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HK1073845A - Macrocyclic peptides active against the hepatitis c virus - Google Patents

Macrocyclic peptides active against the hepatitis c virus Download PDF

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Publication number
HK1073845A
HK1073845A HK05106434.6A HK05106434A HK1073845A HK 1073845 A HK1073845 A HK 1073845A HK 05106434 A HK05106434 A HK 05106434A HK 1073845 A HK1073845 A HK 1073845A
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Hong Kong
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compound
mmol
formula
mixture
item
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HK05106434.6A
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Chinese (zh)
Inventor
尤拉.S.桑特里佐斯
戴尔．R．卡梅伦
安妮-玛丽．福彻
埃莉斯.吉罗
纳撒利．吉德鲁
特迪.哈尔莫斯
蒙西．利纳斯-布鲁内特
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贝林格尔.英格海姆加拿大有限公司
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Publication of HK1073845A publication Critical patent/HK1073845A/en

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Description

Macrocyclic peptides effective against hepatitis C virus
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The application is a divisional application of Chinese patent application with the invention name of 00806046.0 corresponding to the international application No. PCT/CA00/00353 and the publication No. WO 00/59929, 4.3.2000, and the invention is named as a macrocyclic ring which can effectively resist hepatitis C virus.
Technical Field
The present invention relates to compounds, compositions, formulations of such compounds, and methods for treating Hepatitis C Virus (HCV) infection. In particular, the present invention provides novel peptide analogs, pharmaceutical compositions containing such analogs, and methods of using these analogs in the treatment of HCV infection.
Background
Hepatitis C Virus (HCV) is the leading cause of post-transfusion and socio-acquired non-a non-B hepatitis throughout the world. It is estimated that more than 1 million and 7 million people worldwide are infected with the virus. A high percentage of carriers become chronically infected and many progress to chronic liver disease, so-called chronic hepatitis C. This group in turn becomes a high risk group for serious liver diseases such as cirrhosis, hepatocellular carcinoma, and ultimately liver disease leading to death.
The mechanism by which HCV forms viral persistence and causes a high rate of chronic liver disease has not been completely elucidated. It is not known how HCV interacts with and bypasses the host's immune system. Furthermore, the role played by cellular and humoral immune responses in protection against HCV infection and disease is not established. Immunoglobulins have been reported to prevent transfusion-associated viral hepatitis, however, the Center for Disease Control (Center for Disease Control) currently does not suggest immunoglobulin therapy suitable for this purpose. The lack of an effective protective immune response hinders the development of vaccines or appropriate post-exposure preventive measures, and it is therefore desirable to firmly address the intervention of antiviral agents in the short term.
In order to identify pharmaceutical agents that can effectively treat HCV infection in patients with chronic hepatitis C, various clinical studies have been conducted. These studies have involved interferon- α alone and in combination with other antiviral agents. Such studies indicate that a significant number of participants do not respond to the treatment and those who respond favorably, most are found to have relapses after termination of treatment.
Until several years ago, Interferon (IFN) was the only treatment proven to be beneficial and approved clinically for patients with chronic hepatitis C. However, the sustained response rate is low and interferon treatment also causes severe side effects (i.e. retinopathy, thyroiditis, acute pancreatitis, depression) that reduce the quality of life of the treated patients. Initially, interferon in combination with ribavirin was approved for patients who did not respond to interferon alone. The gold standard for use in treatment of HCV is currently approved for use in congenital patients and is currently selected. However, the side effects caused by IFN are not reduced with this combination therapy.
Accordingly, there is a need to develop effective antiviral agents that can be used to treat HCV infection and overcome the limitations of existing drug therapies.
HCV is a positive strand RNA virus enveloped in the yellow fever virus family. The single-stranded HCV RNA gene is approximately 9500 nucleotides in length and has a single Open Reading Frame (ORF) encoding a single large polyprotein of about 3000 amino acids. In infected cells, the polyprotein is cleaved at multiple sites by cellular and viral proteases to produce structural and non-structural (NS) proteins. In the case of HCV, the production of mature nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) is accomplished by two viral proteases. First, as yet incompletely characterized, was cut at the junction NS2-NS 3; the second serine protease contained in the N-terminal region of NS3 (hereinafter referred to as NS3 protease) and regulates all subsequent cleavages downstream of NS3 in cis, at the NS3-NS4A cleavage site, and in trans, the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly contributing to the membrane localization of NS3 and other viral replicase components. Complex formation of the NS3 protein with NS4A appears to be necessary for the event to proceed, promoting proteolytic efficacy at all positions. The NS3 protein also showed nucleoside triphosphatase and RNA helicase activities. NS5B is an RNA-dependent RNA polymerase, which is involved in HCV replication.
Patent application WO 97/06804 describes the (-) enantiomer of the nucleoside analog cytosine-1, 3-oxathiolane (also known as 3TC) having activity against HCV. Although the compound has been reported to be safe in previous clinical trials against HIV and HBV, its activity against HCV has not been clinically demonstrated, and its mechanism of action against viruses has not been reported.
A common strategy for developing antiviral agents is to inactivate the viral-encoded enzymes necessary for replication of the virus.
In this situation, great efforts have been made to find compounds that inhibit the NS3 protease or RNA helicase of HCV, leading to the following findings:
us patent 5,633,388 describes heterocyclic-substituted carboxamides and analogues having activity against HCV. These compounds target the helicase activity of the viral NS3 protein, but no clinical tests have been reported. Phenanthrenequinones have been reported to have activity against the HCV NS3 protease in vitro by Chu et al (Tet. Lett., (1996), 7229-7232). However, no further development of this compound was reported. An article provided at the Ninth International Conference on Antiviral Research (Ninth International Conference on Antiviral Research), Urabandai, Fukyshima, japan (1996) (Antiviral Research, (1996), 30, 1, a23 (abstract 19)) reported that thiazolidine derivatives are inhibitors of HCV protease.
Several studies have reported compounds that inhibit other serine proteases, such as human leukocyte elastase. One group of these compounds is reported in WO 95/33764(Hoechst Marion Roussel, 1995). The peptides disclosed in this application are morpholinylcarbonyl-benzoyl-peptide analogues which differ structurally from the peptides of the invention.
WO 98/17679 from Vertex Pharmaceuticals inc. discloses inhibitors of serine proteases, in particular inhibitors of the hepatitis C virus NS3 protease. Hoffman LaRoche (WO 98/22496; U.S. Pat. No. 5,866,684 and U.S. Pat. No. 6,018,020) has also reported hexapeptides which are useful as protease inhibitors of antiviral agents for the treatment of HCV infections. Inhibition of the NS4A-4B product has been published by Steinkuhler et al and Ingallinella et al (Biochemistry (1998), 37, 8899-8905 and 8906-8914). WO 97/43310 to Schering Corporation discloses peptide sequences of 20 and 21 amino acids having activity against HCV NS3 protease. WO 98/46597 to Emory University discloses peptides and peptide analogs having activity against serine proteases in vitro. WO 98/46630 to Peptide Therapeutics Limited discloses that depsipeptide (depsipeptide) matrices inhibit the HCV NS3 protease. Finally, U.S. Pat. No. 5,869,253 discloses enzymatic PNA molecules that inhibit HCV NS3 protease.
None of the above prior patent applications suggest that the cyclic peptide has activity and selectivity against the hepatitis C virus NS3 protease.
WO 99/07733, WO 99/07734, WO00/09543 and WO 00/09558 disclose hexa-to tetra-peptide and tripeptide analogs that inhibit the NS3 protease. However, these disclosures do not address or guide the design of the macrocyclic analogs of the present invention. WO 99/38888 published by Institute de Richche di biologica molecular (IRBM) at 8.5.1999, discloses small peptide inhibitors of HCV NS3 protease. The cyclic nature of the peptides of the invention is not suggested or indicated in this disclosure. In addition, the PCT application was published after the priority date of the present invention. WO 99/64442 to IRBM was also published after the priority date of the present invention, disclosing oligopeptides with keto acids at the PI. WO 99/50230 to Vertex Pharmaceuticals (published 10.7.1999) was also published after the priority date of the present invention. Even later, this publication does not slightly suggest any cyclic peptide of the present invention.
One advantage of the present invention is that it provides macrocyclic peptides that inhibit the NS3 protease of hepatitis C virus.
Another advantage of an aspect of the invention resides in the fact that these peptides specifically inhibit NS3 protease and do not show significant inhibitory activity against other serine proteases, such as Human Leukocyte Elastase (HLE), Porcine Pancreatic Elastase (PPE) or bovine pancreatic chymotrypsin, or cysteine proteases, such as human liver cathepsin b (cat b).
Another advantage of the present invention is that it provides small peptides of low molecular weight that are capable of penetrating cell membranes and inhibiting the activity of NS3 protease in cell culture.
Furthermore, a further advantage of the compounds of the invention lies in the fact that they are found to be active in two main genotypes in clinical isolates (1a & 1b), strongly suggesting that these compounds will have activity against all currently known HCV genotypes.
Summary of The Invention
Included within the scope of the invention are:
1. a compound of formula (I):
wherein W is a group selected from the group consisting of CH and N,
R21is H, halogen, C1-6Alkyl radical, C3-6Cycloalkyl radical, C1-6Haloalkyl, C1-6Alkoxy radical, C3-6Cycloalkoxy, hydroxy or N (R)23)2Wherein each R is23Are respectively H, C1-6Alkyl or C3-6A cycloalkoxy group; and is
R22Is H, halogen, C1-6Alkyl radical, C3-6A cycloalkyl group; c1-6Haloalkyl, C1-6Sulfanyl, C1-6Alkoxy radical, C3-6Cycloalkoxy, C2-7Alkoxyalkyl group, C3-6Cycloalkyl radical, C6 or 10Aryl or Het, wherein Het is a five-, six-or seven-membered saturated or unsaturated heterocyclic ring containing one to four heteroatoms selected from nitrogen, oxygen and sulfur;
with R24Substituted cycloalkyl, aryl or Het, wherein R24Is H, halogen, C1-6Alkyl radical, C3-6Cycloalkyl radical, C1-6Alkoxy radical, C3-6Cycloalkoxy, NO2、N(R25)2、NH-C(O)-R25(ii) a Or NH-C (O) -NH-R25Wherein each R is25Are respectively H, C1-6Alkyl or C3-6A cycloalkyl group;
or R24Is NH-C (O) -OR26Wherein R is26Is C1-6Alkyl or C3-6A cycloalkyl group;
R3is hydroxy, NH2Or formula-NH-R31Wherein R is31Is C6Or C10Aryl, heteroaryl, -C (O) -R32、-C(O)-OR32or-C (O) -NHR32Wherein R is32Is C1-6Alkyl or C3-6A cycloalkyl group;
d is a saturated or unsaturated alkylene chain of 5 to 10 atoms, optionally containing one to three heteroatoms, selected from O, S or N-R41Wherein R is41Is H, C1-6Alkyl radical, C3-6Cycloalkyl or-C (O) -R42Wherein R is42Is C1-6Alkyl radical, C3-6Cycloalkyl or C6 or 10An aryl group;
R4is H or one to three substituents on any carbon atom of the chain D, each substituent being selected from C1-6Alkyl radical, C1-6Haloalkyl, C1-6Alkoxy, hydroxy, halogen, amino, oxo, thio or C1-6A sulfanyl group, a mercapto group, an alkyl group,
and A is of the formula-C (O) -NH-R5In which R is5Is selected from C1-8Alkyl radical, C3-6Cycloalkyl radical, C6 or 10Aryl or C7-16Aralkyl group;
or A is a carboxylic acid or a pharmaceutically acceptable salt or ester thereof.
2. A compound of formula I according to item 1, wherein R1The moiety is selected from 2 different diastereomers, represented by structures (i) and (ii):
d is the same as the amide (i) or D is the same as A (ii)
3. A compound of formula I according to item 2, wherein D is attached ipsilaterally to a, as represented by structure (ii).
4. A compound of formula I according to item 1, wherein W is N;
R21is H, C1-6Alkyl radical, C3-6Alkoxy, hydroxy, chloro or N (R)23)2Wherein R is23Is H or C1-6An alkyl group;
R22is H, C1-6Sulfanyl, C1-6Alkoxy, phenyl or Het selected from:
or
Wherein R is24Is H, C1-6Alkyl, NH-R25、NH-C(O)-R25、NH-C(O)-NH-R25Wherein each R is25Are respectively H, C1-6Alkyl or C3-6A cycloalkyl group;
OR NH-C (O) -OR26Wherein R is26Is C1-6An alkyl group.
5. A compound of formula I according to item 4, wherein R21Is H or C1-6An alkoxy group.
6. A compound of formula I according to item 4, wherein R22Is C1-4Alkoxy, phenyl, or Het selected from:
wherein R is24Is H, C1-6Alkyl, NH-R25Or NH-C (O) -R25；
Wherein each R25Are respectively C1-6Alkyl or C3-6A cycloalkyl group,
OR NH-C (O) -OR26Wherein R is26As defined in item 4.
7. A compound of formula I according to item 6, wherein R21Is methoxy.
8. A compound of formula I according to item 7, wherein R22Is ethoxy or Het selected from:
or；
Wherein R is24aIs NH-R25Or NH-C (O) -R25Wherein R is25Is C1-6An alkyl group;
or R24aIs NH-C (O) -OR26Wherein R is26Is C1-6Alkyl radical, and
R24bis H or C1-6An alkyl group.
9. A compound of formula I according to item 1, wherein R3Is of the formula NH-C (O) -R32Or of the formula NH-C (O) -NH-R32OR of the formula NH-C (O) -OR32Of (a) a carbamate, wherein R32Is C1-6Alkyl or C3-6A cycloalkyl group.
10. A compound of formula I according to item 9, wherein R3 is urea or carbamate, wherein R32Is C1-6Alkyl or C4-6A cycloalkyl group.
11. A compound of formula I according to item 10, wherein R3Is a carbamate, and R32Is tert-butyl, cyclobutyl or cyclopentyl.
12. A compound of formula I according to item 1, wherein D is a saturated or unsaturated alkylene chain of 6 to 8 atoms, optionally containing one or two heteroatoms, selected from O, S or N-R, respectively41Wherein R is41Is H, C1-6Alkyl or C2-7An acyl group.
13. A compound of formula I according to item 12, wherein D may optionally contain one group selected from NH or N-C2-7A heteroatom of an acyl group.
14. A compound according to item 13, wherein the heteroatom is selected from: NH or N (Ac).
15. A compound according to item 13, wherein the D chain contains 7 atoms.
16. A compound according to item 15, wherein the heteroatom is located at the 10 position of the D chain.
17. A compound according to item 13, wherein the D chain is saturated.
18. A compound of formula I according to item 12, wherein D is a saturated or unsaturated alkylene chain of 6 to 8 atoms, optionally containing one heteroatom selected from O or S.
19. A compound according to item 18, wherein the D chain contains 7 atoms.
20. A compound according to item 19, wherein the heteroatom is located at position 9 of the D chain.
21. A compound according to item 20, wherein the D chain is substituted with R at position 84Is substituted in which R4Is H or C1-6An alkyl group.
22. A compound according to item 21, wherein R is4Is H or methyl.
23. A compound according to item 22, wherein R is4Is H or 8- (S) -Me.
24. A compound according to item 23, wherein the D chain is saturated.
25. A compound according to item 19, wherein the D chain contains a double bond at positions 11, 12.
26. A compound according to item 25 wherein the double bond is trans.
27. A compound of formula I according to item 12, wherein D is a saturated or unsaturated all-carbon alkylene chain of 6 to 8 atoms.
28. A compound of formula I according to item 27, wherein the D chain contains 7 atoms.
29. A compound of formula I according to item 28, wherein the D chain is saturated.
30. A compound according to item 29, wherein R is4Substituted by the D chain, wherein R4Is H, oxo, hydroxy, alkoxy or alkyl.
31. A compound according to item 30, wherein R is4Is H or C1-6An alkyl group.
32. A compound according to item 31, wherein R is4Is H or methyl.
33. A compound according to item 32, wherein R is4Is H or 10- (S) -Me.
34. A compound of formula I according to item 28, wherein D contains one double bond.
35. A compound of formula I according to item 34, wherein the double bond is at position 13, 14 of the D chain.
36. A compound of formula I according to item 35, wherein the double bond is cis.
37. A compound according to item 36, wherein R is4Substituted by the D chain, wherein R4Is H, oxo, hydroxy, C1-6Alkoxy or C1-6An alkyl group.
38. A compound according to item 37, wherein R4Is H or C1-6An alkyl group.
39. A compound according to item 38, wherein R is4Is H or methyl.
40. A compound according to item 39, wherein R4Is H or 10- (S) -Me.
41. A compound of formula I according to item 1, wherein a is a carboxylic acid.
42. A compound of formula I according to item 1, wherein W is N:
R3is of the formula-NH-C (O) -NHR32OR NH-C (O) -OR32Wherein R is32Is C1-4Alkyl or C4-6A cycloalkyl group;
d is a saturated or unsaturated alkylene chain of 6 to 8 atoms, linked to R1The same side as A optionally contains one or two heteroatoms selected from O, S or N-R41Wherein R is41Is H or C2-7An acyl group;
R4is H or one to three substituents independently selected from hydroxy or C1-6An alkyl group; and A is a carboxylic acid, or a pharmaceutically acceptable salt or ester thereof.
43. A compound of formula I according to item 42, wherein R21Is H or methoxy;
R22is C1-6Alkyl or Het selected from:
wherein R is24aIs H, C1-6Alkyl, NH-R25、NH-C(O)-R25Or NH-C (O) -NH-R25，
Wherein R is25Is H, C1-6Alkyl or C3-6A cycloalkyl group;
or R24aIs NH-C (O) -OR26Wherein R is26Is C1-6Alkyl or C3-6A cycloalkyl group; and is
R24bIs H or C1-6An alkyl group;
R3is of the formula-N-C (O) -NHR32OR of the formula N-C (O) -OR32Of (a) a carbamate, wherein R32Is C1-6Alkyl or C3-6A cycloalkyl group;
d is a 7-atom alkylene chain which may optionally contain a double bond in position 11, 12 or 13, 14;
the D chain optionally containing a heteroatom selected from O, S, NH, N (Me) or N (Ac); and is
R4Is H or C1-6An alkyl group.
44. A compound of formula I according to item 43, wherein R21Is methoxy, and R22Is ethoxy or
Wherein R is24aIs NH- (C)1-4Alkyl), NH-C (O) - (C)1-4Alkyl), NH-C (O) -O- (C)1-4Alkyl) or NH-C (O) -NH- (C)1-4Alkyl groups);
d is C7Full carbon chain, saturated or containing a cis double bond at positions 13, 14.
45. A compound of the formula:
it is contained in R1Wherein the double bond, D-R1Bond stereochemistry and R22Is defined as follows:
compound # Double bond D-R1Bond stereochemistry R22：
101 12, 13-trans 1R, D are the same as amide Phenyl radical
102 Is free of 1R, D are the same as the acid Phenyl radical
And 103 Is free of 1R, D are the same as amide Phenyl radical
46. A compound of the formula:
it is contained in R1Wherein R is3、R4Said double bond position, D-R1Bond stereochemistry, R21And R22Are all defined as follows:
compound # R3： R4： Double bond D-R1Bond stereochemistry R21： R22：
202 NH-Boc H 11, 12-trans 1R or 1S, D is the same as the acid H H；
203 NH-acetyl H 11, 12-trans 1R or 1S, D is the same as the acid H H；
205 NH-Boc 11-OH12-OH cis Is free of 1R or 1S, D is the same as the acid H H；
206 NH-Boc H 13, 14-cis form 1R, D are the same as the acid H H；
207 NH-Boc H 13, 14-cis form 1R, D are the same as the acid OMe H；
208 NH-Boc H 13, 14-cis form 1R, D are the same as the acid OMe Phenyl radical
209 NH-C(O)-NH-tBu H 13, 14-cis form 1R, D are the same as the acid OMe Phenyl radical
210 NH-Boc H 13, 14-cis form 1S, D is the same as the acid OMe Phenyl radical
211 NH2 H 13, 14-cis form 1R, D are the same as the acid OMe Phenyl radical
213 OH (one isomer) H 13, 14-cis form 1R, D are the same as the acid OMe H；
214 NH-Boc 10-oxo radical 13, 14-cis form 1R, D are the same as the acid OMe Phenyl radical
47. A compound of the formula:
it is contained in R1The single stereoisomer of (1), wherein R3、D、D-R1Bond stereochemistry, R21And R22Is defined as follows:
48. a compound of the formula:
wherein, the D-R1The bond is as the same as the acid, R4、X9And the 11, 12 double bonds are defined as follows:
49. a compound of the formula:
wherein, the D-R1The bond being on the same side as the acid, X10、X11And X12Is defined as follows:
compound # X10； X11； X12：
501 CH2 O CH2
502 CH2 CH2 CH2
503 CH2 CH2 NH
504 CH2 CH2 N(Me)
505 CH2 CH2 N(CO)Me
506 CH2 CH2 N(CO)Ph
507 NH CH2 CH2
And 508 N(CO)Me CH2 CH2
50. A compound of the formula:
wherein said D-R1The bond is as the same as the acid, R21And R22Is defined as follows:
51. a compound of the formula:
wherein said D-R1The bond being ipsilateral to the acid, R4、X9、X10、X11The 13, 14 double bond and R22Is defined as follows:
52. a compound of the formula:
wherein said D-R1The bond being on the same side as the acid, the 13, 14 double bond being cis, R32、R4And R22Is defined as follows:
53. a compound of the formula:
wherein said D-R1Bond on the same side as acid, R32、R4And R22Is defined as follows:
54. a method of inhibiting the replication of hepatitis C virus by exposing the virus to an inhibitory amount of a compound of formula I of item 1 against hepatitis C virus NS3 protease.
55. A pharmaceutical composition comprising a compound of formula I having an anti-hepatitis C virally effective amount of item 1, or a therapeutically acceptable salt or ester thereof, in admixture with a pharmaceutically acceptable carrier medium or adjuvant.
56. The pharmaceutical composition according to item 55, further comprising an additional immunomodulator.
57. The pharmaceutical composition according to item 56, wherein the additional immunomodulator is selected from alpha-, beta-and delta-interferon.
58. The pharmaceutical composition according to item 55, further comprising an antiviral agent.
59. The pharmaceutical composition according to item 58, wherein the antiviral agent is selected from the group consisting of: ribavirin and amantadine.
60. The pharmaceutical composition according to item 55, further comprising an additional HCV protease inhibitor.
61. The pharmaceutical composition according to item 55, further comprising other inhibitors of interest in the HCV life cycle, such as helicases, polymerases, or metalloproteinases.
62. The pharmaceutical composition according to item 55, for use in the manufacture of a medicament for treating a hepatitis C viral infection in a mammal. 63. The pharmaceutical composition according to item 61, wherein the inhibitor is selected from the group consisting of helicases, polymerases, and metalloproteinases.
64. The pharmaceutical composition of clause 57, wherein the immunomodulator is alpha interferon.
65. The pharmaceutical composition according to item 57, further comprising an antiviral agent.
66. The pharmaceutical composition according to item 65, wherein the antiviral agent is ribavirin.
67. The pharmaceutical composition according to item 64, further comprising an antiviral agent.
68. The pharmaceutical composition according to item 67, wherein the antiviral agent is ribavirin.
69. Use of the pharmaceutical composition according to item 57 for the preparation of a medicament for the treatment of hepatitis C virus infection in a mammal.
70. The use of the pharmaceutical composition according to item 59 for the preparation of a medicament for the treatment of hepatitis C viral infection in a mammal.
71. Use of the pharmaceutical composition according to item 60 for the preparation of a medicament for the treatment of hepatitis C virus infection in a mammal.
72. Use of the pharmaceutical composition according to item 61 for the preparation of a medicament for treating hepatitis C virus infection in a mammal.
73. Use of the pharmaceutical composition according to item 64 for the preparation of a medicament for the treatment of hepatitis C virus infection in a mammal.
74. The use of the pharmaceutical composition according to item 65 in the preparation of a medicament for the treatment of hepatitis C virus infection in a mammal.
75. The use of the pharmaceutical composition according to item 66 for the preparation of a medicament for the treatment of hepatitis C viral infection in a mammal.
76. Use of the pharmaceutical composition according to item 67 in the preparation of a medicament for treating a hepatitis C viral infection in a mammal.
77. The use of the pharmaceutical composition according to item 68 for the preparation of a medicament for the treatment of hepatitis C virus infection in a mammal.
78. A compound according to item 43, wherein R3is-NH-C (O) -OR32Wherein R is32Is C1-4Alkyl or C4-6A cycloalkyl group; d is a C7 all-carbon chain saturated or containing a cis-double bond in the 13, 14 position, R22Is that
Wherein R is24aIs NH- (C)1-4Alkyl groups); NH- (C)3-6Cycloalkyl groups); NH-C (O) - (C)1-4Alkyl groups); NH-C (O) -O- (C)1-4Alkyl groups); or NH-C (O) -NH- (C)1-4Alkyl groups).
79. Compound 208 according to item 46.
80. Compound 209 according to item 46.
81. Compound 214 according to item 46.
82. Compound 217 according to item 46.
83. Compound 408 according to item 48.
84. A compound 508 according to item 49.
85. Compound 601 according to item 50.
86. Compound 603 according to item 50.
87. Compound 702 according to item 51.
88. Compound 703 according to item 51.
89. Compound 709 according to item 51.
90. Compound 714 according to item 51.
91. Compound 715 according to item 51.
92. Compound 719 according to item 51.
93. Compound 725 according to item 51.
94. Compound 736 according to item 51.
95. Compound 738 according to item 51.
96. Compound 801 according to item 52.
97. Compound 809 according to item 52.
98. Compound 810 according to item 52.
99. Compound 811 according to item 52.
100. Compound 812 according to item 52.
101. Compound 814 according to item 52.
102. Compound 818 according to item 52.
103. Compound 819 according to item 52.
104. Compound 821 according to item 52.
105. Compound 822 according to item 52.
106. Compound 823 according to item 52.
107. A compound 904 according to item 53.
108. Compound 909 according to item 53.
109. Compound 914 according to item 53.
110. Compound 916 according to item 53.
111. A pharmaceutical composition comprising an anti-hepatitis c virus effective amount of a compound of formula 1 as defined in any one of claims 79 to 110 or a pharmaceutically acceptable salt or ester thereof in admixture with a pharmaceutically acceptable carrier medium or adjuvant.
112. The pharmaceutical composition of item 111, further comprising an additional immunomodulator.
113. The pharmaceutical composition of item 112, wherein the additional immunomodulator comprises alpha-, beta-, and delta-interferon.
114. The pharmaceutical composition of item 113, wherein the additional immune modulator is an interferon-alpha.
115. The pharmaceutical composition of item 111, further comprising an antiviral agent.
116. The pharmaceutical composition according to item 115, wherein the antiviral agent is ribavirin.
117. The pharmaceutical composition according to item 113, further comprising an antiviral agent.
118. The pharmaceutical composition according to item 117, wherein the antiviral agent is ribavirin.
119. The pharmaceutical composition of item 114, further comprising an antiviral agent.
120. The pharmaceutical composition according to item 119, wherein the antiviral agent is ribavirin.
121. The pharmaceutical composition according to item 111, further comprising another inhibitor of HCV protease.
122. The pharmaceutical composition according to item 111, further comprising an inhibitor of another object in the HCV life cycle.
123. The pharmaceutical composition of item 122, wherein the inhibitor is selected from the group consisting of helicases, polymerases, and metalloproteinases.
124. Use of the pharmaceutical composition according to item 111 for the preparation of a medicament for treating hepatitis c virus infection in a mammal.
125. The use of the pharmaceutical composition according to item 114 in the preparation of a medicament for the treatment of hepatitis C virus infection in a mammal.
126. The use of the pharmaceutical composition according to item 116 in the preparation of a medicament for treating a hepatitis C viral infection in a mammal.
127. Use of the pharmaceutical composition according to item 120 for the preparation of a medicament for treating hepatitis c virus infection in a mammal.
128. A compound having the following formula (A):
wherein X is PG or R2；
Each PG is a protecting group, R2As defined in item 1;
a is a protected carboxylic acid;
n is 2.
129. A process for the preparation of a compound of formula (a) according to item 128, which process comprises reacting a compound of formula (B) with a compound of formula (C):
wherein PG, X, A', and n are as defined in item 128.
130. A compound having the following formula (D):
wherein X is PG or R2；
PG is a protecting group which is a protecting group,
R2as defined in item 1;
a' is a protected carboxylic acid;
n is 2.
131. A process for the preparation of a compound of formula (D) according to item 130, which process comprises cleaving the protecting group PG on the pyrrolidone in the compound of formula (a);
wherein X is PG or R3；
Each PG is a protecting group, respectively,
R2and a' and n are as defined in item 130.
132. A compound having the following formula (E):
wherein X is PG or R2；
PG is a protecting group which is a protecting group,
R2as defined in item 1;
a' is a protected carboxylic acid;
R3as defined in item 1;
n is 0 or 2, and
(1) when N is 0, D' is a 5-atom saturated alkylene chain, optionally containing 1 to 3 groups independently selected from O, S or N-R41A heteroatom of (a);
(2) when N is 2, D' is a 3-atom saturated alkylene chain, optionally containing 1 to 3 groups independently selected from O, S or N-R41A heteroatom of (a);
and R is41As defined in item 1.
133. A process for preparing a compound of formula (E) according to item 132, comprising reacting a compound of formula (D) with a compound of formula (F):
wherein X, A', n, R3And D' are both as defined in item 132.
134. A compound having the following formula (G):
wherein R is2And R3As defined in item 1;
a' is a protected carboxylic acid;
n is 0 or 2, and
(1) when N is 0, D' is a 5-atom saturated alkylene chain, optionally containing 1 to 3 groups independently selected from O, S or N-R41A heteroatom of (1);
(2) when N is 2, D' is a 3-atom saturated alkylene chain, optionally containing 1 to 3 groups independently selected from O, S or N-R41A heteroatom of (1);
and R41As defined in item 1.
135. A process for preparing a compound of formula (G) according to item 134, which comprises causing ring closure of the compound of formula (E) by reacting the compound of formula (E) in the presence of a transition metal-based catalyst to produce a compound of formula (G'):
wherein X is PG or R2；
PG is a protecting group;
and R is2、R3D 'and a' are as defined in item 134;
and when X is PG, the compound of formula (G') is deprotected and then reacted with R3-reacting an OH compound to produce a compound of formula (G):
wherein R is3R3, D ', n and a' are as defined in item 134.
136. A compound having the following formula (H):
wherein R is2And R3As defined in item 1;
a' is a protected carboxylic acid;
n is 0 or 2; and also
(1) When N is 0, D' is a 5-atom saturated alkylene chain, optionally containing 1 to 3 groups independently selected from O, S or N-R41A heteroatom of (1);
(2) when N is 2, D' is a 3-atom saturated alkylene chain, optionally containing 1 to 3 groups independently selected from O, S or N-R41A heteroatom of (a);
R41as defined in item 1.
137. A process for preparing a compound of formula (H) according to item 136, comprising subjecting a compound of formula (G) to hydrogenation:
wherein R is2、R3D ', n and a' are as defined in item 136.
138. A compound having the following formula (J):
wherein R is2And R3As defined in item 1;
m is 1 to 5;
n is 1 to 5;
a is a protected carboxylic acid;
cbz is benzyloxycarbonyl.
139. A compound having the following formula (K):
wherein R is2And R3As defined in item 1;
m is 1 to 5;
n is 1 to 5;
a' is a protected carboxylic acid;
cbz is benzyloxycarbonyl.
140. A process for the preparation of a compound of formula (K) according to item 139, which process comprises subjecting a compound of formula (J) to hydroboration:
wherein R is2、R3M, n, A' and Cbz are as defined in item 139.
141. A compound having the following formula (L):
wherein R is2And R3As defined in item 1;
m is 1 to 5;
n is 1 to 5;
a' is a protected carboxylic acid.
142. A process for the preparation of a compound of formula (L) according to item 141, which process comprises subjecting a compound of formula (K) to hydrogenation in the presence of an acid:
wherein R is2And R3M, n and A' are as defined in item 141.
And Cbz is benzyloxycarbonyl.
Pharmaceutical compositions comprising a compound of formula I, or a therapeutically acceptable salt or ester thereof, in an amount effective against hepatitis C virus, in admixture with a pharmaceutically acceptable carrier medium or adjuvant, are included within the scope of the present invention.
An important aspect of the present invention is directed to a method of treating hepatitis C infection in a mammal by administering to the mammal an amount of a compound of formula I, or a therapeutically acceptable salt or ester thereof, or a composition thereof, effective against hepatitis C virus.
In another important aspect, there is provided a method of inhibiting replication of the hepatitis C virus by exposing the virus to a compound of formula I, or a therapeutically acceptable salt or ester thereof, in an amount that inhibits the hepatitis C NS3 protease, such as a composition described above.
Other aspects are directed to methods of treating a hepatitis C virus infection in a mammal by administering thereto an amount of a compound of formula I, or a therapeutically acceptable salt or ester thereof, effective against the hepatitis C virus. According to a particular embodiment, the pharmaceutical composition of the invention comprises an additional immunomodulator. Examples of additional immunomodulators include, but are not limited to, alpha-, beta-, and delta-interferons.
According to another embodiment, the pharmaceutical composition of the invention may also additionally comprise an antiviral agent. Examples of antiviral agents include ribavirin and amantadine. According to another embodiment, the pharmaceutical composition of the present invention may additionally comprise other HCV protease inhibitors. According to another embodiment, the pharmaceutical composition of the invention may additionally comprise inhibitors of other targets in the HCV life cycle, such as helicases, polymerases, metalloproteinases or IRES.
Detailed description of the preferred embodiments
Definition of：
As used herein, the following definitions apply, unless otherwise noted:
with respect to examples, where (R) or (S) is used to indicate the absolute configuration of a substituent, e.g., R of a compound of formula I4The context of the entire compound is not intended to be the context of a single substituent.
When identified herein using "P1, P2, P3, etc," it is intended to refer to the position of the amino acid residue starting from the C-terminus and extending toward the N-terminus of the peptide analog (that is, P1 represents the 1 st position from the C-terminus, P2: the 2 nd position from the C-terminus, etc.) (see Berger A.& Schechter I.，Transactions of the Royal Society London series B257，249-264(1970))。
When the term "1-aminocyclopropyl-carboxylic acid" (ACCA) is used herein, it is intended to refer to the compound of the formula:
when the term "vinyl-ACCA" is used herein, it is intended to refer to the formula:
the compound of (1).
When the term "homo-allyl-ACCA" is used herein, it is intended to refer to the formula:
the compound of (1).
When the term "halogen" is used herein, it is intended to mean a halogen substituent selected from bromine, chlorine, fluorine or iodine.
"C" as used herein1-6The term "haloalkyl", as used herein, alone or in combination with other substituents, meansRefers to an acyclic, straight or branched chain alkyl substituent containing 1 to 6 carbon atoms with one or more halogen-substituted hydrogens selected from bromine, chlorine, fluorine or iodine.
"C" as used herein1-6The term "thioalkyl" as used herein alone or in combination with other substituents, means an acyclic, straight chain or branched alkyl substituent containing a thiol group, such as, for example, thiopropyl.
"C" as used herein1-6The term "alkyl" or "(lower) alkyl", used alone or in combination with other substituents, means an acyclic, straight or branched chain alkyl substituent containing from 1 to 6 carbon atoms and including, for example, methyl, ethyl, propyl, butyl, hexyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl, 1-dimethylethyl.
"C" as used herein3-6The term "cycloalkyl", alone or in combination with other substituents, means a cycloalkyl substituent containing from 3 to 6 carbon atoms and includes cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
The term "unsaturated cycloalkyl" includes, for example, substituted cyclohexenyl:
as used herein, the term "saturated or unsaturated alkylene" refers to a divalent alkyl substituent derived by removing one hydrogen atom from each end of a saturated or unsaturated straight or branched chain aliphatic hydrocarbon and includes, for example, -CH2CH2C(CH3)2CH2CH2-、-CH2CH2CH＝CHCH2CH2-or-CH2C＝CCH2CH2-. The alkyl chain optionally containing heteroatoms, such as oxygen (e.g. CH)3-CH2-O-CH2-)。
"C" as used herein1-6The term "alkoxy", alone or in combination with other substituents, means the substituent-O-C1-6Alkyl, wherein the alkyl is as defined above, containing up to 6 carbon atoms. Alkoxy groups include methoxy, ethoxy, propoxy, 1-methoxyethoxy, butoxy, and 1, 1-dimethylethoxy. The latter substitution is commonly referred to as t-butoxy.
"C" as used herein3-6The term "cycloalkoxy", used alone or in admixture with other substituents, means a substituent, -O-C, containing from 3 to 6 carbon atoms3-6A cycloalkyl group.
"C" as used herein1-6The term "alkoxyalkyl" means the substituent C1-6alkyl-O-C1-6Alkyl, wherein the alkyl is as defined above, containing up to 6 carbon atoms. Such as methoxymethyl means-CH2-O-CH3。
"C" as used herein2-7The term "acyl", alone or in admixture with other substituents, means C attached through a carbonyl group1-6Alkyl radicals, e.g. C (O) -C1-6An alkyl group.
"C" as used herein6Or C10The term "aryl", alone or in combination with other substituents, means an aromatic monocyclic ring system containing 6 carbon atoms, or an aromatic bicyclic ring system containing 10 carbon atoms. For example, aryl includes phenyl or naphthyl-ring systems.
"C" as used herein7-16The term "aralkyl", alone or in combination with other substituents, means an aryl group as defined above attached through an alkyl group, wherein the alkyl group is as defined above, containing from 1 to 6 carbon atoms. Aralkyl groups include, for example, benzyl and butylphenyl.
The term "Het", as used herein, alone or in combination with other substituents, means a monovalent substituent derived by removal of a hydrogen from a five-, six-or seven-membered saturated or unsaturated (including aromatic) heterocyclic ring containing one to four heteroatoms selected from nitrogen, oxygen and sulfur. Examples of suitable heterocycles include: tetrahydrofuran, thiophene, diazepine, isoxazole, piperidine, dioxane, morpholine, pyrimidine or
The term "Het" also includes heterocycles as defined above fused to one or more other rings which may be heterocyclic or any other ring. One such example includes thiazolo [4, 5-b ] -pyridine.
Although generally included under the term "Het", as used herein, the term "heteroaryl" is precisely defined as an unsaturated heterocyclic ring, the double bond of which forms an aromatic system. Examples of suitable heteroaromatic systems include: quinoline, indole, pyridine,
(ii) a Or
The term "pharmaceutically acceptable ester" as used herein, alone or in combination with other substituents, means an ester of a compound of formula I wherein any carboxyl function, preferably the carboxyl terminus, of the molecule is replaced by an alkoxycarbonyl function:
wherein the R portion of the ester is selected from alkyl (e.g., methyl, ethyl, n-propyl, t-butyl, n-butyl); alkoxyalkyl (e.g., methoxymethyl); alkoxyacyl (e.g., acetoxymethyl); aralkyl (e.g., benzyl);aryloxyalkyl (e.g., phenoxymethyl); aryl (e.g. phenyl), optionally with halogen, C1-4Alkyl or C1-4Alkoxy substitution. Other suitable prodrug esters are found in Design of products, Bundgaard, h.ed.elsevier (1985), which is incorporated herein by reference. Such pharmaceutically acceptable esters are generally hydrolyzed in vivo upon injection into a mammal and converted to the acid form of the compound of formula I.
With respect to the esters described above, unless otherwise specified, any alkyl moiety present advantageously contains from 1 to 6 carbon atoms, particularly from 1 to 6 carbon atoms. Any aryl moiety present in such esters advantageously includes a phenyl group.
In particular, the ester may be C1-16Alkyl esters, unsubstituted benzyl esters, or with at least one halogen, C1-6Alkyl radical, C1-6Alkoxy-, nitro-or trifluoromethyl-substituted benzyl esters.
The term "pharmaceutically acceptable salts" as used herein includes those derived from pharmaceutically acceptable bases. Examples of suitable bases include choline, ethanolamine, and ethylenediamine. Also contemplated is the addition of Na+、K+And Ca++Salts are included within the scope of the present invention (see also Pharmaceutical salts, large, s.m. et al, j.pharm.sci. (1977), 66, 1-19, incorporated herein by reference).
Preferred embodiments
R 1: a preferred embodiment of the present invention comprises compounds of formula I as described above, wherein R is1The moiety is selected from 2 different diastereomers, wherein the 1-carbon center having the R configuration has the configurations represented by structures (i) and (ii):
d is the same side as the amide (i) or D is the same side as A (ii).
More preferably, R is attached in the ipsilateral configuration to A as represented by structure (ii)1The linking group D of (1).R 2：
A preferred embodiment of the present invention comprises compounds of formula I as described above, wherein R is2The part is as follows:
wherein W is preferably N.
Preferably, R is21Is H, C1-6Alkyl radical, C1-6Alkoxy, hydroxy, chloro or N (R)23)2Wherein R is23Preferably H or C1-6An alkyl group. More preferably, R21Is H or C1-6An alkoxy group. Most preferably R21Is methoxy.
Preferably, R is22Is H, C1-6Sulfanyl, C1-6Alkoxy, phenyl or Het selected from the group consisting of:
and
het of (1).
Preferably, R is24Is H, C1-6Alkyl, NH-R25、NH-C(O)-R25Or NH-C (O) -NH-R25OR NH-C (O) -OR26。
More preferably, R22Is C1-4Alkoxy, phenyl, or Het selected from:
and
more preferably, R24Is H, C1-6Alkyl, NH-R25、NH-C(O)-R25OR NH-C (O) -OR26。
Most preferably, R22Is an ethoxy group or a Het selected from the group consisting of,
and
most preferably, R24aIs NH-R25、NH-C(O)-R25OR NH-C (O) -OR26. Most preferably, R24bIs H or C1-6An alkyl group.
Preferably each R25Are respectively H, C1-6Alkyl or C3-6A cycloalkyl group. More preferably, R25Is C1-6Alkyl or C3-6A cycloalkyl group. More preferably R25Is C1-6An alkyl group. Preferably, R is26Is C1-6An alkyl group.R 3：
A preferred embodiment of the present invention comprises compounds of formula I as described above, wherein R is3Part is preferably of the formula NH-C (O) -R32Amides of the formula NH-C (O) -NH-R32OR of the formula NH-C (O) -OR32The carbamate of (1). More preferably R3Is carbamate or urea. Most preferably R3Is a carbamate. It is preferred that R is32Is C1-6Alkyl or C3-6A cycloalkyl group. More preferably R32Is C1-6Alkyl or C4-6A cycloalkyl group. Most preferably R32Is tert-butyl, cyclobutyl or cyclopentyl.D：
Preferred embodiments of the present invention include compounds of formula I wherein the crosslinking agent D is a saturated or unsaturated alkylene chain of 6 to 8 atoms. More preferably, the linking group D is a 7 atom chain.
More preferably, the D chain contains one or two members selected from: o, S, NH, N-C1-6Alkyl or N-C2-7A heteroatom of an acyl group. More preferably, the D chain optionally contains a member selected from the group consisting of: NH or N-C2-7The heteroatom of the acyl group, most preferably N (Ac), is located at atom 10 of the chain. Preferably, the chain contains saturated nitrogen atoms.
In addition, D contains a heteroatom selected from O or S. Preferably, when D is 7 atoms in length, the O or S atom is located at position 9 of the chain. More preferably R4In which R is substituted for the chain4Is H or C1-6An alkyl group. More preferably, R4Is H or methyl. Most preferably R4Is H or 8- (S) -Me. Even more preferably, D is saturated. Alternatively, D contains a double bond at positions 11, 12. Preferably, the double bond is trans.
In addition, D is an alkylene chain which is saturated or unsaturated with all carbons. In this case, D is preferably saturated and 7 atoms in length. Preferably, R is4Substituted D, wherein R4Is H, oxo, thioxo, hydroxy, sulfanyl, alkoxy, or alkyl. More preferably, R is4Is H or C1-6An alkyl group. Most preferably, R4Is H or methyl. R4H or 10- (S) -Me is particularly preferred.
In addition, D is all the alkylene chains containing one double bond and is 7 atoms in length. More preferably the double bond is located at positions 13, 14 of the chain. Most preferably, the double bond is cis. Preferably, with R4Substituted D chain, wherein R4Is H, oxo, hydroxy, alkoxy or alkyl.More preferably, R4Is H or C1-6An alkyl group. More preferably, R4Is H or methyl. Most preferably, R4Is H or 10- (S) -Me.A：
Preferred embodiments of the present invention include compounds of formula I as described above, wherein A is a carboxylic acid.
The specific scheme is as follows:
a preferred embodiment of the present invention comprises compounds of formula I as described above, wherein R is2Is a quinoline substituent (i.e., W is N);
R3is of the formula-NH-C (O) -NHR32OR-NH-C (O) -OR32Wherein R is32Is C1-4Alkyl or C4-6A cycloalkyl group;
d is a saturated or unsaturated alkylene chain of 6 to 8 atoms, in the same configuration as A with R1The connection optionally contains one or two selected from O, S or N-R41Wherein R is41Is C2-7An acyl group;
R4is H or one to three are independently selected from hydroxy or C1-6A substituent of an alkyl group; and is
A is a carboxylic acid or a pharmaceutically acceptable salt or ester thereof.
More preferably wherein R is1As defined above; r21Is H or methoxy;
R22is C1-6Alkoxy, or Het selected from the group consisting of;
and
wherein R is24aIs H, C1-6Alkyl, NH-R25、NH-C(O)-R25、NH-C(O)-NH-R25，
Wherein R is25Is H, C1-6Alkyl or C3-6A cycloalkyl group;
or R24aIs NH-C (O) -OR26Wherein R is26Is C1-6Alkyl or C3-6A cycloalkyl group;
and R is24bIs H or C1-6An alkyl group;
R3is of the formula NH-C (O) -NHR32OR of the formula NH-C (O) -OR32Of (a) a carbamate, wherein R32Is C1-6Alkyl or C3-6A cycloalkyl group;
d is a saturated or unsaturated alkylene chain of C7-atom, optionally containing a double bond in position 11, 12 or 13, 14;
the D chain optionally containing a heteroatom selected from O, S, NH, N (Me) or N (Ac); and R is4Is H or C1-6An alkyl group.
Most preferably, wherein R is21Is methoxy, and R22Is an ethoxy group or:
wherein R is24aIs NH- (C)1-4Alkyl), NH-C (O) - (C)1-4Alkyl) or NH-C (O) -O- (C)1-4Alkyl groups);
and D is saturated or contains a cis double bond at positions 13, 14.
Finally, all compounds of formula I provided in tables 1 to 9 are included within the scope of the present invention.
The pharmaceutical compositions of the present invention may be administered orally, parenterally or via an implanted reservoir. Oral administration or administration by injection is preferred. The pharmaceutical compositions of the present invention may contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle. In some cases, the pH of the formulation may be adjusted using pharmaceutically acceptable acids, bases, or buffer solutions in order to facilitate stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal and intralesional injection or infusion techniques.
The pharmaceutical compositions may be in the form of sterile injectable preparations, for example sterile injectable aqueous or oleaginous suspensions. The suspension may be formulated according to the known art using suitable dispersing or wetting agents, such as tween 80, and suspending agents.
The pharmaceutical compositions of the present invention may be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dehydrated corn starch. When administered orally as an aqueous suspension, the active ingredient is combined with emulsifying and suspending agents. If desired, sweeteners and/or flavors and/or coloring agents may be added.
Other suitable excipients or carriers that may be used with the formulations and compositions mentioned above may be found in standard pharmacological texts, for example in "Remington's Pharmaceutical Sciences", 19 th edition, Mack Publishing Company, Easton, penn, 1995. For the prevention and treatment of diseases caused by HCV, the protease inhibitor compounds described herein are useful in monotherapy in a dosage range of between about 0.01 and about 100 mg/kg of body weight per day, preferably between about 0.5 and about 75 mg/kg of body weight per day. Typically, the pharmaceutical compositions of the present invention will be administered from about 1 to about 5 times per day, or alternatively as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form may vary depending upon the host treated and the particular mode of administration. A representative formulation will contain from about 5% to about 95% active ingredient (weight/weight). Preferably, such formulations contain from about 20% to about 80% of the active compound.
Those skilled in the art will appreciate that lower or higher doses than those mentioned above may be required. The particular dosage and mode of treatment for any particular patient will depend upon a variety of factors including the activity of the particular compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, severity and course of infection, the patient's propensity to develop infection, and the judgment of the treating physician. Generally, treatment is initiated at a small dose that is substantially lower than the optimal dose of the peptide. The dose is then increased by a small increment until the optimum effect is reached in this case. In general, it is desirable to administer the compounds at concentrations that are generally sufficient to produce effective antiviral results, but do not cause any harmful or adverse side effects.
When the compositions of the present invention comprise a compound of formula I in combination with one or more additional therapeutic or prophylactic agents, the compound and additional agent(s) should be present in amounts that provide a dosage level of between about 10 and 100%, more preferably about 10 to 80%, typically in a single therapeutic administration.
When these compounds, or pharmaceutically acceptable salts thereof, are formulated with a pharmaceutically acceptable carrier, the resulting compositions can be administered in vivo to a mammal, such as a human, in order to inhibit the HCV NS3 protease, or to treat or prevent HCV viral infections. Such treatment may also be accomplished using the compounds of the present invention in combination with the following agents, including but not limited to: immunomodulators, such as alpha-, beta-or delta-interferons; other antiviral agents, such as ribavirin, amantadine; other HCV NS3 protease inhibitors; for other target inhibitors in the HCV life cycle, such as helicases, polymerases, metalloproteinases, or Internal Ribosome Entry Sites (IRES); or a combination thereof. Additional formulations may be mixed with the compounds of the present invention to produce a single dosage form. Alternatively, such additional formulations can be administered to the mammal separately as part of a multiple dosage form. Accordingly, other embodiments of the present invention provide a method of inhibiting HCV NS3 protease activity in a mammal by administering a compound of formula I, wherein the substituents are as defined above.
In preferred embodiments, these methods are useful for reducing HCV NS3 protease activity in a mammal. If the pharmaceutical composition comprises only a compound of the invention as an active ingredient, such methods may additionally comprise the step of administering to the mammal an inhibitor selected from an immunomodulator, antiviral, HCV protease inhibitor, or an agent which is an agent in the HCV life cycle, such as a helicase, polymerase, metalloprotease. Such additional formulations can be administered to the mammal prior to, concurrently with, or subsequent to the administration of the compositions of the present invention.
In another preferred embodiment, the methods are useful for inhibiting viral replication in a mammal. Such methods are useful for treating or preventing HCV diseases. If the pharmaceutical composition includes only a compound of the present invention as an active ingredient, such methods may additionally comprise the step of administering to the mammal an agent selected from an immunomodulator, antiviral, HCV protease inhibitor, or other targeted inhibitor in the HCV life cycle. Such additional formulations can be administered to the mammal prior to, concurrently with, or subsequent to the administration of the compositions of the present invention.
The compounds described above are also useful as laboratory reagents. Applicants have for the first time provided compounds having low molecular weight, which have high activity and specificity for the HCV NS3 protease. Some of the compounds of the present invention are useful for providing research tools for structural biology research for designing viral replication assays, approval of animal assay systems, and further advance knowledge of HCV disease mechanisms.
The compounds of the present invention may also be used to treat or prevent viral contaminated materials and thereby reduce the risk of viral infection in laboratories or medical personnel, or patients in contact with such materials (e.g., blood, tissue, surgical instruments and clothing, laboratory equipment and clothing, and blood collection or transfusion devices and materials). Methodology of
Several ways are disclosed in WO00/09543 and WO 00/09558 to accomplish the synthesis of acyclic intermediates of the compounds of formula I.
Compounds of the present invention (where PG is an appropriate protecting group) are synthesized according to the general procedure outlined in schemes I, II and III. [ in all the schemes provided below, D' has the same definition as D, but is shorter, 2 to 5 atoms ].
When the present invention comprises a compound of formula I wherein A is an N-substituted amide, vinyl-ACCA or homo-allyl ACCA (R)1) Prior to coupling to P2, coupling to the appropriate amine was performed. Those skilled in the art will appreciate such coupling. The skilled person will recognize that such amides (A) are not protected, but carry any relevant substituents R as defined above5。
The ring closure reaction (macrocyclization) is accomplished by either alkene metathesis (scheme I), or, when nitrogen atoms are present in the linker, by reductive amination (scheme II), or by peptide bond formation (scheme III).
Details of these methods are provided below:
A. macrocyclization by ene displacement
Scheme I
D is saturated D is unsaturated
Scheme I:
one of skill in the art can readily recognize several methods by which coupling sequences can be accomplished. Starting from 4- (S) -hydroxyproline, substituents may be added at the 4-hydroxy group by a Mitsunobu reaction, either before or after macrocyclization (as described in Mitsunobu Synthesis 1981, January, 1-28; Rano et al Tet. Lett.1994, 36, 377-3792; Krchnak et al Tet. Lett.1995, 36, 6193-6196). In addition, combinations may also be made with the requisite 4- (R) -hydroxy-substituted proline as disclosed in the general procedures of WO00/09543 & WO 00/09558 (see below for specific examples of these fragments).
Step A, B, C: briefly, the P1, P2 and P3 moieties may be linked by well known peptide coupling techniques and as commonly disclosed in WO00/09543 & WO 00/09558. Step D: ru-based catalysts can be used by olefin metathesis, such as those available from Miller, s.j.; blackwell, h.e.; grubbs, r.h.j.am.chem.soc.1996, 118, 9606-9614 (a); kingsbury, j.s.; harrity, j.p.a.; boniatebus, p.j.; hoveyda, A.H.J.am.chem.Soc.1999, 121791-; stevens, e.d.; nolan, s.p.; petersen, j.l.; J.Am.chem.Soc.1999, 121, 2674-2678(c) to complete the formation of macrocycles. It will also be appreciated that the reaction may be carried out using catalysts containing other transition metals, such as Mo.
Grubbs ' Harveda (Horeyda's) Nolan (Nolan's)
Catalyst
Step E: if desired, the double bond is reduced by standard hydrogenation methods well known in the art. When A' is a protected carboxylic acid, it may also be suitably deprotected.
B. Macrocyclization by reductive amination (linker containing N)
When the linker contains a nitrogen atom, macrocyclization can be obtained by reductive amination as shown in scheme II to obtain the inhibitor of general structure II.
Scheme II
A ═ protected carboxylic acids or N-substituted amides
n is 1 to 5
m is 1 to 5
Step A: hydroboration of the double bond (H.C.Brown and B.C.SubbaO, J.Am.Che.Soc.1959, 81, 6434-.
Step B: hydrogenation in the presence of an acid results in the removal of the amino protecting group followed by macrocyclization by reductive amination. The P3 units used in this synthesis are readily available from a variety of amino acids, such as lysine, ornithine, glutamine (after Hofmann reaction: Ber.1881, 14, 2725) and others; such modifications of the synthesis are well known in the art.
Step C: the secondary amine (formed after step D) in linker D can be alkylated with an alkyl halide or acetylated with an alkyl or aryl acid chloride using methods well known in the art to obtain the inhibitor of general structure II, if desired. When A' is a protected carboxylic acid, it may also be suitably deprotected.
C. Macrocyclization by lactam formation
In addition, it is also understood that these macrocyclic compounds having the general structures I and II can be synthesized in other ways. For example, P1 and P3 can be attached to linker D prior to coupling with P2, and the macrocyclization reaction forms the lactam in two possible ways, as will be recognized by those skilled in the art and as shown in scheme III.
Scheme III
Synthesis of P1
The synthesis of the inhibitor with the general structures I and II requires the same fragment of P1:
a) vinyl ACCA, the synthesis and dissociation of which is described in WO00/09543 & WO 00/09558, or b) with allyl ACCA (example 1, compound 1 f).
Synthesis of P2
The synthesis of compounds of formula I using some P2 fragments is described in WO00/09543 & WO 00/09558.
Additional P2 fragments were synthesized as follows:
a. synthesis of 2- "Het" -4-hydroxy-7-methoxyquinoline derivatives
(i) From the corresponding "Het" carboxylic acid 1Vb
Procedure IV
The synthesis was performed according to the modified procedure in Li et al J.Med.chem.1994, 34, 3400-. Wherein R is prepared as described in Brown et al J.Med.chem.1989, 32, 807-82621Intermediate IVa of OMe (example 7, compound 7 b).
Step A: under alkaline conditions, using POCl3The carboxylate group is activated to couple intermediate IVa with heterocyclic carboxylic acid IVb. The inhibitor is prepared by using a mixture ofA carboxylic acid of general structure IVb; these are commercially available, synthesized as shown in schemes V, VI and VII, or individually synthesized using the methods described in the specific examples.
And B: ring closure followed by dehydration under basic conditions to obtain quinolines of general structure IVd.
(i.a.) Synthesis of "Het" -carboxylic acids of general formula IVb
Synthesis of 2- (substituted) -amino-4-carboxy-aminothiazole derivative (Vc)
A modified procedure described in berdiikhina et al chem. heterocyclic. comp. (engl. trans.) -1991, 4427-433 was used.
Procedure V
Using the general synthetic procedure outlined in scheme V, with different alkyl substituents (R)25Alkyl group) and 3-bromopyruvic acid to produce compounds of general structure Vc, various 2-alkylaminothiazolyl-4-carboxylic acids. This type of condensation reaction is well known in the art. In addition, as shown in scheme VI, according to: unangst, p.c.; the procedure of Connor, D.T.J.Heteromyces.chem.29, 5, 1992, 1097-1100, synthesized a P2 fragment containing a 2-amino-substituted-thiazole derivative from the corresponding 2-carboxy derivative.
Scheme VI
Examples of such processes are described in WO00/09543 & WO 00/09558.
Synthesis of 2-carboxy-4-substitutedVIId of aminothiazole derivatives
The general synthetic method outlined in scheme VII is used to make compounds of general structure VIId, various 4-alkylthiazolyl-2-carboxylic acids.
Scheme VII
The procedure described in janussz et al j.med.chem.1998, 41, 3515-: thioxooxamic acid (thioamate) ethyl ester (VIIa) and compounds having the general structure VIIb (R)24Alkyl group) to form a thiazolecarboxylic acid of the general structure VIId. This type of condensation reaction is well known in the art.
Synthesis of 2-carboxy- (substituted) -imidazole derivatives (VIIIb)
The general synthetic method outlined in scheme VIII was used to make compounds of general structure VIIIb, various 4-alkylimidazolyl-2-carboxylic acids.
Scheme VIII
The procedure described by Baird et al J.Amer.chem.Soc.1996, 118, 6141-: deprotonation of alkylimidazoles using strong acids (e.g. nBuLi) followed by CO2React to form carboxylic acid VIIIb. This type of condensation reaction is well known in the art.
b. Synthesis of 4-hydroxy-7-methoxy-2- (imidazolyl or pyrazolyl) quinoline
Using the procedure outlined in scheme IX, 4-hydroxy-7-R with an imidazole or pyrazole moiety at C2 was prepared21A quinolyl group.
Procedure IX
The key intermediates are described in detail in example 6 (where R is21Synthesis of 4-benzyloxy-2-chloro-7-methoxyquinoline IXa (compound 6e) by OMe).
Step A: at elevated temperatures, various imidazoles, alkyl-substituted imidazoles, pyrazoles, or alkyl-substituted pyrazoles may be used to replace the 2-chloro moiety in compound IXa to provide compounds of the general structure IXb.
And B: when the benzyl protecting group is removed from the 4-hydroxy moiety of quinoline by standard hydrogenation methods, quinoline derivatives of the general structure IXc are obtained.
Synthesis of P3
By olefin metathesis, various P3 fragments containing appropriate D linkages for extended macrocyclization were synthesized. P3 units containing a terminal olefin for metathesis are generally synthesized according to the general scheme (scheme X, XI & XII) described below.
Synthesis of type A Binders
The general synthesis for making the binder is entirely carbon based (no heteroatoms) (scheme X).
Procedure X
The synthesis was performed according to the procedure of Evans et al J.Am.chem.Soc.1990, 112, 4011-.
The starting carboxylic acids (Xa) are commercially available or can be prepared by literature procedures known to those skilled in the art.
Step A: carboxylic acid Xa was activated with pivaloyl chloride and then reacted with the anion of the chiral adjuvant 4(S) -4- (benzyl) -2-oxazolidinone of Evens' according to well known chemical methods (see: d.j. ager et al, aldrich Acta 1997, 30, 3-11, and references herein) to give the compound of general structure Xb.
Step B: stereoselective α -azidoations of chiral imide enolates using trileyl azide in the presence of a base such as KHMDS will result in the formation of compounds with the general structure Xb, which is also well known in the art (see: D.J. Ager et al Aldrich mica acta 1997, 30, 3-11, and references therein).
Step C: reduction of alpha-azides by SnCl2Catalysis, followed by amine protection, forms its t-butyl carbamate, yielding an intermediate with the general structure Xc. These reactions are also well known in the art.
Step D: finally, the chiral auxiliary is hydrolyzed under basic conditions, e.g. H2O2With LiOH, an amino acid type linker with the general structure Xe is generated.
Alternatively, the P3 portion of Xe having the same general structure can also be synthesized according to the procedure described in scheme XI, by M.J.Burk et al J.Am.chem.Soc.1998, 120, 657-. These compounds are in the methylene (-CH) group with the linker2-) number of units (m 1 to 5), and in R4The substitution of the alkyl group is changed but no heteroatom is contained.
Scheme XI
Step A: monoacid compound XIb was prepared from commercially available diethyl 2-acetamidomalonate by standard ester hydrolysis under basic conditions.
Step B: the knoevenagel type condensation between the aldehyde of general structure XIc and compound XIb in the presence of a base such as pyridine and acetic anhydride results in the formation of the enamide intermediate XId, which has Z stereochemistry around the newly formed double bond as shown.
Step C: regioselective and enantioselective catalytic hydrogenation of the enamide intermediate XId to the amino acid intermediate XIe was accomplished using Burk's method.
Step D: the P3 moiety of general structure XIf was obtained by adding a tert-butyl carbamate substituent prior to the acetate group, nitrogen-protecting the acetamide derivative XIe, and hydrolyzing the ethyl ester under standard basic conditions.
Synthesis of type B Binders
General structure of B-type bonding agent
X ═ O or S
R1H or CH3
R2H or CH3
This general synthesis is used to make binders containing oxygen or sulfur.
Scheme XII
R1H or CH3
R2H or CH3But not R1＝R2＝CH3
Step A: using allyl iodide in Ag2The alkylation of an appropriately protected amino acid, such as Boc- (L) -serine methyl ester, Boc- (L) -threonine methyl ester or Boc- (L) -allothreonine methyl ester, in the presence of O gives the methyl ester XIIb.
Step B: the methyl ester was hydrolyzed under standard alkaline conditions to produce ether-type binders of general structure XIIc (X ═ O).
Step C: the sulfur analogs are prepared from the same starting amino acid XIIa (as suitably protected previously) and their hydroxyl groups are converted to good leaving groups (e.g. tosylate intermediate XIId) using standard methods well known in the art.
Step D: subsequent replacement of the tosylate moiety with the anion of the thioacetate, by conversion of the chiral center at the β -carbon, results in the formation of the thioester intermediate XIIe.
Step E: the thioester moiety is hydrolyzed under mild basic conditions to yield the free thiol XIIf.
Step F: alkylation of the thiol moiety is readily carried out using allyl iodide under basic conditions.
Step G: finally, after hydrolysis of the methyl ester using standard procedures, the sulfide analogue XIIc (X ═ S) was obtained.
R3Synthesis of fragments
In WO00/09543, the synthesis of compounds wherein R is described3Is NH-RR31Examples of fragments of (a).
Examples
The invention is illustrated in more detail by the following non-limiting examples. Other specific synthetic or isolation methods can be found in WO00/09543 & WO 00/09558.
The temperature is provided in degrees celsius. Unless otherwise stated, solution percentages represent weight to volume and solution ratios represent volume to volume relationships. Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker 400MHz spectrometer; chemical shifts (δ) are reported in parts per million (ppm) and are referenced to the deuterated solvent internally. DMSO-d as its TFA salt unless otherwise specified6The NMR spectra of all final compounds (inhibitors) were recorded. According to the Still's flash chromatography technique (w.c. Still et al, j.org.chem., 1978, 43, 2923), on silica gel (SiO)2) The flash column chromatography was performed.
Abbreviations used in the examples include Bn: a benzyl group; boc: tert-butoxycarbonyl [ Me ]3COC(O)](ii) a BSA: bovine serum albumin; cbz: a benzyloxycarbonyl group; CHAPS: 3- [ (3-Cholamidopropyl) -dimethyl-amino (amonio)]-1-propane sulfonate; DBU: 1, 8-diazabicyclo [5.4.0 ]]Undec-7-ene; CH (CH)2Cl2CDM, with a linear chain; dichloromethane; DEAD; azodicarboxylic diethyl ester; the DIAD: diisopropyl azodicarboxylate; DIPEA: diisopropylethylamine; DMAP: dimethylaminopyridine; DCC: 1, 3-dicyclohexylcarbodiimide; DME: 1, 2-dimethoxyethane; DMF: dimethylformamide; DMSO, DMSO: dimethyl sulfoxide; DTT: dithiothreitol or threo-1, 4-dimercapto-2, 3-butanediol; DPPA: diphenylphosphoryl azide; EDTA ethylene diamine tetraacetic acid; et: an ethyl group; EtOH: ethanol; EtOAc: acetic acid ethyl ester; et (Et)2O: diethyl ether; ESMS: electrospray mass spectrometry: HATU: o- (7-azabenzotriazol-1-yl) -1, 1, 3, 3-tetramethylhexafluorophosphate; HPLC: high performance liquid chromatography; MS: mass spectrometry analysis; MALDI-TOF: matrix referenced Laser absorption ionization (matrix Laser dispersion ionization) effect-time of flight, FAB: fast atom impact; LAH: lithium aluminum hydride; me: a methyl group; MeOH: methanol; MES: (2- [ N-morpholine)]Ethane-sulfonic acid); NaHMDS: sodium bis (trimethylsilyl) amide; NMM: n-methylmorpholine; NMMO: n-methylmorpholine oxide; NMP: n-methylpyrrolidine; pr: propyl; succ: 3-carboxypropionyl; PNA: 4-nitrophenylamino or p-nitrophenylamino; TBAF: tetra-n-butylammonium fluoride; TBME: tert-butyl-methyl ether; tBuOK: potassium tert-butoxide; TBTU: 2- (1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyltetrafluoroborate; TCEP: tris (2-carboxyethyl) phosphine hydrochloride; TFA: a trifluoroacetate salt; THF: tetrahydrofuran; and (3) TIS: triisopropylsilane; TLC: thin layer chromatography; TMSE: trimethylsilylethyl; Tris/HCl: tris (hydroxymethyl) aminomethane hydrochloride.
Part P1
Example 1
Synthesis of tert-butyl- (1R, 2R)/(1S, 2S) -1-amino-2-homoallylcyclopropyl carboxylate (1 f):
A. 1, 2-dibromo-5-hexene (1b, 8.10 g, 33.46 mmol) and di-tert-butyl malonate (1a, 4.82 g, 22.30 mmol) were added successively to a suspension of benzyltriethylammonium chloride (5.08 g, 22.3 mmol) in 50% aqueous NaOH (50 ml). The mixture was stirred vigorously at room temperature for 16 hours, then with H2Diluting with CH2Cl2(3X 50 ml) was extracted. Further with H2O (2X 50 ml), brine/H2The organic layer was washed with O (2/1, 2X 50 mL) and MgSO4Dried and evaporated. The crude residue was purified by flash column chromatography on silica gel using 3 to 5% EtOAc in hexanes as eluent to afford 38% of compound 1c (2.48 g).
1H NMR(CDCl3，400MHz)：δ1.19(bd，J＝7.9Hz，2H)，1.25-1.33(m，1H)，1.46(s，9H)，1.48(s，9H)，1.47-1.60(m，1H)，1.75-1.82(m，1H)，2.14-2.22(m，2H)，4.93-5.50(m，2H)，4.96(dm，J＝10.2Hz，1H)，5.18(dm，J＝17.2Hz，1H)。
ES(+)MS m/z 297(M+H)-。
B. At 0 ℃ adding H2O (203 μ l, 11.27 mmol) was added to a suspension of potassium tert-butoxide (5.75 g, 51.25 mmol) in anhydrous diethyl ether (150 ml) and the reaction mixture was stirred at 0 ℃ for 10 min. An ether solution of compound 1c (2.48 g in 10 ml of diethyl ether, 10.25 mmol) was added and the mixture was stirred at room temperature for 5 hours. With ice-cold H2The mixture was diluted with O and extracted with diethyl ether (3 × 200 ml). The aqueous layer was acidified to ph3.5-4 with ice cold 10% aqueous citric acid and extracted again with EtOAc (3 × 200 ml). With H2The EtOAc layer was washed with O (2X 100mL) and brine (100mL) and MgSO4Drying and evaporation gave compound 1d in 85% yield based on the content of starting material recovered.
1H NMR(CDCl3，400MHz)：δ1.51(s，9H)，1.64-1.68(m，1H)，1.68-1.75(m，1H)，1.77-1.88(m，1H)，1.96-2.01(m，1H)，2.03-2.22(m，3H)，5.01(dm，J＝6.4Hz，1H)，5.03(dm，J＝14.9Hz，1H)，5.72-5.83(m，1H)。
ES(+)MS m/z 241(M+H)-。
C. Adding Et3N (800 μ l, 5.68 mmol) was added to a solution of the acid 1d in dry benzene (1.14 g in 25 ml benzene, 4.74 mmol), followed by diphenylphosphoryl azide (1.13 ml, 5.21 mmol) and the mixture was heated to reflux for 3.5 h. Next, trimethylsilylethanol (1.36 ml, 9.48 mmol) was added and stirring was continued under reflux for another 4 hours. The mixture was cooled to room temperature, evaporated to half its original volume, diluted with diethyl ether (30 ml) and with 5% NaHCO3Aqueous (2X 30 ml), brine (50 ml) and MgSO 24Dried and evaporated. The residual oil was separated on the silica gel layer using 10% EtOAc in hexanes as eluent to give compound 1e (1.49 g) in 88% pure yield.
1H NMR(CDCl3，400MHz)：δ0.03(s，9H)，0.91-0.99(m，2H)，1.18-1.29(m，2H)，1.45(bs，11H)，1.56-1.72(m，2H)，2.02-2.18(m，2H)，4.12(t，J＝8.3Hz，2H)，4.93(dm，J＝10.2Hz，1H)，4.98(dm，J＝17.2Hz，1H)，5.07(bs，1H)，5.71-5.83(m，1H)。
D. Mixing t-Bu4NF (6.7 ml of a 1M solution in THF, 6.7 mmol) was added to a solution of cyclopropyl derivative 1e (1.19 g, 3.35 mmol in 30 ml of THF) and the mixture was first stirred at room temperature for 16 hours and then heated to reflux for 15 minutes. The solvent is carefully evaporated under low pressure (care should be taken during evaporation of the solvent because of the high volatility of the free amine 1 f). The crude residue was redissolved in EtOAc (100ml) and washed with H2O (2X 50 ml), brine (50 ml) and MgSO 24Dry and carefully evaporate the solvent again. The crude product 1f (which is a mixture of the two enantiomers 1 f' and 1f ") was used for coupling with the P2 proline derivative without further purification. At this stage, using flash chromatography, one can easily accomplish the desired stereochemistry at P1
Isolation of the P1P2 fragment (example 21, fragment 21 b).
Part P2
Example 2
Synthesis of Boc-4(R) - [ (7-methoxy-4-quinolyl) oxy ] proline (2 c):
according to Chun, m.w.; olmstead, k.k.; choi, y.s.; lee, c.o.; lee, C. -K.; kim.j.h.; 4-hydroxy-7-methoxyquinoline (2b) was prepared as described by Lee, J.Bioorg.Med.chem.Lett.1997, 7, 789. In N2Next, a solution of Compound 2b (1.88 g, 10.73 mmol) and DEAD (3.4 mL, 21.46 mmol) in dry THF was added to 0 deg.C in dry THF (160 mL)To a stirred solution of protected cis-hydroxyproline 2a (2.63 g, 10.73 mmol) and triphenylphosphine (5.63 g, 21.46 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 14 hours. THF was then evaporated and after flash column chromatography using 5% MeOH in EtOAc as eluent, pure product 2c was isolated in 35% yield (1.5 g).
1H NMR(CDCl3，400MHz)：δ1.44(s，9H)，1.65(2，1H)，2.34-2.43(m，1H)，2.63-2.76(m，1H)，3.78(s，3H)，3.75-3.85 &3.89-3.99(2m, 1H, 2 spiro isomers), 3.95(s, 3H), 4.51&4.60(2t, J ═ 8Hz, 1H, 2 spiro isomers), 5.15(bs, 1H), 6.53-6.59(m, 1H), 7.12-7.18(dd, J ═ 8.9& 2.2Hz，1H)，7.36(d，J＝2.6Hz，1H)，8.03(bd，J＝9.2Hz，1H)，8.65(bs，J＝5.1Hz，1H)。
Example 3
Synthesis of 2-ethoxy-4-hydroxy-7-methoxyquinolyl (3c)
The synthesis of p-methoxyanthranilic methyl ester 3a was carried out as described in Katz et al, J.org.chem., 1953, 18, 1380-1400.
A general synthesis of quinoline derivatives 3c is a procedure for modifying Baccar et al, Indian Journal of chemistry, 1995, Sat. B, 330-332.
A. P-methoxy anthranilic acid methyl ester 3a (3.069 g, 16.96 mmol) was dissolved in triethyl orthoacetate (4.7 ml, 25.4 mmol) and then anhydrous HCl solution (4N/dioxane, 50 μ l, 0.6 mmol) was added. The resulting mixture was heated to reflux for 19 hours. The volatiles were then evaporated under vacuum to give product 3b (4.92 g, amber oil, quantitative yield) which was used in the next step.
B. LiHMDS (1M @) was incubated at-78 ℃ under nitrogenTHF, 22 ml, 1.3 eq) was added to a solution of matrix 3b (16.96 mmol) in THF (34 ml). Shortly after the addition, the cold warm bath was removed and the mixture was left to stir at ambient temperature for 1 hour, followed by the addition of the other portion of LiHMDS (16 ml). The resulting mixture was then stirred until purified by TLC (100% EtOAc, imide salt) Rf0.7, product Rf0.2) was found to have completely disappeared the starting material (1 hour). HCl (4N/dioxane, 10 ml) was then added and the mixture was concentrated under vacuum. From EtOAc (10 mL) with NaH2PO4In a mixture of aqueous solutions (1M, 10 ml), the resulting paste was triturated and shaken with sound. A rich precipitate formed, which was collected by filtration, washed with water and dehydrated to give the desired product 3c as a grey solid (3.117 g, 2 steps yield 84%, purity > 99% by HPLC).
1H NMR(400MHz，DMSO-d)δ(ppm)：7.88(d，J＝8.9Hz，1H)，6.98(br.s，1H)，6.89(br.d，J＝8.6Hz，1H)，5.94(br.s，1H)，4.30(br.s，2H)，3.84(s，3H)，1.34(t，J＝7.0Hz，3H)。
Example 4
Synthesis of 4-hydroxy-7-methoxy-2- (3-methyl-1, 2, 4-oxadiazol-5-yl) quinolyl (4d)
A. NaH (60% in mineral oil, 190 mg, 4.98 mmol) was added to a solution of 2-methoxycarbonyl-4-hydroxy-7-methoxyquinoline 4a (the preparation of which is described in WO00/09543 and WO 00/09558) (1 g, 4.29 mmol) in DMF (10 ml) under nitrogen. The resulting mixture was stirred at ambient temperature for 1 hour, then MEM chloride (455 μ l, 4.98 mmol) was added dropwise and the resulting mixture was stirred at ambient temperature for an additional 19.5 hours. The reaction mixture was diluted with EtOAc (100mL) and washed with H2O (50 mL), brine (50 mL), MgSO4Dried and concentrated in vacuo to afford a crude reaction isolate (1.37 g). Purification by flash column chromatography gave product 4b as a colorless oil (1.04 g, 75% yield).
B. THF (3ml) was added to a mixture of freshly activated 4A molecular sieve (500 mg) and acetamidoxime (248 mg, 3.35 mmol). The resulting mixture was stirred under nitrogen at ambient temperature for 15 minutes, then NaH (60% in mineral oil, 124 mg, 3.24 mmol) was added in portions. The resulting suspension was stirred at ambient temperature for 1 hour. A solution of ester 4b (500 mg, 1.56 mmol) in THF (5 ml) was then added. The resulting mixture was heated to reflux for 1 hour, then filtered over celite, rinsed with EtOAc (3 parts 20 ml), and concentrated in vacuo. The resulting crude mixture was purified by flash column chromatography (100% EtOAc) to give product 4c as a white solid (352 mg, 65% yield).
C. Aqueous HCl (1N, 1ml) was added to MEM ether 4c (170 mg, 0.493 mmol) in THF (4 ml). The resulting mixture was stirred at ambient temperature for 1 hour, then with NaH2PO4Aqueous solution (1M, 50 ml) was diluted. The solid formed was filtered, triturated with EtOAc, filtered and dried to give the desired product (4d) as a white solid (90 mg, 71% yield). MS (ES +)258(M+1)，(ES-)256(M-1)。
1H NMR(400MHz，DMSO-d)δ(ppm)：8.03(d，J＝9.2Hz，1H)，7.38(d，J＝2.2Hz，1H)，7.06(d.J＝8.6Hz，1H)，6.85(br.s，1H)，3.88(s，3H)，2.64(s，3H)。
Example 5
Synthesis of 4-hydroxy-7-methoxy-2- (5-methyl-1, 3, 4-oxadiazol-5-yl) quinoline (5e)
A. Anhydrous hydrazine (57 μ l, 1.8 mmol) was added to the substrate 4b (465 mg, 1.45 mmol) in ethanol (5 ml). The resulting solution was heated to reflux for 4 hours and then concentrated in vacuo to afford product 5a as a yellow solid (704 mg, quantitative crude yield) which was used in the next step.
B. Compound 5a (assumed to be 1.45 mmol) in triethyl orthoacetate (5 ml) was heated to 100-. The resulting mixture was then diluted with EtOAc (100ml) and saturated NaHCO3Aqueous (50 ml), brine (50 ml) rinse with MgSO4Dehydrated, concentrated in vacuo, and purified by flash column chromatography (100% EtOAc). Compound 5b was obtained as a yellow oil (359 mg, 61% yield in two steps). MS (ES +)392(m +1), (ES-)390 (m-1). C. Compound 5b (333 mg, 0.852 mmol) was heated to 140 ℃ for 8.5h under high vacuum and purified by flash column chromatography (100% EtOAc) to give 5b (116 mg, 35%, R)f0.5) and 5c (138 mg, corrected yield 72%, Rf0.3) of a mixture of (B). Aqueous HCl (1N, 1ml) was added to a solution of compound 5c (138 mg, 0.4 mmol) in THF (4 ml) and the resulting mixture was stirred until complete (30 min). THF was evaporated under vacuum and NaH was added2PO4Aqueous solution (1M, 2 ml). The resulting suspension was shaken with sound waves, filtered and the solid was dehydrated under high vacuum to give the desired product 5d as a grey solid (75 mg, 73%). MS (ES +)258(m+1)，(ES-)256(m-1)。1H NMR(400MHz，DMSO-d)：δ8.03(d，J＝9.2Hz，1H)，7.39(d.J＝2.2Hz，1H)，7.06(br.d，J＝8.6Hz，1H)，6.85(br.s，1H)，3.88(s，3H)，2.46(s，3H)。
Example 6
Synthesis of 4-benzyloxy-2- (chloro) -7-methoxyquinoline (6e)
A. The commercially available meta-ammonia in dioxane (80 ml) was addedAnisole (25 g, 0.20 mol) was cooled to 0 ℃ and anhydrous HCl (4N/dioxane, 75 ml, 0.30 mol) was added. Et was then added2O (500 ml) and stirring was continued for 1 hour. The grey solid was then filtered and dried under vacuum to give salt 6a (31.88 g, 98% yield).
B. Ethyl cyanoacetate (21.3 mL, 0.20 mmol) was added to the salt and the mixture was heated to 280-300 ℃ in a flask equipped with a distillation head and a collection flask. The produced ethanol was collected to monitor the progress of the reaction. When 9 ml of ethanol were collected (theoretical content 11.7 ml), heating was stopped, the reaction mixture was cooled to room temperature, diluted with water (200 ml) -EtOAc (200 ml), then stirred and NaH was added2PO4Aqueous solution (300 ml). After stirring for a further 1h, filtration and drying, 6b (19.06 g, 84.5% pure, approx. 50% yield) is obtained as a yellow solid, which can then be used in the next reaction.
C. Compound 6b (11.0 g, 57.8 mmol) in DMF (100ml) at 0 ℃ was added to NaH (60% in mineral oil, 2.78 g, 115.6 mmol). The ice bath was then removed and the mixture was stirred at ambient temperature for 1 hour, then benzyl bromide (7.6 ml, 63.6 mmol) was added and the reaction mixture was stirred for 16 hours. The solution was then diluted with EtOAc (220 ml) -hexanes (220 ml) and the solid formed was filtered with saturated NaHCO3The aqueous solution (110 ml) was triturated with water, hexane-EtOAc (1: 1 ratio, 100ml) and dried under high vacuum. This gave product 6c (5.6 g, 91% purity, 35% yield) as a yellow solid. Isoamyl nitrite (3.8 ml, 28.6 mmol) was added to compound 6c (2.67 g, 9.52 mmol) in acetic acid (21 ml) and the resulting mixture was stirred at ambient temperature and monitored by HPLC. More isoamyl nitrite (1.3 ml, 9.52 mmol) was added after 2 hours and the mixture was kept stirring over 90 hours (HPLC 81% product, 3% matrix). To the resulting suspension was added water (100ml), followed by filtration. Drying the collected brown solid under high vacuum to giveProduct 6d (2.35 g, 92% purity, 72% yield).
D. To compound 6d (1.5 g, 4.39 mmol) was added phosphorus oxychloride (13 ml, 141 mmol) and the resulting mixture was heated to reflux for 1 hour, then diluted with EtOAc (150 ml) and slowly quenched with aqueous NaOH (1N, 150 ml) to pH9 at 0 ℃. The two layers were separated and MgSO4The organic layer was dried and concentrated in vacuo to give a brown solid which was purified by flash column chromatography (15% EtOAc/hexanes). Product 6e was obtained as a yellow solid (819 mg, purity > 9%, 62% yield).
1H NMR(400MHz，CDCl3)：δ8.07(d，J＝9.2Hz，1H)，7.50-7.40(m，5H)，7.29(d，J＝2.5Hz，1H)，7.12(dd，J＝9.2，2.5Hz，1H)，6.73(s，1H)，5.26(s，2H)，3.92(s，3H)。
Example 7
Synthesizing 4-hydroxy-2- (1-imidazolyl) -7-methoxyquinoline (7 b); 4-hydroxy-2- (4-methyl-1-imidazolyl) -7-methoxyquinoline (7 d); 4-hydroxy-7-methoxy-2- (1-pyrazolyl) quinoline (7 f); and 4-hydroxy-2- (3-methyl-1-pyrazolyl) -7-methoxyquinoline (7 h).
A. Compound 6e (423 mg, 1.41 mmol) and imidazole (400 mg, 5.88 mmol) were heated to 110 ℃ for 20 h. The mixture was diluted with EtOAc and then washed with water and brine, over MgSO4Drying and concentration under reduced pressure gave compound 7a as a yellow solid (422 mg, 96% purity, 90% yield). Compound 7a (319 mg, 0.963 mmol) in a mixture of ethanol (5 ml) and THF (5 ml) was purged with Pd (5%/C, 64 mg) and placed under hydrogen at one atmosphere. After stirring at ambient temperature for 7.5 h, the reaction mixture was filtered, washed with chloroform-methanol mixture and concentrated to give 7b as a yellow solid (130 mg, 97.7% pure, 56%)Yield). MS (ES +)242(m +1), (ES-)240(m-1)。
1H NMR(400MHz，DMSO-d)：δ8.51(s，1H)，8.03(d，J＝8.9Hz，1H)，7.93(s，1H)，7.23(d，J＝1.9Hz，1H)，7.15(s，1H)，7.12(dd，J＝9.2，2.2Hz，1H)，6.92(br.s，1H)，3.91(s，3H)。
B. Compound 6e (251 mg, 0.837 mmol) and 4-methylimidazole (344 mg, 4.19 mmol) were heated to 110 ℃ for 20 h. The mixture was then diluted with EtOAc, washed with water and brine, and over MgSO4Drying and concentration under reduced pressure gave the crude product as a 10: 1 mixture containing the 4-methyl and 5-methylimidazolyl isomers, respectively. The predominantly hypothetical desired isomer 11c was isolated by flash column chromatography (100% EtOAc) from a second more polar fraction containing the 4-and 5-methylimidazolyl isomers (76 mg, 23% yield) as a white solid (166 mg, 99% purity, 57% yield). Compound 7C (163 mg, 0.472 mmol) and Pd (5%/C, 33 mg) in a mixture of ethanol (2.4 ml) and THF (5 ml) were purged and placed under hydrogen at one atmosphere. After stirring at ambient temperature for 18 h, the reaction mixture was filtered, washed with chloroform-methanol mixture and concentrated to give 7d as a white solid (118 mg, 99% purity, 98% yield).
1H NMR(400MHz，DMSO-d)：δ8.42(br.s，1H)，8.01(d，J＝9.2Hz，1H)，7.64(br.s，1H)，7.21(br.s，1H)，7.10(d，J＝8.9Hz，1H)，6.89(br.s，1H)，3.90(s，3H)，2.20(s，3H)。
C. Compound 6e (184 mg, 0.614 mmol) and pyrazole (209 mg, 3.07 mmol) were heated at 110 ℃ for 17 hours. The mixture was then diluted with EtOAc and washed with aqueous NaOH (1N) and brine, MgSO4Drying and concentration under reduced pressure gave the crude product which was purified by flash column chromatography (2: 1 hexanes-EtOAc) to give 7e as a light yellow solid (103 mg, 50% yield). Purge compound 7e (103 mg, 0.311 mmol) and Pd (5%/C, 20 mg) in ethanol (2 ml) and THF (2 ml)) And placed under hydrogen gas at one atmosphere. After stirring at ambient temperature for 5.5 h, the reaction mixture was filtered, washed with chloroform-methanol mixture, and concentrated to give 7f as a yellow solid (77 mg, 99% purity, 99% yield). MS (ES +)242(m +1), (ES-)240(m-1)。
1H NMR(400MHz，DMSO-d)：δ8.82(d，J＝2.5Hz，1H)，8.31(s，1H)，8.00(d，J＝8.9Hz，1H)，7.83(br.s，1H)，7.43(br.s，1H)，7.24(br.s，1H)，7.10(d，J＝8.6Hz，1H)，6.59(br.s，1H)，3.90(s，3H)。
D. Compound 6e (217 mg, 0.724 mmol) and 4-methylimidazole (594 mg, 7.24 mmol) were heated at 110 ℃ for 23 hours. The mixture was shown to be a 1: 1 mixture of debenzylated compound 7h and benzylated compound 7g, then diluted with EtOAc (2-3 mL) and filtered to give the product as a pure debenzylated white solid 7h (111 mg, 95% purity, 54% yield).
1H NMR(400MHz，DMSO-d)：δ8.58(d，J＝2.6Hz，1H)，7.98(d，J＝9.2Hz，1H)，7.25(br.s，1H)，7.20(s，1H)，7.04(br.d，J＝9.2Hz，1H)，6.38(s，1H)，3.89(s，3H)，2.30(s，3H)。
Example 8
Synthesis of 4-hydroxy-7-methoxy-2- [4- (2-isopropylaminothiazolyl) ] quinoline (8f)
Note that: [ Using the same synthetic scheme, wherein other alkyl thiourea to replace compounds 8b, make various 2-alkyl amino thiazolyl substituent ].
A. Protocol for the conversion of m-aminoanisole to 8a, with literature: e.j.brown et al j.med.chem.1989, 32, 807-. However, the purification procedure was modified to avoid purification by chromatography. Using MgSO4Activated carbonAnd 5% w/w (based on expected mass) of silica gel to the EtOAc phase containing the desired product. After filtration over celite, the product was triturated with ether. Compound 8a was obtained as a light brown solid with a purity > 99% (confirmed by HPLC).
B. A suspension of isopropylthiourea (8b, 3.55 g, 30 mmol) and 3-bromopyruvic acid (8c, 5g, 1 eq) in dioxane (300 ml, 0.1M) was heated to 80 ℃. When 80 ℃ is reached, the solution becomes clear and soon the product precipitates out as a white solid. After heating for 2 hours, the solution was cooled to room temperature and the white precipitate was filtered to give compound 8d of high purity (> 98%, purity confirmed by NMR) and 94% yield (7.51 g).
C. A mixture of carboxylic acid 8d (4.85 g, 18.2 mmol) and aniline derivative 8a (3 g, 1 eq) in pyridine (150 ml, 0.12M) was cooled to-30 ℃ (upon cooling, the clear solution became partially a suspension). Phosphorus oxychloride (3.56 ml, 2.1 eq) was added slowly over 5 minutes. The reaction was stirred at-30 ℃ for 1 hour, the ice bath was removed and the reaction mixture was allowed to warm to room temperature. After 1.5 hours, the reaction mixture was poured into ice and the pH was adjusted to 11 with 3N aqueous NaOH solution and CH2Cl2Extracting with MgSO 24Dried, filtered and concentrated under vacuum. The grey solid was then purified by flash chromatography (45% EtOAc in hexanes) to give compound 8e as a light yellow solid in 73% yield (6.07 g).
D. A solution of tBuOK (2.42 g, 21.6 mmol) in dry tBuOH (40 mL, 0.14M, distilled from Mg metal) was heated to reflux. Compound 8e (1.8 g, 5.4 mmol) was added portionwise over 5 minutes and the resulting dark red solution was stirred under reflux for a further 20 minutes (completion of reaction monitored by HPLC). The mixture was cooled to room temperature and HCl (4N in dioxane, 1.5 equivalents) was added. The mixture was then concentrated under vacuum to ensure removal of all HCl and dioxane, and the product redissolved in CH2Cl2Twice, dry-burning under vacuum to obtainHCl salt of compound 8f (1.62 g, 93% purity by HPLC) as a gray solid. The product was then poured into phosphoric acid buffer (1 NNaH)2PO4pH4.5) and shaking with sound wave. The grey solid was filtered and dried in vacuo to give compound 8f (1.38 g, 81% yield) as a grey solid (91% purity by HPLC).
1H NMR(400MHz，DMSO)：δ8.27(s，1H)，8.12(d，1H，J＝9.2Hz)，7.97(br.s，1H)，7.94(s，1H)，7.43(s，1H)，7.24(dd，1H，J＝9.2，2.2Hz)，3.97(m，1H)，3.94(s，3H)，1.24(d，2H，J＝6.4Hz)。
Example 9
Synthesis of 4-hydroxy-7-methoxy-2- [2- (4-isopropylthiazolyl) ] quinoline (9f)
Note: various 2- (4-alkyl) -thiazole substituents were prepared using the same synthetic scheme in which the 9b compound was replaced with another 2-bromoketone.
A. Adding Br2(4.79nl, 93mmel, 1 eq.) 3-methyl-butan-2-one (8 g, 93mmol) in MeOH (100ml) was added dropwise over a period of 45 minutes at 30 ℃. The resulting mixture was stirred at room temperature for 90 minutes. Pentane was added and the reaction solution was washed with 5% NaHCO3Washing the solution with an aqueous solution and dissolving the organic phase in anhydrous NaSO4Dried, filtered and concentrated in vacuo to give a crude yellow oil, compound 9b, which was used in the next step without purification. Ethyl thiotranoate (9a, 1.8g, 13.5mmol) and bromoketone derivative 9b (13.5mmol) were stirred at 70 ℃ for 15 h. Then concentrated in vacuo and then purified by flash column chromatography using 15% EtOAc in hexanes as eluent to give compound 9c (740 mg, 28% yield).
B. At room temperature with LiOH2O (148 mg, 3.5mmol, 1 eq.) in THF/MeOH/H2In O (3: 1 ratio, 13 ml)A solution of compound 9c (700 mg, 3.5mmol) in water for 5 hours. The pH was then adjusted to 6 with 0.1N HCl and the mixture was concentrated to dryness under vacuum to give 13d, which was used directly in the next step without further purification.
C. A solution of 4-methoxy-2-amino-acetophenone (intermediate 8a, 570 mg, 3.45 mmol) and carboxylic acid derivative 9d (590 mg, 3.45 mmol, 1 eq) in pyridine (30 ml) was cooled to-20 ℃. POCl was then added dropwise over 5 minutes3(0.35 ml, 3.79 mmol, 1.1 equiv.). The resulting solution was stirred at-10 ℃ for 2 hours. By adding further H2O quench the reaction and concentrate the mixture under vacuum. The residue was poured into saturated NaHCO3Aqueous, and extracted with EtOAc. The organic layer was washed with MgSO4Dried, filtered and concentrated under vacuum. The crude product was purified by flash column chromatography using 25% EtOAc in hexanes as eluent to give compound 9e as a white solid (740 mg, 67% yield).
D. tBuOH (518 mg, 2.1 eq) was added to a suspension of compound 9e (700 mg, 2.2 mmol) in anhydrous tBuOK (11 ml). The resulting mixture was heated to 75 ℃ for 7.5 hours, then the solution was cooled to room temperature and acidified by addition of HCl (4N, HCl in dioxane, 2.5 ml). The mixture was concentrated under vacuum and the resulting residue was poured into 1N NaH2PO4And filtering the solution. The solid was then triturated with a small amount of EtOAc, filtered and dehydrated in vacuo to give compound 9f as a pale grey solid (270 mg, 41% yield).
1H NMR(400MHz，DMSO-d6)：δ8.00(br.s，1H)，7.60(br.s，1H)，7.51(br.s，1H)，7.43(br.s，1H)，7.29(br.s，1H)，7.14(br.s，1H)，6.95(br.s，1H)，3.90(s，3H)，3.15(m，1H)，1.33(d，J＝5.4Hz，6H)。
Example 10
Synthesis of 4-hydroxy-2- (1-methyl-2-imidazolyl) -7-methoxyquinoline (10d)
A. A solution of N-methylimidazole 10a (5 g, 61 mmol) in 100ml THF was cooled to-78 ℃. n-BuLi (24.4 ml of 2.5M/Et) was added dropwise over 15 minutes2Solution O, 1 equivalent). The resulting mixture was stirred at-78 ℃ for 90 minutes and then poured in portions over solid CO2The above. The heterogeneous mixture was stirred for 2 hours and allowed to reach room temperature. 1N HCl was added to pH5 and the aqueous layer was separated and freeze dried. The residue thus obtained was extracted with EtOAc (salts removed) and dried (Na)2SO4) Filtered and concentrated under reduced pressure. 6.2 g (80% yield) of a white solid 10b were obtained.
B. A solution of 4-methoxy-2-amino-acetophenone 8a (394 mg, 2.39 mmol) and carboxylic acid derivative 10b (301 mg, 1 eq) in pyridine (10 ml) was cooled to-20 ℃. POCl was added dropwise over 5 minutes3(244 microliters, 1.1 equivalents). The resulting solution was stirred at-10 ℃ for 2.5 hours. Water was then added and the mixture was concentrated under reduced pressure. The residue was poured into saturated NaHCO3In solution and extracted with EtOAc. The organic phase was dried (MgSO)4) Filtered and concentrated under reduced pressure. The product was purified by chromatography using silica gel (25% EtOAc/Hex) to give 530 mg of 10c as a pale yellow solid (81% yield).
C. tBuOK (431 mg, 2.1 eq) was added to a suspension of substrate 10c (500 mg, 1.8 mmol) in 8 ml tBuOH. The resulting mixture was then heated at 75 ℃ for 7 hours, the solution was allowed to reach room temperature overnight, and 2.5 ml of HCl (4N/dioxane) was added. The mixture was concentrated under reduced pressure and the resulting residue was diluted with EtOAc. 1N NaOH was added until pH7. The organic phase was separated and dried (MgSO)4) And filtered and concentrated under reduced pressure to give 145 mg of 10d as a light grey solid (31% yield).1H NMR(400MHz，DMSO-d)：δ7.99(d，J＝8.9Hz，1H)，7.49(s，1H)，7.37(s，1H)，7.18(s，1H)，6.92(d，J＝8.9Hz，1H)，6.31(s，1H)，3.87(s，3H)，3.84(s，3H)。
Example 11
Synthesis of 4-hydroxy-2- (1-pyrrolyl) -7-methoxyquinoline (11b)
A. A solution of substrate 11a (obtained from compound 6C after hydrogenolysis of the benzyl group with 5% Pd/C in ethanol-THF) (1 g, 5.25 mmol) and 2, 5-dimethoxytetrahydrofuran (0.68 ml, 1 eq) in glacial acetic acid was refluxed for 4.5 h and allowed to reach room temperature. The mixture was then concentrated under reduced pressure. The residue was diluted with methanol and NaOH (aq) 1N was added until pH7. The product was purified by chromatography using silica gel (3% MeOH/CH)2Cl2The residue was first adsorbed on silica gel). 140 mg (13% yield) of 11b was obtained as a white solid.
1H NMR(400MHz，DMSO-d)：δ7.98(d，J＝9.2Hz，1H)，7.64(s，2H)，7.18(d，J＝2.5Hz，1H)，7.05(br.d，J＝7.9Hz，1H)，6.88(br.s，1H)，6.32(s，2H)，3.90(s，3H)。
Example 12
Synthesis of 4-hydroxy-7-methoxy-2- (6-methyl-2-pyridyl) quinoline (12d)
A. 6-Methylpyridinecarboxylic acid 12a (411 mg, 3.0 mmol) and SOCl were refluxed in benzene (5 mL)2(0.520 ml, 7.2 mmol, 2.4 eq.) for 2 hours. The solvent and excess SOCl were removed from the reaction mixture under vacuum2And the residue was triturated with pentane. The solid material formed was filtered off, and the filtrate was concentrated to give acid chloride 12b (500 mg, 2.6 mmol).
B. Will be in CH2Cl2A solution of aniline 8a (344 mg, 2.08 mmol), DIPEA (1.45 mL, 8.35 mmol) and DMAP (61 mg, 0.5 mmol) in (10 mL) was added to 0 deg.C in CH2Cl2(5 ml) of crude acid chloride 12 b. The reaction mixture was stirred at room temperature for 16 hours. Volatile components were removed in vacuo, the residue was dissolved in EtOAc and washed with 5% NaHCO3(2x)、H2O and brine rinse the solution. The organic layer was then washed with MgSO4Dried and concentrated under vacuum. The mixture was purified by flash column chromatography using EtOAc/hexanes (1: 2) as eluent to give amide 12c (490 mg, 82%).
C. tBuOK (410 mg, 3.43 mmol) was added to a suspension of amide 12c (490 mg, 1.71 mmol) in t-BuOH (10 ml) and the mixture was stirred at 75 ℃ for 6h and then at room temperature for 16 h. The mixture was then poured into phosphoric acid buffer solution (175 ml, pH 7) and stirred for 30 minutes. The solid was triturated twice with ethyl acetate. The organic phase was washed with brine, MgSO4Dehydrated and concentrated under vacuum. The resulting solid was triturated with EtOAc to give quinoline derivative 12d (263 mg, 58%).1H NMR(CDCl3，400MHz)：δ2.68(s，3H)，3.94(s，3H)，6.85-6.88(2d，J＝8.6&9.5Hz，2H)，6.94(dd，J＝8.9&2.2Hz，1H)，7.27(dd，J＝6.7&1.9Hz，1H)，7.73-7.79(m，2H)，8.28(d，J＝8.9Hz，1H)，10.3(br.s，1H)。
Example 13
Synthesis of 4-hydroxy-7-methoxy-2- (5-methoxy-2-pyridyl) quinoline (13d)
A. NaOH (2M, 4.70 ml) was added to a solution of compound 13a (623 mg, 3.73 mmol) in MeOH and the mixture was stirred at room temperature for 2 hours. The solution was then acidified with HCl (6N, 2.2 ml) and concentrated to give compound 13b, which was used directly in the next step without purification.
B. Aniline 8a (500 mg, 3.03 mmol) was added to a solution of crude compound 13b (about 3.73 mmol) in pyridine (25 ml) and POCl was added3Before (0.35 ml, 3.73 mmol), the solution was cooled to-25 ℃. The reaction mixture was stirred at-10 ℃ for 1 hour, then at 0 ℃ for 2 hours. The mixture was then poured into H2O, and extracted with EtOAc (2-3 ×). With 5% NaHCO3The combined organic layers were washed with brine, MgSO4Dried and concentrated under vacuum. The crude material was purified by flash column chromatography using EtOAc/hexanes (1: 2) as eluent to give amide 13c (617 mg, 55%).
C. tBuOK (490 mg, 4.11 mmol) was added to a suspension of amide 13c (617 mg, 2.05 mmol) in dry t-BuOH (10 ml) and the mixture was stirred at 75 ℃ for 6h and then at room temperature for 16 h. The reaction mixture was poured into phosphoric acid buffer solution (175 ml, pH 7) and stirred for 30 minutes. The resulting solid material was filtered and triturated with EtOAc to give quinoline derivative 13d (250 mg, 43%).1HNMR(DMSO，400MHz)：δ3.86(s，3H)，3.94(s，3H)，6.72(bs，1H)，6.91(dd，J＝8.9&1.9Hz，1H)，7.54(d，J＝1.9Hz，1H)，7.60(dd，J＝8.9&2.9Hz，1H)，7.97(d，J＝8.9Hz，1H)，8.21(d，J＝8.6Hz，1H)，8.48(d，J＝1.9Hz，1H)。
Example 14
Synthesis of 4-hydroxy-7-methoxy-2- (oxazol-5-yl) quinoline (14 c):
A. the protected quinoline derivative 4b from example 4 (3.8 g, 11.8 mmol) was dissolved in CH2Cl2(60 ml) and cooled to-78 ℃ before adding diisobutylaluminum hydride (7.9 ml, 1 eq, 1.5M in toluene) very slowly over 15 minutes. After stirring for 80 minutes, an additional amount of DIBAL (5.5 mL, 0.7 eq., 1.5M in toluene) was added. After stirring for a further 2 hours at-78 ℃, the reaction was carefully quenched with methanol (4 ml) at-78 ℃ and then poured into an aqueous solution of Rochelle (Rochelle) salt (1N K-Na tartrate). Mixing the viscous paste with CH2Cl2(300 ml) were stirred together for 2 hours until clear. The phases were separated and the organic phase was dried (MgSO4) Filtered and concentrated to give a white solid. Purification by flash column chromatography (SiO)2230- & 400 days) using 50% EtOAc/hexanes gave aldehyde 14a as a white solid (2.5 g, 73%).
B. Tosylmethyl isocyanate (66 mg, 0.34 mmol) was added to K in MeOH (7 mL)2CO3(48 mg, 0.34 mmol) in a stirred suspension. The reaction mixture was heated at 80 ℃ for 16 h and concentrated to dryness in vacuo. By flash chromatography (SiO)2230-: 331.0(M + H)+。
C. The MEM protected hydroquinone 14b was dissolved in THF (3ml) and treated with aqueous HCl (1N, 1 ml). The reaction was stirred at room temperature for 30 minutes before being concentrated to dryness in vacuo. The residue was treated with phosphate buffer (3ml, 1N solution, pH4.5) and stirred before the product was filtered, washed with distilled water and dried overnight under high vacuum (60 ℃, 16 h). Desired hydroxyquinoline 14c (0.065g, 100%) MS to give a tan solid: 242.9(M + H).
1H NMR(DMSO-d6)：δ8.65(s，1H)，8.02(bs，1H)，7.97(d，J＝8.9Hz，1H)，7.19(s，1H)，6.93(d，J＝7.9Hz，1H)，6.42(bs，1H)，3.87(s，3H)，ES(+)MS：m/z 242.9(M+H)+
Peptide linker moiety (P3)
Example 15
(2S) -N-Boc-amino-non-8-enoic acid (15g)
A. Aqueous sodium hydroxide (1M, 1 eq, 460 ml) was added dropwise over 30 to 45 minutes to a solution of commercially available ethylene 2-acetamidomalonate 15a (100 g, 0.46 mol) in dioxane (500 ml). The resulting mixture was stirred continuously for 16.5 hours, then dioxane was evaporated in vacuo and the aqueous solution was extracted with three 300 ml portions of ethyl acetate and acidified to pH1 with concentrated HCl. The solution was allowed to stand to form crystals in an ice-water bath. After a small amount of crystallization occurred, the mixture was sonicated and a rich precipitate appeared. Filtration and concentration in vacuo afforded compound 15b (62.52 g, 72%) as a white solid in yield.
B. An aqueous solution of sodium periodate (40.7 g, 0.190 mol, 1.1 eq. in 475 ml of H) was added over a period of 20 minutes2O) to a1 liter round bottom flask with magnetic stirring of commercially available 7-octene-1, 2-diol 15c (25 g, 0.173 moles) and H2O (100ml) in an emulsion (slightly exothermic). The resulting mixture was stirred at room temperature for an additional 1 hour (reaction completion confirmed by TLC). The mixture was then poured into a separatory funnel and the aqueous layer was separated from the organic layer. Saturated aqueous solution with NaCl was poured out of the organic portion and separated. The two organics were combined, dried over sodium sulfate, and filtered on a cotton plug (in a pasteur pipette) to give compound 15d (15.135 g, colorless oil, 78% yield). With CH2Cl2Extracting the aqueous solution with anhydrous MgSO4Dried and concentrated in vacuo (no heating, heptanal boiling point 153 ℃) to give additional compound 15d (1.957 g, colorless oil, 10% yield). The total yield is 88%.
C. To solid ethyl 2-acetamidomalonate 15b (7.57 g, 40 mmol) was added 6-heptanal 15d (4.48 g) in pyridine (32 ml, 10 eq.) over 1 minute40 mmol) of the solution. The resulting solution was cooled in a 10 ℃ bath and acetic anhydride (12 ml, 3.2 eq) was added over 4 minutes. The resulting organic solution was stirred at room temperature and another portion of ethyl 2-acetamidomalonate 15b (2.27 g) was added. The resulting mixture was stirred at room temperature for an additional 11 hours. Ice (60 ml) was then added and the solution was stirred for 1.5 hours, then the mixture was diluted with 250 ml of water and extracted with two portions of ether. With 1N HCl, saturated NaHCO3Washing the solution of ether with Na2SO4Dried, concentrated, and purified by flash chromatography (EtOAc 40%/hexanes) to give compound 15e as a light yellow oil (4.8 g, 50 yield).
D. To a solution of degassed (argon sparged for 30 minutes) 2-acetamido-2, 8-nonadienoic acid Z-ethyl ester 15e (8.38 g, 35 mmol) in anhydrous ethanol (70 ml) was added (S, S) -Et-DUPHOS Rh (COD) OTf (51 mg, S/C ═ 496). The mixture was placed under 30 psig of hydrogen (under 4 vacuums-H)2After circulation) and stirred on a Parr (Parr) shaker for 2 hours. The resulting mixture was evaporated to dryness to obtain crude compound 15f, which was used in the next step without purification.
E. Reacting Boc2O (13.2 g, 2 eq) and DMAP (740 mg, 0.2 eq) were added to a solution of crude 2-acetamido-8-nonenoic acid (S) -ethyl ester 15f (7.3 g, 30.3 mmol) in THF (100ml) and the reaction mixture was heated at reflux for 2.5 h. Subsequently, most of the THF solvent was evaporated to CH2Cl2The crude mixture was diluted and washed with 1N HCl to remove DMAP. Further saturated NaHCO3Extracting the organic layer with an aqueous solution of anhydrous Na2SO4Dried and concentrated under vacuum. The crude product was then diluted with THF (50 mL) and water (30 mL) and LiOH H was added2O (2.54 g, 2 equivalents), and the resulting mixture was stirred at room temperature for 25 hours (completion of hydrolysis was confirmed by TLC). The reaction mixture was concentrated under vacuum to remove most of the THF solvent and taken up with CH2Cl2And (6) diluting. Washing the resulting solution with 1N HCl, anhydrousNaSO4Dried and concentrated under vacuum. To remove small amounts of impurities and excess Boc2O, the crude product was purified by flash chromatography (using a gradient from 100% hexane to 100% EtOAc in solution as eluent). 15g (5.82 g, 71% yield) of the title compound was obtained as a pale yellow oil with high purity.1H NMR(DMSO，400MHz)：δ7.01(d，J＝8Hz，1H)，5.79(tdd，Jt＝6.7Hz，Jd＝17.0，10.2Hz，1H)，5.00(md，Jd＝17.0Hz，1H)，4.93(md，Jd＝10.2Hz，1H)，3.83(m，1H)，2.00(q，J＝6.9Hz，2H)，1.65-1.5(m，2H)，1.38(s，9H)，1.35-1.21(m，6H)。
Example 15A
Another synthesis method of (2S) -N-Boc-aminononan-8-enoic acid (15 g):
A. 8-bromo-1-octene (15h, 2.52 ml, 15 mmol) was added dropwise over a period of 15 min to a stirred suspension of finely cut Mg bands (0.55 g, 22.5 mmol) in anhydrous THF (30 ml) containing dibromoethane (0.1 ml) [ the reaction was slightly exothermic]. After 30 minutes, the mixture was heated to 38 ℃ for 1 hour and then added to excess solid CO by intubation2Before neutralization, cool to-78 ℃. The mixture was diluted with diethyl ether (100ml) and the solution was washed with brine (2X 50 ml) and MgSO4Dried and evaporated. The crude oil was obtained and purified by chromatography on silica gel using 15% EtOAc in hexanes as eluent to give compound 15i (1.44 g) in 62% yield.
1H NMR(CDCl3，400MHz)：δ1.31-1.42(m，6H)，1.60-1.69(m，2H)，2.02-2.09(m，2H)，2.35(t，J＝8.3Hz，2H)，4.99(dm，J＝10.0Hz，1H)，5.04(dm，J＝17.0Hz，1H)，5.75-5.86(m，1H)。
B. Et to be newly distilled3N (1.6 mL, 11.3 mmol) and neopentyl glycolAcid chloride (1.18 ml, 9.58 mmol) was added by syringe to a vigorously stirred solution of carboxylic acid 15i (1.36 g, 8.7 mmol) in anhydrous THF (70 ml) at-78 ℃ under anhydrous conditions. The mixture was stirred at-78 ℃ for 15 minutes and then at 0 ℃ for 45 minutes. The mixture was cooled to-78 ℃ and then passed through a cannula to-78 ℃ in anhydrous solution of lithium 4(S) -4- (benzyl) -2-oxazolidinone in THF, the lithium salt of the oxazolidinone reagent was first prepared by slowly adding n-BuLI (2.00M in hexane, 7.85 ml, 15.7 mmol) to a solution of oxazolidinone (2.78 g, 15.7 mmol) in THF (20 ml) at-78 ℃. The reaction mixture was stirred at-78 ℃ for 15 minutes and then at room temperature for 1.5 hours. Finally, it was quenched with an aqueous solution of sodium bisulfate (100ml 1M) and THF was evaporated to its original volume of 3/4. The residue was extracted with EtOAc (2 × 150 ml) and 5% NaHCO3The combined organic layers were washed with brine (2X 50 mL), 3X 50 mL, MgSO4Dried and evaporated. The resulting crude oil was chromatographed on silica gel using 15% EtOAc in hexanes to give compound 15j (1.88 g) in 68% yield.
1H NMR(CDCl3，400MHz)：δ1.35-1.47(m，6H)，1.67-1.74(m，2H)，2.02-2.09(m，2H)，2.65(dd，J＝13.4&9.9Hz，1H)，2.84-3.02(m，2H)，3.31(dd，J＝13.4&3.2Hz，1H)，4.13-4.22(m，2H)，4.62-4.71(m，1H)，4.93(d，J＝10.2Hz，1H)，5.00(dd，J＝17.2&1.6Hz，1H)，5.75-5.84(m，1H)，7.18-7.38(m，5H)。
C. A solution of acid derivative 15j (3.25 g, 10.30 mmol) in anhydrous THF (40 ml) at-78 ℃ was cannulated into a stirred solution of KHMDS (0.8M THF, 22 ml, 17.5 mmol) in anhydrous THF (50 ml). The mixture was stirred at-78 ℃ for 45 minutes. To this mixture was added a solution of trilylazide (3.67 g, 11.85 mmol) in anhydrous THF (40 ml) at-78 ℃. The mixture was stirred at-78 ℃ for 3 minutes and then quenched with acetic acid (5 ml). Then, the mixture was stirred at room temperature for 1 hour and 45 minutesAnd finally stirred at 40 ℃ for 15 minutes. Most of the THF was evaporated. The residue was dissolved in EtOAc (100ml) as H2O (50 mL), 5% NaHCO3The organic solution was washed (3X 50 ml) with brine (50 ml), (MgSO)4) And evaporated. Chromatography of the obtained oil on silica gel, using hexane/CH2Cl2(1/1) was used as eluent to give compound 15k (2.47 g, 67% yield).
1H NMR(CDCl3400 MHz): δ 1.32-1.45(m, 6H), 1.45-1.6(m, 1H), 1.75-1.88(2, 2H, rotamer), 2.01-2.11(m, 2H), 2.82-2.87(m, 1H), 3.33(dd, J ═ 13.4)&3.2Hz，1H)，4.10-4.28(m，2H)，4.62-4.72(m，1H)，4.90-5.05(m，3H)，5.73-5.88(m，1H)，7.17-7.38(m，5H)。
D. A solution of azide 15k (2.45 g, 6.9 mmol) in anhydrous MeOH (20 mL) at 0 deg.C was cannulated with anhydrous SnCl in anhydrous MeOH (80 mL)2(2.61 g, 13.8 mmol) in a stirred solution. The mixture was stirred at room temperature for 4 hours. MeOH was evaporated and the resulting foamy mass was dissolved in dioxane/H2In O (100. mu.l/20. mu.l) and Boc2O (3.0 g, 13.8 mmol) and NaHCO3(2.89 g, 34.5 mmol) treatment (with more NaHCO if necessary)3The pH was adjusted to 8) and the mixture was stirred at room temperature for 16 hours. Fractions of dioxane (. about.50%) were evaporated and the residue was extracted twice with EtOAc. The organic solution was washed with brine (2 × 50 ml), dried and evaporated. The resulting residue was chromatographed on silica gel using 20-25% EtOAc in hexanes as the eluent to give compound 151(1.75 g, 60% yield).
1H NMR(CDCl3，400MHz)：δ1.27-1.53(m，6H)，1.46(s，9H)，1.80(m，1H)，2.00-2.08(m，1H)，2.80(t，J＝12.1Hz，1H)，3.34(d，14.3Hz，1H)，4.17-4.23(m，2H)，4.60-4.66(m，1H)，4.93(d，J＝10.2Hz，1H)，5.05(dd，J＝17.2&1.9Hz，1H)，5.13(bs，1H)，5.38-5.43(m，1H)，5.74-5.84(m，1H)，7.22-7.36(m，5H)。
E. H is to be2O2(30% v/w, 2.05 ml, 16.2 mmol) and LiOH. H2O (0.34 g, 8.1 mmol) was added to 90 ℃ in THF/H2N-Boc derivative 151(1.74 g, 4.04 mmol) in O (75 ml/15 ml) and the solution was stirred at 0 ℃ for 1 hour. With Na2SO3(2.24 g, H)2O in, 15 ml, 17.8 mmol) to quench the reaction. The pH was adjusted to 4-5 with 10% aqueous citric acid and the mixture was diluted with EtOAc. The aqueous portion was re-extracted once with EtOAc and the organic solution was washed twice with brine, dried and evaporated. Chromatography of the residue on silica gel using 20% hexane in EtOAc as eluent gave 15g (0.76 g, 70% yield) of the free carboxylic acid. The compound is in all respects identical to that obtained in example 15.
Example 16
Synthesis of (2S) -N-Boc-amino-5-oxo-non-8-enoic acid methyl ester (16 d):
the synthesis is based on the method of T.Tsuda et al, J.Am.chem.Soc.1980, 102, 6381-6384.
A. N at-78 deg.C2Next, n-Bu was added over a period of 5 minutes2Mg (0.9M/hexane, 5.8 ml, 5.2 mmol) was added dropwise to a thoroughly stirred solution of monoallyl malonate (1.50 g, 10.4 mmol) in anhydrous THF (20 ml). The viscous suspension is then stirred at room temperature for 1 hour and evaporated to dryness (under N)2Lower vacuum release). The solid Mg salt 16b was dried under vacuum for 1 hour.
Glutamic acid derivative 16a was first mixed with 1, 1' -carbonyldiimidazole (1.65 g, 10.21 mmol) in anhydrous THF and the mixture was stirred at room temperature for 1 hour to activate the free acid moiety. Next, the glutamic acid derivative which had been activated was injected into the solution of Mg salt 16b with a cannula, and the resulting reaction mixture was stirred at room temperature for 16 hours. Then diluted with EtOAc and the organic solution washed with 0.5N ice cold HCl, brine, dried and evaporated. The resulting residue was chromatographed on silica gel using 35-40% EtOAc in hexanes as the eluent to give compound 16c (1.85 g, 53% yield).
1H NMR(CDCl3，400MHz)：δ1.44(s，9H)，1.85-1.95(m，1H)，2.12-2.22(m，1H)，2.58-2.74(m，2H)，3,48(s，2H)，3.74(s，3H)，4.24-4.34(m，1H)，4.52(dm，J＝5.7Hz，2H)，5.09(m，1H)，5.25(dm，J＝10.2Hz，1H)，5.34(m，J＝17.2Hz，1H)，5.91(m，1H)。
B. Diester 16c (0.687g 2 mmol) in anhydrous DMF (3ml) was added (by syringe and in N2Next), to a stirred solution of tetrakis (triphenylphosphine) Pd (0) (0.116 g, 5 mol%, 0.1 mmol) in anhydrous DMF (7 ml). The mixture was stirred at room temperature for 3.5 hours. DMF was evaporated under reduced pressure and the residue was diluted with EtOAc (20 ml). The EtOAc solution was washed with 0.5N ice cold HCl (5 ml), brine (10 ml), dried and evaporated. Chromatography of the residue on silica gel using 15-20% EtOAc in hexanes as eluent gave compound 16d (0.253 g, 42% yield).
1H NMR(CDCl3，400MHz)：δ1.44(s，9H)，1.84-1.94(m，1H)，2.08-2.22(m，1H)，2.33(dd，J＝14.0&7.3Hz，2H)，2.45-2.55(m，4H)，3.74(s，3H)，4.28(bm，1H)，4.98(dm，J＝10.2Hz，1H)，5.03(dm，J＝17.2Hz，1H)，5.00-5.10(m，1H)，5.74-5.85(m，1H)。
Example 17
Synthesis of (2S, 5R) -N-Boc amino-5-methyl-non-8-enoic acid (17f)
A, B, C, D commercially available (R) - (+) -citronellal 17a was first converted to amino acid derivative 17B according to the same synthetic procedure as described above in example 15 for the conversion of aldehyde 15D to amino acid intermediate 15 f.
E. Compound 17b (0.675 g, 5.6 mmol) was dissolved in tBuOH/acetone/H2O (1: 1, 18 ml) in an ice bath (0 ℃). NMMO (0.789 g, 6.74 mmol, 1.2 eq.) and OsO were added successively4(2.5 w/w in tBuOH, 0.7 ml, 0.067 mmol, 0.012 eq) and the reaction mixture was stirred at room temperature for 4 h. Most of the acetone was removed by evaporation under vacuum, and the mixture was then extracted with EtOAc. With H2The organic layer was further washed with brine, anhydrous MgSO4Dried and evaporated to dryness. After flash column chromatography using 1% EtOH in EtOAc as eluent, diol 17c was obtained in high purity in 77% yield (0.575 g).
F. NaIO was heated at 0 deg.C4(0.48 g, 2.25 mmol, 1.3 eq.) was added to the reaction mixture in THF/H2To a solution of diol 17c (0.575 g, 1.73 mmol) in O (1: 1, 20 mL) and the reaction mixture was stirred at room temperature for 3.5 h. Most of the THF solvent was then removed by evaporation under vacuum and the remaining mixture was extracted with EtOAc (2 × 100 ml). With 5% aqueous citric acid (2X 20 ml), 5% NaHCO3The combined organic layers were further washed with aqueous (20 ml) and brine (2 × 50 ml), then the EtOAc solution was washed with anhydrous MgSO4Dried and evaporated to dryness under vacuum. The aldehyde intermediate 17d (0.47 g crude) was used directly in the next step without further purification.
G. KHMDS (0.5M in toluene, 5.2 mL, 2.6 mmol) was added to Ph in dry toluene (15 mL)3PCH3Br (925 mg, 2.6 mmol) at room temperature, N2The resulting yellow suspension was stirred for 30 minutes. After this period, the suspension was first cooled to 0 ℃ and aldehyde 17d (0.47 g, 1.73 mmol, dissolved in 15 ml of anhydrous THF) was added via syringeMedium), and allowing the mixture to warm to room temperature. After stirring at room temperature for 1 hour, most of the THF was removed by evaporation in vacuo, EtOAc (100ml) was added to the mixture and washed with H2O (30 mL), 5% NaHCO3The organic layer was washed with aqueous solution (30 ml) and brine (30 ml). The EtOAc solution was then extracted with anhydrous MgSO4Dried and evaporated to dryness under vacuum and purified by flash column chromatography on silica gel using hexane: after EtOAc (3: 2) was used as eluent, pure compound 17e was isolated in 63% yield (0.29 g) in the last two steps.
Hydrolysis of the ethyl ester, while simultaneously exchanging the Boc in the intermediate with an N-acetyl protecting group, gave compound 17f, using the same procedure reported for converting compound 15f to 15g (17f, 310 mg, quantitative).1H NMR(CDCl3，400MHz)：δ0.88(d，J＝6.4Hz，3H)，1.18-1.28(m，2H)，1.35-1.48(m，3H)，1.45(s，9H)，1.64-1.74(m，1H)，1.81-1.89(m，1H)，1.94-2.12(m，2H)，4.28(bd，J＝～3.2Hz，1H)，4.83(dm，J＝11.1Hz，1H)，5.00(dm，J＝16.8Hz，1H)，5.74-5.84(m，1H)。
Example 18
Synthesis of N-Boc-O-allyl- (L) -threonine (18d)
A. Boc- (L) -threonine 18a (500 mg, 2.28 mmol) was partially dissolved in CH at 0 deg.C2Cl2In MeOH (8 ml/0.5 ml each). A solution of diazomethane in diethyl ether was added slowly until the yellow color remained, indicating the presence of excess diazomethane. Upon evaporation of the solvent, crude methyl ester 18b (0.534 g) was obtained as a cloudy white oil.
B. Intermediate 18b (311 mg, 1.33 mmol) was then dissolved in anhydrous diethyl ether (8 ml) and Ag was added2O (341 mg, 1.47 mmol) and novaActivated 4 * molecular sieve (1 g). Finally, allyl iodide (134 μ l, 1.47 mmol) was added to the reaction flask and the mixture was stirred under reflux. After 20 hours and 30 hours, two additional portions of allyl iodide (0.50 mmol per 45 μ l) were added and stirring was continued for a total of 36 hours. The mixture was then filtered through celite and purified by flash column chromatography on silica gel using EtOAc/hexanes (1: 4) as eluent to give 73 mg (27% yield) of compound 18c as a clear oil.
1H NMR(CDCl3，400MHz)：δ1.21(d，J＝6.0Hz，3H)，1.45(s，9H)，3.75(s，3H)，3.82-3.87(m，1H)，3.99-4.07(m，2H)，4.29(dd，J＝9.5&2.5Hz，1H)，5.14(dm，J＝10.5Hz，1H)，5.21(dm，J＝17.2Hz，1H)，5.75-5.84(m，1H)。
C. Ester compound 18c (99 mg, 0.362 mmol) was dissolved in THF/MeOH/H2O (2: 1, 4 ml) mixture, and LiOH. H2O (61 mg, 1.45 mmol). The solution was stirred at room temperature for 2 hours and then acidified to pH-3 with 1N HCl before the solvent was removed under vacuum. The oil thus obtained, compound 18d, was used to synthesize a macrocyclic inhibitor.
Example 19
Synthesis of (2S, 3S) -N-Boc-2-amino-3- (mercaptoallyl) butanoic acid (19e)
A. Compound 19a (9.1 mmol) was dissolved in pyridine (5 ml) and the solution was cooled to 0 ℃ in an ice bath, tosyl chloride (2.3 g, 11.8 mmol, 1.3 eq) was added in small portions and the reaction mixture was stirred at room temperature for 24 h. After this period, the reaction mixture was distributed between diethyl ether (300 ml) and H2O (100 ml). The ether layer was further washed with 0.2M HCl (6X 100mL) and brine (100mL) and dried over anhydrous MgSO4Drying and filteringAnd concentrated to dryness under vacuum. Purification of the crude material by flash column chromatography using hexane/EtOAc (gradient from 8: 2 to 7: 3 ratio) as eluent resulted in isolation of tosyl derivative 19b in 85% yield (3.05 g).
B. Potassium thioacetate (365 mg, 3.2 mmol, 1.6 eq) was added to a solution of intermediate 19b (7.75 mg, 2 mmol) in anhydrous DMF (2.5 ml) and the mixture was stirred at room temperature for 24 h. Most of the DMF was then evaporated under vacuum and the remaining mixture was distributed in EtOAc and H2And O is between. The aqueous layer was extracted with EtOAc and the combined organic layers were washed with brine, anhydrous MgSO4Dried and evaporated to dryness. Purification of the crude material by flash column chromatography using hexane/EtOAc (4: 1 ratio) as eluent resulted in isolation of compound 19c in 80% yield (465 mg).
C. An aqueous solution of 0.2M NaOH (2.4 ml) was added to the reaction solution in H2To a solution of thioester 19c (465 mg) in O/EtOH (3: 5 ratio, 8 mL) and the mixture was stirred at room temperature for 1.5 hours. Allyl iodide (0.292 ml, 3.2 mmol, 2 eq) was then added and stirring continued for another 30 minutes at room temperature. The reaction mixture was concentrated to half its original volume and then extracted with EtOAc. The liquid layer was acidified to pH-3 with cooled 0.5NHCl aqueous solution and back extracted with EtOAc. The combined organic layers were washed with brine, anhydrous MgSO4Dried and evaporated to dryness under vacuum. The crude reaction mixture contains at least four products: after flash column chromatography on silica gel with hexane/EtOAc (gradient from 9: 1 to 3: 1 ratio), the entire product was isolated. Structure of the least polar Compound (TLC R)f0.68 in hexanes/EtOAc 4: 1) consistent with the desired product 19d (83 mg, 18% yield).
1HNMR(CDCl3，400MHz)：δ1.24(d，J＝7Hz，3H)，1.46(s，9H)，3.13-3.19(m，2H)，3.24-3.29(m，1H)，3.77(s，3H)，4.50(dd，J＝8.6&3.8Hz，1H)，5.12(d，J＝12.4Hz，1H)，5.15(dd，J＝18.4&1.3Hz，1H)，5.22(bd，J＝7.6Hz，1H)，5.75-5.85(m，1H)。
D. Will be in MeOH/H at room temperature2A solution of methyl ester 19d (83 mg, 0.287 mmol) in O (3: 1, 4 mL) was mixed with aqueous NaOH (0.2N, 1.3 mL, 0.26 mmol) for 24h and 1h at 40 ℃. The reaction mixture was acidified with cooled aqueous HCl (0.5N HCl at 0 ℃, pH 4-5), MeOH was removed in vacuo, and the remaining aqueous mixture was extracted with EtOAc. The organic solution was washed with MgSO4Dried and evaporated to dryness to give compound 19 e. Finally compound 19e was used to synthesize the inhibitor without any further purification.
Example 20
(S) -N-Boc-2-amino-3-methyl-3- (1-mercapto-4-butenyl) butanoic acid (20c)
A. L-Penicilliamine 20a (448 mg, 3mmol) was dissolved in DMF/DMSO (5: 1 ratio, 6 mL), 4-bromopentene (0.46 mL, 4.5 mmol, 1.5 equiv) and CsOH. H.H.2O (1.0 g, 6 ml, 2 eq) and the reaction mixture was stirred at room temperature. After 24 hours, Boc was added2O (820 mg, 3.75 mmol, 1.25 eq) was added to the mixture and stirring was continued for an additional 12 hours. DMF was then removed under vacuum, the remaining mixture was diluted with cooled 0.5N aqueous HCl, adjusted to pH-4-5, and extracted with EtOAc (2 × 50 ml). The organic layer was washed with brine (2 ×), and anhydrous MgSO4Dried and evaporated to dryness to give crude carboxylic acid 20 b.
B. Purification of 20b was difficult, so the crude product was first treated with diazomethane to form the corresponding methyl ester 20c, which was then purified by flash column chromatography using hexane/EtOAc (9: 1) as eluent to yield 190 mg (20% yield) of pure methyl ester 20 c.
1H NMR(400MHz，CDCl3)：δ1.35(s，3H)，1.37(s，3H)，1.44(s，9H)，1.59-1.67(m，2H)，2.11-2.17(m，2H)，2.51-2.60(m，2H)，3.74(s，3H)，4.29(d，J＝8.6Hz，1H)，4.98(dm，J＝10.5Hz，1H)，5.03(dm，J＝19Hz，1H)，5.35(bd，J＝7Hz，1H)，5.72-5.83(m，1H)。
C. The ester was then dissolved in THF/MeOH/H2Adding LiOH & H into O (2: 1, 5 ml)2O (50 mg, 2.0 mmol, 2 eq) and the reaction mixture was stirred at 40 ℃ for 4 hours to hydrolyze ester 20c back to acid 20 b. The reaction mixture was acidified to pH4-5 with 0.5N HCl, THF and MeOH were evaporated to dryness, and the remaining aqueous solution was extracted with EtOAc. The EtOAc layer was washed with anhydrous MgSO4Drying and evaporation to dryness gave compound 20b, which was used in the subsequent synthesis of the macrocyclic inhibitor without further purification.
Acyclic dipeptide and tripeptide intermediates
The general procedure for the coupling reaction carried out in solution, and specific examples thereof, is described in WO00/09543 and WO 00/09558.
These procedures have been used to synthesize the intermediate dipeptides 26c, 30a and the tripeptides 23a, 24a, 31a, 32a and 33 a.
Example 21
Synthesis of acyclic tripeptide 21e
A. NMM (1.21 ml, 10.05 mmol) and HATU (1.53 ml, 4.02 mmol) were added sequentially to CH2Cl2Proline derivative 21a (prepared from commercially available Boc-4(R) -hydroxyproline and 4-chloro-quinoline as described in WO00/09543 and WO 00/09558) (1.32 g, 3.68 mmol) in (10 ml) and a solution of crude homoallyl ACCA1f (-3.35 mmol) and the suspension was stirred at room temperature for 18 h. In this sectionAfter this time, the solvent was evaporated and the crude reaction mixture was redissolved in EtOAc (30 ml). With 5% NaHCO3The solution was washed with aqueous (2X 10 mL), brine (10 mL) and MgSO4Dried and evaporated. The crude product was purified by chromatography on silica gel using 8% diethyl ether in EtOAc as eluent to give the desired diastereomer of compound 21b in 20% yield (absolute stereochemistry not determined).
1H NMR(CDCl3，400MHz)：δ0.93&1.01(t, J ═ 8.3Hz, 1H, rotamers in the ratio 3: 7), 1.14-1.35(m, 2H), 1.44(s, 9H), 1.45(s, 9H), 1.50-1.82(m, 4H), 2.08-2.24(m, 2H), 2.32(bs 0.7H), 2.63(bs, 0.75H), 2.93(bs, 0.75H), 3.16(m, 0.25H), 3.77(bs, 1.5H), 3.88(bs, 0.5H), 4.4-4.55(m, 1H), 4.98(d, J ═ 10.2Hz, 1H), 5.03(dd, J ═ 17.2 Hz, 1H), 1.14-1.35 (bs, 0.5H), 1.4.4 (bs, 0.5H), 4.4.4 (m, 1H), 4.98(d, J&1.6Hz，1H)，5.24(bs，1H)，5.75-5.88(m，1H)，6.57&6.78(2bs, 1H, 2 rotamers), 7.42-7.58(m, 3H), 7.63-7.73(m, 2H), 8.04(d, J ═ 8.3Hz, 1H), 8.11(d, J ═ 8.3Hz, 1H), 8.74(d, J ═ 5.1Hz, 1H).
B. A solution of HCl in dioxane (4M, 4 mL) was added to anhydrous CH2Cl2To a solution of dipeptide 21b (137 mg, 0.248 mmol) in (a), and the mixture was stirred at room temperature for 1.5 hours. The solvent is then evaporated and the residue is dried under high vacuum to give the free amino acid. The mixture was dissolved in diethyl ether/MeOH (3 μ l/2 μ l) and treated with a slight excess of diazomethane dissolved in diethyl ether. After 30 minutes, the excess diazomethane was destroyed by addition of HCl (4M in dioxane) and the mixture was evaporated to dryness to give the HCl salt of compound 21c, which can be used in the next step without any purification.
C. (2S) -N-Boc-amino-hept-6-enoic acid 21d (0.151 g, 0.62 mmol), NMM (210. mu.l, 1.91 mmol) and HATU (0.236 g, 0.62 mmol) were added sequentially to CH2Cl2(25 ml) in a stirred suspension of crude dipeptide 21c (0.23 g, 0.48 mmol), andthe mixture was stirred at room temperature for 16 hours (after 1 hour, if necessary, the pH was adjusted to-8 with NNM). Evaporation of CH2Cl2The residue was dissolved in EtOAc (50 ml) and washed with 5% NaHCO3The organic solution was washed with brine (2 × 20 ml), dried and evaporated (2 × 20 ml). The crude product obtained was chromatographed on silica gel (50 ml, 2% EtOH/EtOAc) to give compound 21e (0.139 g, 46% yield).
1H NMR(CDCl3400MHz, 6: 1 ratio rotamers) chemical shifts δ 1.21-1.27(m, 1H), 1.36(s, 9H), 1.45-1.81(4m, 7H), 2.20-2.22(m, 4H), 2.28-2.37(m, 1H), 2.90-2.99(m, 1H), 3.66(s, 3H), 3.94-3.98(m, 1H), 4.29(bdJ ═ 9.9Hz, 1H, 4.46-4.50(m, 1H), 4.81(dd, J ═ 8.3&5.4Hz，1H)，4.92-5.06(m，4H)，5.16(d，J＝8.3Hz，1H)，5.37(m，1H)，5.70-5.84(m，2H)，6.82(d，J＝5.1Hz，1H)，7.47-7.55(m，2H)，7.71(dt，J＝7.0&1.3Hz，1H)，8.03(d，J＝8.6Hz，1H)，8.17(d，J＝8.0Hz，1H)，8.78(d，J＝5.1Hz，1H)。
Macrocyclic peptides
Example 22
General procedure for macrocyclization by olefin metathesis
In all cases, the tripeptide diene was dissolved in CH at a concentration of 0.01M2Cl2After deoxygenation of the solution by bubbling argon (volume 500 ml about 1 hour). A solution of the catalyst (5-30 mol%, dissolved in a small amount of degassed CH) is added2Cl2Medium), and the reaction mixture was refluxed until all starting material was converted to product as indicated by TLC and HPLC. The crude reaction mixture was then concentrated to near dryness and filtered through a short pad of silica gel, first with CH2Cl2Elution removed most of the catalyst, followed by elution with EtOAc to elute all of the macrocyclic product(s) (mostly single diastereomer). By chiral HPLC on CHIRALCEL OJ-R column (fromChiral Technologies Inc., 0.46 v.15 cm) using 70% H at 205 nm2O+0.06％ TFA-30％ CH3The crude product from each reaction was analyzed in an isocratic mixture of CN + 0.06% TFA. By passing1H, COSY, TOCSY and ROESY NMR data fully characterize the major macrocyclic products in order to confirm their structure and stereochemistry.
Example 23
Intermediate of synthetic macrocycle (23b)
By bubbling Ar for 2 hours, the reaction solution is added to anhydrous CH2Cl2A solution of diene 23a (4.0 g, 7.88 mmol) in (800 ml, Aldrich-anhydrous) was deoxygenated. Havida's catalyst (262 mg, 0.434 mmol, 5.5 mol%) was added as a solid and refluxed under an Ar balloon. After 28 hours, the red-orange solution was evaporated to an amorphous solid, which was then purified by flash column chromatography on silica gel. The initial solvent system was 10% in CH2Cl2EtOAc in (iv). Once the catalyst was eluted from the column, the solvent was changed to pure EtOAc. The catalyst eluted from the column was evidenced by its color. The macrocyclic product 23b of the colourless foam is isolated and redissolved in CH2Cl2In hexane (. about.1: 2). The solvent was evaporated to give a white powder (3.362 g, 89% yield).
1H NMR(CDCl3，400MHz)：δ1.20-1.50(m，6H)，1.43(s，9H)，1.53(dd，J＝9.5&5.4，1H)，1.61-1.70(m，1H)，1.76-1.90(m，2H)，2.05-2.26(m，4H)，2.45(d，J＝14.3，1H)，3.67(s，3H)，3.71(d，J＝11.1，1H)，3.90(dd，J＝11.1&4.3，1H)，4.43-4.53(m，2H)，4.76(d，J＝8.6，1H)，4.86(bd，J＝9.8，1H)，5.20-523(m，2H)，5.57(dt，J＝7.0&9.8，1H)，7.32(bs，1H)。
Example 24
Intermediate of synthetic macrocycle (24b)
Bubbling through Ar for 2 hours, in anhydrous CH2Cl2A solution of diene 24a (2.76 g, 3.82 mmol) in (600 ml, anhydrous) was deoxygenated. Adding anhydrous and degassed CH via cannula2Cl2A solution of havida catalyst (117 mg, 0.19 mmol, 0.05 eq) in (8 ml) and the reaction stirred under reflux under an Ar balloon. After 20 hours, the reaction mixture was approximately 50% complete, at which point a second portion of catalyst (117 mg) was added and stirring continued for an additional 16 hours. The solution was then concentrated to about 100ml, applied over a pad of silica gel (6X 10 cm) and passed first with CH2Cl2And (4) eluting and recovering the catalyst. Compound 26b was washed out of the silica gel pad with 3% MeOH in EtOAc and repurified by flash column chromatography using EtOAc/hexanes (2: 1) to give a 70% yield of a slightly olive white solid (1.85 g, 94% purity by HPLC).
1H NMR(400MHz，DMSO-d)：δ8.69(s，1H)，8.13(d，J＝9.2Hz，1H)，7.50-7.44(m，2H)，7.17(dd，J＝9.2，2.2Hz，1H)，7.04(d，J＝6.4Hz，1H)，5.60-5.56(m，1H)，5.52(dd，J＝9.2Hz，1H)，5.25(dd，J＝9.2Hz，1H)，4.59(d，J＝11Hz，1H)，4.44(dd，J＝9.2Hz，1H)，4.05-3.98(m，1H)，3.94(s，3H)，3.92(s，3H)，3.89-3.82(m，1H)，3.55(s，3H)，2.64-2.53(m，1H)，2.46(d，J＝7.3Hz，1H)，2.40-2.31(m，1H)，2.21(dd，J＝8.9Hz，1H)，1.78-1.65(m，2H)，1.55(dd，J＝4.8Hz，1H)，1.485(dd，J＝4.8Hz，1H)，1.41-1.30(m，7H)，1.16(s，9H)。
MS：es-：795.4(M+H)-。
Example 25
Synthesis of Compounds 202&203 (Table 2)
A. Under reflux, using in CH2Cl2Catalytic content of bis- (tricyclohexylphosphine) dichloromethylbenzene ruthenium IV (grubbs catalyst, supra) (52 mg, 0064 mmol) in (ml), cyclization of diene compound 21e (0.130 g, 0.205 mmol) for 2h gave compound 25a (60.1 mg, 48% yield) after chromatography on silica gel (50 ml, 3% EtOH/EtOAc).
1H NMR(CDCl3，400MHz)：δ1.22-1.30(m，2H)，1.35(2，9H)，1.44-2.35(m，13H)，3.07-3.14&31.6-3.24(2m, 1H, rotamers in a ratio of 1: 3), 3.69(s, 3H), 3.96-4.04(m, 1H), 4.42-4.50(m, 1H), 4.95-5.04(m, 1H), 5.05-5.15(m, 1H), 5.20-5.30(m, 1H), 5.55-5.65(m, 1H), 6.75-6.79(2d, J ═ 5.4Hz, 1H, rotamers in a ratio of 1: 3), 7.36(s, 1H), 7.46-7.50(m, 1H), 8.03(d, J ═ 8.3Hz, 1H), 8.13&8.17(2d, J ═ 8.0Hz, 1H, the rotamer in a 1: 3 ratio), 8.77(d, J ═ 5.1Hz, 1H).
B. In THF/MeOH/H2LiOH. H in O (4 ml/2 ml)2O (8.7 mg, 0.206 mmol) partially hydrolyzes the ester of macrocycle compound 25a (0.0156 g, 0.026 mmol). Reverse phase HPLC through C18 on a Whatman (Partisil10, 0DS3)50/2.4cm column using from 5% CH3CN aqueous solution to 100% CH3The crude product was purified by a solvent gradient of CN to afford pure compound 202(11.8 mg) as an amorphous white solid.
1H NMR(DMSO，400MHz)：δ1.12(s，9H)，1.20-1.24(m，2H)，1.32-1.40(m，3H)，1.58-1.62(m，2H)，1.68-1.78(m，3H)，1.95-2.02(m，1H)，2.08-2.18(m，2H)，2.42-2.59(m，2H)，3.97-4.00(bd，J＝9.8Hz，2H)，4.47(t，J＝8.6Hz，1H)，4.58(d，J＝11.8Hz，1H)，5.22-5.29(m，1H)，5.46-5.54(m，1H)，5.66(s，1H)，7.12(d，J＝6.0Hz，1H)，7.49(d，J＝3.5Hz，1H)，7.68(dd，J＝7.3Hz，1H)，7.98(dd，J＝7.0Hz，1H)，8.08(d，J＝8.3Hz，1H)，8.21(s，1H)，8.35(d，J＝8.3Hz，1H)，9.08(d，J＝5Hz，1H)。
C. In the presence of 4M HCl/dioxane (5 ml), stirred in anhydrous CH2Cl2Macrocyclic compound 25a (20 mg, 0.033 mmol) in (1 ml) for 1 hour. The mixture was evaporated. And carefully dried. Redissolving the residue in CH2Cl2DMF (3 ml/1 ml) and treated with NMM (14.5 μ l, 0.132 mmol) and acetic anhydride (7.0 μ l, 0.073 mmol) and stirred at room temperature for 14 h. The mixture was evaporated and dried under high vacuum. The residue was then dissolved in THF/MeOH/H2O (4 ml/2 ml) in a mixture with LiOH 2H2O (11 mg, 0.264 mmol) was stirred together overnight. After acidification to pH3 with 1N ice cold HCl the residue was isolated and purified by C18 reverse phase HPLC using from 0-40% CH3A solvent gradient of aqueous CN (0.06% TFA) to isolate pure compound 203 as an amorphous white solid (12 mg).
1H NMR(50mM Na2PO4Buffer solution, pH 6.0, 600 MHz): δ 1.22-1.27(m, 2H), 1.38-1.43(m, 2H), 1.58-1.64(m, 2H), 1.67-1.76(m, 2H), 1.77-1.84(m, 1H), 1.92-1.99(m, 1H), 2.22-2.08(m, 1H), 2.12-2.27(m, 1H), 2.22-2.27(m, 1H), 2.60-2.67(m, 1H, Pro- β'), 2.83-2.89(m, 1H, Pro- β), 4.32(dd, J ═ 12.1&3.5Hz，1H，Pro-δ′)，4.41(dd，J＝12.1&7.3Hz，1H)，4.56(bd，J＝8.0Hz，1H，Pro-δ)，4.62(dd，J＝8.9Hz，1H，Pro-α)，5.40-5.46(m，1H)，5.55-5.61(m，1H)，5.73(bs，1H，Pro-γ)，7.41(d，J＝6.3Hz，1H)，7.64(bs，1H，Acca-NH)，7.80(dd，J＝7.9Hz，1H)，8.03(dd，J＝8.0Hz，1H)，8.07(d，J＝9.5Hz，1H)，8.16(d，J＝7Hz，1H，AcNH)，8.36(d，J＝8.3Hz，1H)，8.90(d，J＝6.0Hz，1H)。
Example 26
Synthesis of Compound 508 (Table 5)
A. Stirring at 16 ℃ in EtOAc/CH3/CN/H2A solution of Boc-protected L-glutamine 26a (4.93 g, 20 mmol) iodophenyl diacetate (7.73 g, 24 mmol 1.2 equiv.) in O (2: 1, 60 mL) was stirred for 1 hour at 20 deg.C for 3 hours. Then with H2The reaction mixture was diluted with O (20 mL) and the EtOAc and CH were removed under vacuum3CN solvent and the remaining aqueous mixture was extracted with diethyl ether (3 × 50 ml) and EtOAc (50 ml) to remove most of the impurities. The aqueous layer (containing the amine intermediate) was then concentrated to dryness and the remaining material redissolved in 10% Na2CO3To (30 ml) was cooled to 0 ℃ in an ice bath and a solution of benzyl chloroformate (3.3 ml, 20.4 mmol, 1.02 eq) in dioxane (40 ml) was added slowly (-10 min). The reaction mixture was stirred at 0 ℃ for 1 hour and at room temperature for 2 hours. Then with H2The mixture was diluted with O (50 ml), extracted with cold (-5 ℃) diethyl ether (3 × 50 ml), acidified to pH 3-4 with 4N HCl, and extracted with EtOAc (3 × 50 ml). The combined organic layers were dried over anhydrous MgSO4Dried and evaporated to dryness under vacuum. The crude material was purified by flash column chromatography using EtOAc/hexanes/AcOH (7: 2.9: 0.1) to afford compound 26b (3.04 g) in 43% overall yield.
B. Dipeptide intermediate 26c (250 mg, 0.41 mmol), compound 26b (171 mg, 0.49 mmol, 1.2 equivalents), and HATU (185 mg, 0.49 mmol, 1.2 equivalents) were dissolved in CH2Cl2(6 ml) and DIPEA (0.29 ml, 1.62 mmol, 4 eq) was added. The reaction mixture was stirred at room temperature for 14 hours, then CH was evaporated under vacuum2Cl2And the crude material was redissolved in EtOAc. With 5% NaHCO3The EtOAc solution was washed with aqueous solution and brine, anhydrous MgSO4Dried and evaporated to dryness. In thatThe crude material was purified by flash column chromatography using EtOAc/hexanes (4: 1) as eluent to afford compound 26d (338 mg) in 98% yield.
C. A solution of compound 26d (335 mg, 0.394 mmol) in THF (5 ml) was cooled to 0 ℃ and BH in dimethyl sulfide was added3Solution (0.12 ml of 10M solution, 1.2 mmol, 3 equiv). The reaction mixture was allowed to warm to room temperature and stirred for 1 hour. It was then cooled to 0 ℃ again, after which an aqueous solution of NaOH (0.8 ml of a 2.5M solution, 1.97 mmol, 5 eq) was added slowly over a period of 15 minutes, followed by H addition slowly (-15 minutes)2O20.8 ml of an 8.8M solution, 6.9 mmol, 17.5 eq. The reaction mixture was allowed to warm to room temperature and stirred for 1 hour. After this period, the reaction mixture was acidified to pH4 to allow excess BH3Quenching, then adding NaHCO3Adjusting the pH of the aqueous solution to 9-10, removing THF under vacuum and distributing the crude material in H2O2And EtOAc. The aqueous layer was re-extracted with EtOAc, and the combined organic layers were washed with brine, anhydrous MgSO4Dried and evaporated to dryness under vacuum. The crude material was purified by flash column chromatography using EtOAc/hexanes/NH4OH (8: 2: 0.5) was used as eluent to give pure compound 26e (192 mg) in 57% yield.
D. Des-Martin periodate (195 mg, 97%, 0.33 mmol, 1.5 equiv.) was added to CH2Cl2(8 ml) of compound 26e and stirring the reaction mixture at room temperature for 1.5 h. By adding Na2S2O3The reaction was quenched with aqueous solution (3ml of 5% solution) and then saturated NaHCO was added3Aqueous solution (5 ml) and the mixture was stirred at room temperature for 15 minutes. Finally, the crude reaction product was extracted with EtOAc and 5% NaHCO3The organic layer was washed with aqueous solution and brine, dried over anhydrous MgSO4Dehydration and evaporation under vacuum gave 188 mg of aldehyde 28f, which was used in the next step without further purificationAnd (5) further purifying.
E. At H2Next, compound 26f (188 mg, 0.22 mmol), CH in ethanol (5 mL) was stirred at atmospheric pressure3CO2H (38. mu.l) and Pd (OH)2(25 mg) of the solution was brought to room temperature for 16 hours. After this period, more H was added to the flask2Gas, Pd (OH)2(180 mg) and CH3CO2H (154 μ l) and stirring was continued for a further 24 hours. The mixture was then filtered and the solvent was evaporated to dryness and the crude macrocyclic product was purified by flash column chromatography using CHCl3MeOH/AcOH (10: 2: 1) gave 26g (48 mg) of compound in 30% yield.
F. Stirring at room temperature in CH2Cl2A mixture of compound 26g (22 mg, 0.031 mmol), DIPEA (27 μ l, 0.155 mmol, 5 equivalents) and acetic anhydride (8.7 μ l, 0.093 mmol, 3 equivalents) in (5 ml) was 16 h. CH is then removed under vacuum2Cl2Addition of THF/MeOH/H2O (2: 1, 5 ml) and LiOH 2H2A mixture of O (13 mg, 0.31 mmol, 10 equivalents), and the hydrolysis reaction was allowed to proceed at room temperature for 68 hours and at 50 ℃ for 2 hours. The reaction mixture was then acidified (pH ═ 4) and purified by reverse phase HPLC to afford final product 508 (-6 mg, final two steps yield ∼ 26%).
508 of1H NMR (DMSO, 400MHz) (confirmed as a mixture of rotamers by COSY, TOCSY and ROESY NMR data): δ 1.18(s, 9H), 1.09-1.85 (overlapping m, 11H), 1.95(s, 3H), 2.30(m, 1H), 2.63(m, 1H), 3.18-4,14 (overlapping m, 6H), 3.96(m, 3H), 4.44(m, 1H), 4.62&4.69(2d, J ═ 11.8Hz, 1H, rotamer), 5.82(bs, 1H), 7.20(m, 2H), 7.53(bs, 1H), 7.67(bs, 4H), 8.19(bs, 3H), 8.61(s, 1H).
Example 27
Synthesis of saturated macrocyclic intermediate (27a)
A. The unsaturated macrocyclic intermediate 23b (3.50 g, 7.30 mmol) was dissolved in EtOAc (30 ml) and 700 mg (20% w/w) 5% Rh on alumina was added. At atmospheric pressure and room temperature in H2The mixture was stirred under gas for 1.5 hours. After this period, HPLC analysis confirmed complete conversion of the starting material to two products, the desired product 27a and a minor product (8% of the total mass), which later was confirmed to be compound 27b, formed by opening of the cyclopropane ring. The reaction mixture was filtered and concentrated to give a pale green solid (3.47 g). The solid was co-evaporated twice with EtOH to remove all EtOAc (the presence of EtOAc interferes with the next step). It has proven very difficult to isolate compounds 27a and 27b by chromatography, and therefore another method based on the relative ratios of hydrolysis of the individual methyl ester moieties was devised.
B. A crude mixture of compounds 27a and 27b (3.47 g) was dissolved in THF: MeOH (1: 1, 20 mL) and LiOH. H was added2O in water (24 mg in 5 ml H)2O, 8% equivalent) and the reaction mixture was stirred at room temperature for 16 hours (complete hydrolysis of by-product 27b to its corresponding acid 27c was confirmed by HPLC). The reaction mixture was concentrated under vacuum to remove most of the THF and MeOH, and distributed in H2Between O (100mL) and EtOAc (300 mL), the organic layer was washed with 0.5N NaOH (3X 100mL), brine (100mL), 10% aqueous citric acid (2X 100mL), brine (100mL), and anhydrous MgSO4Dried, filtered and concentrated to dryness. The desired product 27a was obtained in high purity as a pale green foam (> 90% by HPLC), with a total yield of 93% (3.28 g) in both steps.
1H NMR(400MHz，CDCl3)：δ1.1-1.38(m，13H)，1.42(s，9H)，1.51-1.57(m，1H)，1.63-1.67(dd，J＝8.0&5.1Hz，1H)，1.81-1.87(m，1H)，1.92-1.99(m，1H)，2.02-2.08(m，1H)，2.62(d，J＝14Hz，1H)，3.4(d，J＝8.3Hz，1H)，3.65(s，3H)，4.01(dd，J＝10.8&4.1Hz，1H)，4.42-4.48(m，1H)，4.51-4.55(m，1H)，4.87(d，J＝8.6Hz，1H)，5.14(d，J＝8.6Hz，1H)，7.97(br s，1H)。
Example 28
Synthetic Compound #741 (Table 7)
The quinoline derivative 8f is linked to the pre-formed macrocycle compound 23b by a Mitsunobu reaction. Quinoline derivative 8f (30 mg, 0.095 mmol) was dissolved in THF, then macrocycle 23b (45.6 mg, 1 eq.) and PPh were added3(49.8 mg, 2 eq). The resulting mixture was cooled to 0 ℃. DIAD (37.4 μ l, 2 equivalents) was then added dropwise. The solution was stirred at 0 ℃ for 1 hour and then at room temperature overnight. The mixture was then diluted with EtOAc (15 ml) and saturated NaHCO3The solution (15 ml) was washed, followed by a saline wash. With MgSO4The solution was dried, filtered and concentrated in vacuo. 202 mg of a yellow oil are obtained. The product was purified by flash chromatography on silica gel (100% EtOAc). After purification, the product still contains DIAD by-products. The product obtained contained 55% w/w of the desired product, so a yield of 62% was declared.
The ester intermediate (46 mg, 0.06 mmol) was dissolved in THF/MeOH/H2To a mixture of O (2: 1, 2 ml) was added LiOH. H2O (20 mg, 0.48 mmol), and the solution was stirred at room temperature. After 16 hours, the reaction mixture was analyzed by HPLC indicating that hydrolysis was complete. The organic solvent was removed in vacuo and the remaining crude material was dissolved in DMSO and purified by C18 reverse phase HPLC to afford pure inhibitor 741.
1H NMR(400MHz，DMSO-d)：δ(ppm)：8.67(s，1H)，8.29-8.14(m，2H)，8.08-7.97(m，1H)，7.91-7.78(m，1H)，7.74(s，1H)，7.31-7.20(m，1H)，7.10(d，J＝5.7Hz，1H)，5.82-5.71(m，1H)，5.58-5.47(m，1H)，5.32-5.23(m，1H)，4.74-4.64(m，1H)，4.55-4.47(m，1H)，4.23-4.06(m，1H)，4.04-3.94(m，1H)，3.97(s，3H)，3.92-3.85(m，1H)，2.70-2.55(m，2H)，2.53-2.36(m，2H)，2.20-2.09(m，1H)，1.80-1.62(m，2H)，1.56-1.43(m，2H)，14.2-1.29(m，6H)，1.27(d，J＝3.2Hz，3H)，1.25(d，J＝2.9Hz，3H)，1.12(s，9H)。MS：763.1(M+1)，761.1(M-1)。
Example 29
Synthesis of Compound 205 (Table 2)
OsO in t-butanol at 0 deg.C4To a solution of (0.36 ml of 35% w/v, 0.035 mmol) in t-butanol/H2To a solution of macrocycle compound 25a (21 mg, 0.035 mmol) in O (1.5 ml/1.5 ml) and the mixture was stirred at room temperature for 1 hour. The mixture was diluted with EtOAc (20 ml) and 5% NaHCO3The organic solution was washed with aqueous solution (2 × 10 ml), brine (2 × 10 ml), dehydrated and evaporated to dryness. The crude compound was dissolved in THF/MeOH/H2O (3 ml/1.5 ml) and in LiOH. H2O (13 mg, 0.28 mmol) was stirred in the presence of O for 16 hours. The mixture was acidified to pH with 0.5N ice cold HCl4Evaporated and purified by C18 reverse phase HPLC using free H2O (0.06% TFA) to 40% CH3Solvent gradient of aqueous CN (0.06% TFA). Cis-diol 205 was isolated as a high purity, amorphous white solid.
Compound # 205:1H NMR(DMSO，400MHz)：δ1.01(s，9H)，1.06-1.30(m，9H)，1.48-1.68(m，3H)，1.78-1.88(m，1H)，≈2.2-2.5(2m，2H)，3.78-3.82(m，1H)，3.86-3.90(m，1H)，4.39(t，J＝8.9Hz，1H)，4.61(d，J＝11.4Hz，1H)，5.60(bs，1H，Pro-γ)，7.03(d，J＝6.0Hz，1H)，7.40(bs，1H)，7.58-7.62(m，1H) 7.87-7.91(m, 1H), 8.00(d, J ═ 8.3Hz, 1H), 8.24(d, J ═ 8.6Hz, 1H), 8.60(s, 1H), 8.99(bs, 1H). EMS (negative ionization mode): m/z 625(M-H)-。
Example 30
Synthesis of Compounds 214&218 (Table 2)
A. In LiOH. H2O (50 mg, 1.2 mmol) in the presence of MeOH/H with stirring at room temperature2A solution of keto-nonenoic acid ester 16d (0.180 g, 0.6 mmol) in O (5 ml/2 ml) for 1 hour. The solution was acidified to pH6 with 0.5N ice cold HCl and most of the MeOH was evaporated. The residue was then dissolved in EtOAc (30 ml) and the solution was washed with 0.5N ice cold HCl (10 ml), brine (10 ml), dried and evaporated. The crude residue is then redissolved in CH2Cl2(10 ml) and reacted with P1-P2 fragment 30a (0.337 g, 0.6 mmol) in the presence of HATU (233 mg, 0.612 mmol) and DIPEA (420 μ l, 2.4 mmol) at room temperature over a period of 16 hours. The reaction mixture was chromatographed on a layer of silica gel using EtOAc/hexanes (1/1) as the eluent, to isolate pure compound 30b (0.370 g, 83% yield, > 95% purity by HPLC).
1H NMR(CDCl3，400MHz)：δ1.41(s，9H)，1.45-1.54(m，1H)，1.58-1.62(m，1H)，1.73-1.77(m，1H)，1.86-1.91(m，1H)，2.16(dd，J＝17.8&8.6Hz，1H)，2.26-2.43(2m，2H)，2.46-2.58(m，2H)，2.64-2.81(m，1H)，2.85-2.92&2.95-3.03(2m, 1H, rotamers in a ratio of 1: 3), 3.67(s, 3H), 3.95(s, 3H), 4.10-4.18(m, 1H), 4.20-4.30(m, 1H), 4.40-4.55(m, 1H), 4.80-4.88(m, 1H), 4.92-5.10(m, 2H), 5.14(dd, J ═ 10.2)&1.6Hz，1H)，5.24-5.38(m，4H)，5.42-5.54(m，1H)，5.68-5.86(m，2H)，7.04-7.14(m，2H)，7.42-7.64(m，5H)，7.92-8.12(m，3H)。
B. In a period of 2 hours, under reflux, in CH2Cl2(from CaH)2Distilled and degassed with argon for 30 minutes) in the presence of bis- (tricyclohexylphosphine) dichloromethylbenzene ruthenium IV (catalyst (0.125 mg, 0.15 mmol), diene 30b (0.370 g, 0.49 mmol) was cyclized. After flash column chromatography on silica gel with EtOAc/hexanes (3/1), the compound was obtained as a mixture of stereoisomers (30c and 30d, 1: 1 ratio) in 35% yield (0.124 g).
Mixture 30c&30d of1H NMR(CDCl3，400MHz)：δ1.44(s，4H)，1.37(s，4H)，1.60(m，2H)，1.83(m，0.5H)，2.01(m，1H)，2.09(m，1H)，2.42(m，5H)，2.73(m，2H)，3.26(m，0.5H)，3.69(s，1.5H)，3.76(s，1.5H)，3.96(s，3H)，4.10(m，1H)，4.24(m，0.5H)，4.10(m，0.5H)，4.58(m，1H)，4.73(m，1H)，4.89(m，0.5H)，4.97(m，0.5H)，5.30(m，0.5H)，5.44(m，2H)，5.64(m，1H)，7.1-7.0(m，3H)，7.47(m，4H)，8.08-7.98(m，3H)。
C, D. in the presence of LiOH. H at room temperature for a period of 16 hours2HTF/MeOH/H of O (11 mg, 0.246 mmol)2Hydrolysis of methyl esters 30c and 30d (24 mg, 0.033 mmol) in O (1 ml/0.5 ml) was performed. After this period, the reaction mixture was acidified to pH4-5 and chromatographed on a C18 reverse phase HPLC column using a column from H2O (0.06% TFA) to 50% CH3Solvent gradient of aqueous CN (0.06% TFA). The desired compounds 214 and 218 were isolated in high purity (94% purity by HPLC) from a mixture of the two compounds, yielding 15% (3 mg).
Compound 214:1H NMR(DMSO，400MHz)：δ1.15(s，9H)，1.48-1.54(m，2H)，1.65-1.74(m，1H)，1.77-1.85(m，1H)，2.12-2.25(m，4H)，2.27-2.34(m，1H)，2.61-2.68(m，1H)，2.87(bt，J＝11.5Hz，1H)，3.92(dd，J＝9.2&1.5Hz，1H，Pro-δ)，3.97(s，3H，-OCH3)，4.14-4.20(m，1H)，4.52(t，J＝7.8Hz，1H，Pro-α)，4.66(d，J＝11.8Hz，1H，Pro-δ)，5.45(t，J＝9.9Hz，1H)，5.51-5.58(m，1H)，5.82(bs，1H，Pro-γ)，7.09(d，J＝6.0Hz，1H，BocNH)，7.26(bs，1H)，7.53(s，1H)，7.67(bs，3H)，8.16(d，J＝2Hz，1H)，8.18(s，1H)，8.83(s，1H，ACCA-NH)。
compound 218:1h NMR (DMSO, 400 MHz): δ 1.06-1.10(m, 1H), 1.18(s, 9H), 1.52-1.55(m, 1H), 1.62-1.80(m, 1H), 2.10-2.68 (overlapping, 9H), 3.90(bd, J ═ 8.3Hz, 1H), 3.96(s, 3H, OCH)3)，4.20-4.27(m，1H)，4.58-4.63(m，1H，Pro-δ)，4.66(dd，J＝8.3Hz，1H，Pro-α)，4.88(dd，J＝10.2Hz，1H)，5.18-5.26(m，1H)，5.73-5.79(m，1H，Pro-γ)，7.01(d，J＝6.4Hz，1H)，7.23(bs，1H)，7.50(bs，1H)，7.66(bs，3H)，8.20(bs，2H)，8.53(s，1H)。
Example 31
Synthesis of Compound 209 (Table 2)
A. Diene 31a (249 mg, 0.330 mmol) was dissolved in 30 ml of anhydrous CH2Cl2And the solution was degassed with hydrogen for 15 minutes. The catalyst ruthenium IV bis- (tricyclohexylphosphine) dichloromethylbenzene (82 mg, 0.100 mmol) was dissolved in 3ml of anhydrous degassed CH2Cl2And added to the solution of the diene. In N2The reaction mixture was refluxed for 2 hours. The solution was concentrated and purified by flash column chromatography to give compound 31b as a brown solid in 71% yield (171 mg).
1H NMR(CDCl3，400MHz)：δ1.22-1.44(m，10H)，1.42(s，9H)，1.66-1.74(m，1H)，1.87-1.97(m，2H)，2.13-2.38(m，3H)，2.32-2.39(m，1H)，3.08-3.16(m，1H)，3.41(s，3H)，4.07-4.22(m，3H)，4.28-4.34(m，1H)，4.58-4.64(m，1H)，4.95-4.99(m，1H)，5.22-5.29(m，2H)，5.38-5.43(m，1H)，5.48-5.56(m，1H)，7.00-7.12(m，3H)，7.43-7.55(m，4H)，7.97-8.11(m，3H)。
ES(+)MS：m/z 727.4(M+H)+。
B. Compound 31b (0.117 mmol) was stirred in a solution of HCl (1 ml of 4N in dioxane) for 30 minutes and concentrated to dryness. Dissolving the solid in CH2Cl2(2 ml), Et was added successively3N (82 μ l, 0.585 mmol) and tert-butyl isocyanate (35 mg, 0.351 mmol). After stirring at room temperature for 20 h, the mixture was concentrated to dryness and the crude compound 31c was used in the final hydrolysis step without further purification.
D. Compound 31c (85 mg, 0.117 mmol) was dissolved in THF/MeOH/H2To O (2 ml/1 ml), LiOH. H was added2O (39 mg, 0.936 mmol), and the solution was stirred at room temperature for 20 hours. After this time, acetic acid (1 ml) was added and the solution was concentrated to remove MeOH and THF. After purification of the crude product by C18 reverse phase HPLC, pure compound 209 was isolated (25 mg, 31 yield).
1H NMR(MDSO，400MHz)：δ1.04(s，9H)，1.15-1.24(m，2H)，1.30-1.40(m，5H)，1.44-1.51(m，2H)，1.54-1.68(m，1H)，1.75-1.88(m，1H)，2.18(dd，J＝17.2&8.5Hz，1H)2.32-2.45(m，1H，Pro-β)，2.54-2.62(m，1H)，2.65-2.68(m，1H，Pro-β)，3.91(dd，J＝11.1&3.5Hz，1H，Pro-δ)，3.96(s，3H，-OCH3)，4.17-4.23(m，1H)，4.47(dd，J＝8.6，1H，Pro-α)，4.67(bd，J＝7.9Hz，1H，Pro-δ)，5.30(dd，J＝9.5Hz，1H)，5.52(bdd，J＝19&8.3，1H)，5.68(s，1H)，5.78(bs，1H，Pro-γ)，5.94(bs，1H)，7.21(bs，1H)，7.51(bs，1H)，7.66(bs，4H)，8.19(s，2H)，8.40(d，J＝7Hz，1H)，8.61(s，1H，ACCA-NH)。
ES(+)(MS：m/z 698 .3(M+H)-。
Example 32
Synthesis of Compounds #404 and #407 (Table 4)
A. Diene 32a (84 mg, 0.11 mmol) was dissolved in anhydrous CH2Cl2(11 ml) and the solution was degassed with a stream of argon during 15 minutes. First, bis- (tricyclohexylphosphine) dichloromethylbenzene ruthenium IV catalyst (19 mg, 0.023 mmol) was dissolved in 1ml degassed CH2Cl2Then transferred via cannula into the reaction flask. The reaction mixture was stirred under reflux for 2 hours. The solvent was then removed in vacuo and the reaction mixture was purified by flash column chromatography on silica gel using EtOAc/hexanes (1: 1) as eluent to give macrocycle compound 32b (33 mg, 41% yield) as a yellow oil.
B. Ester intermediate 32b (33 mg, 0.045 mmol) was dissolved in THF/MeOH/H2To a mixture of O (2: 1, 2 ml) was added LiOH. H2O (8 mg, 0.18 mmol), and the solution was stirred at room temperature. After 16 hours, the reaction mixture was analyzed by HPLC indicating that hydrolysis was not complete. Thus, an additional amount of LiOH H was added2O (4 mg, 0.09 mmol), and the solution was stirred at room temperature for a total of 36 hours. Finally, the solution was acidified with a small aliquot of acetic acid, the organic solvent was removed under vacuum, and the remaining crude material was purified by C18 reverse phase HPLC to give pure inhibitor 404.
1H NMR(DMSO，400MHz)：δ1.21(d，J＝6.0Hz，3H，Me)，1.36(s，9H，Boc)，1.1-1.4(3m，3H)，1.66(m，1H)，1.80(m，1H)，2.10(m，2H)，2.57(m，2H)，3.90(m，4H)，4.47(bd，J＝12.7Hz，1H)，4.58(bd，J＝7.3Hz，1H)，4.66(dd，J＝8.0Hz，1H)，5.57(m，1H)，5.66(m，1H)，5.83(bs，1H)，6.18(bd，J＝6.9Hz，1H)，7.25(bd，J＝7.3Hz，1H)，7.56(bs，1H)，7.70(m，4H)，8.22(bd，J＝2.9Hz，2H)，8.29(bs，9.2Hz，1H)。
C. Inhibitor 404(15 mg, 0.021 mmol) was dissolved in ethanol (2 ml) and Pd 10%/C (2 mg) was added. The mixture was stirred under hydrogen at room temperature for 16 hours. After filtration, the mixture was purified by C18 reverse phase HPLC to afford inhibitor 407 as a white solid (10 mg, 66% yield).
1H NMR(DMSO，400MHz)：δ1.04(m，1H)，1.17(d，J＝6.0Hz，3H)，1.35(s，9H)，1.25-1.75(m，12H)，2.32-2.45(m，1H)，3.40-3.50(m，2H)，3.74-3.83(m，1H)，3.85-3.93(m，1H)，3.97(s，3H)，4.27-4.36(dd，J＝21.1&8.6Hz，1H)，4.54(dd，J＝7.95&7.95Hz，1H)，5.64(d，J＝8.3Hz，1H)，5.82(br s，1H)，7.27-7.33(m，1H)，7.53-7.57(bs，1H)，7.60-7.74(m，4H)，8.13-8.27(m，3H)，8.30-8.35(br，s，1H)。
Example 33
Synthesis of Compound #824 (Table 8)
A. Compound 33a (. about.0.55 mmol) was dissolved in CH2Cl2(100ml) and carefully degassed before adding a sample of haveda catalyst (17 mg, 0.028 mmol, 0.05 eq). The solution was then stirred under reflux for 5 hours. The reaction mixture was concentrated and purified by flash column chromatography using CH2Cl2A solvent gradient/EtOAc (ratio from 3: 2 to 2: 3) afforded compound 33b in 72% yield (194 mg).
B. Over a period of 20 minutes, DIAD (140 μ l, 0.71 mmol, 5 equivalents) was slowly added to compound 33b (70 mg, 0.142 mmol), 2-ethoxy-4-hydroxy-7-methoxyquinoline 3c (63 mg, 0.284 mmol, 2 equivalents) and Ph in anhydrous THF (15 ml)3P (186 mg, 0.71 mmol, 5 eq). The reaction mixture was allowed to warm to room temperature and stirred at room temperature for 2.5 hours. Then, in trueTHF was evaporated under air and the crude product was purified by flash column chromatography using a solvent gradient of hexane/EtOAc (from 7: 3 to 1: 1). Compound 33c (72 mg) was isolated in 73% yield.
C. Compound 33c (72 mg, 0.104 mmol) was reacted with CH2Cl2(5 ml) and 4M HCl in dioxane (5 ml) were mixed and the mixture was allowed to stir at room temperature for 1.5 hours to cleave the Boc protecting group to obtain HCl salt of intermediate 33 d. The crude reaction mixture was evaporated to dryness under vacuum and dried under vacuum to ensure removal of all HCl and was used in the next step without purification.
D. A solution of phosgene in toluene (1.93M, 274 μ l, 0.528 mmol) was added dropwise to a solution of cyclopentanol (29 μ l, 0.32 mmol) in THF (10 ml) and the mixture was stirred at room temperature for 2 hours to form the cyclopentyl chloroformate reagent. After this period, about half of the solvent was removed by evaporation under vacuum, by addition of CH2Cl2The remaining pale yellow solution was diluted (5 ml) and re-concentrated to half its original volume to ensure removal of all excess phosgene. The reagent solution of cyclopentyl chloroformate was further diluted with THF (10 ml), cooled to 0 deg.C, and solid compound 33d (0.104 mmol) at 0 deg.C was added. Et was added to the reaction mixture3N (75 μ l, 0.534 mmol, 5.2 eq) and stirring was continued at 0 ℃ for 1.5 h. The solvent was removed under vacuum and the crude material was purified by flash column chromatography using EtOAc/hexane (1: 1) as eluent to give compound 33e (75 mg) in almost quantitative yield.
E. By reacting compound 33e (75 mg, 0.11 mmol) with LiOH. H at 50 deg.C2O (35 mg, 0.84 mmol, 8 equiv.) in THF/MeOH/H2O (2: 1 ratio, 7.5 ml) in a solvent mixture for 2.5 hours. When the hydrolysis was complete, the mixture was acidified to pH4.5 and the solvent was evaporated to dryness under vacuum. The crude product was purified by C18 reverse phase preparative HPLC using H2O to 58% CH3Solvents for aqueous CN solutionsGradient (with 0.06% TFA) gave inhibitor #824 as a white amorphous solid (45 mg, 65% yield).
#824 of Na+Of salt1H NMR (DMSO, 400 MHz): δ 0.88(d, J ═ 7.6Hz, 3H), 0.95-1.70 (superposed resonance, 17H), 1.37(t, J ═ 7Hz, 3H), 2.00-2.10(m, 1H), 2.10-2.33(m, 3H), 2.38-2.44(m, 1H), 3.80-3.85(m, 1H), 3.85(s, 3H), 4.02-4.08(m, 1H), 4.42(q, J ═ 7Hz, 2H), 4.35-4.44(m, 1H), 4.50(d, J ═ 10.8Hz, 1H), 4.63(bs, 1H), 5.28(dd, J ═ 9.5Hz, 1H), 5.38(bs, 1H), 5.42-5.5H (m, 1H), 37.6 (s, 6.87(dd, 8H), 5.9.5H, 9.5.5H, 1H, 9(bs, 1H), 5.8 (dd, 1H), 5.8H, 1&2.2Hz，1H)，7.07(d，J＝2.2Hz，1H)，7.28(d，J＝7.0Hz，1H)，7.90(d，J＝8.9Hz，1H)，8.57(s，1H)。
Example 34
Synthesis of Compound #812 (Table 8)
A. Over a period of 15 minutes, DIAD (10.75 ml, 54.6 mmol, 2.0 equiv.) was added dropwise to the macrocyclic intermediate 23b (13.05 g, 27.2 mmol, 1.0 equiv.), Ph in THF (450 ml) at 0 ℃, Ph3P (14.28 g, 54.5 mmol, 2.0 equiv.) and 2-carboxymethoxy-4-hydroxy-7-methoxyquinoline (WO 00/09543)&WO 00/09558) (6.67 g, 28.6 mmol, 1.05 eq), then the ice bath was removed and the reaction mixture was stirred at room temperature for 3 hours. After complete conversion of the starting material to product, the solvent was evaporated under vacuum, the remaining mixture was diluted with EtOAc, and saturated NaHCO3Washed with (2X) and brine (1X), and the organic layer was washed with anhydrous MgSO4Dried, filtered and evaporated to dryness. After flash column chromatography, pure compound 34a was obtained: the column was eluted first with hexane/EtOAc (50: 50) followed by CHCl3EtOAc (95: 5) elution to remove Ph3PO and DIAD byproducts, and elution of impurities was monitored by TLC. Finally, with CHCl3EtOAc (70: 30) elutes the desired product 34a from the column. Typically, the chromatography step is repeated 2-3 times before isolating the high purity white solid compound 34a, with a total yield of 68% (12.8 g, 99.5% purity by HPLC). B. 4N HCl in dioxane (12 mL) was added to CH2Cl2(15 mL) of a solution of Boc-protected intermediate 34a (1.567 g) and the reaction mixture was stirred at room temperature for 1 hour [ resulting in the formation of a viscous gel midway during the reaction and an additional 10 mL of CH was added2Cl2]. When the deprotection was complete, the solvent was evaporated to dryness to give a yellow solid, and a paste-like material. Redissolving the mixture in CH2Cl2About 5% MeOH and re-evaporated to dryness under vacuum to give compound 34b as a yellow solid, which was used in the next step without any purification.
C. A solution of phosgene in toluene (1.93M, 5.96 ml, 11.502 mmol) was added dropwise to a solution of cyclopentanol (614 μ l, 6.76 mmol) in THF (15 ml) and the mixture was stirred at room temperature for 2 hours to form cyclopentyl chloroformate reagent (z). After this period, about half of the solvent was removed by evaporation under vacuum, with additional CH2Cl2The remaining pale yellow solution was diluted (5 ml) and concentrated to half its original volume to ensure removal of all excess phosgene. The solution of the cyclopentyl ester of chloroformic acid reagent was further diluted with THF (15 ml) and added to amine-2 HCl salt 34 b. The mixture was cooled to 0 ℃ in an ice bath and Et was used3Addition of N (dropwise addition) the pH was adjusted to 8.5-9 and the reaction mixture was stirred at 0 ℃ for 1 hour. After this time, the mixture was diluted with EtOAc, saturated NaHCO, and water (1 ×)3(2x)、H2O (2x) and brine (1 x). The organic layer was dried over anhydrous MgSO4Dried, filtered and evaporated under vacuum to give a yellow-amber foam. After purification by flash column chromatography (using from 30% in EtOAc)Solvent gradient hexane to 20% hexane as eluent), compound 34c was obtained in 80% yield (1.27 g) and purity > 93%.
D. Dimethyl ester 34c (1.17 g) was dissolved in THF/MeOH/H2To a mixture of O (20 ml, 2: 1 ratio) was added an aqueous solution of NaOH (1.8 ml, 1N, 1 equiv.). The reaction mixture was stirred at room temperature for 1 hour before it was evaporated to dryness to give the sodium salt 34d as a white solid (-1.66 mmol). Compound 34d was used in the next step without purification.
E. Crude sodium salt 34d (1.66 mmol) was dissolved in THF (17 ml) and Et was added3N, and the mixture was cooled to 0 ℃ in an ice bath. Isobutyl chloroformate (322 μ l, 2.5 mmol) was added dropwise and the mixture was stirred at 0 ℃ for 75 minutes. After this period diazomethane (15 ml) was added and stirring was continued at 0 ℃ for 30 minutes and then at room temperature for 1 hour. The bulk of the solvent was evaporated to dryness under vacuum, the remaining mixture was diluted with EtOAc, and saturated NaHCO3(2x)、H2O (2x) and brine (1x) rinse with anhydrous MgSO4Dry, filter and evaporate to dryness to give compound 34e as a pale yellow foam (1.2 g,. about.1.66 mmol). The diazoketone intermediate 34e is used in the next step without purification.
F. Diazoketone 34e (1.2 g, 1.66 mmol) was dissolved in THF (17 ml) and cooled to 0 ℃ in an ice bath. Aqueous HBr (48%, 1.24 ml) was added dropwise and the reaction mixture was stirred at 0 ℃ for 1 hour. The mixture was then diluted with EtOAc and saturated NaHCO3(2x)、H2The organic layer was washed with O (2X) and brine (1X) and dried over anhydrous MgSO4Dried, filtered and evaporated to dryness to give the β -bromoketone intermediate 34f (1.657 mmol) as a pale yellow foam.
G. Thiourea (118 mg, 1.55 mmol) was added to a solution of bromoketone 34f (600 mg, 0.779 mmol) in isopropanol (5 ml) and the reaction mixture was placed in an oil bath pre-heated to 75 ℃, allowed to standThe mixture was stirred for 1 hour. The isopropanol was then removed under vacuum and the product was dissolved in EtOAc (100 ml). With saturated NaHCO3And brine to wash the solution and remove the organic layer with anhydrous Na2SO4Drying, filtration and evaporation gave 34g (522 mg) of the crude product as a red-brown solid. This material was used in the final step without any further purification.
H. Crude methyl ester 34g (122 mg, 0.163 mmol) was dissolved in THF/MeOH/H2O solution (2: 1 ratio, 4 ml) using LiOH. H2O (89 mg, 2.14 mmol) saponifies it. The hydrolysis reaction is carried out at room temperature over a period of 12-15 hours. The solvent was then removed under vacuum and the crude product was purified by C18 reverse phase HPLC using from 10% in H2CH in O3CN to 100% CH3CN solvent gradient to give HCV protease inhibitor #812 as a yellow solid (24 mg, 20% overall yield of intermediate 34f to inhibitor # 812).
1H NMR(400MHz，DMSO-d6)：δ8.63(s，1H)，8.26-8.15(m，2H)，7.79(bs，1H)，7.72(bs，1H)，7.50(bs，2H)，7.33-7.25(m，2H)，5.77(bs，1H)，5.52(dd，J＝8.3Hz，1H)，5.27(dd，J＝9.2Hz，1H)，4.64(d，J＝10.8Hz，1H)，4.50(dd，J＝8.3Hz，1H)，4.39-4.31(m，1H)，4.08-3.99(m，2H)，3.94(s，3H)，3.87(d，J＝9.5Hz，2H)，2.65-2.53(m，2H)，2.46-2.36(m，2H)，2.20-2.12(dd，J＝8.6Hz，1H)，1.80-1.64(m，2H)，1.63-1.06(m，14H)。MS：es+：733.2(M+H)+，es-：731.2(M-H)-。
Example 34A
Using the same procedure as described in example 34, but reacting bromoketone 34f with commercially available N-methylthiourea, #811 (Table 8)
1H NMR(400MHz，DMSO-d6)：δ8.63(s，1H)，8.20(s，1H)，8.18(s，1H)，8.12-7.93(m，1H)，7.88-7.69(m，2H)，7.32-7.24(m，2H)，5.82-5.75(m，1H)，5.52(ddd，J＝8.1Hz，1H)，5.28(d，J＝9.9Hz，1H)，4.67-4.61(m，1H)，4.51(dd，J＝8.8Hz，1H)，4.44-4.37(m，1H)，4.08-4.00(m，1H)，3.96(s，3H)，3.89(m，1H)，3.04(d，J＝4.1Hz，3H)，2.65-2.37(m，3H)，2.16(m，1H)，1.77-1.65(m，2H)，1.63-1.11(m，17H)。
MS；es+：747.2(M+H)+，es-：745.3(M-H)-。
Example 34B
The same procedure as described in example 34 was used, but bromoketone 34f was reacted with commercially available N-ethylthiourea to give #810 (table 8).
1H NMR(400MHz，DMSO-d6)：δ8.63(s，1H)，8.27(bs，1H)，8.20(d，J＝9.0Hz，1H)，8.13-8.07(m，1H)，7.86(bs，1H)，7.78(s，1H)，7.33-7.25(m，2H)，5.81(bs，1H)，5.54(dd，J＝8.8Hz，1H)，5.28(dd，J＝9.7Hz，1H)，4.65(d，J＝12.4Hz，1H)，4.51(dd，J＝8.8Hz，1H)，4.38(bs，1H)，4.03(m，1H)，3.97(s，3H)，3.92-3.87(m，1H)，3.54-3.46(m，2H)，2.68-2.65(m，2H)，2.47-2.38(m，1H)，2.15(dd，J＝8.6Hz，1H)，1.78-1.65(m，2H)，1.60-1.12(m，17H)，1.25(t，J＝7.3Hz，3H)。
MS；es+：783.2(M+Na)+，es-：761.2(M+H)-。
Example 34C
The same procedure as described in example 34 was used, but bromoketone 34f was reacted with commercially available N-isopropyl thiourea to give # 822.
1HNMR(400MHz，DMSO-d6): δ 8.63(s, 1H), 8.33-8.23(bs, 1H), 8.21(d, J ═ 9.2Hz, 1H), 8.04(d, J ═ 8.3Hz, 1H), 7.86(bs, 1H), 7.77(s, 1H), 7.35-7.23(m, 2H), 5.81(bs, 1H), 5.52(dd, J ═ 8.5Hz, 1H), 5.27(dd, J ═ 9.2Hz, 1H), 4.65(d, J ═ 11.8Hz, 1H), 4.51(dd, J ═ 7.6Hz, 1H), 4.37(bs, 1H), 4.15(bs, 1H), 4.07-3.98(m, 2H), 3.97(s, 3H), 3.88(d, 8.88 (b, 8H), 1H), 13.47 (dd, 13.8H), 1H, 13.8.8 (1H), 1H-13.8.8 (d, 1H), j ═ 6.5Hz, 6H), 1.23-1.09(m, 2H), MS: es-： 775.0(M+H)+，es-：772.9(M-H)-。
Example 34D
Using the same procedure as described in example 34, but reacting bromoketone 34f with commercially available N-acetylthiourea, #809 was obtained
1H NMR(400MHz，DMSO-d6)：δ8.62(s，1H)，8.30(bs，1H)，8.17(d，J＝8.9Hz，1H)，7.62(bs，1H)，7.52(bs，1H)，7.28(d，J＝6.4Hz，1H)，7.21(bs，1H)，5.63(bs，1H)，5.54(dd，J＝8.1Hz，1H)，5.28(dd，J＝9.5Hz，1H)，4.62(d，J＝12.1Hz，1H)，4.56-4.46(m，2H)，4.11-4.04(m，1H)，3.95(s，3H)，3.93-3.88(m，1H)，2.62-2.54(m，1H)，2.45-2.36(m，1H)，2.22(s，3H)，2.21-2.13(m，1H)，1.79-1.69(m，2H)，1.65-1.30(m，16H)，1.26-1.12(m，2H)。MS；es+：775.3(M+H)+，es-：773.3(M-H)-。
Example 34E
DIPEA (0.55 mL, 3.18 mmol, 10)Eq) and chloromethyl methyl ester (0.13 ml, 1.6 mmol, 5 eq) were added at room temperature in CH2Cl2(5 ml) of a stirred solution of 34g (0.24 g, 0.32 mmol) of 2-amino-4-thiazolyl intermediate. The reaction mixture was stirred for 6.5 hours before being concentrated in vacuo. The crude isolated product was then hydrolyzed to the desired carboxylic acid as described in example 34 to afford compound # 818.
1H NMR(400MHz，DMSO-d6)：δ8.61(s，1H)，8.21-8.07(m，2H)，7.61-6.38(m，2H)，7.26(d，J＝6.4Hz，1H)，7.19-7.10(m，1H)，5.60-5.47(m，2H)，5.27(dd，J＝9.2Hz，1H)，4.63-4.53(m，1H)，4.47(d，J＝7.9Hz，1H)，4.13-4.04(m，1H)，3.93(s，3H)，3.92-3.87(m，2H)，3.79(s，3H)，4.42-2.30(m，2H)，2.17(dd，J＝9.3Hz，1H)，1.81-1.68(m，2H)，1.63-1.29(m，16H)，1.23-1.10(m，2H)。MS；es+：791.1(M+H)+，es-：789.1(M-H)-。
Example 34F
Similar substituted carbamate intermediates were obtained according to the conditions described above in example 34 but using isobutyl chloroformate. The crude miscellaneous materials were then hydrolyzed to the desired compound # 819.
1H NMR(400MHz，DMSO-d6)：δ8.62(s，1H)，8.47-8.27(bs，1H)，8.18(d，J＝8.6Hz，1H)，7.69-7.60(m，1H)，7.60-7.51(m，1H)，7.28(d，J＝6.7Hz，1H)，7.28-7.19(m，1H)，5.70-5.60(m，1H)，5.52(dd，J＝8.3Hz，1H)，5.27(dd，J＝9.8Hz，1H)，4.63(d，J＝11.8Hz，1H)，4.53-4.44(m，2H)，4.10-3.99(m，1H)，4.04(d，J＝6.7Hz，2H)，3.95(s，3H)，3.94-3.87(m，1H)，2.65-2.53(m，1H)，2.46-2.34(m，1H)，2.16(dd，J＝8.1Hz，1H)，2.03-1.91(m，1H)，1.79-1.09(m，20H)，0.95(d，J＝6.7Hz，6H)。MS：es+：833.2(M+H)+，es-：831.2(M-H)-。
Example 35
Synthesis of Compound #908
Starting from derivative 27a, using the same chemistry as described in example 34, the following saturated macrocycle, compound #908 (table 9) was obtained
1H NMR(400MHz，DMSO-d6)：δ8.47(s，1H)，8.16(d，J＝10Hz，1H)，8.15-8.07(m，1H)，7.82-7.63(m，2H)，7.53-7.43(m，2H)，7.33-7.22(m，1H)，7.13(d，J＝7Hz，1H)，5.77-5.65(m，1H)，4.62-4.52(m，2H)，4.50-4.4(m，1H)，4.20-4.10(m，1H)，3.94(s，3H)，3.89-3.83(m，1H)，2.59-2.53(m，1H)，2.48-2.40(m，1H)，1.79-1.0(m，25H)。MS：es+：735.2(M+H)+，es-：733.2(M-H)-。
Example 35A
Synthesis of Compound #909
Using the same procedure as described in example 35, but using commercially available N-acetylthiourea, compound #909 (Table 9) was obtained
1H NMR(400MHz，DMSO-d6)：δ8.53-8.41(m，2H)，8.20(d，J＝9.2Hz，1H)，7.68(bs，1H)，7.27(dd，J＝9.2Hz，1H)，7.15(d，J＝6.4Hz，1H)，5.67(bs，1H)，4.65-4.50(m，3H)，4.44-4.37(m，1H)，4.21-4.13(m，1H)，3.96(s，3H)，3.99-3.86(m，1H)，2.62-2.39(m，2H)，2.24(s，3H)，1.78-1.67(m，3H)，1.67-1.01(m，22H)。
MS：es+：798.0(M+Na)+，es-：777.0(M+H)+。
Example 35B
Synthesis of Compound #910
Using the same procedure as described in example 35, but using commercially available N-ethylthiourea, compound #910 (Table 9) was obtained
1H NMR(400MHz，DMSO-d6)：δ8.47(s，1H)，8.29(bs，1H)，8.20(d，J＝9.2Hz，1H)，8.09(bs，1H)，7.87(s，1H)，7.77(s，1H)，7.32(dd，J＝9.2Hz，1H)，7.14(dd，J＝6.7Hz，1H)，5.78(bs，1H)，4.58(dd，J＝8.1Hz，2H)，4.43(bs，1H)，4.18-4.12(m，1H)，3.97(s，3H)，3.87(d，J＝8.9Hz，1H)，3.55-3.46(m，2H)，2.63-2.53(m，1H)，2.47-2.41(m，1H)，1.78-1.00(m，25H)，1.25(t，J＝7.3Hz，3H)。MS：es+：763.1(M+H)+，es-：761.1(M-H)-。
Example 35C
Synthesis of Compound #911
Using the same procedure as described in example 35, but using commercially available N-isopropylthiourea, compound #911 (Table 9) was obtained
1H NMR(400MHz，DMSO-d6)：δ8.47(s，1H)，8.29-8.19(m，1H)，8.19(d，J＝9.2Hz，1H)，8.09-8.0(m，1H)，7.83(bs，1H) 7.74(bs, 1H), 7.31 (d, J ═ 8Hz, 1H), 7.14(d, J ═ 6.4Hz, 1H), 5.76(bs, 1H), 4.64-4.53(m, 2H), 4.44(bs, 1H), 4.22-4.09(m, 3H), 3.97(s, 3H), 3.87(d, J ═ 8.6Hz, 1H), 2.63-2.58(m, 1H), 2.46-2.41(m, 1H), 1.79-1.10(m, 24H), 1.27 and 1.26(2xd, J ═ 6.5Hz, 6H). MS: es+：777.0(M+H)+，es-：775.0(M-H)-。
Example 36
Synthesis of Compound #716
1H NMR(400MHz，DMSO-d6)：δ(ppm)8.62(s，1H)，8.13(d，J＝9.2Hz，1H)，7.64-7.54(m，2H)，7.47(d，J＝2.6Hz，1H)，7.16(dd，J＝9.2，2.2Hz，1H)，7.03(d，J＝6.0Hz，1H)，5.63(s，1H)，5.52(q，J＝9.9Hz，1H)，5.26(t，J＝8.9Hz，1H)，4.62(d，J＝11.45Hz，1H)，4.45(dd，J＝9.2，8.27Hz，1H)，4.02(m，1H)，3.93(s，3H)，3.7(dd，J＝7.6，1.0Hz，1H)，2.66(s，3H)，2.55-2.65(m，1H)，2.35-2.45(m，1H)，2.17(q，J＝8.6Hz，1H)，1.65-1.75(m，2H)，1.5-1.35(m，7)，1.15(s，9H)。
MS：705(M+1)，703(M-1)
Example 37
Synthesis of Compound #717
1H NMR(400MHz，DMSO-d6)：8(ppm)8.62(s，1H)，8.15(d，J＝8.9Hz，1H)，7.62(s，1H)，7.49(s，1H)，7.19(dd，J＝9.2，2.2Hz，1H)，7.02(d，J＝5.4Hz，1H)，5.64(s，1H)，5.52(q，J＝9.9Hz，1H)，5.26(t，J＝9.2Hz，1H)，4.63(d，J＝11.44Hz，1H)，4.45(t，J＝9.2Hz，1H)，3.94(s，3H)，3.9-3.8(m，1H)，2.7-2.55(m，1H)，2.4-2.3(m，1H)，2.18(q，J＝8.9Hz，1H)，1.75-1.65(m，2H)，1.5-1.2(m，7H)，1.14(s，9H)。
MS：705(M+1)，703(M-1)
Example 38
Synthesis of Compound #718
1HNMR(400MHz，DMSO-d6)：δ(ppm)：9.55(s，1H)，8.63(s，1H)，8.43(s，1H)，8.13(d，J＝9.2Hz，1H)，7.66(s，1H)，7.46(s，1H)，7.32(d，J＝2.6Hz，1H)，7.10-7.07(m，2H)，5.64-5.54(m，1H)，5.59-5.48(m，1H)，5.33-5.23(m，1H)，4.73-4.61(m，1H)，4.45(dd，J＝7.5，9.1Hz，1H)，4.09-4.00(m，1H)，3.92(s，3H)，3.93-3.83(m，1H)，2.67-2.55(m，2H)，2.53-2.43(m，1H)，2.42-.2.31(m，1H)，2.23-2.12(m，1H)，1.81-1.66(m，2H)，1.52-1.52(m，2H)，1.42-1.25(m，6H)，1.21(s，1H)。
MS：689.3(M+1)，687.3(M-1)
Example 39
Synthesis of Compound #722
1H NMR(400MHz，DMSO-d6)：δ(ppm)：9.70(s，1H)，8.64(s，1H)，8.26(s，1H)，8.14(d，J＝9.2Hz，1H)，7.45(s，1H)，7.30(d，J＝2.5Hz，1H)，7.14-7.06(m，2H)，5.60-5.54(m，1H)，5.58-5.48(m，1H)，5，31-5.23(m，1H)，4.71-4.62(m，1H)，4.49-4.40(m，1H)，4.08-3.99(m，1H)，3.92(s，3H)，3.92-3.84(m，1H)，2.69-2.54(m，2H)，2.53-2.46(m，1H)，2.42-2.31(m，1H)，2.37(s，3H)，2.22-2.13(m，1H)，1.81-1.64(m，2H)，1.54-1.42(m，2H)，1.42-1.27(m，6H)，1.22(s，9H)。
Example 40
Synthesis of Compound #733
1H NMR(400MHz，DMSO-d6)：δ(ppm)：8.75(m，1H)，8.62(s，1H)，8.06(d，J＝9.2Hz，1H)，7.88-7.87(m，1H)，7.48(s，1H)，7.28(d，J＝2.6Hz，1H)，7.05-7.00(m，2H)，6.64-6.63(m，1H)，5.62-5.58(m，1H)，5.55-5.49(m，1H)，5.28-5.24(m，1H)，4.64-4.61(m，1H)，4.48-4.44(m，1H)，4.07-4.03(m，1H)，3.91(s，3H)，3.92-3.85(m，1H)，2.67-2.54(m，2H)，2.53-2.45(m，1H)，2.41-2.34(m，1H)，2.20-2.14(m，1H)，1.75-1.69(m，2H)，1.50-1.43(m，2H)，1.41-1.32(m，6H)，1.17(s，9H)。
MS：689.3(M+1)，687.2(M-1)
EXAMPLE 41
Synthesis of Compound #703
1H NMR(400MHz，DMSO-d6)：δ：8.50(s，1H)，8.19(s，1H)，8.17(s，1H)，8.11-8.00(m，1H)，7.88-7.77(m，1H)，7.73(s，1H)，7.25(d，J＝8.6Hz，1H)，6.93(d，J＝6Hz，1H)，5.89-5.68(m，1H)，4.62(d，J＝11Hz，1H)，4.53(dd，J＝8.3Hz，1H)，4.16-4.07(m，1H)，3.96(s，3H)，3.88(bd，J＝9.5Hz，1H)，3.53-3.43(m，2H)，2.63-2.51(m，1H)，2.46-2.36(m，1H)，1.81-1.62(m，2H)，1.60-1.01(m，15H)，1.24(t，J＝7.4Hz，3H)，1.17(s，9H)。MS：es+：75 1.1(M+H)+，es-：749.1-(M-H)-。
Example 42
Synthesis of Compound #734
1H NMR(400MHz，DMSO-d6)：δ(ppm)：8.62(s，1H)，8.54(s，1H)，8.04(d，J＝9.2Hz，1H)，7.70(s，1H)，7.43(s，1H)，7.24(d，J＝2.6Hz，1H)，7.05-6.98(m，2H)，5.57-5.54(m，1H)，5.55-5.48(m，1H)，5.28-5.24(m，1H)，4.63-4.59(m，1H)，4.47-4.43(m，1H)，4.13-3.99(m，1H)，3.09(s，3H)，3.92-3.83(m，1H)，2.67-2.55(m，2H)，2.53-2.46(m，1H)，2.43-2.31(m，1H)，2.22-2.15(m，1H)，2.15(3H)，1.75-1.70(m，2H)，1.51-1.42(m，2H)，1.41-1.28(m，6H)，1.17(s，9H)。
MS：703.2(M+1)，701.3(M-1)。
Example 43
Synthesis of Compound #738
1H NMR(400MHz，DMSO-d6)：δ(ppm)：8.64(d，J＝2.5Hz，1H)，8.62(s，1H)，8.04(d，J＝9.2Hz，1H)，739(s，1H)，7.24(d，J＝2.5Hz，1H)，7.04(d，J＝6.0Hz，1H)，6.99(dd，J＝2.2，9.2Hz，1H)，6.43(d，J＝2.2Hz，1H)，5.62-5.57(m，1H)，5.56-5.47(m，1H)，5.31-5.22(m，1H)，4.65-4.56(m，1H)，4.45(dd，J＝7.6，8.9Hz，1H)，4.07-4.00(m，1H)，3.90(s，3H)，3.88-3.84(m，1H)，2.68-2.56(m，2H)，2.54-2.43(m，1H)，2.24-2.31(m，1H)，2.34(s，3H)，2.24-2.14(m，1H)，1.80-1.64(m，2H)，1.52-1.43(m，2H)，1.43-1.27(m，6H)，1.18(s，9H)。
MS：703.2(M+1)，701.2(M-1)。
Example 44
Synthesis of Compound #725
1H NMR(400MHz，DMSO-d6)：δ(ppm)：8.62(s，1H)，8.10(d，J＝9.2Hz，1H)，7.57(s，1H)，7.49(s，1H)，7.35(d，J＝2.2Hz，1H)，7.09-7.03(m，2H)，5.65-5.61(m，1H)，5.55-5.49(m，1H)，5.28-5.24(m，1H)，4.62-4.57(m，1H)，4.49-4.45(m，1H)，4.08-4.01(m，1H)，3.93(s，3H)，3.92-3.86(m，1H)，3.20-3.14(m，1H)，2.65-2.56(s，1H)，2.53-2.47(m，1H)，2.42-2.35(m，1H)，2.22-2.15(m，1H)，1.79-1.68(m，2H)，1.50-1.43(m，2H)，1.41-1.28(m，12H)，1,18(s，9H)。
MS：748.2(M+1)，746.2(M-1)。
Example 45
Synthesis of Compound #726
1H NMR(400MHz，DMSO-d6)：δ(ppm)：8.64(s，1H)，8.10(d，J＝9.5Hz，1H)，7.83-7.76(m，2H)，7.60(s，1H)，7.44-7.42(m，1H)，7.18-7.01(m，2H)，5.56-5.49(m，2H)，5.29-5.24(m，1H)，4.66-4.63(m，1H)，4.47-4.42(m，1H)，4.28(s，3H)，4.06-4.02(m，2H)，3.94(s，3H)，3.93-3.86(m，1H)，2.66-2.55(m，2H)，2.42-2.31(m，2H)，2.22-2.14(m，1H)，1.79-1.65(m，2H)，1.52-1.27(m，7H)，1.22(s，9H)。
MS：703.2(M+1)，701.3(M-1)。
Example 46
Synthesis of Compound #906
1H NMR(400MHz，DMSO-d6)：δ(ppm)：8.46(s，1H)，8.06(d，J＝9.2Hz，1H)，7.57(s，1H)，7.49(s，1H)，7.34(m，1H)，7.14-7.05(m，2H)，5.63-5.58(m，1H)，4.66-4.61(m，1H)，4.54-4.44(m，2H)，4.23-4.18(m，1H)，3.93(s，3H)，3.92-3.88(m，1H)，3.21-3.14(m，1H)，2.44-2.33(m，1H)，1.35(d，J＝7Hz，6H)，1.73-1.01(m，26H)。
MS：762.0(M+1)，759.9(M-1)。
Example 47
Synthesis of Compound #907
1H NMR(400MHz，DMSO-d6)：δ(ppm)：8.46(s，1H)，7.98(d，J＝8.9Hz，1H)，7.91-7.89(m，2H)，7.23-7.21(m，2H)，7.07-7.00(m，2H)，6.35-6.32(m，2H)，5.64-5.58(m，1H)，4.65-4.61(m，1H)，4.53-4.47(m，2H)，4.24-4.19(m，1H)，3.90(s，3H)，3.86-3.84(m，1H)，2.40-2.33(m，1H)，1.73-1.01(m，26H)。
MS：702.0(M+1)，699.9(M-1)。
Example 48
Fluorescent Gene (fluorogenic) assay for full-Length NS3-NS4 heterodimer proteins
NS2-NS 5B-3' noncoding region was cloned into pCR by RT-PCR using RNA extracted from serum of HCV genotype 1b infected individuals (supplied by Dr. Bernard Wilems, Hopital St-Luc, Montreal, Quebec, Canada)*3 vector (Invitrogen). The NS3-NS4 region (NS3-NS4AFL) was then subcloned by PCR into pFastBacTMHTa baculovirus expression vector (Gibco/BRL). Sequence of vectorsThe column includes a region encoding a 28-residue N-terminal sequence containing a hexahistidine tag. Using Bac-to-BacTMBaculovirus expression system (Gibco/BRL) to produce recombinant baculovirus. Multiplicity of infection at 27 ℃ by infection with recombinant baculovirus 10 with an infection multiplicity of 0.1-0.26Sf21 cells/ml to express His-NS3-NS4 AFL. True auto-proteolysis occurs during expression, resulting in a non-covalent and stable NS3-NS4A protein complex (termed full-length "FL"). After 48 to 64 hours, the infected culture was harvested by centrifugation at 4 ℃. At 50mM NaPO4The cell pellet was homogenized in 2mM beta-mercaptoethanol, pH7.5, 40% glycerol (weight/volume), in the presence of a mixture of protease inhibitors. His-NS3-NS4AFL extracted from cell lysates was then treated with 1.5% NP-40, 0.5% Triton X-100, 0.5M NaCl and DNase. After ultracentrifugation, the soluble extract was diluted four-fold and bound on a Pharmacia Hi-Trap Ni-chelating column. His-NS3-NS4AFL was eluted in a > 90% pure form using a gradient of 50 to 400mM imidazole (judged by SDS-PAGE). His-NS3-NS4AFL was stored at-80 ℃ in 50mM sodium phosphate, pH7.5, 10% (w/v) glycerol, 0.5M NaCl, 0.25M imidazole and 0.1% NP-40. Melted on ice and diluted before use.
The protease activity of His-NS3-NS4AFL was determined in 50mM Tris-HCl, pH8.0, 0.25M sodium citrate, 0.01% (w/v) n-dodecyl-. beta. -D-maltoside, 1mM TECP. Five (5) μ M internally quenched matrix anthranoyl (anthraniloyl) -ddivparbu [ c (O) -O ] at 23 ℃ in the presence of various concentrations of inhibitor]-AMY(3-NO2) TW-OH and 1.5 nMHI-NS 3-NS4AFL were incubated for 45 minutes. The final DMSO concentration did not exceed 5.25%. The reaction was quenched by the addition of 1M MES, pH 5.8. The N-terminal product was monitored for fluorescence on a Perkin-Elmer LS-50B fluorometer equipped with a 96-well plate reader (excitation wavelength: 325 nm; emission wavelength: 423 nm).
The% inhibition was calculated using the following formula:
100- [ (fluorescent light)inh-fluorescenceblank) /(fluorescence)ctl-fluorescenceblank)×100]
A non-linear curve fitting Hill mode was applied to the inhibition-concentration data and 50% effective concentration (IC.C.) was calculated by using SAS Software (Statistical Software System: SAS Institute, Inc. Cary, N.C.)50)。
Example 49
Radioactive assay for recombinant HCV NS3 protease
Radioactive assay for HCV NS3 protease uses a substrate, DDIVPC-SMSYTW, which is enzymatically cleaved between cysteine and serine residues. The sequence DDIVPC-SMSYTW corresponds to the natural cut position of NS5A/NS5B, in which the cysteine residue in P2 has been replaced by proline. Peptide matrix DDIVPC-SMSYTW and tracer biotin DDIVPC-SMS [2 ] in the presence or absence of an inhibitor125I-Y]TW was cultured with recombinant NS3 protease. Separation of the matrix from the product is accomplished by adding avidin-coated agarose beads to the sample mixture, followed by filtration of the assay mixture. Allowing SMS [2 ] to be found in the filtrate (with or without inhibitor)125I-Y]The amount of TW product was used to calculate the percent substrate conversion and percent inhibition.
A. Reagent
Tris and Tris-HCl (ultrapure) were obtained from Life Technologies. Glycerol (ultrapure), MES and BSA were purchased from Sigma*. TCEP from Pierce and DMSO from Aldrich*And NaOH is obtained from Anachemia*。
Determination of buffer solution: 50mM Tris-HCl, pH7.5, 30% (w/v) glycerol, 2% (w/v) CHAPS, 1 mg/ml BSA, 1mM TCEP (TCEP added before use, from 1M stock solution in water).
Matrix: DDIVPC-SMSYTW, 25 microliter final concentration (obtained from 2mM stock in DMSO, stored at-20 ℃ to avoid oxidation). Tracer: reduced mono-iodinated substrate (Biotin-DDIVPC-SMS [2 ]125I-Y]TW) (≈ 1nM final concentration).
HCV NS3 protease type 1b at a final concentration of 25nM (from 50mM sodium phosphate, pH7.5, 10% glycerol, 300mM NaCl, 5mM DTT, 0.01% NP-40).
B. Draft table
The assay was performed in 96-well polypropylene plates. Each well contains:
20 μ l matrix/tracer in assay buffer;
10 microliters of + -inhibitor in 20% DMSO/assay buffer;
10 microliters of NS3 protease 1 b.
Blanks (no inhibitor and no enzyme) and controls (no inhibitor) were prepared on the same assay tray.
The enzyme reaction was started by adding the enzyme solution and incubating the assay mixture at 23 ℃ for 60 minutes under gentle agitation. Twenty (20) microliters of 0.025 NaOH was added to quench the enzyme reaction.
In Millipore*Twenty (20) microliters of avidin-coated agarose beads (from Pierce) were added to the MADP N65 filter disks*). The quenched assay mixture was transferred to the filter tray and incubated at 23 ℃ for 60 minutes with gentle agitation.
Use of Millipore*The MultiScreen Vacuum manufactured Filtration device filters the plate and transfers 40 microliters of filtrate to an opaque 96-well culture plate containing 60 microliters of scintillation fluid per well.
In Packard*On a TopCount device, use125I-liquid procedure (protocol) for 1 minute to calculate the filtrate.
The% inhibition was calculated using the formula:
100- [ (count)inh-countingblank) /(count)ctl-countingblank)×100]
Will fit into Hill modeWas applied to the inhibition-concentration data, and 50% effective concentration (ic.c.) was calculated by using SAS Software (Statistical Software System: sasigntite, inc50)。
Example 50
Specific assay
Determination of the specificity of the compounds against various serine proteases: human leukocyte elastase, porcine pancreatic elastase and bovine pancreatic alpha-chymotrypsin and one cysteine protease: human liver cathepsin B. In all cases, the 96-well plate format procedure uses a chromogen matrix specific for each enzyme. Each assay included pre-incubation of the enzyme-inhibitor for 1 hour at room temperature, followed by addition of the matrix and hydrolysis to approximately 30% conversion by UV Thermomax*Microtiter plate reader or fluorescent Perkin-Elmer*LS50B were measured on a culture dish reader. Keeping the concentration of the substrate as much as possible like KMAs low as to reduce competition by the matrix. The compounds varied from 300 to 0.06uM depending on their potency.
The final conditions for each assay were as follows: 50mM Tris-HCl pH8, 0.5M Na2SO450mM NaCl, 0.1mM EDTA, 3% DMSO, 0.01% Tween, containing
[ 100. mu.M Succ-AAPF-pNA and 250pM α -chymotrypsin ], [ 133. mu.M Succ-AAA-pNA and 8nM porcine elastase ], [ 133. mu.M Succ-AAV-pNA and 8mM leukocyte elastase ]; or
[100mM NaHPO4pH6, ImM EDTA, 3% DMSO, 1mM TCEP, 0.01% Tween 20, 4. mu. M Z-FR-AMC (7-amino-4-methylcoumarin) and 0.5nM cathepsin B (activation of the starting enzyme in a buffer containing 20mM TCEP just prior to use)]。
Representative examples of porcine pancreatic elastase are summarized below:
the following were added to a flat-bottomed 96-well culture dish of polystyrene (Cellwells, Corning) using a Biomek liquid manipulator (Beckman):
40 microliter assay buffer (50mM Tris-HCl, pH8, 1M Na2 SO)4，50mM NaCl，0.1mMEDTA)；
20 microliters of enzyme solution (50mM Tris-HCl, pH8, 50mM NaCl, 0.1mM EDTA, 0.02% Tween-20, 40nM porcine pancreatic elastase); and
20 microliters of inhibitor solution (50mM Tris-HCl, pH8, 50mM NaCl, 0.1mM EDTA, 0.02% Tween-20, 1.5 mM-0.3. mu.M inhibitor, 15% v/v DMSO).
After pre-incubation for 60 minutes at room temperature, 20. mu.l of matrix solution (50mM Tris-HCl pH8, 0.5M Na) was added to each well2SO450mM NaCl, 0.1mM EDTA, 665. mu. MSucc-AAA-pNA) and further incubation of the reaction at room temperature for 60 minutes, followed by UVThermomax*The absorbance was read on an incubation plate reader. One row of wells is control (no inhibitor) and blank (no inhibitor and no enzyme).
On different plates, serial 2-fold dilutions of inhibitor solutions were done by liquid manipulator using 50mM Tris-HCl pH8, 50mM NaCl, 0.1mM EDTA, 0.02% Tween-20, 15% (v/v) DMSO. All other specific assays were done in a similar manner.
The% inhibition was calculated using the formula:
[1-((UVinh-UVblank)/(UVctl-UVblank))]×100
a non-linear curve fitting Hill mode was applied to the inhibition-concentration data and 50% effective concentration (IC.C.) was calculated by using SAS Software (Statistical Software System: SAS Institute, Inc. Cary, N.C.)50)。
Example 51
Cell-based assay for the NS3 protease
The assay was performed with Huh-7 cells, a human cell line derived from liver cancer, co-transfected with 2 DNA constructs:
one (designated NS3) expressed a partial HCV non-structural polyprotein fused in the following order to the tTA protein via the NS5A-NS5B cleavage site; NS3-NS4A-NS4B-NS5A- (NS5B) -tTA, wherein (NS5B) represents the first 6 amino acids of NS 5B. The polyprotein is expressed under the control of the CMV promoter, and the other (called SEAP) under the regulation of the tTA-responsive promoter, the reporter protein, secreted alkaline phosphatase (SEAP).
The first construction resulted in the expression of the polyprotein, from which a different mature protein was released by cleavage with NS3 protease. It is believed that the mature viral proteins form complexes on the membrane of the endoplasmic reticulum. tTA is a fusion protein described by Gossen and Bujard (Proc. Natl. Acad. Sci. USA89 (1992); 5547-. the release of tTA protein requires NS 3-dependent cleavage at the NS5A-NS5B cleavage site between NS5A and itself. The final cleavage allows the tTA to move to the nucleus and transactivates the SEAP gene. Thus, reduction of NS3 proteolytic activity resulted in the confinement of tTA in the cytoplasm with concomitant reduction of SEAP activity.
To control cellular activities beyond the inhibition of NS3 protease by this compound, parallel co-transfection was performed using constructs expressing tTA alone, and the same reporter construct, to render SEAP activity independent of NS3 protease.
Protocol of the assay: constructed with two DNAs, Huh-7 cells grown in CHO-SFMII (Life technologies) + 10% FCS (fetal calf serum) were co-transfected at the following ratio; every 4X 1067 micrograms of NS3-500 nanograms of SEAP +800 microliters of FuGENE (Boehringer Mannheim) were used for each Huh-7 cell. After 5 hours at 37 ℃, cells were washed, trypsinized, and plated (at 80,000 cells/well) onto 96-well plates containing a range of concentrations of test compound. After 24 hours incubation, SEAP activity in the medium was measured using the Phospha-Light tool kit (Tropix).
Using SASThe software performs an analysis of% inhibition of SEAP activity at the compound concentration concerned to obtain EC50。
List of compounds
Representative compounds of the invention are listed below.
All compounds listed in tables 1 to 9 were found to be active in the enzyme assay provided in example 48. The numbers with asterisks indicate the enzyme activity obtained with the radioactivity assay provided in example 49, with an IC at 50. mu.M50. In these enzyme assays, the following fractionation was used: a is more than or equal to 1 mu M; 1 mu M > B > 0.1 mu M; and C is less than or equal to 0.1 mu M.
Several compounds were tested in the specificity assay provided in example 50 and found to be specific for NS3 protease. In general, the results from the different specificity assays are as follows: HLE > 300 μ M; PPE > 300. mu.M; alpha-Chym > 300. mu.M; Cat.B > 300. mu.M; indicating that these compounds are highly specific for hcv ns3 protease and are not expected to show serious side effects. In addition, some of these compounds were tested in the cell-based assay described in example 51 and found to have an EC of less than 10 μ M50It is strongly suggested that these compounds can cross cell membranes. In particular the compounds of tables 7, 8 and 9, have been evaluated in cellular assays and the results indicated in the last column. In cellular assays, the following codes were used: a is more than 1 mu M; b is less than or equal to 1 mu M.
The following abbreviations are used in the following tables:
MS: electrospray mass spectrometry data: m/z MH+Unless otherwise indicated by an asterisk ═ m/z MH-: ac; acetyl; bn: a benzyl group; boc: tert-butoxycarbonyl; ph: a phenyl group; pr: and (4) propyl.
TABLE 1
At R1A single stereoisomer of (A)
Compound number Double bond D-R1Stereochemistry of bond R22 MS Enzyme activity
101 12, 13-trans 1R, D are the same as amide Phenyl radical 685.8 A*
102 Is free of 1R, D are the same as the acid Phenyl radical 687.2 C
103 Is free of 1RD is the same as amide Phenyl radical 687.2 A*
TABLE 2
At R1A single stereoisomer of (A)
Compound number R3 R4 Double bond D-R1Bond stereochemistry R21 R22 MS Enzyme activity
202 NH-Boc H 11, 12-trans 1R or 1S, D is the same as the acid H H 593.7 B
203 NH-acetyl H 11, 12-trans 1R or 1S, D is the same as the acid H H 535.6 A
205 NH-Boc 11-OH12-OH cis Is free of 1R or 1S, D is the same as the acid H H 627.7 B
206 NH-Boc H 13, 14-cis form 1R, ID is the same as the acid H H 593.7 C
207 NH-Boc H 13, 14-cis form 1R, ID is the same as the acid OMe H 623.7 C
208 NH-Boc H 13, 14-cis form 1R, D are the same as the acid OMe Phenyl radical 699.8 C
209 NH-C(O)-NH-tBu H 13, 14-cis form 1R, D are the same as the acid OMe Phenyl radical 698.8 C
TABLE 3
At R1A single stereoisomer of (A)
TABLE 4
D-R1Bond on the same side as acid
TABLE 5
D-R1Bond on the same side as acid
Compound # 10-X 11-X 12-X MS Enzyme activity
501 CH2 O CH2 703.2 C
502 CH2 CH2 CH2 701 C
503 CH2 CH2 NH 702.3 A
504 CH2 CH2 N(Me) 716.3 A*
505 CH2 CH2 N(CO)Me 744.3 B
506 CH2 CH2 N(CO)Ph 806.3 B
507 NH CH2 CH2 702.3 C
508 N(CO)Me CH2 CH2 744.3 C
TABLE 6
D-R1Bonding to the same side as the acid
TABLE 7
D-R1Bond on the same side as acid
TABLE 8
D-R1Bond ipsilateral to acid, double bond 13, 14: cis form
TABLE 9
D-R1Bond on the same side as acid

Claims (9)

1. A compound having the following formula (A):
wherein X is PG or R2；
PG is a protecting group, or PG on pyrrolidone is H,
R2is composed of
W is CH or N;
R21is H, halogen, C1-6Alkyl radical, C3-6Cycloalkyl radical, C1-6Haloalkyl, C1-6Alkoxy radical, C3-6Cycloalkoxy, hydroxy or N (R)23)2Wherein each R is23Are respectively H, C1-6Alkyl or C3-6A cycloalkyl group;
R22is H, halogen, C1-6Alkyl radical, C3-6Cycloalkyl radical, C1-6Haloalkyl, C1-6Sulfanyl, C1-6Alkoxy radical, C3-6Cycloalkoxy, C2-7Alkoxyalkyl group, C3-6Cycloalkyl radical, C6 or 10Aryl or Het, wherein Het is a five-, six-or seven-membered saturated or unsaturated heterocyclic ring containing one to four heteroatoms selected from nitrogen, oxygen and sulfur; said cycloalkyl, aryl or Het is substituted by R24Is substituted in which R24Is H, halogen, C1-6Alkyl radical, C3-6Cycloalkyl radical, C1-6Alkoxy radical, C3-6Cycloalkoxy, NO2、N(R25)2、NH-C(O)-R25(ii) a Or NH-C (O) -NH-R25Wherein each R is25Are respectively H, C1-6Alkyl or C3-6A cycloalkyl group; or R24Is NH-C (O) -OR26Wherein R is26Is C1-6Alkyl or C3-6A cycloalkyl group;
a' is a protected carboxylic acid;
n is 2.
2. The compound of claim 1, wherein PG on the pyrrolidone is H is:
3. a process for the preparation of a compound of formula (a) according to claim 1, which process comprises reacting a compound of formula (B) with a compound of formula (C):
wherein PG, X, A' and n are as defined in claim 1.
4. A process according to claim 3, which comprises converting the compound of formula (a) into a compound of formula (D) by cleaving the protecting group represented by PG on pyrrolidone into H:
wherein PG is a protecting group, and PG is a protecting group,
X，R2and a' and n are as defined in claim 1.
5. A compound having the following formula (E):
wherein X is PG or R2；
PG is a protecting group;
R2as defined in claim 1;
a' is a protected carboxylic acid;
R3is hydroxy, NH2Or formula-NH-R31Wherein R is31Is C6 or 10Aryl, heteroaryl, -C (O) -R32、-C(O)-NHR32OR-C (O) -OR32，
Wherein R is32Is C1-6Alkyl or C3-6A cycloalkyl group; (ii) a
n is 0 or 2, and
(1) when N is 0, D' is a 5-atom saturated alkylene chain, optionally containing 1 to 3 groups independently selected from O, S or N-R41A heteroatom of (a);
(2) when N is 2, D' is a 3-atom saturated alkylene chain, optionally containing 1 to 3 groups independently selected from O, S or N-R41The heteroatom(s) of (a),
wherein R is41Is H, C1-6Alkyl radical, C3-6Cycloalkyl or-C (O) -R42Wherein R is42Is C1-6Alkyl radical, C3-6Cycloalkyl or C6 or 10And (4) an aryl group.
6. A process according to claim 5, which comprises reacting a compound of formula (A) wherein PG on the pyrrolidone is H, with a compound of formula (F):
wherein X, A', n, R3And D' are both as defined in claim 5.
7. A compound having the following formula (J):
wherein R is2、R3As defined in claim 5;
m is 1 to 5;
n is 1 to 5;
a' is a protected carboxylic acid;
cbz is benzyloxycarbonyl.
8. A compound having the following formula (K):
wherein R is2、R3As defined in claim 5;
m is 1 to 5;
n is 1 to 5;
a' is a protected carboxylic acid;
cbz is benzyloxycarbonyl.
9. A process for the preparation of a compound of formula (K) according to claim 8, which process comprises subjecting a compound of formula (J) to hydroboration:
wherein R is2、R3M, n, A' and Cbz are as defined in claim 8.
HK05106434.6A 1999-04-06 2005-07-27 Macrocyclic peptides active against the hepatitis c virus HK1073845A (en)

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Application Number Priority Date Filing Date Title
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