U.S. patent application number 10/828771 was filed with the patent office on 2004-10-21 for n-alkanoylphenylalanine derivatives.
Invention is credited to Chen, Li, Guthrie, Robert William, Huang, Tai-Nang, Hull, Kenneth Gregory, Sidduri, Achytharao, Tilley, Jefferson Wright.
Application Number | 20040210051 10/828771 |
Document ID | / |
Family ID | 27369129 |
Filed Date | 2004-10-21 |
United States Patent
Application |
20040210051 |
Kind Code |
A1 |
Chen, Li ; et al. |
October 21, 2004 |
N-alkanoylphenylalanine derivatives
Abstract
Compounds of the formula: 1 are disclosed which have activity as
inhibitors of binding between VCAM-1 and cells expressing VLA-4.
Such compounds are useful for treating diseases whose symptoms
and/or damage are related to the binding of VCAM-1 to cells
expressing VLA-4.
Inventors: |
Chen, Li; (Westfield,
NJ) ; Guthrie, Robert William; (Saddle Brook, NJ)
; Huang, Tai-Nang; (Lexington, MA) ; Sidduri,
Achytharao; (Livingston, NJ) ; Tilley, Jefferson
Wright; (North Caldwell, NJ) ; Hull, Kenneth
Gregory; (Cambridge, MA) |
Correspondence
Address: |
HOFFMANN-LA ROCHE INC.
PATENT LAW DEPARTMENT
340 KINGSLAND STREET
NUTLEY
NJ
07110
|
Family ID: |
27369129 |
Appl. No.: |
10/828771 |
Filed: |
April 21, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10828771 |
Apr 21, 2004 |
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10117616 |
Apr 5, 2002 |
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10117616 |
Apr 5, 2002 |
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09138353 |
Aug 21, 1998 |
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6455550 |
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60056929 |
Aug 22, 1997 |
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60094591 |
Jul 29, 1998 |
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Current U.S.
Class: |
544/59 ; 544/162;
544/386; 546/225; 548/200; 548/236; 548/333.5; 548/530 |
Current CPC
Class: |
C07C 2601/08 20170501;
C07C 255/57 20130101; C07D 257/04 20130101; C07C 317/44 20130101;
C07D 211/64 20130101; C07C 275/60 20130101; C07C 335/08 20130101;
C07C 271/22 20130101; C07C 235/40 20130101; C07D 277/14 20130101;
C07C 275/30 20130101; C07C 2601/14 20170501; C07C 275/24 20130101;
C07D 213/89 20130101; C07D 213/81 20130101; C07D 487/04 20130101;
C07D 261/18 20130101; C07D 249/06 20130101; C07C 255/41 20130101;
C07D 401/06 20130101; C07C 235/64 20130101; C07C 275/18 20130101;
C07C 323/60 20130101; C07D 231/14 20130101; C07D 295/088 20130101;
C07D 317/30 20130101; C07C 237/24 20130101; C07D 317/68 20130101;
C07D 217/26 20130101; C07C 235/82 20130101; C07C 233/87 20130101;
C07D 233/32 20130101 |
Class at
Publication: |
544/059 ;
544/162; 544/386; 546/225; 548/200; 548/236; 548/333.5;
548/530 |
International
Class: |
C07D 279/12; C07D
277/04; C07D 263/34; C07D 211/32 |
Claims
1. A compound of the formula: 597wherein: one of X and X' is
hydrogen, halogen, or lower alkyl, and the other is a group of the
formula: 598wherein 599is a 5- or 6-membered heteroaromatic ring
containing 1, 2 or 3 heteroatoms selected from N, O, and S; or
600is a 9- or 10-membered bicyclic heteroaromatic ring containing
1, 2, 3 or 4 heteroatoms selected from O, S, and N, a is 0 or 1,
R.sub.1 is hydrogen or lower alkyl, R.sub.15 is hydrogen, halogen,
nitro, lower alkyl sulfonyl, cyano, lower alkyl, lower alkoxy,
lower alkoxycarbonyl, carboxy, lower alkyl aminosulfonyl,
perfluorolower alkyl, lower alkylthio, hydroxy lower alkyl, alkoxy
lower alkyl, alkylthio lower alkyl, alkylsulfinyl lower alkyl,
alkylsufonyl lower alkyl, lower alkylsulfinyl, lower alkanoyl,
aroyl, aryl, aryloxy or a group of the formula
R.sub.17--C.ident.C--, and R.sub.16 is hydrogen, halogen, nitro,
cyano, lower alkyl, OH, perfluorolower alkyl, or lower alkylthio,
and R.sub.30 is hydrogen or lower alkyl, or is absent; and Y is a
group of the formula: 601wherein: R.sub.22 and R.sub.23 are
independently aryl, heteroaryl or lower alkyl which is
unsubstituted or substituted by one or more chloro, bromo, nitro,
hydroxy, lower alkoxy, aryl, lower alkanoyl, aroyl or cyano,
R.sub.24 is aryl, cyano, alkylsulfonyl or lower alkyl or alkenyl
unsubstituted or substituted by an aryl or heteroaryl ring, and
when R.sub.22 is aryl and R.sub.23 is aryl or lower alkyl,
hydrogen, and the total number of carbon atoms in R.sub.22,
R.sub.23 and R.sub.24 is from 6 to 14; or Y is a 3-7 membered ring
of the formula: 602wherein: R.sub.25 is lower alkyl, unsubstituted
or fluorine substituted lower alkenyl, or a group of formula
R.sub.26--(CH.sub.2).sub.e--, R.sub.26 is aryl, heteroaryl, azido,
cyano, hydroxy, lower alkoxy, lower alkoxycarbonyl, lower alkanoyl,
lower alkylthio , lower alkyl sulfonyl, lower alkyl sulfinyl,
perfluoro lower alkanoyl, nitro, or R.sub.26 is a group of formula
--NR.sub.28R.sub.29, wherein: R.sub.28 is hydrogen or lower alkyl,
R.sub.29 is hydrogen, lower alkyl, lower alkoxycarbonyl, lower
alkoxycarbonylaminocarbonyl, lower alkanoyl, aroyl, heteroaroyl,
perfluoro lower alkanoyl, lower alkyl sulfonyl, lower
alkylaminocarbonyl, arylaminocarbonyl, heterocycloalkyl carbonyl,
lower alkylaminothiocarbonyl, or R.sub.28 and R.sub.29 taken
together with the nitrogen atom to which they are attached form a
4, 5 or 6-membered saturated heterocyclic ring containing one or
two heteroatoms with the second heteroatom being O, S, or
N--R.sub.27; Q is --(CH.sub.2).sub.fO--, --(CH.sub.2).sub.fS--,
--(CH.sub.2).sub.f--, or when f=0, a bond, R.sub.27 is hydrogen,
lower alkyl, aryl, lower alkanoyl, aroyl, or lower alkoxycarbonyl;
the carbon atoms in said ring are unsubstituted or substituted by
lower alkyl or halogen, e is an integer from 0 to 4, and f is an
integer from 0 to 3; and Z is hydrogen or lower alkyl; and the
pharmaceutically acceptable salts and esters thereof.
2. The compound of claim 1 wherein X' is hydrogen.
3. The compound of claim 2 having the formula: 603wherein Q' is
unsubstituted or lower alkyl substituted --(CH.sub.2).sub.f--, f is
1, 2 or 3, and X and R.sub.25 are as in claim 1.
4. The compound of claim 3 wherein f is 2 whereby said compound is
of the formula: 604wherein R.sub.m1, R.sub.m2 and R.sub.m3 are
independently hydrogen or lower alkyl, and R.sub.25 is lower alkyl,
lower alkenyl which is unsubstituted or substituted by fluorine, or
a group of the formula R.sub.26--(CH.sub.2).sub.e-- wherein
R.sub.26 and e are as in claim 1.
5. The compound of claim 4 wherein said compound is of the formula:
605wherein R.sub.m1, R.sub.m2 and R.sub.m3 and R.sub.25 are as in
claim 4.
6. The compound of claim 4 wherein X is a group of the formula:
606
7. The compound of claim 6 wherein a is 0.
8. The compound of claim 4 wherein said compound is of the formula:
607wherein R.sub.m1, R.sub.m2 and R.sub.m3 and R.sub.25 are as in
claim 4.
9. The compound of claim 8 wherein said compound is of the formula:
608
10. The compound of claim 9 wherein X is a group of the formula:
609
11. The compound of claim 10 wherein a is 0.
12. The compound of claim 11 having the formula: 610
13. The compound of claim 8 wherein R.sub.m1, R.sub.m2 and R.sub.m3
are all hydrogen, and R.sub.25 is lower alkyl or lower alkenyl
which is unsubstituted or substituted by fluorine.
14. The compound of claim 4 having the formula: 611wherein X,
R.sub.26 and e are as in claim 1.
15. The compound of claim 14 wherein e is 0 whereby said compound
is of the formula: 612
16. The compound of claim 15 wherein R.sub.26 is cyano or aryl.
17. The compound of claim 16 wherein R.sub.26 is cyano or phenyl
which is unsubsituted or mono-substituted by halogen, lower alkyl
or lower alkoxy.
18. The compound of claim 17 wherein said compound is of the
formula: 613
19. The compound of claim 18 wherein X is a group of the formula:
614wherein R.sub.1 and a are as in claim 1.
20. The compound of claim 19 wherein a is 0.
21. The compound of claim 20 having the formula: 615
22. The compound of claim 17 wherein R.sub.26 is phenyl which is
unsubsituted or mono-substituted by halogen, lower alkyl or lower
alkoxy.
23. The compound of claim 22 wherein X is a group of the formula:
616
24. The compound of claim 23 wherein a is 0.
25. The compound of claim 24 having the formula: 617
26. The compound of claim 24 having the formula: 618
27. The compound of claim 24 having the formula: 619
28. The compound of claim 24 having the formula: 620
29. The compound of claim 24 having the formula: 621
30. The compound of claim 22 wherein X is a group of the formula:
622wherein R.sub.1 and a are as in claim 1.
31. The compound of claim 30 wherein a is 0.
32. The compound of claim 31 having the formula: 623
33. The compound of claim 14 wherein e is 1 whereby said compound
is of the formula: 624
34. The compound of claim 33 wherein R.sub.26 is lower alkoxy or
aryl.
35. The compound of claim 34 wherein R.sub.26 is methoxy, or is
phenyl which is unsubsituted, mono-substituted by halogen, lower
alkoxy, cyano or tetrazolyl which tetrazolyl is unsubstituted or
monosubstituted by methyl, or disubstituted by lower alkoxy.
36. The compound of claim 35 wherein R.sub.26 is methoxy.
37. The compound of claim 35 wherein R.sub.26 is phenyl which is
mono- or di-substituted by lower alkoxy.
38. The compound of claim 37 wherein R.sub.26 is phenyl which is
mono-substituted by lower alkoxy.
39. The compound of claim 38 wherein R.sub.26 is a group of the
formula: 625
40. The compound of claim 39 wherein X is a group of the formula:
626
41. The compound of claim 40 wherein a is 0.
42. The compound of claim 41 having the formula: 627
43. The compound of claim 35 wherein R.sub.26 is unsubstituted
phenyl.
44. The compound of claim 43 wherein X is a group of the formula:
628
45. The compound of claim 44 wherein a is 0.
46. The compound of claim 45 having the formula: 629
47. The compound of claim 37 wherein R.sub.26 is phenyl which is
di-substituted by lower alkoxy.
48. The compound of claim 47 wherein R.sub.26 is a group of the
formula: 630
49. The compound of claim 35 wherein R.sub.26 is phenyl
monosubstituted by chloro, tetrazolyl, which is unsubstituted or
mono-subsituted by methyl, or cyano.
50. The compound of claim 49 wherein R.sub.26 is a group of the
formula: 631
51. The compound of claim 33 wherein R.sub.26 is tetrazolyl or
cyano.
52. The compound of claim 14 wherein e is 2 whereby said compound
is of the formula: 632
53. The compound of claim 52 wherein R.sub.26 is
--NR.sub.28R.sub.29 where R.sub.28 and R.sub.29 are as in claim
1.
54. The compound of claim 53 wherein R.sub.28 and R.sub.29 are
independently hydrogen or lower alkyl.
55. The compound of claim 53 wherein R.sub.28 is hydrogen and
R.sub.29 is lower alkoxycarbonyl, lower alkylsulfonyl,
heterocycloalkyl, aroyl or heteroaroyl.
56. The compound of claim 53 wherein R.sub.28 is lower alkyl and
R.sub.29 is lower akanoyl, lower alkoxycarbonyl or lower
alkylaminocarbonyl.
57. The compound of claim 53 wherein R.sub.28 and R.sub.29 taken
together with the nitrogen atom to which they are attached form a
morpholinyl group.
58. The compound of claim 52 wherein R.sub.26 is lower alkyl
sulfonyl, lower alkyl thio, lower alkyl sulfinyl, lower alkylthio,
azido, cyano, hydroxy, lower alkoxy, lower alkanoyl or lower
alkanoylamino.
59. The compound of claim 14 wherein e is 3 whereby said compound
is of the formula: 633
60. The compound of claim 59 wherein R.sub.26 is lower alkyl
sulfonyl, lower alkyl sulfinyl, lower alkylthio, azido, cyano,
hydroxy, lower alkoxy, lower alkanoyl or lower alkanoylamino.
61. The compound of claim 14 wherein e is 4 whereby said compound
is of the formula: 634
62. The compound of claim 61 wherein R.sub.26 is lower alkyl
sulfonyl, lower alkyl sulfinyl, lower alkylthio, azido, cyano,
hydroxy, lower alkoxy, lower alkanoyl or lower alkanoylamino.
63. The compound of claim 62 wherein a is 0, R.sub.1 is hydrogen
and R.sub.26 is lower alkyl sulfonyl.
64. The compound of claim 63 wherein Het is a 6-membered
heteroaromatic ring of the formula: 635
65. The compound of claim 64 having the formula: 636
66. The compound of claim 64 having the formula: 637
67. The compound of claim 64 having the formula: 638
68. The compound of claim 63 wherein Het is a 5-membered
heteroaromatic ring of the formula: 639
69. The compound of claim 68 having the formula: 640
70. The compound of claim 68 having the formula: 641
71. The compound of claim 68 having the formula: 642
72. The compound of claim 63 wherein Het is a 9-membered
heteraromatic ring of the formula: 643
73. The compound of claim 72 having the formula: 644
74. The compound of claim 3 wherein f is 1 whereby said compound is
of the formula: 645wherein R.sub.25 is lower alkyl or a group of
the formula R.sub.26--(CH.sub.2).sub.e-- wherein R.sub.26 and e are
as in claim 1.
75. The compound of claim 74 wherein R.sub.25 is a group of the
formula R.sub.26--(CH.sub.2).sub.e--.
76. The compound of claim 75 wherein e is 1 whereby said compound
is of the formula: 646
77. The compound of claim 76 wherein R.sub.26 is lower alkoxy or
aryl.
78. The compound of claim 77 wherein R.sub.26 is methoxy or phenyl
which is unsubsituted, mono-substituted by halogen, lower alkoxy,
cyano or tetrazolyl which tetrazolyl is unsubstituted or
monosubstituted by methyl, or disubstituted by lower alkoxy.
79. The compound of claim 78 wherein R.sub.26 is phenyl which is
monosubstituted by tetrazolyl which terazolyl is monosubstituted by
methyl.
80. The compound of claim 79 wherein R.sub.26 is of the formula:
647
81. The compound of claim 3 wherein f is 3 and said compound is of
the formula: 648wherein R.sub.25 and X are as in claim 1.
82. The compound of claim 81 wherein said compound is of the
formula: 649wherein R.sub.25 and X are as in claim 1.
83. The compound of claim 82 wherein R.sub.25 is lower alkyl.
84. The compound of claim 83 wherein X is a group of the formula:
650
85. The compound of claim 84 wherein a is 0.
86. The compound of claim 85 having the formula: 651
87. The compound of claim 81 wherein said compound is of the
formula: 652wherein R.sub.25 and X are as in claim 1.
88. The compound of claim 87 wherein R.sub.25 is lower alkyl.
89. The compound of claim 88 wherein X is a group of the formula:
653
90. The compound of claim 89 wherein a is 0.
91. The compound of claim 90 having the formula: 654
92. The compound of claim 87 wherein R.sub.25 is a group of the
formula R.sub.26--(CH.sub.2).sub.e-- wherein R.sub.26 and e are as
in claim 1.
93. The compound of claim 92 wherein e is 0 whereby said compound
is of the formula: 655
94. The compound of claim 93 wherein R.sub.26 is lower alkoxy or
aryl.
95. The compound of claim 94 wherein R.sub.26 is methoxy, or is
phenyl which is: 1) unsubstituted, 2) mono-substituted by halogen,
lower alkoxy, cyano or tetrazolyl which tetrazolyl is unsubstituted
or monosubstituted by methyl, or 3) disubstituted by lower
alkoxy.
96. The compound of claim 95 wherein R.sub.26 is phenyl which is
unsubsituted or mono-substituted by lower alkoxy.
97. The compound of claim 96 wherein X is a group of the formula:
656
98. The compound of claim 97 wherein a is 0.
99. The compound of claim 98 having the formula: 657
100. The compound of claim 98 having the formula: 658
101. The compound of claim 92 wherein e is 1 whereby said compound
is of the formula: 659
102. The compound of claim 101 wherein R.sub.26 is lower alkoxy or
aryl.
103. The compound of claim 102 wherein R.sub.26 is methoxy, or is
phenyl which is: 1) unsubstituted, 2) mono-substituted by halogen,
lower alkoxy, cyano or tetrazolyl which tetrazolyl is unsubstituted
or monosubstituted by methyl, or 3) disubstituted by lower
alkoxy.
104. The compound of claim 103 wherein R.sub.26 is phenyl which is
unsubsituted or mono-substituted by lower alkoxy.
105. The compound of claim 1 having the formula: 660wherein X,
R.sub.22, R.sub.23 and R.sub.24 are as in claim 1.
106. The compound of claim 105 wherein R.sub.22 and R.sub.23 are
independently unsubstituted phenyl or lower alkyl, and R.sub.24 is
hydrogen or unsubstituted lower alkyl or alkenyl.
107. The compound of claim 106 wherein R.sub.22, R.sub.23 and
R.sub.24 are lower alkyl.
108. The compound of claim 107 wherein X is a group of the formula:
661
109. The compound of claim 108 wherein a is 0.
110. The compound of claim 108 wherein R.sub.22 is methyl, R.sub.23
is ethyl and R.sub.24 is n-butyl.
111. The compound of claim 110 having the formula: 662
112. The compound of claim 106 wherein R.sub.22 is unsubstituted
phenyl, R.sub.23 is lower alkyl and R.sub.24 is hydrogen.
113. The compound of claim 112 wherein X is a group of the formula:
663
114. The compound of claim 113 wherein a is 0.
115. The compound of claim 114 having the formula: 664
116. The compound of claim 114 having the formula: 665
117. The compound of claim 106 wherein R.sub.22 and R.sub.23 are
unsubstituted phenyl and R.sub.24 is hydrogen.
118. The compound of claim 117 wherein X is a group of the formula:
666
119. The compound of claim 118 wherein a is 0.
120. The compound of claim 119 having the formula: 667
121. The compound of claim 1 which is an ester of the formula:
668wherein: R.sub.31, is lower alkyl; or R.sub.31 a group of
formula: 669 wherein: R.sub.32 is hydrogen or lower alkyl, R.sub.33
is hydrogen, lower alkyl, or aryl, R.sub.34 is hydrogen or lower
alkyl, h is an integer from 0 to 2, g is an integer from 0 to 2,
and the sum of h and g is 1 to 3; or R.sub.31 is a group of
formula: 670 wherein: R.sub.32, g, and h are as above for P-1, T is
O, S, --(CH.sub.2).sub.j--, a group of the formula N--R.sub.35, or
when j=0, a bond, R.sub.35 is hydrogen, lower alkyl, lower
alkanoyl, or lower alkoxycarbonyl, and j is 0, 1 or 2 and wherein
X, X' Y and Z are as in claim 1.
122. The compound of claim 121 having the formula: 671wherein Q" is
unsubstituted or lower alkyl substituted --(CH.sub.2).sub.f--, f is
1, 2 or 3, and X and R.sub.25 are as in claim 1.
123. The compound of claim 122 having the formula: 672wherein X,
R.sub.26 and e are as in claim 1.
124. The compound of claim 123 wherein e is 4 whereby said compound
is of the formula: 673
125. The compound of claim 124 wherein R.sub.26 is lower alkyl
sulfonyl, lower alkyl thio, lower alkyl sulfinyl, lower alkylthio,
azido, cyano, hydroxy, lower alkoxy, lower alkanoyl or lower
alkanoylamino.
126. The compound of claim 125 wherein a is 0, R.sub.1 is hydrogen,
R.sub.26 is lower alkyl sulfonyl or lower alkoxy and Het is
pyridyl.
127. The compound of claim 126 wherein R.sub.31 is ethyl,
2-(N,N-diethylamino)ethyl or 2-(4-morpholinyl)ethyl.
128. The compound of claim 127 having the formula: 674
129. The compound of claim 127 having the formula: 675
130. The compound of claim 1 wherein Z is lower alkyl.
131. The compound of claim 130 having the formula: 676wherein Q'"
is unsubstituted or lower alkyl substituted --(CH.sub.2).sub.f--, f
is 1, 2 or 3, and X and R.sub.25 are as in claim 1.
132. The compound of claim 131 having the formula: 677
Description
[0001] This application is a divisional of Ser. No. 10/117,616,
filed Apr. 5, 2002, which is a divisional of Ser. No. 09/138,353,
filed Aug. 21, 1998, now U.S. Pat. No. 6,455,550 issued Sep. 24,
2002, which claims benefit of Provisional Application No.
60/056,929 filed Aug. 22, 1997 and Provisional Application No.
60/094,591 filed Jul. 28, 1998.
BACKGROUND OF THE INVENTION
[0002] Vascular cell adhesion molecule-1 (VCAM-1), a member of the
immunoglobulin (Ig) supergene family, is expressed on activated,
but not resting, endothelium. The integrin VLA-4 (a.sub.4b.sub.1),
which is expressed on many cell types including circulating
lymphocytes, eosinophils, basophils, and monocytes, but not
neutrophils, is the principal receptor for VCAM-1. Antibodies to
VCAM-1 or VLA-4 can block the adhesion of these mononuclear
leukocytes, as well as melanoma cells, to activated endothelium in
vitro. Antibodies to either protein have been effective at
inhibiting leukocyte infiltration and preventing tissue damage in
several animal models of inflammation. Anti-VLA-4 monoclonal
antibodies have been shown to block T-cell emigration in
adjuvant-induced arthritis, prevent eosinophil accumulation and
bronchoconstriction in models of asthma, and reduce paralysis and
inhibit monocyte and lymphocyte infiltration in experimental
autoimmune encephalitis (EAE). Anti-VCAM-1 monoclonal antibodies
have been shown to prolong the survival time of cardiac allografts.
Recent studies have demonstrated that anti-VLA-4 mAbs can prevent
insulitis and diabetes in non-obese diabetic mice, and
significantly attenuate inflammation in the cotton-top tamarin
model of colitis.
[0003] Thus, compounds which inhibit the interaction between
.alpha..sub.4-containing integrins, such as VLA-4 and VCAM-1, will
be useful as therapeutic agents for the treatment of chronic
inflammatory diseases such as rheumatoid arthritis (RA), multiple
sclerosis (MS), pulmonary inflammation (e.g., asthma), and
inflammatory bowel disease (IBD).
SUMMARY OF THE INVENTION
[0004] It has been discovered that compounds of the formula: 2
[0005] and the pharmaceutically acceptable salts and esters thereof
wherein X, X', Z and Y are as defined below, inhibit the binding of
VCAM-1 to VLA-4 and so would be useful in treating inflammatory
diseases in which such binding acts to bring on the disease.
DETAILED DESCRIPTION OF THE INVENTION
[0006] As used in this specification, the term "lower alkyl", alone
or in combination (for example, as part of "lower alkanoyl,"
below), means a straight-chain or branched-chain alkyl group
containing a maximum of six carbon atoms, such as methyl, ethyl,
n-propyl, isopropyl, n-butyl, sec.butyl, isobutyl, tert.butyl,
n-pentyl, n-hexyl and the like. Lower alkyl groups may be
unsubstituted or substituted by one or more groups selected
independently from cycloalkyl, nitro, aryloxy, aryl, hydroxy,
halogen, cyano, lower alkoxy, lower alkoxycarbonyl, lower alkanoyl,
lower alkylthio, lower alkyl sulfinyl, lower alkyl sulfonyl, and
substituted amino, e.g., lower alkoxycarbonyl amino. Examples of
substituted lower alkyl groups include 2-hydroxyethyl,
2-methoxypropyl, 3-oxobutyl, cyanomethyl, trifluoromethyl,
2-nitropropyl, benzyl, including p-chloro-benzyl and
p-methoxy-benzyl, and 2-phenyl ethyl.
[0007] The term "cycloalkyl" means an unsubstituted or substituted
3- to 7-membered carbacyclic ring. Substitutents useful in
accordance with the present invention are hydroxy, halogen, cyano,
lower alkoxy, lower alkanoyl, lower alkyl, aroyl, lower alkylthio,
lower alkyl sulfinyl, lower alkyl sulfonyl, aryl, heteroaryl and
substituted amino.
[0008] The term "heterocycloalkyl" means an unsubstituted or
substituted 5- to 6-membered carbacyclic ring in which one or two
of the carbon atoms has been replaced by heteroatoms independently
selected from O, S and N. Preferred heterocycloalkyl groups are
pyrrolidinyl and morpholinyl.
[0009] The term "lower alkoxy" means a lower alkyl group (as
defined above) bonded through an oxygen atom. Examples of
unsubstituted lower alkoxy groups are methoxy, ethoxy, n-propoxy,
isopropoxy, n-butoxy, tert-butoxy and the like.
[0010] The term "lower alkylthio" means a lower alkyl group bonded
through a divalent sulfur atom, for example, a methyl mercapto or a
isopropyl mercapto group.
[0011] The term "aryl" means a mono- or bicylic aromatic group,
such as phenyl or naphthyl, which is unsubstituted or substituted
by conventional substituent groups. Preferred subsituents are lower
alkyl, lower alkoxy, hydroxy lower alkyl, hydroxy, hydroxyalkoxy,
halogen, lower alkylthio, lower alkylsulfinyl, lower alkylsulfonyl,
cyano, nitro, perfluoroalkyl, alkanoyl, aroyl, aryl alkynyl,
heteroaryl (especially tetrazolyl), lower alkynyl and lower
alkanoylamino. Examples of aryl groups that may be used in
accordance with this invention are unsubstituted phenyl, m- or
o-nitrophenyl, p-tolyl, m- or p-methoxyphenyl, 3,4-dimethoxyphenyl,
p-chlorophenyl, p-cyanophenyl, m-methylthiophenyl,
2-methyl-5-nitrophenyl, 2,6-dichlorophenyl, m-perfluorophenyl,
1-naphthyl, m- or p-2-methyltetraozolyl, and the like.
[0012] The term "arylalkyl" means a lower alkyl group as
hereinbefore defined in which one or more hydrogen atoms is/are
replaced by an aryl or heteroaryl group as herein defined. Any
conventional arylalkyl may be used in accordance with this
invention, such as benzyl, 2-phenyl ethyl, and the like.
[0013] The term "aryloxy" means an aryl group, as hereinbefore
defined which is bonded via an oxygen atom. The preferred aryloxy
group is phenoxy.
[0014] The term "heteroaryl" means an unsubstituted or substituted
5- or 6-membered monocyclic hetereoaromatic ring or a 9- or
10-membered bicyclic hetereoaromatic ring containing 1, 2, 3 or 4
hetereoatoms which are independently N, S or O. Examples of
hetereoaryl rings are pyridine, benzimidazole, indole, imidazole,
thiophene, isoquinoline, quinzoline, tetrazole, and the like.
Substitutents as defined above for "aryl" are included in the
definition of heteroaryl. The wide variety of heteroaryl groups
useful in accordance with the invention is illustrated by Examples
56, 57, 74, 364 and 381-386.
[0015] The term "lower alkoxycarbonyl" means a lower alkoxy group
bonded via a carbonyl group. Examples of alkoxycarbonyl groups are
methoxycarbony, ethoxycarbonyl, t-butoxycarbonyl and the like.
[0016] The term "lower alkylcarbonyloxy" means lower alkylcarbonyl
groups bonded via an oxygen atom, for example an acetoxy group.
[0017] The term "lower alkanoyl" means lower alkyl groups bonded
via a carbonyl group and embraces in the sense of the foregoing
definition groups such as acetyl, propionyl and the like. Where the
lower alkyl portion of the lower alkanoyl group is substituted, the
preferred substitutents are methoxy, trifluoro, phenyl,
cyclopentyl, methoxycarbonyl, amino and t-butoxycarbonylamino.
[0018] The term "lower alkylcarbonylamino" means lower
alkylcarbonyl groups bonded via a nitrogen atom, such as
acetylamino.
[0019] The term "aroyl" means an mono- or bicyclic aryl or
heteroaryl group bonded via a carbonyl group. Examples of aroyl
groups are benzoyl, 3-cyanobenzoyl, m-perfluromethyl-benzoyl,
p-methoxy-benzoyl, 2-naphthoyl, groups of the formula: 3
[0020] and the like.
[0021] The present invention comprises a compound of the formula:
4
[0022] and the pharmaceutically acceptable salts and esters
thereof.
[0023] In accordance with the invention, Z is hydrogen or lower
alkyl (preferably hydrogen), one of X and X' is hydrogen, halogen,
or lower alkyl (X' is preferably hydrogen), and the other
(preferably X) is a group X-6, X-7 or X-10 as described below. Y is
a group Y-1 or Y-2 as described below.
[0024] Y-1 is a group of the formula: 5
[0025] wherein:
[0026] R.sub.22 and R.sub.23 are independently aryl, heteroaryl or
lower alkyl which is unsubstituted or substituted by one or more
chloro, bromo, nitro, hydroxy, lower alkoxy, aryl, lower alkanoyl,
aroyl or cyano,
[0027] R.sub.24 is aryl, cyano, alkylsulfonyl or lower alkyl or
alkenyl unsubstituted or substituted by an aryl or heteroaryl ring,
and when R.sub.22 is aryl and R.sub.23 is aryl or lower alkyl,
hydrogen, and
[0028] the total number of carbon atoms in R.sub.22, R.sub.23 and
R.sub.24 is from 6 to 14.
[0029] In Y-1, R.sub.22 and R.sub.23 are preferably lower alkyl or
phenyl, and R.sub.24 is preferably lower alkyl except when R.sub.22
is aryl and R.sub.23 is aryl or lower alkyl, then R.sub.24 is
preferably hydrogen.
[0030] However, Y is preferably the group Y-2 which is a 3-7
membered ring of the formula: 6
[0031] wherein:
[0032] R.sub.25 is lower alkyl, unsubstituted or fluorine
substituted lower alkenyl, or a group of formula
R.sub.26--(CH.sub.2).sub.e--,
[0033] R.sub.26 is aryl, heteroaryl, azido, cyano, hydroxy, lower
alkoxy, lower alkoxycarbonyl, lower alkanoyl, lower alkylthio ,
lower alkyl sulfonyl, lower alkyl sulfinyl, perfluoro lower
alkanoyl, nitro, or R.sub.26 is a group of formula
--NR.sub.28R.sub.29, wherein:
[0034] R.sub.28 is hydrogen or lower alkyl,
[0035] R.sub.29 is hydrogen, lower alkyl, lower alkoxycarbonyl,
lower alkoxycarbonylaminocarbonyl, lower alkanoyl, aroyl,
heteroaroyl, perfluoro lower alkanoyl, lower alkyl sulfonyl, lower
alkylaminocarbonyl, arylaminocarbonyl, or lower
alkylaminothiocarbonyl, or
[0036] R.sub.28 and R.sub.29 taken together with the nitrogen atom
to which they are attached form a 4, 5 or 6-membered saturated
heterocyclic ring containing one or two heteroatoms with the second
heteroatom being O, S, or N--R.sub.27;
[0037] Q is --(CH.sub.2).sub.fO--, --(CH.sub.2).sub.fS--,
--(CH.sub.2).sub.f--, or when f=0, a bond,
[0038] the dotted line is a second bond which is present or
absent,
[0039] R.sub.27 is hydrogen, lower alkyl, aryl, lower alkanoyl,
aroyl, or lower alkoxycarbony, the carbon atoms in the ring are
unsubstituted or substituted by lower alkyl or halogen,
[0040] e is an integer from 0 to 4, and
[0041] f is an integer from 0 to 3.
[0042] Q is preferably --(CH.sub.2).sub.f-- or, when f=0, a bond.
When R.sub.25 is a group of formula R.sub.26--(CH.sub.2).sub.e--,
R.sub.26 is preferably aryl, heteroaryl, azido, cyano, hydroxy,
lower alkoxy, lower alkoxycarbonyl, lower alkanoyl, lower
alkylthio, lower alkyl sulfonyl, lower alkyl sulfinyl, nitro, or
R.sub.26 is a group of formula --NR.sub.28R.sub.29 wherein R.sub.28
is hydrogen or lower alkyl,
[0043] R.sub.29 is hydrogen, lower alkyl, lower alkoxycarbonyl,
lower alkanoyl, aroyl, perfluoro lower alkanoyl, lower alkyl
sulfonyl, lower alkylaminocarbonyl, heterocycloalkyl carbonyl,
arylaminocarbonyl, or R.sub.28 and R.sub.29 taken together with the
nitrogen to which they are attached form a 4, 5 or 6-membered
saturated heterocyclic ring which can contain one oxygen atom. When
R.sub.26 is aryl, it is especially phenyl unsubstituted,
mono-substituted by chloro, methoxy, cyano, or tetrazolyl which is
unsubstituted or substituted by methyl, or is phenyl di-substituted
by methoxy.
[0044] The group X-6 is of the formula: 7
[0045] wherein:
[0046] R.sub.1 is hydrogen or lower alkyl,
[0047] R.sub.15 is hydrogen, halogen, nitro, lower alkyl sulfonyl,
cyano, lower alkyl, lower alkoxy, lower alkoxycarbonyl, carboxy,
lower alkyl aminosulfonyl, perfluorolower alkyl, lower alkylthio,
hydroxy lower alkyl, alkoxy lower alkyl, alkylthio lower alkyl,
alkylsulfinyl lower alkyl, alkylsufonyl lower alkyl, lower
alkylsulfinyl, lower alkanoyl, aroyl, aryl, aryloxy or a group of
the formula R.sub.17--C.ident.C--,
[0048] R.sub.16 is hydrogen, halogen, nitro, cyano, lower alkyl,
OH, perfluorolower alkyl, or lower alkylthio,
[0049] R.sub.17 is hydrogen, aryl, heteroaryl, or lower alkyl which
is unsubstituted or substituted by OH, aryl, or heteroaryl, and
[0050] a is 0 or 1.
[0051] The groups R.sub.15 and R.sub.16 are preferably
independently hydrogen, lower alkyl, nitro, halogen (especially
chloro or fluoro), perfluoromethyl, cyano or phenoxy. R.sub.1 is
preferably hydrogen and a is preferably 0.
[0052] X-7 is a group of the formula: 8
[0053] wherein Het is a 5- or 6-membered heteroaromatic ring
containing 1, 2 or 3 heteroatoms selected from N, O, and S, or
[0054] Het is a 9- or 10-membered bicyclic heteroaromatic ring
containing 1, 2, 3 or 4 heteroatoms selected from O, S, and N;
[0055] a, R.sub.1, R.sub.15 and R.sub.16 are as above, and
[0056] R.sub.30 is absent or is hydrogen or lower alkyl.
[0057] Het is preferably a 5- or 6-membered monocyclic
heteroaromatic ring containing 1, 2 or 3 nitrogens, or a nitrogen
and a sulfur, or a nitrogen and an oxygen. When Het is a bicyclic
heteroaromatic ring, it preferably contains from 1 to 3 nitrogens
as the heteroatoms. R.sub.15 is preferably, nitro, lower alkyl
sulfonyl, cyano, lower alkyl, lower alkoxy, perfluorolower alkyl,
lower alkylthio, lower alkanoyl, or aryl (especially unsubstituted
phenyl); R.sub.16 is preferably hydrogen, halogen, nitro, cyano,
lower alkyl, perfluoro lower alkyl; and R.sub.30, when present, is
preferably hydrogen or lower alkyl.
[0058] The group X-10 is of the formula: 9
[0059] wherein:
[0060] R.sub.18 is hydrogen, substituted or unsubstituted lower
alkyl, aryl, heteroaryl, arylalkyl, heteroaryl alkyl,
[0061] R.sub.19 is substituted or unsubstituted lower alkyl, aryl,
heteroaryl, arylalkyl, heteroaryl alkyl, and
[0062] R.sub.20 is substituted or unsubstituted lower alkyl,
substituted or unsubstituted lower alkanoyl, carboxyl lower
alkanoyl, aroyl, aryloxy lower alkanoyl.
[0063] R.sub.18 is preferably phenyl wherein the phenyl ring is
unsubstituted or monosubstituted by lower alkoxy or halogen, or is
phenyl lower alkyl. R.sub.19 is preferably lower alkyl, which is
unsubstituted or substituted by pyridyl or phenyl wherein the
phenyl ring is unsubstituted or monosubstituted by lower alkoxy or
halogen. R.sub.20 is preferably lower alkanoyl
[0064] The compounds of the invention include the pharmaceutically
acceptable salts and esters thereof. Certain prefered esters of the
invention were discovered which are useful to improve
bioavailabilty of compounds of this invention. These preferred
esters are of the formula: 10
[0065] wherein X, X', Z and Y are as described above, and R.sub.31
is lower alkyl, or R.sub.31 is a group of formula P-1: 11
[0066] wherein:
[0067] R.sub.32 is hydrogen or lower alkyl,
[0068] R.sub.33 is hyrogen, lower alkyl, aryl,
[0069] R.sub.34 is hydrogen or lower alkyl,
[0070] h is an integer from 0 to 2,
[0071] g is an integer from 0 to 2,
[0072] the sum of h and g is 1 to 3; or
[0073] R.sub.31 is a group of formula P-2: 12
[0074] wherein:
[0075] R.sub.32, g, and h are as previously defined,
[0076] T is O, S, --(CH.sub.2).sub.j--, a bond (when j=0) or a
group of the formula N--R.sub.35,
[0077] R.sub.35 is hydrogen, lower alkyl, lower alkanoyl, lower
alkoxycarbonyl, and
[0078] j is 0, 1 or 2.
[0079] R.sub.31 is preferably ethyl or 2-(4-morpholinyl)ethyl.
[0080] The compounds of the invention can exist as stereoisomers
and diastereomers, all of which are encompassed within the scope of
the present invention.
[0081] The especially preferred groups Y-1 are of the formula:
13
[0082] The especially preferred groups Y-2 are of the formulas
shown in the following table:
1TABLE 1 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
55 56 57 58 59 60 61 62 63 64 65
[0083] The especially preferred groups X-6 are of the formula:
66
[0084] The especially preferred groups X-7 are of the formula:
67
[0085] The especially preferred groups X-10 are of the formula:
2 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89
90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108
109 110 111 112 113 114 115 116 117 118 119 120 121 122
[0086] The compounds of the invention inhibit the binding of VCAM-1
and fibronectin to VLA-4 on circulating lymphocytes, eosinophils,
basophils, and monocytes ("VLA-4-expressing cells"). The binding of
VCAM-1 and fibronectin to VLA-4 on such cells is known to be
implicated in certain disease states, such as rheumatoid arthritis,
multiple sclerosis, inflammatory bowel disease, and particularly in
the binding of eosinophils to pulmonary endothelium which is the
cause of the pulmonary inflammation which occurs in asthma Thus,
the compounds of the present invention can be used as medicaments
for the treatment of disorders which are known to be associated
with such binding, and especially would be useful for the treatment
of asthma.
[0087] Furthermore, compounds of the invention also inhibit the
binding of VCAM-1 and MadCAM to the cellular receptor alpha4-beta7,
also known as LPAM, which is expressed on lymphocytes, eosinophiles
and T-cells. While the precise role of alpha4-beta7 interaction
with various ligands in inflammatory conditions such as asthma is
not completely understood, compounds of the invention which inhibit
both alpha4-beta1 and alpha4-beta7 receptor binding are
particularly effective in animal models of asthma.
[0088] Furthermore, work with monoclonal antibodies to alpha4-beta7
indicate that compounds which inhibit alpha4-beta7 binding to
MadCAM or VCAM are useful for the treatment of inflammatory bowel
disease. They would also be useful in the treatment of other
diseases in which such binding is implicated as a cause of disease
damage or symptoms.
[0089] The compounds of the invention can be administered orally,
rectally, or parentally, e.g., intravenously, intramuscularly,
subcutaneously, intrathecally or transdermally; or sublingually, or
as opthalmalogical preparations, or as an aerosol for the treatment
of pulmonary inflammation. Capsules, tablets, suspensions or
solutions for oral administration, suppositories, injection
solutions, eye drops, salves or spray solutions are examples of
administration forms.
[0090] Intravenous, intramuscular, oral or inhalation
administration is a preferred form of administration. The dosages
in which the compounds of the invention are administered in
effective amounts depend on the nature of the specific active
ingredient, the age and the requirements of the patient and the
mode of administration. Dosages may be determined by any
conventional means, e.g., by dose-limiting clinical trials. Thus,
the invention further comprises a method of treating a host
suffering from a disease in which VCAM-1 or fibronectin binding to
VLA-4-expressing cells is a causative factor in the disease
symptoms or damage by administering an amount of a compound of the
invention sufficient to inhibit VCAM-1 or fibronectin binding to
VLA-4-expressing cells so that said symptoms or said damage is
reduced. In general, dosages of about 0.1-100 mg/kg body weight per
day are preferred, with dosages of 1-25 mg/kg per day being
particularly preferred, and dosages of 1-10 mg/kg body weight per
day being espeically preferred.
[0091] The invention further comprises pharmaceutical compositions
which contain a pharmaceutically effective amount of a compound of
the invention and a pharmaceutically acceptable carrier. Such
compositions may be formulated by any conventional means. Tablets
or granulates can contain a series of binders, fillers, carriers or
diluents. Liquid compositions can be, for example, in the form of a
sterile water-miscible solution. Capsules can contain a filler or
thickener in addition to the active ingredient. Furthermore,
flavour-improving additives as well as substances usually used as
preserving, stabilizing, moisture-retaining and emulsifying agents
as well as salts for varying the osmotic pressure, buffers and
other additives can also be present.
[0092] The previously mentioned carrier materials and diluents can
comprise any conventional pharmaceutically acceptable organic or
inorganic substances, e.g., water, gelatine, lactose, starch,
magnesium stearate, talc, gum arabic, polyalkylene glycols and the
like.
[0093] Oral unit dosage forms, such as tablets and capsules,
preferably contain from 25 mg to 1000 mg of a compound of the
invention.
[0094] The compounds of the present invention may be prepared by
any conventional means. In reaction Scheme 1, a compound of formula
1 in which R.sub.1 is hydrogen or lower alkyl, and which is a known
compound or can be prepared by standard methodology, is treated
with a reducing agent capable of selectively reducing a nitro group
in the presence of a benzylic alcohol. This procedure is
advantageously carried out in the presence of a derivatizing agent
of the formula R.sub.2--OCOX wherein X is a leaving group and
R.sub.2 is tert-alkyl, benzyl or the like so as to form a readily
cleavable protecting group, thus leading directly to a compound of
formula 2. For example, this procedure can be conveniently carried
out by catalytic hydrogenation of 1 over Pd(C) in ethyl acetate in
the presence of di-tert-butyl dicarbonate to give a derivative of 2
in which R.sub.2 is tert-butyl.
[0095] Conversion to an aldehyde of formula 3 can be carried out
using an one of a variety of oxidizing agents capable of oxidizing
a benzylic alcohol to the corresponding aldehyde, for example
activated manganese dioxide in a suitable solvent, for example
dichloromethane. Reaction of 3 to give a dehydroamino acid of
formula 5 can be effected by treatment with a Wittig reagent of
formula 4 in which R.sub.3 is lower alkyl and R.sub.4 is an alkoxy
group, for example benzyloxy- or tert-butoxy- or represents a
portion of one of the acyl groups of the compounds of the
invention, for example substituted lower alkyl or substituted
cycloalkyl. For example treatment of 3 with
(.+-.)-N-(benzyloxycarbonyl)-.alpha.-phos- phonoglycine trimethyl
ester in the presence of a suitable base for example tetramethyl
guanidine leads directly to a dehydroamino acid of formula 5,
R.sub.3=methyl and R.sub.4=benzyloxy. Enantioselective reduction of
5 to the L-amino acid 6 can be effected by use of a number of
reducing agents suitable for the purpose, for example, the recently
described ethyl-DuPHOS rhodium reagent (Burk, M. J., Feaster, J.
E.; Nugent, W. A.; Harlow, R. L. J Am. Chem. Soc. 1993, 115, 10125)
using essentially the literature procedure. 123
[0096] One process for the conversion of compounds of structure 6
into compounds of the invention is shown in Reaction Scheme 2. The
protecting group incorporating R.sub.2 can be removed under
conditions dependent on the particular choice of R.sub.2 as well as
R.sub.3 and R.sub.4. The choice of these groups will be dependent
on the particular target compound. A variety of common protecting
groups and their use are described in "T. W. Green and P. G. M.
Wuts, Protective Groups in Organic Synthesis, 2nd edition, Wiley
Interscience, New York, 1991" For example when R.sub.2 is a
tert-butyl group and R.sub.3 is lower alkyl and R.sub.4 is either a
benzyloxy group or represents a portion of one of the acyl groups
of the compounds of the invention, for example substituted lower
alkyl or substituted cycloalkyl, treatment with trifluoroacetic
acid either neat or in dichloromethane solution in the presence of
suitable scavengers, for example, triethylsilane or anisol leads to
a compound of formula 7. This compound can be coupled with a
carboxylic acid of formula 8 using standard peptide coupling
conditions, for example HBTU in the presence of DIPEA in a polar,
aprotic solvent such as DMF at a temperature between 0.degree. C.
and room temperature to give a compound of formula 9. In the
carboxylic acid of formula 8, R.sub.5 may represent a substituted
alkyl group, a substituted aromatic ring, or a substituted
heteroaromatic ring. R.sub.5 may also incorporate suitably
protected reactive functionalities to permit final conversion into
compounds of the invention. The choice and use of such groups will
be apparent to those skilled in the art. 124125
[0097] Depending on the choice of R.sub.4 and whether an ester or
acid is the final goal of the synthesis, compound 9 may be a
compound of the invention or in the case that R.sub.4 is a
protecting group, for example, a benzyloxy group, it may be removed
under appropriate conditions, for example by catalytic
hydrogenation over Pd(C) in a suitable solvent such as a lower
alcohol to give a compound of formula 10. This intermediate can be
coupled with a carboxylic acid of formula 11 using standard peptide
coupling conditions, for example HBTU in the presence of DIPEA in a
polar, aprotic solvent such as DMF at a temperature between
0.degree. C. and room temperature to give a compound of formula 12.
In the carboxylic acid of formula 11, R.sub.6 may represent a
portion of a compound of the invention, for example, a substituted
alkyl or substituted cycloalkyl. These compounds are known
compounds or can be prepared by known methods. R.sub.6 may also
incorporate suitably protected reactive functionalities to permit
final conversion into compounds of the invention. The choice and
use of such groups will be apparent to those skilled in the art.
General methods for the preparation of such compounds are
illustrated in Reaction Scheme 13. If the acid 13 is the target
compound, conversion of a compound of formula 12 can be effected
using standard hydrolysis conditions appropriate for the particular
choice of R.sub.3 and any functional groups present as part of
R.sub.5 and R.sub.6. In the case where R.sub.3 is lower alkyl,
treatment with an alkali metal hydroxide, for example lithium
hydroxide in aqueous THF is generally effective.
[0098] In reaction Scheme 3, a compound of formula 14 in which
R.sub.7 is a lower alkyl group which may serve as a protecting
group or a group suitable for use in a prodrug for example methyl,
ethyl, tert-butyl or the like or represents a connection to a solid
phase resin, for example a Wang resin, is coupled with a carboxylic
acid of formula 11 using standard peptide coupling conditions, for
example HBTU in the presence of DIPEA in a polar, aprotic solvent
such as DMF at a temperature between 0.degree. C. and room
temperature to give a compound of formula 15. Reduction of the
nitro group of 15 can be effected by catalytic hydrogenation for
example using Pd(C) as a catalyst or by treatment with a standard
reducing agent, for example SnCl.sub.2. The resulting compound of
structure 16 is useful as a key intermediate for several series of
compounds. In the instance highlighted in Scheme 3, it can be
coupled with an acid of formula 8 using standard peptide coupling
conditions, for example HBTU in the presence of DIPEA in a polar,
aprotic solvent such as DMF at a temperature between 0.degree. C.
and room temperature to give a compound of formula 17. Compound 17
may be a compound of the invention depending on the nature of
R.sub.7 or may be converted to a compound of the invention by an
appropriate hydrolysis procedure, for example in the case where
R.sub.7 is lower alkyl, by hydrolysis by treatment with excess
alkali metal hydroxide, such as lithium hydroxide in aqueous
alcohol. When R.sub.7 represents a resin suitable for solid phase
synthesis, appropriate hydrolysis conditions will depend on the
choice of resin. In the case of Wang resin, treatment with
trifluoroacetic acid in the presence of appropriate scavengers will
lead to an acid of formula 18. 126
[0099] In a method particularly well suited for solid phase
synthesis, an N'-Alloc-amino-N.sup..alpha.-Fmoc protected
phenylalanine derivative of formula 19 can be coupled to a resin
suitable for solid phase synthesis, for example, a Wang resin using
standard coupling procedures, for example, by forming a mixed
anhydride with 2,6-dichlorobenzoyl chloride and carrying out the
coupling reaction in a polar, aprotic solvent such as N-methyl
pyrrolidinone to give a compound of structure 20 in which R.sub.7'
represents the resin. The Alloc group may be removed by standard
methods, for example by treatment with a reducing agent such as
nBu.sub.3SnH in the presence of a catalyst which is a source of
Pd.sup.o, for instance, Pd(Ph.sub.3P).sub.2Cl.sub.2 to give an
amine derivative of structure 21. This compound can be coupled with
a carboxylic acid of formula 8 using standard peptide coupling
conditions, for example HBTU in the presence of DIPEA in a polar,
aprotic solvent such as DMF at a temperature between 0.degree. C.
and room temperature to give a compound of formula 22. The Fmoc
protecting group may be removed from 22 using standard base
treatment well known to those practicing peptide chemistry, for
example with piperidine in DMF, to afford an amine of formula 23.
The resulting compound 23 can be coupled with a carboxylic acid of
formula 11 using standard peptide coupling conditions, for example
HBTU in the presence of DIPEA in a polar, aprotic solvent such as
DMF at a temperature between 0.degree. C. and room temperature to
give a compound of formula 24. Finally the compound of structure 24
can be cleaved from the resin under conditions dependent on the
particular choice of resin. For example, in the case of a Wang
resin, acid treatment with trifluoroacetic acid in dichloromethane
in the presence of scavengers as necessary will afford a compound
of formula 18.
[0100] Depending on the particular synthetic target, the order of
removal of the protecting groups from 19 may be altered so that the
Fmoc group is first removed, coupling of the resulting amine with
an acid of formula 11 is carried out followed by removal of the
Alloc group and coupling of the product with an acid of formula 8
and cleavage from the resin. Also the choice of protecting groups
can be modified to reflect the reactivities of the resin and the
nature of any functional groups incorporated into R.sub.5 and
R.sub.6. 127128
[0101] Compounds derived from 3- or 4-(alkylamino)phenylalanine
derivatives can be prepared as outlined in Reaction Scheme 5. A
compound of formula 16 or 7 may be treated with diazomethyane in a
suitable solvent, for example, ethyl ether to give products of
formulas 25 and 26 repectively in which R.sub.8 is methyl.
Alternatively, the compound of structure 16 or 7 may be treated
with an lower alkyl aldehyde or ketone, for example acetone, to
give an intermediate Schiff's base which is in turn subjected to
catalytic hydrogenation or reduction with sodium cyanoborohydride
in the presence of an organic acid, for example acetic acid to give
a compound of formula 25 or 26 in which R.sub.8 is lower alkyl
other than methyl. Conversion of compounds 25 or 26 to prodrug
esters 27 or 28 or to the corresponding acids 29 or 30 respectively
can be carried out as described above in Reaction Schemes 2 and 3.
129
[0102] For the preparation of 3- or 4-sulfonylamino phenylalanine
derivatives, compounds of formula 7, 16, 25 or 26 may be reacted
with a sulfonyl chloride of formula 31, in which R.sub.9 is a
substituted aryl or heteroaryl moiety, in an inert solvent, for
example dichloromethane in the presence of a non-nucleophilic base,
for example triethylamine or pyridine at about 0.degree. C. to room
temperature to give compounds of structure 32 or 33 respectively as
illustrated in Reaction Scheme 6 for compounds 7 and 26. These can
be further converted to compounds of formulas 34 and 35 if desired
using the general methods described above in Reaction Schemes 2 and
3. Furthermore, the group R.sub.4CO-- may replaced by a group
R.sub.6CO-- using the general chemistry described in scheme 2.
[0103] For the preparation of compounds derived from 3- or
4-aminomethylphenylalanine, the procedure shown in Reaction Scheme
7 may be employed. A 3- or 4-hydroxymethyl benzoate of formula 36
in which R.sub.10 is lower alkyl, which are known compounds, or can
be prepared by known methods, is treated with a silylating agent in
which R.sub.11-R.sub.13 are lower alkyl or phenyl, for example
tert-butyldimethylsilyl chloride in an inert solvent, for example
dimethylformamide in the presence of imidazole at about 0.degree.
C. to give a silyl protected compound of formula 37. Reduction of
37 may be carried out using a variety of suitable reducing agents,
for example, lithium aluminum hydride in an inert solvent such as
ether or tetrahydrofuran at a temperature of about 0.degree. C.
followed by an aqueous workup to give an intermediate alcohol which
can be oxidized by any of several oxidizing agents suitable for
oxidizing benzyl alcohols to the corresponding aldehydes, for
example activated manganese dioxide, to give an aldehyde of formula
38. Monosilyl protected diols are alternatively available from 3-
or 4-hydroxymethylbenzylalcohols by monosilylation and separation
of the side products. Alternatively, an ester of formula 37 may be
reduced directly to an aldehyde of formula 38 using
diisobutylaluminum hydride at low temperature, for example at
-78.degree. C.
[0104] Reaction of 38 to give a dehydroamino acid of formula 39 can
be effected by treatment with a Wittig reagent of formula 4 in
which R.sub.3 is lower alkyl and R.sub.4 is an alkoxy group, for
example benzyloxy- or tert-butoxy- or represents a portion of one
of the acyl groups of the compounds of the invention, for example
substituted lower alkyl or substituted cycloalkyl. For example
treatment of 38 with
(.+-.)-N-(benzyloxycarbonyl)-.alpha.-phosphonoglycine trimethyl
ester in the presence of a suitable base for example tetramethyl
guanidine leads directly to a dehydroamino acid of formula 39,
R.sub.3=methyl and R.sub.4=benzyloxy. Enantioselective reduction of
39 to the L-amino acid 40 can be effected by use of one of a number
of reducing agents suitable for the purpose, for example, the
recently described ethyl-DuPHOS rhodium reagent. It will be readily
apparent to those skilled in the art that the optimal procedure for
the further conversion of 40 into compounds of the invention will
depend on the choices of R.sub.4 and R.sub.3. For the case wherein
R.sub.3 is lower alkyl and R.sub.4 is benzyloxy, conversion to an
amine of formula 41 can be conveniently effected by catalytic
transfer hydrogenation of 40 over Pd(C) in a suitable solvent, for
example, methanol in the presence of ammonium formate as the
reducing agent. Acylation of 41 with a carboxylic acid of formula
11 can be carried as described above in Reaction Scheme 2 to give a
compound of formula 42. Conditions for removal of the silyl
protecting group will depend on the particular choice of
R.sub.11-R.sub.13. In the case of R.sub.11, R.sub.12=methyl and
R.sub.13=tert-butyl, this group is readily removed by treatment
with a strong acid, for example hydrochloric acid in an appropriate
solvent for the choice of R.sub.3, for example where R.sub.3 is
methyl, methanol.
[0105] The resulting benzylic alcohol of formula 43 can be
converted to an amine of formula 45 using procedures well
established for similar transformations. For example, the alcohol
of formula 43 can be converted to a leaving group, for example a
mesylate by treatment with methane sulfonyl chloride in the
presence of a proton acceptor, for example pyridine, followed by
displacement with an alkali metal azide, for example sodium azide
in a polar aprotic solvent such as dimethylformamide.
Alternatively, the transformation from 43 to an azide of formula 44
can be carried out directly by treatment with diphenyl
phosphorazidate as described in: Thompson, A. S.; Humphrey, G R.;
DeMarco, A. M.; Mathre, D. J.; Grabowski, E. J. J. J Org. Chem.
1993, 58, 5886-5888. Reduction of the azide 44 to an amine of
formula 45 can be carried out by a number of means suitable for the
conversion of azides to amines, for example by treatment with a
phosphine, for example triphenyl phosphine in an inert solvent such
as dichloromethane or THF followed by an aqueous workup or by
catalytic hydrogenation over an appropriate catalyst, for example
Pd(C) in a solvent suitable for catalytic hydrogenations such as a
lower alkanol or tetrahydrofuran. The resulting amine of formula 45
can be converted into the corresponding compounds of the invention
using the procedures applicable to free amines described in the
other reaction schemes. For example, coupling of 45 with a
carboxylic acid of formula 8 under the conditions described in
Reaction Scheme 2 leads to an amide of formula 46 which may be
further converted to an acid of formula 47 if desired by base
catalyzed hydrolysis as described in Reaction Scheme 2. 130
131132133
[0106] For the synthesis of urea derivatives, a compound of formula
26 can be treated with an isocyanate of formula 49, wherein
R.sub.14 is substituted aryl, substituted heteroaryl or substituted
lower alkyl with potentially reactive substituents protected as
appropriate using conventional protecting group strategies, in a
suitable inert solvent, for example dichloromethane, to give a urea
of formula 50. More generally, a compound of formula 26 can be
treated with a phosgene equivalent, for example, triphosgene in an
inert solvent such as dichloromethane in the presence of a
non-nucleophilic proton acceptor, for example
diisopropylethylamine, to give an intermediate of formula 48.
Subsequent treatment of a compound of formula 48 with an amine of
formula 51 in which R.sub.15 and R.sub.16 are independently
hydrogen, substituted lower alkyl, substituted aryl, substituted
heteroaryl or taken together form a substituted 5, 6 or 7 membered
ring leads to a compound of formula 52. Further conversion, if
necessary, of 50 or 52 to compounds of the invention can be carried
out as described in Reaction Scheme 5. 134
[0107] For the synthesis of imides, an aminophenylalanine
derivative of structure 53 in which R.sub.1 is hydrogen or lower
alkyl, R.sub.6 is as previously defined and R.sub.7" is hydrogen or
a readily cleavable group such as substituted benzyl, tert-butyl,
allyl, or the like, or in the event that a prodrug ester is desired
as the final product, is that ester group, for example ethyl, is
employed. Compounds of formula 53 can be readily obtained from
intermediates described above in Reaction Scheme 2. Reaction of a
compound of formula 53 with a cyclic anhydride of formula 54 in an
inert solvent, for example dichloromethane leads to a ring opened
intermediate of formula 55. The structure implied by 54 includes
bicyclic molecules which may incorporate fused aromatic or
heteroaromatic rings. In place of 54, it is also possible to use
dicarboxylic acids which are capable of forming cyclic imides. In
the latter case, a condensing agent must be employed in the first
step, for example carbonyl diimidazole. Treatment of the compound
of formula 55 with a reagent such as carbonyl diimidazole capable
of effecting cyclodehydration leads to an imide of formula 56.
Further manipulation of functional groups which were present on the
anhydride of formula 54 and modification of R.sub.7" may be carried
out on compound 56 as desired to obtain further analogs using
standard chemistry which is compatible with the presence of the
imide functionality.
[0108] For the synthesis of compounds of the invention in which
R.sub.1 is halogen, preferably chloro, the appropriate halogen atom
can be incorpoarted into the starting material or inserted at
various points during the course of the synthesis depending on the
nature of the additional functionality in the molecule. A chlorine
atom can be incorporated into the compound of structure 1, shown in
scheme 1 and carried through to the compounds of the invention by
avoiding reagents which would be expected to react with a halogen
atom For example a compound of formula 6 in which R.sub.1 is
hydrogen can be treated with a mild chlorinating agent, for
example, N-chlorosuccinimide in the presence of a proton acceptor,
for example, sodium acetate to give the corresponding compound of
formula 6 in which R.sub.1 is chloro. In the case where 6 is
derived from 3-amino-L-phenylalanine, a mixture of regioisomers may
ensue which may be separated at a convenient point in the overall
synthesis. Other intermediates described in the above schemes may
be more suitable starting materials for halogenation for a
particular target molecule. The particular merits of individual
candidate starting materials will be apparent to those skilled in
the art. 135
[0109] For the synthesis of the thiazolidinones of formula 62
described in reaction scheme 10, an aminophenylalanine derivative
of structure 16, in which R.sub.6 and R.sub.7 are as previously
defined may be employed. Reaction of 16 with an .alpha.-mercapto
carboxylic acid of formula 59 in which R.sub.20 can be hydrogen,
lower alkyl or aryl, for example .alpha.-mercapto acetic acid, and
an aldehyde of formula 60 in which R.sub.21 can be lower alkyl,
arylalkyl or a substituted aryl group, for example benzaldehyde, in
an appropriate solvent such as benzene, THF or a lower alcohol, for
example methanol, in the presence of a water scavenger such as 4
.ANG. molecular sieves at 60 to 80.degree. C. provides compound of
formula 61. Compound 61 may be a compound of the invention
depending on the nature of R.sub.7 or may be converted to a
compound of the invention by an appropriate hydrolysis procedure,
for example in the case where R.sub.7 is lower alkyl, by treatment
with excess alkali metal hydroxide, such as sodium hydroxide in
aqueous alcohol. When R.sub.7 represents a resin suitable for solid
phase synthesis, the appropriate hydrolysis conditions will depend
on the choice of resin. In the case of Wang resin, treatment with
trifluoroacetic acid in the presence of appropriate scavengers will
lead to an acid of formula 62. The sequence may be initiated with
related anilines, for example a compound of formula 7 in which
R.sub.1 is lower alkyl or halogen to give the corresponding
thiazolidinones. 136
[0110] For the synthesis of imidazolidinones of formula 67 shown in
reaction scheme 11, an aminophenylalanine derivative of structure
16 in which R.sub.6 and R.sub.7 are as previously defined may be
employed. Compound 16 can be readily obtained through the synthesis
described in reaction scheme 3. This compound can be coupled with a
N-protected .alpha.-amino acid of formula 63, in which R.sub.22 can
be a lower alkyl or an aryl group, R.sub.23 can be a natural or
unnatural D- or L-.alpha.-amino acid side chain or R.sub.22 and
R.sub.23 together can form a ring, for example a proline or
pipicolinic acid ring and R.sub.24 may be a standard amine
protecting group suitable for the particular selection of R.sub.6,
R.sub.7, R.sub.22, and R.sub.23 for example tert-butoxycarbonyl.
The coupling reaction can be effected using standard peptide
coupling conditions, for example HBTU in the presence of DIPEA in a
polar, aprotic solvent such as DMF at a temperature between
0.degree. C. and room temperature to give a compound of formula 64.
Depending on the nature of protecting group R.sub.24, an
appropriate deprotection method is employed to give a compound of
formula 65. In the event that the protecting group R.sub.24 is a
Boc group, the deprotection can be carried out by the reaction of
64 with HCl in dioxane at room temperature. Reaction of compound 65
with an aldehyde of formula 60, in which the R.sub.21 is as defined
above, in the presence of a water scavenger such as 4 .ANG.
molecular sieves at 60 to 80.degree. C. in an appropriate solvent,
for example THF, provides a compound of formula 66. Compound 66 may
be a compound of the invention depending on the nature of R.sub.7
or may be converted to a compound of the invention by an
appropriate hydrolysis procedure, for example in the case where
R.sub.7 is lower alkyl, by hydrolysis by treatment with an alkali
metal hydroxide, such as sodium hydroxide in aqueous alcohol to
give a carboxylic acid of formula 67. 137
[0111] For the synthesis of imidazolidinones of formula 68
described in reaction scheme 12, an aminophenylalanine derivative
of structure 16 in which R.sub.6 and R.sub.7 are as previously
defined is employed. Compound 16 can be readily obtained through
the synthesis described in reaction scheme 3 in the case of R.sub.7
is lower alkyl. This compound can be coupled with a N-protected
.alpha.-amino acid of formula 69, in which R.sub.25 can be a
natural or unnatural, D- or L-.alpha.-amino acid side chain and
R.sub.26 is a nitrogen protecting group of the type conventionally
used in peptide chemistry, for example, a Fmoc group, using
standard peptide coupling conditions, for example HBTU in the
presence of DIPEA in a polar, aprotic solvent such as DMF at a
temperature between 0.degree. C. and room temperature to give a
compound of formula 70. Depending on the nature of protecting group
R.sub.26, an appropriate deprotection method is employed to give
compound of formula 71. In the case of the protecting group
R.sub.26 is Fmoc group, it may be removed from 70 using standard
base treatment well known to those practicing peptide chemistry,
for example with piperidine in DMF, to afford an amine of formula
71. The compound 71 can then react with an aldehyde 60, in which
R.sub.21 is as previously defined, in the presence of a water
scavenger such as 4 .ANG. molecular sieves in an appropriate
solvent such as dichloromethane or THF at 25-80.degree. C. (bath
termperature) to give an imine of formula 72. The imine 72 may then
be treated with an acylating agent such as the acyl chloride of
formula 74 in which R.sub.27 can be an alkyl or aryl group in the
presence of a base such DIPEA or DBU in an appropriate solvent such
as dichloromethane or THF at 25-80.degree. C. (bath temperature) to
give an acyl imidazolidinone of formula 73. Other acylating groups
may be employed for example, acid anhydrides, and where
appropriate, 74 may bear protected substituents which can later be
removed at the neccessary point in the synthesis. Compound 73 may
be a compound of the invention, or depending on the nature of
R.sub.7 may be converted to a compound of the invention by an
appropriate hydrolysis procedure, for example in the case where
R.sub.7 is lower alkyl, by hydrolysis by treatment with an alkali
metal hydroxide, for example sodium hydroxide in aqueous alcohol to
give, after acidification, a carboxylic acid of formula 68. The
sequence may be initiated with related anilines, for example a
compound of formula 7 in which R.sub.1 is lower alkyl or halogen to
give the corresponding 3-acyl imidazolidinones. 138139
[0112] The acids of formula 11 are known compounds or can be
prepared using standard methodologies. For the preparation of
substituted alkyl- or cycloalkylcarboxylic acids, alkylation
reactions can be employed using an alkali metal dianion of the acid
or monoanion of the corresponding ester. For example, a cycloalkyl
carboxylic acid ester of formula 75 can be treated with a strong
base, for example, lithium diisopropylamide in an inert solvent,
for example THF followed by addition of group R.sub.28--Lv wherein
R.sub.28 represents a desired side chain, such as a substituted
benzyl, lower alkyl, lower alkoxy alkyl, azidolower alkyl and the
like and Lv represents a leaving group such as a bromide, iodide,
mesylate or similar group known to participate in ester enolate
alkylation reactions. The product ester 76 may be hydrolyzed to the
acid 77 using alkali metal hydroxide in a suitable solvent, for
example aqueous alcohol. Depending on the nature of R.sub.28 and
the eventual target, the compound 77 may be a coupled to an amine
such as compound 23 and converted to the target directly or
R.sub.28 may be subject to further manipulation at a suitable point
in the synthesis. For example, if R.sub.28 is an azido lower alkyl
moiety, the azide may be reduced using for example a trialkyl
phosphine reagent followed by flnctionalization of the product
amine by alkylation, acylation, sulfonylation and related
procedures well known to those skilled in the art. If R.sub.28
incorparates a leaving group, for example, a terminal bromine atom,
this group may be displaced by an appropriate nucleophile, for
example, sodium methyl mercaptide to give in this case, a thioether
which may be the desired product or can be itself further
manipulated, for example by oxidation to a sulfoxide or sulfone
using standard reaction conditions. Other nucleophiles which may be
employed to produce intermediates leading to compounds of this
invention include: sodium cyanide, sodium methoxide, sodium azide,
morpholine and others. When R.sub.28 incorporates a ketal group,
this group may be hydrolzyed at a convenient point in the synthesis
to provide a keto group. This group in turn may be further
manipulated, for example by reduction to an alcohol or conversion
to derivative such as an oxime. 140
[0113] General Melting points were taken on a Thomas-Hoover
apparatus and are uncorrected. Optical rotations were determined
with a Perkin-Elmer model 241 polarimeter. .sup.1H-NMR spectra were
recorded with Varian XL-200 and Unityplus 400 MHz spectrometers,
using tetramethylsilane (TMS) as internal standard. Electron impact
(EI, 70 ev) and fast atom bombardment (FAB) mass spectra were taken
on VG Autospec or VG 70E-HF mass spectrometers. Silica gel used for
column chromatography was Mallinkrodt SiliCar 230-400 mesh silica
gel for flash chromatography; columns were run under a 0-5 psi head
of nitrogen to assist flow. Thin layer chromatograms were run on
glass thin layer plates coated with silica gel as supplied by E.
Merck (E. Merck #1.05719) and were visualized by viewing under 254
nm UV light in a view box, by exposure to I.sub.2 vapor, or by
spaying with either phosphomolybdic acid (PMA) in aqueous ethanol,
or after exposure to Cl.sub.2, with a
4,4'-tetramethyldiaminodiphenylmethane reagent prepared according
to E. Von Arx, M. Faupel and M Brugger, J. Chromatography, 1976,
120, 224-228.
[0114] Reversed phase high pressure liquid chromatography (RP-HPLC)
was carried out using either a Waters Delta Prep 4000 employing a
3.times.30 cm, Waters Delta Pak 15 .mu.M C-18 column at a flow of
40 mL/min employing a gradient of acetonitrile:water (each
containing 0.75% TFA) typically from 5 tp 95% acetonitrile over
35-40 min or a Rainin HPLC employing a 41.4 mm.times.30 cm, 8
.mu.M, Dynamax.TM. C-18 column at a flow of 49 mL/min and a similar
gradient of acetonitrile:water as noted above.
[0115] Dichloromethane (CH.sub.2Cl.sub.2), 2-propanol, DMF, THF,
toluene, hexane, ether, and methanol, were Fisher reagent grade and
were used without additional purification except as noted,
acetonitrile was Fisher hplc grade and was used as is.
[0116] Definitions:
[0117] THF is tetrahydrofuran,
[0118] DMF is N,N-dimethylformamide,
[0119] HOBT is 1-hydroxybenzotriazole,
[0120] BOP is
[(benzotriazole-1-yl)oxy]tris-(dimethylamino)phosphonium
hexafluorophosphate,
[0121] HATU is
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
[0122] HBTU is O-benzotriazole-N,N,N',N',-tetramethyluronium
hexafluorophosphate,
[0123] DIPEA is diisopropylethylamine,
[0124] DMAP is 4-(N,N-dimethylamino)pyridine
[0125] DPPA is diphenylphosphoryl azide
[0126] DBU is 1,8-diazabicyclo[5.4.0]undec-7-ene
[0127] NaH is sodium hydride
[0128] brine is saturated aqueous sodium chloride solution
[0129] TLC is thin layer chromatography
[0130] LDA is lithium diisopropylamide
[0131] BOP-Cl is bis(2-oxo-3-oxazolidinyl)phosphinic chloride
[0132] NMP is N-methyl pyrrolidinone
EXAMPLES
Example 1
[0133] Synthesis of 1-Benzylcyclopentane Carboxylic Acid Ethyl
Ester. 141
[0134] A solution of diisopropylamine (1.0 mL, 7.7 mmol) in 20 mL
of dry THF was cooled to -78.degree. C. in a dry ice-acetone bath
under argon. n-Butyl lithium in hexanes (2.5 M, 3.6 mL, 7.7 mmol)
was added all at once and the mixture was stirred for 0.5 hour and
transferred to a precooled (-30.degree. C.) solution of 1.0 g (7.0
mmol) of ethyl cyclopentane carboxylate in 10 mL of THF. After a
further 45 min, benzyl bromide (0.92 mL, 7.7 mmol) was added and
the mixture was allowed to warm to room temperature over night. The
resulting mixture was concentrated, and the residue was taken up in
70 mL of ether, washed with water, 1 N HCl, water, saturated brine
and was dried (MgSO.sub.4). The residue obtained after filtration
and evaporation was purified by flash chromatography over 100 g of
silica gel, eluting with 3% ethyl acetate-hexane to give
1-benzylcyclopentane carboxylic acid ethyl ester (0.80 g, 45%) as a
colorless oil.
Example 2
[0135] Synthesis of 1-benzylcyclopentane Carboxylic Acid. 142
[0136] A solution of 1-benzylcyclopentane carboxylic acid ethyl
ester (0.40 g, 1.7 mmol) in 10 mL of THF and 5 mL of methanol was
treated with a solution of 200 mg of lithium hydroxide hydrate in 5
mL of water and the mixture was stirred for 3 days at room
temperature and at 40.degree. C. for 3 days. The mixture was
diluted with water, washed with ether and acidified with excess 6 N
HCl. The aqueous layer was extracted with ether and the combined
extracts were washed with water and brine and were dried
(MgSO.sub.4). Evaporation gave 0.34 g (96%) of 1-benzylcyclopentane
carboxylic acid as a thick yellow oil.
Example 3
[0137] Synthesis of 1-[(4-methoxyphenyl)methyl]cyclopentane
Carboxylic Acid. 143
[0138] A solution of diisopropylamine (58 mL, 0.44 mol) in THF 400
mL was cooled to below 0.degree. C. and n-butyl lithium in hexane
(170 mL, 2.5 N, 0.43 mol) was added dropwise maintaining the
temperature below 0.degree. C. Upon completion of the addition,
ethyl cyclopentylcarboxylate (55 g, 0.39 mol) in THF (175 mL) was
added dropwise maintaining the internal temperature between -60 and
-70.degree. C. Upon completion of the addition, the internal
temperature was allowed to rise to -40.degree. C. and was held
there for 20 min and lowered back to -70.degree. C. A solution of
4-methoxybenzyl bromide (75 g, 0.48 mol) in 175 mL of THF was added
dropwise and the mixture was allowed to warm to room temperature
overnight. A solution of 20% ammonium chloride in water (225 mL)
was added followed by 450 mL of ethyl acetate, the layers were
separated, the aqueous layer was extracted with ethyl acetate (450
mL) and the combined organic layers were washed with saturated
brine (2.times.450 mL) and were dried (MgSO.sub.4). The crude
product was chromatographed on silica gel, eluting with 1-5% ether
in hexane to give 1-[(4-methoxyphenyl)methyl]cyclo-pentane
carboxylic acid ethyl ester (80.8 g, 79%) as an oil.
[0139] The material obtained above was dissolved in methanol (620
mL) and 2 N sodium hydroxide (350 mL) and the mixture was heated to
reflux overnight. The mixture was allowed to cool and was
concentrated. The yellow, basic residue was washed with ether
(2.times.500 mL) and was acidified with excess 6 N hydrochloric
acid to pH <2. This solution was extracted with dichloromethane
(2.times.500 mL), the extracts were combined, dried (MgSO.sub.4)
and evaporated to a white solid (69.6 g, 89%), mp 63.5-64.5.degree.
C.
Example 4
[0140] Synthesis of 1-(2-azidoethyl)cyclopentane Carboxylic Acid.
144
[0141] To an ice cold solution of diisopropylamine (56 mL, 0.396
mol) in THF 85 (mL) was added n-butyl lithium in hexane solution
(240 mL, 1.6 M, 0.393 mol) over 20 min. The mixture was stirred at
0.degree. C. for 30 min, cooled to a bath temperature of
-65.degree. C. and ethyl cyclopentane carboxylate (37.4 g, 0.263
mol) in THF (50 mL) was added over 20 min. After 1 hr, a solution
of 1,2-dibromoethane (47 mL, 0.545 mol) in THF (50 mL) was added,
the mixture was held at -65.degree. C. for 3 hr and allowed to warm
to room temperature overnight. The reaction was quenched by
addition of saturated ammonium chloride solution (200 mL), the
layers were separated and the aqueous layer was extracted with
ethyl acetate (100 mL). The combined extracts were washed with 1:1
brine:water (250 mL) and were dried (Na.sub.2SO.sub.4). The
solution was filtered and concentrated, diluted with toluene (100
mL) and concentrated. The dilution and concentration was repeated
twice to give ethyl 1-(2-bromoethyl)cyclopentane carboxylate (52.5
g).
[0142] A solution of the above bromide (52.5 g, 0.211 mol) and
sodium azide (54 g, 0.831 mol) in DMF (200 mL) was stirred at
50.degree. C. for 5 hr under a nitrogen atmosphere and was
filtered. The filtrate was concentrated to near dryness, diluted
with ethyl acetate (500 mL), filtered and concentrated to give
crude ethyl 1-(2-azidoethyl)cyclopentan- e carboxylate (40.9 g) as
a brown oil. This material was combined with product from a
previous run (total 63.5 g) and was purified by chromatography over
250 g of silica gel, eluting with 5% ethyl acetate in hexane to
give 50.3 g of product as a light brown oil.
[0143] The oil from above (50.3 g, 0.238 mol) was dissolved in THF
(750 mL) and methanol (375 mL) and a solution of LiOH hydrate (15
g, 0.357 mol) in water (300 mL) was added. The resulting solution
was stirred at 40.degree. C. overnight and concentrated. The
residue was dissolved in 2 L of water containing 40 mL of 1N NaOH
and was washed with hexane (1 L). The aqueous layer was acidified
with 1 N HCl (375 mL) and was extracted with ether (2.times.1 L).
The combined extracts were dried (Na.sub.2SO.sub.4) and
concentrated to give 1-(2-azidoethyl)cyclopentane carboxylic acid
(37.5 g) as an amber liquid.
Example 5
[0144] Synthesis of 4-(chloromethyl)-N-methylbenzamide. 145
[0145] To a solution of 4-chloromethyl benzoic acid (17.05 g, 100
mmol) in toluene (dried over molecular sieves 4A) was added thionyl
chloride (11 mL, 150 mmol). The mixture was heated to 80.degree. C.
and stirred overnight and then 3 h at 105.degree. C. The reaction
mixture was cooled to room temperature and excess thionyl chloride
and toluene were removed under vacuum. The resulting oily residue
was azeotroped with toluene (50 mL), then dried under high vacuum
for 45 min to give the crude acid chloride.
[0146] To the crude acid chloride in dichloromethane (200 mL, dried
over molecular sieves 4A) was added methylamine hydrochloride (7.5
g, 110 mmol) at -10.degree. C. in one portion. To the mixture
diisopropylamine (35 mL, 201 mmol) was added dropwise over 15 min
while maintaining the temperature of the reaction mixture below
2.degree. C. After addition, the suspension was allowed to warm to
room temperature and stirred for 30 min. Then, the reaction mixture
was diluted with water (125 mL) and the layers were separated. The
aqueous layer was extracted with dichloromethane (2.times.60 mL)
and the combined extracts were washed successively with water (150
mL) and brine solution (150 mL). After drying over anhydrous
magnesium sulfate, the solution was concentrated to 50 mL. The
precipitated white solid was collected by filtration and washed
with dichloromethane and hexane to obtain 4-(chloromethyl)-N-methy-
lbenzamide (12.02 g) as a white solid. A second crop material (3.05
g) was obtained from mother liquor by concetration and dilution
with hexane to give a total of 15.07 g, 82%, mp 138-139.5.degree.
C.
Example 6
[0147] Synthesis of 4-(1-methyltetrazol-5-yl)benzyl Chloride.
146
[0148] To a suspension of 4-(chloromethyl)-N-methylbenzamide (12 g,
65.3 mmol) in toluene (dried over molecular sieves 4A) was added
thionyl chloride (7.15 mL, 98 mmol). The mixture was heated to
reflux (90.degree. C.) and the resulting light yellow solution was
stirred at reflux overnight. The reaction mixture was cooled to
room temperature and excess thionyl chloride and toluene were
removed under vacuum. The resulting oily residue was azeotroped
with toluene (50 mL), then dried under high vacuum for 1.5 h to
give the crude imidoyl chloride.
[0149] To a suspension of sodium azide (5.1 g, 78.5 mmol) in
acetonitrile (62 mL) was added chlorotrimethylsilane (10.5 mL, 82.5
mmol) and the mixture was stirred for 1.5 h at room temperature.
After cooling to 0.degree. C., a solution of the crude imidoyl
chloride prepared above in acetonitrile (20 mL) was added. This
mixture was stirred for 1.5 h at 0.degree. C., then allowed to warm
to room temperature and stirred for 18 h. TLC analysis indicated
the presence of traces of starting amide. Then, the reaction
mixture was diluted with water (70 mL) and ethyl acetate (70 mL)
and was poured into a mixture of saturated ammonium chloride (70
mL) and ethyl acetate (70 mL). The two layers were separated and
the aqueous layer was extracted with ethyl acetate (2.times.50 mL).
The combined extracts were washed successively with water (90 mL)
and brine solution (90 mL). After drying over anhydrous magnesium
sulfate, the solution was concentrated to 40 mL to afford a white
precipitate. The solid was collected by filtration washing with
hexane. Attempted purification by crystallization in various
solvents were unsuccessful. Thus, it was purified by preparative
HPLC using ethyl acetate and hexane in 1:2 ratio as eluent to
afford 4-(1-methyltetrazol-5-yl)benzyl chloride (11.35 g, 83%) as a
white solid; mp 90-92.degree. C.
Example 7
[0150] Synthesis of
1-[[4-(1-methyltetrazol-5-yl)phenyl]methyl]cyclobutane Carboxylic
Acid. 147
[0151] A solution of diisopropylamine (1.05 mL, 7.5 mmol) in THF (5
mL) was colled to -10.degree. C. and a solution of n-butyl lithium
(2.9 mL, 7.25 mmol) in hexanes was added dropwise while maintaining
the temperature below 0.degree. C. After addition, the solution was
stirred for 30 min at 0.degree. C. The solution was cooled to
-70.degree. C. and a solution of methyl cyclobutane carboxylate
(0.57 g, 5 mmol) in THF (2 mL) was added dropwise maintaining the
internal temperature between -60 to -70.degree. C. After addition,
the reaction mixture was stirred for 30 min at -50 to -60.degree.
C. Then, a solution of 4-(1-methyltetrazol-5-yl- )benzyl chloride
(0.94 g, 4.5 mmol) in THF (5 mL) was added dropwise and the
reaction mixture was stirred for 1 h at -60 to -70.degree. C. Then,
it was allowed to warm to room temperature and stirred overnight at
which point TLC analysis indicated the absence of
4-(1-methyltetrazol-5-yl)benz- yl chloride (Note: the product and
4-(1-methyltetrazol-5-yl)benzyl chloride has the same Rf value,
however they were differentiated by spraying with PMA). The mixture
was poured into a mixture of water (70 mL) and ethyl acetate (70
mL). An emulsion formed and was filtered through celite. The
resulting two layers were separated and the aqueous layer was
extracted with ethyl acetate (2.times.50 mL). The combined extracts
were washed with saturated brine and dried over anhydrous magnesium
sulfate. After filtration, the solution was concentrated under
vacuum and the residue was purified by silica gel chromatography
eluting with 1:2 ethyl acetate:hexane to give methyl
1-[[4-(1-methyltetrazol-5-yl- )phenyl]methyl]cyclobutane
carboxylate (0.42 g, 32%) as a syrup. HR MS: obs. mass, 301.1668.
Calcd mass, 301.1664.
Example 8-14
[0152] Using the procedure described in example 7, the cyclopentane
carboxylate derivatives shown below were prepared.
3 148 Example R Yield % HR MS Obs. HR MS Calcd 8 149 66 248.1406
248.1412 9 150 99 252.0921 252.0917 9 151 59 243.1253 243.1259 10
152 71 278.1518 278.1518 11 153 53 301.1669 301.1664 12 154 18
301.1668 301.1664 13 CH3O(CH2)2-- 36 186.1257 187.1256 14 CH3OCH2--
45
Example 15
[0153] Synthesis of
1-[[4-(1-methyl-5-tetrazolyl)phenyl]methyl]cyclobutane Carboxylic
Acid. 155
[0154] To a solution of methyl
1-[[4-(1-methyltetrazol-5-yl)phenyl]methyl]- cyclobutane
carboxylate (0.33 g, 1.15 mmol) in a mixture of THF (7 mL) and
methanol (7 mL) was added 1 N sodium hydroxide (7 mL). The mixture
was heated to 55.degree. C. and stirred for 4 h at which point TLC
analysis indicated the absence of starting material. After cooling
to room temperature, the solvent was removed under vacuum and the
residue was diluted with water and extracted with ethyl acetate to
remove any neutral impurities. Then, the aqueous layer was
neutralized with 1 N hydrochloric acid and the product was
extracted with ethyl acetate (2.times.50 mL). The combined extracts
were washed with saturated brine and dried over sodium sulfate.
After filtration, the solution was concentrated under vacuum and
the residue was dried under high vacuum to afford
1-[[4-(1-methyl-5-tetrazolyl)phenyl]methyl]cyclobutane carboxylic
acid (180 mg, 57%) as a light yellow syrup.
Examples 16-23
[0155] Using the procedure described in example 15, the following
cyclopentane carboxylic acids were prepared from the corresponding
methyl esters:
4 156 Example R Yield % HR MS Obs. HR MS Calcd 16 157 90 234.1264
234.1256 17 158 73* 238.0757 238.0761 18 159 79 230.1173 230.1181
19 160 96 264.1361 264.1362 20 161 95 267.1508 267.1508 21 162 100
286.1433 286.1430 22 CH3O(CH2)2-- 92 172.1095 172.1099 23 CH3OCH2--
73 158.0948 158.0943
[0156] yield is the two steps according to the procedures described
in examples 13 and 15.
Example 24
[0157] Synthesis of 1-[(4-methoxyphenyl)methyl]cyclohexane
Carboxylic Acid 163
[0158] Using the procedures described in examples 7 and 15,
starting with 4-methoxybenzyl chloride,
1-[(4-methoxyphenyl)methyl]cyclohexane carboxylic acid was prepared
in 23% overall yield. HRMS: obs. mass, 248.1426. Calcd. mass,
248.1412 (M+).
Example 25
[0159] Synthesis of
1-[3-(1-methyl-5-tetrazolyl)phenyl]methyl]cyclohexane Carboxylic
Acid 164
[0160] Using the procedures described in examples 7 and 15,
starting with 1-[3-(1-methyl-5-tetrazolyl)benzyl chloride,
1-[3-(1-methyl-5-tetrazolyl)- phenyl]methyl]cyclohexane carboxylic
acid was prepared in 77% overall yield. HRMS: obs. mass, 301.1667.
Calcd. mass, 301.1664 (M+H).
Example 26
[0161] Synthesis of
N-[(1-phenylcyclopentyl)carbonyl]-4-amino-L-phenylalan- ine Methyl
Ester 165
[0162] To a solution of 4-nitrophenylalanine hydrochloride methyl
ester (3.90 g, 15 mmol) and 1-phenylcyclopentane carboxylic acid
(3.4 g, 18 mmol) in 30 mL of DMF was added HBTU (6.8 g, 18 mmol)
and diisopropylethyl amine (6.4 mL, 30 mmol) at room temperature.
The mixture was then stirred at this temperature for 8 hr. The
reaction was then diluted with 250 mL of ethyl acetate and was
washed with 0.5 N HCl (40 mL), saturated NaHCO3 (2.times.40 mL) and
saturated brine (2.times.40 mL). After removal of the solvent, the
residue was purified on a silica gel column eluting with ethyl
acetate:hexane (1:3) to give
N-[(1-phenylcyclopentyl)carbonyl]-4-nitro-L-phenylalanine methyl
ester (4.5 g, 75.7%).
[0163] A suspension of
N-[(1-phenylcyclopentyl)carbonyl]-4-nitro-L-phenyla- lanine methyl
ester (3.5 g, 8.88 mmol) and stannous chloride (10 g, 44 mmol) in
60 mL of ethanol was refluxed for 50 min under argon. The ethanol
was then removed under reduced pressure and the residue was treated
with 50 mL of saturated NaHCO3 followed by sodium carbonate to
adjust pH above 9. The white slurry was extracted with ethyl
acetate (3.times.300 mL). The combined extracts were washed with
water (100 mL) and brine (100 mL) and were dried (MgSO4). Removal
of the solvent afforded
4-amino-N-[(1-phenylcyclopentyl)carbonyl]-L-phenylalanine methyl
ester (2.9 g, 89%).
Example 27
[0164] Synthesis of
N-[(1-phenylcyclopentyl)carbonyl]-4-[(4-quinolinylcarb-
onyl)amino]-L-phenylalanine Sodium Salt 166
[0165] To a solution of
4-amino-N-[(1-phenylcyclopentyl)carbonyl]-L-phenyl- alanine methyl
ester (81 mg, 0.2 mmol) and 4-quinolinecarboxylic acid (43.3 mg,
0.25 mmol) in 1 mL of DMF was added HBTU (95 mg, 0.25 mmol) and
diisopropylethylamine (65 .mu.L, 0.5 mmol) at room temperature. The
mixture was then stirred at this temperature for overnight. The
reaction was then diluted with 15 mL of ethyl acetate and was
washed with water (2 mL), saturated NaHCO3 (2.times.2 mL) and
saturated brine (2.times.2 mL). The solution was dried (MgSO4) and
concentrated. The residue was hydrolyzed with 0.5 mL of 1N NaOH in
5 mL of ethanol at 25.degree. C. overnight. The crude product was
purified by passing through an open C-18 column eluting with water
(200 mL), 30% methanol in water (200 mL), 40% methanol in water
(200 mL) and pure methanol (200 mL). The fractions containing
product were concentrated and lyophilized to give
N-[(1-phenylcyclopentyl)carbonyl]-4-[(4-quinolinylcarbonyl)amino]-L-pheny-
lalanine sodium salt (79.5 mg, 75%), HR-FABMS: obs. mass, 530.2056.
Calcd. mass, 530.2058.
Examples 28-31
[0166] Using the general method described in Example 27, the
following analogs were prepared starting with the product from
example 26 and the appropriate benzoic or hetereoaromatic
carboxylic acids:
5 167 HRMS (M + H) Example R' calcd. found 28 168 457.2127 457.2135
29 169 496.1848 496.1844 30 170 572.2161 572.2144 31 171 530.2056
530.2057
Example 32
[0167] Synthesis of
4-nitro-N-[[1-(phenylmethyl)cyclopentyl]carbonyl]-L-ph- enylalanine
Methyl Ester 172
[0168] A solution of 1-benzylcyclopentane carboxylic acid (0.135 g,
0.66 mmol), 4-nitro-L-phenylalanine methyl ester (0.187 g, 0.72
mmol), and HBTU (0.272 g, 0.72 mmol) in 2 mL of DMF was treated
with diisopropylethylamine (0.35 mL, 2 mmol). The mixture was
stirred over night, concentrated, diluted with ethyl acetate,
washed with water, 1 N HCl, water, saturated NaHCO3 and dried
(MgSO4). The residue obtained after evaporation was purified by
chromatography over 30 g of silica gel, eluting with 40% ethyl
acetate:hexane to give 4-nitro-N-[[1-(phenylmethyl-
)cyclopentyl]carbonyl]-L-phenylalanine methyl ester (194 mg, 71%),
as a white foam.
Example 33
[0169] Synthesis of
4-amino-N-[[1-(phenylmethyl)cyclopentyl]carbonyl]-L-ph- enylalanine
Methyl Ester 173
[0170] A solution of
4-nitro-N-[[1-(phenylmethyl)cyclopentyl]carbonyl]-L-p- henylalanine
methyl ester (185 mg, 0.45 mmol) in 10 mL of ethanol was
hydrogenated at atmospheric pressure over 47 mg of 10% Pd(C) for 3
hours. The reaction mixture was filtered through a pad of celite
and evaporated to dryness to give
4-amino-N-[[1-(phenylmethyl)cyclopentyl]carbonyl]-L-ph- enylalanine
methyl ester (170 mg, 99%) of as a white solid suitable for use in
the next step.
Example 34
[0171] Synthesis of
N-[[1-(phenylmethyl)cyclopentyl]carbonyl]-4-[(4-quinol-
inylcarbonyl)amino)-L-phenylalanine 174
[0172] A solution of
4-amino-N-[[1-(phenylmethyl)cyclopentyl]carbonyl]-L-p- henylalanine
methyl ester (29.5 mg, 0.078 mmol), quinoline-4-carboxylic acid (17
mg, 0.10 mmol), and HBTU (38 mg, 0.10 mmol) in 1 mL of DMF was
treated with diisopropylethylamine (30 .mu.L, 0.17 mmol). The
mixture was stirred over night and was diluted with 15 mL of ethyl
acetate and 10 mL of ether and was washed with portions of water
(2.times.10 mL), saturated NaHCO3 (10 mL) and was dried (MgSO4).
Concentration gave 48 mg which was dissolved in 3 mL of methanol.
Sodium hydroxide solution (0.10 mL, 4 N, 0.4 mmol) was added and
the mixture was stirred for 2 hr. The excess base was quenched by
addition of 0.1 mL of acetic acid, the solution was filtered
through a 0.2.mu. nylon filter and the filtrate was purified by
RP-HPLC on a 4.times.30 cm Rainin C-18 column using a gradient of 5
to 95% acetonitrile:water containing 0.75% trifluoroacetic acid at
a flow of 49 mL/min over 30 min. The peak eluting at 74.5%
acetonitrile was the acid
N-[[1-(phenylmethyl)cyclopentyl]carbonyl]-4-[(4-quinolinylcarbonyl)a-
mino)-L-phenylalanine (15 mg), HR-FAB-MS: obs. mass 522.2393.
Calcd. mass 522.2393 (M+H), the peak eluting at 83% acetonitrile
was recovered
N-[[1-(phenylmethyl)cyclopentyl]carbonyl]-4-[(4-quinolinylcarbonyl)amino]-
-L-phenylalanine methyl ester (15 mg), HR-FAB-MS: obs. mass
536.2556. Calcd. mass 536.2549 (M+H).
Example 35
[0173] Synthesis of
4-[[(2-nitrophenyl)carbonyl]amino]-N-[[1-(phenylmethyl-
)cyclopentyl]carbonyl]-L-phenylalanine 175
[0174]
4-Amino-N-[[1-(phenylmethyl)cyclopentyl]carbonyl]-L-phenylalanine
methyl ester (31.7 mg, 0.083 mmol) and 2-nitrobenzoic acid (38 mg,
0.10 mmol) were reacted as described in example 34 to give 26.7 mg
of
4-[[(2-nitrophenyl)carbonyl]amino]-N-[[1-(phenylmethyl)cyclopentyl]carbon-
yl]-L-phenylalanine, HR-FAB-MS: obs. mass 516.2113. Calcd. mass
516.2134 (M+H).
Example 36
[0175] Synthesis of
4-[[(2-methyl-5-nitrophenyl)carbonyl]amino]-N-[[1-(phe-
nylmethyl)cyclopentyl]carbonyl]-L-phenylalanine. 176
[0176]
4-Amino-N-[[1-(phenylmethyl)cyclopentyl]carbonyl]-L-phenylalanine
methyl ester (35.3 mg, 0.093 mmol) and 2-methyl-5-nitrobenzoic acid
20 mg, 0.11 mmol) were reacted as described in example 34 to give
4-[[(2-methyl-5-nitrophenyl)carbonyl]amino]-N-[[1-(phenylmethyl)cyclopent-
yl]carbonyl]-L-phenylalanine (34 mg, 69%), HR-FAB-MS: obs. mass
530.2298. Calcd. mass 530.2291 (M+H).
Example 37
[0177] Synthesis of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(phenylm-
ethyl)cyclopentyl]carbonyl]-L-phenylalanine 177
[0178]
4-Amino-N-[[1-(phenylmethyl)cyclopentyl]carbonyl]-L-phenylalanine
methyl ester (67 mg, 0.176 mmol) and 2,6-dichlorobenzoyl chloride
(50 mg, 0.23 mmol) were dissolved in 5 mL of dichloromethane and
2,6-lutidine (50 .mu.L, 0.43 mmol) were added. After 4 hours, the
mixture was diluted with ether and dichloromethane and washed with
1 N HCl, water, and saturated NaHCO3 and was dried (MgSO4). The
crude product was dissolved in 4 mL of methanol and treated with 4
N NaOH (0.1 mL). After 2 hours, the excess base was quenched with
0.1 mL of acetic acid, the solution was filtered through a 0.2.mu.
nylon filter and the filtrate was purified by RP-HPLC on a
4.times.30 cm Rainin C-18 column using a gradient of 5 to 95%
acetonitrile:water containing 0.75% trifluoroacetic acid at a flow
of 49 mL/min over 30 min. The peak eluting at 87% acetonitrile was
concentrated and lyophilized to give
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(ph-
enylmethyl)cyclopentyl]carbonyl]-L-phenylalanine (46 mg),
LR(+)LSIMS : m/z 539 (M+H) (2 Cl)
Example 38
[0179] Synthesis of
N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-4-
-nitro-L-phenylalanine Methyl Ester. 178
[0180] A solution of 1-(4-methoxy)benzylcyclopentane carboxylic
acid (0.0.20 g, 0.86 mmol), 4-nitro-L-phenylalanine methyl ester
(0.24 g, 0.94 mmol), and HBTU (0.36 g, 0.94 mmol) in 3 mL of DMF
was treated with 0.52 mL (3 mmol) of diisopropylethylamine. The
mixture was stirred over night, concentrated, diluted with ethyl
acetate, washed with water, 1 N HCl, water, saturated NaHCO3 and
dried (MgSO4). The residue obtained after evaporation was purified
by chromatography over 30 g of silica gel, eluting with 40% ethyl
acetate:hexane to give N-[[1-[(4-methoxyphenyl)met-
hyl]cyclopentyl]carbonyl]-4-nitro-L-phenylalanine methyl ester (256
mg, 68%), as a white foam, HR-FAB-MS: Obs mass, 441.2027. Calcd
mass, 441.2025 (M+H).
Example 39
[0181] Synthesis of
4-amino-N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]car-
bonyl]-L-phenylalanine Methyl Ester 179
[0182] A solution of
N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]--
4-nitro-L-phenylalanine methyl ester (253 mg, 0.575 mmol) in 10 mL
of ethanol was hydrogenated at atmospheric pressure over 45 mg of
10% Pd(C) for 3 hours. The reaction mixture was filtered through a
pad of celite and evaporated to dryness to give
4-amino-N-[[1-[(4-methoxyphenyl)methyl]-
cyclopentyl]carbonyl]-L-phenylalanine methyl ester (225 mg, 95%) as
a white solid suitable for use in the next step, HR-FAB-MS: Obs
mass, 410.2196. Calcd mass, 410.2200 (M+).
Example 40
[0183] Synthesis of
N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-4-
-[(4-quinolinyl carbonyl)amino]-L-phenylalanine. 180
[0184] A solution of
4-amino-N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]ca-
rbonyl]-L-phenylalanine methyl ester (30.7 mg, 0.075 mmol),
quinoline-4-carboxylic acid (17 mg, 0.10 mmol), and HBTU (38 mg,
0.10 mmol) in 1 mL of DMF was treated with diisopropylethylamine
(30 .mu.L, 0.17 mmol). The mixture was stirred over night and was
diluted with 15 mL of ethyl acetate and 10 mL of ether and was
washed with water (2.times.10 mL), saturated NaHCO3 (1.times.10 mL)
and was dried (MgSO4). Concentration gave 42 mg which was dissolved
in 3 mL of methanol. Sodium hydroxide (0.10 mL, 4 N, 0.4 mmol) was
added and the mixture was stirred for 2 hours. The excess base was
quenched by addition of acetic acid (0.1 mL), the solution was
filtered through a 0.2.mu. nylon filter and the filtrate was
purified by RP-HPLC on a 4.times.30 cm Rainin C-18 column using a
gradient of 5 to 95% acetonitrile:water containing 0.75%
trifluoroacetic acid at a flow of 49 mL/min over 30 min. The peak
eluting at 74% acetonitrile was concentrated and lyophilized to
give
N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-4-[(4-quinolinylcarb-
onyl)amino]-L-phenylalanine (22.8 mg), HR-FAB-MS: obs. mass
552.2482. Calcd. mass 552.2498 (M+H). The peak eluting at 82.6%
acetonitrile was recovered
N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-4-[(4-quin-
olinylcarbonyl)amino]-L-phenylalanine methyl ester (11.2 mg),
HR-FAB-MS: obs. mass 566.2675. Calcd. mass 566.2655 (M+H).
Example 41
[0185] Synthesis of
N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-4-
-[[(2-nitrophenyl)carbonyl]amino]-L-phenylalanine. 181
[0186]
4-Amino-N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-L-phen-
ylalanine methyl ester (32.3 mg, 0.079 mmol) and 2-nitrobenzoic
acid (17 mg, 0.10 mmol) were reacted as described in example 40 to
give 22 mg of
N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-4-[[(2-nitrophenyl)c-
arbonyl]amino]-L-phenylalanine, HR-FAB-MS: obs. mass 546.2235.
Calcd. mass 546.2240 (M+H).
Example 42
[0187] Synthesis of
N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-4-
-[[(2-methyl-5-nitrophenyl)carbonyl]amino]-L-phenylalanine. 182
[0188]
4-Amino-N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-L-phen-
ylalanine methyl ester (23.4 mg, 0.057 mmol) and
2-methyl-5-nitrobenzoic acid 13 mg, 0.07 mmol) were reacted as
described in example 40 to give 23 mg (69%) of
N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-4-[[(2-m-
ethyl-5-nitrophenyl)carbonyl]amino]-L-phenylalanine, HR-FAB-MS:
obs. mass 560.2413. Calcd. mass 560.2397 (M+H).
Example 43
[0189] Synthesis of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[(4-meth-
oxyphenyl)methyl]cyclopentyl]carbonyl]-L-phenylalanine. 183
[0190]
4-Amino-N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-L-phen-
ylalanine methyl ester (66 mg, 0.17 mmol) and 2,6-dichlorobenzoyl
chloride (50 mg, 0.23 mmol) were dissolved in 5 mL of
dichloromethane and 2,6-lutidine (50 .mu.L, 0.43 mmol) were added.
After 4 hours, the mixture was diluted with ether and
dichloromethane and washed with 1 N HCl, water, and saturated
NaHCO3 and was dried (MgSO4). The crude product was dissolved in
methanol (4 mL) and treated with 4 N NaOH (0.1 mL). After 2 hours,
the excess base was quenched with acetic acid (0.1 mL), the
solution was filtered through a 0.2.mu. nylon filter and the
filtrate was purified by RP-HPLC on a 4.times.30 cm Rainin C-18
column using a gradient of 5 to 95% acetonitrile:water containing
0.75% trifluoroacetic acid at a flow of 49 mL/min over 35 min. The
peak eluting at 79% acetonitrile was concentrated and lyophilized
to give
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[(4-methoxyphenyl)methyl]cy-
clopentyl]carbonyl]-L-phenylalanine (28.2 mg), HR-FABMS:
[0191] obs. mass 591.1445. Calcd. mass 591.1430 (M+Na).
Example 44
[0192] Synthesis of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(1,1-dimeth-
ylethoxy)carbonyl]-L-phenylalanine Methyl Ester 184
[0193] To a solution of
4-(amino)-N-[(1,1-dimethylethoxy)carbonyl]-L-pheny- lalanine methyl
ester (2.6 g, 8.6 mmol) in dichloromethane (20 mL) were added
diisopropylethylamine (2.3 mL, 13 mmol) followed by
2,6-dichlorobenzoyl chloride (1.99 g, 9.5 mmol) at room
temperature. The mixture was stirred for 15 h at which time a white
precipitate was formed. The mixture was diluted with 30 mL of
dichloromethane and 50 mL of water. The layers were separated and
the aqueous layer was extracted with 100 mL (2.times.50 mL) of
dichloromethane. The combined organic extracts were washed with
brine and dried over anhydrous magnesium sulfate. Filtration and
concentration of the solvent gave 4.03 g (quantitative) of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(1,1-dimethy-
lethoxy)-carbonyl]-L-phenylalanine methyl ester as a white solid,
mp 148-151.degree. C.
Example 45
[0194] Synthesis of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalanin- e Methyl
Ester Hydrochloride Salt 185
[0195]
4-[[(2,6-Dichlorophenyl)carbonyl]amino]-N-[(1,1-dimethylethoxy)carb-
onyl]-L-phenylalanine methyl ester (1.86 g, 4.0 mmol) was treated
with 4 N hydrochloric acid in dioxane (10 mL) at room temperature.
After 5 minutes, the solids went into solution and the mixture was
stirred for 1 h. Then, 25 mL of ethyl ether was added to
precipitate the product. The solids were collected by filtration
and washed with hexane. The resulting solid was very hydroscopic
and became gummy. This material was dissolved in 50 mL of methanol
and concentrated. After drying under high vacuum,
4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalanine methyl
ester hydrochloride salt. (1.64 g, 97%) was obtained as a light
yellow solid: mp 158-161.degree. C.
Example 46
[0196] Synthesis of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(4-metho-
xyphenyl-methyl)cyclohexyl]carbonyl]-L-phenylalanine Methyl Ester.
186
[0197] To a solution of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylal- anine methyl
ester hydrochloride salt (0.2 g, 0.5 mmol) and
1-(4-methoxyphenylmethyl)cyclohexane-carboxylic acid (0.15 g, 0.60
mmol) in DMF (2 mL) were added HBTU (0.23 g, 0.60 mmol) and
diisopropylethylamine (0.20 mL, 1.2 mmol) at room temperature. The
mixture was stirred overnight and diluted with 25 mL of ethyl
acetate. The ethyl acetate layer was washed successively with 0.5N
hydrochloric acid (2.times.20 mL), saturated sodium bicarbonate
solution (2.times.20 mL), brine (1.times.20 mL) and dried over
anhydrous magnesium sulfate. Filtration and concentration gave 350
mg of white solid which was purified by column chromatography over
15 g of silica gel, eluting with 20-30% ethyl acetate in hexane to
give 4-[[(2,6-dichlorophenyl)carbonyl]a-
mino]-N-[[1-(4-methoxyphenylmethyl)cyclohexyl]carbonyl]-L-phenylalanine
methyl ester (0.25 g, 84% ) as a white solid, mp 85-87.degree. C.
HR MS: Obs mass, 597.1913. Calcd mass, 597.1923 (M+H).
Example 47
[0198] Synthesis of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(4-metho-
xyphenylmethyl)cyclohexyl]carbonyl]-L-phenylalanine. 187
[0199] To a suspension of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(4-
-methoxyphenyl-methyl)cyclohexyl]carbonyl]-L-phenylalanine methyl
ester (0.15 g, 0.25 mmol) in ethanol (2 mL) was added aqueous 1.0 N
sodium hydroxide (1.5 mL, 3 mmol) at room temperature. The mixture
was heated to 50.degree. C. and the resulting clear solution was
stirred overnight. The mixture was concentrated, the residue was
diluted with 5 mL of water and extracted with 25 mL of ether to
remove any neutral impurities. The aqueous layer was acidified with
1 N HCl and the precipitated white solid was collected by
filtration and washed with water (20 mL) and hexane (20 mL). After
air-drying, 4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(4-m-
ethoxyphenylmethyl)cyclohexyl]carbonyl]-L-phenylalanine (0.12 g,
82%) was obtained as a white solid, mp 136-140.degree. C. HR MS:
Obs mass, 583.1763. Calcd mass, 583.1766 (M+H).
Examples 48 to 60
[0200] The compounds shown below were prepared according to the
procedures given examples 46 and 47.
6 188 Starting Material From HRMS (M + H) Example Example R Yield %
calcd. obs. 48 20 189 92 621.1784 621.1758 49 commercial 190 93
517.1297 517.1285 50 25 191 89 635.1940 635.1967 51 17 192 84
573.1114 573.1113 52 16 193 99 569.1610 569.1620 53 18 194 91
586.1277 586.1285 54 19 195 87 599.1715 599.1714 55 21 196 91
621.1784 621.1782 56 401 197 95 607.1627 607.1644 57 15 198 80
607.1627 607.1640 58 4 199 99 518.1362 518.1345 59 22 200 93
507.1454 507.1464 60 23 201 99 493.1297 493.1300
Example 61
[0201] Coupling of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-4-[[(2-propenyloxy-
)carbonyl]amino]-L-phenylalanine to Wang Resin 202
[0202] A 250 mL cylindrical glass vessel equipped with a coarse
glass frit was charged with 10 g of Wang resin, (loading factor:
1.15 mmol/g, 300 mesh). The resin was washed with dichloromethane
(2.times.100 mL), methanol (2.times.100 mL) and dimethylformamide
(2.times.100 mL). To the swollen resin was added
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-4-[[(2-prope-
nyloxy)carbonyl]amino]-L-phenylalanine (11.2 g, 23 mmol) and
2,6-dichlorobenzoyl chloride (8.06 mL, 57.5 mmol) in
N-methylpyrrolidone (70 mL) and the mixture was agitated for 30
minutes. Pyridine (6.45 mL, 80.5 mmol) was added and the resulting
mixture was agitated for 24 hr. The substitution was determined to
be 0.75 mmol of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-4-[[(2-propenyloxy)carbonyl]amino]-L-
-phenylalanine per gram of resin by quantitative UV measurement of
the fmoc present on the resin.
Example 62
[0203] Synthesis of
4-amino-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-phenyla- lanine on
Wang Resin 203
[0204] A 500 mL cylindrical glass vessel equipped with a coarse
glass frit was charged with
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-4-[[(2-propenyloxy)-
carbonyl]amino]-L-phenylalanine substituted Wang resin (10 g, 7.5
mmol) obtained from Example 61 and a solution prepared from
bis(triphenylphosphine)palladium dichloride (1.6 g, 2.3 mmol) and
acetic acid (5 mL, 83 mmol) in dried dichloromethane (150 mL). The
resulting mixture was agitated for 30 minutes followed by the
addition of tri-n-butyl tin hydride (20 mL, 74.3 mmol). The
resulting mixture was agitated for 1 hr. To the mixture was added
tri-n-butyl tin hydride (10 mL, 37 mmol). Agitation was continued
for 1 hour and the mixture was filtered. To the resulting resin was
added a solution prepared from bis(triphenylphosphine)palladium
dichloride (1.6 g, 2.3 mmol) and acetic acid (5 mL, 83 mmol) in dry
dichloromethane (150 mL). The mixture was agitated for 30 minutes
followed by the addition of tri-n-butyl tin hydride (20 mL, 74.3
mmol). The resulting mixture was agitated 1 hour. To the mixture
was added additional tri-n-butyl tin hydride (10 mL, 37.15 mmol).
Agitation was continued for 1 hour. After the second deprotection
cycle, the mixture was washed with dichloromethane (2.times.100
mL), methanol (2.times.100 mL) and dimethylformamide (2.times.100
mL) to give
4-amino-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-phenylalanine on
Wang resin suitable for use in subsequent steps.
Example 63
[0205] Synthesis of 4-[(4-quinolinylcarbonyl)amino]-L-phenylalanine
on Wang Resin 204
[0206] A 250 mL cylindrical glass vessel equipped with a coarse
glass frit was charged with
4-amino-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-phenylala- nine (10
g) obtained in Example 62 and a solution prepared from
quinoline-4-carboxylic acid (5.2 g, 30 mmol), BOP (13.75 g, 30
mmol) and diisopropylethylamine (6.8 mL) in 70 mL of
N-methylpyrrolidinone. The slurry was agitated for 4 hr. The
mixture was filtered and washed with dichloromethane (2.times.100
mL), methanol (2.times.100 mL) and dimethylformamide (2.times.100
mL). To the washed resin was added a solution of 25% piperidine in
N-methylpyrrolidinone (80 mL), the mixture was agitated at room
temperature for 20 minutes and filtered. The process was repeated
and the resulting slurry was filtered and washed with
dichloromethane (2.times.100 mL), methanol (2.times.100 mL) and
dimethylformamide (2.times.100 mL). Filtration afforded
4-[(4-quinolinylcarbonyl)amino]-L-phenylalanine on Wang resin
suitable for use in the next step.
Example 64
[0207] Synthesis of
N-[(2,2-dichloro-1-methylcyclopropyl)carbonyl]-4-[(4-q-
uinolinylcarbonyl)amino]-L-phenylalanine 205
[0208] 4-[(4-Quinolinylcarbonyl)amino]-L-phenylalanine on Wang
resin (300 mg) obtained from Example 63 was washed with
dichloromethane (2.times.10 mL), methanol (2.times.10 mL) and
dimethylformamide (2.times.10 mL). To the resin was added a
solution prepared from 2,2-dichloro-1-methylcyclopr- opylcarboxylic
acid (180 mg, 1.02 mmol), BOP (450 mg, 1.02 mmol) and
diisopropylethylamine (0.23 mL) in 4 mL of N-methylpyrrolidinone at
room temperature. The resulting mixture was agitated for 2 hours.
The reaction mixture was then filtered and washed with
dichloromethane (2.times.10 mL), methanol (2.times.10 mL) and
dichloromethane (2.times.10 mL). Cleavage was effected with 90%
trifluoroacetic acid (TFA) in dichloromethane for 5 minutes. The
mixture was filtered and the TFA was removed under high vacuum.
Addition of ether (25 mL) effected precipitation of
N-[(2,2-dichloro-1-methylcyclopropyl)carbonyl]-4-[(4-qui-
nolinylcarbonyl)amino]-L-phenylalanine. The compound was purified
by reverse phase HPLC (DuPont, Rx C18, 7 .mu.M, 2.12 cm.times.25
cm) using acetonitrile and water as the mobile phase with a linear
gradient from 20-50% of acetonitrile over 180 minutes. LRMS (M+H)
obs. mass, 486.5. Calcd. mass 486.3.
Examples 65-81
[0209] Using the procedure described in Example 64, the compounds
shown below were prepared.
7 206 Example R 65 207 66 208 67 209 68 210 69 211 70 212 71 213 72
214 73 215 74 216 75 217 76 218 77 219 78 220 79 221 80 222 81
223
Examples 82 to 106
[0210] Using the method described in examples 63 and 64, the
following derivatives were prepared:
8 224 Example Y X 82 225 226 83 227 228 84 229 230 85 231 232 86
233 234 87 235 236 88 237 238 89 239 240 90 241 242 91 243 244 92
245 246 93 247 248 94 249 250 95 251 252 96 253 254 97 255 256 98
257 258 99 259 260 100 261 262 101 263 264 102 265 266 103 267 268
104 269 270 105 271 272 106 273 274
Example 107
[0211] Synthesis of
N-[(1,1-dimethylethoxy)carbonyl]-4-[[(9H-fluoren-9-ylm-
ethoxy)carbonyl]amino]-L-phenylalanine Phenylmethyl Ester 275
[0212]
N-[(1,1-Dimethylethoxy)carbonyl]-4-[[(9H-fluoren-9-ylmethoxy)carbon-
yl]amino]-L-phenylalanine (5.02 g, 10 mmol) and benzyl bromide (3.5
mL, 29 mmol) were stirred in DMF (25 mL) over KHCO3 (1.75 g, 17.5
mmol). After 18 hr, a white precipitate had formed. Most of the DMF
was evaporated under reduced pressure, the residue was taken up in
100 mL of dichloromethane and was washed with water (2.times.50
mL). Most of the dichloromethane was evaporated and ether (100 mL)
was added to precipitate the product. Filtration, washing with
ether afforded
N-[(1,1-dimethylethoxy)carbonyl]-4-[[(9H-fluoren-9-ylmethoxy)carbonyl]ami-
no]-L-phenylalanine phenylmethyl ester (5.3 g), mp 186-187.degree.
C.
Example 108
[0213] Synthesis of
4-amino-N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]car-
bonyl]-L-phenylalanine 276
[0214]
4-Amino-N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-L-phen-
ylalanine methyl ester (280 mg, 0.68 mmol) in THF (12 mL) was
treated with a solution of lithium hydroxide hydrate (100 mg, 2.4
mmol) in water (2 mL) and the mixture was stirred for 3 hr. The
mixture was concentrated and the residue was acidified with 6 H HCl
to give a white sticky solid. Trituration with water and drying
under high vacuum afforded crude
4-amino-N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-L-phenylalan-
ine (200 mg) suitable for use in the next step.
Example 109
[0215] Synthesis of
4-(2,3-dihydro-1,3-dioxo-1H-pyrrolo[3,4-c]pyridin-2-yl-
)-N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-L-phenylalanine
277
[0216] A solution of 4-amino
-N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]c-
arbonyl]-L-phenylalanine (98 mg, 0.23 mmol), DIPEA (30 .mu.L, 0.23
mmol) and 3,4-pyridinedicarboxylic acid anhydride (114 mg, 0.77
mmol) in dichloromethane (10 mL) was stirred 18 hr at room
temperature. The mixture was concentrated to remove most of the
dichloromethane and the residue was taken up in DMF (3 mL).
Carbonyl diimidazole (103 mg, 0.64 mmol) was added to the resulting
solution and the reaction was allowed to proceed for 18 hr. The
resulting mixture was purified directly by RP-HPLC on a 4.times.30
cm Rainin C-18 column using a gradient of 5 to 95%
acetonitrile:water containing 0.75% trifluoroacetic acid at a flow
of 49 mL/min over 35 min. The peak eluting at 45.6% acetonitrile
was concentrated and lyophilized to give
4-(2,3-dihydro-1,3-dioxo-1H-pyrrolo[-
3,4-c]pyridin-2-yl)-N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-L-
-phenylalanine (28 mg) HR FABMS: obs. mass 528.2146. Calcd. mass
528.2134 (M+H).
Example 110
[0217] Synthesis of
4-(1,3-dioxo-2H-isoindol-2-yl)-N-[[1-[(4-methoxyphenyl-
)methyl]cyclopentyl]carbonyl]-L-prolyl]-L-phenylalanine 278
[0218] Using the procedure described in example 109, starting with
phthalic anhydride,
4-(1,3-dioxo-2H-isoindol-2-yl)-N-[[1-[(4-methoxypheny-
l)methyl]cyclopentyl]carbonyl]-L-prolyl]-L-phenylalanine was
obtained, HR FABMS: obs. mass 527.2172. Calcd. mass 527.2182
(M+H).
Example 111
[0219] Synthesis of
4-[(RS)-2,3,5,6,7,7a-hexahydro-1,3-dioxo-1H-pyrrolo[3,-
4-c]pyridin-2-yl]-N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-L-p-
henylalanine and
N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-4-[(-
3aRS,7aRS)-(octahydro-1,3-dioxo-1H-pyrrolo[3,4-c]pyridin-2-yl)]-L-phenylal-
anine 279
[0220] A solution of
4-(2,3-dihydro-1,3-dioxo-1H-pyrrolo[3,4-c]pyridin-2-y-
l)-N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-L-phenylalanine
(30 mg, 0.057 mmol) in ethanol (4 mL) containing a few drops of TFA
was hydrogenated over 10% Pd(C) (6 mg) for 18 hours. The mixture
was filtered and evaporated and the residue was purified by RP-HPLC
on a 4.times.30 cm Rainin C-18 column using a gradient of 5 to 95%
acetonitrile:water containing 0.75% trifluoroacetic acid at a flow
of 49 mL/min over 35 min. The peak eluting at 59.5% acetonitrile
was concentrated and mixture was lyophilized to give
N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]--
4-[(3aRS,7aRS)-(octahydro-1,3-dioxo-1H-pyrrolo[3,4-c]pyridin-2-yl)]-L-phen-
ylalanine (4.2 mg), HR FABMS: obs. mass 534.2602. Calcd. mass
534.2604 (M+H). The peak eluting at 62% acetonitrile was
concentrated and the mixture lyophilzed to give
4-[(RS)-2,3,5,6,7,7a-hexahydro-1,3-dioxo-1H-py-
rrolo[3,4-c]pyridin-2-yl]-N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbo-
nyl]-L-phenylalanine (4.9 mg), HR FABMS: obs. mass 532.2452. Calcd.
mass 532.2447 (M+H).
Example 112
[0221] Synthesis of
4-[[(2,4,6-trimethylphenyl)sulfonyl]amino]-L-phenylala- nine on
Wang Resin 280
[0222] 4-Amino-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-phenylalanine
on Wang resin (3.0 g, 2.28 mmol) obtained from Example 62 was
suspended in pyridine (15 mL), the slurry was cooled to 0.degree.
C. and 2,4,6-trimethylphenylsulfonyl chloride (2.49 g, 11.4 mmol)
was added. The resulting mixture was agitated for 2 hr. The mixture
was filtered and washed with dichloromethane and methanol. The
coupling procedure was repeated. The resulting resin was treated
with 25% piperidine in N-methylpyrrolidinone (2.times.15 min) and
was washed with dichloromethane and methanol to give
4-[[(2,4,6-trimethylphenyl)sulfonyl]- amino]-L-phenylalanine on
Wang resin.
Example 113
[0223] Synthesis of
N-[(2,2-dichloro-1-methylcyclopropyl)carbonyl]-4-[[(2,-
4,6-trimethylphenyl)carbonyl]amino]-L-phenylalanine. 281
[0224] 4-[[(2,4,6-Trimethylphenyl)sulfonyl]amino]-L-phenylalanine
on Wang resin (0.30 g, 0.23 mmol),
2,2-dichloro-1-methylcyclopropane carboxylic acid (0.19 g, 1.14
mmol), BOP ((0.50 g, 1.14 mmol) and DIPEA (0.26 mL, 1.5 mmol) in
N-methylpyrrolidinone (3 mL) was agitated for 3 hr. The mixture was
filtered and the resin was washed with dichloromethane and methanol
and was air dried. Treatment with 90% TFA in dichloromethane for 3
min effected cleavage from the resin. The mixture was filtered, the
filtrate was concentrated and lyophilized from water to give
N-[(2,2-dichloro-1-methylcyclopropyl)carbonyl]-4-[[(2,4,6-trimethylphenyl-
)carbonyl]amino]-L-phenylalanine as a white solid.
Example 114
[0225] Synthesis of
N-[[1-(4-methoxyphenyl)cyclohexyl]carbonyl]-4-[[(2,4,6-
-trimethylphenyl)sulfonyl]amino]-L-phenylalanine 282
[0226]
N-[[1-(4-methoxyphenyl)cyclohexyl]carbonyl]-4-[[(2,4,6-trimethylphe-
nyl)sulfonyl]amino]-L-phenylalanine was prepared using the
procedure described in Example 113, starting with
1-(4-methoxyphenyl)cyclohexane carboxylic acid.
Example 115
[0227] Synthesis of
N-[(1-adamantyl)carbonyl]-4-[[(2,4,6-trimethylphenyl)s-
ulfonyl]amino]-L-phenylalanine.
[0228]
N-[(1-Adamantyl)carbonyl]-4-[[(2,4,6-trimethylphenyl)sulfonyl]amino-
]-L-phenylalanine was prepared using the procedure described in
Example 113, starting with 1-adamantane carboxylic acid.
Example 116
[0229] Synthesis of
4-[[(4-cyano-4-phenyl-1-piperidinyl)carbonyl]amino]-L--
phenylalanine on Wang Resin 283
[0230] 4-Amino-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-phenylalanine
on Wang resin (300 mg, 0.228 mmol) obtained from Example 62 was
placed in a vessel fitted with a glass frit and was washed with
dichloromethane (2.times.10 mL), methanol (2.times.10 mL) and
dichloromethane (2.times.10 mL). To the resin was added carbonyl
diimidazole (0.22 g, 1.4 mmol) and triethylamine (0.38 mL, 2.7
mmol) in 3 mL of dichloromethane. The mixture was agitated over
night. The mixture was then filtered and washed with
dichloromethane (3.times.5 mL). To the resin was added
4-cyano-4-phenylpiperidine hydrochloride (0.25 g, 1.14 mmol) and
DMAP (0.14 g, 1.14 mmol) in 3 mL of N-methylpyrrolidinone. The
resulting mixture was agitated for 3 hours. The reaction mixture
was then filtered and washed with dichloromethane (2.times.10 mL),
methanol (2.times.10 mL), dimethylformamide (2.times.10 mL) and
methanol (2.times.10 mL). Cleavage of the Fmoc group was effected
with 25% piperidine in N-methylpyrrolidinone (2.times.15 min to
give 4-[[(4-cyano-4-phenyl-1-pip-
eridinyl)carbonyl]amino]-L-phenylalanine on Wang resin.
Example 117
[0231] Synthesis of
4-[[(4-cyano-4-phenyl-1-piperidinyl)carbonyl]amino]-N--
[(2,2-dichloro-1-methylcyclopropyl)carbonyl]-L-phenylalanine
284
[0232] A suspension of
4-[[(4-cyano-4-phenyl-1-piperidinyl)carbonyl]amino]-
-L-phenylalanine on Wang resin (0.30 g, 0.228 mmol),
2,2-dichloro-1-methylcyclopropane carboxylic acid (0.19 g, 1.14
mmol), BOP (0.50 g, 1.14 mmol) and DIPEA (0.26 mL, 1.5 mmol) in
N-methylpyrrolidinone (3 mL) was agitated for 3 hr. The mixture was
filtered and washed with dichloromethane and methanol. Cleavage
from the resin was effected by treatment with 90% TFA in
dichloromethane for 3 min. The mixture was filtered and the
filtrate evaporated to give
4-[[(4-cyano-4-phenyl-1-piperidinyl)carbonyl]amino]-N-[(2,2-dichloro-1-me-
thylcyclopropyl)carbonyl]-L-phenylalanine. FAB MS m/z 543
(M+H).
Examples 118-122
[0233] Using the procedures described in Examples 116 and 117, the
following compounds were prepared:
9 285 Example Y X 118 286 287 119 288 289 120 290 291 121 292 293
122 294 295
Example 123
[0234] General Procedure for the preparation of ethyl esters from
4-substituted-N-acyl-L-Phenylalanine Derivatives.
[0235] To a suspension of N-(acyl)-4-[(aroyl)amino]-L-phenylalanine
(10 mmol) and powdered sodium bicarbonate (4.2 g, 50 mmol) in DMF
(75 mL) was added excess iodoethane (7.8 g, 50 mmol) at room
temperature. The resulting suspension was stirred until TLC
analysis of the mixture indicated the absence of staring material,
typically 20 h. The excess iodoethane and some DMF was removed on a
rotary evaporator under vaccum. The residue was diluted with 100 mL
of ethyl acetate and washed successively with water (2.times.70
mL), brine solution (70 mL) and dried over MgSO4. Filtration of the
drying agent and removal of the solvent afforded a residue which
was purified by crystallization or silica gel chromatography.
Example 124
[0236] Wang Resin Linked
N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-4-nit-
ro-L-phenylalanine 296
[0237] A mixture of Wang resin linked
N-Fmoc-4-nitro-L-phenylalanine (2.30 g, 1.35 mmol) in 27 mL of 25%
piperidine in NMP (N-methylpyrrolidinone) was shaken for 30 minutes
at room temperature. Solvent was filtered through sintered funnel.
With resin still on the funnel, another 27 mL of 25% piperidine in
NMP was added and the suspension was allowed to stand at room
temperature for 30 minutes. After removal of the solvent by
filtration, the resin was then washed with dichloromethane, DMF,
isopropyl alcohol, dichloromethane sequentially and was dried under
vaccum.
[0238] The above resin was placed into a 50 mL round bottom flask
containing 13 mL of NMP. To it was added,
1-(2-methoxyethyl)cyclopentane carboxylic acid (930 mg, 5.4 mmol),
diisopropylethylamine (DIEA, 1.3 mL, 7.4 mmol), and BOP reagent
(2.4 g, 5.4 mmol). Reaction was shaken overnight. A small aliquot
was removed and analyzed by the Kaiser test which showed negative
for amine. Resin was collected by filtration and was washed with
dichloromethane, DMF, isopropyl alcohol, dichloromethane and dried
under reduced pressure to give 1.97 g of Wang resin linked
N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-4-nitro-L-phenylalanine.
Example 125
[0239] Synthesis of Wang Resin Linked
4-amino-N-[[1-(2-methoxyethyl)cyclop-
entyl]-carbonyl]-L-phenylalanine. 297
[0240] In a 20 mL scintillation vial was placed Wang resin linked
N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-4-nitro-L-phenylalanine
(1.91 g, 1.12 mmol) and a 2 M solution of SnCl2.2H2O in DMF (8 mL).
The reaction mixture was shaken overnight at room temperature. The
resin was collected by filtration and was washed with DMF,
isopropyl alcohol, dichloromethane, and Et2O to give Wang resin
linked
4-amino-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalanine
(2.21 g). A small sample was cleaved from the resin with 50%
TFA/dichloromethane and was analyzed by ESPMS which showed presence
of product but no starting material, m/z 335 (M+H).
Example 126
[0241] Synthesis of Wang Resin Liked
4-[((2R)-2-amino-4-methyl-1-oxopentyl-
)amino]-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalanine.
298
[0242] In a 20 mL scintillation vial was placed Wang resin linked
4-amino-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalanine
(200 mg, 0.118 mmol), Fmoc-D-Leu-OH (201 mg, 0.571 mmol), DIEA (164
uL, 0.95 mmol), HBTU (360 mg, 0.95 mmol) in DMF (5 mL). The
reaction mixture was shaken overnight at room temperature. The
resin was filtered and washed with DMF, isopropyl alcohol,
dichloromethane. A Kaiser test was negative for amine.
[0243] The resin obtained above was treated with 5 mL of 25%
piperidine in NMP for 45 minutes at room temperature. After it was
filtered through sintered funnel and washed with DMF, the resin was
re-suspended in 5 mL of 25% piperidine/NMP in the funnel and was
allowed to stand for 15 minutes at room temperature. This process
was repeated once more and the resin was then washed with DMF,
isopropyl alcohol, dichloromethane. Wang resin linked
4-[((2R)-2-amino-4-methyl-1-oxopentyl)amino]-N-[[1-(2-methox-
yethyl)cyclopentyl]carbonyl]-L-phenylalanine (215 mg) was
obtained.
Example 127
[0244] Synthesis of Wang Resin Linked
4-[(2S,4R)-3-acetyl-4-(2-methylpropy-
l)-5-oxo-2-phenyl-1-imidazolidinyl]-N-[[1-(2-methoxyethyl)cyclopentyl]carb-
onyl]-L-phenylalanine and Wang Resin Linked
4-[(2R,4R)-3-acetyl-4-(2-methy-
lpropyl)-5-oxo-2-phenyl-1-imidazolidinyl]-N-[[1-(2-methoxyethyl)cyclopenty-
l]carbonyl]-L-phenylalanine 299
[0245] The resin obtained in Example 126 (0.59 mmol/g, 0.202 g,
0.11 mmol) was reacted with benzaldehyde (1.78 mmol, 182 .mu.L) in
4 mL of a mixed solvent THF/trimethylorthoformate (1/1) at room
temperature and was shaken for 4 days. To the above suspension was
then added 3 mL of acetic anhydride and the mixture was stirred at
95.degree. C. overnight. After the suspension was cooled to room
temperature, the solvent was removed by filtration and the resin
was washed with dichloromethane, THF and then dichloromethane.
[0246] Treatment of resulting resin with 6 mL of
TFA/dichloromethane (1/1) at room temperature for 3.5 hr resulted
in a crude mixture containing products. The crude products gave the
correct mass by ESPMS (M+H)=578. RP-HPLC (41.4mm.times.30 mm
Dynamax C18 column, 5:95 to 95:5 acetonitrile:water gradient over
30 min monitoring the effluent at 214 .ANG.) separation gave two
diastereomeric products tentatively assigned as:
4-[(2S,4R)-3-acetyl-4-(2-methylpropyl)-5-oxo-2-phenyl-1-imidazolidiny-
l]-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenylalanine
mention time 25.06 min, 25% yield and
4-[(2R,4R)-3-acetyl-4-(2-methylpropyl)-5-ox-
o-2-phenyl-1-imidazolidinyl]-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-
-phenylalanine, retention time 26.98 min, 33% yield.
Example 128-139
[0247] Using procedure described in Example 126-127, the compounds
shown below were prepared
10 300 HRMS HRMS Example R (calcd) (obs) 128 301 578.3230 578.3222
129 302 578.3230 578.3233 130 303 608.3336 608.3325 131 304
608.3336 608.3317 132 305 612.2840 612.2826 133 306 612.2840
612.2838 134 307 578.3230 578.3210 135 308 578.3230 578.3225 136
309 608.3336 608.3320 137 310 608.3336 608.3331 138 311 612.2840
612.2835 139 312 612.2840 612.2864
Example 140
[0248] Synthesis of Wang Resin Linked
N-[[1-(4-methoxyphenyl)cyclohexyl]ca-
rbonyl]-4-nitro-L-phenylalanine 313
[0249] A mixture of Fmoc-4-nitro-L-phenylalanine on Wang resin (581
mg, 0.36 mmol) in 10 mL of 25% piperidine in NMP
(N-methylpyrrolidinone) was shaken for 30 minutes at room
temperature. Solvent was filtered through a sintered funnel. With
the resin still on the funnel, another 27 mL of 25% piperidine in
NMP was added and the suspension was allowed to stand at room
temperature for 30 minutes. After removal of the solvent by
filtration, the resin was then washed with dichloromethane, DMF,
isopropyl alcohol, dichloromethane sequentially and was dried under
vacuum.
[0250] The above resin was placed into a 25 mL round bottom flask
containing 4 mL of NMP. To it was added,
1-(4-methoxyphenyl)cyclohexaneca- rboxylic acid (337 mg, 1.44
mmol), diisopropylethylamine (DIEA, 343 .mu.L, 1.98 mmol), and BOP
reagent (637 mg, 1.44 mmol). The reaction mixture was shaken
overnight. A small aliquot was removed and analyzed by the Kaiser
test which was negative for amine. The resin was collected by
filtration and was washed with dichloromethane, DMF, isopropyl
alcohol, dichloromethane and was dried under reduced pressure to
give 580 mg (0.59 mmol/g) of Wang resin linked
N-[[1-(4-methoxyphenyl)cyclohexyl]carbonyl]--
4-nitro-L-phenylalanine.
Example 141
[0251] Synthesis of
4-[2-(4-hydroxyphenyl)-4-oxo-3-thiazolidinyl]-N-[[1-(4-
-methoxyphenyl)cyclohexyl]carbonyl]-L-phenylalanine. 314
[0252] In a 20 mL scintillation vial was added Wang resin linked
N-[[1-(4-methoxyphenyl)cyclohexyl]carbonyl]-4-nitro-L-phenylalanine
(241 mg, 0.14 mmol) prepared in Example 140 and 2 mL of a solution
of 2M SnCl2.2H2O in DMF. The reaction mixture was shaken overnight
at room temperature. The resulting resin was collected by
filtration and was washed with DMF, isopropyl alcohol,
dichloromethane, and ether to yield 243 mg (0.14 mmol) of
4-amino-N-[[(1-(4-methoxyphenyl)cyclohexyl]carbonyl-
]-L-phenylalanine on Wang resin.
[0253] The
4-amino-N-[[(1-(4-methoxyphenyl)cyclohexyl]carbonyl]-L-phenylal-
anine on Wang resin (243 mg, 0.149 mmol) prepared above was reacted
with 4-hydroxybenzaldehyde (153 mg, 1.25 mmol) and mercapto acetic
acid (174 .mu.L, 2.5 mmol) in the presence of 3 .ANG. molecular
sieves (100 mg) in THF (5 mL) at 90.degree. C. for 4 hr. After it
was cooled to room temperature, the reaction mixture was filtered
and the resin was washed with THF, dichloromethane, DMF, MeOH and
ether (3.times.30 mL for each solvent). The resin was then treated
with 50% of TFA in dichloromethane at room temperature for 1 hr.
The suspension was then filtered and the resin was washed with
acetonitrile (2.times.10 mL). The combined filtrates were
concentrated to dryness and purified by RP-HPLC to give
4-[2-(4-hydroxyphenyl)-4-oxo-3-thiazolidinyl]-N-[[1-(4-methoxyphenyl)cycl-
ohexyl]carbonyl]-L-phenylalanine (40 mg, 50% yield), HRMS
(C32H34N2O6S) obs mass, 575.2199. Calcd mass, 575.2216 (M+H).
Example 142
[0254] Using procedure described in Example 141,
N-[[1-(4-methoxyphenyl)cy-
clohexyl]carbonyl]-4-[2-(2-naphthyl)-4-oxo-3-thiazolidinyl]-L-phenylalanin-
e HR MS (C36H36N2O5S) Obs mass, 608.1910. Calcd mass, 608.1904 (M+)
was prepared. 315
Example 143
[0255] Synthesis of
(S)-4-(3,4-dimethyl-2,5-dioxo-1-imidazolidinyl)-N-[[1--
(4-methoxyphenyl)cyclohexyl]carbonyl]-L-phenylalanine 316
[0256] In a 20 mL scintillation vial was added
4-amino-N-[[(1-(4-methoxyph-
enyl)cyclohexyl]carbonyl]-L-phenylalanine on Wang resin (188 mg,
0.11 mmol, 0.59 mmol/g loading) as prepared in Example 141,
Fmoc-N-methyl-L-alanine-OH (114 mg, 0.341 mmol), DIEA (100 uL, 0.58
mmol), HBTU (220 mg, 0.58 mmol) in DMF (2 mL). The reaction mixture
was shaken overnight at room temperature. The resin was filtered
and rinsed with DMF, isopropyl alcohol, dichloromethane and dried
under reduced pressure. A Kaiser test was negative for amine.
[0257] The resin obtained above (200 mg) was treated with 5 mL of
25% piperidine in NMP for 45 minutes at room temperature. After it
was filtered through a sintered glass funnel and rinsed with DMF,
the resin was re-suspended in 5 mL of 25% piperidine/NMP in the
funnel and was allowed to stand for 15 minutes at room temperature
and filtered. This process was repeated and the resin was then
rinsed with DMF, isopropyl alcohol, dichloromethane.
[0258] The above resin was placed in a 20 mL scintillation vial
with carbonyl diimidazole (CDI, 201 mg, 1.24 mmol) and DIEA (64
.mu.L, 0.374 mmol) in dichloromethane (5 mL). The reaction mixture
was shaken overnight at room temperature. Resin was then filtered
and washed with dichloromethane, DMF, isopropyl alcohol,
dichloromethane.
[0259] The resulting resin was transferred to a 50 mL round bottom
flask and stirred in 50% TFA/dichloromethane (10 mL) for 3 hours at
room temperature. The resin was filtered and rinsed with MeCN. The
mother liquor was concentrated. The residue was purified by
reversed phase HPLC using a linear gradient of 10:95 to 95:5
(MeCN:H2O) over 40 minutes.
(S)-4-(3,4-dimethyl-2,5-dioxo-1-imidazolidinyl)-N-[[1-(4-methoxyphenyl)cy-
clohexyl]carbonyl]-L-phenylalanine (22 mg, 0.043 mmol) was obtained
in 39% overall yield calculated based on the loading of
4-nitro-N-Fmoc-phenyalan- ine on Wang resin as 0.059 mmol/g. HRMS
(C28H33N3O6) Obs mass, 508.2456. Calcd mass, 508.2448 (M+H).
Example 144
[0260] Using procedure described in Example 143.
N-[[1-(4-methoxyphenyl)cy-
clohexyl]carbonyl]-4-[(4S)-3-methyl-4-(2-methylpropyl)-2,5-dioxo-1-imidazo-
lidinyl]-L-phenylalanine (46 mg, 0.082 mmol) was synthesized in 52%
yield. HRMS (C31H39N3O6): Obs mass, 550.2904. Calcd mass, 550.2917
(M+H). 317
Example 145
[0261] 2,6-Dimethyl-4-trifluoromethyl-3-pyridinecarboxylic Acid.
318
[0262] A solution of
2,6-dimethyl-4-trifluoromethyl-3-pyridinecarboxylic acid ethyl
ester in 40 mL of THF and 10 mL of 1 N sodium hydroxide solution
was heated to reflux for 48 h. TLC of the mixture (3:7
methanol:dichloromethane) indicated that starting material was
consumed. The mixture was acidified with acetic acid (5 mL) and
evaporated to dryness. The residue was triturated with THF and the
solution was concentrated to give 0.7 g of material containing some
THF and acetic acid as indicated by NMR. This material was combined
with the product of a similar experiment and was chromatographed on
90 g of silica gel, eluting with (3:7) methanol:dichloromethane to
give 1.05 g of a solid. This material was diluted with toluene (6
mL) and evaporated several times to remove most of the acetic acid
to afford after drying under high vacuum, 0.9 g of a white foam.
LR-ES-MS (C9H6F3NO2): 218 (M-H).
Example 146
[0263] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-azi-
doethyl)cyclopentyl]carbonyl]-L-phenylalanine. 319
[0264] Using the general procedure described in example 46,
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-azidoethyl)cyclopentyl]c-
arbonyl]-L-phenylalanine methyl ester was prepared in 78% overall
yield. HR MS: (C25H27C12N5O4): Obs mass, 532.1519. Calcd mass,
532.1518, (M+H).
Example 147
[0265] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-ami-
noethyl)cyclopentyl]carbonyl]-L-phenylalanine Methyl. 320
[0266] To a suspension of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[l-(2-
-azidoethyl)cyclopentyl]carbonyl]-L-phenylalanine methyl ester
(15.47 mmol, 8.24 g) in THF (70 mL) was added a solution of 1.0 M
trimethylphosphine in toluene (25 mmol, 25 mL) at 0.degree. C. The
mixture was stirred 7 h at room termperature, at which time TLC
analysis indicated the absence of starting material. Then, 3 equiv.
of water (45 mmol, 0.82 mL) were added and the mixture was stirred
for 15 h at room temperature. The solvent was removed under vacuum
and the residue was azeotrophed two times with toluene to give a
pasty material which was dissolved in THF:dichloromethane (250 mL)
and dried over anhydrous sodium sulfate. The solution was filtered
through a plug of celite and the celite was washed with THF (100
mL). The combined filtrates were evaporated under vacuum to obtain
5 g (64%) of methyl
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-aminoethyl)cyclopentyl]c-
arbonyl]-L-phenylalanine ester as a pink solid. HR MS:
(C25H29C12N3O4) Obs mass, 506.1625. Calcd mass, 506.1613,
(M+H).
Example 148
[0267] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[(1-
-oxoethyl)amino]ethyl]cyclopentyl]carbonyl]-L-phenylalanine Methyl
Ester. 321
[0268] To a solution of methyl
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[-
[1-(2-aminoethyl)cyclopentyl]carbonyl]-L-phenylalanine ester (9.6
mmol, 5 g) in pyridine (70 mL) was added 2 equiv. of acetic
anhydride (20 mmol, 2.04 g) at room temperature. The colored
solution was stirred for 15 h at room temperature and then diluted
with 200 mL of ethyl acetate. The ethyl acetate layer was washed
successively with 1N hydrochloric acid (2.times.100 mL), brine
solution (100 mL) and dried over anhydrous magnesium sulfate.
Filtration of the drying agent and concentration of the solvent
afforded a fluffy solid which was only 85% pure by HPLC. This solid
was triturated with ethyl acetate (40 mL) and then hexane (20 mL)
was added. The solid was collected by filtration and dried at air
to afford 3.38 g (63%) of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[-
(1-oxoethyl)amino]ethyl]cyclopentyl]carbonyl]-L-phenylalanine
methyl ester as a yellow solid, mp 190-194.degree. C. HR MS
(C27H31C12N3O5): Obs mass, 548.1696. Calcd mass, 548.1719
(M+H).
Example 149
[0269] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalan- ine
Hydrochloride Salt. 322
[0270] To a solution of methyl
4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-p- henylalanine
hydrochloride salt (30 mmol, 12.1 g) in ethanol (300 mL) was added
1 N sodium hydroxide (90 mL). The mixture was heated to
40-45.degree. C. for 1 h at which point TLC analysis of the
reaction mixture indicated the absence of starting material and it
was cooled to room temperature. The solvent was removed under
vacuum and the residue was extracted with ether (2.times.100 mL) to
remove neutral impurities. Then, the basic aqueous layer was
acidified with 1 N hydrochloric acid to pH 2 to give a clear
solution. The solution was lyopholized under high vacuum to afford
11.7 g (100%) of the title compound as a white amorphous solid. HR
MS (C16H15C13N2O3): Obs. mass, 354.2014. Calcd. mass, 354.2053
(M+H).
Example 150
[0271] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(9H-fluor-
en-9-ylmethoxy)carbonyl]-L-phenylalanine. 323
[0272] To a mixture of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylala- nine
hydrochloride salt (30 mmol, 10.59 g),
N-(9-fluorenylmethoxycarbonylo- xy)succinimide (30 mmol, 10.12 g)
and sodium carbonate (300 mmol, 31.8 g) were added dioxane (75 mL)
and water (25 mL) at room temperature. The suspension was stirred
for 15 h at room temperature at which time TLC analysis of the
mixture indicated the absence of starting material. The inorganic
solids were filtered through celite and washed with ethyl acetate.
While washing with ethyl acetate some organic compound was
precipitated out from filtrate which was collected and air dried.
The filtrate was concentrated under vacuum and the residue was
combined with the above organic solid compound and the product was
taken up in hot THF and precipatated with ether to obtain 14.1 g
(81%), as a white solid, mp 230-234.degree. C. LR MS
(C31H24C12N2O5): Obs mass, 597. Calcd mass, 597, (M+Na).
Example 151
[0273]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(9H-fluoren-9-ylmethoxy)-
carbonyl]-L-phenylalanine on Wang resin was prepared from the
product of example 150 with a loading of 0.765 mmol/g of resin
using the general procedure described in example 61. 324
Example 152
[0274] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalan- ine on Wang
Resin. 325
[0275] To the
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(9H-fluoren-9-ylm-
ethoxy)carbonyl]-L-phenylalanine on Wang resin prepared above (12.1
mmol, 17.4 g) was added 20% of piperidine in NMP at room
temperature. The mixture was shaken for 1.5 h at room temperature
and the resin was filtered and washed with DMF (2.times.20 mL).
Then, the resin was suspended in 20% piperidine in NMP (100 mL) and
the solvent was decanted. The resin was washed with dichloromethane
(2.times.20 mL) and dried under high vacuum to obtain 13.5 g of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-L- -phenylalanine on Wang
resin.
Example 153
[0276]
N-[[1-(2-Azidoethyl)cyclopentyl]carbonyl]-4-[[(2,6-dichlorophenyl)c-
arbonyl]-amino]-L-phenylalanine on Wang resin was prepared from the
product of example 152 using the general method described in
example 64. The loading was 0.695 mmol/g of resin. 326
Example 154
[0277] Preparation of
N-[[1-(2-aminoethyl)cyclopentyl]carbonyl]-4-[[(2,6-d-
ichlorophenyl)carbonyl]amino]-L-phenylalanine on Wang Resin.
327
[0278] To a suspension of
N-[[1-(2-azidoethyl)cyclopentyl]carbonyl]-4-[[(2-
,6-dichlorophenyl)carbonyl]amino]-L-phenylalanine on Wang resin
(9.33 mmol, 13.43 g, 0.695 mmol per g) in THF (60 mL) was added a
solution of 1.0 M trimethylphosphine in THF (37.3 mmol, 37.3 mL) at
0.degree. C. The mixture was allowed to warm to room temperature
and stir for 4 h at which time a Kaiser test was possitve for
amine. The resin was filtered and was washed with DMF (4.times.20
mL), dichloromethane (4.times.10 mL), isopropanol (4.times.10 mL),
DMF (2.times.20 mL) and dichloromethane (2.times.20 mL),
respectively. After drying under high vacuum, 13.43 g of
N-[[1-(2-aminoethyl)cyclopentyl]carbonyl]-4-[[(2,6-dichlorophenyl)carbony-
l]amino]-L-phenylalanine on Wang resin was obtained.
Example 155
[0279] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[[(-
1,1-dimethyl-ethyl)carbonyl]amino]ethyl]cyclopentyl]carbonyl]-L-phenylalan-
ine on Wang Resin. 328
[0280] To a mixture of
N-[[1-(2-aminoethyl)cyclopentyl]carbonyl]-4-[[(2,6--
dichlorophenyl)carbonyl]-amino]-L-phenylalanine on Wang resin
(0.069 mmol, 100 mg, 0.695 mmol per gram loading) in pyridine (2
mL) was added excess of 2,2,2-trimethylacetyl chloride (0.28 mmol,
33 mg) at 0.degree. C. The mixture was allowed to warm to room
temperature and shaken for 3 h at which time a Kaiser test was
negative for amine. The resin was filtered and washed with
dichloromethane (2.times.10 mL) and was dried under high vacuum to
afford 4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[[N-(1,-
1-dimethylethyl)carbonyl]amino]-ethyl]cyclopentyl]carbonyl]-L-phenylalanin-
e on Wang resin.
Example 156
[0281]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[[N-(1,1-dimethyle-
thyl)-carbonyl]amino]ethyl]cyclopentyl]carbonyl]-L-phenylalanine
was prepared by cleavage of the product of example 155 from the
resin with TFA using the general procedure described in example 64
to give 25 mg (62%) of a white solid. HR MS (C29H35C12N3O5): Obs
mass, 576.2019. Calcd mass, 576.2032 (M+H). 329
Example 157
[0282] Preparation of
N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-N-methyl-
-4-nitro-L-phenylalanine Methyl Ester. 330
[0283] To a suspension of
N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-4-ni-
tro-L-phenylalanine methyl ester (2.73 mmol, 1.03 g) and silver
oxide (10.9 mmol, 2.53 g) in DMF (25 mL) was added methyl iodide
(160 mmol, 10 mL) at room temperature. The suspesion was stirred
for 2 days at room temperature at which time TLC analysis of the
mixture indicated the presence of starting material. An additional
10 mL (160 mmol) of methyl iodide and 2 g (8.6 mmol) of silver
oxide were added, respectively. The suspension was stirred for 24 h
and the solid was filtered through a pad of celite and was washed
with ethyl acetate (30 mL) and methanol (30 mL). The filtrate was
concentrated and the residue was extracted with ethyl acetate
(3.times.30 mL).The organic layer was washed with water (20 mL) and
brine solution (20 mL) and was dried over anhydrous magnesium
sulfate. Filtration of the drying agent and removal of the solvent
gave a crude compound which was purified by silica gel column
chromatography to obtain 530 mg (50%) as a light brown oil. HR MS
(C20H28N2O6): Obs. mass, 392.1940. Calcd. mass, 392.1947 (M+).
[0284] Example 158 to 167 using the general procedure described in
examples 155 and 156, the analogues shown below were prepared from
N-[[1-(2-aminoethyl)cyclopentyl]carbonyl]-4-[[(2,6-dichlorophenyl)carbony-
l]amino]-L-phenylalanine on Wang resin.
11 331 HRMS.sup.1 Example R Formula Calcd. Obs. 158 332
C28H28Cl2N4O6: 609.1284 609.1275 159 333 C31H33Cl2F3N4O6 685.1807
685.1784 160 334 C30H30Cl2N4O5: 597.1691 597.1661 161 335
C32H41Cl2N3O5 618.2501 618.2499 162 336 C27H31Cl2N3O6 564.1668
564.1641 163 337 C29H33Cl2N3O7 606.1774 606.1752 164 338
C31H37Cl2N3O5 602.2188 602.2165 165 339 C32H31Cl2N3O7 640.1617
640.1613 166 340 C32H33Cl2N3O5 609.2631 609.2558 167 341
C33H39Cl2N5O5 656.2406 656.2420 M + H ion unless otherwise
indicated
Example 168
[0285] Preparation of 1-(4-bromobutyl)cyclopentane Carboxylic Acid
Methyl Ester. 342
[0286] To a solution of diisopropylamine (150 mmol, 21 mL) in THF
(100 mL) was added dropwise a solution of n-butyl lithium (145
mmol, 58 mL, 2.5M) in hexanes at -10.degree. C. while maintaining
the temperature below 0.degree. C. After addition, the solution was
stirred for 30 min at 0.degree. C. To this, a solution of methyl
cyclopentane carboxylate (100 mmol, 13.1 g) in THF (20 mL) was
added dropwise at -70.degree. C. maintaining the internal
temperature between -60 to -70.degree. C. After addition, the
reaction mixture was stirred for 1 h at -50 to -60.degree. C. Then,
a solution of 1,4-dibromobutane (100 mmol, 21.59 g) in THF (20 mL)
was added dropwise and the light brown suspension was stirred for 1
h at -60 to -70.degree. C. Then, it was allowed to warm to room
temperature and stirred overnight. The reaction mixture was poured
into a saturated solution of ammonium chloride (200 mL) and the
organic compound was extracted into ether (2.times.100 mL). The
combined extracts were washed with a saturated brine solution (150
mL) and dried over anhydrous magnesium sulfate. After filtration of
the drying agent, the solution was concentrated under vacuum and
the resulting residue was distilled at 120-133.degree. C./2.5 mm Hg
to obtain 1-(4-bromobutyl)cyclopentane carboxylic acid methyl ester
as a colorless oil (12.8 g, 48%). HR MS (C11H19BrO2): Obs mass,
262.0565. Calcd mass, 262.0568 (M+).
Example 169
[0287] Preparation of 1-(4-cyanobutyl)cyclopentane Carboxylic Acid
Methyl Ester 343
[0288] A solution of 1-(4-bromobutyl)cyclopentane carboxlic acid
methyl ester was treated with a solution of 0.5 molar lithium
cyanide in DMF (5 mmol, 10 mL) and stirred overnight at room
temperature followed by heating at 75.degree. C. for two hrs. The
reaction mixture was evaporated to dryness, the residue was
dissolved in ethyl acetate (30 mL) and washed with sat'd sodium
bicarbonate (2.times.10 mL), and brine (5 mL) and dried over
magnesium sulfate to give a yellow oil (860 mg). Silica gel
chromtography eluting with 1:3 ethyl acetate:hexane yielded a pale
oil (83%, 693 mg). LR MS (C12H19NO2): Obs mass, 227 (M+NH4), 248
(M+K).
Example 170
[0289] Preparation of 1-(3-bromopropyl)cyclopentane Carboxylic Acid
Methyl Ester 344
[0290] To a solution of 1.5 molar LDA in cyclohexane (60 mmol, 40
mL) cooled to -70.degree. C. was added a solution of cyclopentane
carboxylic acid methyl ester (42 mmol, 5.6 g) in THF (15 mL) over
30 min. The mixture was stirred for 1 hr at -70.degree. C. Then a
solution of 1,3-dibromopropane (60 mmol, 11.9 g) in THF (30 mL)
which had been pre-cooled to -70.degree. C. was added all at once
and the reaction mixture was stirred at -70.degree. C. for one hr
then at room temperature overnight. The reaction mixture was poured
into brine (200 mL), the layers were separated, the aqueous layer
was extracted with ether (3.times.30 mL) and the combined organic
layers were washed with brine (20 mL), dried (magnesium sulfate)
and concentrated to give a yellow oil (15 g). Distillation through
a short path apparatus with a 3" vigreux column gave a yellow oil,
7.96 g (76%), bp 76-80.degree. C. at 0.3 mm.
Example 171
[0291] 1-(3-Cyanopropyl)cyclopentane carboxylic acid methyl ester
was prepared from 1-(3-bromopropyl)cyclopentane carboxylic acid
methyl ester using the general method described in example 169 to
give a 79% yield of a pale yellow oil. LR MS (C11H17NO2): Calcd
mass, 196. Obs mass, 196 (M+H). 345
Example 172
[0292] Preparation of 1-[4-(methylthio)butyl]cyclopentane
Carboxylic Acid Methyl Ester. 346
[0293] To a solution of 1-(4-bromobutyl)cyclopentane carboxylic
acid methyl ester (38 mmol, 10 g) in DMF (100 mL) was added sodium
thiomethoxide (72.6 mmol, 5.09 g). After addition of sodium
thiomethoxide, the reaction was exothermic and the mixture became a
light brown cloudy solution. The mixture was stirred for 15 h at
room temperature and was poured into water (200 mL). The organic
compound was extracted with ether (2.times.150 mL). The combined
extracts were washed with brine (150 mL) and dried over anhydrous
magnesium sulfate. After filtration of the drying agent, the
solution was concentrated under vacuum and the resulting residue
was purified by silica gel column chromatography to afford 4.43 g
(51%) of a colorless oil. LR MS (C12H22O2S): 230 (M+).
Example 173
[0294] 1-(3-Methylthiopropyl)cyclopentane carboxylic acid methyl
ester was prepared from 1-(3-bromopropyl)cyclopentane carboxylic
acid methyl ester using the general procedure described in example
172 to give a 54% yield of a pale yellow oil. 347
Example 174
[0295] Preparation of 1-[(4-(methylsulfonyl)butyl]cyclopentane
Carboxylic Acid Methyl Ester 348
[0296] To a solution of 1-[4-(methylthio)butyl]cyclopentane
carboxylic acid methyl ester (19.2 mmol, 4.43 g) in AcOH (20 mL)
was added 30% hydrogen peroxide (10 mL). The reaction mixture was
heated to 70.degree. C. and stirred for 15 h at which time the TLC
of the mixture indicated the absence of starting material. The
reaction mixture was cooled to room temperature and was
concentrated under vacuum. The residue was poured into saturated
sodium bicarbonate solution and was extracted with ether
(3.times.100 mL). The combined extracts were washed with a
saturated solution of sodium chloride (200 mL) and dried over
anhydrous magnesium sulfate. After filtration of the drying agent,
the solvent was removed under vacuum and the resulting residue was
purified by silica gel column chromatography to afford 4.94 g (98%)
as a colorless oil. LR MS (C12H22O4S): 263 (M+H).
Example 175
[0297] Preparation of 1-[4-(methylsulfonyl)butyl]cyclopentane
Carboxylic Acid 349
[0298] To a solution of 1-[4-(methylsulfonyl)butyl]cyclopentane
carboxylic acid methyl ester (18.8 mmol, 4.94 g) in a mixture of
THF (38 mL) and methanol (38 mL) was added 1 N sodium hydroxide (38
mL). The mixture was heated to 50-55.degree. C. for 15 h at which
point TLC analysis of the reaction mixture indicated the absence of
starting material and the mixture was allowed to cool to room
temperature. The solvent was removed under vacuum and the residue
was diluted with water (100 mL) and extracted with ether
(2.times.50 mL) to remove any neutral impurities. Then, the basic
aqueous layer was acidified with 1 N hydrochloric acid and the
product was extracted with ethyl acetate (2.times.75 mL). The
combined extracts were washed with brine solution and dried over
anhydrous sodium sulfate. After filtration of the drying agent, the
solution was concentrated under vacuum and the residue was dried
under high vacuum to afford 4.31 g (92%) of the title compound as a
low melting white solid. LR MS (C11H20O4S): 249 (M+H).
Example 176
[0299] Preparation of 1-(2-bromoethyl)cyclopentane Carboxylic Acid
Methyl Ester. 350
[0300] To a solution of diisopropylamine (150 mmol, 21 mL) in THF
(100 mL) was added dropwise a solution of n-butyl lithium (145
mmol, 58 mL 2.5M) in hexanes at -10.degree. C. while maintaining
the temperature below 0.degree. C. After addition, the solution was
stirred for 30 min at 0.degree. C. To this, a solution of
cyclopentane carboxylic acid methyl ester (100 mmol, 13.1 g) in THF
(20 mL) was added dropwise at -70.degree. C. maintaining the
internal temperature between -60 to -70.degree. C. After addition,
the reaction mixture was stirred for 1 h at -50 to -60.degree. C.
Then, a solution of 1,2-dibromoethane (90 mmol, 16.91 g) in THF (20
mL) was added dropwise and the light brown suspension was stirred
for 1 h at -60 to -70.degree. C. Then, it was allowed to warm to
room temperature and was stirred overnight. The reaction mixture
was poured into a saturated solution of ammonium chloride (200 mL)
and the organic compound was extracted into ether (2.times.100 mL).
The combined extracts were washed with a saturated solution of
sodium chloride (150 mL) and dried over anhydrous magnesium
sulfate. After filtration of the drying agent, the solution was
concentrated under vacuum and the resulting residue was distilled
at 95-105.degree. C./2.5 mm Hg to obtain 11.5 g (49%) of a
colorless oil.
Example 177
[0301] Preparation of 1-[2-(4-morpholino)ethyl]cyclopentane
Carboxylic Acid Methyl Ester. 351
[0302] To a solution of 1-(2-bromoethyl)cyclopentane carboxylic
acid methyl ester (2 mmol, 0.47 g) in DMF (10 mL) was added sodium
iodide (0.3 mmol, 45 mg) and morpholine (10 mmol, 0.87 g). The
reaction mixture was stirred for 3 days at room temperature at
which time the TLC of the mixture indicated the absence of starting
material. The mixture was diluted with ethyl acetate (100 mL) and
washed successively with water (2.times.50 mL) and a saturated
solution of sodium chloride (100 mL) and was dried over anhydrous
magnesium sulfate. After filtration of the drying agent, the
solution was concentrated under vacuum to afford 0.44 g (92%) of a
colorless oil. HR MS (C13H23NO3): Obs mass, 241.1675. Calcd mass,
241.1678 (M+).
Example 178
[0303] Preparation of 1-[2-(4-morpholino)ethyl]cyclopentane
Carboxylic Acid. 352
[0304] To a solution of 1-[2-(4-morpholino)ethyl]cyclopentane
carboxylic acid methyl ester (1.75 mmol, 0.42 g) in a mixture of
THF (5 mL) and methanol (5 mL) was added 1 N sodium hydroxide (3.5
mL). The mixture was heated to 50-55.degree. C. for 40 h at which
point TLC analysis of the reaction mixture indicated the absence of
starting material and the mixture was allowed to cool to room
temperature. The solvent was removed under vacuum and the residue
was diluted with water (100 mL) and extracted with ether
(2.times.50 mL) to remove any neutral impurities. Then, the basic
aqueous layer was neutralized with 1 N hydrochloric acid and
extracted with ethyl acetate (2.times.75 mL). The aqueous layer was
neutralized with saturated sodium carbonate solution and extracted
with ethyl acetate (3.times.50 mL). TLC of the aqueous layer
indicated the presence of some more product. Thus, all ethyl
acetate extracts were combined with aqueous layer and concentrated.
The solid residue was triturated with methanol. The undissolved
solids were filtered and the filtrate was concentrated under
vacuum. The resulting solid was dissolved again in methanol and
concentrated HCl was added to form a salt. Then, the methanol was
removed to obtain the HCl salt of
1-[2-(morpholino)ethyl]cyclopentane carboxylic acid (1.09 g, may
contain some NaCl) as a white solid. LR MS (C12H21NO3): 228
(M+H).
Example 179
[0305] Preparation of Methyl 1-(3,3
difluoro-2-propylene)cyclopentane Carboxylate 353
[0306] A solution of 0.89 M LDA (0.24 mmol, 27 mL) was prepared
from diisipropylamine (75 mmol, 7.58 g) in THF (50 mL) and 2.5 M
n-butyl lithium in hexane (72 mmol, 29 mL).
[0307] The solution was cooled to -70.degree. C. and cycylopentane
carboxylic acid methyl ester (15 mmol, 1.92 g) in THF (10 mL) was
added dropwise over 20 min while maintaining the temperature at
-70.degree. C. The mixture was stirred an additional 1 h at
-70.degree. C. and a solution of trifluoropropyl bromide (15 mmol,
2.65 g) in THF (10 mL) was added over 15 min. The reaction mixture
was stirred 1 h at -70.degree. C. and allowed to warm to room
temperature overnight. The reaction mixture was poured into brine
(150 mL) and the organic layer was separated. The aqueous layer was
extracted with ether (30 mL), the combined organic layers were
washed with brine (10 mL), dried over anhydrous magnesium sulfate
and evaporated to dryness to give a yellow oil ((4.1 g) which gave
after chromotography on silica gel (120 g, 10% ethyl acetate in
hexane) pale yellow oil (56%, 1.72 g).
Example 180
[0308] Preparation of 5-Iodo-2-pentanone Ethylene Ketal. 354
[0309] A solution of 5-chloro-2-pentanone (40 mmol, 6.59 g) in
acetone (40 mL) was treated with NaI (60 mmol, 9 g) and refluxed
overnight. After cooling to room temperature, the solids were
filtered off and the supernatant was concentrated to a dark gummy
solid. A 1:1 mixture of ether and pet. ether (20 mL) was added, the
mixture was stirred for 30 min., filtered and evaporated to dryness
to give 7.2 g of a red oil which upon distillation (34-36.degree.
at 0.3 mm) gave 5 g of the ketone. To a solution of this ketone
(23.6 mmol, 5 g) in toluene (40 mL) in a 100 mL flask fitted with a
Dean-Stark trap was added ethylene glycol (27 mmol, 1.67 g ) and
100 mg of para-toluenesulfonic acid and the reaction mixture was
reluxed 6 h. After cooling, the toluene solution was washed with 1N
NaOH (20 mL), water (5.times.20 mL), and brine (5 mL) was dried
over potassium carbonate, filtered and evaporated to dryness to
yield, upon distillation, 4.8 g (47%) of 5-Iodo-2-pentanone
ethylene ketal as a colorless oil bp 44-48.degree. C. at 0.3
mm.
Example 181
[0310] Preparation of 1-(4-(ethylenedioxy)pentyl)cyclopentane
Carboxylic Acid Methyl Ester 355
[0311] A solution of lithium diisopropylamidetetrahydrofuran 1.5M
solution in cyclohexane (15 mmol, 10 mL) was cooled to -70.degree.
C., and a solution of cyclopentane carboxylic acid methyl ester (10
mmol, 1.28 g) in THF (10 mL) was added dropwise over 15 min,
maintaining the internal temperature at -60 to -70.degree. C. The
yellow solution was stirred 1 h at -70.degree. C. and a solution of
5-iodo-2-pentanone ethylene ketal (10 mmol, 1.28 g) in THF (10 mL)
was added over 15 min, while maintaining temperature of -70.degree.
C. After stirring for 1 h at -70.degree. C., the reaction mixture
was allowed to warm to room temperature overnight. The reaction
mixture was poured into brine (150 mL) and the organic layer was
separated. The aqueous layer was extracted with ether (20 mL), the
combined organic layers were washed with brine (10 mL), dried over
anhydrous magnesium sulfate and evaporated to dryness to give 2.5 g
(97%) of a pale yellow oil.
Example 182
[0312] Preparation of 4-Iodo-2-butanone Ethylene Ketal. 356
[0313] A solution of 4 bromo-2-butanone ethylene ketal (16.4
mmol,3.2 g) in acetone (30 mL) was treated with sodium iodide (24
mmol, 3.7 g) and sodium carbonate (50 mmol, 5.1 g) and was reluxed
overnight. The resulting mixture was filtered, washed with acetone
(10 mL) and evaporated to dryness to a white solid. The residue was
triturated with a mixture of 1:1 ether: pet ether (20 mL), stirred
30 min., filtered and evaporated to give 3.74 g (94%) of a pale
yellow oil. LR-ES (C13H22O4): 243 (M+H).
Example 183
[0314] 1-[3-(Ethylenedioxy)butyl]cyclocarboxylic acid methyl ester
was prepared using the general method described in example 181 to
give a 25% yield of a colorless oil. LR-ES MS (C13H22O4): 243
(M+H). 357
Example 184
[0315] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-(me-
thylsulfonyl)propyl]cyclopentyl]carbonyl]-L-phenylalanine Methyl
Ester 358
[0316] A solution of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-[(me-
thylthio)propyl]cyclo-pentyl]carbonyl]-L-phenylalanine methyl ester
(110 mg, 0.2 mmol) in dichloromethane (10 mL) was cooled in ice
bath and treated with meta-chloroperbenzoic acid (0.7 mmol, 150
mg). After stirring at room temperature for 3 h, the reaction
mixture was diluted with dichloromethane (30 mL) and washed with
sat'd sodium bicarbonate (10 mL), and brine (5 mL) and dried over
magnesium sulfate. Evaporation to a yellow oil and silica gel
chromtography eluting with 5% methanol in dichloromethane yielded a
white pasty solid (96%, 113 mg). LR-ES MS (C27H32N2O6C12S): 583
(M+H).
Example 185
[0317] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-[(m-
ethyl-sulfinyl)propyl]cyclopentyl]carbonyl]-L-phenylalanine Methyl
Ester 359
[0318] A solution of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-(met-
hylthio)propyl]cyclopentyl]carbonyl]-L-phenylalanine methyl ester
(110 mg, 0.2 mmol) in a mixture of ethyl acetate (8 mL) and THF (3
mL) was treated with a solution of oxone (0.05 mmol, 31 mg) in
water (2 mL) and the two phase system was stirred vigorously for 2
h at room temperature followed by further addition of oxone (0.05
mmol, 31 mg) and continued stirring overnight. After separation of
the layers, the aqueous phase was extracted with ethyl acetate (5
mL), and the combined organic layers were washed with brine (3 mL),
dried over magnesium sulfate and evaporated to dryness to give a
white solid. Chromotography on silica gel eluting with methanol
(7.5%) in dichloromethane gave a white solid (68%, 78 mg). LR-ES MS
(C27H32N2O5C12S): 567 (M+H).
Example 187
[0319] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[[(-
methylamino)carbonyl]amino]ethyl]cyclopentyl]carbonyl]-L-phenylalanine.
360
[0320] To a solution of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-a-
minoethyl)cyclopentyl]carbonyl]-L-phenylalanine methyl ester (40
mg, 0.080 mmol) in dichloromethane (1 mL) was added methyl
isocyanate (5 mg, 0.095 mmol). The resultant mixture was stirred
for 18 h. The reaction mixture was concentrated in vacuo and the
crude urea was used in the next step without purification.
[0321] To a solution of the crude methyl ester (50 mg, 0.080 mmol)
in MeOH (1 mL) was added a solution of LiOH (8 mg, 0.19 mmol) in
water (0.5 mL). The mixture was stirred for 2 h and then it was
acidified (pH 1-2) with 0.5M HCl. The reaction mixture was poured
into a round bottom flask and concentrated in vacuo. Purification
by reversed-phase HPLC, using a 15-95% acetonitrile-water gradient
over 25 min., provided 30 mg (68%). HR MS (C26H30C12N4O5): Calcd
mass, 549.1671. Obs mass, 549.1677 (M+H).
Examples 188 to 191
[0322] Using the general procedure described in example 187, and
starting with
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-aminoethyl)cyclopen-
tyl]carbonyl]-L-phenylalanine methyl ester, the analogues listed
below were prepared:
12 361 HRMS1 Example R Yield % Formula calcd. found 188 362
33.sup.1 C25H28Cl2N4O5 535.1515 535.1510 189 363 60.sup.2
C27H30Cl2N4O7 615.1390 (M + Na) 613.1372 (M + Na) 190 364 65
C31H31Cl2N5O7 678.1499 (M + Na) 678.1496 (M + Na) 191 365 62
C32H34Cl2N4O5 647.1804 (M + Na) 647.1818 (M + Na) 192 366 50.sup.3
C26H30N4O4Cl2S 587.1263 (M + Na) 587.1251 (M + Na)
[0323] Starting material=trimethylsilylisocyanate
[0324] Starting material=methoxycarbonylisocyanate
[0325] Starting material=methyl isothiocyanate
Example 193
[0326]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[2-[[(methoxy)carbonyl]a-
mino]ethyl]cyclopentyl]carbonyl]-L-phenylalanine. 367
[0327] To a solution of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-a-
minoethyl)cyclopentyl]-carbonyl]-L-phenylalanine methyl ester (40
mg, 0.080 mmol) in dichloromethane (1 mL) was added
diisopropylethylamine (20 mg, 0.16 mmol) and methyl chloroformate
(7 mg, 0.080 mmol). The resultant mixture was stirred for 18 h. The
reaction mixture was concentrated in vacuo and the crude carbamate
was used in the next step without purification.
[0328] To a solution of the crude methyl ester (54 mg, 0.080 mmol)
in MeOH (1 mL) was added a solution of LiOH (8 mg, 0.19 mmol) in
water (0.5 mL). The mixture was stirred for 2 h and then it was
acidified (pH 1-2) with 0.5M HCl. The reaction mixture was poured
into a round bottom flask and concentrated in vacuo. Purification
by reversed-phase HPLC, using a 15-95% acetonitrile-water gradient
over 25 min., provided 25 mg (57%). HR MS (C26H29N3O6C12): Calcd
mass, 550.1511. Obs mass, 550.1524 (M+H).
Example 194
[0329] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[(m-
ethylsulfonyl)amino]ethyl]cyclopentyl]carbonyl]-L-phenylalanine.
368
[0330] To a solution of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-a-
minoethyl)cyclopentyl]-carbonyl]-L-phenylalanine methyl ester (40
mg, 0.080 mmol) in dichloromethane (1 mL) was added
diisopropylethylamine (12 mg, 0.095 mmol) and methanesulfonyl
chloride (9 mg, 0.080 mmol). The resultant mixture was stirred for
18 h. The reaction mixture was concentrated in vacuo and the crude
sulfonamide was used in the next step without purification.
[0331] To a solution of the crude methyl ester (55 mg, 0.080 mmol)
in MeOH (1 mL) was added a solution of LiOH (8 mg, 0.19 mmol) in
water (0.5 mL). The mixture was stirred for 2 h and then it was
acidified (pH 1-2) with 0.5M HCl. The reaction mixture was poured
into a round bottom flask and concentrated in vacuo. Purification
by reversed-phase HPLC, using a 15-95% acetonitrile-water gradient
over 25 min., provided 28 mg (61%). HR MS (C25H29N3O6C12S): Calcd
mass, 592.1052. Obs mass, 592.1068 (M+Na).
Example 195
[0332] Preparation of
1-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl]cycl- opentyl
Carboxylic Acid Methyl Ester. 369
[0333] To a solution of 1-(2-azidoethyl)cyclopentane carboxylic
acid methyl ester (5.00 g, 25.4 mmol) in ethyl acetate (100 mL) was
added di-tert-butyl-dicarbonate (55.4 g, 254 mmol) and 10% Pd on
carbon (1.5 g). The reaction mixture was shaken for 3 h under
hydrogen gas (50 psi) on a Parr shaker apparatus. The reaction
mixture was filtered through a pad of celite and the pad was washed
with ethyl acetate (2.times.150 mL). The combined organic phase was
transferred to a round bottom flask and concentrated in vacuo.
Purification by flash column chromatography, using hexane-ethyl
acetate (9:1), afforded 5.30 g (76%) as a light yellow oil. HR MS
(C14H25NO4): Calcd: 272.1862. Obs mass, 272.1856 (M+H).
Example 196
[0334] Preparation of
1-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl]cycl- opentane
Carboxylic Acid. 370
[0335] To a solution of the
1-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethy- l]cyclopentane
carboxylic acid methyl ester (10.6 g, 39.0 mmol) in THF/MeOH (3:1,
40 mL) was added a solution of LiOH (4.20 g, 98.0 mmol) in water
(10 mL). The mixture was stirred for 2.5 h at 50.degree. C. and
then cooled to room temperature. The reaction mixture was poured
into a round bottom flask and concentrated in vacuo. The mixture
was diluted with H.sub.2O (60 mL) and acidified with 1M HCl. The
aqueous phase was extracted with ethyl acetate (3.times.200 mL) and
the combined organic layer was dried over MgSO.sub.4, filtered and
concentrated in vacuo. Purification by flash column chromatography,
using hexane-ethyl acetate (3:1), afforded 5.80 g (58%) as a white
powder. HR MS (C13H23NO4): Calcd mass, 258.1705. Obs mass, 258.1700
[(M+H).
Example 197
[0336] Preparation of
1-[2-[[(1,1-dimethylethoxy)carbonyl](methyl)amino]et-
hyl]-cyclopentane Carboxylic Acid Methyl Ester. 371
[0337] To a slurry of NaH (1.20 g, 47.5 mmol) in DMF (20 Obs mass,
mL) at 0.degree. C. was added dropwise a solution of
1-[2-[[(1,1-dimethylethoxy)- carbonyl]amino]ethyl]cyclopentane
carboxylic acid (5.8 g, 22.6 mmol) in THF (25 mL). The resultant
mixture was stirred for 1 h at 0.degree. C. and methyl iodide (3.3
Obs mass, mL, 52.3 mmol) was added dropwise. The reaction mixture
was stirred for 1 h at 0.degree. C. and 5 h at room temperature.
The reaction was quenched with sat. ammonium chloride (20 Obs mass,
mL) solution and transferred to a separatory funnel. The aqueous
phase was extracted with ethyl acetate (3.times.100 mL) and the
combined organic layer was dried over MgSO.sub.4, filtered and
concentrated in vacuo. Purification of the residue by flash column
chromatography, using hexane-ethyl acetate (9:1), afforded 5.40 g
(84%). HR MS (C15H27NO4): Calcd: 286.2018. Obs mass, 286.2021
(M+H).
Example 198
[0338] Preparation of
1-[2-[[(1,1-dimethylethoxy)carbonyl](methyl)amino]-e-
thyl]cyclopentane Carboxylic Acid. 372
[0339] To a solution of
1-[2-[[(1,1-dimethylethoxy)carbonyl](methyl)amino]-
ethyl]cyclopentane carboxylic acid methyl ester (4.00 g, 14.0 mmol)
in THF/MeOH (2:1, 24 mL) was added a solution of LiOH (1.50 g, 35.0
mmol) in water (8 mL). The mixture was stirred overnight at
50.degree. C. and then cooled to room temperature. The reaction
mixture was poured into a round bottom flask and concentrated in
vacuo. The mixture was diluted with H.sub.2O (50 mL) and acidified
with 1M HCl. The aqueous phase was extracted with ethyl acetate
(3.times.150 mL) and the combined organic layer was dried over
MgSO.sub.4, filtered and concentrated in vacuo. Purification by
flash column chromatography, using hexane-ethyl acetate (5:1),
afforded (3.60 g, 95%) of a white powder. HR MS (C14H25NO4): Calcd
mass, 272.1862. Obs mass, 272.1872 (M+H).
Example 199
[0340] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-(me-
thylamino)-ethyl]cyclopentyl]carbonyl]-L-phenylalanine methyl ester
was prepared by coupling of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenyla- lanine methyl
ester as prepared in example 45 with the product from example 198
using the general procedure described in example 146. The
protecting Boc group was removed from the product by treatment with
4 N HCl in dioxane as described in example 45 and the product was
used as is in subsequent steps. 373
Example 200
[0341] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[(a-
cetyl)(methyl) amino]ethyl]cyclopentyl]carbonyl]-L-phenylalanine.
374
[0342] Acetylation of the product of example 199 using the general
procedure described in example 148 followed by ester hydrolysis
using the general procedure described in example 47 gave the title
compound in 75% yield. HR MS (C27H31C12N3O5): Calcd mass, 548.1719.
Obs mass, 548.1716 (M+H).
Example 201
[0343] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[[(-
methylamino)carbonyl](methyl)amino]ethyl]cyclopentyl]carbonyl]-L-phenylala-
nine. 375
[0344] Reaction of the product of example 199 with methyl
isocyanate using the general procedure described in example 187
followed by ester hydrolysis using the general procedure described
in example 47 gave the title compound in 69% yield. HR MS
(C27H32C12N4O5): Calcd mass, 563.1828. Obs mass, 563.1816
(M+H).
Example 202
[0345] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[(m-
ethoxycarbonyl)-(methyl)amino]ethyl]cyclopentyl]carbonyl]-L-phenylalanine.
376
[0346] Reaction of the product of example 199 with methyl
chloroformate using the general procedure described in example 193
followed by ester hydrolysis using the general procedure described
in example 47 gave the title compound in 70% yield. HR MS
(C.sub.27H.sub.31C.sub.12N.sub.3O.sub.- 6): Calcd mass, 586.1488.
Obs mass, 586.1465 (M+Na).
Example 203
[0347]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[(trifluoroacetyl)-
amino]ethyl]cyclopentyl]carbonyl]-L-phenylalanine. 377
[0348] To a solution of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-a-
minoethyl)-cyclopentyl]-carbonyl]L-phenylalanine (0.041 mmol, 20
mg) in DMF (2 mL) was added 2 equiv. of trifluoroacetic anhydride
(0.082 mmol, 17.2 mg) at room temperature. The mixture was stirred
for 15 h at room temperature at which time HPLC analysis of the
reaction mixture indicated the absence of staring material. Then,
without any work-up, it was purified by reverse phase HPLC to
afford 5.7 mg (21%) of a white solid. HR MS:
[0349] (C26H26Cl2N3O5): Obs. mass, 588.1280. Calcd. mass, 588.1269
(M+H).
Example 204
[0350] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-(di-
methylamino)ethyl]cyclopentyl]carbonyl]-L-phenylalanine Methyl
Ester 378
[0351] To a mixture of
N-[[1-(2-aminoethyl)cyclopentyl]carbonyl]-4-[[(2,6--
dichlorophenyl)-carbonyl]amino]-L-phenylalanine methyl ester (0.339
mmol, 0.17 g), zinc chloride (1.36 mmol, 0.185 g) and
paraformaldehyde (1.36 mmol, 40.7 mg) was added dichloromethane (2
mL) at room temperature and the mixture was stirred for 1.5 h.
Then, sodium borohydride (1.36 mmol, 51.3 mg) was added and the
resulting mixture was stirred for 15 h at room temperature. The
mixture was poured into NH.sub.4OH (10 mL) and extracted with
dichloromethane (2.times.20 mL). The combined organic layer was
washed with brine solution (20 mL) and dried over anhydrous
magnesium sulfate. Filtration of the drying agent and removal of
the solvent gave a crude product which was purified by HPLC to
afford 18 mg (10%) of a light yellow solid. HR MS (C27H33C12N3O4):
Obs. mass, 534.1927. Calcd. mass, 534.1926 (M+H).
Example 205
[0352] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[[(-
4-methoxyphenyl)carbonyl]amino]ethyl]cyclopentyl]carbonyl]-L-phenylalanine
Methyl Ester 379
[0353] To a solution of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-a-
minoethyl)cyclopentyl]-carbonyl]-L-phenylalanine methyl ester (0.2
mmol, 101 mg) and 4-methoxybenzoyl chloride (0.25 mmol, 52.1 mg) in
dichloromethane (1 mL) was added DIPEA (0.3 mmol, 38.7 mg) at room
temperature. The reaction mixture was stirred for 15 h at room
temperature and then diluted with 20 mL of dichloromethane. The
dichloromethane layer was washed successively with water (20 mL)
and brine solution (20 mL) and was dried over anhydrous magnesium
sulfate. Filtration of the drying agent and concentration of the
solvent afforded a crude product which was purified by reversed
phase HPLC to obtain 0.1 g (78%) of a yellow syrup. HR MS
(C33H35C12N3O6): Obs. mass, 662.1778. Calcd. mass, 662.1801
(M+Na).
Example 206
[0354]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[[(3-trifluorometh-
ylphenyl)-carbonyl]amino]ethyl]cyclopentyl]carbonyl]-L-phenylalanine
methyl ester was prepared from
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N--
[[1-(2-aminoethyl)cyclopentyl]carbonyl]-L-phenylalanine methyl
ester and 3-trifluoromethylbenzoyl chloride using the general
method described in example 205 to give a 99% yield of a white
solid. HR MS C33H32C12N3O5): Obs. mass, 700.1596. Calcd. mass,
700.1569 (M+Na). 380
Example 207
[0355] 1-[4-(Azido)butyl]cyclopentane carboxylic acid methyl ester
was prepared from 1-[4-(bromobutyl)]cyclopentane carboxylic acid
methyl ester and sodium azide using the general procedure described
in example 4 to give a syrup in 87% overall yield. HR MS
(C11H19N3O2): Obs mass, 225.1523. Calcd mass, 225.1536 (M+).
381
Example 208
[0356] 1-[4-(Azido)butyl]cyclopentane carboxylic acid was prepared
by hydrolysis of the ester prepared in example 207 using the
general procedure described in example 15 to give a brown syrup in
quantitative yield. HR MS (C10H17N3O2): Obs mass, 211.1285. Calcd
mass, 211.1267 (M+). 382
Example 209
[0357] 1-(3-Azidopropyl)cyclopentane carboxylic acid was prepared
from 1-(3-bromopropyl)cyclopentane carboxylic acid methyl ester
using the general procedure described in example 4. The acid was
isolated as an oil. LR ES MS (C9H15N3O2): 196.1 (M-H). 383
Example 210
[0358]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(4-azidobutyl)cyclope-
ntyl]-carbonyl]-L-phenylalanine methyl ester was prepared from
1-[4-(azido)butyl]cyclopentane carboxylic acid and of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalanine methyl
ester hydrochloride salt using the procedure described in example
46 to give a 99% yield of a white solid, mp 195-199.degree. C. HR
MS (C27H31C12N5O4): Obs mass, 560.1833. Calcd mass, 560.1831 (M+H).
384
Example 211
[0359]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(4-aminobutyl)cyclope-
ntyl]-carbonyl]-L-phenylalanine methyl ester was prepared from
4-[[(2,6-dichlorophenyl)-carbonyl]-amino]N-[[1-(4-azidobutyl)cyclopentyl]-
carbonyl]-L-phenylalanine methyl ester using the general procedure
described in example 147 to give a 30% yield of a white solid. HR
MS: (C27H33C12N3O4): Obs mass, 534.1352. Calcd mass, 534.1368
(M+H). 385
Example 212
[0360]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-[(acetyl)amino]but-
yl]-cyclopentyl]carbonyl]-L-phenylalanine methyl ester was prepared
from
4-[[(2,6-dichlorophenyl)-carbonyl]amino]-N-[[1-(4-aminobutyl)cyclopentyl]-
carbonyl]-L-phenylalanine methyl ester using the procedure
described in example 148 in 80% overall yield as a white solid. HR
MS (C29H35Cl2N3O5): Obs mass, 576.1690. Calcd mass, 576.1714 (M+H).
386
Example 213
[0361] Preparation of 1-[(5-tetrazolyl)methyl]cyclopentane Methyl
Ester. 387
[0362] To a solution of methyl 1-(1-cyanomethyl)cyclopentane
carboxylate (5.5 mmol, 0.9 g) in toluene (15 mL) were added
trimethylsilyl azide (11 mmol, 1.26 g) and dibutyltin oxide (0.55
mmol, 137 mg) at room temperature. The mixture was heated to
110.degree. C. and stirred for 15 h. Then, the reaction mixture was
cooled to room temperature and toluene was removed under vacuum.
The brown residue was diluted with ethyl acetate (100 mL) and
washed with saturated sodium bicarbonate solution (2.times.50 mL)
and the starting material and some impurities remained in ethyl
acetate. The aqueous sodium bicarbonate layer was neutralized with
3N HCl and extracted with ethyl acetate (2.times.50 mL). The
combined extracts were washed with brine solution (50 mL) and dried
over anhydrous magnesium sulfate. After filtration of the drying
agent, the solution was concentrated under vacuum and the residue
was dried under high vacuum to afford 0.31 g (27%) of a low melting
white solid. HR MS (C9H14N4O2): Obs mass, 210.0218. Calcd mass,
210.0252 (M+).
Example 214
[0363] Preparation of 1-[(1-tetrazolyl)methyl]cyclopentane
Carboxylic Acid 388
[0364] Using the procedure described in example 15,
1-[(5-tetrazolyl)methyl]cyclopentane carboxylic acid was prepared
from the corresponding ester in 67% overall yield as a white solid:
mp 192-196.degree. C. HR MS (C8H12N4O2): Obs mass, 196.0329. Calcd
mass, 196.0318 (M+).
Example 215
[0365] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[-1-(buty-
l)cyclopentyl]carbonyl]-L-phenylalanine Methyl Ester. 389
[0366] A mixture of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(3-buten-
yl)cyclopentyl]carbonyl]-L-phenylalanine methyl ester (0.19 mmol,
100 mg) and 10% palladium on carbon (200 mg) in EtOH (2 mL) was
stirred under a hydrogen atmosphere at room temperature for 15 h.
Then, the charcoal was filtered and washed with EtOH (10 mL). The
filtrate was removed under vacuum to obtain 37.5 mg (37%) of as a
white solid: mp 193-196.degree. C. HR MS: Obs. mass, 519.1818.
Calcd. mass, 519.1817 (M+H).
Example 216
[0367]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[[2-[[(1,1-dimethy-
lethoxy-carbonyl]amino]-1-oxoethyl]amino]ethyl]cyclopentyl]carbonyl]-L-phe-
nylalanine was prepared by coupling of the product from example 147
with Boc-glycine using the general HBTU protocol described in
example 46, followed by treatment with NaOH to effect ester
hydrolysis as described in example 47 to give a 75% yield. HR MS
(C31H38Cl2N4O7): Calcd mass, 671.2016. Obs mass, 671.2002 (M+Na).
390
Example 217
[0368]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[(2-amino-1-oxoeth-
yl)amino]-ethyl]cyclopentyl]carbonyl]-L-phenylalanine was prepared
by treatment of the product of example 216 with 4 N HCl in dioxane.
HR MS (C26H30Cl2N4O5): Calcd mass, 571.1419. Obs mass, 571.1491
(M+Na). 391
Example 218
[0369] 1-(Cyanomethyl)cyclopentane carboxylic acid methyl ester was
prepared from cyclopentane carboxylic acid methyl ester and
chloroacetonitrile using the procedure described in example 7 to
give a 38% yield. HR MS (C9H13NO2): Obs mass, 167.0173. Calcd mass,
167.0146 (M+). 392
Examples 219-229
[0370] Using the procedure described in example 15, the following
cyclopentane carboxylic acids were prepared:
13 393 Example R Yield % Formula MS 219 394 90 C9H16O2S HR MS
Calcd, 188.0871 Obs, 188.0871 220 395 90 C11H20O2S LR-ES MS 214 (M
- H) 221 396 86 C11H17NO2 LR-ES MS 196 (M + H) 222 397 73 C13H22O4
LR-ES MS 243 (M + H) 223 398 90 C10H18O2S LR-ES MS 201 (M - H) 224
399 90 C10H14F2O2 225 400 95 C10H19NO2 LR-ES 182 226 401 25
C12H20O4 LR-ES 229 (M + H) 227 402 90 C10H16O2 HR MS Calcd,
168.2459 Obs, 168.2434 228 403 98 C8H11NO2 HR MS Calcd, 153.0198
Obs, 153.0274
Example 229
[0371] Preparation of Methyl 1-(4-chlorobutyl)cyclopentane
Carboxylate. 404
[0372] 1-(4-Chlorobutyl)cyclopentane carboxylic acid methyl ester
was prepared from cyclopentane carboxylic acid methyl ester and
4-chloro-1-bromobutane using the procedure described in example 7
to give a 64% yield. HR MS (C11H19ClO2): Obs. mass, 218.1072.
Calcd. mass, 218.1074, (M+).
Example 230
[0373] Preparation of 1-(3-butenyl)cyclopentane Carboxylic Acid
Methyl Ester 405
[0374] To a solution of diisopropylamine (225 mmol, 31.6 mL) in THF
(150 mL) was added dropwise a solution of n-butyl lithium (217.5
mmol, 87 mL, 2.5M) in hexanes at -10.degree. C. while maintaining
the temperature below 0.degree. C. After addition, the solution was
stirred for 30 min at 0.degree. C. To this, a solution of methyl
cyclopentane carboxylate (150 mmol, 19.23 g) in THF (30 mL) was
added dropwise at -70.degree. C. maintaining the internal
temperature between -60 to -70.degree. C. After addition, the
reaction mixture was stirred for 1 h at -50 to -60.degree. C. Then,
a solution of 4-bromo-1-butene (142.2 mmol, 19.2 g) in THF (30 mL)
was added dropwise and the light brown suspension was stirred for 1
h at -60 to -70.degree. C. Then, it was allowed to warm to room
temperature and stirred overnight. The reaction mixture was poured
into a saturated solution of ammonium chloride (250 mL) and the
mixture was extracted with ether (2.times.150 mL). The combined
extracts were washed with a saturated solution of sodium chloride
(150 mL) and dried over anhydrous magnesium sulfate. After
filtration of the drying agent, the solution was concentrated under
vacuum and the residue was distilled at 63-67.degree. C./2.5 mm Hg
to give 13.77 g (53%) of a colorless oil. HR MS (C11H16O2): Obs
mass, 182.1311. Calcd mass, 182.1307 (M+).
Example 231
[0375] Preparation of 1-[2-(methylthio)ethyl]cyclopentane
Carboxylic Acid Methyl Ester 406
[0376] To a solution of 1-(2-bromoethyl)cyclopentane carboxylic
acid methyl ester (2.0 mmol, 472 mg) in DMF (5 mL) was added sodium
thiomethoxide (2.65 mmol, 186 mg). The reaction mixture was stirred
for 15 h at room temperature and was poured into water (30 mL). The
organic compound was extracted into diethyl ether (2.times.20 mL).
The combined extracts were washed with a saturated solution of
sodium chloride (50 mL) and dried over anhydrous magnesium sulfate.
After filtration of the drying agent, the solution was concentrated
under vacuum and the resulting residue was purified by silica gel
column chromatography to afford 334 mg (82%) of a colorless oil. HR
MS C10H18O2S): Obs mass, 202.1024. Calcd mass, 202.1028 (M+).
Example 232
[0377] Preparation of 1-[4-(methoxy)butyl]cyclopentane Carboxylic
Acid. 407
[0378] To a solution of 1-(4-chlorobutyl)cyclopentane carboxylic
acid methyl ester (30 mmol, 6.56 g) in a mixture of THF (60 mL) and
methanol (60 mL) was added 1 N sodium hydroxide (60 mL). The
mixture was heated to 40-45.degree. C. for 15 h at which point TLC
analysis of the reaction mixture indicated the absence of starting
material and it was cooled to room temperature. The solvent was
removed under vacuum and the residue was diluted with water (100
mL) and extracted with ether (2.times.100 mL) to remove any neutral
impurities. Then, the basic aqueous layer was acidified with 1 N
hydrochloric acid and the product was extracted with ethyl acetate
(2.times.75 mL). The combined extracts were washed with brine
solution and were dried over anhydrous sodium sulfate. After
filtration of the drying agent, the solution was concentrated under
vacuum and the residue was purified by silica gel column
chromatography to afford 4.2 g (68%) of
1-(4-chlorobutyl)cyclopentane carboxylic acid as a liquid and 1.3 g
(22%) of 1-[4-(methoxy)butyl]cyclopentane carboxylic acid as a
viscous oil. HR MS (C11H20O3): Obs mass, 200.0175. Calcd mass,
200.0143 (M+).
Examples 233-248
[0379] Using the general coupling procedure described in example
46, the following analogues were prepared:
14 408 Starting material from HRMS.sup.1 Example Example R Yield %
Formula calcd. found 233 219 409 91 C26H30Cl2N2O4S 537.1381
537.1393 234 232 410 67 C28H34Cl2N2O5 549.1923 549.1903 235 209 411
C26H29Cl2N5O4 568.1 (M + Na) 568.1 (M + Na) 236 220 412 81
C28H34N2O6Cl2S 619.1413 (M + Na) 619.1404 (M + Na) 237 221 413 74
C28H31N3O4Cl2 544.1770 544.1765 238 175 414 80 C28H34N2O6Cl2S
619.1413 (M + Na) 619.1404 (M + Na) 239 222 415 67 C30H36N2O6Cl2
591.2028 591.2034 240 223 416 76 C27H32N2O4Cl2S 573.1358 (M + Na)
573.1343 (M + Na) 241 224 417 52 C26H26N2O4Cl2F2 242 225 418 76
C27H29N3O4Cl2 530.1613 530.1603 243 226 419 73 C29H34N2O6Cl2
577.1852 577.1854 244 227 420 99 C27H30Cl2N2O4 517.1661 517.1674
245 228 421 67 C25H25Cl2N3O4 502.1300 502.1299 246 214 422 85
C25H26Cl2N6O4 545.1471 545.1454 M + H ion unless otherwise
indicated
Example 247
[0380]
4-[[(2,6-Dichlorophenyl)carbonyl]amino]-N-[[1-[(2-morpholinyl)ethyl-
]cyclopentyl]carbonyl]-L-phenylalanine methyl ester was prepared
from 4-[[(2,6-Dichlorophenyl)carbonyl]amino]-L-phenylalanine methyl
ester and 1-[(2-morpholinyl)ethyl]cyclopentane carboxylic acid
using the general coupling procedure described in example 46 to
provide a 62% yield. HRMS (C29H35Cl2N3O5): Obs. mass, 576.2531.
Calcd. 6.2582, (M+H). 423
Example 248
[0381] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(4-hyd-
roxybutyl)-cyclopentyl]carbonyl]-L-phenylalanine Methyl Ester.
424
[0382] To a suspension of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(3-
-butenyl)cyclopentyl]-carbonyl]-L-phenylalanine methyl ester (3.13
mmol, 1.62 g) and anhydrous copper (I) chloride (5.0 mmol, 500 mg)
in THF (30 mL) was added solid sodium borohydride (5.0 mmol, 200
mg) at -5.degree. C. over 5 min. After the addition, the reaction
mixture was allowed to warm to room temperature and the brown
reaction mixture was stirred for 36 h at which time TLC analysis of
the mixture indicated the absence of starting material. Then, the
excess hydride was quenched by the addition of water (5 mL) and the
reaction mixture was cooled to 0.degree. C. To this, a solution of
sodium acetate (20 mL, 3.0N) was added dropwise maintaining the
temperature below 10.degree. C. this was followed by H.sub.2O.sub.2
(25 mL, 30%). After addition of hydrogen peroxide, the reaction
mixture was allowed to warm to room temperature and was stirred for
3 h and followed by 1 h at 40-45.degree. C. to complete the
hydrolysis. Then, it was poured into a mixture of water (50 mL) and
ethyl acetate (50 mL). The two layers were separated and the
aqueous layer was extracted with ethyl acetate (2.times.30 mL). The
comined extracts were washed with brine solution and dried over
anhydrous magnesium sulfate. Filtration of the drying agent and
removal of the solvent gave a crude product which was purified by
silica gel column chromatography to afford 1.04 g (62%) of a white
amorphous solid. HR MS (C27H32Cl2N2O5): Obs. mass, 535.1758. Calcd.
Mass, 535.1766, M+H).
Examples 249-274
[0383] The compounds shown below were prepared from the
corresponding methyl esters according to the procedure given in
example 47.
15 425 Starting material from HRMS1 Example Example R Yield %
Formula calcd. found 249 233 426 84 C25H28Cl2N2O4S 523.1225
523.1230 250 234 427 83 C27H32Cl2N2O5 535.1766 535.1750 251 148 428
66 C25H29Cl2N3O3 534.1562 534.1577 252 147 429 30 C24H27Cl2N3O4
492.1458 492.1460 253 204 430 53 C26H31Cl2N3O4 520.1770 520.1756
254 252 431 42 C29H35Cl2N3O6 614.1801 (M + Na) 614.1786 (M + Na)
255 248 432 67 C26H30Cl2N2O5 521.1610 521.1598 256 247 433 9
C28H33Cl2N3O5 562.1875 562.1880 257 205 434 60 C32H33Cl2N3O6
648.1644 (M + Na) 648.1649 (M + Na) 258 206 435 55 C32H30Cl2F3N3O5
686.1413 (M + Na) 686.1441 (M + Na) 259 212 436 52 C28H33Cl2N3O5
562.1875 562.1858 260 236 437 81 C27H32N2O4ClS 551.1538 551.1528
261 210 438 73 C26H29Cl2N5O4 546.1675 546.1678 262 237 439 73
C27H29N3O4Cl2 530.1613 530.1609 263 238 440 74 C27H32N2O6Cl2S
583.1436 583.1423 264 239 441 50.sup.1 C27H30N2O5Cl2 533.1610
533.1590 265 240 442 67 C26H30N2O4Cl2S 537.1381 537.1355 266 241
443 73 C25H24N2O4Cl2F2 547.0984 (M + Na) 547.0979 (M + Na) 267 184
444 27 C26H30N2O6Cl2S 569.1272 569.1280 268 242 445 45
C26H27N3O4Cl2 538.1277 (M + Na) 538.1275 (M + Na) 269 243 446 82
C26H28N2O5Cl2 541.1273 (M + Na) 541.1277 (M + Na) 270 215 447 69
C26H30Cl2N2O4 505.1661 505.1674 271 244 448 83 C26H28Cl2N2O4
503.1505 503.1516 272 245 449 51 C24H23Cl2N3O4 488.1144 488.1150
273 246 450 44 C24H22Cl2N6Na2O4 575.0954 575.0949 274 185 451 65
C26H30N2O5Cl2S 553.1329 553.1330 M + H ion unless otherwise
indicated
Example 275
[0384]
N-[[1-(3-(Acetylamino)propyl]cyclopentyl]carbonyl]-4-[[(2,6-dichlor-
ophenyl)-carbonyl]amino]-L-phenylalanine methyl ester was prepared
in 50% yield from N-[[1-(3-(azido
propyl)cyclopentyl]carbonyl]-4-[[(2,6-dichloro-
phenyl)carbonyl]amino]-L-phenylalanine methyl ester using the
general procedure described in examples 21 1 and 212. 452
Example 276
[0385]
N-[[1-(3-(Acetylamino)propyl]cyclopentyl]carbonyl]-4-[[(2,6-dichlor-
ophenyl)-carbonyl]amino]-L-phenylalanine was prepared from
N-[[1-(3-(Acetylamino)
propyl]-cyclopentyl]carbonyl]-4-[[(2,6-dichlorophe-
nyl)carbonyl]amino]-L-phenylalanine methyl ester by hydrolysis
using the general procedure described in example 47 to give a 70%
yield. HR MS (C27H31Cl2N3O5): Obs mass, 570.1533. Calcd mass,
570.1539 (M+Na). 453
Example 277
[0386] General Method for the Preparation of Morpholinoethyl Esters
from 4-(substituted)-N-acyl-L-phenylalanine Derivatives.
[0387] To a solution of a 4-(substituted)-N-acyl-L-phenylalanine
(0.5 mmol) and 2-morpholinoethanol (0.131 g, 1.0 mmol) in THF (5
mL) was added diisopropylcarbodimide (94.6 mg, 0.75 mmol) and
4-dimethylaminopyridine (30.5 mg, 0.25 mmol) at room temperature.
The resulting mixture was stirred at room temperature until time
TLC analysis of the reaction mixture indicated the absence of acid,
typically 15 h. Then, the mixture was diluted with water (50 mL)
and the THF was removed under vaccum and the residue was extracted
with dichloromethane (3.times.25 mL). The combined extracts were
washed with water (2.times.50 mL), brine solution (50 mL) and dried
over MgSO.sub.4. Filtration of the drying agent and concentration
of the solvent gave a white residue which was purified by silica
gel column chromatography eluting with dichloromethane-ethyl
acetate mixtures to obtain the target product.
Examples 278-356
[0388] Procedure for the Preparation of
4-[(4R)-3-acyl-5-oxo-2-substituted-
-4-substituted-1-imidazolidinyl]-N-[[1-[2-(acetylamino)ethyl]cyclopentyl]c-
arbonyl]-L-phenylalanines 454
[0389] A 250 mL flask was charged with
4-nitro-N-Fmoc-L-phenylalanine (20.7 g, 47.8 mmol) and NMP (30 mL).
The mixture was warmed up to accelerate dissolution. After it was
cooled to room temperature, the mixture was treated with
2,6-dichlorobenzoyl chloride (20 g, 95.6 mmol) and pyridine (12 mL,
143.4 mmol). This mixture was shaken for 5 min and was added to a
suspension of Wang resin (21.7 g, 1.1 mmol/g) in NMP (60 mL). The
mixture was then shaken at room temperature overnight. After
removal of solvent by filtration, the resin was washed with DMF
(4.times.60 mL), MeOH (4.times.60 mL), DMF (4.times.60 mL) and
finally dichloromethane (4.times.60 mL). The resin was then dried
under vacuum at room temperature overnight to give 34.27 g of resin
with loading of 0.668 mmol/g determined by the UV method (Barry
Bunin, The Combinatorial Index, p. 219 (Academic Press, 1998)).
455
[0390] The above resin (25.5 g) was treated with 160 mL of 20%
piperidine in NMP and shaken for 15 min and was filtered. This
process was then repeated 2 times. The resin was then washed with
DMF (4.times.100 mL), MeOH (4.times.100 mL), DMF (4.times.100 mL)
and finally dichloromethane (4.times.100 mL). The resin was then
dried under vacuum at room temperature overnight to give 22 g of
resin.
[0391] A portion of this free amine resin (13 g) was suspended in
80 mL of NMP and was treated with 1-1-(2-azidoethyl)cyclopentane
carboxylic acid (4.8 g, 26.05 mmol) followed by DIEA (15mL) and BOP
reagent (15.4 g, 34.2 mmol). The reaction was shaken at room
temperature overnight. After filtration, the resin was washed with
DMF (4.times.60 mL), MeOH (4.times.60 mL), DMF (4.times.60 mL) and
finally dichloromethane (4.times.60 mL). The resin was then dried
under vacuum at room temperature overnight to give azide resin.
This resin was reduced by treatement with trimethylphosphine (1.0 M
in THF, 30 mL) in 20 mL of THF at room temperature for 4 hr and
then was treated with water (3 mL) for 30 min. After filtration,
the resin was washed with DMF (4.times.60 mL), MeOH (4.times.60
mL), DMF (4.times.60 mL) and finally dichloromethane (4.times.60
mL). The resin was then dried under vacuum at room temperature
overnight to give free aminoethyl resin (13.19 g).
[0392] A portion of the above resin (6.3 g) was suspended in
dichloromethane (40 mL) and was treated with acetic anhydride (2
mL, 21 mmol) and DIEA (3.6 mL, 21 mmol). The above mixtrue was
shaken at room temperature overnight. After filtration, the resin
was washed with DMF (4.times.40 mL), MeOH (4.times.40 mL), DMF
(4.times.40 mL) and finally dichloromethane (4.times.40 mL). The
resin was then dried under vacuum at room temperature overnight to
give acetamide resin (6.27 g). A small sample of resin was
collected and treated with 50% TFA in dichloromethane to give the
cleavage product, N-[[1-[2-(acetylamino)ethyl]cyclopentyl]car-
bonyl]-4-nitro-L-phenylalanine. LS MS (M-H, m/z: 391). The
remainder of the resin was then treated with SnCl2 (2M in DMF, 40
mL) at room temperature overnight. After filtration, the resin was
washed with DMF (4.times.40 mL), MeOH (4.times.40 mL), DMF
(4.times.40 mL) and finally dichloromethane (4.times.40 mL). It was
then dried under vacuum at room temperature overnight to give
4-amino-N-[[1-[2-(acetylamino)ethyl]cyclope-
ntyl]carbonyl]-L-phenylalanine on resin (6.1 g). 456
[0393] The above free
4-amino-N-[[1-[2-(acetylamino)ethyl]cyclopentyl]carb-
onyl]-L-phenylalanine on resin was split into 5 reaction vessels.
To each vessel was added, in parallel, 8 mL of DMF followed by a
Fmoc-D-amino acid (selected from: Fmoc-D-phenylalanine (C1),
Fmoc-D-4-chlorophenylalan- ine (C2), Fmoc-D-3-pyridinylalanine
(C3), Fmoc-D-4-methoxyphenylalanine (C4) and Fmoc-D-alanine (C5),
2.25 mmol), HBTU (1.4 g, 3.75 mmol) and DIEA (0.75 mL). The above
mixture was shaken at room temperature overnight. The resin from
each vessel was filtered, washed with DMF (4.times.20 mL), MeOH
(4.times.20 mL), DMF (4.times.20 mL) and finally dichloromethane
(4.times.20 mL). These resins were then individually dried under
vacuum at room temperature overnight to give 5 individual
Fmoc-D-amino acid containing resins. Each of the five individual
batches of resin obtained above was treated in parallel with 10 mL
of 20% piperidine in NMP under shaking for 15 min and was filtered.
This process was then repeated two times to give the following five
derivatives after individual drying:
N-[[1-[2-(acetylamino)ethyl]cyclopentyl]carbonyl]-4-[[-
(2R)-2-amino-1-oxo-3-phenylpropyl]amino]-L-phenylalanine on Wang
resin,
N-[[1-[2-(acetylamino)ethyl]cyclopentyl]carbonyl]-4-[[(2R)-2-amino-3-(4-c-
hlorophenyl)-1-oxopropyl]amino]-L-phenylalanine on Wang resin,
N-[[1-[2-(acetylamino)ethyl]cyclopentyl]carbonyl]-4-[[(2R)-2-amino-1-oxo--
3-(3-pyridinyl)propyl]amino]-L-phenylalanine on Wang resin,
N-[[1-[2-(acetylamino)ethyl]-cyclo-pentyl]carbonyl]-4-[[(2R)-2-amino-3-(4-
-methoxyphenyl)-1-oxopropyl]amino]-L-phenylalanine on Wang resin,
and
N-[[1-[2-(acetylamino)-ethyl]cyclopentyl]carbonyl]-4-[((2R)-2-amino-1-oxo-
propyl)amino]-L-phenylalanine on Wang resin.
[0394] A library of imidazolidin-4-ones was prepared as discrete
compounds using the IRORI AccuTag-100 Combinatorial Chemistry
System (IRORI AccuTag-100 Combinatorial Chemistry System The
technique for labeling a reaction vessel and use of the reaction
vessels referred to herein as Microkans is described in the User's
Guide, 1996, IRORI, 11025 North Torrey Pines Road, La Jolla, Calif.
92037). IRORI is registered trademark of IRORI. IRORI, AccuTag,
Microkan and Synthesis Manager are trademarks of IRORI.
[0395] Each of the five resin derivatives was split into 16
Microkans containing radio frequency tags to give a total of 80
Microkan reaction vessels. Using the Synthesis Manager to read the
radio frequency tags in each, these were then sorted into 4 groups
of 20 so that each group had 4 Microkans containing each of the
five resin bound D-aminoacids prepared above. Each group of 20
microkans was individually placed in a reaction vessel containing a
mixture of dry solvent (THF/methyl orthoformate=1/1, 80 mL) and one
of four aldehydes (benzaldehyde (A1); 4-pyridylaldehyde (A2),
4-chlorobenzaldehyde (A3) or phenylpropionaldehyde (A4)
(20.times.1.33 mmol ). The above mixtures were shaken at room
temperature for 3 days to form imine intermediates. After removal
of the solvent by decantation, the groups of Microkans were
individually washed with dry THF (2.times.20 mL).
[0396] The resulting 80 Microkans were then sorted into 4 groups of
20 with each group incorporating one example of each of the D-amino
acids (C1to C5) combined with each of the four aldehydes (A1 to A4)
according to the table shown below:
16 457 C'-1/A'-1 C'-2/A'-1 C'-3/A'-1 C'-4/A'-1 C'-5/A'-1 C'-1/A'-2
C'-2/A'-2 C'-3/A'-2 C'-4/A'-2 C'-5/A'-2 C'-1/A'-3 C'-2/A'-3
C'-3/A'-3 C'-4/A'-3 C'-5/A'-3 C'-1/A'-4 C'-2/A'-4 C'-3/A'-4
C'-4/A'-4 C'-5/A'-4 A'-1 = phenyl A'-2 = 4-pyridyl A'-3 =
4-chlorophenyl A'-4 = 2-phenylethyl C'-1 = benzyl C'-2 =
4-chlorobenzyl C'-3 = 3-pyridinylmethyl C'-4 = 4-methoxybenzyl C'-5
= methyl
[0397] Each group of 20 Microkans was individually placed in a
reaction vessel containing dry solvent (THF/methyl
orthoformate=1/1, 10 mL). To the first reaction vessel was then
added acetic anhydride (B1) (5.5 mmol) and the resulting mixture
was shaken at 90.degree. C. for 4 hr. The remaining three reaction
vessels were individually treated with an anhydride: butryric
anhydride (B2), succinic anhdride (B3) and phenoxyacetic anhyride
(B4) and subjected to the same reaction conditions in parallel.
After filtration, each of the four groups of Microkans was
individually washed with DMF (4.times.40 mL), MeOH (4.times.40 mL),
DMF (4.times.40 mL) and finally dichloromethane (4.times.40 mL).
The Microkans were then sorted into separate vials using the
Synthesis Manager to identify each by means of the individual radio
freqency tags. Each vial was treated with cleavaging reagent 50%
TFA/dichloromethane (2.5 mL). The vials were shaken for 2 hr at
room temperature and the resulting mixtures were filtered. The
filtrate from each reaction was concentrated to dryness to give the
crude product which was then treated with ether under shaking at
room temperature for 20 min. The suspension was allowed to stand at
room temperature for 15-30 min and the ether was removed. The ether
wash was repeated and residues were dissolved in MeCN/H2O (2/1). A
portion of this solution was set aside for analysis and the balance
was freeze-dried to give crude products. The analytical samples
were analyzed by LCMS using a Micromass platform II with a 5 min
linear gradent (5% MeCN in water to 95% MeCN in water contain 0.01%
of TFA) to determine purity while recording the MS spectra to
verify the identity of the major peak. The subset of this library
derived from 4-pyridylaldehide (A-2) was removed from the
collection because of low purity scores. The remaining 60 members
were purified by reversed-phase HPLC to give a library of
imidazolidinones with purity greater than 90%. During the
separation, some of the diastereomers could be separated to give
the diastereomers at the 2-position of the imidazolidine ring
designated diastereomers 1 and 2(based upon the order of elution)
while others were assayed as mixtures of diastereomers. The
molecular weights of these purified products were comfirmed by ESMS
(M-H, M, or M+H) as shown in table below.
17 Example# MOLSTRUCTURE MS MW LOT# 278 458 667.0 (M - H) 668.79
30073-72A Diastereomer 1 279 459 667.0 (M - H) 668.79 30073-72B
Diastereomer 2 280 460 695.0 (M - H) 696.85 30073-73A Diastereomer
1 281 461 695.0 (M - H) 696.85 30073-73B Diastereomer 2 282 462
725.0 (M - H) 726.83 30073-74A Diastereomer 1 283 463 725.0 (M - H)
726.83 30073-74B Diastereomer 2 284 464 758.9 (M - H) 760.87
30073-75A Diastereomer 1 285 465 758.9 (M - H) 760.87 30073-75B
Diastereomer 2 286 466 700.9 (M - H) 703.24 30073-76A Diastereomer
1 287 467 700.9 (M - H) 703.24 30073-76B Diastereomer 2 288 468
729.0 (M - H) 731.30 30073-77A Diastereomer 1 289 469 729.0 (M - H)
731.30 30073-77B Diastereomer 2 290 470 758.9 (M - H) 761.28
30073-78 Diastereomeric Mixture 291 471 793.0 (M - H) 795.34
30073-79A Diastereomer 1 292 472 793.0 (M - H) 795.34 30073-79B
Diastereomer 2 293 473 695.0 (M - H) 696.85 30073-80A Diastereomer
1 294 474 695.0 (M - H) 696.85 30073-80B Diastereomer 2 295 475
723.0 (M - H) 724.91 30073-81 Diastereomeric Mixture 296 476 753.2
(M - H) 754.89 30073-82 Diastereomeric Mixture 297 477 786.9 (M -
H) 788.95 30073-83 Diastereomeric Mixture 298 478 640.0 (M + H)
639.76 30073-84A Diastereomer 1 299 479 640.0 (M + H) 639.76
30073-84B Diastereomer 2 300 480 666.1 (M - H) 667.81 30073-85
Diastereomeric Mixture 301 481 696.1 (M - H) 697.80 30073-86
Diastereomeric Mixture 302 482 729.9 (M - H) 731.86 30073-87
Diastereomeric Mixture 303 483 671.9 (M - H) 674.20 30073-88
Diastereomeric Mixture 304 484 (M) 702.26 30073-89 Diastereomeric
Mixture 305 485 731.9 (M) 732.24 30073-90 Diastereomeric Mixture
306 486 764.0 (M - H) 766.30 30073-91 Diastereomeric Mixture 307
487 668.0 (M + H) 667.81 30073-92 Diastereomeric Mixture 308 488
696.1 (M + H) 695.87 30073-93 Diastereomeric Mixture 309 489 726.1
(M + H) 725.85 30073-94 Diastereomeric Mixture 310 490 760.1 (M +
H) 759.91 30073-95 Diastereomeric Mixture 311 491 639.0 (M + H)
638.77 30073-96A Diastereomer 1 312 492 639.0 (M + H) 638.77
30073-96B Diastereomer 2 313 493 665.0 (M - H) 666.82 30073-97A
Diastereomer 1 314 494 667.0 (M + H) 666.825 30073-97B Diastereomer
2 315 495 695.0 (M - H) 696.81 30073-98A Diastereomer 1 316 496
695.0 (M - H) 696.81 30073-97B Diastereomer 2 317 497 729.0 (M - H)
730.87 30073-99A Diastereomer 1 318 498 729.0 (M - H) 730.87
30073-99B Diastereomer 2 319 499 670.9 (M - H) 673.22 30073-100A
Diastereomer 1 320 500 670.9 (M - H) 673.22 30073-100B Diastereomer
2 321 501 698.9 (M - H) 701.27 30073-101A Diastereomer 1 322 502
699.0 (M - H) 701.27 30073-101B Diastereomer 2 323 503 728.9 (M -
H) 731.25 30073-102A Diastereomer 1 324 504 728.8 (M - H) 731.256
30073-102B Diastereomer 2 325 505 765.31 30073-103A Diastereomer 1
326 506 762.9 (M - H) 765.31 30073-103B Mixture of Diastereomer 327
507 665.1 (M - H) 666.82 30073-104A Diastereomer 1 328 508 665.0 (M
- H) 666.82 30073-104B Diastereomer 2 329 509 693.0 (M - H) 694.88
30073-105 Diastereomeric Mixture 330 510 723.1 (M - H) 724.86
30073-144 Diastereomeric Mixture 331 511 757.0 (M - H) 758.92
30073-145 Diastereomeric Mixture 332 512 670.9 (M - H) 673.22
30073-146A Diastereomer 1 333 513 670.9 (M - H) 673.22 30073-146B
Diastereomer 2 334 514 669.0 (M - H) 701.27 30073-147
Diastereomeric Mixture 335 515 729.0 (M - H) 731.25 30073-148
Diastereomeric Mixture 336 516 762.7 (M - H) 765.31 30073-149A
Diastereomer 1 337 517 762.7 (M - H) 765.31 30073-149B Diastereomer
2 338 518 704.8 (M - H) 707.66 30073-150 Diastereomeric Mixture 339
519 732.9 (M - H) 735.71 30073-151 Diastereomeric Mixture 340 520
764.3 (M - H) 765.70 30073-152 Diastereomeric Mixture 341 521 796.9
(M - H) 799.76 30073-153 Diastereomeric Mixture 342 522 560.9 (M -
H) 562.67 30073-154A Diastereomer 1 343 523 561.0 (M - H) 562.67
30073-154B Diastereomer 2 344 524 589.0 (M - H) 590.73 30073-155A
Diastereomer 1 345 525 589.0 (M - H) 590.73 30073-155B Diastereomer
2 346 526 619.2 (M - H) 620.71 30073-156A Diastereomer 1 347 527
619.0 (M - H) 620.71 30073-156B Diastereomer 2 348 528 653.0 (M -
H) 654.77 30073-157A Diastereomer 1 349 529 653.0 (M - H) 654.77
30073-157B Diastereomer 2 350 530 594.9 (M - H) 597.1 30073-144
Diastereomic Mixture 351 531 622.9 (M - H) 625.17 30073-159A
Diastereomer 1 352 532 623.0 (M - H) 625.17 30073-159B Diastereomer
2 353 533 652.9 (M - H) 655.15 30073-160A Diastereomer 1 354 534
653.0 (M - H) 655.15 30073-160B Diastereomer 2 355 535 687 (M - H)
689.21 30073-161A Diastereomer 1 356 536 686.9 (M - H) 689.21
30073-161B Diastereomer 2
Example 357
[0398] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[(2-(m-
ethylsulfinyl)ethyl]cyclopentyl]carbonyl]-L-phenylalanine 537
[0399] To a suspension of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[(-
2-(methylthio)ethyl]cyclopentyl]carbonyl]-L-phenylalanine (0.095
mmol, 50 mg) in ethyl acetate (4 mL) was added THF (1.5 mL) to
afford a clear solution. Then, water (3 mL) and oxone (0.048 mmol,
30 mg) was added at room temperature. The mixture was stirred for
15 h at which time TLC analysis of the mixture indicated the
absence of starting material. The solid was collected by filtration
and washed with water. This material was purified by reverse phase
HPLC to afford 26.3 mg (51%) of a white solid: mp 255-258.degree.
C. HR MS (C25H28C12N2O5S): Obs mass, 539.1187. Calcd mass, 539.1174
(M+H).
Example 358
[0400] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[(2-[(-
methylsulfonyl)ethyl]cyclopentyl]carbonyl]-L-phenylalanine. 538
[0401] To a suspension of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[(-
2-(methylthio)ethyl]cyclopentyl]carbonyl]-L-phenylalanine (0.095
mmol, 50 mg) in ethyl acetate (4 mL) was added THF (1 mL) to afford
a clear solution. Then, water (2 mL) and oxone (0.019 mmol, 12 mg)
was added at room temperature. The mixture was stirred for 15 h and
the precipitated sulfoxide was collected by filtration and washed
with water. Then, the solid was redissolved in acetic acid (2 mL)
and treated with 30% hydrogen peroxide (0.7 mL). The mixture was
stirred for 15 h at room temperature at which time the TLC analysis
indicated the absence of sulfoxide. This mixture was directly
purified by reverse phase HPLC to afford 14 mg (66%) of a white
solid, mp 184-187.degree. C. HR MS (C25H28Cl2O6S): Obs mass,
577.0928. Calcd mass, 577.0944 (M+Na).
Example 359
[0402] Preparation of
4-[(2-chloro-5-bromophenylcarbonyl)amino]-N-[(1,1-di-
methylethyoxy)carbonyl]-L-phenylalanine Methyl Ester. 539
[0403] To a mixture of
4-amino-N-[(1,1-dimethylethoxy)carbonyl)-L-phenylal- anine methyl
ester (20 mmol, 5.88 g), 2-chloro-5-bromobenzoic acid (22 mmol,
5.18 g) and HBTU (22 mmol, 8.34 g) in DMF (70 mL) was added
diisopropylethylamine (50 mmol, 8.7 mL) at room temperature. The
suspension was stirred for 48 h at which time TLC analysis of the
mixture indicated the absence of starting material. Then, the
mixture was diluted with water (100 mL) and the solids were
collected by filtration and washed with water (150 mL). After air
drying, the crude product was purified by silica gel column
chromatography to obtain 1.02 g (10%) of a white solid: mp
158-161.degree. C. HR MS (C22H24BrClN2O5): Ob, 533.0442. Calcd
mass, 533.0455 (M+Na).
Example 360
[0404] Preparation of
4-[(2-chloro-5-cyanophenylcarbonyl)amino]-N-[(1,1-di-
methylethoxy)carbonyl]-L-phenylalanine Methyl Ester. 540
[0405] To a mixture of
4-[(2-chloro-5-bromophenylcarbonyl)amino]-N-[(1,1-d-
imethylethoxy)carbonyl]-L-phenylalanine methyl ester (2 mmol, 1.02
g), zinc cyanide (1.3 mmol, 152 mg) and Pd(PPh.sub.3).sub.4 (0.2
mmol, 231 mg) was added distilled and deoxygenated DMF (8 mL) at
room temperature. The suspension was heated to 80-85.degree. C. and
stirred for 15 h at which time TLC analysis of the mixture
indicated the absence of starting material. Then, the reaction
mixture was cooled to room temperature and diluted with ethyl
acetate (70 mL) and washed with 20% aqueous ammonium hydroxide (50
mL), brine solution (50 mL) and was dried over anhydrous magnesium
sulfate. Filtration of the drying agent and concentration of the
solvent gave a crude product which was purified by silica gel
column chromatography to obtain 555 mg (61%) of a white solid, mp
185-187.degree. C. HR MS (C23H24ClN3O5): Obs mass, 480.1301. Calcd
mass, 480.1302 (M+Na).
Example 361
[0406] Preparation of
4-[(2-chloro-5-cyanophenylcarbonyl)amino]-L-phenylal- anine Methyl
Ester TFA Salt. 541
[0407] To a solution of
4-[(2-chloro-5-cyanophenylcarbonyl)amino]-N-[(1,1--
dimethylethoxy)carbonyl]-L-phenylalanine methyl ester (1.2 mmol,
0.55 g) in dichloromethane (12 mL) was added trifluoroacetic acid
(3 mL) at room temperature. The reaction mixture was stirred for 15
h at room temperature at which time TLC analysis of the mixture
indicated the absence of starting material. Then, the solvent was
removed under vacuum and the residue was azeotroped with toluene
(2.times.10 mL) and dried under high vacuum to afford 0.43 g (100%)
of a yellow solid. HR MS (C18H16ClN3O3): Obs mass, 358.0963. Calcd
mass, 358.0959 (M+H).
Example 362
[0408] Preparation of
4-[(2-chloro-5-cyanophenylcarbonyl)amino]-N-[[1-(2-m-
ethoxy-ethyl)cyclopentyl]carbonyl]-L-phenylalanine Methyl Ester.
542
[0409] To a solution of
4-[(2-chloro-5-cyanophenylcarbonyl)amino]-L-phenyl- alanine methyl
ester TFA salt (0.55 mmol, 0.35 g), HBTU (0.65 mmol, 0.24 g) and
1-(2-methoxyethyl)cyclopentane carboxylic acid (0.65 mmol, 0.11 g)
in DMF (3 mL) was added diisopropylethylamine (1.65 mmol, 0.29 mL)
at room temperature. The clear solution was stirred for 15 h at
room temperature and diluted with 50 mL of ethyl acetate. Then, the
ethyl acetate layer was washed successively with 0.5N hydrochloric
acid (2.times.20 mL), saturated sodium bicarbonate solution
(2.times.20 mL) and brine solution and was dried over anhydrous
magnesium sulfate. Filtration of the drying agent and concentration
of the solvent gave a crude product which was purified by silica
gel column chromatography to afford 0.25 g (87%) of a white solid:
mp 172-175.degree. C. HR MS (C27H30ClN3O5): Obs mass, 512.1949.
Calcd mass, 512.1953 (M+H).
Example 363
[0410] Preparation of
4-[(2-chloro-5-cyanophenylcarbonyl)amino]-N-[[1-(2-m-
ethoxyethyl)cyclopentyl]carbonyl]-L-phenylalanine. 543
[0411] To a mixture of
4-[(2-chloro-5-cyanophenylcarbonyl)amino]-N-[[1-(2--
methoxy-ethyl)cyclopentyl]carbonyl]-L-phenylalanine methyl ester
(0.1 mmol, 51 mg) and lithium iodide (1.0 mmol, 133 mg) was added
pyridine (2 mL) at room temperature. The solution was refluxed for
15 h at which time TLC Paralysis of the mixutre indicated the
absence of starting material. Then, the mixture was cooled to room
temperature and diluted with water (15 mL) and the bulk of the
pyridine was removed under reduced pressure. Then it was extracted
with ether (2.times.15 mL) to remove any neutral impurities. The
aqueous layer was acidified with 1N HCl and the precipitated white
solid was collected by filtration and washed with 20 mL of water
and 20 mL of hexane. After air-drying, the crude product was
crystallized from acetonitrile to afford 20 mg (40%) of a white
solid: mp 169-172.degree. C. HR MS (C26H26ClN3O5): Obs mass,
498.1802. Calcd mass, 498.1795, M+H).
Example 364
[0412] Preparation of
4-[[(2,4-dimethylpyridin-3-yl)carbonyl]amino]-N-[[1--
[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-L-phenylalanine.
544
[0413] To a solution of 2,4-dimethylpyridinecarboxylic acid (0.6
mmol, 102 mg) in dichloromethane (3 mL) was added a drop of DMF and
oxalyl chloride (0.78 mmol, 99 mg) at 0.degree. C. (ice bath). The
solution was stirred at this temperature for 30 min, warmed to room
temperature and stirred for an additional 1 h. Then, the solvent
and excess oxalyl chloride was removed under vacuum and the residue
was dried under high vacuum. To this
4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-L-phenylalan-
ine methyl ester (0.5 mmol, 212 mg) was added and the mixture was
dissolved in dichloromethane (5 mL). To this clear solution was
added DIPEA (2.0 mmol, 0.258 g) at room temperature. The mixture
was stirred for 15 h at which time TLC analysis of the mixture
indicated the absence of starting material. Then, the mixture was
diluted with dichloromethane (20 mL) and water (100 mL). The two
layers were separated and the organic layer was washed with
saturated sodium bicarbonate solution (20 mL), brine solution (30
mL) and dried over anhydrous magnesium sulfate. Filtration of the
drying agent and removal of the solvent gave a crude product which
was purified by silica gel column chromatography to afford 0.232 g
(80%) of a white solid. HR MS (C29H39N3O6S): Obs. mass, 558.2629.
Calcd. mass, 558.2638, M+H).
[0414] Preparation of
4-[[(2,4-dimethyl-3-pyridinyl)carbonyl]amino]-N-[[1--
[4-(methylsulfonyl)-butyl]cyclopentyl]carbonyl]-L-phenylalanine.
545
[0415] Using the procedure described in example 47,
4-[[(2,4-dimethyl-3-pyridyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)buty-
l]cyclopentyl]carbonyl]-L-phenylalanine methyl ester prepared above
was hydrolyzed in 88% yield to give a white solid. HR MS
(C28H37N3O6S): Obs. mass, 544.2471. Calcd. mass, 544.2481
(M+H).
Example 365
[0416]
4-[(4R)-3-acetyl-5-oxo-2-phenyl-4-(phenylmethyl)-1-imidazolidinyl]--
N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-L-phenylalanine.
[0417] Synthesis of
N-[(1,1-dimethylethoxy)carbonyl]-4-[[(2R)-2-amino-1-ox-
o-3-phenylpropyl]amino]-L-phenylalanine methyl ester 546
[0418] To the solution of
4-amino-N-[(1,1-dimethylethoxy)carbonyl]-L-pheny- lalanine methyl
ester (5.09 g, 17 mmol) in DMF (60 mL) was added
Fmoc-D-Phenylalanine (8.70 g, 22.5 mmol), DIPEA (12 mL, 69 mmol)
and HBTU (8.50 g, 22.5 mmol). The mixture was then stirred at room
temperature for 4 h. The reaction mixture was diluted with water
(150 mL) and the light yellow solid which precipitated was
collected by filtration. This solid was then redissolved in 60 mL
of acetone and the solution was treated with 100 mL of water. The
solid was collected by filtration and was washed with 1N HCl,
H.sub.2O. After drying at 60.degree. C. under vaccum overnight, a
light yellow solid was obtained (13.2 g). A portion of this solid
(2.51 g, 3.78 mmol) was dissolved in 15 mL of DMF and to the
solution was added 1.5 mL of piperidine. The above solution was
stirred at room temperature for 45 min. After removal of the
solvent, the residue was recrystillized from ethyl acetate-hexane
to give
N-[(1,1-dimethylethoxy)carbonyl]-4-[[(2R)-2-amino-1-oxo-3-phenylpropyl]am-
ino]-L-phenylalanine methyl ester (1.36 g, 3.0 mmol ) in 81.5%
yield. LR MS 442 (M+H).
[0419] Synthesis of
4-(3-acetyl-5-oxo-2-phenyl-4-phenylmethyl-1-imidazolid-
inyl)-N-[(1,1,-dimethylethoxy)carbonyl]-L-phenylalanine methyl
ester 547
[0420] A solution of above amine (1.48, 3.35 mmol) and benzaldehyde
(376 .mu.l, 3.7 mmol) in dichloromethane (10 mL) and methyl
orthoformate (10 mL) was stirred at room temperature for 3 days.
The reaction flask was then warmed to 90.degree. C. and acetic
anhydride (neat, 1.8 mL) was added. The resulting mixture was
stirred at 110.degree. C. for 4 hr. The solvent was then evaporated
and crude product was purified by silica gel chromatography (ethyl
acetate:hexane=1:1) to give 4-(3-acetyl-5-oxo-2-phe-
nyl-4-phenylmethyl-1-imidazolidinyl)-N-[(1,1,-dimethylethoxy)carbonyl]-L-p-
henylalanine methyl ester diatereomer 1 (417 mg) and diastereomer 2
(1.25 g) These compounds are diastereomeric at the 2-position of
the imidazolidinone ring. Both diastereomers gave LR MS
(C33H37N3O6): 572 (M+H). 548
[0421]
4-(3-acetyl-5-oxo-2-phenyl-4-phenylmethyl-1-imidazolidinyl)-N-[(1,1-
,-dimethylethoxy)carbonyl]-L-phenylalanine methyl ester
(Diastereomer 1) (415 mg, 0.7 mmol) was treated with 10 mL of 4N
HCl in dioxane at room temperature for 2 hr. After removal of
solvent, the residue was dried overnight under vacuum. The residue
(241 mg, 0.471 mmol) was dissolved in DMF (4 mL) and was treated
with 1-(4-methylsulfonyl)butyl)cyclopentane carboxylic acid (153
mg, 0.617 mmol), HBTU (234 mg, 0.617 mmol) and DIEA (246 .mu.L,
1.42 mmol) at room temperature for 4 hr. The mixture was diluted
with 30 mL of ethyl acetate, the mixture was washed with 1N HCR,
water and brine (8 mL each), After it was dried over MgSO4, the
solvent was removed and the residue was filtered through silica gel
eluting with ethyl acetate:hexane=4:1 to give
4-(3-acetyl-5-oxo-2-phenyl-4-phenylmethy-
l-1-imidazolidinyl)-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-L-
-phenylalanine methyl ester diastereomer 1 (147 mg, 0.2 mmol) in
44% yield. LR MS: 702 (M+H). 549
[0422]
4-(3-acetyl-5-oxo-2-phenyl-4-phenylmethyl-1-imidazolidinyl)-N-[[1-[-
4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-L-phenylalanine
methyl ester diastereomer 1 (90 mg, 0.128 mmol) in EtOH (3 mL) was
treated with NaOH (1N, 0.3 mL) at room temperature for 30 min. The
resulting solution was acidified with 1 drop of HOAc and was
purified by HPLC (C-18, linear gradent from 5% acetonitrile to 95%
in water over 30 min) to give 84 mg (95%, 0.122 mmol) of
4-(3-acetyl-5-oxo-2-phenyl-4-phenylmethyl-1-imidazol-
idinyl)-N-[[1-[4-(methyl-sulfonyl)butyl]cyclopentyl]carbonyl]-L-phenylalan-
ine diastereomer 1. LR MS: 688 (M+H).
Example 366
[0423]
4-[(4R)-3-Acetyl-5-oxo-2-phenyl-4-(3-pyridinylmethyl)-1-imidazolidi-
nyl]-N-[(1-phenylcyclopentyl)carbonyl]-L-phenylalanine was prepared
from Fmoc-D-3-pyridinylalanine, benzaldehyde and
4-amino-N-[(1,1-dimethylethox- y)carbonyl]-L-phenylalanine methyl
ester using the general procedure described in example 365. MS: 631
(M+H) 550
Example 367
[0424] Preparation of
4-(5-bromo-1,3-dioxo-2H-isoindol-2-yl)-N-[(1,1-dimet-
hylethoxy)carbonyl]-L-phenylalanine Methyl Ester. 551
[0425] To a suspension of
4-amino-N-[(1,1-dimethylethoxy)carbonyl]-L-pheny- lalanine methyl
ester (6.78 mmol, 1.99 g) in dichloromethane (90 mL) was added a
solution of 4-bromophthalic anhydride (6.78 mmol, 1.54 g) in
dichloromethane (30 mL) and 1,1'-carbonyldiimidazole (6.78 mmol,
1.1 g) at room temperature. The resulting solution was stirred for
15 h at which time TLC analysis of the mixture indicated the
absence of starting material. The mixture was diluted with water
(100 mL) and layers were separated. The aqueous layer was extracted
with dichloromethane (2.times.100 mL) and the combined extracts
were washed with brine solution and dried over anhydrous magnesium
sulfate. Filtration of the drying agent and concentration of the
solvent under vacuum gave a crude product which was purified by
silica gel column chromatography to obtaine 2.4 g (70%) of a white
solid: mp 168-170.degree. C. HR MS C23H23BrN2O6): Obs mass,
525.0656. Calcd mass, 525.0637 (M+Na).
Example 368
[0426] Preparation of
4-(5-cyano-1,3-dioxo-2H-isoindol-2-yl)-N-[(1,1-dimet-
hylethoxy)carbonyl]-L-phenylalanine Methyl Ester. 552
[0427] To a mixture of
4-(5-bromo)1,3-dioxo-2H-isoindol-2-yl)-N-[(1,1-dime-
thylethoxy)carbonyl]-L-phenylalanine methyl ester (0.5 mmol, 0.25
g), zinc cyanide (0.3 mmol, 35 mg) and Pd(PPh.sub.3).sub.4 (0.05
mmol, 57.7 mg) was added distilled and deoxygenated DMF (2 mL) at
room temperature. The suspension was heated to 80-85.degree. C. and
was stirred for 15 h under argon an atmosphere. At this time TLC
analysis of the mixture indicated the absence of starting material.
The reaction mixture was cooled to room temperature and was diluted
with ethyl acetate (50 mL) and was washed with 20% aqueous ammonium
hydroxide (50 mL) and brine solution (50 mL) and was dried over
anhydrous magnesium sulfate. Filtration of the drying agent and
concentration afforded a crude product which was purified by silica
gel column chromatography to afford 170 mg (75%) of a white solid.
HR MS (C24H23N3O6): Obs mass, 472.1472. Calcd mass, 472.1485
(M+Na).
Example 369
[0428] Preparation of
4-(5-cyano-1,3-dioxo-2H-isoindol-2-yl)-L-phenylalani- ne Methyl
Ester TFA Salt. 553
[0429] To a solution of
4-(5-cyano-1,3-dioxo-2H-isoindol-2-yl)-N-[(1,1-dim-
ethylethoxy)carbonyl]-L-phenylalanine methyl ester (1.33 mmol, 0.6
g) in dichloromethane (12 mL) was added trifluoroacetic acid (3 mL)
at room temperature. The reaction mixture was stirred for 15 h at
room temperature at which time TLC analysis of the mixture
indicated the absence of starting material. The solvent was removed
under vacuum and the residue was azeotrophed with toluene
(2.times.10 mL) and dried under high vacuum to afford 0.46 g (100%)
of a yellow solid. HR MS (C19H15N3O4): Obs mass, 350.0156. Calcd
mass, 350.0183 (M+H).
Example 370
[0430] Preparation of
4-[(4-cyano)1,3-dioxo-2H-isoindol-2-yl]-N-[[1-(2-met-
hoxyethyl-cyclopentyl]carbonyl]-L-phenylalanine Methyl Ester.
554
[0431] To a solution of
4-(5-cyano-1,3-dioxo-2H-isoindol-2-yl)-N-[(1,1-dim-
ethylethoxy)carbonyl]-L-phenylalanine methyl ester TFA salt (0.65
mmol, 0.22 g), HBTU (0.7 mmol, 0.26 g) and
1-(2-methoxyethyl)cyclopentane carboxylic acid (0.7 mmol, 0.12 g)
in DMF (3 mL) was added diisopropylethylamine (2.0 mmol, 0.35 mL)
at room temperature. The clear solution was stirred 24 h at room
temperature and was diluted with 50 mL of ethyl acetate. The ethyl
acetate layer was washed successively with 0.5N hydrochloric acid
(2.times.20 mL), saturated sodium bicarbonate solution (2.times.20
mL), brine solution and was dried over anhydrous magnesium sulfate.
Filtration of the drying agent and concentration of the solvent
gave a crude product which was purified by silica gel column
chromatography to obtain 0.25 g (77%) of a white solid: mp
122-126.degree. C. HR MS (C26H29N3O6): Obs mass, 504.2135. Calcd
mass, 504.2134 (M+H).
Example 371
[0432] Preparation of
4-(5-cyano-1,3-dioxo-2H-isoindol-2-yl)-N-[[1-(2-meth-
oxyethyl)cyclopentyl]carbonyl]-L-phenylalanine. 555
[0433] To a mixture of
4-(5-cyano-1,3-dioxo-2H-isoindol-2-yl)-N-[[1-(2-met-
hoxyethyl)cyclopentyl]carbonyl]-L-phenylalanine methyl ester (0.3
mmol, 151 mg) and lithium iodide (3.0 mmol, 397 mg) was added
pyridine (6 mL) at room temperature. The solution was refluxed for
15 h at which time TLC analysis of the mixutre indicated the
absence of starting material. Then, it was cooled to room
temperature and diluted with water (15 mL) and the bulk of the
pyridine was removed by concentration under reduced pressure. The
residue was extracted with ether (2.times.15 mL) to remove any
neutral impurities. The aqueous layer was acidified with 1N HCl and
the precipitated white solid was collected by filtration and washed
with 20 mL of water and 20 mL of hexane. After air-drying, the
crude product was purified by reverse phase HPLC to afford 50 mg
(34%) of a white solid, mp 143-146.degree. C. HR MS (C27H27N3O6):
Obs mass, 490.1990. Calcd mass, 490.1978 (M+H).
Example 372
[0434]
4-[[(2,4-Dimethyl-3-pyridinyl)carbonyl]amino]-N-[[1-[4-(methylsulfo-
nyl)-butyl]cyclopentyl]carbonyl]-L-phenylalanine ethyl ester was
prepared using the general method described in example 123 starting
with
4-[[(2,4-dimethylpyridin-3-yl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)bu-
tyl]cyclopentyl]carbonyl]-L-phenylalanine. 556
Example 373
[0435]
4-[[(2,6-Dichlorophenyl)carbonyl]amino]-N-[[1-(2-methoxyethyl)cyclo-
pentyl]-carbonyl]-L-phenylalanine ethyl ester was prepared using
the general method described in example 123 starting with
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-methoxyethyl)cyclopentyl-
]carbonyl]-L-phenylalanine. 557
Example 374
[0436]
4-[[(2,6-Dichlorophenyl)carbonyl]amino]-N-[[-1-[4-(methylsulfonyl)b-
utyl]-cyclopentyl]carbonyl]-L-phenylalanine ethyl ester was
prepared using the general method described in example 123 starting
with
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[-1-[4-(methylsulfonyl)butyl]c-
yclopentyl]carbonyl]-L-phenylalanine. 558
Example 375
[0437]
4-[(4R)-3-acetyl-4-(phenylmethyl)-5-oxo-2-phenyl-1-imidazolidinyl]--
N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-L-phenylalanine
ethyl ester was prepared using the method described in example 123
from
4-[(4R)-3-acetyl-4-(phenylmethyl)-5-oxo-2-phenyl-1-imidazolidinyl]-N-[[1--
[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-L-phenylalanine.
559
Example 377
[0438]
4-[[(2,6-Dichlorophenyl)carbonyl]amino]-N-[[1-(2-methoxyethyl)cyclo-
pentyl]-carbonyl]-L-phenylalanine 2-(4-morpholino)ethyl ester was
prepared using the general method described in example 277 starting
with
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-methoxyethyl)cyclopentyl-
]carbonyl]-L-phenylalanine. 560
Example 378
[0439]
4-[(2,4-Dimethyl-3-pyridinyl)carbonyl]amino]-N-1-[(4-methylsulfonyl-
)butyl]-cyclopentyl]carbonyl-L-phenylalanine 2-(4-morpholino)ethyl
ester was prepared from
4-[(2,4-dimethyl-3-pyridinyl)carbonyl]amino]-N-1-[(4-me-
thylsulfonyl)butyl]cyclopentyl]carbonyl-L-phenylalanine using the
procedure described in example 277. 561
Example 379
[0440]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)bu-
tyl]-cyclopentyl]carbonyl]-L-phenylalanine 2-(4-morpholino)ethyl
ester was prepared from
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsul-
fonyl)butyl]cyclopentyl]carbonyl]-L-phenylalanine using the
procedure described in example 277. 562
Example 380
[0441]
4-[(4R)-3-acetyl-4-(phenylmethyl)-5-oxo-2-phenyl-1-imidazolidinyl]--
N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-L-phenylalanine
2-(4-morpholino)ethyl ester was prepared from
4-[(4R)-3-acetyl-4-(phenylm-
ethyl)-5-oxo-2-phenyl-1-imidazolidinyl]-N-[[1-[4-(methylsulfonyl)butyl]cyc-
lopentyl]carbonyl]-L-phenylalanine using the procedure described in
example 277. 563
Example 380a
[0442]
4-[[(2,6-Dichlorophenyl)carbonyl]amino]-N-[[1-(2-methoxyethyl)cyclo-
pentyl-]carbonyl]-L-phenylalanine 2-(N,N-diethylamino)ethyl ester
was prepared using the general method described in example 277
starting with
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-methoxyethyl)cyclopentyl-
]carbonyl]-L-phenylalanine. 564
Example 381
[0443]
4-[(2,6-Dimethyl-4-trifluoromethyl-3-pyridinyl)carbonyl]amino]-N-[[-
1-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-L-phenylalanine
was prepared from
4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl-
]-L-phenylalanine methyl ester and
2,6-dimethyl-4-trifluoromethyl-3-pyridi- ne carboxylic acid using
the general procedure described in example 364. 565
Example 382
[0444] Preparation of 4-trifluoromethyl-5-pyrimidine Carboxylic
Acid. 566
[0445] A solution of 2-chloro-4-trifluoromethyl-5-pyrimidine
carboxylic acid benzyl ester in cyclohexene (3 mL, 30 mmole) and
ethanol (9 mL) was treated with 10% palladium on carbon and the
resulting mixture was heated to reflux for 1 h. The mixture was
cooled to room temperature and filtered through a pad of Celite and
concentrated to give a quantitative yield of a gummy, off-white
solid. LR ES MS (C6H3F3N2O2): 191 (M-H).
Examples 383-387
[0446] The
4-[[(heteroaryl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]-
-cyclopentyl]carbonyl]-L-phenylalanine derivatives shown in the
table below were prepared from
4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclopent-
yl]carbonyl]-L-phenylalanine methyl ester and the appropriate
heteroaromatic carboxylic acids using the general procedure
described in example 34
18 567 Ex- am- Yield HRMS1 IC50 ple R % Formula calcd. found nM 383
568 384 569 385 570 386 571 387 572
Example 388
[0447] Preparation of
4-amino-N-methyl-N-[[1-(2-methoxyethyl)cyclopentyl]c-
arbonyl]-L-phenylalanine Methyl Ester. 573
[0448] To a mixture of
N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-N-methy-
l-4-nitro-L-phenylalanine methyl ester (1.35 mmol, 530 mg), zinc
dust (325 mesh, 13.5 mmol, 0.88 g, 10 equiv.) and ammonium chloride
(20.2 mmol, 1.08 g, 15 equiv.) was added methanol (10 mL) and water
(5 mL) at room temperature. After addition of water, the reaction
was exothermic. The suspension was stirred for 2 h at room
temperature at which time TLC analysis of the mixture indicated the
absence of starting material, the reaction mixture was filtered
through a pad of celite and the filter cake was washed with
methanol (50 mL) and water (40 mL). The mixture was concentrated
and extracted with ethyl acetate (3.times.30 mL). The combined
extracts were washed with brine solution (30 mL) and dried over
anhydrous magnesium sulfate. Filtration of the drying agent and
concentration of the solvent afforded 490 mg (100%) of a yellow
oil. HR MS (C20H30N2O4): Obs. mass,362.2202. Calcd. mass, 362.2206
(M+).
Example 389
[0449]
4-[[(2,6-Dichlorophenyl)carbonyl]amino]-N-methyl-N-[[1-(2-methoxyet-
hyl)-cyclopentyl]carbonyl]-L-phenylalanine methyl ester was
prepared from of
4-amino-N-methyl-N-[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-L-phenyla-
lanine methyl ester and 1-(2-methoxyethyl)cyclopentane carboxylic
acid using the general procedure described in example 44 to afford
a 64% yield of a white solid. HR MS (C27H32Cl2N2O5): Obs. mass,
535.1742. Calcd. mass, 535.1766 (M+H). 574
Example 390
[0450]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-methyl-N-[[1-(2-methoxyet-
hyl)-cyclopentyl]carbonyl]-L-phenylalanine was prepared from
4-[[(2,6-dichlorophenyl)
carbonyl-amino]-N-[[1-(2-methoxyethyl)cyclopenty-
l]carbonyl]-N-methyl-L-phenylalanine methyl ester using the general
procedure described in example 47 to give a 43% yield of a white
solid. HR MS (C.sub.26H.sub.30Cl.sub.2N.sub.2O.sub.5): Obs. mass,
519. 1453. Calcd mass, 519. 1454 (M-H). 575
Example 391
[0451] Preparation of 1-(2-methoxyethyl)cyclopentane Carboxylate
Methyl Ester 576
[0452] To a solution of diisopropylamine (21 mL, 150 mmol) in THF
(100 mL) was added dropwise a solution of n-butyl lithium (58 mL,
145 mmol) in hexanes at -10.degree. C. while maintaining the
temperature below 0.degree. C. After addition, the solution was
stirred for 30 min at 0.degree. C. To this, a solution of methyl
cyclopentane carboxylate (12.8 g, 100 mmol) in THF (20 mL) was
added dropwise at -70.degree. C. maintaining the internal
temperature between -60 to -70.degree. C. After addition, the
reaction mixture was stirred for 30 min at -50 to -60.degree. C.
Then, a solution of 2-methoxy ethyl bromide (12.5 g, 90 mmol) in
THF (20 mL) was added dropwise and the light brown suspension was
stirred for 30 min at -60 to -70.degree. C. Then, it was allowed to
warm to room temperature and stirred overnight. The reaction
mixture was poured into a saturated solution of ammonium chloride
(250 mL) and was extracted with ether (2.times.100 mL). The
combined extracts were washed with a saturated solution of sodium
chloride (100 mL) and dried over anhydrous magnesium sulfate. After
filtration of the drying agent, the solution was concentrated under
vacuum to afford 16.55 g of crude product as a black liquid.
Distillation at 70-75.degree. C./1.5 mm Hg afforded 7.98 g of a
colorless oil and a further 2.76 g as a light yellow oil for a
total yield of 10.74 g (64%). HR MS (C10H18O3): Obs. mass,
186.1257. Calcd. mass, 186.1256 (M+).
Example 392
[0453] Preparation of 1-(2-methoxyethyl)cyclopentane Carboxylic
Acid. 577
[0454] To a solution of 1-(2-methoxyethyl)cyclopentane carboxylate
methyl ester (7.987 g, 42.9 mmol) in a mixture of THF (170 mL) and
methanol (170 mL) was added 1 N sodium hydroxide (170 mL). The
mixture was heated to 40.degree. C. for 2 h at which point TLC
(ether:hexane 1:1, iodine chamber) analysis indicated the absence
of starting material and the mixture was cooled to room
temperature. The solvent was removed under vacuum and the residue
was diluted with water (100 mL) and was extracted with ether
(2.times.200 mL) to remove any neutral impurities. The basic
aqueous layer was acidified with 1 N hydrochloric acid and the
product was extracted with ethyl acetate (2.times.100 mL). The
combined extracts were washed with brine solution and dried over
sodium sulfate. After filtration of the drying agent, the solution
was concentrated under vacuum and the residue was dried under high
vacuum to afford 6.183 g (82%) of a light brown oil. HR MS
(C9H16O3): Obs. mass, 172.0154. Calcd. mass, 172.0126 (M+).
Example 393
[0455] Preparation of
N-[(1,1-dimethylethoxy)carbonyl]-4-nitro-L-phenylala- nine Methyl
Ester. 578
[0456] To suspesion of of
4-nitro-N-[(1,1-dimethylethoxy)carbonyl]-L-pheny- lalanine (226.2
mmol, 70.2 g) and sodium carbonate (1.13 mol, 95 g) in DMF (500 mL)
was added methyl iodide (1.13 mol, 70.4 mL) at room temperature.
The suspesion was stirred for 15 h at room temperature at this time
TLC analysis of the mixture indicated the absence of starting acid
and the excess methyl iodide and some DMF were removed under high
vacuum. The residue was poured into water (2 L) and stirred at room
temperature as a precipitate formed slowly over 72 h. The
precipitated solids were collected by filtration and washed with
water (2 L). After air and vacuum drying, 72 g (98%) of a light
yellow solid, mp 95-96.degree. C. were obtained. .sup.1H-NMR,
(DMSO-d.sub.6) (400 MHz) .delta. 8.16 (d, 2H, J=20 Hz), 7.53 (d,
2H, J=20 Hz), 7.39 (d, 1H, J=22 Hz), 4.26-4.28 (m, 1H), 3.6 (s,
3H), 2.96-3.19 (m, 2H), 1.25 (s, 9H). .sup.13C NMR, CDCl.sub.3 (100
Mhz) .delta. 172.04, 155.29, 146.27, 145.96, 130.48, 123.18, 78.36,
54.44, 51.9, 36.1, 27.99. HR MS: Obs. mass, 325.1404. Calcd. mass,
325.1400 (M+H).
Example 394
[0457] Preparation of of
4-amino-N-[(1,1-dimethylethoxy)carbonyl]-L-phenyl- alanine Methyl
Ester. 579
[0458] To a mixture of of
N-[(1,1-dimethylethoxy)carbonyl]-4-nitro-L-pheny- lalanine methyl
ester (222 mmol, 72 g), zinc dust (325 mesh, 2.2 mol, 145.2 g, 10
equiv) and ammonium chloride (3.3 mol, 178.1 g, 15 equiv) was added
methanol (1 L) and water (500 mL) at room temperature. After
addition of water, an exothermic reaction ensued and the internal
temperature rose to 45 to 50.degree. C. The suspension was stirred
for 30 min to 1 h at room temperature, at which time TLC analysis
of the mixture indicated the absence of starting material, and the
reaction mixture was filtered through a pad of celite and the
filtered cake was washed with methanol (1 L) and water (500 mL).
Concentration to remove most of the methanol afforded white solid
which was collected by filtration and washed with water. After air
drying, 65.5 g of a white solid, mp 86-89.degree. C. was obtained.
.sup.1H-NMR (DMSO-d.sub.6) (400 MHz) .delta. 6.9 (d, 2H, J=20 Hz),
6.62 (d, 2H, J=20 Hz), 7.39 (d, 1H, J=22 Hz), 4.26-4.28 (m, 1H),
3.68 (s, 3H), 2.96-3.19 (m, 2H), 1.25 (s, 9H). HR MS: Obs. mass,
294.1614. Calcd. mass, 294.1621 (M+).
Example 395
[0459] Preparation
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(1,1-dimethy-
lethoxy)carbonyl]-L-phenylalanine Methyl Ester. 580
[0460] To a solution of
4-amino-N-[(1,1-dimethylethoxy)carbonyl]-L-phenyla- lanine methyl
ester (127.6 mmol, 37.57 g) and 2,6-dichlorobenzoyl chloride (140.6
mmol, 29.45 g) in dichloromethane (350 mL) was added
diisopropylethylamine (192 mmol, 33.4 mL) at room temperature. The
brown solution was stirred for 15 h at room temperature to afford a
white suspension. At this time, TLC analysis of the mixture
indicated the absence of starting material. The solids were
collected by filtration and were washed with dichloromethane (150
mL) and air dried to obtain 52.75 g (88.4%) of a white solid: mp
192-194.degree. C. .sup.1H NMR (DMSO-d.sub.6) (400 MHz) .delta.
10.68 (s, 1H), 7.47-7.6 (m, 5H), 7.2-7.29 (m, 3H), 4.12-4.17 (m,
1H), 3.62 (s, 3H), 2.79-2.99 (m, 2H), 1.33 (s, 9H). .sup.13C NMR
CDCl.sub.3 (100 Mhz) d 172.49, 161.82, 155.37, 136.99, 136.36,
131.28, 131.16, 129.48, 128.19, 119.31, 78.27, 55.3, 51.76, 35.9,
27.77. HR MS: Obs. mass, 466.1069. Calcd. mass, 466.1062).
Example 396
[0461] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalan- ine Methyl
Ester Hydrochloride Salt. 581
[0462] Solid
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(1,1-dimethylethox-
y)carbonyl]-L-phenyl-alanine methyl ester (92.97 mmol, 43.45 g) in
dioxane (90 mL) was treated with 166 mL of 4.0 N hydrochloric acid
in dioxane at room temperature. After 5 minutes, the solids went
into solution and the mixture was stirred for 2 h. The reaction
mixture was concentrated a yellow syrup and 250 mL of ethyl ether
was added. A gum was formed which was dissolved in THF (100 mL) and
methanol (100 mL). The solvent was removed under vacuum to obtain
43.7 (100%) of a white solid: mp 180-195.degree. C. .sup.1H NMR
(DMSO-d.sub.6) (400 MHz) .delta. 10.81 (s, 1H), 7.76 (d, 2H, J=22
Hz), 7.58 (d, 2H, J=18 Hz), 7.51 (t, 1H, J=15 Hz), 7.24 (d, 2H,
J=22 Hz),4.23-4.26 (m, 1H), 3.56 (s, 3H), 3.14-3.17 (m, 2H).
.sup.13C NMR, CDCl.sub.3 (100 Mhz) d 169.03, 161.72, 137.56,
136.11, 131.19, 130.95, 129.93, 129.79, 128.06, 119.46, 53.17,
52.6, 35.13. HR MS (C17H16Cl2N2O3.HCl): Obs. mass, 367.0611. Calcd.
mass, 367.0616 (M+H).
Example 397
[0463] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-met-
hoxyethyl)-cyclopentyl]carbonyl]-L-phenylalanine Methyl Ester.
582
[0464] To a solution of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylal- anine methyl
ester hydrochloride salt (43.03 mmol, 11.75 g) and
1-(2-methoxyethyl)cyclopentane carboxylic acid (43.5 mmol, 7.5 g)
in DMF (130 mL) was added HBTU (43.5 mmol, 16.5 g) and
diisopropylethylamine (108.8 mmol, 19.02 mL) at room temperature.
The clear solution was stirred 23 h at room temperature and was
diluted with 200 mL of ethyl acetate. The ethyl acetate layer was
washed successively with 0.5 N hydrochloric acid (2.times.100 mL),
saturated sodium bicarbonate solution (2.times.100 mL), brine
solution and was dried over anhydrous magnesium sulfate. Filtration
of the drying agent and concentration of the solvent gave 18.86 g
(84%) of a white solid, mp 85-87.degree. C. .sup.1H NMR
(DMSO-d.sub.6) (400 MHz) .delta. 10.65 (s, 1H), 7.88 (d, 1H, J=19
Hz), 7.47-7.59 (m, 5H), 7.21 (d, 2H, J=19 Hz), 4.47-4.53 (m, 1H),
3.64 (s, 3H), 2.88-3.1 (m, 7H), 1.76-1.98 (m, 4H), 1.23-1.47 (m,
6H). HRMS C26H30Cl2N2O5): Obs. mass, 521.1586. Calcd. mass,
521.1610 (M+H).
Example 398
[0465] Preparation of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-met-
hoxyethyl)cyclopentyl]carbonyl]-L-phenylalanine. 583
[0466] To a suspension of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-
-methoxyethyl)-cyclopentyl]carbonyl]-L-phenylalanine methyl ester
(20.17 mmol, 10.52 g) in ethanol (80 mL) and tetrahydrofuran (10
mL) was added aqueous 1.0 N sodium hydroxide (80 mL) at room
temperature. The mixture was heated to 50.degree. C. and the
resulting clear solution was stirred overnight. Then, the ethanol
solution was concentrated and diluted with 50 mL of water and was
extracted with 200 mL of ether to remove neutral impurities. The
aqueous layer was acidified with 1N HCl and the precipitated white
solid was collected by filtration and washed with 200 mL of water
and 200 mL of hexane. After air-drying, 8.4 g (82%) of as a white
solid: mp 136-140.degree. C. was obtained. .sup.1H NMR,
DMSO-d.sub.6 (400 MHz) .delta. 10.64 (s, 1H), 7.73 (d, 1H, J=22
Hz), 7.46-7.59 (m, 5H), 7.22 (d, 2H, J=22 Hz), 4.43-4.48 (m, 1H),
2.87-3.11 (m, 7H), 1.76-2.01 (m, 4H), 1.23-1.47 (m, 6H). HR MS
(C25H28Cl2N2O5): Obs. mass, 507.1464. Calcd. mass, 507.1454
(M+H).
Example 399
[0467] Preparation of 3-(1-methyltetrazol-5-yl)benzyl Chloride.
584
[0468] Preparation of 3-chloromethylbenzoic Acid.
[0469] A 2 L, three-necked, round bottom flask equipped with a
mechanical stirrer, thermometer and condenser with an open end (no
nitrogen inlet tube was connected to avoid pressure build-up in the
apparatus) was charged with 109 g (800 mmol) of m-toluic acid and
320 mL of chlorobenzene. The mixture was heated to ca. 90.degree.
C. with a steam bath to give a homogeneous solution and 53.4 g (400
mmol, 0.5 equiv) of N-chlorosuccinimide (NCS) and 800 mg (3.3 mmol)
of benzoyl peroxide (BPO) were added. The yellow solution was
stirred at ca. 95.degree. C. for 2.5 h. Then, 26.7 g (200 mmol,
0.25 equiv) of NCS and 400 mg (1.65 mmol) of BPO were added and the
mixture was stirred at 95.degree. C. for 2.5 h. Then, a further
26.7 g (200 mmol, 0.25 equiv) of NCS and 400 mg (1.65 mmol) of BPO
were added and the mixture was stirred at 95.degree. C. for 2.5 h.
To the reaction mixture was added 480 mL of water and the resulting
suspension was allowed to cool to room temperature with stirring
overnight. To the yellow slurry was added 480 mL of hexane. The
resulting suspension was stirred at room temperature for 30 min,
then filtered through a coarse sintered glass filter. The collected
solid was washed thoroughly with 2.times.130 mL of water and then
with 2.times.130 mL of hexane, and dried by suction for 2.5 h. The
solid was then suspended in 800 mL of water and the mixure was
heated on a steam bath for 30 min. After standing at room
temperature overnight, the white solid was collected by filtration
and dried by suction for 1.5 h. Further drying at 55.degree. C.
under high vacuum overnight yielded 73.7 g (54.0%) of
3-chloromethylbenzoic acid; mp 134-136.degree. C. 585
[0470] Preparation of 3-(chloromethyl)-N-methylbenzamide.
[0471] A 250 mL, round bottom flask equipped with a magnetic
stirrer, reflux condenser and calcium chloride drying tube was
charged with 34.1 g (200 mmol) of 3-chloromethylbenzoic acid and
125 mL of toluene (dried over molecular sieves 4A). To this
suspension was added 21.9 mL (300 mmol) of thoinyl chloride and the
mixture was heated to 85-90.degree. C. for 15 h. While heating,
evolution of gas, presumably hydrogen chloride and sulfur dioxide,
was observed. The reaction mixture was cooled to room temperature
and excess thionyl chloride and toluene were removed under vacuum.
The resulting oily residue was azeotroped with 100 mL of toluene,
then dried under high vacuum for 1 h to give the crude acid
chloride.
[0472] A 1 L, three necked, round bottom flask equipped with a
magnetic stirrer, addition funnel, thermometer and argon bubbler
was charged with the crude acid chloride obtained above, and 400 mL
of dichloromethane (dried over molecular sieves 4A). After the
solution was cooled to -5 to 0.degree. C. (using an ice-sodium
chloride bath), 14.9 g (220 mmol) of methylamine hydrochloride was
added in one portion. To this mixture was added 69.6 mL (400 mmol)
of diisopropylethylamine (DIPEA) dropwise over 15-20 min, while
maintaining the temperature of the reaction mixture below 2.degree.
C. After completion of the addition, the mixture was stirred for 45
min at 0 to 5.degree. C., then allowed to warm to room temperature.
After stirring for 15 min at room temperature, TLC analysis
indicated complete reaction. The reaction mixture was diluted with
250 mL of water, and stirred for 5 minutes. The two layers were
separated and the aqueous phase was extracted with 2.times.100 mL
of dichloromethane. The combined organic layers were washed
successively with 300 mL of water, and 300 mL of saturated sodium
chloride solution. After drying over anhydrous magnesium sulfate,
the solution was concentrated by rotary evaporation under house
vacuum. The residue was further dried in vacuo to give a light
yellow solid. This solid was dissolved in 110 mL of toluene at
.about.60-70.degree. C. The resulting solution was allowed to cool
to room temperature and seeded with crystals of product, then
stored in a refrigerator overnight. The resulting precipitate was
collected by filtration and washed with 30 mL of hexane. After
drying under high vacuum, 29.4 g (80.0% yield) of
3-(chloromethyl)-N-methylbenzamide was obtained as a light yellow
solid; mp 59-61.degree. C. 586
[0473] Preparation of 3-(1-methyltetrazol-5-yl)benzyl Chloride.
[0474] A 250 mL round bottom flask equipped with a magnetic
stirrer, reflux condenser and calcium chloride drying tube was
charged with 27.2 g (148 mmol) of
3-(chloromethyl)-N-methylbenzamide, 100 mL of toluene (dried over
molecular sieves 4A). To this solution was added 16.2 mL (222 mmol)
of thionyl chloride and the mixture was heated to 85-90.degree. C.
for 15 h (Note 6). While heating, evolution of gas, presumably
hydrogen chloride and sulfur dioxide, was observed. After cooling
to room temperature, excess thionyl chloride and toluene were
removed under vacuum. The resulting residue was azeotroped with 100
mL of toluene, then dried under high vacuum for 1 h to give the
crude imidoyl chloride. A 500 mL, three necked, round bottom flask
equipped with a magnetic stirrer, thermometer and argon bubbler was
charged with 11.6 g (178 mmol) of sodium azide and 140 mL of
acetonitrile (freshly opened bottle). To this suspension was added
23.7 mL (187 mmol) of chlorotrimethylsilane and the mixture was
stirred for 1.5 h at room temperature. After cooling to 0.degree.
C., a solution of the crude imidoyl chloride, prepared above, in 40
mL of acetonitrile was added. This heterogeneous mixture was
stirred for 1-2 h at 0.degree. C., then allowed to warm to room
temperature and stirred for 15 h. TLC analysis indicated complete
reaction. The reaction mixture was quenched by the addition of 150
mL of water, then diluted with 150 mL of ethyl acetate. The two
layers were separated and the aqueous phase was extracted with
2.times.100 mL of ethyl acetate. The combined organic layers were
washed successively with 200 mL of water, and 200 mL of saturated
sodium chloride solution. After drying over anhydrous magnesium
sulfate, the solution was concentrated. The residue was further
dried in vacuo to give a light yellow solid (29.7 g). This solid
was dissolved in 220 mL of 5.5:4.5 hexane:ethyl acetate at
.about.60-70.degree. C. The resultant solution was allowed to cool
to room temperature and was seeded with crystals of product, then
stored in a refrigerator overnight. The resulting precipitate was
collected by filtration and washed with 50 mL of hexane. After
drying by suction, 24.5 g (79.5% yield) of
3-(1-methyltetrazol-5-yl)benzyl chloride was obtained as a white
amorphous solid; mp 63-65.degree. C.
Example 400
[0475] 1-[[3-(1-Methyltetrazol-5-yl)phenyl]methyl]cyclobutane
carboxylic acid methyl ester was prepared from
3-(1-methyltetrazol-5-yl)benzyl chloride using the general method
described in example 7 to give a 77% yield of a viscous oil. HR MS:
obs. mass, 287.1514. Calcd. mass, 287.1508 (M+H). 587
Example 401
[0476] 1-[[3-(1-Methyltetrazol-5-yl)phenyl]methyl]cyclobutane
carboxylic acid was prepared from
1-[[3-(1-methyltetrazol-5-yl)phenyl]methyl]cyclobu- tane carboxylic
acid methyl ester using the general procedure described in example
15 to give an 83% yield of a viscous oil. HR MS: obs. mass,
273.1226. Calcd. mass, 273.1238 (M+H) 588
Example 402
[0477]
4-Amino-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-L-phen-
ylalanine methyl ester was prepared from 4-nitro-L-phenylalanine
methyl ester and 4-(methylsulfonyl)butyl]cyclopentane carboxylic
acid using the general procedure described in example 26. HR MS
(C21H32N2O5S): Obs mass, 425.2121. Calcd mass, 425.2110 (M+H).
589
Example 403
[0478] 1-(4-Bromobutyl)cyclobutane carboxylic acid methyl ester was
prepared from 1,4-dibromobutane and cyclobutane carboxylic acid
methyl ester using the general procedure described in example 168.
590
Example 404
[0479] 1-[4-(Methylthio)butyl]cyclobutane carboxylic acid methyl
ester was prepared from 1-(4-bromobutyl)cyclobutane carboxylic acid
methyl ester and sodium methylmercaptan using the procedure
described in example 172. 591
Example 405
[0480] 1-[4-(methylsulfonyl)butyl]cyclobutane carboxylic acid was
prepared from 1-[4-(methylthio)butyl]cyclobutane carboxylic acid
methyl ester using the general procedures described in examples 174
and 175. 592
Example 406
[0481]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)bu-
tyl]-cyclobutane]carbonyl]-L-phenylalanine was prepared from
4-[[(2,6-dichlorophenyl)-carbonyl]amino]-L-phenylalanine methyl
ester and 1-[4-(methylsulfonyl)butyl]cyclobutane carboxylic acid
using the procedure described in examples 46 and 47. 593
Example 407
[0482]
4-[(4R)-3-Acetyl-4-(phenylmethyl)-5-oxo-2-phenyl-1-imidazolidinyl]--
N-[(1-phenylcyclopentyl)carbonyl]-L-phenylalanine was prepared from
Fmoc-D-phenylalanine, benzaldehyde and
4-amino-N-[(1,1-dimethylethoxy)car- bonyl]-L-phenylalanine methyl
ester using the general procedure described in example 365. 594
Example 408
[0483]
4-[3-Acetyl-5-oxo-2-[(3-pyridinyl)methyl]-4-phenylmethyl-1-imidazol-
idinyl]-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-L-phenylalani-
ne methyl ester was prepared from
4-amino-N-[(1,1-dimethylethoxy)carbonyl]- -L-phenylalanine methyl
ester and Fmoc-D-pyridinylalanine using the general method
described in example 365. 595
Example 409
[0484]
4-[(2,6-Dimethyl-4-trifluoromethyl-3-pyridinyl)carbonyl]amino]-N-[[-
1-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-L-phenylalanine
methyl ester was prepared from
4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclopenty-
l]carbonyl]-L-phenylalanine methyl ester. 596
[0485] To a solution of
2,6-dimethyl-4-trifluoromethyl-5-pyridinecarboxyli- c acid (0.42
mmol, 92 mg) in dichloromethane (5 mL) was added 3 drops of DMF and
oxalyl chloride (0.57 mmol, 73 mg) at 0.degree. C. (ice bath). The
solution was stirred at this temperature for 30 min, warmed to room
temperature and stirred for an additional 30 min. Then, the solvent
and excess oxalyl chloride was removed under vacuum and the residue
was dried under high vacuum and dissoved in 3 mL of ethyl acetate
(dried over 4A sieves). The acid chloride solution was then added
to a solution of
4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-L-phenylalan-
ine methyl ester (0.35 mmol, 148 mg) in ethyl acetate (dried over
4A sieves) (5 mL) containing Amberlyst A-21 ion exchange resin (2
meq, 450 mg). The mixture was then sonicated with a Ace High
Intensity Ultrasonic Processor, 600 watts at 40% power with a
miniprobe for total of 9 hrs. Then the mixture was diluted with
ethyl acetate (20 mL) and washed with saturated sodium bicarbonate
solution (10 mL), brine solution (5 mL) and dried over anhydrous
magnesium sulfate. Filtration of the drying agent and removal of
the solvent gave a crude product which was purified by silica gel
column chromatography to afford 0.152 g (70%) of a yellow
solid.
Example 410
[0486] VLA-4/VCAM-1 Screening Assay
[0487] VLA-4 antagonist activity, defined as ability to compete for
binding to immobilized VCAM-1, was quantitated using a solid-phase,
dual antibody ELISA. VLA-4 (.alpha.4.beta.1 integrin) bound to
VCAM-1 was detected by a complex of anti-integrin .beta.1 antibody:
HRP-conjugated anti-mouse IgG: chromogenic substrate (K-Blue).
Initially, this entailed coating 96 well plates (Nunc Maxisorp)
with recombinant human VCAM-1 (0.4 .mu.g in 100 .mu.l PBS), sealing
each plate and then allowing the plates to stand at 4.degree. C.
for .sup..about.18 hr. The VCAM-coated plates were subsequently
blocked with 250 .mu.l of 1% BSA/0.02% NaN.sub.3 to reduce
non-specific binding. On the day of assay, all plates were washed
twice with VCAM Assay Buffer (200 .mu.l/well of 50 mM Tris-HCl, 100
mM NaCl, 1 mM MnCl.sub.2, 0.05% Tween 20; pH 7.4). Test compounds
were dissolved in 100% DMSO and then diluted 1:20 in VCAM Assay
Buffer supplemented with 1 mg/mL BSA (i.e., final DMSO=5%). A
series of 1:4 dilutions were performed to achieve a concentration
range of 0.005 nM-1.563 .mu.M for each test compound. 100 .mu.l per
well of each dilution was added to the VCAM-coated plates, followed
by 10 .mu.l of Ramos cell-derived VLA-4. These plates were
sequentially mixed on a platform shaker for 1 min, incubated for 2
hr at 37.degree. C., and then washed four times with 200 .mu.l/well
VCAM Assay Buffer. 100 .mu.l of mouse anti-human integrin .beta.1
antibody was added to each well (0.6 .mu.g/mL in VCAM Assay
Buffer+1 mg/mL BSA) and allowed to incubate for 1 hr at 37.degree.
C. At the conclusion of this incubation period, all plates were
washed four times with VCAM Assay Buffer (200 .mu.l/well). A
corresponding second antibody, HRP-conjugated goat anti-mouse IgG
(100 .mu.l per well (1:800 dilution in VCAM Assay Buffer+1 mg/mL
BSA), was then added to each well, followed by a 1 hr incubation at
room temperature and concluded by three washes (200 .mu.l/well)
with VCAM Assay Buffer. Color development was initiated by addition
of 100 .mu.l K-Blue per well (15 min incubation, room temp) and
terminated by addition of 100 .mu.l Red Stop Buffer per well. All
plates were then read in a UV/Vis spectrophotometer at 650 nM.
Results were calculated as % inhibition of total binding (i.e.,
VLA-4+VCAM-1 in the absence of test compound). The results are
provided in the following table:
19 TABLE 2 conc. % Inh. @ Example nM conc. IC.sub.50 nM 27 2.7 28
22.6 29 3.6 30 6.1 31 9.8 35 5.0 36 4.6 37 5.0 40 2.1 41 1.7 42
0.61 43 0.63 47 2.5 48 0.44 49 4.7 50 1.3 51 1.6 52 1.1 53 0.5 54
0.82 55 0.47 56 0.94 57 0.42 58 0.58 59 0.26 60 1.05 64 3.0 65 100
92" 2.4 10 72 66 100 85" 10 47 67 100 81 10 41 68 100 86 8.2 10 49
69 100 87 5.8 10 49 70 100 86 7.0 10 49 71 100 86 2.5 10 51 72 100
80 10 39 73 100 90 10 64 74 100 86 13.6 10 50 75 100 92 3.8 10 68
76 100 93 4.6 10 73 77 100 93 5.6 10 67 78 100 93 9.0 10 63 79 100
95 1.2 10 77 80 100 89 6.4 10 53 81 100 93 1.4 10 73 82 7.3 83 1.1
84 4.4 85 0.43 86 6.8 87 1.5 88 3.9 89 81 90 1.04 91 40 92 3.0 93
1.9 94 0.96 95 0.69 96 17.4 97 0.50 98 11.7 99 2.1 100 0.26 101
18.3 102 0.95 103 0.70 104 10.4 105 0.83 106 13.5 109 62 110 8.2
111* 9.4 113 43 114 155 115 22.7 116 0.88 118 0.35 119 5.0 120 2.9
121 66 122 2.1 128 5.0 129 153 130 6.6 131 63 132 2.8 133 60 134
0.19 135 17 136 0.73 137 19 138 0.42 139 38 141 3.9 142 1.7 143
0.85 144 0.75 156 1.15 158 5 159 6.4 160 5.8 161 9.4 162 1.2 163
2.4 164 6.2 165 11 166 2.5 167 9.2 187 0.26 192 0.14 193 0.55 194
0.33 200 0.81 201 1.2 202 1.0 203 0.64 216 2.9 217 2.0 249 0.29 250
0.32 251 0.37 252 5.1 253 3.4 254 2.0 255 0.5 256 2.4 257 40 258 14
259 3.6 270 1.1 271 0.55 272 1.0 273 1.5 363 0.64 371 1.65
[0488]
4-[(RS)-2,3,5,6,7,7a-hexahydro-1,3-dioxo-1H-pyrrolo[3,4-c]pyridin-2-
-yl]-N-[[1-[(4-methoxyphenyl)methyl]cyclopentyl]carbonyl]-L-phenylalanine
Example 411
[0489] Ramos (VLA-4)/VCAM-1 Cell-Based Screening Assay Protocol
Materials:
[0490] Soluble recombinant human VCAM-1 (mixture of 5- and 7-Ig
domain) was purified from CHO cell culture media by immunoaffinity
chromatography and maintained in a solution containing 0.1 M
Tris-glycine (pH 7.5), 0.1 M NaCl, 5 mM EDTA, 1 mM PMSF, 0.02%
0.02% NaN.sub.3 and 10 1 g/mL leupeptin. Calcein-AM was purchased
from Molecular Probes Inc.
[0491] Methods:
[0492] VLA-4 (.alpha.4.beta.1 integrin) antagonist activity,
defined as ability to compete with cell-surface VLA-4 for binding
to immobilized VCAM-1, was quantitated using a Ramos-VCAM-1 cell
adhesion assay. Ramos cells bearing cell-surface VLA-4, were
labeled with a fluorescent dye (Calcein-AM) and allowed to bind
VCAM-1 in the presence or absence of test compounds. A reduction in
fluorescence intensity associated with adherent cells (%
inhibition) reflected competitive inhibition of VLA-4 mediated cell
adhesion by the test compound.
[0493] Initially, this entailed coating 96 well plates (Nunc
Maxisorp) with recombinant human VCAM-1 (100 ng in 100 .mu.l PBS),
sealing each plate and allowing the plates to stand at 4.degree. C.
for .sup..about.18 hr. The VCAM-coated plates were subsequently
washed twice with 0.05% Tween-20 in PBS, and then blocked for 1 hr
(room temperature) with 200 .mu.l of Blocking Buffer (1% BSA/0.02%
thimerosal) to reduce non-specific binding. Following the icubation
with Blocking Buffer, plates were inverted, blotted and the
remaining buffer aspirated. Each plate was then washed with 300
.mu.l PBS, inverted and the remaining PBS aspirated.
[0494] Test compounds were dissolved in 100% DMSO and then diluted
1:25 in VCAM Cell Adhesion Assay Buffer (4 mM CaCl.sub.2, 4 mM
MgCl.sub.2 in 50 mM TRIS-HCl, pH 7.5) (final DMSO=4%). A series of
eight 1:4 dilutions were performed for each compound (general
concentration range of 1 nM -12,500 nM). 100 .mu.l/well of each
dilution was added to the VCAM-coated plates, followed by 100 .mu.l
of Ramos cells (200,000 cells/well in 1% BSA/PBS). Plates
containing test compounds and Ramos cells were allowed to incubate
for 45 min at room temperature, after which 165 .mu.l/well PBS was
added. Plates were inverted to remove non-adherent cells, blotted
and 300 .mu.L/well PBS added. Plates were again inverted, blotted
and the remaining buffer gently aspirated. 100 .mu.l Lysis Buffer
(0.1% SDS in 50 mM TRIS-HCl, pH 8.5) was added to each well and
agitated for 2 min on a rotary shaking platform. The plates were
then read for fluorescence intensity on a Cytofluor 2300
(Millipore) fluorecence measurement system (excitation=485 nm,
emission=530 nm). The results are shown in the following table:
20 TABLE 3 IC.sub.50 in Ramos Cell Conc. % Inh Based Assay Example
(nM) @ Conc. (nM) 43 36.5 53 37.5 55 18.5 95 34.5 100 54.5 56 31.5
57 27 58 47 59 13.5 156 28 158 124 159 313 160 328 162 23 163 85
187 5.7 188 36 189 130 190 650 191 322 192 22 193 40 194 27 200 19
201 19 202 31 203 47 216 356 217 211 249 62 250 28 251 9 252 228
253 89 254 360 255 27 256 151 257 635 258 1,034 259 129 260 130 276
22 260 94 261 184 262 39 263 5 270 150 271 67 272 96 273 104 278 2
280 4.2 282 1.8 283 10 32 284 2.9 286 24 288 10 92 291 10 48 293 40
299 1.7 300 10 32 303 10 70 304 10 35 305 10 37 311 3 313 10 68 315
10 71 316 10 22 317 10 17 319 10 68 321 10 59 323 10 76 324 10 20
322 5 342 10 24 344 19 346 10 23 349 10 58 350 13 351 13 357 25 358
75 363 11
Example 412
[0495] Oral Dosage Form
21 Item Ingredients mg/tablet 1 Compound of invention 25 100 250
500 2 Anhydrous lactose 83 35 19 38 3 Croscarmellose sodium 6 8 16
32 4 Povidone K30 5 6 12 24 5 Magnesium stearate 1 1 3 6 Total
weight (mg) 120 150 300 600
[0496] Manufacturing Procedure:
[0497] 1. Mix items 1,2 ,3 in a suitable mixer for 15 minutes.
[0498] 2. Granulate the powder mix from step 1 with 20% PVP K30
solution.
[0499] 3. Dry the granulation in step 2 at 50.degree. C.
[0500] 4. Pass the granulation from step 3 through a suitable
milling equipment.
[0501] 5. Add the item 5 to the milled granulation from Step 4 and
mix for 3 minutes.
[0502] 6. Compress the granulation from Step 5 on a suitable
press.
Example 413
[0503] Aerosol Administration Formulation
22 Ingredients Qty/mL Compound of invention 3-150 mg* Sodium
chloride 8.0 mg Phosphate buffer (20 mM)pH 7.0* q.s. 1.0 mL
[0504] Depending upon activity of the compound
[0505] pH can be adjusted with Sodium hydroxide solution (1 N) or
HCl solution (10%w/v)
[0506] Procedure:
[0507] Dissolve the drug substance in the buffer.
[0508] Filter the solution through a 0.22 micron filter.
[0509] The particle size distribution after nebulizing the above
solution (as measured using Malvern Mastersizer X) is in the range
of 1-6 microns.
* * * * *