500 ml) was added and the reaction was let to stir at room temperature for 90 minutes. TLC in 2:3 ethylacetate: hexanes confirmed the completion of the reaction (Rf=0.25). The reaction was quenched with the slow addition of water (100 ml) at 0° C. The methanol was removed under reduced pressure and the remaining aqueous phase was diluted with ethylacetate. The organic layer was washed with water (3×500 ml), saturated sodium bicarbonate (3×500 ml) and brine (500 ml). The organic layer was dried over sodium sulfate, filtered and concentrated to a white solid (21.70 g, 90.5%). NMR &dgr; ppm (CD3OD): 7.8 (m, 2H), 7.7 (m, 2H), 7.4 (m, 2H), 7.3 (m, 2H), 4.3-4.5 (m, 2H), 4.2 (m, 1H), 3.6 (s, 1H), 3.5 (s, 2H), 1.5 (m, 1H), 1.3-1.4 (m, 3H), 0.99 (m, 3H).
[0457] c) Synthesis of 9-Fluorenylmethoxycarbonyl-Norvalinal (Fmoc-nVal-CHO): 580
[0458] To a solution of Fmoc-norValinol (21.70 g, 66.77 mmol) in dichloromethane (668 ml) was added triethylamine (37.23 ml, 267 mmol) and the solution was cooled to 0° C. A suspension of pyridine sulfur trioxide complex (42.51 g, 267 mmol) in dimethylsulfoxide (96 ml) was added to the chilled solution. After one hour, TLC in 2:3 ethylacetate: hexanes confirmed the completion of the reaction. The dichloromethane was removed under reduced pressure and the remaining residue was dissolved in ethylacetate and washed with water (2×50 ml), 1N saturated sodium bisulfate (2×50 ml), saturated sodium bicarbonate (2×50 ml) and brine (50 ml). The organic layer was concentrated to yield a white solid. Theoretical yield (21.57 g) was assumed and the reaction was taken to the next step without further purification.
[0459] C) Synthesis of Diphenylmethyl Semicarbazide (dpsc) Trifluoroacetate Salt (Steps a-b Below):
[0460] a) Synthesis of Boc-Semicarbazid-4-yl Diphenylmethane 581
[0461] To a solution of carbonyldiimidazole (16.2 g, 0.10 mole) in dimethylformamide (225 ml) was added a solution of t-butyl carbazate (13.2 g, 0.100 mol) in dimethylformamide (225 ml) dropwise over 30 minutes. Diphenylmethylamine (18.3 g, 0.10 mol) was added next over 30 minutes. The reaction was allowed to stir at room temperature for one hour. Water (10 mL) was added and the mixture was concentrated to about 150 mL under reduced pressure. This solution was poured into water (500 mL) and extracted with ethyl acetate (400 mL). The ethylacetate phase was washed two times each with 75 mL 1N HCl, water, saturated sodium bicarbonate solution and sodium chloride, and dried with magnesium sulfate. The mixture was filtered and the solution was concentrated to give 29.5 g (85% yield) of a white foam. This material could be purified by recrystallization from ethyl acetate/hexane, but was pure enough to use directly in the next step: mp 142-143° C. 1H NMR (CDCl3) d 1.45 (s, 9H), 6.10 (dd, 2H), 6.42 (s, 1H), 6.67 (bs, 1H), 7.21-7.31 (m, 10H). Anal calculated for C19H23N3O3: C, 66.84; H, 6.79; N, 12.31. Found: C, 66.46; H, 6.75; N; 12.90.
[0462] b) Synthesis of Diphenylmethyl Semicarbazide (dpsc) Trifluoroacetate Salt 582
[0463] A solution of Boc-semicarbazid-4-yl diphenylmethane (3.43 g, 10 mmol) in dichloromethane (12.5 mL) was treated with 12.5 mL of trifluoroacetic acid at room temperature and stirred for 30 min. The solution was added dropwise to 75 mL of ether and the resulting solid (2.7 g, 80%) was collected by filtration. mp 182-184° C. 1H NMR (CD3OD) d 6.05 (s, 1H), 7.21-7.35 (m, 10H). 13C NMR (CD3OD) d 57.6, 118.3 (q, CF3), 126.7, 127.9, 141.6, 156.9,160.9 (q, CF3CO2H).
[0464] D) Synthesis of Fmoc—nVal—(CHOH)—Gly—Oallyl: 583
[0465] To a solution of Fmoc-nVal-CHO (Step IB) (5.47 g, 16.90 mmol) in dichloromethane (170 ml) was added allyl isocyanoacetate (Step IA) (2.46 ml, 20.28 mmol) and pyridine (5.47 ml, 67.61 mmol). The reaction mixture was cooled to 0° C. and trifluoroacetic acid (3.38 ml, 33.80 mmol) was added dropwise. The reaction was stirred at 0° C. for 1 h, and then at room temperature for 48 hours. TLC taken in ethylacetate confirmed the completion of the reaction. The reaction mixture was concentrated and subjected to flash chromatography using 20% to 70% ethylacetate in hexanes. Fractions containing the desired product were pooled and concentrated to a white foam (6.88 g, 87.3%). TLC in 50:50 ethylacetate shows one spot (Rf=0.37). NMR &dgr; ppm (CD3OD): 7.8 (m, 2H), 7.65 (m, 2H), 7.4 (m, 2H), 7.3 (m, 2H), 5.9 (m, 1H), 5.1-5.4 (m, 2H), 4.55-4.65 (m, 2H), 4.3-4.4 (m, 2H), 4.15-4.25 (m, 1H), 4.01 (s, 1H), 3.9-4.0 (m, 3H), 1.5-1.6 (m, 2H), 1.35-1.45 (m, 3H), 0.9 (m, 3H).
[0466] E) Synthesis of Fmoc—nVai—(CO)—Gly—Oallyl: 584
[0467] to a solution of Fmoc—nVal—(CHOH)—Gly-Oallyl (Step D) (5.01 g, 10.77 mmol) in dimethylsulfoxide (100 ml) and toluene (100 ml) was added EDC (20.6 g 107.7 mmol). The reaction mixture was cooled to 0° C. and dichloroacetic acid (4.44 ml, 53.83 mmol) was added dropwise. The reaction was stirred for 15 minutes at 0° C. and 1 h at room temperature. After cooling back to 0 C, water (70 ml) was added and the toluene was removed under reduced pressure. The remaining residue was diluted with ethylacetate and washed several times with a saturated sodium bicarbonate solution followed by 1N sodium bisulfate and brine. The organic layer was dried over sodium sulfate, filtered and concentrated. The theoretical yield of 4.99 g was assumed and the reaction was taken to the next step without further purification. TLC in 50:50 ethylacetate shows one spot (Rf=0.73).
[0468] F) Synthesis of Fmoc—nVal—(dpsc)—Gly-Oallyl: 585
[0469] To a solution of Fmoc—nVal—(CO)—Gly-Oallyl (Step E) (4.99 g, 10.75 mmol) in ethanol (130 ml) and water (42 ml) was added diphenylmethyl semicarbazide (dpsc) trifluoroacetate salt (Step IC) (7.6 g, 21.5 mmol) and sodium acetate .3H2O (1.76 g, 12.9 mmol), successively. The reaction mixture was heated at reflux for 90 minutes. The completion of reaction was confirmed by TLC taken in 1:1 ethylacetate:hexane. Ethanol was removed under reduced pressure and the remaining residue was dissolved in ethylacetate and washed with 1N sodium bisulfate (2×10 ml), saturated sodium bicarbonate (2×10 ml), followed by brine (10 ml). The organic layer was dried, filtered and concentrated. The resulting residue was purified by flash chromatography in 20% to 50% ethylacetate in hexanes to give a white solid (5.76 g, 78%). TLC in 50:50 ethylacetate: hexanes showed two spots (cis and trans isomers) with Rf=0.42 and 0.5.
[0470] G) Synthesis of Fmoc—nVal—(dpsc)—Gly—OH: 586
[0471] To a solution of Fmoc—nVal—(dpsc)—Gly-Oallyl (Step IG) (4.53 g, 6.59 mmol) in tetrahydrofuran (300 ml) was added dimedone (4.62 g, 32.97 mmol) followed by tetrakis(triphenylphosphine) palladium(0) catalyst (0.76 g, 0.66 mmol). The completion of the reaction was confirmed by TLC after 90 minutes using 9:1 dichloromethane: methanol. The reaction mixture was concentrated and the remaining residue was dissolved in ethylacetate and washed three times with 50 ml portions of 0.1M potassium biphosphate. The organic layer was then treated with 50 ml sodium bisulfite and the two phase system was stirred for 15 minutes. The phases were separated and the procedure was repeated twice more. The organic layer was dried and concentrated and subjected to flash chromatography with 20% to 100% ethylacetate in hexanes. This was followed with 9:1 dichloromethane: methanol solution. The fractions corresponding to the pure product were pooled and concentrated to obtain a white solid (3.99 g, 94%). TLC in 9:1 dichloromethane: methanol showed two spots (cis and trans isomers). NMR &dgr; ppm (CD3OD): 7.75 (m, 2H), 7.6 (m, 3H), 7.2-7.4 (m, 14H), 6.1-6.2 (m, 1H), 4.25-4.4 (m, 2H), 4.1-4.2 (m, 2H), 3.85 (s, 2H), 1.6-1.8 (m, 2H), 1.3-1.5 (m, 2H), 0.95 (t, 3H).
[0472] Step 2. Synthesis H-Phg-MBHA Resin: 587
[0473] The commercially available MBHA resin (2.6 g, 1.12 mmol/g, 2.91 mmol) was transferred to a 250 mL fritted solid phase reaction vessel equipped with a nitrogen inlet. It was then washed thoroughly with 30 ml portions of dichloromethane, methanol, dimethylformamide and dichloromethane and coupled over 18 hours to the commercially available Fmoc—Phg—OH (2.17 g, 5.82 mmol) according Procedure A with 99.82% efficiency. The resin was then subjected to Fmoc deprotection according to procedure B. A qualitative ninhydrin assay on a small aliquot gave dark blue resin and solution, indicating a successful reaction.
[0474] Step 3. Synthesis of H-nVal(dpsc)-Gly-Phg-MBHA Resin: 588
[0475] The resin obtained in step II (2.6 g, 0.8 mmol/g, 2.91 mmol) was reacted with Fmoc—nVal—(dpsc)—Gly-Oallyl (Step IG) (5.82 mmol, 3.77 g) according to Procedure A. After 18 hours, quantitative ninhydrin analysis indicated 99.91% coupling efficiency. The resin was subjected to Fmoc deprotection according to procedure B. A qualitative ninhydrin assay on a small aliquot gave dark blue resin and solution, indicating a successful reaction.
[0476] Step 4. Synthesis of Boc-Pro(4T-NHFmoc)-nVal(dpsc)-Gly-Phg-MBHA Resin: 589
[0477] The compound H-nVal(dpsc)-Gly-Phg-MBHA resin (Step 3 above) (600 mg, 0.8 mmol/g, 0.67 mmol) was transferred to a fritted polypropylene tube and was coupled to Boc-Pro(4t-NHFmoc)—OH (Example VI, Step 3) (610 mg, 1.34 mmol) according to procedure A. After 18 hours, quantitative ninhydrin analysis indicated 99.96% coupling efficiency.
[0478] Step 5. Synthesis of Boc-Pro(4T-NH2)-nVal(dpsc)-Gly-Phg-MBHA Resin: 590
[0479] The resin from the previous step (Boc-Pro(4t-NHFmoc)-nVal(dpsc)-Gly-Phg-MBHA resin) was subjected to Fmoc deprotection according to procedure B. A qualitative ninhydrin assay on a small aliquot gave dark blue resin and solution, indicating a successful reaction.
[0480] Step 6. Synthesis of Boc-Pro(4T-NHSO2Bn)-nVal(dpsc)-Gly-Phg-MBHA Resin: 591
[0481] To the resin obtained from the previous step (Boc-Pro(4t-NH2)-nVal(dpsc)-Gly-Phg-MBHA resin) (0.2 g, 0.22 mmol) suspended in NMP (2 ml) was added 2,4,6-collidine (0.24 ml, 1.79 mmol) and benzenesulfonyl chloride and the reaction was shaken for 18 hours. The solvent was drained and the resin was washed thoroughly with 2 ml portions of dichloromethane, methanol, dimethylformamide and dichloromethane. Qualitative ninhydrin analysis showed colorless beads and solution indicating a successful reaction.
[0482] Step 7. Synthesis of Fmoc-G(Chx)-Pro(4t-NHSO2Bn)-nVal(dpsc)-Gly-Phg-MBHA Resin: 592
[0483] The resin obtained in the previous step (Boc-Pro(4t-NHSO2Bn)-nVal(dpsc)-Gly-Phg-MBHA resin) was subjected to the Boc deprotection procedure according to Procedure C. Fmoc-G(Chx) (0.17 g, 0.45 mmol) was then coupled according to procedure A. After 18 hours qualitative ninhydrin analysis showed colorless beads and the quantitative ninhydrin analysis indicated 99.79% coupling efficiency.
[0484] Step 8. Synthesis of iBoc-G(Chx)-Pro(4t-NHSO2Bn)-nVal(dpsc)-Gly-Phg-MBHA Resin: 593
[0485] The resin obtained in the previous step (Fmoc-G(Chx)-Pro(4t-NHSO2Bn)-nVal(dpsc)-Gly-Phg-MBHA resin) was subjected to Fmoc deprotection according to procedure B. A ninhydrin assay on a small aliquot gave dark blue resin and solution, indicating a successful reaction. To the resin (0.2 g, 0.22 mmol) suspended in 2 ml NMP was added isobutylchloroformate (0.12 ml, 0.90 mmol) followed by diisopropylethylamine (0.31 ml, 1.79 mmol), and the reaction mixture was shaken for 18 hours at room temperature. Qualitative ninhydrin analysis showed colorless beads and solution indicating a successful reaction.
[0486] Step 9. Synthesis of iBoc-G(Chx)-Pro(4T-NHSO2Bn)-nVal(CO)-Gly-Phg-MBHA Resin: 594
[0487] The compound of the previous step (iBoc-G(Chx)-Pro(4t-NHSO2Bn)-nVal(dpsc)-Gly-Phg-MBHA resin) (200 mg) was subjected to semicarbazone hydrolysis Procedure D.
[0488] Step 10. Synthesis of Synthesis of iBoc-G(Chx)-Pro(4T-NHSO2Bn)-nVal(CO)-Gly-Phg-NH2: 595
[0489] The resin of the previous step (iBoc-G(Chx)-Pro(4t-NHSO2Bn)-nVal(CO)-Gly-Phg-MBHA resin) (100 mg) was subjected to HF cleavage condition (Procedure E) to yield the desired crude product. The material was purified by HPLC using a 2.2×25 cm reverse phase column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a gradient using 20-50% acetonitrile in water. Analytical HPLC using a 4.6×250 mm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size, eluting with 25-75% acetonitrile (containing 0.1% trifluoroacetic acid) showed one peak at 13.5 minutes. Low resolution mass spectrum confirmed the desired mass (MH+826.4).
[0490] IV. Additional Compounds Prepared by Solution Phase Synthesis:
[0491] Representative procedures to prepare additional inventive compounds are shown below, and the compounds prepared by such procedures are listed in Table 5.
Example XXIII Preparation of a Compound of Formula XXIII[0492] 596
[0493] Step 1. 597
[0494] A stirred solution of ketimime XXIIIa (50 g, 187.1 mmol) under N2 in dry THF (400 mL) was cooled to −78° C. and treated with 1 M solution of K-tBuO (220 mL, 1.15 equiv.) in THF. The reaction mixture was warmed to 0° C. and stirred for 1 h and treated with bromomethyl cyclobutane (28 mL, 249 mmol). The reaction mixture was stirred at room temperature for 48 h and concentrated in vacuo. The residue was dissolved in Et2O (300 mL) and treated with aq. HCl (2 M, 300 mL) The resulting solution was stirred at room temperature for 5 h and extracted with Et2O (1 L). The aqueous layer was made basic to pH ˜12-14 with NaOH (50% aq.) and extracted with CH2Cl2 (3×300 mL). The combined organic layers were dried (MgSO4), filtered, and concentrated to give pure amine (XXIIIb, 18 g) as a colorless oil.
[0495] Step 2. 598
[0496] A solution of amine XXIIIb (18 g, 105.2 mmol) at 0° C. in CH2Cl2 (350 mL) was treated with di-tert-butyldicarbonate (23 g, 105.4 mmol) and stirred at rt. for 12 h. After the completion of the reaction (TLC), the reaction mixture was concentrated in vacuo and the residue was dissolved in THF/H2O (200 ml, 1:1) and treated with LiOH.H2O (6.5 g, 158.5 mmol) and stirred at room temperature for 3 h. The reaction mixture was concentrated and the basic aqueous layer was extracted with Et2O. The aqueous layer was acidified with conc. HCl to pH˜1-2 and extracted with CH2Cl2. The combined organic layers were dried (MgSO4), filtered, and concentrated in vacuo to yield XXIIIc as a colorless viscous oil which was used for next step without any further purification.
[0497] Step 3. 599
[0498] A solution of acid XXIIIc (15.0 g, 62 mmol) in CH2Cl2 (250 mL) was treated with BOP reagent (41.1 g, 93 mmol), N-methyl morpholine (27 mL), N,O-dimethyl hydroxylamine hydrochloride (9.07 g, 93 mmol) and stirred overnight at rt. The reaction mixture was diluted with 1 N aq. HCl (250 mL), and the layers were separated and the aqueous layer was extracted with CH2Cl2 (3×300 ml). The combined organic layers were dried (MgSO4), filtered and concentrated in vacuo and purified by chromatography (SiO2, EtOAc/Hex 2:3) to yield the amide XXIIId (15.0 g) as a colorless solid.
[0499] Step 4. 600
[0500] A solution of amide XXIIId (15 g, 52.1 mmol) in dry THF (200 mL) was treated dropwisely with a solution of LiAlH4 (1M, 93 mL, 93 mmol) at 0° C. The reaction mixture was stirred at room temperature for 1 h and carefully quenched at 0° C. with a solution of KHSO4 (10% aq.) and stirred for 0.5 h. The reaction mixture was diluted with aq. HCl (1 M, 150 mL) and extracted with CH2Cl2 (3×200 mL), The combined organic layers were washed with aq. HCl (1 M), saturated NaHCO3, brine, and dried (MgSO4). The mixture was filtered and concentrated in vacuo to yield XXIIIe as a viscous colorless oil (14 g).
[0501] Step 5. 601
[0502] A solution of the aldehyde XXIIIe (14 g, 61.6 mmol) in CH2Cl2 (50 mL), was treated with Et3N (10.73 mL, 74.4 mmol), and acetone cyanohydrin (10.86 g, 127.57 mmol) and stirred at room temperature for 24 hrs. The reaction mixture was concentrated in vacuo and diluted with aq. HCl (1 M, 200 mL) and extracted into CH2Cl2 (3×200 mL). The combined organic layer were washed with H2O, brine, dried (MgSO4), filtered, concentrated in vacuo and purified by chromatography (SiO2, EtOAc/Hex 1:4) to yield XXIIIf (10.3 g) as a colorless liquid
[0503] Step 6. 602
[0504] Methanol saturated with HCl*, prepared by bubbling HCl gas to CH3OH (700 ml) at 0° C., was treated with cyanohydrin XXIIIf and heated to reflux for 24 h. The reaction was concentrated in vacuo to yield XXIIIg, which was used in the next step without purification.
[0505] * Alternatively 6M HCl prepared by addition of AcCl to dry methanol can also be used.
[0506] Step 7. 603
[0507] A solution of the amine hydrochloride XXIIIg in CH2Cl2 (200 mL) was treated with Et3N (45.0 mL, 315 mmol) and Boc2O (45.7 g, 209 mmol) at −78° C. The reaction mixture was then stirred at room temperature overnight and diluted with HCl (2 M, 200 mL) and extracted into CH2Cl2 The combined organic layer were dried (MgSO4) filtered, concentrated in vacuo and purified by chromatography (EtOAc/Hex 1:4) to yield hydroxy ester XXIIh.
[0508] Step 8. 604
[0509] A solution of methyl ester XXIIIh (3 g, 10.5 mmol) in THF/H2O (1:1) was treated with LiOH.H2O (645 mg, 15.75 mmol) and stirred at rt. for 2 h. The reaction mixture was acidfied with aq HCl (1 M, 15 mL) and concentrated in vacuo. The residue was dried in vacuum.
[0510] A solution of the acid in CH2Cl2 (50 mL) and DMF (25 mL) was treated with NH4Cl (2.94 g, 55.5 mmol), EDCl (3.15 g, 16.5 mmol), HOOBt (2.69 g, 16.5 mmol), and NMM (4.4 g, 44 mmol). The reaction mixture was stirred at room temperature for 3 d. The solvents were removed under vacuo and the residue was diluted with aq. HCl (250 mL) and extracted with CH2Cl2. The combined organic layers were washed with aq. Sat'd. NaHCO3, dried (MgSO4) filtered concentrated in vacuo to obtain XXIIIi, which was used as it is in the following steps. (Alternatively XXIIIi can also be obtained directly by the reaction of XXIIIf (4.5 g, 17.7 mmol) with aq. H2O2 (10 mL), LiOH.H2O (820 mg, 20.8 mmol) at 0° C. in 50 mL of CH3OH for 0.5 h.)
[0511] Step 9. 605
[0512] A solution of XXIIIi obtained in the previous step was dissolved in 4 N HCl in dioxane and stirred at rt. for 2 h. The reaction mixture was concentrated in vacuo to give XXIIIj as a solid, which was used without further purification.
[0513] Step 10. 606
[0514] The amino ester XXIIII was prepared following the method of R. Zhang and J. S. Madalengoitia (J. Org. Chem. 1999, 64, 330), with the exeception that the Boc group was cleved by the reaction of the Boc-protected amino acid with methanolic HCl.
[0515] A solution of commercial amino acid Boc—Chg—OH, XXIIk (Senn chemicals, 6.64 g, 24.1 mmol) and amine hydrochloride XXIIII (4.5 g, 22 mmol) in CH2Cl2 (100 mL) at 0° C. was treated with BOP reagent and stirred at rt. for 15 h. The reaction mixture was concentrated in vacuo, then it was diluted with aq. 1 M HCl and extracted into EtOAc (3×200 mL). The combined organic layers were washed with sat'd. NaHCO3 (200 mL), dried (MgSO4), filtered and concentrated in vacuo, and chromatographed (SiO2, EtOAc/Hex 3:7) to obtain XXIIIm (6.0 g) as a colorless solid.
[0516] Step 11. 607
[0517] A solution of methyl ester XXIIIm (4.0 g, 9.79 mmol) in THF/H2O (1:1) was treated with LiOH.H2O (401 mg, 9.79 mmol) and stirred at rt. for 3 h. The reaction mixture was acidified with aq. HCl and concentrated in vacuo to obtain the free acid.
[0518] A solution of acid (1.5 g, 3.74 mmol) in DMF/CH2Cl2 (1:1 50 mL) was treated with amine XXIIIj (772 mg, 3.74 mmol), EDCl (1.07 g, 5.61 mmol), HOOBt (959 mg, 5.61 mmol) and NMM (2.15 mL, 14.96 mmol) at −10° C. The reaction mixture was stirred at 0° C. for 48 h and concentrated in vacuo. The residue was diluted with aq. 1 M HCl and extracted with CH2Cl2, The combined organic layers were extracted with aq. NaHCO3, aq. HCl, brine, dried (MgSO4), filtered and concentrated in vacuo to obtain XXIIIn (2.08 g) as a tan colored solid.
[0519] Step 12. 608
[0520] A solution of amide XXIIIn (2.08 g, 3.79 mmol) in toluene and DMSO (1:1 20 mL) at 0° C. was treated with EDCl (7.24 g, 37.9 mmol) and dichloroacetic acid (2.42 g, 19.9 mmol) and stirred at rt. for 4 h. The reaction mixture was diluted with CH2Cl2, washed with sat'd. NaHCO3, and brine. The organic layer were dried (MgSO4) filtered, concentrated, in vacuo and purified by chromatography (SiO2, Acetone/Hexanes 3:7) to yield XXIII as a colorless solid.
Example XXIV Preparation of a Compound of Formula XXIV[0521] 609
[0522] Step 1. 610
[0523] A solution of Boc-tert-Lue XXIVa (Fluka, 5.0 g 21.6 mmol) in dry CH2Cl2/DMF (50 mL, 1:1) was cooled to 0° C. and treated with the amine XXIIII (5.3 g, 25.7 mmol), NMM (6.5 g, 64.8 mmol) and BOP reagent (11.6 g, 25.7 mmol). The reaction was stirred at rt. for 24 h, diluted with aq. HCl (1 M) and extracted with CH2Cl2. The combined organic layers were washed with HCl (aq, 1 M), sat'd. NaHCO3, brine, dried (MgSO4), filtered and concentrated in vacuo and purified by chromatography (SiO2, Acetone/Hexane 1:5) to yield XXIVb as a colorless solid.
[0524] Step 2. 611
[0525] A solution of methyl ester XXIVb (4.0 g, 10.46 mmol) was dissolved in HCl (4 M soln. dioxane) and stirred at rt. for 3 h. The reaction mixture was concentrated in vacuo to obtain the amine hydrochloride salt used in the next step.
[0526] A solution of the amine hydrochloride salt (397 mg, 1.24 mmol) in CH2Cl2 (10 mL) was cooled to −78° C. and treated with tert-butyl isocyanate (250 mg, 2.5 mmol) and stirred at rt. overnight. The reaction mixture was concentrated in vacuo and the residue was diluted with aq. HCl (1M) and extracted with CH2Cl2. The combined organic layers were washed with aq. HCl (1M), sat'd. NaHCO3 and brine. The organic layers were dried, filtered and concentrated in vacuo and the residue was purified by chromatography (SiO2, acetone/Hex 1:4) to yield XXIVc as a colorless solid.
[0527] Step 3. 612
[0528] A solution of methyl ester XXIVc (381 mg, 1.0 mmol) in THF/H2O (1:1, 5 mL) was treated with LiOH.H2O (62 mg, 1.5 mmol) and stirred at rt. for 3 h. The reaction mixture was acidified with aq. HCl and concentrated in vacuo to obtain the free acid.
[0529] A solution of acid (254.9 mg, 0.69 mmol) in DMF/CH2Cl2 (1:1, 5.0 mL) was treated with amine XXIIIj (159 mg, 0.763 mmol), EDCl (199 mg, 1.04 mmol), HOOBt (169.5 mg, 1.04 mmol) and NMM (280 mg, 2.77 mmol) at −20° C. The reaction mixture was stirred at −20° C. for 48 h and concentrated in vacuo. The residue was diluted with aq. 1M HCl and extracted with EtOAc, The combined organic layers were extracted with aq. NaHCO3, aq. HCl, brine, dried (MgSO4) filtered concentrated in vacuo to obtain XXIVd (470 mg) as a tan colored solid.
[0530] Step 4. 613
[0531] A solution of amide XXIVd (470 mg, 0.9 mmol) in toluene and DMSO (1:1 20 mL) at 0° C. was treated with EDCl (1.72 g, 9.0 mmol) and dichloroacetic acid (0.37 mL, 4.5 mmol) and stirred at 0° C. for 4 h. The reaction mixture was diluted with CH2Cl2, and washed with satd. NaHCO3, and brine. The organic layer was dried (MgSO4), filtered, concentrated, in vacuo and purified by chromatography (SiO2, Acetone/Hexanes 3:7) to yield XXIV as a colorless solid.
Example XXV Prepration of a Compound of Formula XXV[0532] 614
[0533] Step 1. 615
[0534] A solution of Fmoc-glycine (Bachem, 2.0 g, 6.87 mmol) in CH2Cl2 (20 mL) was treated with 2-phenyl-2-propanol (Aldrich, 3.36 g, 24.7 mmol), DCC (1M soin CH2Cl2, 8.24 mL), DMAP (167 mg, 1.37 mmol) and stirred at rt. for 24 h. The reaction mixture was concentrated in vacuo and diluted with Et2O (100 mL). The solid seperating out was filtered and the filterate was washed with satd. NaHCO3. The organic layer was dried (MgSO4), filtered, concentrated in vacuo, and purified by chromatography (SiO2, EtOAc/Hex 1:5) to yield ester XXVc (1.1 g) as a colorless viscous liquid.
[0535] Step 2. 616
[0536] A solution of XXVc in CH2Cl2 (16.0 mL) was treated with piperidine (4.0 mL) and stirred at rt. for 0.5 h. The reaction mixture was concentrated in vacuo and purified by chromatography (SiO2, Acetone/Hexanes 1:10 to 1:1) to yield the amine XXVd (420 mg) as a colorless liquid.
[0537] Step 3. 617
[0538] A solution of methyl ester XXIVc (381 mg, 1.0 mmol) in THF/H2O (1:1, 5 mL) was treated with LiOH.H2O (62 mg, 1.5 mmol) and stirred at rt. for 3 h. The reaction mixture was acidified with aq. HCl and concentrated in vacuo to obtain the free acid.
[0539] A solution of acid (2.0 g, 5.5 mmol) in DMF/CH2Cl2 (1:1, 40.0 mL) at −10° C. was treated with amine XXIIIg (1.51 g, 6.8 mmol), EDCl (1.57 g, 8.25 mmol), HOOBt (1.41 g, 8.25 mmol) and NMM (2.5 g, 24.7 mmol). The reaction mixture was stirred at 0° C. for 48 h and concentrated in vacuo. The residue was diluted with aq. 1 M HCl (100 mL) and extracted with CH2Cl2 (3×100 mL). The combined organic layers were extracted with aq. NaHCO3, aq. HCl, brine, dried (MgSO4) filtered, concentrated in vacuo to obtain XXVe (3.17 g) as a tan colored solid used further without any purification.
[0540] Step 4. 618
[0541] A solution of methyl ester XXVe (2.5 g, 4.66 mmol) in THF/H2O/CH3OH (1:1:1, 60 mL) was treated with LiOH.H2O (200 mg, 4.87 mmol) and stirred at rt. for 4 h. The reaction mixture was acidified with aq. HCl and concentrated in vacuo to obtain the free acid.
[0542] A solution of acid (200.0 mg, 0.38 mmol) in DMF/CH2Cl2 (1:1, 6.0 mL) at −10° C. was treated with amine XXVd (78 mg, 0.4 mmol), EDCl (105 mg, 0.55 mmol), HOOBt (95 mg, 0.55 mmol) and NMM (150 mg, 1.48 mmol). The reaction mixture was stirred at 0° C. for 48 h and concentrated in vacuo. The residue was diluted with aq. 1 M HCl (30 mL) and extracted with CH2Cl2 (3×30 mL). The combined organic layers were extracted with aq. NaHCO3 (2×30 mL), aq. HCl, brine (30 mL), dried (MgSO4) filtered, concentrated in vacuo to obtain XXVf (240 mg) as a tan colored solid.
[0543] Step 5. 619
[0544] A solution of XXVf (240 mg, 0.28 mmol) in CH2Cl2 (10 mL) was treated with Dess-Martin reagent (Omega, 242 mg, 0.56 mmol) and stirred at rt. for 2 h. After the oxidation was complete (TLC, Acetone/Hex 1:4) the reaction mixture was diluted with satd. NaHCO3 (20 mL) and Na2S2O3 (10% aq soln, 20 mL). The reaction mixture was stirred for 30 min and extractred with CH2Cl2 (3×30 mL). The combined organic layers were extracted with satd. NaHCO3, brine, dried (MgSO4) filtered concentrated in vacuo and purified by chromatography (SiO2, acetone/Hexanes 1:5) to yield XXV (122 mg) as a colorless solid.
Example XXVI Preparation of a Compound of Formula XXVI[0545] 620
[0546] Step 1: 621
[0547] To a stirred solution of N-Boc-3,4-dehydroproline XXVIa (5.0 g, 23.5 mmol), di-tert-butyl dicarbonate (7.5 g, 34.4 mmol), and 4-N,N-dimethylaminopyridine (0.40 g, 3.33 mmol) in acetonitrile (100 mL) at room temperature was added triethylamine (5.0 mL, 35.6 mmol). The resulting solution was stirred at this temperature for 18 h before it was concentrated in vacuo. The dark brown residue was purified by flash column chromatography eluting with 10-25% EtOAc/hexane to give the product XXVIb as a pale yellow oil (5.29 g, 84%).
[0548] Step 2: 622
[0549] To a stirred solution of dehydroproline XXVIb (10.1 g, 37.4 mmol), benzyltriethylammonium chloride (1.60 g, 7.02 mmol) in chloroform (120 mL) at room temperature was added 50% aqueous sodium hydroxide (120 g). After vigorously stirred at this temperature for 24 h, the black mixture was diluted with CH2Cl2 (200 mL) and diethyl ether (600 mL). After the layers were separated, the aqueous solution was extracted with CH2Cl2/Et2O (1:2, 3×600 mL). The organic solution was dried (MgSO4) and concentrated. The residue was purified by flash column chromatography using 5-20% EtOAc/hexane to afford 9.34 g (71%) of XXVIc as an off-white solid.
[0550] Step 3: 623
[0551] The solution of XXVIc (9.34 g, 26.5 mmol) in CH2Cl2 (25 mL) and CF3CO2H (50 mL) was stirred at room temperature for 4.5 h before it was concentrated in vacuo to give a brown residue which was used in Step 4 without further purification.
[0552] Step 4 624
[0553] Commercial concentrated hydrochloric acid (4.5 mL) was added to a solution of the residue from Step 3 in methanol (70 mL) and the resulting mixture was warmed to 65° C. in an oil bath. After 18 h, the mixture was concentrated in vacuo to give a brown oil XXVIe, which was used in Step 5 without further purification.
[0554] Step 5: 625
[0555] To a stirred solution of proline methyl ester XXVIe from Step 4, commercial N-Boc-cyclohexylglycine XXVIf (10.2 g, 40.0 mmol) and [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate] (HATU) (16.0 g, 42.1 mmol) in DMF (200 mL) at 0° C. was added diisopropylethylamine (18.0 mL, 104 mmol). After allowed to warm to room temperature along with the ice bath over night (18 h), the reaction mixture was diluted with EtOAc (600 mL), 5% H3PO4 (150 mL) and brine (150 mL). The organic solution was washed with 5% H3PO4 (150 mL), saturated NaHCO3 (2×200 mL) before it was dried (MgSO4), filtered and concentrated in vacuo. The residue was purified by flash column chromatography using 5-20% EtOAc/hexane to afford 3.84 g (32%, three steps) of XXVIg as an off-white solid.
[0556] Step 6: 626
[0557] The solution of methyl ester XXVIg (5.87 g, 13.1 mmol) and LiOH (1.65 g, 39.3 mmol) in THF/MeOH/H2O (1:1:1, 90 mL) was stirred at room temperature for 4 h. Methanol and THF were removed under reduced pressure. The aqueous solution was acidified to PH˜2 using 1 N aqueous HCl solution (50 mL) and saturated with solid sodium chloride before it was extracted with EtOAc (3×150 mL). The organic solutions were combined, dried (MgSO4), filtered and concentrated in vacuo to give a white solid XXVIh (5.8 g, quantitative).
[0558] Step 7: 627
[0559] The desired product XXIIIi was prepared according to the procedure in Example XXIII, Step 11.
[0560] Step 8: 628
[0561] The desired product XXVI was prepared according to the procedure in Example XXIII, Step 12.
Example XXVII Preparation of Compound of Formula XXVII[0562] 629
[0563] Step 1 630
[0564] The desired product XXVIIa was prepared according to the procedure in Example XXIII, Step 9.
[0565] Step 2 631
[0566] The desired product XXVIIb was prepared according to the procedure in Example XXIV, Step 2.
[0567] Step 3 632
[0568] The desired product XXVII was prepared according to the procedure in Example XXIII, Step 12.
Example XXVIII Preparation of a Compound of Formula XXVIII[0569] 633
[0570] Step 1: 634
[0571] The intermediate XXVIIIb was prepared according to the procedure in Example XXIII, Steps 3-6.
[0572] Step 2: 635
[0573] The acid from Example XXIV, Step 2 (XXVIIIc) (0.7 g) was reacted with product from Step 1 above (0.436 g), HATU (0.934 g) and DIPEA (1.64 mL) in the manner previously described in Example IX, Step 2a to afford 0.66 g of the desired product XXVIIId.
[0574] Step 3: 636
[0575] The product of Step 2 (0.5 g) was reacted with Dess-Martin reagent (1 g) in the manner previously described in Example XX, Step 7. Purification by flash column chromatography (40% EtOAc, Hexane, silica) furnished 0.35 g of product XXVIIIe. Mass spectrum (LCMS) 522 (M+H+).
[0576] Step 4: 637
[0577] The product of Step 4 (0.3 g) was added a 1/1 H2O/MeOH solution (20 mL) and NaHCO3 solid (242 mg, 5 equiv.). After being stirred for 18 hours at room temperature, the reaction was diluted with EtOAc and layers were separated. The aqueous layer was acidified to pH 2 with HCl 1.0 N and extracted with EtOAc. The EtOAc layer was washed with brine then dried over MgSO4, filtered and concentrated in vacuo to afford product XXVIIIf as a white powder (0.26 g). Mass spectrum (LCMS) 508 (M+H+).
[0578] Step 5: 638
[0579] The product of Step 5 (0.15 g) was dissolved in CH2Cl2 and reacted with HATU (0.137 g), NH4Cl (0.08 g, 5equiv.) and DIPEA (0.53 mL). After 2 hours at room temperature, the reaction was diluted with EtOAc, washed with a 10% citric acid solution, then a saturated NaHCO3 solution. The EtOAc layer was washed with brine then dried over MgSO4, filtered and concentrated in vacuo to afford a crude mixture. Purification by flash column chromatography (30% Acetone, Hexane, silica) furnished the desired product XXVIII (0.096 g). Mass spectrum (LCMS) 507 (M+H+).
Example XXIX Preparation of a Compound of Formula XXIX[0580] 639
[0581] Step 1: 640
[0582] To a 0° C. solution of the starting aldehyde (4.0 g) in CH2Cl2 (75 mL) was added acetic acid (2.0 equiv., 2.15 mL) followed by methylisocyanoacetate (1.1 equiv., 1.9 mL). The reaction was then gradually warmed-up to room temperature. After 18 hours (overnight), the reaction was diluted with EtOAc and washed with a saturated NaHCO3 solution. The EtOAc layer was washed with brine then dried over MgSO4, filtered and concentrated in vacuo to afford a crude mixture. Purification by flash column chromatography (30 to 40% EtOAc, Hexane, silica) furnished the product XXIXa (4.5 g).
[0583] Step 2: 641
[0584] To a 0° C. solution of XXIXa (4.4 g) in THF (100 mL) was added 26 mL (2.2 equiv.) of a 1.0 N LiOH solution. The reaction was stirred at this temperature for 2 hours then warmed-up to room temperature. After 2 hours, reaction mixture was acidified to pH 2 with a 1.0 N HCl solution. EtOAc was added and layers were separated. The EtOAc layer was washed with brine then dried over MgSO4, filtered and concentrated in vacuo to afford product XXIXb (3.7 g).
[0585] Step 3: 642
[0586] The acid XXIXb was reacted with the amine from Example XV in the manner previously described in Example XXI, Step 4. The resulting intermediate was then treated with HCl in the manner previously described in Example XXIII, Step 9 to afford product XXIXc.
[0587] Step 4: 643
[0588] The acid XXVIIIc (2.43 g) was dissolved in CH2Cl2 and was reacted with amine XXIXc (2.47 g), HATU (2.5 g) and DIPEA (5.8 mL) in the manner previously described in Example IX, Step 2a to afford, after purification by flash column chromatography (4% MeOH, CH2Cl2, silica), the desired product XXIXd (4.35 g). Mass spectrum (LCMS) 727 (M+H+).
[0589] Step 5: 644
[0590] The product of Step 4 (4.2 g) was reacted with Dess-Martin reagent (6.4 g) in the manner previously described in preparative Example XX, Step 7. Purification by flash column chromatography (100% EtOAc, silica) furnished 3 g of the final product XXIX. Mass spectrum (LCMS) 725 (M+H+).
Example XXX Preparation of a Compound of Formula XXX[0591] 645
[0592] Step 1: 646
[0593] The alcohol 2-(trifluoromethyl)propan-2-ol (1.28 g) was reacted with N,N-disucciminidyl carbonate (3.84 g) and Et3N (4.2 mL) in dry CH3CN (50 mL) for 18 hours. The mixture was diluted with EtOAc (200 mL) and filtered. The filtrate was washed with NaHCO3, brine then dried over MgSO4, filtered and concentrated in vacuo to afford a crude mixture. Purification by flash column chromatography (50% EtOAc, Hexane, silica) furnished the desired product XXXa (0.3 g).
[0594] Step 2: 647
[0595] The product from Example XXIX (0.3 g) was treated with 100 mL of 4.0 N HCl in dioxane. After 1 h, 200 mL of Et2O were added and the resulting precipitate was filtered off and dried under vacuo to afford the product XXXb (0.27 g) as a white powder. Mass spectrum (LCMS) 625 (M−HCl+H+).
[0596] Step 3: 648
[0597] To a room temperature solution of XXXb (0.05 g) in CH2Cl2 (5 mL) was added DIPEA (0.040 mL) XXXa (1.5 equiv., 0.030 g), followed by 1 crystal of DMAP. After 30 minutes, reaction was diluted with EtOAc (20 mL) and washed with HCl 1.5 N then NaHCO3 then brine. EtOAc layer was dried over MgSO4, filtered and concentrated in vacuo to afford a crude mixture. Purification by preparative chromatography (40% Acetone, Hexane, silica) furnished the desired product XXX (0.044 g). Mass spectrum (LCMS) 779 (M+H+).
Example XXXI Preparation of a Compound of Formula XXXI[0598] 649
[0599] Step 1: 650
[0600] To a solution of XXXb (0.05 g) in CH2Cl2 (5 mL) at room temperature was added DIPEA (0.040 mL) and tert-butylisocyanate (1.2 equiv., 0.01 mL). After 18 hours, reaction was diluted with EtOAc (20 mL) and washed with HCl 1.5 N, NaHCO3 and brine. EtOAc layer was dried over MgSO4, filtered and concentrated in vacuo to afford a crude mixture. Purification by preparative chromatography (100% EtOAc, silica) furnished the final product XXXI (0.021 g). Mass spectrum (LCMS) 724 (M+H+).
Example XXXII Preparation of a Compound of Formula XXXII[0601] 651
[0602] Step 1: 652
[0603] The product from Example XXVIII was treated in the manner previously described in preparative Example XXX, Step 2 to afford product XXXIIa. Mass spectrum (LCMS) 407 (M−HCl+H+).
[0604] Step 2: 653
[0605] The amine XXXIIa was reacted with XXXa in the manner previously described in preparative Example XXX, Step 3 to afford the desired product XXXII. Mass spectrum (LCMS) 508 (M+H+).
Example XXXIII Preparation of a Compound of Formula XXXIII[0606] 654
[0607] Step 1: 655
[0608] The amine XXXIIa was reacted with tert-butylisocyanate in the manner previously described in Example XXXI, Step 1, to afford the product XXXIII. Mass spectrum (LCMS) 561 (M+H+).
Example XXXIV Preparation of a Compound of Formula XXXIV[0609] 656
[0610] Step 1: 657
[0611] To the mixture of ester (6.0 g) and molecular sieve (5.2 g) in anhydrous methylene chloride (35 mL) was aded pyrrolidine (5.7 mL, 66.36 mmol.). The resulting brown slurry was stirred at room temperature under N2 for 24 h, filtered and washed with anhydrous CH3CN. The combined filtrate was concentrated to yield the desired product.
[0612] Step 2: 658
[0613] To a solution of the product from proceeding step in CH3CN (35 mL) was added anhydrous K2CO3, methallyl chloride (2.77 g, 30.5 mmol.), Nal (1.07 g, 6.7 mmol.). The resulting slurry was stirred at ambient temperature under N2 for 24 h. 50 mL of ice-cold water was added followed by 2N KHSO4 solution until pH was 1. EtOAc (100 mL) was added and the mixture was stirred for 0.75 h. Combined organic layer was collected and washed with brine, dried over MgSO4, and evaporated to yield the desired product.
[0614] Step 3: 659
[0615] The product from preceding step (2.7 g, 8.16 mmol.) was dissolved in dioxane (20 mL) and treated with freshly prepared 1N LiOH (9 mL). The reaction mixture was stirred at ambient temperature under N2 for 20 h. The reaction mixture was taken in EtOAc and washed with H2O. The combined aqueous phase was cooled to 0° C. and acidifed to pH 1.65 using 1N HCl. The turbid mixture was extracted with EtOAc (2×100 mL). Combined organic layer was washed with brine, dried over MgSO4, concentrated to give the desired acid (3.40 g).
[0616] Step 4: 660
[0617] To a suspension of NaBH(OAc)3 (3.93 g, 18.5 mmol.) in CH2Cl2 (55 mL) was added a solution of product from preceding step in anhydrous CH2Cl2 (20 mL) and acetic acid (2 mL). The slurry was stirred at ambient temperature for 20 h. Ice cold water (100 mL) was added to the slurry and stirred for ½ hr. Organic layer was separated, filtered, dried and evaporated to yield the desired product.
[0618] Step 5: 661
[0619] To a solution of the product from preceding step (1.9 g) in MeOH (40 mL) was treated with excess of CH2N2/Et2O solution and stirred for overnight. The reaction mixture was concentrated to dryness to yield a crude residue. The residue was chromatographed on silica gel, eluting with a gradient of EtOAc/hexane to afford 1.07 g of the pure desired product.
[0620] Step 6: 662
[0621] To a solution of product from preceding step (1.36 g) in anhydrous CH2Cl2 (40 mL) was treated with BF3.Me2O (0.7 mL). The reaction mixture was stirred at ambient temperature for 20 h and quenched with sat. NaHCO3 (30 mL) ad stirred for ½ hr. Organic layer was separated and combined organic layer was washed with brine, dried over MgSO4, concentrated to give crude residue. The residue was chromotagraphed on silica gel eluting with a gradient of EtOAc/hexane to afford 0.88 g of the desired compound.
[0622] Step 7: 663
[0623] To a solution of the product (0.92 g) from preceding step in MeOH (30 mL) was added 10% Pd/C (0.16 g) at room temperature and hydrogenated at ambient temperature under 1 atm. Pressure. The reaction mixture was stirred for 4 h and concentrated to dryness to yeild the desired compound.
[0624] Step 8: 664
[0625] The desired product was prepared according to the procedure in Example XXIII, Step10.
[0626] Step 9: 665
[0627] The desired acid product was prepared according to the procedure in Example XXIV, Step 3.
[0628] Step 10: 666
[0629] The desired product XXXIV was prepared according to the procedure in Example XXIX, Steps 4-5.
Example XXXV Preparation of a Compound of Formula XXXV[0630] 667
[0631] Step 1: 668
[0632] A solution of triethyl phosphonate (44.8 g) in THF (30 mL) at 0° C. was treated with a 1M solution (200 mL) of sodium bis(trimethylsilylamide) in THF. The resulting mixture was stirred at RT for 0.5 hour, and then cooled to 0° C. A solution of 1,4-cyclohexanedione ethylene ketal (15.6 g) in THF (50 mL) was added dropwise, and the resulting solution was stirred at RT for 18 hours. The reaction mixture was then cooled to 0° C., treated with cold aqueous citric acid, and the mixture was extracted with EtOAc. The extract was washed with saturated aqueous NaHCO3, then brine; then dried over anhydrous Na2SO4, filtered, and the filtrate evaporated. The residue was chromatographed on silica gel, eluting with a gradient of CH2Cl2/EtOAc to afford the title compound (21 g), 92% yield. Mass spectrum (FAB) 227.3 (M+H+).
[0633] Step 2: 669
[0634] The product of the preceding step (20 g) was dissolved in EtOH (150 mL) and treated with 10% Pd/C under 1 atm of hydrogen for 3 days. The mixture was filtered and the filtrate evaporated to afford the title compound (20.39), 100% yield. Mass spectrum (FAB) 229.2 (M+H+).
[0635] Step 3: 670
[0636] The product of the preceding step (20 g) was dissolved in MeOH (150 mL) and treated with a solution of LiOH (3.6 g) in water (50 mL). The mixture was stirred for 18 hours, and concentrated under vacuum. The residue was dissolved in cold water (100 mL), the solution was acidified to pH 2-3 with 5N HCl, and the resulting mixture was extracted with EtOAc. The extract was dried over anhydrous Na2SO4, filtered, and the filtrate evaporated to afford the title compound (17.1 g), 97% yield. Mass spectrum (FAB) 201.2 (M+H+).
[0637] Step 4: 671
[0638] 1. The product of the preceding step (3.0 g) was dissolved in Et2O (150 mL), treated with Et3N (2.1 mL), and the solution cooled to −78° C. Pivaloyl chloride (1.85 mL) was added dropwise, and after 0.25 hour additional stirring, the reaction was allowed to warm to 0° C. over 0.75 hour, and then cooled again to −78° C. to afford a solution of mixed anhydride for reaction in part 2.
[0639] 2. A solution of (S)-4-benzyl-2-oxazolidinone (2.66 g) in THF (22 mL) was cooled to −78° C., and a 1.6 M solution (9.38 mL) of n-butyllithium in hexane was added dropwise. After an additional 0.33 hour stirring at this temperature, the solution was transferred via canula to the cold solution of part 1. The mixture was stirred at −78° C., then warmed to 0° C., and stirred at this temperature for 0.5 hour. The organic layer was separated, the aqueous layer was extracted with Et2O, the combined organics were washed with brine, dried over anhydrous Na2SO4, filtered, and the filtrate evaporated. The residue was chromatographed on silica gel, eluting with a gradient of hexane/EtOAc (9:1) to afford the title compound (5.0 g), 93% yield. Mass spectrum (FAB) 360.4 (M+H+).
[0640] Step 5: 672
[0641] The product of the preceding step (2.7 g) was dissolved in THF (25 mL), cooled to −78° C., transferred by canula to a solution of 0.5 M potassium bis(trimethylsilyl)amide/toluene (16.5 mL) in THF (25 mL) at −78° C., and the resulting solution was stirred at −78° C. for 0.75 hour. To this solution was added via canula a solution of trisyl azide (3.01 g) in THF (25 mL) pre-cooled to −78° C. After 1.5 minutes, the reaction was quenched with acetic acid (1.99 mL), the reaction was warmed to RT, and then stirred for 16 hours. The reaction was diluted with EtOAc (300 mL), and washed with 5% aqueous NaCl. The aqueous phase was extracted with EtOAc, the combined organic phases were washed with saturated aqueous NaHCO3, then brine; then dried over anhydrous Na2SO4, filtered, and the filtrate evaporated. The residue was chromatographed on silica gel, eluting with EtOAc/hexane (1:3) to afford the title compound (2.65 g), 88% yield.
[0642] Step 6: 673
[0643] The product of the preceding step (11.4 g) was dissolved in 95% formic acid (70 mL) and heated at 70° C. for 0.5 hour while stirring. The solution was evaporated under vacuum, and the residue was taken up in EtOAc. The solution was washed with saturated aqueous NaHCO3, then brine; then dried over anhydrous Na2SO4, filtered, and the filtrate evaporated. The residue was chromatographed on silica gel to afford the title compound (8.2 g).
[0644] Step 7: 674
[0645] The product of the preceding step (8.2 g) was dissolved in CH2Cl2 (16 mL) and treated with diethylaminosulfur trifluoride (DAST, 7.00 mL) at RT for 3 hours. The reaction was poured over ice/water (200 cc), and extracted with CH2Cl2. The extract was washed with saturated aqueous NaHCO3, then brine; then dried over anhydrous Na2SO4, filtered, and the filtrate evaporated. The residue was chromatographed on silica gel, eluting with EtOAc/hexane (15:85) to afford the title compound (4.5 g), 52% yield.
[0646] Step 8: 675
[0647] The product of the preceding step (3.7 g) was dissolved in a mixture of THF (150 mL) and water (48 mL), cooled to 0° C., treated with 30% H2O2 (3.95 mL), and then with LiOH.H2O (0.86 g). The mixture was stirred for 1 hour at 0° C., then quenched with a solution of Na2SO3 (5.6 g) in water (30 mL), followed by a solution of 0.5 N NaHCO3 (100 mL). The mixture was concentrated under vacuum to ½ volume, diluted with water (to 500 mL), and extracted with CH2Cl2 (4×200 mL). The aqueous phase was acidified to pH 1-2 with 5N HCl, and extracted with EtOAc (4×200 mL). The extract was washed brine; then dried over anhydrous Na2SO4, filtered, and the filtrate evaporated to afford the title compound (1.95 g), 91% yield, which was used directly in the next step.
[0648] Step 9: 676
[0649] The product of the preceding example (2.6 g) was dissolved in Et2O (50 mL) and treated dropwise with a solution of CH2N2 in Et2O until the solution remained yellow. The solution was stirred for 18 hours, then evaporated under vacuum to afford the title compound (2.8), which was used directly in the next step.
[0650] Step 10: 677
[0651] The product of the preceding step (1.95 g) was dissolved in MeOH (150 mL), treated with formic acid (1.7 mL), then treated with 10% Pd/C (3.3 g, Degussa type E101) under 1 atm of hydrogen for 1.5 hours. The mixture was filtered and the filtrate evaporated to afford the title compound (2.1 g) as the formic acid salt, which was used directly in the next step.
[0652] Step 11: 678
[0653] The product of the preceding step (2.1 g) was dissolved in 1,4-dioxane (100 mL) and di-tert-butyl dicarbonate (1.9 g) was added, followed by diisopropylethylamine (2.9 mL). The solution was stirred for 18 hours, and concentrated under vacuum. The residue was treated with aqueous 5% KH2PO4 and the mixture extracted with EtOAc. The extract was washed with brine; then dried over anhydrous MgSO4, filtered, and the filtrate evaporated. The residue was chromatographed on silica gel, eluting with a gradient of CH2Cl2/Et2O to afford the title compound (2.5 g), 99% yield. Mass spectrum (FAB) 307.9 (M+H+).
[0654] Step 12: 679
[0655] The product of the preceding step (2.5 g) was dissolved in 1,4-dioxane (35 mL), treated with aqueous 1M LiOH (17 mL), and stirred for 2 hours. The mixture was quenched with ice/water (125 cc), the mixture was acidified to pH 3-4 with 3N HCl, and extracted with EtOAc. The extract was dried over anhydrous MgSO4, filtered, and the filtrate evaporated to afford the title compound (2.3 g), 96% yield. Mass spectrum (FAB) 294.0 (M+H+).
[0656] Step 13: 680
[0657] The desired product was prepared according to the procedure in Example XXIII, Step 10.
[0658] Step 14: 681
[0659] The desired acid product was prepared according to the procedure in Example XXIV, Step 3.
[0660] Step 15: 682
[0661] The desired acid product was prepared according to the procedure in Example XXIX, Step 4.
Example XXXVI Preparation of Compounds of Formulas XXXVI and XXXVIII[0662] Compounds of formulas XXXVI and XXXVIII were prepared according to the scheme below and utilizing preparative Examples 11 through 15 discussed above. 683
[0663] The compound of formula XXXVIb was prepared from a compound of formula XXXVIa as follows by known procedures: 684
[0664] To a solution of Compound XXXVIa (6.58 g, 22 mmol) in 100 mL of MeOH was added 10% Pd/C (0.8 g) and p-toluene sulfonic acid (4.2 g). The reaction mixture was subjected to hydrogenation at room temperature overnight. The reaction mixture was filtered through celite and washed with excess MeOH. The combined filtrate was concentrated in-vacuo to provide the title compound XXXVIb as a gummy. Conversion of XXXVIb to XXXVI and XXXVII followed the route as shown in the scheme above and according to preparative examples 11-15.
Example XXXVIII Preparation of a Compound of Formula XXXVIII[0665] A compound of the formula XXXVIII was prepared utilizing the following scheme and following preparative Examples 11 through 15 discussed earlier. 685 686
Example XXXIX Synthesis of the Compound of Formula XXXIX[0666] 687
[0667] Step 1: 688
[0668] A solution of the sulfonyl chloride XXXIXa prepared by the procedure of H. Mcklwain (J. Chem. Soc 1941, 75) was added dropwise to a mixture of 1.1. equiv of t-butylmethylamine and triethylamine at −78° C. and stirred at rt for 2 h. The reaction mixture was concentrated in vacuo and purified by chromatography (SiO2, Hex/Acetone 4:1) to yield sulfonamide XXXIXb as a colorless oil.
[0669] Step 2: 689
[0670] A solution of the Cbz-protected amine XXXIXb was dissolved in methanol and treated with 5 mol % of Pd/C (5% w/w) and hydrogenated at 60 psi. The reaction mixture was filtered through a plug of celite and concentrated in vacuo to obtain the free amine XXXIXc which solidfied on standing.
[0671] Step 3: 690
[0672] The hydroxy sulfonamide XXXIXd was synthesized similar to the procedure for the synthesis of XXVf except replacing the amine XXVd with XXXIXc. The crude reaction mixture directly used for the next reaction.
[0673] Step 4: 691
[0674] The hydroxy amide XXXIXd was oxidized to compound XXXIX using the Dess Martin reagent following the procedure for the synthesis of XXV (step 5). The crude mixture was purified by chromatography (SiO2, Acetone/Hexane 3:7) to obtain XXXIX as a colorless solid.
[0675] Assay for HCV Protease Inhibitory Activity:
[0676] Spectrophotometric Assay: Spectrophotometric assay for the HCV serine protease was performed on the inventive compounds by following the procedure described by R. Zhang et al, Analytical Biochemistry, 270 (1999) 268-275, the disclosure of which is incorporated herein by reference. The assay based on the proteolysis of chromogenic ester substrates is suitable for the continuous monitoring of HCV NS3 protease activity. The substrates were derived from the P side of the NS5A-NS5B junction sequence (Ac-DTEDWX(Nva), where X=A or P) whose C-terminal carboxyl groups were esterified with one of four different chromophoric alcohols (3- or 4-nitrophenol, 7-hydroxy-4-methyl-coumarin, or 4-phenylazophenol). Presented below are the synthesis, characterization and application of these novel spectrophotometric ester substrates to high throughput screening and detailed kinetic evaluation of HCV NS3 protease inhibitors.
[0677] Materials and Methods:
[0678] Materials: Chemical reagents for assay related buffers were obtained from Sigma Chemical Company (St. Louis, Mo.). Reagents for peptide synthesis were from Aldrich Chemicals, Novabiochem (San Diego, Calif.), Applied Biosystems (Foster City, Calif.) and Perseptive Biosystems (Framingham, Mass.). Peptides were synthesized manually or on an automated ABI model 431A synthesizer (from Applied Biosystems). UV/VIS Spectrometer model LAMBDA 12 was from Perkin Elmer (Norwalk, Conn.) and 96-well UV plates were obtained from Corning (Corning, N.Y.). The prewarming block was from USA Scientific (Ocala, Fla.) and the 96-well plate vortexer was from Labline Instruments (Melrose Park, Illinois). A Spectramax Plus microtiter plate reader with monochrometer was obtained from Molecular Devices (Sunnyvale, Calif.).
[0679] Enzyme Preparation: Recombinant heterodimeric HCV NS3/NS4A protease (strain 1a) was prepared by using the procedures published previously (D. L. Sali et al, Biochemistry, 37 (1998) 3392-3401). Protein concentrations were determined by the Biorad dye method using recombinant HCV protease standards previously quantified by amino acid analysis. Prior to assay initiation, the enzyme storage buffer (50 mM sodium phosphate pH 8.0, 300 mM NaCl, 10% glycerol, 0.05% lauryl maltoside and 10 mM DTT) was exchanged for the assay buffer (25 mM MOPS pH 6.5, 300 mM NaCl, 10% glycerol, 0.05% lauryl maltoside, 5 &mgr;M EDTA and 5 &mgr;M DTT) utilizing a Biorad Bio-Spin P-6 prepacked column.
[0680] Substrate Synthesis and Purification: The synthesis of the substrates was done as reported by R. Zhang et al, (ibid.) and was initiated by anchoring Fmoc—Nva—OH to 2-chlorotrityl chloride resin using a standard protocol (K. Barlos et al., Int. J. Pept. Protein Res., 37 (1991), 513-520). The peptides were subsequently assembled, using Fmoc chemistry, either manually or on an automatic ABI model 431 peptide synthesizer. The N-acetylated and fully protected peptide fragments were cleaved from the resin either by 10% acetic acid (HOAc) and 10% trifluoroethanol (TFE) in dichloromethane (DCM) for 30 min, or by 2% trifluoroacetic acid (TFA) in DCM for 10 min. The combined filtrate and DCM wash was evaporated azeotropically (or repeatedly extracted by aqueous Na2CO3 solution) to remove the acid used in cleavage. The DCM phase was dried over Na2SO4 and evaporated.
[0681] The ester substrates were assembled using standard acid-alcohol coupling procedures (K. Holmber et al, Acta Chem. Scand., B33 (1979) 410-412). Peptide fragments were dissolved in anhydrous pyridine (30-60 mg/ml) to which 10 molar equivalents of chromophore and a catalytic amount (0.1 eq.) of para-toluenesulfonic acid (PTSA) were added. Dicyclohexylcarbodiimide (DCC, 3 eq.) was added to initiate the coupling reactions. Product formation was monitored by HPLC and found to be complete following 12-72 hour reaction at room temperature. Pyridine solvent was evaporated under vacuum and further removed by azeotropic evaporation with toluene. The peptide ester was deprotected with 95% TFA in DCM for two hours and extracted three times with anhydrous ethyl ether to remove excess chromophore. The deprotected substrate was purified by reversed phase HPLC on a C3 or C8 column with a 30% to 60% acetonitrile gradient (using six column volumes). The overall yield following HPLC purification was approximately 20-30%. The molecular mass was confirmed by electrospray ionization mass spectroscopy. The substrates were stored in dry powder form under desiccation.
[0682] Spectra of Substrates and Products: Spectra of substrates and the corresponding chromophore products were obtained in the pH 6.5 assay buffer. Extinction coefficients were determined at the optimal off-peak wavelength in 1-cm cuvettes (340 nm for 3-Np and HMC, 370 nm for PAP and 400 nm for 4-Np) using multiple dilutions. The optimal off-peak wavelength was defined as that wavelength yielding the maximum fractional difference in absorbance between substrate and product (product OD—substrate OD)/substrate OD).
[0683] Protease Assay: HCV protease assays were performed at 30° C. using a 200 &mgr;l reaction mix in a 96-well microtiter plate. Assay buffer conditions (25 mM MOPS pH 6.5, 300 mM NaCl, 10% glycerol, 0.05% lauryl maltoside, 5 &mgr;M EDTA and 5 &mgr;M DTT) were optimized for the NS3/NS4A heterodimer (D. L. Sali et al, ibid.)). Typically, 150 &mgr;l mixtures of buffer, substrate and inhibitor were placed in wells (final concentration of DMSO 4% v/v) and allowed to preincubate at 30° C. for approximately 3 minutes. Fifty &mgr;ls of prewarmed protease (12 nM, 30° C.) in assay buffer, was then used to initiate the reaction (final volume 200 &mgr;l).The plates were monitored over the length of the assay (60 minutes) for change in absorbance at the appropriate wavelength (340 nm for 3-Np and HMC, 370 nm for PAP, and 400 nm for 4-Np) using a Spectromax Plus microtiter plate reader equipped with a monochrometer (acceptable results can be obtained with plate readers that utilize cutoff filters). Proteolytic cleavage of the ester linkage between the Nva and the chromophore was monitored at the appropriate wavelength against a no enzyme blank as a control for non-enzymatic hydrolysis. The evaluation of substrate kinetic parameters was performed over a 30-fold substrate concentration range (˜6-200 &mgr;M). Initial velocities were determined using linear regression and kinetic constants were obtained by fitting the data to the Michaelis-Menten equation using non-linear regression analysis (Mac Curve Fit 1.1, K. Raner). Turnover numbers (kcat) were calculated assuming the enzyme was fully active.
[0684] Evaluation of Inhibitors and Inactivators: The inhibition constants (Ki*) for the competitive inhibitors Ac—D—(D—Gla)—L—I—(Cha)—C—OH (27), Ac-DTEDWA(Nva)—OH and Ac-DTEDWP(Nva)—OH were determined experimentally at fixed concentrations of enzyme and substrate by plotting vo/vi vs. inhibitor concentration ([I] o) according to the rearranged Michaelis-Menten equation for competitive inhibition kinetics: vo/vi=1+[I] o/(Ki* (1+[S] o/Km)), where vo is the uninhibited initial velocity, vi is the initial velocity in the presence of inhibitor at any given inhibitor concentration ([I]o) and [S]o is the substrate concentration used. The resulting data were fitted using linear regression and the resulting slope, 1/(Ki*(1+[S] o/Km), was used to calculate the Ki* value.
[0685] The obtained Ki* values for the various compounds of the present invention are given in the afore-mentioned Tables wherein the compounds have been arranged in the order of ranges of Ki* values. From these test results, it would be apparent to the skilled artisan that the compounds of the invention have excellent utility as NS3-serine protease inhibitors.
[0686] While the present invention has been described with in conjunction with the specific embodiments set forth above, many alternatives, modifications and other variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are intended to fall within the spirit and scope of the present invention. 3 TABLE 2 molecular Ex. # STRUCTURE weight 1 692 691.7853 2 693 627.7441 3 694 754.8883 4 695 527.6259 5 696 698.7799 6 697 631.7352 7 698 381.476 8 699 540.6626 9 700 498.5813 10 701 633.7482 11 702 641.7249 12 703 641.7249 13 704 683.8061 14 705 637.7802 15 706 637.7802 16 707 637.7802 17 708 625.769 18 709 613.6707 19 710 613.6707 20 711 627.6978 21 712 609.726 22 713 609.726 23 714 609.726 24 715 611.742 25 716 600.7183 26 717 554.7361 27 718 478.5937 28 719 546.7132 29 720 562.7562 30 721 699.8519 31 722 643.7435 32 723 509.6077 33 724 637.7802 34 725 637.7802 35 726 579.6995 36 727 537.6619 37 728 539.6342 38 729 597.7149 39 730 493.6055 40 731 632.8044 41 732 747.8965 42 733 523.6348 43 734 598.7024 44 735 578.712 45 736 495.6214 46 737 627.7878 47 738 541.6501 48 739 543.666 49 740 501.5847 50 741 656.7394 51 742 578.712 52 743 725.8901 53 744 584.6782 54 745 538.6467 55 746 685.8248 56 747 527.6695 57 748 810.9557 58 749 552.6737 59 750 592.7391 60 751 534.702 61 752 653.8232 62 753 696.892 63 754 606.7662 64 755 643.7435 65 756 742.8771 66 757 747.8965 67 758 747.8965 68 759 761.9236 69 760 747.8965 70 761 733.913 71 762 746.9118 72 763 646.7935 73 764 746.9118 74 765 668.8782 75 766 628.8129 76 767 760.9792 77 768 818.0723 78 769 761.964 79 770 844.0702 80 771 753.9443 81 772 844.0702 82 773 753.9443 83 774 747.8965 84 775 804.0049 85 776 879.2858 86 777 823.1774 87 778 832.0994 88 779 775.9911 89 780 725.8901 90 781 698.9483 91 782 642.84 92 783 853.0995 93 784 789.9778 94 785 809.9682 95 786 878.8583 96 787 772.006 97 788 761.9672 98 789 728.85 99 790 828.0239 100 791 789.0334 101 792 775.0063 102 793 886.1102 103 794 880.8306 104 795 855.0718 105 796 790.7047 106 797 821.0543 107 798 685.7812 108 799 891.8973 109 800 775.0063 110 801 785.0452 111 802 789.0334 112 803 803.0605 113 804 862.4689 114 805 884.1323 115 806 889.5384 116 807 887.1794 117 808 831.071 118 809 830.0863 119 810 858.1405 120 811 874.1399 121 812 904.1227 122 813 929.195 123 814 873.0867 124 815 872.1019 125 816 900.1561 126 817 860.11 127 818 804.0016 128 819 803.0169 129 820 831.071 130 821 806.0612 131 822 749.9528 132 823 748.9681 133 824 777.0223 134 825 842.1382 135 826 786.0299 136 827 813.0994 137 828 829.0988 138 829 788.0022 139 830 815.0717 140 831 846.1265 141 832 790.0181 142 833 817.0876 143 834 833.087 144 835 911.2017 145 836 931.1921 146 837 844.1106 147 838 788.0022 148 839 815.0717 149 840 817.0876 150 841 831.1147 151 842 819.0599 152 843 833.087 153 844 829.0988 154 845 845.0981 155 846 816.0784 156 847 773.0125 157 848 787.0396 158 849 850.0959 159 850 807.03 160 851 821.0571 161 852 793.9876 162 853 759.9701 163 854 767.9714 164 855 711.863 165 856 712.8506 166 857 712.8506 167 858 817.0876 168 859 817.0876 169 860 817.0876 170 861 817.0876 171 862 777.0223 172 863 777.0223 173 864 801.0882 174 865 919.9515 175 866 919.9515 176 867 892.8821 177 868 892.8821 178 869 818.0723 179 870 761.964 180 871 789.0334 181 872 789.0334 182 873 820.0883 183 874 763.9799 184 875 791.0494 185 876 791.0494 186 877 791.0494 187 878 809.0674 188 879 809.0674 189 880 823.0945 190 881 823.0945 191 882 865.1758 192 883 865.1758 193 884 817.0876 194 885 817.0876 195 886 1606.121 887 196 888 1606.121 889 197 890 1638.12 891 198 892 1638.12 893 199 894 775.0063 200 895 775.0063 201 896 763.887 202 897 707.7786 203 898 734.848 204 899 774.9659 205 900 800.0139 206 901 687.7971 207 902 714.8666 208 903 853.0774 209 904 853.0774 210 905 811.0398 211 906 811.0398 212 907 811.0398 213 908 817.0876 214 909 817.0876 215 910 835.1057 216 911 630.8288 217 912 616.8018 218 913 742.9208 219 914 744.9367 220 915 735.9694 221 916 853.0774 222 917 809.0862 223 918 749.9965 224 919 612.7703 225 920 598.7432 226 921 758.9638 227 922 684.8401 228 923 758.9638 229 924 758.9638 230 925 795.0404 231 926 795.0404 232 927 624.7815 233 928 610.7544 234 929 770.9749 235 930 612.7703 236 931 722.8369 237 932 598.7432 238 933 795.0592 239 934 758.9638 240 935 839.0414 241 936 729.8375 242 937 756.0443 243 938 701.9518 244 939 734.0159 245 940 715.9789 246 941 715.9789 247 942 741.9951 248 943 821.0786 249 944 626.7974 250 945 612.7703 251 946 698.8672 252 947 674.842 253 948 584.7162 254 949 735.9694 255 950 772.9909 256 951 776.9383 257 952 626.7974 258 953 835.0189 259 954 835.0189 260 955 612.7703 261 956 686.856 262 957 686.856 263 958 686.856 264 959 686.856 265 960 742.9236 266 961 738.9325 267 962 738.9325 268 963 817.0444 269 964 738.9325 270 965 772.9909 271 966 795.0592 272 967 758.9638 273 968 810.9966 274 969 610.7544 275 970 596.7273 276 971 756.9479 277 972 756.9479 278 973 744.9799 279 974 698.8672 280 975 698.8672 281 976 709.8471 282 977 598.7432 283 978 810.9966 284 979 758.9638 285 980 742.9236 286 981 817.0444 287 982 817.0444 288 983 759.9526 289 984 494.6367 290 985 719.9263 291 986 731.938 292 987 677.8887 293 988 612.7703 294 989 612.7703 295 990 716.9261 296 991 717.9109 297 992 950.0884 298 993 729.9221 299 994 578.712 300 995 564.6849 301 996 703.8838 302 997 553.7021 303 998 703.8838 304 999 552.7173 305 1000 523.6756 306 1001 731.9783 307 1002 509.6485 308 1003 508.6638 309 1004 731.9783 310 1005 667.8503 311 1006 667.8503 312 1007 567.7292 313 1008 724.9054 314 1009 724.9054 315 1010 762.9736 316 1011 764.9896 317 1012 764.9896 318 1013 764.9896 319 1014 908.0734 320 1015 724.9054 321 1016 508.6638 322 1017 522.6909 323 1018 522.6909 324 1019 731.938 325 1020 744.9367 326 1021 727.9102 327 1022 567.7292 328 1023 584.8029 329 1024 726.9214 330 1025 726.9214 331 1026 726.9214 332 1027 740.9484 333 1028 688.8284 334 1029 564.6849 335 1030 550.6578 336 1031 820.9918 337 1032 710.8784 338 1033 746.9089 339 1034 710.8784 340 1035 590.6823 341 1036 716.9261 342 1037 539.675 343 1038 772.9473 344 1039 731.938 345 1040 731.938 346 1041 731.938 347 1042 546.7132 348 1043 606.7662 349 1044 578.712 350 1045 564.7722 351 1046 548.7291 352 1047 562.7562 353 1048 642.8432 354 1049 536.718 355 1050 574.7673 356 1051 726.9214 357 1052 726.9214 358 1053 580.7279 359 1054 639.799 360 1055 538.6902 361 1056 562.7562 362 1057 566.7444
[0687] 4 TABLE 4 Ki* STRUCTURE NAME Range 1058 iBoc-G(Chx)-P(4t- NHiBoc)-nV-(CO)- G-G(Ph)—Am A 1059 2-CO2)PhCO- G(Chx)-P(4t- MeNHCOPh(3- OPh)-nV-(CO)-G- G(Ph)—Am A 1060 iBoc-G(Chx)-P(4t- NHSO2Ph)-nV- (CO)-G-G(Ph)—Am A 1061 iBoc-G(Chx)-P(4t- UreaPh)-nV-(CO)- G-G(Ph)—Am A 1062 iBoc-G(Chx)-P(4t- MeNHCOPh)-nV- (CO)-G-G(Ph)—Am A 1063 iBoc-G(Chx)-P(4t- MeNHSO2Ph)-nV- (CO)-G-G(Ph)—Am A 1064 iBoc-G(Chx)-P(4t- MeNHCOPh(3- OPh))-nV-(CO)-G- G(Ph)—Am A 1065 2-CO2)PhCO- 3(chx)-P(4t- UreaPh)-nV-(CO)- G-G(ph)—Am C 1066 iBoc-G(Chx)-P(4t- NHSO2-(4Me)Ph)- nV(CO)-G-G(Ph)—Am B 1067 iBoc-G(Chx)-P(4t- NHSO2-(3Cl)Ph)- nV-(CO)-G-G(Ph)—Am B 1068 iBoc-G(Chx)-P(4t- NHSO2-(4- NHAc)Ph)-nV- (CO)-G-G(Ph)—Am A 1069 iBoc-G(Chx)-P(4t- NHSO2-(3,4- diCl)Ph)-nV-(CO)- G-G(Ph)—Am B 1070 iBoc-G(Chx)-P(4t- Urea-1-Np)-nV- (CO)-G-G(Ph)—Am B 1071 iBoc-G(Chx)-P(4t- NHSO2-2-Np)-nV- (CO)-G-G(Ph)—Am B 1072 iBoc-G(Chx)-P(4t- NHSO2-(4Cl)Ph)- nV-(CO)-G-G(Ph)—Am B 1073 iBoc-G(Chx)-P(4t- NHSO2-5(2,3- dihydrobenzofuran))- nV-(CO)-G- G(Ph)—Am B 1074 iBoc-G(Chx)-P(4t- NHSO2-6(4- OMe)Courmarin)- nV-(CO)-G-G(Ph)—Am B 1075 iBoc-G(Chx)-P(4t- Urea-Ph(4-OMe))- nV-(CO)-G-G(Ph)—Am A 1076 iBoc-G(Chx)-P(4t- Urea-Ph(4-Cl))-nV- (CO)-G-G(Ph)—Am B 1077 iBoc-G(Chx)-P(4t- Urea-Ph(4-Cl))-nV- (CO)-G-G(Ph)—Am C 1078 iBoc-G(Chx)-P(4t- Urea-Ph(4-Ac))- nV-(CO)-G-G(Ph)—Am B 1079 iBoc-G(Chx)-P(4t- Urea-Ph(4-Ac))- nV-(CO)-G-G(Ph)—Am B 1080 iBoc-G(Chx)-P(4t- NHSO2-Ph(4- OMe))-nV-(CO)-G- G(Ph)—Am B 1081 iBoc-V-P(4t- NHSO2-Ph)-nV- (CO)-G-G(Ph)—Am B 1082 iBoc-G(Chx)-P(4t- NHSO2-1Np)-nV- (CO)-G-G(Ph)—Am B 1083 iBoc-G(Chx)-P(4t- NHSO2-8- Quinoline)-nV- (CO)-G-G(Ph)—Am B 1084 (2,5-diF-6- CO2)PhCO- G(Chx)-P(4t-NH- iBoc)-nV-(CO)-G- G(Ph)—Am A 1085 (2,5-diF-6- CO2)PhCO- G(Chx)-P(4t- NHSO2-Ph)-nV- (CO)-G-G(Ph)—Am A 1086 3,4-diCl-6- CO2)PhCO- G(Chx)-P(4t-NH- iBoc)-nV-(CO)-G- G(Ph)—Am A 1087 3,4-diCl-6- CO2)PhCO- G(Chx)-P(4t- UreaPh)-nV(CO)- G-G(Ph)—Am A 1088 iBoc-G(Chx)-P(4t- Urea-(3-Cl)Ph)-nV- CO)-G-G(Ph)—Am B 1089 (3,4-diCl-6- CO2)PhCO- G(Chx)-P(4t- NHSO2-Ph)-nV- (CO)-G-G(Ph)—Am A 1090 iBoc-G(Chx)-P(3,4- iPr)-nV-(CO)-G- G(Ph)—OH A 1091 iBoc-G(Chx)-P(4t- Chx)-nV-(CO)-G- G(Ph)—Am B 1092 iBoc-G(Chx)-P(4- diMe)-nV-(CO)-G- G(Ph)—Am A 1093 iBoc-G(Chx)-P(4- Bn,4-Me)-nV-(CO)- G-G(Ph)—Am B 1094 iBoc-G(Chx)-P(4- spirocyclopentane)- nV-(CO)-G- G(Ph)—OH A 1095 iBoc-G(Chx)-2- Azabicyclo[2.2.2]octane- 3-CO-nV- (CO)-G-G(Ph)—Am B 1096 iPrOCO-G(Chx)- P(4-OtBu)-nV- (CO)-G-G(Ph)—OH A 1097 Neopentoxy(CO)- G(Chx)-P(4-OtBu)- nV-(CO)-G-G(Ph)—OH B 1098 Neopentoxy(CO)- G(Chx)-P(OH)-nV- (CO)-G-G(Ph)—OH B 1099 Ethoxy(CO)- G(Chx)-P(OH)-nV- (CO)-G-G(Ph)—OH B 1100 iBoc-G(Chx)-P(4,4- diMe)-nV-(CO)-G- G(Ph)—N(Me)2 A 1101 iBoc-G(Chx)-P(3,4- iPr)-nV-(CO)-G- G(Ph)—N(Me)2 A 1102 iBoc-G(Chx)-P(4- spirocyclopentane)- nV-(CO)-G- G(Ph)—N(Me)2 A 1103 iBoc-G(Chx)-P(4c- Me, 4t-Pr)-nV- (CO)-G-G(Ph)—N(Me)2 A 1104 iBoc-G(Chx)-P(4,4- diMe)-nV-(CO)-G- G(Ph)—OMe A 1105 iBoc-G(Chx)-P(4- spirocyclopentane)- nV-(CO)-G- G(Ph)—OMe A 1106 iBoc-G(Chx)-P(3t- Me)-nV-(CO)-G- G(Ph)—N(Me)2 A 1107 iBoc-G(Chx)-P(4,4- diMe)-nV-(CO)—S(Me)- G(Ph)—OH A 1108 iBoc-G(Chx)-P(4,4- diMe)-nV-(CO)—S- G(Ph)—OH B 1109 iBoc-G(Chx)-P(4,4- diMe)-nV-(CO)- G(Ac)-G(Ph)—OH C 1110 N—Me-G(Chx)- P(4,4-diMe)-nV- (CO)-G-G(Ph)—CO2H C 1111 iBoc-G(tBu)-P(4,4- diMe)-nV-(CO)-G- G(Ph)—N(Me)2 A 1112 iBoc-G(Chx)-P(3,4- (diMe-cyclopropyl))- G((S,S)—Me- cyclopropyl)-(CO)- G-G(Ph)—N(Me) A 1113 iBoc-G(Chx)-P(6S—CEM)- nV-(CO)-G- G(Ph)—N(Me)2 A 1114 iPoc-G(tBu)-P(4,4- diMe)-nV-(CO)-G- G(Ph)—N(Me)2 A 1115 iBoc-G(Chx)-P(6R- CEM)-nV-(CO)-G- G(Ph)—N(Me)2 A 1116 iBoc-G(tBu)-P(4,4- diMe)-L-(CO)-G- G(Ph)—N(Me)2 A 1117 ((R)-1-Me-iBoc)- G(Chx)-P(4,4- diMe)-nV-(CO)-G- G(Ph)—N(Me)2 A 1118 iBoc-G(Chx)-P(5- c/t-Me)-nV-(CO)- G-G(Ph)—CO2H A 1119 iBoc-G(Chx)-P(5- cis-Ph)-nV-(CO)- G-G(Ph)—CO2H B 1120 iBoc-G(4,4- diMeChx)-P(4,4- diMe)-nV-(CO)-G- G(Ph)—N(Me)2 A 1121 iBoc-G(1-MeChx)- P(4,4-diMe)-nV- (CO)-G-G(Ph)—N(Me)2 A 1122 iBoc-G(Chx)-P(3,4- CH2)-nV-(CO)-G- G(Ph)—N(Me)2 A 1123 iBoc-Chg-Pip-nV- (CO)-G-G(Ph)—N(Me)2 C 1124 iBoc-G(Chx)-P(4,4- diMe)-L-(CO)-G- G(Ph)—N(Me)2 A 1125 iPoc-G(tBu)-P(4,4- diMe)-L-(CO)-G- G(Ph)—N(Me)2 A 1126 iPoc-G(tBu)-P(5- c/t-Me)-nV-(CO)- G-G(Ph)—N(Me)2 A 1127 ((R)-1-Me-iBoc)- G(tBu)-P(4,4- diMe)-nV-(CO)-G- G(Ph)—N(Me)2 A 1128 (S)-1-MeiBoc- G(Chx)-P(4,4- diMe)-nV-(CO)-G- G(Ph)—N(Me)2 A 1129 iBoc-G(tBu)-P(4- cis-Me)-nV-(CO)- G-G(Ph)—N(Me)2 A 1130 iBoc-G(Chx)-P(4- cis-Me)-nV-(CO)- G-G(Ph)—N(Me)2 A 1131 iBoc-G(tBu)-P(5- cis-Me)-nV-(CO)- G-G(Ph)—N(Me)2 A 1132 iBoc-G(Chx)-P(5- cis-Me)-nV-(CO)- G-G(Ph)—N(Me)2 A 1133 iBoc-G(Chx)-P(t- 3Ph)-nV-(CO)-G- G(Ph)—N(Me)2 B 1134 iBoc-allo(Ile)-P(4,4- diMe)-nV-(CO)-G- G(Ph)—N(Me)2 A 1135 iBoc-G(Chx)-Pip(4- morpholino)-nV- (CO)-G-G(Ph)—N(Me)2 B 1136 iBoc-G(1-MeChx)- P[3,4-(diMe- cyclopropyl)]-nV- (CO)-G-G(Ph)—N(Me)2 A 1137 iBoc-G(1-MeChx)- P[3,4-(diMe- cyclopropyl)]-L- (CO)-G-G(Ph)—N(Me)2 A 1138 iBoc-G(tBu)-P[3,4- (diMe-cyclopropyl)]-L- (CO)-G-G(Ph)—N(Me)2 A 1139 iBoc-erythro-D,L- F(beta-Me)-P(4,4- diMe)-nV-(CO)-G- G(Ph)—N(Me)2 A 1140 ((R)-1-Me)iBoc- G(1-MeChx)-P[3,4- (diMe-cyclorpropyl)]-nV- (CO)-G-G(Ph)—N(Me)2 A 1141 iPoc-G(tBu)-P[3,4- (diMe-cyclopropyl)]-nV- (CO)-G-G(Ph)—N(Me)2 A 1142 iPoc-G(tBu)-P[3,4- (diMe-cyclopropyl)]-L- (CO)-G-G(Ph)—N(Me)2 A 1143 iBoc-G(tBu)-P(3,4- CH2)-nV-(CO)-G- G(Ph)—N(Me)2 A 1144 iBoc-G(Chx)-P(3,4- CH2)-nV-(CO)-G- G(Ph)—N(Me)2 A 1145 iPoc-G(tBu)-P(3,4- CH2)-nV-(CO)-G- G(Ph)—N(Me)2 A 1146 ((R)-1-Me)iBoc- G(tBu)-P(3,4- CH2)-nV-(CO)-G- G(Ph)—N(Me)2 A 1147 ((R)-1-Me)iBoc- G(1-MeChx)-P(3,4- CH2)-nV-(CO)-G- G(Ph)—N(Me)2 A
[0688] 5 TABLE 5 Ki* Structure MW Range 1148 507 B 1149 481 B 1150 473 C 1151 586 B 1152 497 C 1153 483 C 1154 481 C 1155 479 B 1156 507 A 1157 521 A 1158 612 A 1159 533 A 1160 569 A 1161 557 B 1162 521 C 1163 555 A 1164 497 C 1165 569 B 1166 533 B 1167 519 C 1168 621 B 1169 392 C 1170 418 C 1171 509 B 1172 493 C 1173 507 B 1174 567 A 1175 519 A 1176 519 B 1177 535 B 1178 523 C 1179 547 B 1180 547 B 1181 519 A 1182 505 C 1183 494 B 1184 480 B 1185 466 C 1186 493 B 1187 505 B 1188 491 B 1189 541 B 1190 478 C 1191 555 B 1192 554 B 1193 465 C 1194 520 A 1195 558 A 1196 532 A 1197 547 B 1198 547 B 1199 553 A 1200 520 B 1201 521 A 1202 543 C 1203 569 B 1204 507 B 1205 522 B 1206 606 C 1207 493 B 1208 467 C 1209 507 B 1210 572 A 1211 718 C 1212 547 A 1213 666 B 1214 540 C 1215 554 B 1216 540 B 1217 632 B 1218 580 B 1219 552 A 1220 592 A 1221 518 A 1222 506 A 1223 532 A 1224 581 B 1225 566 C 1226 599 B 1227 553 B 1228 568 B 1229 566 A 1230 566 A 1231 644 A 1232 543 C 1233 574 A 1234 534 C 1235 549 B 1236 562 A 1237 662 A 1238 563 B 1239 518 B 1240 492 B 1241 533 A 1242 510 C 1243 504 A 1244 530 B 1245 516 B 1246 574 B 1247 561 B 1248 533 B 1249 493 C 1250 546 A 1251 561 A 1252 505 B 1253 490 B 1254 539 C 1255 532 A 1256 561 A 1257 573 A 1258 567 A 1259 581 A 1260 608 A 1261 587 B 1262 561 B 1263 581 A 1264 573 A 1265 624 A 1266 547 A 1267 583 A 1268 545 B 1269 609 C 1270 549 C 1271 575 C 1272 613 A 1273 573 A 1274 561 A 1275 625 A 1276 666 C 1277 588 A 1278 599 A 1279 573 A 1280 587 A 1281 615 A 1282 535 B 1283 561 A 1284 531 A 1285 651 A 1286 506 A 1287 520 A 1288 546 A 1289 602 A 1290 549 B 1291 587 A 1292 561 A 1293 517 B 1294 491 B 1295 533 B 1296 507 A 1297 598 A 1298 535 A 1299 561 A 1300 633 A 1301 497 C 1302 607 A 1303 574 B 1304 518 B 1305 580 C 1306 544 B 1307 562 A 1308 561 A 1309 587 A 1310 533 A 1311 559 A 1312 557 C 1313 535 A 1314 535 B 1315 547 A 1316 546 A 1317 546 B 1318 523 B 1319 663 C 1320 637 C 1321 521 B 1322 573 B 1323 559 A 1324 533 A 1325 573 B 1326 595 B 1327 575 A 1328 560 B 1329 534 C 1330 727 A 1331 727 A 1332 753 C 1333 753 B 1334 745 A 1335 745 A 1336 759 C 1337 759 B 1338 669 B 1339 669 A 1340 554 C 1341 610 B 1342 711 A 1343 713 A 1344 713 A 1345 732 A 1346 733 A 1347 733 A 1348 737 A 1349 667 A 1350 612 C 1351 745 C 1352 745 C 1353 745 C 1354 759 C 1355 759 C 1356 759 C 1357 668 C 1358 636 B 1359 733 A 1360 767 B 1361 626 B 1362 715 C 1363 715 A 1364 699 B 1365 725 A 1366 781 B 1367 743 B 1368 743 C 1369 743 A 1370 757 B 1371 757 C 1372 757 B 1373 715 A 1374 715 A 1375 701 C 1376 701 A 1377 713 A 1378 739 A 1379 741 C 1380 715 C 1381 837 B 1382 751 A 1383 725 C 1384 711 C 1385 737 A 1386 775 A 1387 729 A 1388 729 A 1389 715 A 1390 775 A 1391 739 A 1392 713 A 1393 719 A 1394 719 A 1395 719 A 1396 773 A 1397 727 A 1398 727 A 1399 727 A 1400 787 A 1401 809 C 1402 709 A 1403 769 B 1404 723 C 1405 713 A 1406 723 A 1407 723 B 1408 771 C 1409 741 A 1410 725 A 1411 745 A 1412 716 A 1413 733 A 1414 713 A 1415 753 A 1416 726 A 1417 712 A 1418 771 B 1419 804 A 1420 726 A 1421 746 A 1422 752 A 1423 741 A 1424 727 A 1425 699 A 1426 739 A 1427 712 A 1428 698 A 1429 757 B 1430 790 A 1431 712 A 1432 732 A 1433 738 A 1434 869 A 1435 785 A 1436 785 A 1437 785 A 1438 785 A 1439 781 A 1440 780 A 1441 697 C 1442 671 C 1443 780 A 1444 884 A 1445 855 A 1446 757 B 1447 741 B 1448 779 B 1449 725 A 1450 787 A 1451 785 A 1452 737 A 1453 737 A 1454 739 A 1455 855 A 1456 826 A 1457 857 A 1458 826 A 1459 765 A 1460 792 A 1461 799 A 1462 784 A 1463 750 A 1464 771 A 1465 771 A 1466 536 C 1467 508 B 1468 601 C 1469 587 B 1470 494 C 1471 512 C 1472 538 C 1473 538 C 1474 522 C 1475 496 C 1476 522 C 1477 540 C 1478 598 C 1479 480 C 1480 508 B 1481 548 C 1482 534 B 1483 584 C 1484 570 B 1485 558 C 1486 433 C 1487 407 C 1488 393 C 1489 433 C 1490 419 C 1491 534 C 1492 520 B 1493 534 C 1494 520 B 1495 550 C 1496 536 C 1497 538 C 1498 568 B 1499 582 C 1500 570 C 1501 584 C 1502 418 C 1503 554 C 1504 508 C 1505 494 B 1506 562 C 1507 548 A 1508 520 C 1509 506 C 1510 540 C 1511 562 C 1512 548 B 1513 480 C 1514 466 C 1515 568 C 1516 554 B 1517 508 B 1518 482 C 1519 496 C 1520 522 C 1521 535 C 1522 539 B 1523 563 B 1524 567 C 1525 561 C 1526 567 C 1527 581 C 1528 495 C 1529 654 B 1530 549 C 1531 567 C 1532 581 C 1533 654 C 1534 626 B 1535 654 A 1536 535 C 1537 535 B 1538 523 C 1539 523 C 1540 561 B 1541 511 C 1542 537 C 1543 654 B 1544 654 A 1545 626 B 1546 652 B 1547 525 C 1548 539 C 1549 549 C 1550 641 B 1551 630 C 1552 653 B 1553 653 B 1554 553 C 1555 655 C 1556 629 C 1557 539 C 1558 521 C 1559 521 C 1560 547 C 1561 547 C 1562 590 B 1563 590 B 1564 641 B 1565 565 C 1566 579 C 1567 644 C 1568 587 C 1569 654 B 1570 716 B 1571 668 B 1572 670 A 1573 666 C 1574 666 C 1575 630 B 1576 531 C 1577 563 C 1578 537 C 1579 575 B 1580 591 B 1581 586 C 1582 586 C 1583 585 B 1584 563 B 1585 547 B 1586 519 C 1587 640 B 1588 546 B 1589 646 B 1590 594 C 1591 592 B 1592 533 C 1593 545 C 1594 659 B 1595 609 A 1596 635 B 1597 685 B 1598 519 C 1599 621 B 1600 521 B 1601 547 B 1602 573 B 1603 609 B 1604 547 B 1605 719 B 1606 719 C 1607 653 B 1608 597 B 1609 697 A 1610 619 B 1611 651 C 1612 592 B 1613 587 C 1614 563 B 1615 589 C 1616 621 C 1617 519 C 1618 597 B 1619 549 C 1620 535 C 1621 521 B 1622 519 C 1623 689 C 1624 611 C 1625 600 C 1626 595 B 1627 541 C 1628 549 B 1629 593 C 1630 680 B 1631 559 C 1632 559 C 1633 573 B 1634 644 C 1635 537 C 1636 627 C 1637 609 B 1638 664 B 1639 650 C 1640 661 B 1641 571 C 1642 661 B 1643 607 B 1644 625 C 1645 575 B 1646 575 B 1647 575 B 1648 575 B 1649 559 B 1650 573 B 1651 637 B 1652 473 C 1653 559 B 1654 549 C 1655 587 C 1656 547 C 1657 547 B 1658 573 C 1659 573 C 1660 607 C 1661 595 B 1662 581 B 1663 609 B 1664 629 C 1665 694 C 1666 605 C 1667 579 C 1668 627 C 1669 563 C 1670 571 C 1671 572 B 1672 551 C 1673 609 C 1674 593 B 1675 593 C 1676 613 C 1677 593 B 1678 581 C 1679 571 B 1680 577 C 1681 615 C 1682 571 C 1683 571 C 1684 545 C 1685 633 C 1686 585 B 1687 587 B 1688 647 B 1689 512 C 1690 575 C 1691 658 C 1692 621 C 1693 565 C 1694 572 A 1695 587 A 1696 587 B 1697 509 C 1698 533 C 1699 587 B 1700 644 C 1701 594 B 1702 695 B 1703 650 B 1704 600 B 1705 628 A 1706 556 B 1707 674 B 1708 579 C 1709 637 C 1710 671 C 1711 583 C 1712 587 B 1713 601 B 1714 623 B 1715 621 A 1716 645 C 1717 664 B 1718 573 C 1719 559 C 1720 847 B 1721 651 B 1722 547 C 1723 561 B 1724 561 B 1725 546 C 1726 545 C 1727 633 B 1728 681 C 1729 561 C 1730 598 B 1731 583 C 1732 567 C 1733 539 C 1734 519 C 1735 708 B 1736 649 C 1737 561 B 1738 461 C 1739 531 C 1740 606 A 1741 606 A 1742 592 A 1743 666 C 1744 626 B 1745 640 B 1746 654 B 1747 698 B 1748 654 B 1749 758 C 1750 638 A 1751 683 B 1752 593 A 1753 621 A 1754 607 B 1755 627 B 1756 586 A 1757 534 B 1758 560 C 1759 621 A 1760 616 B 1761 572 A 1762 547 C 1763 561 C 1764 521 C 1765 620 B 1766 578 B 1767 560 A 1768 620 A 1769 618 B 1770 632 B 1771 662 B 1772 592 B 1773 590 B 1774 690 B 1775 609 B 1776 749 B 1777 648 A 1778 783 B 1779 783 B 1780 634 C 1781 648 C 1782 634 C 1783 649 C 1784 629 C 1785 657 C 1786 614 A 1787 702 B 1788 702 A 1789 675 B 1790 647 B 1791 568 C 1792 619 C 1793 482 C 1794 576 C 1795 617 B 1796 651 C 1797 637 C 1798 684 B 1799 685 B 1800 698 B 1801 605 B 1802 620 B 1803 672 C 1804 620 B 1805 594 B 1806 606 B 1807 580 C 1808 532 B 1809 572 B 1810 738 A 1811 718 B 1812 664 B 1813 614 B 1814 624 B 1815 558 B 1816 633 B 1817 770 C 1818 535 C 1819 533 C 1820 677 C 1821 563 B 1822 651 A 1823 634 A 1824 706 C 1825 757 A 1826 662 A 1827 660 A 1828 648 A 1829 648 C 1830 668 B 1831 618 A 1832 660 B 1833 601 B 1834 673 B 1835 662 A 1836 602 A 1837 681 A 1838 681 C 1839 655 C 1840 689 B 1841 660 A 1842 538 C 1843 764 A 1844 816 C 1845 780 B 1846 560 C 1847 602 C 1848 625 B 1849 685 B 1850 587 A 1851 587 A 1852 601 A 1853 625 B 1854 601 A 1855 627 B 1856 679 A 1857 628 A 1858 587 A 1859 641 A 1860 659 A 1861 674 A 1862 615 B 1863 641 B 1864 641 B 1865 627 A 1866 665 A 1867 614 A 1868 737 B 1869 666 A 1870 660 A 1871 591 C 1872 615 C 1873 754 B 1874 577 C 1875 694 A 1876 702 A 1877 701 A 1878 546 B 1879 520 B 1880 546 B 1881 723 B 1882 675 A 1883 771 B 1884 847 C 1885 641 A 1886 613 A 1887 651 C 1888 700 A 1889 569 A 1890 756 B 1891 786 A 1892 669 B 1893 601 A 1894 601 B 1895 683 A 1896 673 A 1897 680 A 1898 602 A 1899 735 A 1900 743 A 1901 655 B 1902 692 A 1903 639 A 1904 639 A 1905 675 A 1906 621 A 1907 668 A 1908 642 A 1909 654 A 1910 601 C 1911 663 B 1912 641 A 1913 702 A 1914 701 A 1915 588 B 1916 638 A 1917 630 A 1918 697 A 1919 621 A 1920 608 B 1921 682 A 1922 667 B 1923 520 B 1924 645 B 1925 669 C 1926 575 A 1927 709 B 1928 652 B 1929 714 A 1930 561 B 1931 561 B 1932 685 B 1933 580 A 1934 606 A 1935 653 A 1936 667 A
Claims
1. A compound, including enantiomers, stereoisomers, rotamers, tautomers, racemates and prodrug of said compound, and pharmaceutically acceptable salts or solvates of said compound, or of said prodrug, said compound having the general structure shown in Formula I:
- 1937
- wherein:
- Y is selected from the group consisting of the following moieties: alkyl, alkyl-aryl, heteroalkyl, heteroaryl, aryl-heteroaryl, alkyl-heteroaryl, cycloalkyl, alkyloxy, alkyl-aryloxy, aryloxy, heteroaryloxy, heterocycloalkyloxy, cycloalkyloxy, alkylamino, arylamino, alkyl-arylamino, arylamino, heteroarylamino, cycloalkylamino and heterocycloalkylamino, with the proviso that Y maybe optionally substituted with X11 or X12;
- X11 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocyclyl, heterocyclylalkyl, aryl, alkylaryl, arylalkyl, heteroaryl, alkylheteroaryl, or heteroarylalkyl, with the proviso that X may be additionally optionally substituted with X12;
- X12 is hydroxy, alkoxy, aryloxy, thio, alkylthio, arylthio, amino, alkylamino, arylamino, alkylsulfonyl, arylsulfonyl, alkylsulfonamido, arylsulfonamido, carboxy, carbalkoxy, carboxamido, alkoxycarbonylamino, alkoxycarbonyloxy, alkylureido, arylureido, halogen, cyano, or nitro, with the proviso that said alkyl, alkoxy, and aryl may be additionally optionally substituted with moieties independently selected from X12;
- R1 is COR5 or B(OR)2, wherein R5 is H, OH, OR8, NR9R10, CF3, C2F5, C3F7, CF2R6, R6, or COR7 wherein R7 is H, OH, OR8, CHR9R10, or NR9R10, wherein R6, R8, R9 and R10 are independently selected from the group consisting of H, alkyl, aryl, heteroalkyl, heteroaryl, cycloalkyl, cycloalkyl, arylalkyl, heteroarylalkyl, [CH(R1′)]pCOOR11, [CH(R1′)] pCONR12R13, [CH(R1′)]pSO2R11, [CH(R1′)]pCOR11, [CH(R1′)]pCH(OH)R11, CH(R1′)CONHCH(R2′)COO R11, CH(R1′)CONHCH(R2′)CONR12R13, CH(R1′)CONHCH(R2′)R11, CH(R1′)CONHCH(R2′)CONHCH(R3′)COO R11, CH(R1′)CONHCH(R2′)CONHCH(R3′)CONR12R13, CH(R1′)CONHCH(R′2)CONHCH(R3′)CONHCH(R4′)COO R11, CH(R1′)CONHCH(R2′)CONHCH(R3′)CONHCH(R4′)CONR12R13, CH(R1′)CONHCH(R2′)CONHCH(R3′)CONHCH(R4′)CONHCH(R5′)COO R11 and CH(R1′)CONHCH(R2′)CONHCH(R3′)CONHCH(R4′)CONHCH(R5′) CONR12R13, wherein R1′, R2′, R3′, R4′, R5′, R11, R12, R13, and R′ are independently selected from the group consisting of H, alkyl, aryl, heteroalkyl, heteroaryl, cycloalkyl, alkyl-aryl, alkyl-heteroaryl, aryl-alkyl and heteroaralkyl;
- Z is selected from O, N, CH or CR;
- W maybe present or absent, and if W is present, W is selected from C═O, C═S, C(═N—CN), or SO2;
- Q maybe present or absent, and when Q is present, Q is CH, N, P, (CH2)p, (CHR)p, (CRR′)p, O, NR, S, or SO2; and when Q is absent, M may be present or absent; when Q and M are absent, A is directly linked to L;
- A is O, CH2, (CHR)p, (CHR—CHR′)p, (CRR′)p, NR, S, SO2 or a bond;
- E is CH, N, CR, or a double bond towards A, L or G;
- G may be present or absent, and when G is present, G is (CH2)p, (CHR)p, or (CRR′)p; and when G is absent, J is present and E is directly connected to the carbon atom in Formula I as G is linked to;
- J maybe present or absent, and when J is present, J is (CH2)p, (CHR)p, or (CRR′)p, SO2, NH, NR or O; and when J is absent, G is present and E is directly linked to N shown in Formula I as linked to J;
- L may be present or absent, and when L is present, L is CH, CR, O, S or NR; and when L is absent, then M may be present or absent; and if M is present with L being absent, then M is directly and independently linked to E, and J is directly and independently linked to E;
- M may be present or absent, and when M is present, M is O, NR, S, SO2, (CH2)p, (CHR)p (CHR—CHR′)p, or (CRR′)p;
- p is a number from 0 to 6; and
- R, R′, R2, R3 and R4 are independently selected from the group consisting of H; C1-C10 alkyl; C2-C10 alkenyl; C3-C8 cycloalkyl; C3-C8 heterocycloalkyl, alkoxy, aryloxy, alkylthio, arylthio, amino, amido, ester, carboxylic acid, carbamate, urea, ketone, aldehyde, cyano, nitro, halogen; (cycloalkyl)alkyl and (heterocycloalkyl)alkyl, wherein said cycloalkyl is made of three to eight carbon atoms, and zero to six oxygen, nitrogen, sulfur, or phosphorus atoms, and said alkyl is of one to six carbon atoms; aryl; heteroaryl; alkyl-aryl; and alkyl-heteroaryl;
- wherein said alkyl, heteroalkyl, alkenyl, heteroalkenyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl moieties may be optionally and chemically-suitably substituted, with said term “substituted” referring to optional and chemically-suitable substitution with one or more moieties selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heterocyclic, halogen, hydroxy, thio, alkoxy, aryloxy, alkylthio, arylthio, amino, amido, ester, carboxylic acid, carbamate, urea, ketone, aldehyde, cyano, nitro, sulfonamido, sulfoxide, sulfone, sulfonyl urea, hydrazide, and hydroxamate; further wherein said unit N—C—G—E—L—J—N represents a five-membered or six-membered cyclic ring structure with the proviso that when said unit N—C—G—E—L—J—N represents a five-membered cyclic ring structure, or when the bicyclic ring structure in Formula I comprising N, C, G, E, L, J, N, A, Q, and M represents a five-membered cyclic ring structure, then said five-membered cyclic ring structure lacks a carbonyl group as part of the cyclic ring.
2. The compound of claim 1, wherein R1 is COR5, and R5 is H, OH, COOR8, CONR9R10.
3. The compound of claim 2, wherein R1 is COCONR9R10, and R9 is H, R10 is H, R14, [CH(R1′)]pCOOR11, [CH(R1′)]pCONR12R13, [CH(R1′)]pSO2R11, [CH(R1′)]pSO2N R12R13, [CH(R1′)]pCOR11, CH(R1′)CONHCH(R2′)COOR11, CH(R1′)CONHCH(R2′) CONR12R13, or CH(R1′)CONHCH(R2′)(R′), wherein R14 is H, alkyl, aryl, heteroalkyl, heteroaryl, cycloalkyl, alkyl-aryl, alkyl-heteroaryl, aryl-alkyl, alkenyl, alkynyl or heteroaralkyl.
4. The compound of claim 3, wherein R10 is H, R14, CH(R1′)COOR11, CH(R1′)CH(R1′)COOR11, CH(R1′)CON R12R13, CH(R1′)CH(R1′)CONR12R13, CH(R1′)CH(R1′)SO2R11, CH(R1′)CH(R1′)SO2N R12R13, CH(R1′)CH(R1′)COR11, CH(R1′)CONHCH(R2)COOR11, CH(R1′)CONHCH(R)CONR12R13, or CH(R′1)CONHCH(R2′)(R′), wherein R1′ is H or alkyl, and R2′ is phenyl, substituted phenyl, hetero atom-substituted phenyl, thiophenyl, cycloalkyl, piperidyl or pyridyl.
5. The compound of claim 4, wherein R1′ is H.
6. The compound of claim 5, wherein R11 is H, methyl, ethyl, allyl, tert-butyl, benzyl, &agr;-methylbenzyl, &agr;,&agr;-dimethylbenzyl, 1-methylcyclopropyl or 1-methylcyclopentyl;
- R′ is hydroxymethyl or CH2CONR12R13;
- R2′ is independently selected from the group consisting of:
- 1938
- wherein:
- U1 and u2 maybe same or different and are selected from H, F, CH2COOH, CH2COOMe, CH2CONH2, CH2CONHMe, CH2CONMe2, azido, amino, hydroxyl, substituted amino, substituted hydroxyl;
- U3 and U4 maybe same or different and are selected from O and S;
- U5 is selected from the moieties consisting of alkyl sulfonyl, aryl sulfonyl, heteroalkyl sulfonyl, heteroaryl sulfonyl, alkyl carbonyl, aryl carbonyl, heteroalkyl carbonyl, heteroaryl carbonyl, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl or a combination thereof;
- and NR12R13 is selected from the group consisting of:
- 1939
- wherein U6 is H, OH, or CH2OH, and
- R14 is selected from the group consisting of: H, Me, Et, n-propyl, methoxy, cyclopropyl, n-butyl, 1-but-3-ynyl, benzyl, &agr;-methylbenzyl, phenethyl, allyl, 1-but-3-enyl, OMe, cyclopropylmethyl.
7. The compound of claim 2, wherein R2 is selected from the group consisting of the following moieties:
- 1940 1941
8. The compound of claim 7, wherein R3 is selected from the group consisting of:
- 1942 1943
- wherein R31═OH or O-alkyl;
- Y19 is selected from the following moieties:
- 1944
- and Y20 is selected from the following moieties:
- 1945
9. The compound of claim 8, wherein R3 is selected from the group consisting of the following moieties:
- 1946
10. The compound of claim 9, wherein Z is N and R4 is H.
11. The compound of claim 10, wherein W is C═O.
12. The compound of claim 11, wherein Y is selected from the following moieties:
- 1947 1948 1949 1950 1951 1952 1953
- wherein:
- Y11 is selected from H, COOH, COOEt, OMe, Ph, OPh, NHMe, NHAc, NHPh, CH(Me)2, 1-triazolyl, 1-imidazolyl, and NHCH2COOH;
- Y12 is selected from H, COOH, COOMe, OMe, F, Cl, or Br;
- Y13 is selected from the following moieties:
- 1954
- Y14 is selected from MeSO2, Ac, Boc, iBoc, Cbz, or Alloc;
- Y15 and Y16 are independently selected from alkyl, aryl, heteroalkyl, and heteroaryl;
- Y17 is CF3, NO2, CONH2, OH, COOCH3, OCH3, OC6H5, C6H5, COC6H5, NH2, or COOH; and
- Y18 is COOCH3, NO2, N(CH3)2, F, OCH3, CH2COOH, COOH, SO2NH2, or NHCOCH3.
13. The compound of claim 12, wherein Y is selected from the group consisting of:
- 1955 1956 1957
- wherein:
- Y17═CF3, NO2, CONH2, OH, NH2, or COOH;
- Y18═F, COOH,
14. The compound of claim 13, wherein Y is selected from the group consisting of:
- 1958 1959
15. The compound of claim 14, wherein L and M are absent, and J is directly linked to E.
16. The compound of claim 14, wherein L, J and M are absent and E is directly linked to N.
17. The compound of claim 14, wherein G and M are absent.
18. The compound of claim 14, wherein the moiety:
- 1960
19. The compound of claim 18, wherein structure a is selected from the following structures:
- 1961
20. The compound of claim 18, wherein structure a is:
- 1962
- wherein R20 is selected from the following structures:
- 1963
21. The compound of claim 18, wherein structure a is:
- 1964
- wherein R21 and R22 may be the same or different and are independently selected from the following structures:
- 1965 1966
22. The compound of claim 18, wherein structure a is selected from the following structures:
- 1967
23. The compound of claim 14, wherein:
- 1968
- wherein Q may be present or absent, and if Q is absent, M is directly linked to A.
24. The compound of claim 23, wherein structure b is selected from the following structures:
- 1969
25. The compound of claim 14, wherein:
- 1970
- wherein G and J are independently selected from the group consisting of (CH2)p, (CHR)p, (CHR—CHR′)p, and (CRR′)p; A and M are independently selected from the group consisting of O, S, SO2, NR, (CH2)p, (CHR)p, (CHR—CHR′)p, and (CRR′)p; and Q is CH2, CHR, CRR′, NH, NR, O, S, SO2, NR, (CH2)p, (CHR)p, and (CRR′)p.
26. The compound of claim 25, wherein structure c is selected from the following structures:
- 1971 1972
27. The compound of claim 14, wherein:
- 1973
- is selected from the following structures:
- 1974 1975 1976
28. The compound of claim 27, wherein:
- 1977
- is selected from the following structures:
- 1978 1979
29. A pharmaceutical composition comprising as an active ingredient a compound of claim 1.
30. The pharmaceutical composition of claim 29 for use in treating disorders associated with HCV.
31. The pharmaceutical composition of claim 29 additionally comprising a pharmaceutically acceptable carrier.
32. The pharmaceutical composition of claim 31, additionally containing an antiviral agent.
33. The pharmaceutical composition of claim 32, still additionally containing an interferon.
34. The pharmaceutical composition of claim 33, wherein said antiviral agent is ribavirin and said interferon is a-interferon or pegylated interferon.
35. A method of treating disorders associated with the HCV, said method comprising administering to a patient in need of such treatment a pharmaceutical composition which comprises therapeutically effective amounts of a compound of claim 1.
36. The method of claim 35, wherein said administration is oral or subcutaneous.
37. The use of a compound of claim 1 for the manufacture of a medicament to treat disorders associated with the HCV.
38. A method of preparing a pharmaceutical composition for treating the disorders associated with the HCV, said method comprising bringing into intimate contact a compound of claim 1 and a pharmaceutically acceptable carrier.
39. A compound exhibiting HCV protease inhibitory activity, including enantiomers, stereoisomers, rotamers, tautomers, racemates and prod rug of said compound, and pharmaceutically acceptable salts or solvates of said compound, or of said prodrug, said compound being selected from the compounds of structures listed below:
- 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2025 2026 2027 2028 2029 2030 2031 2032 2033 2034 2035 2036 2037
40. A pharmaceutical composition for treating disorders associated with the HCV, said composition comprising therapeutically effective amount of one or more compounds in claim 39 and a pharmaceutically acceptable carrier.
41. The pharmaceutical composition of claim 40, additionally containing an antiviral agent.
42. The pharmaceutical composition of claim 41, still additionally containing an interferon or PEG-interferon alpha conjugate.
43. The pharmaceutical composition of claim 42, wherein said antiviral agent is ribavirin and said interferon is &agr;-interferon.
44. A method of treatment of a hepatitis C virus associated disorder, comprising administering an effective amount of one or more compounds of claim 39.
45. A method of modulating the activity of hepatitis C virus (HCV) protease, comprising contacting HCV protease with one or more compounds of claim 39.
46. A method of treating, preventing, or ameliorating one or more symptoms of hepatitis C, comprising administering an effective amount of one or more compounds of claim 39.
47. The method of claim 45, wherein the HCV protease is the NS3/NS4a protease.
48. The method of claim 47, wherein the compound or compounds inhibit HCV NS3/NS4a protease.
49. A method of modulating the processing of hepatitis C virus (HCV) polypeptide, comprising contacting a composition containing the HCV polypeptide under conditions in which the polypeptide is processed with one or more compounds of claim 39.
Type: Application
Filed: Jul 19, 2001
Publication Date: Dec 16, 2004
Inventors: Anil K. Saksena (Upper Montclair, NJ), Viyyoor Moopil Girijavallabhan (Parsippany, NJ), Raymond G. Lovey (West Caldwell, NJ), Edwin Jao (Warren, NJ), Frank Bennett (Piscataway, NJ), Jinping L. McCormick (Edison, NJ), Haiyan Wang (Cranbury, NJ), Russell E. Pike (Stanhope, NJ), Stephane L. Bogen (Somerset, NJ), Tin-Yau Chan (Edison, NJ), Yi-Tsung Liu (MorrisTownship, NJ), Zhaoning Zhu (East Windsor, NJ), F. George Njoroge (Warren, NJ), Ashok Arasappan (Bridgewater, NJ), Tejal Parekh (Mountain View, CA), Ashit K. Ganguly (Upper Montclair, NJ), Kevin X. Chen (Iselin, NJ), Srikanth Venkatraman (Fords, NJ), Henry A. Vaccaro (South Plainfield, NJ), Patrick A. Pinto (Morris Plains, NJ), Bama Santhanam (Bridgewater, NJ), Scott Jeffrey Kemp (San Diego, CA), Odile Esther Levy (San Diego, CA), Marguerita Lim-Wilby (La Jolla, CA), Susan Y. Tamura (Santa Fe, NM), Wanli Wu (Edison, NJ), Siska Hendrata (Edison, NJ), Yuhua Huang (Scotch Plains, NJ)
Application Number: 09908955
International Classification: A61K038/07; A61K038/06; C07K005/08; C07K005/10;
