U.S. patent application number 09/863579 was filed with the patent office on 2001-11-15 for diephenyl carbocyclic thioamide derivatives.
Invention is credited to Hull, Kenneth G., Sidduri, Achytharao, Tilley, Jefferson W..
Application Number | 20010041799 09/863579 |
Document ID | / |
Family ID | 22390545 |
Filed Date | 2001-11-15 |
United States Patent
Application |
20010041799 |
Kind Code |
A1 |
Hull, Kenneth G. ; et
al. |
November 15, 2001 |
Diephenyl carbocyclic thioamide derivatives
Abstract
It has been discovered that compounds of the formula: 1 and the
pharmaceutically acceptable salts and esters thereof wherein X and
Y are as defined below, inhibit the binding of VCAM-1 to VLA-4 and
are useful in treating inflammation associated with chronic
inflammatory diseases such as rheumatoid arthritis (RA), multiple
sclerosis, (MS), asthma, and inflammatory bowel disease (I BD).
Inventors: |
Hull, Kenneth G.;
(Marlborough, MA) ; Sidduri, Achytharao;
(Livingston, NJ) ; Tilley, Jefferson W.; (North
Caldwell, NJ) |
Correspondence
Address: |
HOFFMANN-LA ROCHE INC.
PATENT LAW DEPARTMENT
340 KINGSLAND STREET
NUTLEY
NJ
07110
|
Family ID: |
22390545 |
Appl. No.: |
09/863579 |
Filed: |
May 23, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09863579 |
May 23, 2001 |
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09505903 |
Feb 17, 2000 |
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60120475 |
Feb 18, 1999 |
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Current U.S.
Class: |
546/314 ;
548/530; 558/414; 562/426 |
Current CPC
Class: |
C07C 2601/08 20170501;
A61P 11/06 20180101; C07C 327/46 20130101; C07D 213/56 20130101;
C07D 233/38 20130101; C07D 401/06 20130101; A61P 43/00 20180101;
C07C 2601/04 20170501; A61P 29/00 20180101; C07D 213/82 20130101;
C07C 327/48 20130101; C07D 239/28 20130101; A61P 1/00 20180101 |
Class at
Publication: |
546/314 ;
548/530; 558/414; 562/426 |
International
Class: |
C07D 213/81; C07D
27/44 |
Claims
1. A compound of the formula: 183wherein X is a group of the
formula 184wherein: R.sub.15 is 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, lower alkylthio
lower alkyl, lower alkylsulfinyl lower alkyl, lower alkylsulfonyl
lower alkyl, lower alkylsulfinyl, lower alkanoyl, aroyl, aryl,
aryloxy; R.sub.16 is hydrogen, halogen, nitro, cyano, lower alkyl,
OH, perfluorolower alkyl, or lower alkylthio; or X is a group of
formula X-2 185wherein Het is a 5- or 6-membered heteroaromatic
ring containing 1, 2 or 3 heteroatoms selected from N,O, and S, or
Het is a 9- or 10-membered bicyclic heteroaromatic ring containing
1, 2, 3 or 4 heteroatoms selected from O, S, and N; R.sub.15 and
R.sub.16 are as above; R.sub.30 is hydrogen or lower alkyl; and p
is an integer from 0 to 1 or X is a group of formula X-3
186wherein: R.sub.18 is aryl, heteroaryl, aryl lower alkyl,
heteroaryl lower alkyl R.sub.19 is substituted or unsubstituted
lower alkyl, aryl, heteroaryl, arylalkyl, heteroaryl alkyl, and
R.sub.20 is substituted or unsubstituted lower alkanoyl or aroyl;
and Y is a group of formula Y-1 187wherein: R.sub.22 and R.sub.23
are independently hydrogen, lower alkyl, lower alkoxy, cycloalkyl,
aryl, arylalkyl, nitro, cyano, lower alkylthio, lower
alkylsulfinyl, lower alkyl sulfonyl, lower alkanoyl, halogen, or
perfluorolower alkyl and at least one of R.sub.22 and R.sub.23 is
other than hydrogen, and R.sub.24 is hydrogen, lower alkyl, lower
alkoxy, aryl, nitro, cyano, lower alkyl sulfonyl, or halogen; or
Y-2 is a group of the formula: 188Het is a five or six membered
heteroaromatic ring bonded via a carbon atom wherein said ring
contains one, two or three heteroatoms selected from the group
consisting of N, O and S and R.sub.30 and R.sub.3, are
independently hydrogen, lower alkyl, cycloalkyl, halogen, cyano,
perfluoroalkyl, or aryl and at least one of R.sub.30 and R.sub.31
is adjacent to the point of attachment; p is an integer of from 0
to 1; or Y is a group of formula Y-3 189wherein: 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 H or lower alkyl, R.sub.29 is hydrogen, lower alkyl,
lower alkoxycarbonyl, lower alkanoyl, aroyl, perfluoro lower
alkanoylamino, lower alkyl sulfonyl, lower alkylaminocarbonyl,
arylaminocarbonyl, or R.sub.28 and R.sub.29 taken together form a
4, 5 or 6-membered saturated carbacyclic ring optionally containing
one heteroatom selected from 0, S, and N; with the carbon atoms in
the ring being unsubstituted or substituted by lower alkyl or
halogen, Q is --(CH.sub.2).sub.fO--, --(CH.sub.2).sub.fS--,
--(CH.sub.2).sub.fN(R.sub.27)--, or --(CH.sub.2).sub.f--, R.sub.27
is H, lower alkyl, aryl, lower alkanoyl, aroyl or lower
alkoxycarbonyl, e is an integer from 0 to 4, and f is an integer
from 0 to 3; and the dotted bond is optionally hydrogenated; and
pharmaceutically acceptable salts and esters thereof.
2. A compound of claim 1 wherein X is a group of the formula 190and
Y, R.sub.15 and R.sub.16 are as in claim 1.
3. A compound of claim 2 wherein R.sub.15 is lower alkyl, nitro,
halogen, perfluoromethyl, or cyano and R.sub.16 is hydrogen, lower
alkyl, nitro, halogen, perfluoromethyl, or cyano.
4. A compound of claim 3 wherein R.sub.15 and R.sub.16 are
independently chloro or fluoro.
5. A compound of claim 3 wherein X-1 is selected from the group of
191
6. A compound of claim 1 wherein X is a group of the formula X-2
192and p, Y, R,.sub.5, R.sub.16, and R.sub.30 are as in claim
1.
7. A compound of claim 6 wherein Het is 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.
8. A compound of claim 6 wherein Het is a bicyclic heteroaromatic
ring containing from 1 to 3 nitrogens.
9. A compound of claim 6 wherein R.sub.1 5 is nitro, lower alkyl
sulfonyl, cyano, lower alkyl, lower alkoxy, perfluorolower alkyl,
lower alkylthio, lower alkanoyl, or aryl.
10. A compound of claim 9 wherein R.sub.15 is unsubstituted
phenyl.
11. A compound of claim 6 wherein R.sub.16 is hydrogen, halogen,
nitro, cyano, lower alkyl, perfluoro lower alkyl; and R.sub.30 is
hydrogen or lower alkyl.
12. A compound of claim 6 wherein Het is a 6 membered monocyclic
heteroaromatic ring containing 1 or 2 nitrogens or a 10 membered
bicyclic heteroaromatic ring containing one nitrogen, R.sub.15 is
lower alkyl, or perfluoroalkyl and R.sub.16 is hydrogen, lower
alkyl, or perfluoroalkyl, and R.sub.30 is absent.
13. A compound of claim 6 wherein X-2 is selected from the group of
193
14. A compound of claim 1 wherein X is a group of formula X-3
194and Y, R.sub.18, R.sub.19, and R.sub.20 are as in claim 1.
15. A compound of claim 14 wherein R.sub.18 is phenyl.
16. A compound of claim 14 wherein R.sub.19 is lower alkyl which is
unsubstituted or substituted by pyridyl or phenyl.
17. A compound of claim 14 wherein R.sub.20 is substituted or
unsubsituted lower alkanoyl.
18. A compound of claim 14 wherein R.sub.18 is phenyl, R.sub.19 is
lower alkyl which is unsubstituted or substituted by pyridyl or
phenyl and R.sub.20 is lower alkanoyl.
19. A compound of claim 14 wherein R.sub.18 is phenyl which is
unsubstituted or substituted by halogen or lower alkoxy; R.sub.19
is phenyl lower alkyl which is unsubstituted or substituted by
lower alkoxy, pyridyl lower alkyl, or lower alkyl; and R.sub.20 is
substituted or unsubstituted lower alkanoyl.
20. A compound of claim 19 wherein X-3 is selected from the group
of 195
21. A compound of claim 1 wherein Y is a group of formula 196and X,
R.sub.22, R.sub.23, and R.sub.24 are as in claim 1.
22. A compound of claim 21 wherein R.sub.22 and R.sub.23 are lower
alkyl, trifluoromethyl, or halogen and R.sub.24 is hydrogen, lower
alkyl, lower alkoxy, or halogen.
23. A compound of claim 22 wherein Y-1 is selected from the group
of 197
24. A compound of claim 1 wherein Yis a group of the formula Y-2
198and p, X, Het, R.sub.30 and R.sub.3,, are as in claim 1.
25. A compound of claim 24 wherein Het is a 6 membered
heteroaromatic ring.
26. A compound of claim 25 wherein the heteroatom is N.
27. A compound of claim 26 wherein Y-2 is selected from the group
of 199
28. A compound of claim 1 wherein Y is a group of formula Y-3
200and Y, R.sub.25 and Q are as in claim 1, and the dotted bond can
be optionally hydrogenated.
29. A compound of claim 28 wherein Y-3 is selected from the group
of 201
30. A compound of claim 1 wherein X is a group of the formula X-1
202and Y is a group of the formula Y-1 203
31. A compound of claim 1 wherein X is a group of the formula X-1
204and Y is a group of the formula Y-2
32. A compound of claim 1 wherein X is a group of the formula X-1
205and Y is a group of the formula Y-3 206wherein R.sub.15,
R.sub.16, R.sub.25 and Q are as above; and the dotted bond can be
optionally hydrogentated.
33. A compound of claim 1 wherein X is a group of the formula X-2
207and Y is a group of the formula Y-1 208
34. A compound of claim 1 wherein X is a group of the formula X-2
209and Y is a group of the formula Y-2
35. A compound of claim 1 wherein X is a group of the formula X-2
210and Y is a group of the formula Y-3 211wherein R.sub.15,
R.sub.16, R.sub.25, R.sub.30, Q and p are as above and the dotted
bond can be optionally hydrogenated.
36. A compound of claim 1 wherein X is a group of the formula X-3
212and Y is a group of the formula Y-1 213
37. A compound of claim 1 wherein X is a group of the formula X-3
214and Y is a group of the formula Y-2 215
38. A compound of claim I wherein X is a group of the formula X-3
216and Y is a group of the formula Y-3 217wherein R.sub.18,
R.sub.19, R.sub.20, R.sub.25, and Q are as above and the dotted
bond can be optionally hydrogenated.
39. A compound of claim 1 wherein X is a group of the formula X-1
218wherein R.sub.16 is in the ortho position and is hydrogen, lower
alkyl, nitro, cyano, halogen, lower alkylthio, perfluoroloweralkyl
and R,5 is lower alkyl, nitro, cyano, halogen, lower alkylsulfonyl,
perfluoroloweralkyl, and Y is a group of the formula Y-1 219where
R.sub.22 is hydrogen, halogen, trifluoroalkyl, or lower alkyl and
R.sub.23 is halogen, trifluoroalkyl, or lower alkyl, and R.sub.24
is hydrogen or Y is a group of the formula Y-3 220wherein Q is as
above and the dotted bond can be optionally hydrogenated; R.sub.25
is R.sub.26--(CH.sub.2)e-; e is 2-4 and R.sub.26 is azido, cyano,
hydroxy, lower alkoxy, lower alkoxycarbonyl, lower alkanoyl, lower
alkyl sulfonyl, lower alkyl sulfinyl, perfluoro lower alkanoyl,
nitro, or lower alkylthio or R.sub.25 is NHR.sub.29 where R.sub.29
is lower alkanoyl or lower alkylamino carbonyl.
40. A compound of claim 39 wherein X is a group of the formula X-1
221wherein R.sub.16 is in the ortho position and is hydrogen, lower
alkyl, nitro, cyano, halogen, lower alkylthio, perfluoroloweralkyl
and R.sub.15 is lower alkyl, nitro, cyano, halogen, lower
alkylsulfonyl, perfluoroloweralkyl; and Y is a group of the formula
Y-1 222where R.sub.22 is hydrogen, halogen, or lower alkyl and
R.sub.23 is halogen or lower alkyl, and R.sub.24 is hydrogen.
41. A compound of claim 40 wherein R.sub.16 is hydrogen or halogen
and R.sub.15 is halogen; R.sub.22 is hydrogen, halogen, ethyl, or
methyl and R.sub.23 is halogen, ethyl, or methyl.
42. A compound of claim 41 wherein R.sub.16 is in the ortho
position and R.sub.15 and R.sub.16 are both chlorine, and R.sub.22
is methyl and R.sub.23 is chlorine or ethyl.
43. A compound of claim 42 which is
4-[[(2,6-dichlorophenyl)carbonyl]amino-
]-N-[(2-chloro-6-methylphenyl)thioxomethyl]-L-phenylalanine.
44. A compound of claim 39 wherein X is a group of the formula X-1
223wherein R.sub.16 is in the ortho position and is hydrogen, lower
alkyl, nitro, cyano, halogen, lower alkylthio, perfluoroloweralkyl
and R.sub.15 is lower alkyl, nitro, cyano, halogen, lower
alkylsulfonyl, perfluoroloweralkyl; and Y is a group of the formula
Y-3 224which is a four to six membered cycloalkyl ring, R.sub.25 is
R.sub.26--(CH.sub.2)e-; e is 2-4 and R.sub.26 is azido, id cyano,
hydroxy, lower alkoxy, lower alkoxycarbonyl, lower alkanoyl, lower
alkyl sulfonyl, lower alkyl sulfinyl, perfluoro lower alkanoyl,
nitro, or lower alkylthio; and the dotted bond is hydrogenated.
45. A compound of claim 44 wherein R.sub.16 is hydrogen or halogen
and R.sub.15 is halogen; and Y-3 is a four or five membered ring
and R.sub.26 is lower alkoxy, lower alkyl sulfonyl, lower alkyl
sulfinyl, or lower alkylthio.
46. A compound of claim 45 wherein R.sub.16 is in the ortho
position and R.sub.15 and R.sub.16 are both chlorine, and R.sub.26
is lower alkyl sulfonyl or lower alkylthio.
47. A compound of claim 46 which is
4-[[(2,6-dichlorophenyl)carbonyl]amino-
]-N-[[1-[(4-methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanine-
.
48. A compound of claim 39 wherein X is a group of the formula X-1
225where R.sub.16 is hydrogen or halogen and R.sub.15 is halogen
and Y is a group of the formula Y-1 226where R.sub.22 is hydrogen,
halogen, ethyl, or methyl and R.sub.23 is halogen, ethyl, or methyl
and R.sub.24 is hydrogen or Y is a group of the formula Y-3
227where Y-3 is a four or five membered ring, R.sub.25 is as in
claim 39 and R.sub.26 is lower alkoxy, lower alkyl sulfonyl, lower
alkyl sulfinyl, or lower alkylthio, and the dotted bond is
optionally hydrogenated.
49. A compound of claim 48 wherein R.sub.16 is in the ortho
position and R.sub.15 and R.sub.16 are both chlorine, and when Y is
Y-1 then R.sub.22 is methyl and R.sub.23 is chlorine or ethyl and
when Y is Y-3, Y-3 is a four or five membered ring and R.sub.26 is
lower alkyl sulfonyl or lower alkylthio.
50. A compound of claim 1 wherein Y is as in formula 1 and X is X-1
228where R.sub.15 is ortho and is halogen, lower alkyl, or
perfluoroalkyl and R.sub.16 is hydrogen, halogen, lower alkyl, or
perfluoroalkyl.
51. A compound of claim 50 wherein R.sub.15 is chlorine and
R.sub.16 is hydrogen or chlorine.
52. A compound of claim 1 wherein Y is as in formula 1 and X is X-2
229where Het is pyridine or pyrimidine and R,.sub.5 is lower alkyl
or perfluoroalkyl R.sub.16, and R.sub.20 are hydrogen, lower alkyl,
or perfluoroalkyl.
53. A compound of claim 1 wherein Y is as in formula 1 and X is X-3
230where R.sub.19 is pyridinyl lower alkyl or phenyl lower alkyl,
R.sub.20 is lower alkanoyl, and R.sub.18 is phenyl.
54. A compound of claim 1 wherein X is as in formula 1 and Y is Y-1
231where R.sub.22 is hydrogen or lower alkyl, R.sub.23 is halogen,
lower alkyl, or perfluoroalkyl, and R.sub.24 is hydrogen.
55. A compound of claim 54 wherein R.sub.22 is hydrogen or methyl
and R.sub.23 is halogen, ethyl, or trifluoromethyl.
56. A compound of claim 1 wherein X is as in formula 1 and Y is Y-3
232which is a four to six membered cycloalkyl ring, R.sub.25 is
R.sub.26--(CH.sub.2)e-, e is 2-4, and R.sub.26 is alkoxy, lower
alkyl sulfonyl, loweralkylthio, or NHR.sub.29 where R.sub.29 is
loweralkoxycarbonyl or loweralkylaminocarbonyl, and the dotted bond
is hydrogenated.
57. A compound of claim 56 wherein R.sub.26 is methoxy, methyl
sulfonyl, or methylthio.
58. A compound of claim 50 wherein Y is Y-1 233where R.sub.22 is
hydrogen or lower alkyl, R.sub.23 is halogen, lower alkyl, or
perfluoroalkyl, and R.sub.24 is hydrogen.
59. A compound of claim 58 wherein R.sub.15 is chlorine and
R.sub.16 is hydrogen or chlorine.
60. A compound of claim 59 which is
4-[[(2,6-dichlorophenyl)carbonyl]amino-
]-N-[(2-bromophenyl)thioxomethyl]-L-phenylalanine.
61. A compound of claim 59 which is
4-[[(2,6,-dichlorophenyl)carbonyl]amin-
o]-N-[(2-ethyl-6-methylphenyl)thioxomethyl] L-phenylalanine.
62. A compound of claim 59 which is
4-[[(2,6,-dichlorophenyl)carbonyl]amin-
o]-N-[(2-fluorophenyl)thioxomethyl]-L-phenylalanine.
63. A compound of claim 59 which is
4-[[(2,6,-dichlorophenyl)carbonyl]amin-
o]-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalanine.
64. A compound of claim 52 wherein Y is Y-1 234where R.sub.22 is
hydrogen or lower alkyl, R.sub.23 is halogen, lower alkyl, or
perfluoroalkyl, and R.sub.24 is hydrogen.
65. A compound of claim 53 wherein Y is Y-1 235where R.sub.22 is
hydrogen or lower alkyl, R.sub.23 is halogen, lower alkyl, or
perfluoroalkyl, and R.sub.24 is hydrogen.
66. A compound of claim 65 which is
4-[(2S,4R)-3-acetyl-2-phenyl-4-[(3-pyr-
idinyl)methyl]-5-oxo-imidazolidin-1-yl]-N-[(2-ethyl-6-methylphenyl)thioxom-
ethyl]-L-phenylalanine.
67. A compound of claim 50 wherein Y is Y-3 236which is a four to
six membered cycloalkyl ring, R.sub.25 is R.sub.26--(CH.sub.2)e-, e
is 2-4, and R.sub.26 is alkoxy, lower alkyl sulfonyl,
loweralkylthio, or NHR.sub.29 where R.sub.29 is loweralkoxycarbonyl
or loweralkylaminocarbonyl, and the dotted bond is
hydrogenated.
68. A compound of claim 67 wherein R,, is chlorine and R.sub.16 is
hydrogen or chlorine.
69. A compound of claim 68 which is
4-[[2,6-dichlorophenyl)carbonyl]amino--
N-[[1-[2-(acetylamino)ethyl]cyclopentyl]thioxomethyl]-L-phenylalanine.
70. A compound of claim 68 which is [[1-[2-[[(methylamino)carbonyl]
amino]ethyl]
cyclopentyl]thioxomethyl]-4-[[(2,6-dichlorophenyl)carbonyl]a-
mino]L-phenylalanine.
71. A compound of claim 68 which is
4-[[(2,6,-dichlorophenyl)carbonyl]amin-
o]-N-[[1-(2-methoxyethyl)cyclopentyl]thioxomethyl]-L-phenylalanine.
72. A compound of claim 68 which is
4-[[(2,6,-dichlorophenyl)carbonyl]amin-
o]-N-[[1-[(4-methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine-
.
73. A compound of claim 68 which is
4-[[(2,6,-dichlorophenyl)carbonyl]amin-
o]-N-[[1-(3-methylthio)propyl]cyclobutyl]thioxomethyl]-L-phenylalanine.
74. A compound of claim 68 which is
4-[[(2,6,-dichlorophenyl)carbonyl]amin-
o]-N-[[1-(3-methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-L-phenylalanine-
.
75. A compound of claim 67 wherein R.sub.26 is methoxy, methyl
sulfonyl, or methyl thio.
76. A compound of claim 68 wherein R.sub.26 is methoxy, methyl
sulfonyl, or methyl thio.
77. A compound of claim 52 wherein Y is Y-3 237which is a four to
six membered cycloalkyl ring, R.sub.25 is R.sub.26--(CH.sub.2)e-, e
is 2-4, and R.sub.26 is alkoxy, lower alkyl sulfonyl,
loweralkylthio, or NHR.sub.29 where R.sub.29 is loweralkoxycarbonyl
or loweralkylaminocarbonyl, and the dotted bond is
hydrogenated.
78. A compound of claim 77 wherein R.sub.26 is methoxy, methyl
sulfonyl, or methyl thio.
79. A compound of claim 78 which is
4-[(2,6-dimethyl-3-pyridinylcarbonyl)a-
mino]-N-[[1-[(4-methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylala-
nine.
80. A compound of claim 78 which is
4-[[[4-(trifluoromethyl)-5-pyrimidinyl- ]carbonyl]amino]-N-[[l
-(4-methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-
-phenylalanine.
81. A compound of claim 78 which is
4-[[(2,4-dimethyl-6-trifluoromethyl-3--
pyridinyl)carbonyl]amino]-N-[[l
-[(4-methylsulfonyl)butyl]cyclobutyl]thiox-
omethyl]-L-phenylalanine.
82. A compound of claim 53 wherein Y is Y-3 238which is a four to
six membered cycloalkyl ring, R.sub.25 is R.sub.26--(CH.sub.2)e-, e
is 2-4, and R.sub.26 is alkoxy, lower alkyl sulfonyl,
loweralkylthio, or NHR.sub.29 where R.sub.29 is loweralkoxycarbonyl
or loweralkylaminocarbonyl, and the dotted bond is optionally
hydrogenated.
83. A compound of claim 82 wherein R.sub.26 is methoxy, methyl
sulfonyl, or methyl thio.
84. A compound of claim 83 which is
4-[(2S,4R)-3-acetyl-2-phenyl-4-[(3-phe-
nyl)methyl]-5-oxo-imidazolidin-1-yl]-N-[[(4-methylsulfonyl)butyl]cyclopent-
yl]thioxomethyl]-L-phenylalanine.
85. A compound of claim 83 which is
4-[(2R,4R)-3-acetyl-2-phenyl-4-[(3-phe-
nyl)methyl]-5-oxo-imidazolidin-1-yl]-N-[[(4-methylsulfonyl)butyl]cyclopent-
yl]thioxomethyl]-L-phenylalanine.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to:
[0002] Chen, et al. U.S. Ser. No. 09/138,353 filed Aug. 21, 1998,
Title: N-ALKANOYLPHENYLALANINE DERIVATIVES; and
[0003] Chen, et al. U.S. Ser. No. 09/137,798 filed Aug. 21, 1998,
Title: N-AROYLPHENYLALANINE DERIVATIVES.
BACKGROUND OF THE INVENTION
[0004] 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.
[0005] Thus, compounds which inhibit the interaction between
.alpha..sub.4-containing integrins and VCAM-1 will be useful as
therapeutic agents for the treatment of inflammation resulting from
chronic inflammatory diseases such as rheumatoid arthritis,
multiple sclerosis (MS), asthma, and inflammatory bowel disease
(IBD).
SUMMARY OF THE INVENTION
[0006] It has been discovered that compounds of the formula: 2
[0007] and the pharmaceutically acceptable salts and esters thereof
wherein X and Y are as defined below, inhibit the binding of VCAM-1
to VLA-4 and are useful in treating inflammation associated with
chronicinflammatory diseases such as rheumatoid arthritis (RA),
multiple sclerosis, (MS), asthma, and inflammatory bowel disease (I
BD).
DETAILED DESCRIPTION OF THE INVENTION
[0008] The present invention is directed to the discovery that
compounds of the formula: 3
[0009] and the pharmaceutically acceptable salts and esters
thereof, inhibit the binding of VCAM-1 to VLA-4 and are useful in
treating inflammation associated with chronic inflammatory diseases
such as rheumatoid arthritis (RA), multiple sclerosis, (MS),
asthma, and inflammatory bowel disease (I BD).
[0010] In accordance with the invention, X is a group X-1, X-2 or
X-3 as described below. Y is a group Y-1, Y-2 or Y-3 as described
below.
[0011] The group X-1 is of the formula: 4
[0012] wherein:
[0013] R.sub.15 is 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, lower alkylthio lower alkyl, lower
alkylsulfinyl lower alkyl, lower alkylsulfonyl lower alkyl, lower
alkylsulfinyl, lower alkanoyl, aroyl, aryl, aryloxy;
[0014] R.sub.16 is hydrogen, halogen, nitro, cyano, lower alkyl,
OH, perfluorolower alkyl, or lower alkylthio.
[0015] The groups R.sub.15 and R.sub.16 are preferably
independently hydrogen, lower alkyl, nitro, halogen (especially
chloro or fluoro), perfluoromethyl, or cyano for R.sub.16, and
lower alkyl, nitro, halogen (especially chloro or fluoro),
perfluoromethyl, or cyano for R.sub.15.
[0016] It is preferred that groups selected as R.sub.15, or
R.sub.15 and R.sub.16, be electron-deficient as defined below.
[0017] X-2 is a group of the formula: 5
[0018] wherein Het is a 5- or 6-membered heteroaromatic ring
containing 1, 2 or 3 heteroatoms selected from N,O, and S, or
[0019] Het is a 9- or 10-membered bicyclic heteroaromatic ring
containing 1, 2, 3 or 4 heteroatoms selected from O, S, and N;
[0020] R.sub.15 and R.sub.16 are as above, and
[0021] R.sub.30 is hydrogen or lower alkyl; and p is an integer
from 0 to 1.
[0022] 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.
[0023] The group X-3 is of the formula: 6
[0024] wherein:
[0025] R.sub.18 is aryl, heteroaryl,
[0026] R.sub.19 is substituted or unsubstituted lower alkyl, aryl,
heteroaryl, arylalkyl, heteroaryl alkyl, and
[0027] R.sub.20 is substituted or unsubstituted lower alkanoyl or
aroyl
[0028] R.sub.18 is preferably phenyl. R.sub.19 is preferably lower
alkyl, which is unsubstituted or substituted by pyridyl or phenyl.
R.sub.20 is preferably lower alkanoyl
[0029] Y is a group of formula Y-1, Y-2, or Y-3 wherein:
[0030] Y-1 is a group of the formula: 7
[0031] wherein:
[0032] R.sub.22 and R.sub.23 are independently hydrogen, lower
alkyl, lower alkoxy, cycloalkyl, aryl, arylalkyl, nitro, cyano,
lower alkylthio, lower alkylsulfinyl, lower alkyl sulfonyl, lower
alkanoyl, halogen, or perfluorolower alkyl and at least one of
R.sub.22 and R.sub.23 is other than hydrogen, and
[0033] R.sub.24 is hydrogen, lower alkyl, lower alkoxy, aryl,
nitro, cyano, lower alkyl sulfonyl, or halogen
[0034] Y-2 is a group of the formula 8
[0035] Het is a five or six membered heteroaromatic ring bonded via
a carbon atom wherein said ring contains one, two or three
heteroatoms selected from the group consisting of N, O and S and
R.sub.30 and R.sub.31 are independently hydrogen, lower alkyl,
cycloalkyl, halogen, cyano, perfluoroalkyl, or aryl and at least
one of R.sub.30 and R.sub.31 is adjacent to the point of
attachment, p is an integer of from 0 to 1.
[0036] Y-3 is a 3-7 membered ring of the formula: 9
[0037] wherein:
[0038] 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,
[0039] wherein
[0040] R.sub.28 is H or lower alkyl,
[0041] R.sub.29 is hydrogen, lower alkyl, lower alkoxycarbonyl,
lower alkanoyl, aroyl, perfluoro lower alkanoylamino, lower alkyl
sulfonyl, lower alkylaminocarbonyl, arylaminocarbonyl, or
[0042] R.sub.28 and R.sub.29 taken together form a 4, 5 or
6-membered saturated carbocyclic ring optionally containing one
heteroatom selected from O, S, and N with the carbon atoms in the
ring being unsubstituted or substituted by lower alkyl or
halogen,
[0043] Q is --(CH.sub.2).sub.fO-, --(CH.sub.2).sub.fS- ,
--(CH.sub.2).sub.fN(R.sub.27)-, or --(CH.sub.2).sub.f-,
[0044] R.sub.27 is H, lower alkyl, aryl, lower alkanoyl, aroyl or
lower alkoxycarbonyl,
[0045] e is an integer from 0 to 4, and f is an integer from 0 to
3; the dotted bond is optionally hydrogenated.
[0046] This invention is directed to a compound of the formula:
10
[0047] wherein X is a group of the formula 11
[0048] wherein:
[0049] R.sub.15 is halogen, nitro, lower alkyl sulfonyl, cyano,
lower alkyl, lower alkoxy, lower O alkoxycarbonyl, carboxy, lower
alkyl aminosulfonyl, perfluorolower alkyl, lower alkylthio, hydroxy
lower alkyl, alkoxy lower alkyl, lower alkylthio lower alkyl, lower
alkylsulfinyl lower alkyl, lower alkylsulfonyl lower alkyl, lower
alkylsulfinyl, lower alkanoyl, aroyl, aryl, aryloxy;
[0050] R.sub.16 is hydrogen, halogen, nitro, cyano, lower alkyl,
OH, perfluorolower alkyl, or lower alkylthio; or
[0051] X is a group of formula X-2 12
[0052] wherein Het is a 5- or 6-membered heteroaromatic ring
containing 1, 2 or 3 heteroatoms selected from N,O, and S, or
[0053] Het is a 9- or 10-membered bicyclic heteroaromatic ring
containing 1, 2, 3 or 4 heteroatoms selected from O, S, and N;
[0054] R.sub.15 and R.sub.16 are as above;
[0055] R.sub.30 is hydrogen or lower alkyl; and p is an integer
from 0 to 1
[0056] or X is a group of formula X-3 13
[0057] wherein:
[0058] R.sub.18 is aryl, heteroaryl, aryl lower alkyl, heteroaryl
lower alkyl
[0059] R.sub.19 is substituted or unsubstituted lower alkyl, aryl,
heteroaryl, arylalkyl, heteroaryl alkyl, and
[0060] R.sub.20 is substituted or unsubstituted lower alkanoyl or
aroyl;
[0061] and Y is a group of formula Y-1 14
[0062] wherein:
[0063] R.sub.22 and R.sub.23 are independently hydrogen, lower
alkyl, lower alkoxy, cycloalkyl, aryl, arylalkyl, nitro, cyano,
lower alkylthio, lower alkylsulfinyl, lower alkyl sulfonyl, lower
alkanoyl, halogen, or perfluorolower alkyl and at least one of
R.sub.22 and R.sub.23 is other than hydrogen, and
[0064] R.sub.24 is hydrogen, lower alkyl, lower alkoxy, aryl,
nitro, cyano, lower alkyl sulfonyl, or halogen; or Y-2 is a group
of the formula: 15
[0065] Het is a five or six membered heteroaromatic ring bonded via
a carbon atom wherein said ring contains one, two or three
heteroatoms selected from the group consisting of N, O and S and
R.sub.30 and R.sub.31 are independently hydrogen, lower alkyl,
cycloalkyl, halogen, cyano, perfluoroalkyl, or aryl and at least
one of R.sub.30 and R.sub.31 is adjacent to the point of
attachment; p is an integer of from 0 to 1;
[0066] or Y is a group of formula Y-3 16
[0067] wherein:
[0068] 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,
[0069] wherein
[0070] R.sub.28 is H or lower alkyl,
[0071] R.sub.29 is hydrogen, lower alkyl, lower alkoxycarbonyl,
lower alkanoyl, aroyl, perfluoro lower alkanoylamino, lower alkyl
sulfonyl, lower alkylaminocarbonyl, arylaminocarbonyl, or R.sub.28
and R.sub.29 taken together form a 4, 5 or 6-membered saturated
carbacyclic ring optionally containing one heteroatom selected from
O, S, and N; with the carbon atoms in the ring being unsubstituted
or substituted by lower alkyl or halogen,
[0072] Q is --(CH.sub.2).sub.fO-, --(CH.sub.2).sub.fS-,
--(CH.sub.2).sub.fN(R.sub.27)-, or --(CH.sub.2).sub.f-,
[0073] R.sub.27 is H, lower alkyl, aryl, lower alkanoyl, aroyl or
lower alkoxycarbonyl,
[0074] e is an integer from 0 to 4, and
[0075] f is an integer from 0 to 3; and the dotted bond is
optionally hydrogenated; and pharmaceutically acceptable salts and
esters thereof.
[0076] Preferred compounds are as follows:
[0077] Compounds where X is a group of the formula 17
[0078] and Y, R.sub.15 and R.sub.16 are as in formula 1.
[0079] Such compounds where R.sub.15 is lower alkyl, nitro,
halogen, perfluoromethyl, or cyano and R.sub.16 is hydrogen, lower
alkyl, nitro, halogen, perfluoromethyl, or cyano, especially where
R.sub.15 and R.sub.16 are independently chloro or fluoro are
preferred, especially where X-1 is selected from the group of
18
[0080] Compounds of formula I wherein X is a group of the formula
X-2 19
[0081] and p, Y, R.sub.15, R.sub.16, and R.sub.30 are as in formula
1 (compound A) especially where Het is 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 or where Het is
a bicyclic heteroaromatic ring containing from 1 to 3 nitrogens or
where R.sub.15 is nitro, lower alkyl sulfonyl, cyano, lower alkyl,
lower alkoxy, perfluorolower alkyl, lower alkylthio, lower
alkanoyl, or aryl, especially where aryl is unsubstituted phenyl.
In compound A R.sub.16 may be hydrogen, halogen, nitro, cyano,
lower alkyl, perfluoro lower alkyl; and R.sub.30 is hydrogen or
lower alkyl, or in compound A Het may be a 6 membered monocyclic
heteroaromatic ring containing 1 or 2 nitrogens or a 10 membered
bicyclic heteroaromatic ring containing one nitrogen, R.sub.15 is
lower alkyl or perfluoroalkyl and R.sub.16 is hydrogen, lower
alkyl, or perfluoroalkyl, and R.sub.30 is absent. In compound A,
X-2 may be selected from the group of 20
[0082] Compounds of formula 1 wherein X is a group of formula X-3
21
[0083] and Y, R.sub.18, R.sub.19, and R.sub.20 are as in formula 1
(compound B). In compound B, it is preferred that R.sub.18 is
phenyl. In compound B it is also preferred that R.sub.19 is lower
alkyl which is unsubstituted or substituted by pyridyl or phenyl.
In compound B it is also preferred that R.sub.20 is substituted or
unsubsituted lower alkanoyl. In compound B it is also preferred
that R.sub.18 is phenyl, R.sub.19 is lower alkyl which is
unsubstituted or substituted by pyridyl or phenyl and R.sub.20 is
lower alkanoyl. In compound B it is preferred that R.sub.18 is
phenyl which is unsubstituted or substituted by halogen or lower
alkoxy; R.sub.19 is phenyl lower alkyl which is unsubstituted or
substituted by lower alkoxy, pyridyl lower alkyl, or lower alkyl;
and R.sub.20 is substituted or unsubstituted lower alkanoyl.
[0084] In this latter compound, it is preferred that X-3 is
selected from the group of 22
[0085] Compounds of formula 1 where Y is a group of formula 23
[0086] and X, R.sub.22, R.sub.23, and R.sub.24 are as in formula 1
(compound C). It is preferred for compound C that R.sub.22 and
R.sub.23 are lower alkyl, trifluoromethyl, or halogen and R.sub.24
is hydrogen, lower alkyl, lower alkoxy, or halogen, especially when
Y-1 is selected from the group of 24
[0087] Compounds of formula 1 wherein Y is a group of the formula
Y-2 25
[0088] and p, X, Het, R.sub.30 and R.sub.31, are as in formula 1
(compound D). It is preferred for compound D that Het is a 6
membered heteroaromatic ring, especially where the heteroatom is N,
and preferably where Y-2 is selected from the group of 26
[0089] Compounds of formula 1 where Y is a group of formula Y-3
27
[0090] and Y, R.sub.25 and Q are as in formula 1, and the dotted
bond can be optionally hydrogenated (compound E). It is preferred
for compound E that Y-3 is selected from the group of 28
[0091] A compound of formula 1 wherein X is a group of the formula
X-1 and Y is a group of the formula Y-1.
[0092] A compound of formula I wherein X is a group of the formula
X-1 and Y is a group of the 29
[0093] formula Y-2.
[0094] A compound of formula 1 wherein X is a group of the formula
X-1 Y is a group of the formula Y-3 wherein R.sub.15, R.sub.16,
R.sub.25 and Q are as above; and the dotted bond can be optionally
hydrogenated.
[0095] A compound of formula 1 wherein X is a group of the formula
X-2 and Y is a group of the formula Y- 1.
[0096] A compound of formula 1 wherein X is a group of the formula
X-2 and Y is a group of the formula Y-2
[0097] A compound of formula 1 wherein X is a group of the formula
X-2 and Y is a group of the 30
[0098] formula Y-3 wherein R.sub.15, R.sub.16, R.sub.25, R.sub.30,
Q and p are as above and the dotted bond can be optionally
hydrogenated.
[0099] A compound of formula I where X is a group of the formula
X-3 and Y is a group of the formula Y-1.
[0100] A compound of formula I wherein X is a group of the formula
X-3 and Y is a group of the formula Y-2.
[0101] A compound of formula 1 wherein X is a group of the formula
X-3 and Y is a group of the formula Y-3 where R.sub.18, R.sub.19,
R.sub.20, R.sub.25, and Q are as above and the dotted bond can be
optionally hydrogenated.
[0102] A compound of claim 1 wherein X is a group of the formula
X-1 31
[0103] wherein R.sub.16 is in the ortho position and is hydrogen,
lower alkyl, nitro, cyano, halogen, lower alkylthio,
perfluoroloweralkyl and R.sub.15 is lower alkyl, nitro, cyano,
halogen, lower alkylsulfonyl, perfluoroloweralkyl, and Y is a group
of the formula Y-1 32
[0104] where R.sub.22 is hydrogen, halogen, trifluoroalkyl, or
lower alkyl and R.sub.23 is halogen, trifluoroalkyl, or lower
alkyl, and R24 is hydrogen or Y is a group of the formula Y-3
33
[0105] wherein Q is as above and the dotted bond can be optionally
hydrogenated; R.sub.25 is R.sub.26--(CH.sub.2)e-; e is 2-4 and
R.sub.26 is azido, cyano, hydroxy, lower alkoxy, lower
alkoxycarbonyl, lower alkanoyl, lower alkyl sulfonyl, lower alkyl
sulfinyl, perfluoro lower alkanoyl, nitro, or lower alkylthio or
R.sub.25 is NHR.sub.29 where R.sub.29 is lower alkanoyl or lower
alkylamino carbonyl (compound E).
[0106] In compound E it is preferred that X is a group of the
formula X-1 34
[0107] wherein R.sub.16 is in the ortho position and is hydrogen,
lower alkyl, nitro, cyano, halogen, lower alkylthio,
perfluoroloweralkyl and R.sub.15 is lower alkyl, nitro, cyano,
halogen, lower alkylsulfonyl, perfluoroloweralkyl; and Y is a group
of the formula Y-1 35
[0108] where R.sub.22 is hydrogen, halogen, or lower alkyl and
R.sub.23 is halogen or lower alkyl, and R24 is hydrogen, especially
where ,.sub.16 is hydrogen or halogen and R.sub.15 is halogen; R22
is hydrogen, halogen, ethyl, or methyl and R.sub.23 is halogen,
ethyl, or methyl, and additionally where R.sub.16 is in the ortho
position and R.sub.15 and R.sub.16 are both chlorine, and R.sub.22
is methyl and R.sub.23 is chlorine or ethyl. An example of such a
compound is
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-chloro-6-methylphenyl)thiox-
omethyl]-L-phenylalanine.
[0109] In compound E it is also preferred that X is a group of the
formula X-1 36
[0110] wherein R.sub.16 is in the ortho position and is hydrogen,
lower alkyl, nitro, cyano, halogen, lower alkylthio,
perfluoroloweralkyl and R.sub.15 is lower alkyl, nitro, cyano,
halogen, lower alkylsulfonyl, perfluoroloweralkyl; and Y is a group
of the formula Y-3 37
[0111] which is a four to six membered cycloalkyl ring, R.sub.25 is
R.sub.26-(CH.sub.2)e-; e is 2-4 and R.sub.26 is azido, cyano,
hydroxy, lower alkoxy, lower alkoxycarbonyl, lower alkanoyl, lower
alkyl sulfonyl, lower alkyl sulfinyl, perfluoro lower alkanoyl,
nitro, or lower alkylthio; and the dotted bond is hydrogenated. In
such a compound it is preferred that R.sub.16 is hydrogen or
halogen and R.sub.15 is halogen; and Y-3 is a four or five membered
ring and R.sub.26 is lower alkoxy, lower alkyl sulfonyl, lower
alkyl sulfinyl, or lower alkylthio, especially where R.sub.16 is in
the ortho position and R.sub.15 and R.sub.16 are both chlorine, and
R.sub.26 is lower alkyl sulfonyl or lower alkylthio. An example of
such a compound is 4-[[(2,6-dichlorophenyl)carbo-
nyl]amino]-N-[[1-[(4-methylsulfonyl)butyl]
cyclopentyl]thioxomethyl]-L-phe- nylalanine.
[0112] For another preferred compound E X is a group of the formula
X- 1 38
[0113] where R.sub.16 is hydrogen or halogen and R.sub.15 is
halogen and Y is a group of the formula Y-1 39
[0114] where R.sub.22 is hydrogen, halogen, ethyl, or methyl and
R.sub.23 is halogen, ethyl, or methyl and R.sub.24 is hydrogen or Y
is a group of the formula Y-3 40
[0115] where Y-3 is a four or five membered ring, R.sub.25 is as in
claim 39 and R.sub.26 is lower alkoxy, lower alkyl sulfonyl, lower
alkyl sulfinyl, or lower alkylthio, and the dotted bond is
optionally hydrogenated. For such a compound it is preferred that
R.sub.16 is in the ortho position and R.sub.15 and R.sub.16 are
both chlorine, and when Y is Y-1 then R.sub.22 is methyl and
R.sub.23 is chlorine or ethyl and when Y is Y-3, Y-3 is a four or
five membered ring and R.sub.26 is lower alkyl sulfonyl or lower
alkylthio.
[0116] A compound of formula 1 wherein Y is as in formula 1 and X
is X-1 41
[0117] where R.sub.15 is ortho and is halogen, lower alkyl, or
perfluoroalkyl and R.sub.16 is hydrogen, halogen, lower alkyl, or
perfluoroalkyl (compound F).
[0118] For compound F it is preferred that R.sub.15 is chlorine and
R.sub.16 is hydrogen or chlorine.
[0119] A compound of formula 1 wherein Y is as in formula 1 and X
is X-2 where Het is pyridine or pyrimidine and R.sub.15 is lower
alkyl or perfluoroalkyl R.sub.16, and R.sub.20 are hydrogen, lower
alkyl, or perfluoroalkyl (compound G).
[0120] A compound of formula 1 wherein Y is as in formula 1 and X
is X-3 where R.sub.19 is pyridinyl lower alkyl or phenyl lower
alkyl, R.sub.20 is lower alkanoyl, and R18 is phenyl (compound A
compound of formula 1 where X is as in formula 1 and Y is Y-1 where
R.sub.22 is hydrogen or lower alkyl, R.sub.23 is halogen, lower
alkyl, or perfluoroalkyl, and R24 is hydrogen, especially where
R.sub.22 is hydrogen or methyl and R.sub.23 is halogen, ethyl, or
trifluoromethyl.
[0121] A compound of formula 1 wherein X is as in formula 1 and Y
is Y-3 which is a four to six membered cycloalkyl ring, R.sub.25 is
R.sub.26-(CH.sub.2)e-, e is 2-4, and R.sub.26 is alkoxy, lower
alkyl sulfonyl, loweralkylthio, or NHR.sub.29 where R.sub.29 is
loweralkoxycarbonyl or loweralkylaminocarbonyl, and the dotted bond
is hydrogenated. It is preferred that R.sub.26 is methoxy, methyl
sulfonyl, or methylthio.
[0122] A preferred compound F has Y is Y- 1 where R.sub.22 is
hydrogen or lower alkyl, R.sub.23 is halogen, lower alkyl, or
perfluoroalkyl, and R.sub.24 is hydrogen. It is preferred that
R.sub.15 is chlorine and R.sub.16 is hydrogen or chlorine. Examples
are
[0123] 4-[[(2
,6-dichlorophenyl)carbonyl]amino]-N-[(2-bromophenyl)thioxome-
thyl]-L-phenylalanine;
[0124]
4-[[(2,6,-dichlorophenyl)carbonylamino]-N-[(2-ethyl-6-methylphenyl)-
thioxomethyl]L-phenylalanine.
[0125]
4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[(2-fluorophenyl)thioxom-
ethyl]-L-phenylalanine.
[0126]
4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[[2-(trifluoromethyl)phe-
nyl]thioxomethyl]-L-phenylalanine.
[0127] In a preferred compound G, Y is Y-1 where R.sub.22 is
hydrogen or lower alkyl, R.sub.23 is halogen, lower alkyl, or
perfluoroalkyl, and R.sub.24 is hydrogen. In a preferred compound
H, Y is Y-1 where R.sub.22 is hydrogen or lower alkyl, R.sub.23 is
halogen, lower alkyl, or perfluoroalkyl, and R.sub.24 is hydrogen.
An example of such a compound is 4-[(2S,4R)-3-acetyl-2-phenyl-4-
[(3-pyridinyl)methyl]-5-oxo-imidazolid- in- 1
-yl]-N-[(2-ethyl-6-methylphenyl) thioxomethyl]-L-phenylalanine.
[0128] In a preferred compound F, Y is Y-3 which is a four to six
membered cycloalkyl ring, R.sub.25 42
[0129] is R.sub.26--(CH.sub.2)e-, e is 2-4, and R.sub.26 is alkoxy,
lower alkyl sulfonyl, loweralkylthio, or NHR.sub.29 where R.sub.29
is loweralkoxycarbonyl or loweralkylaminocarbonyl, and the dotted
bond is hydrogenated. It is preferred that R.sub.15 is chlorine and
R,.sub.6 is hydrogen or chlorine. Examples of such compounds
are
[0130] 4-[[2,6-dichlorophenyl)carbonyl]amino-N-[[1
-[2-(acetylamino)ethyl]-
cyclopentyl]thioxomethyl]-L-phenylalanine.
[0131]
[[1-[2-[[(methylamino)carbonyl]amino]ethyl]cyclopentyl]thioxomethyl-
]-4-[[(2,6-dichlorophenyl)carbonyl]amino]L-phenylalanine.
[0132] 4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[[1
-(2-methoxyethyl)cyclopentyl]thioxomethyl]-L-phenylalanine.
[0133] 4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[[1
-[(4-methylsulfonyl)butyl]cyclobutyl]
thioxomethyl]-L-phenylalanine.
[0134] 4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[[1
-(3-methylthio)propyl]cyclobutyl]thioxomethyl]-L-phenylalanine.
[0135] 4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[[1
-(3-methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-L-phenylalanine.
[0136] It is preferred that R26 is methoxy, methyl sulfonyl, or
methyl thio, especially where R.sub.15 is chlorine and R.sub.16 is
hydrogen or chlorine.
[0137] A preferred compound G wherein Y is Y-3 which is a four to
six membered cycloalkyl ring, R.sub.25 is R.sub.26--(CH.sub.2)e-, e
is 2-4, and R.sub.26 is alkoxy, lower alkyl sulfonyl,
loweralkylthio, or NHR.sub.29 where R.sub.29 is loweralkoxycarbonyl
or loweralkylaminocarbonyl, and the dotted bond is hydrogenated.
Preferably R.sub.26 is methoxy, methyl sulfonyl, or methyl thio.
Examples are
[0138] 4- [(2,6-dimethyl-3-pyrimidinyl]carbonyl)amino]-N- [[1
[(4-methylsulfonyl)butyl]cyclopentyl]
thioxomethyl]-L-phenylalanine.
[0139] 4-[[[4-(trifluoromethyl)-5-pyrimidinyl]carbonyl]amino]-N-[[1
-(4-methylsulfonyl)butyl]
cyclobutyl]thioxomethyl]-L-phenylalanine.
[0140]
4-[[(2,4-dimethyl-6-trifluoromethyl-3-pyridinyl)carbonyl]amino]-N-[-
[1
-[(4-methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine.
[0141] In a preferred compound H, Y is Y-3 which is a four to six
membered cycloalkyl ring, R.sub.25 43
[0142] is R.sub.26--(CH.sub.2)e-, e is 2-4, and R.sub.26 is alkoxy,
lower alkyl sulfonyl, loweralkylthio, or NHR.sub.29 where R.sub.29
is loweralkoxycarbonyl or loweralkylaminocarbonyl, and the dotted
bond is optionally hydrogenated, especially where R.sub.26 is
methoxy, methyl sulfonyl, or methyl thio. Examples are
4-[(2S,4R)-3-acetyl-2-phenyl-4-[(3-
-phenyl)methyl]-5-oxo-imidazolidin-1-yl]-N-[[(4-methylsulfonyl)butyl]cyclo-
pentyl]thioxomethyl]-L-phenylalanine
[0143]
4-[(2R,4R)-3-acetyl-2-phenyl-4-[(3-phenyl)methyl]-5-oxo-imidazolidi-
n-1-yl]-N-[[(4-methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalan-
ine.
[0144] As used in this specification, the terms are defined as
follows:
[0145] The term "halogen" means bromine, chlorine, fluorine, or
iodine, and the term "halo" means a halogen substituent.
[0146] The term "perfluoro" means complete substitution of all
hydrogen atoms with fluoro substituted, as in perfluoro lower
alkyl, perfluoroloweralkanoyl and perfluoroalkanoylamino. An
example is trifluoromethyl.
[0147] 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 (preferably phenyl or pyridyl), hydroxy (lower alkylhydroxy or
hydroxylower alkyl), halogen, cyano, lower alkoxy (alkoxy lower
alkyl or lower alkyl alkoxy), lower alkanoyl, lower alkylthio
(lower alkylthio lower alkyl) sulfinyl (lower alkyl sulfinyl),
sulfinyl lower alkyl (lower alkyl sulfinyl lower alkyl) sulfonyl
(lower alkyl sulfonyl), sulfonyl lower alkyl (lower alkyl sulfonyl
lower alkyl) perfluoro (perfluoro lower alkyl) and substituted
amino such as aminosulfonyl (lower alkyl aminosulfonyl) or
aminocarbonyl (lower alkyl aminocarbonyl). Examples of substituted
lower alkyl groups include 2-hydroxylethyl, 3-oxobutyl,
cyanomethyl, and 2-nitropropyl. 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.
[0148] The term "cycloalkyl" means an unsubstituted or substituted
3- to 7-membered carbacyclic ring. Substituents 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.
[0149] The term "lower alkoxy" means a lower alkyl group as defined
above, bonded through an oxygen atom. Examples are methoxy, ethoxy,
n-propoxy, isopropoxy, n-butoxy, tert-butoxy and the like.
[0150] The term "lower alkenyl" means a nonaromatic partially
unsaturated hydrocarbon chain containing at least one double bond,
which is preferably 1-10 and more preferably 1-6 carbons in length.
The group may be unsubstituted, or substituted with conventional
substituents, preferably fluoro. Examples are vinyl, allyl,
dimethylallyl, butenyl, isobutenyl, pentenyl.
[0151] 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 substituents are
lower alkyl, lower alkoxy, hydroxy lower alkyl, hydroxy,
hydroxyalkoxy, halogen, lower alkylthio, lower alkylsulfinyl, lower
alkylsulfonyl, cyano, nitro, perfluoroalkyl, alkanoyl, aroyl, aryl
alkynyl, lower alkynyl, aminoalkylcarbonyl (arylaminocarbonyl) and
lower alkanoylamino. The especially preferred substituents are
lower alkyl, hydroxy, and perfluoro lower alkyl. Examples of aryl
groups that may be used in accordance with this invention are
phenyl, p-tolyl, p-methoxyphenyl, p-chlorophenyl, m-hydroxy phenyl,
m-methylthiophenyl, 2-methyl-5-nitrophenyl, 2,6-dichlorophenyl, 1
-naphthyl and the like.
[0152] The term "arylalkyl" means a lower alkyl group as
hereinbefore defined in which one or more hydrogen atoms is/are
replaced by an aryl group as herein defined. Any conventional
aralkyl may be used in accordance with this invention, such as
benzyl and the like. Similarly, the term "heteroarylalkyl" is the
same as an arylalkyl group except that there is a heteroaryl group
as defined below in place of an aryl group. Either of these groups
may be unsubstituted, or may be substituted on the ring portion
with conventional substituents such as
[0153] 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 0. Examples of
hetereoaryl rings are pyridine, benzimidazole, indole, imidazole,
thiophene, isoquinoline, quinzoline and the like. Substituents as
defined above for "aryl" apply equally here in the definition of
heteroaryl. The term "heteroaromatic ring" may be used
interchangeably with the term heteroaryl.
[0154] The term "lower alkoxycarbonyl" means a lower alkoxy group
bonded via a carbonyl group. Examples of alkoxycarbonyl groups are
ethoxycarbonyl and the like.
[0155] The term "lower alkylcarbonyloxy" means lower
alkylcarbonyloxy groups bonded via an oxygen atom, for example an
acetoxy group.
[0156] 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. Lower
alkanoyl groups may be unsubstituted, or substituted with
conventional substituents such as alkoxy, lower alkyl, hydroxy,
aryl, and hetereoaryl.
[0157] The term "lower alkylcarbonylamino" means lower
alkylcarbonyl groups bonded via a nitrogen atom, such as
acetylamino.
[0158] 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, 2-naphthyl and the like. Aroyl
groups may be unsubstituted, or substituted with conventional
substituents such as
[0159] The term "aryloxy" means an aryl group, as hereinbefore
defined, which is bonded via an oxygen atom. The preferred aryloxy
group is phenoxy.
[0160] The term "electron-deficient substituent" means a
substituent on an aromatic or heteroaromatic ring which has a
positive Hammett sigma valus as defined for example in Jerry March,
Advanced Organic Chemistry, 2.sup.nd Edition, McGraw Hill, 1977,
page 246-253. Typical electron withdrawing groups are cyano, nitro,
chloro, alkoxycarbonyl lower alkyl sulfonyl, and aminocarbonyl.
[0161] In the compound of formula 1, Y is preferably the group Y-1
whereby the invention comprises a compound of the formula: 44
[0162] wherein X, R.sub.22, R.sub.23 and R.sub.24 are as above.
[0163] In the group Y-1, R.sub.22 and R.sub.23 are preferably lower
alkyl or halogen and R24 is preferably hydrogen.
[0164] Among the groups Y-1, when R.sub.23 is lower-alkyl or
halogen, Y-1 is preferably: 45
[0165] When Y is a group Y-2, Y is preferably: 46
[0166] When Y is a group Y-3, Y is preferably:
1 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68
69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87
[0167] The especially preferred groups X-1 are of the formula:
88
[0168] The especially preferred groups X-2 are of the formula:
89
[0169] The especially preferred groups X-3 are of the formula:
90
[0170] The compounds of the invention can exist as stereoisomers
and diastereomers, all of which are encompassed within the scope of
the present invention.
[0171] 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 endotheliumn which
contributes to the cause of the pulmonary inflammation which occurs
in asthma. Thus, the compounds of the present invention would be
useful for the treatment of asthma.
[0172] On the basis of their capability of inhibiting binding of
VCAM- 1 and fibronectin to VLA-4 on circulating lymphocytes,
eosinophils, basophils, and monocytes, the compounds of the
invention can be used as medicament for the treatment of disorders
which are known to be associated with such binding. Examples of
such disorders are rheumatoid arthritis, multiple sclerosis,
asthma, and inflammatory bowel disease. The compounds of the
invention are preferably used in the treatment of diseases which
involve pulmonary inflammation, such as asthma. The pulmonary
inflammation, which occurs in asthma, is related to eosinophil
infiltration into the lungs wherein the eosinophils bind to
endothelium which has been activated by some asthma-triggering
event or substance.
[0173] 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, eosinophils
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. 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.
[0174] 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 in the case 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.
[0175] Intravenous, intramuscular, oral or inhalation
administration is a preferred form of use. The dosages in which the
compounds of the invention are administered in effective amounts
depending 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 of 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-l 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 especially
preferred.
[0176] 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,
flavor-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.
[0177] The previously mentioned carrier materials and diluents can
comprise any conventional pharmaceutically acceptable organic or
inorganic substances, e.g., water, gelatin, lactose, starch,
magnesium stearate, talc, gum arabic, polyalkylene glycols and the
like.
[0178] Oral unit dosage forms, such as tablets and capsules,
preferably contain from 25 mg to 1000 mg of a compound of the
invention.
[0179] The compounds of the present invention may be prepared by
any conventional means. In reaction Scheme 1, a
4-nitro-L-phenylalanine derivative of formula 1 in which R.sub.1 is
lower alkyl, which is a known compound or readily prepared by
conventional means, is acylated with a benzoic acid derivative of
formula 2 in which R.sub.2 hydrogen, lower alkyl, lower alkoxy,
cycloalkyl, aryl, arylalkyl, nitro, cyano, lower alkylthio, lower
alkylsulfinyl, lower alkyl sulfonyl, lower alkanoyl, halogen, or
perfluorolower alkyl, R.sub.3 is hydrogen, halogen or lower alkyl
and R.sub.4 is hydrogen, lower alkyl, lower alkoxy, aryl, nitro,
cyano, lower alkyl sulfonyl, or halogen, using conventional means
for amide bond formation. For example, a compound of formula 2 may
be converted to the corresponding acid chloride and condensed with
a compound of formula 1 in the presence of a proton acceptor such
as a tertiary alkylamine. Alternatively compound 1 can be coupled
with a carboxylic acid of formula 2 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 3.
[0180] Conversion of the compound of formula 3 to the corresponding
thioamide of formula 4 can be carried out by treatment with
Lawesson's reagent which is
[2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2-
,4-disulfide]. The procedure is standard and has been described in
detail. See for example, Scheibey, S., Pedersen, B. S., Lawesson,
S. -O. Bull Soc. Chim. Belg. 1978 87, 229 and Cava, M. P.,
Levinson, M. I., Tetrahedron 1985, 41, 5061. The nitro group of the
compound of formula 4 may be reduced to the corresponding amine by
any of the conventional means which are compatible with thioamides.
One convenient procedure employs zinc dust as the reducing agent in
the presence of methanol, ammonium chloride and water at a
temperature of from 35 to 60.degree. C. to give a compound of
formula 5. Acylation of this compound with an aryl- or heteroaryl
carboxylic acid of formula 6 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 gives a compound of formula 7. In certain
cases, for example with hindered carboxylic acids 6, it may be
advantageous to form the corresponding acid halide and react it
with the amine of formula 5, typically in the presence of a slight
excess of a base such as a tertiary amine or
4-(dimethylamino)pyridine. The carboxylic acid of formula 6 may be
substituted by 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. Where appropriate, it may also
incorporate suitably protected reactive functionalities which must
be removed 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. Guidance for the selection and use of
protecting groups is provided in standard reference works, for
example: "T. W. Green and P. G. M. Wuts, Protective Groups in
Organic Synthesis, 2.sup.nd edition, Wiley Interscience, New York,
1991. The ester moiety of compound 7 can generally be cleaved to
the corresponding carboxylic acid by treatment with an alkali metal
hdyroxide, for example, lithium hydroxide in aqueous methanol at a
temperature of from room temperature to 50.degree. C. Depending on
the nature of R.sub.1, alternative procedures may be preferred. The
choice of conditions for ester cleavage in the presence of
functionalities such as thioamides is well known to those skilled
in the art. 91
[0181] Ortho-substituted benzoic acid derivatives which are not
commercially available can be prepared by conventional means. For
example ortho-substituted aryl iodides or triflates may be
carbonylated in the presence of carbon monoxide and a suitable
palladium catalyst. The preparation of such iodide or triflate
intermediates is dependent on the particular substitution pattern
desired and they may be obtained by direct iodination or
diazotization of an aniline followed by treatment with a source of
iodide for example, potassium iodide. Triflates may be derived from
the corresponding phenols by conventional means such as treatment
with trifluoromethane sulfonic anhydride in the presence of a base
such as triethylamine or diisopropylethylamine in an inert solvent.
Other means of obtaining ortho-substituted benzoic acids involves
treatment of an 2-methoxyphenyloxazoline derivative such as 9 with
an alkyl Grignard reagent followed by hydrolysis of the oxazoline
ring following the general procedure described by Meyers, A. I.,
Gabel, R., Mihelick, E. D, J. Org. Chem. 1978, 43, 1372-1379., to
give an acid of formula 10. 2- or 2,6-Disubstituted benzonitriles
also serve as convenient precursors to the corresponsing benzoic
acids. In the case of highly hindered nitrites, for example
2-chloro-6-methylbenzonitrile, conventional hydrolysis under acidic
or basic conditions is difficult and better results are obtained by
DIBAL reduction to the corresponding benzaldehyde followed by
oxidation using a sodium chlorite/hydroperoxide oxidizing reagent.
92
[0182] Employing essentially the same procedures described in
Scheme 1, utilizing a heteroaromatic carboxylic acid in place of 2,
one can prepare compounds of formula 11. 93
[0183] For the synthesis of analogues a branched chain or
cycloalkyl moiety, a similar procedure to that described in scheme
1 can be employed starting with the appropriate branched chain or
cycloalkyl carboxylic acid of formula 12. In this case, R.sub.6
represents is lower alkyl, unsubstituted or fluorine substituted
lower alkenyl, or a substituted lower alkyl group wherein the
substituents may be chosen from aryl, heteroaryl, azido, cyano,
hydroxy, lower alkoxy, lower alkoxycarbonyl, lower alkylthio, lower
alkyl sulfonyl, perfluoro lower alkanoyl, nitro, or a protected
amino group. The amine protecting group must be chosen to be
compatible with the reagents needed to convert carboxamides to
thioamides. Carbamates, for example, the tert-butoxycarbonyl moiety
are suitable. As appropriate, these protecting groups may be
removed by conventional means later in the synthesis and the
resulting free amine can be further functionalized utilizing
standard methods. For example, the amine can be acylated by
treatment with the appropriate anhydride, isocyanate or acid
halide. 94
[0184] The synthesis of imidazolidinones of formula 21 is described
in reaction scheme 3. An aminophenylalanine derivative of structure
13 in which R.sub.6 is aryl, heteroaryl, branched chain alkyl or
derived from a compound of formula 12, and R.sub.7 is lower alkyl,
is coupled with a N-protected alpha-amino acid of formula 14, in
which R.sub.8 can be a natural or unnatural, D- or L-.alpha.-amino
acid side chain and R.sub.9 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 15. Depending on the nature of protecting group
R.sub.9, an appropriate deprotection method is employed to give
compound of formula 16. In the case of the protecting group R.sub.9
is Fmoc group, it may be removed from 15 using standard base
treatment well known to those practicing peptide chemistry, for
example with piperidine in DMF, to afford an amine of formula 16.
The compound 16 can then react with an aldehyde 17, in which
R.sub.10 is lower alkyl, aryl, or aryl lower alkyl, in the presence
of a water scavenger such as 4 .ANG. molecular sieves in an
appropriate solvent such as dichloromethane or THF at 25-60.degree.
C. to give an imine of formula 18. The imine 18 may then be treated
with an acylating agent such as the acyl chloride of formula 19 in
which R.sub.11 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 25-60.degree. C. to give an acyl
imidazolidinone of formula 20. Alternatively, other reactive
acylating groups such as acid anhydrides or mixed anhydrides may be
employed in this reaction. Compound 20 may be converted to a
compound of the invention by an appropriate hydrolysis procedure,
for example 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 21. 95
[0185] 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
(El, 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 12 vapor, or by spraying
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.
[0186] 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 JIM C-18 column at a flow of 40
mL/min employing a gradient of acetonitrile:water (each containing
0.75% TFA) typically from 5 to 95% acetonitrile over 35-40 min or a
Rainin HPLC employing a 41.4.times.300 mm, 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. HPLC conditions are typically
described in the format (5-95-35-214); this refers to a linear
gradient of from 5% to 95% acetonitrile in water over 35 min while
monitoring the effluent with a UV detector set to a wavelength of
214 nM.
[0187] Methylene chloride (dichloromethane), 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.
[0188] Definitions:
[0189] THF is tetrahydrofuran,
[0190] DMF is N,N-dimethylformamide,
[0191] HOBT is 1-hydroxybenzotriazole,
[0192] BOP is
[(benzotriazole-1-yl)oxy]tris-(dimethylamino)phosphonium
hexafluorophosphate,
[0193] HATU is
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
[0194] HBTU is O-benzotriazole-N,N,N',N',-tetramethyluronium
hexafluorophosphate,
[0195] DIPEA is diisopropylethylamine,
[0196] DMAP is 4-(N,N-dimethylamino)pyridine
[0197] DPPA is diphenylphosphoryl azide
[0198] DPPP is 1,3-bis(diphenylphosphino)propane
[0199] DBU is 1,8-diazabicyclo[5.4.0]undec-7-ene
[0200] NaH is sodium hydride
[0201] brine is saturated aqueous sodium chloride solution
[0202] TLC is thin layer chromatography
[0203] LDA is lithium diisopropylamide
[0204] BOP-Cl is bis(2-oxo-3-oxazolidinyl)phosphinic chloride
[0205] NMP is N-methyl pyrrolidinone
[0206] Lawesson's reagent is [2,4-bis(4-methoxyphenyl)-
1,3-dithia-2,4-diphosphetane-2,4-disulfide]
EXAMPLES
Example 1.
N-[[1-(2-methoxyethyl)cyclopentyl]thioxomethyl]-4-nitro-L-pheny-
lalanine methyl ester.
[0207] 96
[0208] To a solution of
[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-4-nitro-- L-phenylalanine
methyl ester (4.30 g, 11.4 mmol) in toluene (20 mL) was added
Lawesson's reagent (2.60 g, 6.27 mmol). The resultant mixture was
warmed to 50.degree. C. and stirred for 18 h. The reaction mixture
was filtered through a sintered glass funnel and the filtrate was
concentrated in vacuo. The residue was by flash column
chromatography over silica gel (hexane-ethyl acetate, 9:1 then 8:
1) to afford N-[[1 -(2-methoxyethyl)cyclopentyl]thioxomethyl]
-4-nitro-L-phenylalanine methyl ester (2.44 g, 54%; 70% based on
recovered starting material) as a light yellow oil. HR MS: Obs.
mass, 395.1639. Calcd. mass, 395.1640 (M+H).
Example 2.
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-(methoxyethyl)-
-cyclopentyl]thioxomethyl]-L-phenylalanine methyl ester.
[0209] 97
[0210] To a suspension of
N-[[1-(2-methoxyethyl)cyclopentyl]thioxomethyl]--
4-nitro-L-phenylalanine methyl ester (4.58 g, 11.6 mmol), zinc dust
(7.50 g, 116 mmol) and ammonium chloride (9.20 g, 174 mmol) in
methanol (200 mL) was added H.sub.2O (100 mL) slowly over 5 min.
After stirring for 20 min, the reaction mixture was partitioned
between ethyl acetate (400 mL) and sat. ammonium chloride solution
(150 mL).The separated aqueous layer was back-extracted with ethyl
acetate (3.times.100 mL) and the organic layers were combined,
dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo.
The residual oil was dried under high vacuum for 2 h to give crude
4-amino-N-[[1-(2-methoxyethyl)cyclopentyl]thioxomethyl]-L-
-phenylalanine methyl ester (4.5 g).
[0211] To a solution of the crude amine obtained above (3.40 g,
.about.8.77 mmol based on 94% purity) and diisopropylethylamine
(1.70 mL, 9.65 mmol) in dichloromethane (15 mL) was added a
solution of 2,6-dichlorobenzoyl chloride (1.9 g, 9.21 mmol) in
dichloromethane (5 mL). The resultant mixture was stirred
overnight, then was concentrated in vacuo and transferred to a
separatory funnel containing ethyl acetate (150 mL) and water (40
mL). The aqueous layer was separated and back extracted with ethyl
acetate (1.times.50 mL). The combined organic layer was washed with
a sat. solution of Na.sub.2CO.sub.3 followed by brine, dried over
MgSO.sub.4, filtered and concentrated in vacuo. The reaction
product by silica gel flash column chromatography (hexane-ethyl
acetate, 3:1) to give
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-methoxyethy-
l)cyclopentyl]thioxomethyl]-L-phenylalanine methyl ester (4.50 g,
95%). HR MS: Obs. mass, 559.1201. Calcd. mass, 559.1201 (M+Na).
Example 3.
[0212]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-methoxyethyl)cyclo-
pentyl]thioxomethyl]-L-phenylalanine. 98
[0213] To a solution of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-m- ethoxyethyl)
cyclopentyl]thioxomethyl]-L-phenylalanine methyl ester (4.00 g,
7.44 mmol) in methanol (18 mL) was added a solution of NaOH (421
mg, 10.5 mmol) in water (3 mL). The mixture was stirred for 2 h and
then acidified (pH .about.1-2) with 0.5N HCl. The reaction mixture
was poured into a separatory funnel containing ethyl acetate (150
mL) and water (25 mL). The separated aqueous layer was
back-extracted with ethyl acetate (2.times.50 mL). The combined
organic layers was washed with brine, dried over MgSO.sub.4,
filtered and concentrated in vacuo. Purification of the residual
material by RP-HPLC (15-95% acetonitrile-water gradient over 25
min) provided 4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1
-(2-methoxyethyl)cyclopentyl]thioxomethyl]-L-phenylalanine (3.05 g,
78%). HR MS: Obs. mass, 545.1043. Calcd. mass, 545.1045 (M+Na).
Example 4.
[0214] 1-(2-azidoethyl)cyclopentanecarboxylic acid. 99
[0215] 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 cyclopentanecarboxylate (37.4 g, 0.263
mol) in THF (50 mL) was added over 20 min. After 1 h, 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 h and then was allowed to
warm to room temperature overnight. After 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, concentrated in vacuo then the residue was
diluted with toluene (100 mL) and concentrated. The dilution and
concentration cycle was repeated twice to give ethyl
1-(2-bromoethyl)cyclopentanecarboxylate (52.5 g).
[0216] 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 h 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- ecarboxylate (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
(5% ethyl acetate in hexane) to give 50.3 g of product as a light
brown oil.
[0217] The oil from above (50.3 g, 0.238 mol) was dissolved in THF
(750 mL) and methanol (375 mL) a 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 treated
with 1 N HCl (375 mL) and was extracted with diethyl ether
(2.times.1 L). The combined extracts were dried (Na.sub.2SO.sub.4),
filtered and concentrated under reduced pressure to give
1-(2-azidoethyl)cyclopentane carboxylic acid (37.5 g) as an amber
liquid.
Example 5.
[0218]
N-[[1-(2-azidoethyl)cyclopentyl]carbonyl]-4-nitro-L-phenylalanine
methyl ester. 100
[0219] A solution of 4-nitro-L-phenylalanine methyl ester
hydrochloride (3.0 g, 11.5 mmol), 1-(2-azidoethyl)cyclopentane
carboxylic acid (2.3 g, 12.7 mmol) and BOP (5.34 g, 12.1 mmol) in
dichloromethane (6 mL) and DMF (4 mL) was treated with
diisopropylethylamine (4.2 mL, 24.2 mmol). The mixture was stirred
overnight at which time TLC (1:1 hexane:ethyl acetate) indicated no
more starting material. The mixture was diluted with water,
extracted with ethyl acetate. The extracts were washed with water
and brine, then were dried (Na.sub.2SO.sub.4), filtered and
evaporated in vacuo. The residue was purified by chromatography
over silica gel (3:1 hexane:ethyl acetate) to afford 4.26 g of
N-[[1-(2-azidoethyl)cyclopentyl]carbonyl]-4-nitro-L-phenylalanine
methyl ester.
Example 6.
[0220]
N-[[1-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl]cyclopentyl]car-
bonyl]-4-nitro-L-phenylalanine methyl ester. 101
[0221] a. A solution of
N-[[1-(2-azidoethyl)cyclopentyl]carbonyl]-4-nitro-- L-phenylalanine
methyl ester (1.92 g, 4.93 mmol) in THF (20 mL) was treated
dropwise with a 1 M solution of trimethylphosphine in THF. After
the addition was complete, the mixture was stirred for 20 min and
water (0.17 mL) was added. The reaction was stirred a further 2 h,
then a little trifluoroacetic acid was added and the mixture was
dried over sodium sulfate and concentrated. 102
[0222] b. To a solution of
N-[[1-(2-aminoethyl)cyclopentyl]carbonyl]-4-nit- ro-L-phenylalanine
methyl ester trifluoroacetic acid salt (2.35 g, 4.93 mmol) in
dioxane (25 mL) was added diisopropylethylamine (0.860 mL, 4.93
mmol) and di-tert-butyl dicarbonate (1.08 g, 4.93 mmol). The
resultant mixture was stirred for 18 h. The reaction mixture was
filtered through a sintered glass funnel and the filtrate was
concentrated in vacuo. Purification of the residual material by
silica gel flash column chromatography (hexane-ethyl acetate 3:1)
afforded
N-[[1-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl]cyclopentyl]carbonyl]-
-4-nitro-L-phenylalanine methyl ester (2.20 g, 95%). HR MS: Obs.
mass, 464.2397. Calcd. mass, 464.2397 (M+H).
Example 7.
[0223]
N-[[1-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl]cyclopentyl]thi-
oxomethyl]-4-nitro-L-phenylalanine methyl ester. 103
[0224] To a solution of
N-[[1-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethy-
l]cyclopentyl]carbonyl]-4-nitro-L-phenylalanine methyl ester (1.00
g, 2.16 mmol) in toluene/dioxane (1:1, 10 mL) was added Lawesson's
reagent (0.524 g, 1.29 mmol). The resultant mixture was warmed to
50 .degree. C and was stirred for 24 h. The reaction mixture was
filtered through a sintered glass funnel and the filtrate was
concentrated in vacuo. Purification of the crude product by silica
gel flash column chromatography (hexane-ethyl acetate, 6:1 then
4:1), afforded N-[[1-[2-[[(1,1-dimethylethoxy)carbonyl]-
amino]ethyl]cyclopentyl]thioxomethyl]-4-nitro-L-phenylalanine
methyl ester (460 mg, 44%; 65% based on recovered starting
material) as a light yellow oil. HR MS: Obs. mass, 478.2014. Calcd.
mass, 478.2012 (M-H).
Example 8.
[0225]
N-[[1-[2-(acetylamino)ethyl]cyclopentyl]thioxomethyl]-4-nitro-L-phe-
nylalanine methyl ester. 104
[0226] To a solution of N-[[1-[2-[[(1
,1-dimethylethoxy)carbonyl]amino]eth-
yl]-cyclopentyl]thioxomethyl]-4-nitro-L-phenylalanine methyl ester
(1.26 g, 2.63 mmol) in dichloromethane (15 mL) was added dropwise
trifluoroacetic acid (7 mL) and the resultant mixture was stirred
for 2 h at room temperature. The reaction mixture was concentrated
in vacuo to afford the trifluoroacetic acid salt of crude
N-[[1-(2-aminoethyl)-cyclop-
entyl]thioxomethyl]-4-nitro-L-phenylalanine methyl ester as a
yellow oil (1.4 g).
[0227] To a solution of the salt obtained above (1.4 g, 2.63 mmol)
in dichloromethane (10 mL) was added diisopropylethylamine (1.37
mL, 7.88 mmol) and acetic anhydride (0.250 mL, 2.63 mmol). The
resultant mixture was stirred overnight. The reaction mixture was
concentrated in vacuo and transferred to a separatory funnel
containing ethyl acetate (100 mL) and water (40 mL). The aqueous
layer was separated and back extracted with ethyl acetate
(1.times.50 mL). The combined organic layer was washed with brine,
dried over MgSO.sub.4, filtered and concentrated in vacuo. The
resulting residue was purified by flash column chromatography
(dichloromethane-acetone, 5:1) to furnish N-[[1
-[2-(acetylamino)ethyl]cy-
clopentyl]thioxomethyl]-4-nitro-L-phenylalanine methyl ester (743
mg, 67%). HR MS: Obs. mass, 422.1744. Calcd. mass, 422.1750
(M+H).
Example 9.
[0228]
4-amino-N-[[1-[2-acetylamino)ethyl]cyclopentyl]thioxomethyl]-L-phen-
ylalanine methyl ester. 105
[0229] To a suspension of
N-[[1-[2-(acetylamino)ethyl]cyclopentyl]thioxome-
thyl]-4-nitro-L-phenylalanine methyl ester (740 mg, 1.75 mmol),
zinc dust (1.14 g, 17.5 mmol) and ammonium chloride (1.41 g, 26.3
mmol) in methanol (20 mL) was added H.sub.2O (10 mL) slowly over 5
min. After stirring for 20 min, the reaction mixture was diluted
with ethyl acetate (80 mL) and sat. ammonium chloride solution (25
mL). The separated aqueous layer was back-extracted with ethyl
acetate (3.times.25 mL) and the organic layers were combined, dried
over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The
residual oil was dried under high vacuum for 2 h to give the crude
amine (750 mg)which was by flash silica gel column chromatography
(dichloromethane-acetone 2:1) to furnish
4-amino-N-[[l-[2-acetylamino)eth-
yl]cyclopentyl]thioxomethyl]-L-phenylalanine methyl ester (650 mg,
95%). HR MS: Obs. mass, 392.2016. Calcd. mass, 392.2008 (M+H).
Example 10.
[0230] N-[[1-[2-(acetylamino)
ethyl]cyclopentyl]thioxomethyl]-4-[[(2,6-dic-
hlorophenyl)carbonyl]amino]-L-phenylalanine. 106
[0231] To a solution of
4-amino-N-[[1-[2-(acetylamino)ethyl]cyclopentyl]th-
ioxomethyl]-L-phenylalanine methyl ester (195 mg, 0.498 mmol) and
diisopropylethylamine (0.0950 mL, 0.548 mmol) in dichloromethane (1
mL) was added a solution of 2,6-dichlorobenzoyl chloride (110 mg,
0.523 mmol) in dichloromethane (1 mL). The resultant mixture was
stirred overnight then was concentrated in vacuo and transferred to
a separatory funnel containing ethyl acetate (50 mL) and water (10
mL). The separated aqueous layer back-extracted with ethyl acetate
(1.times.25 mL). The combined organic layer was washed in turn sat.
solution of Na.sub.2CO.sub.3 and brine, then was dried over
MgSO.sub.4, filtered and concentrated in vacuo to provide crude
N-[[1-[2-(acetylamino) ethyl]cyclopentyl]thioxomethyl]-4-
-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalanine methyl ester
(300 mg).
[0232] To a solution of the above methyl ester (300 mg, 0.498 mmol)
in methanol (1 mL) was added a solution of NaOH (64 mg, 14.9 mmol)
in water (1 mL). The mixture was stirred for 2 h and was then
acidified (pH 1-2) with 0.5M HCl. The reaction mixture was poured
into a separatory funnel containing ethyl acetate (50 mL) and water
(10 mL) and the separated aqueous layer was back-extracted with
ethyl acetate (2.times.25 mL). The combined organic layers were
washed with brine, dried over MgSO.sub.4, filtered and concentrated
in vacuo. Purification of the crude by RP-HPLC (15-95%
acetonitrile-water gradient over 25 min) and lyophylization of the
appropriate fractions, provided N-[[1-[2-(acetylamino)
ethyl]cyclopentyl]thioxomethyl]-4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-
-phenylalanine (126 mg, 46%) as a colorless solid. HR MS: Obs.
mass, 550.1330. Calcd. mass, 550.1334 (M+H).
Example 11.
[0233]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[[(methylamino)car-
bonyl]amino]ethyl]cyclopentyl]thioxomethyl]-L-phenylalanine.
107
[0234] 4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1
-[2-[[(methylamino)carbonyl]amino]ethyl]cyclopentyl]thioxomethyl]-L-pheny-
lalanine was prepared from
N-[[1-[2-[[(1,1-dimethylethoxy)carbonyl]amino]e-
thyl]cyclopentyl]thioxomethyl]-4-nitro-L-phenylalanine methyl ester
and methyl isocyanate by using the general procedure described in
examples 8 to 10. HR MS: Obs. mass, 565.1436. Calcd. mass, 565.1443
(M+H).
Example 12.
[0235] 2-chloro-6-methylbenzaldehyde. 108
[0236] A 500 mL, three-necked, round bottomed flask equipped with a
magnetic stirrer, thermometer, addition funnel, and argon inlet was
charged with 75 g (494 mmol) of 2-chloro-6-methylbenzonitrile and
400 mL of toluene (stored over 4 A molecular sieves). The mixture
was cooled to -2.degree. C. (ice /acetone bath) and a 1M solution
of DIBAL-H in hexanes (593 mmol, 593 mL) was added dropwise over a
period of 30 min while maintaining the temperature below 0 .degree.
C. After the addition was completed, the reaction mixture was
stirred for 1 h at 0.degree. C. and then allowed to warm to room
temperature. After 2 h at room temperature, TLC analysis indicated
the absence of starting material (4:1 hexane:diethyl ether,
phosphomolybdic acid spray, as analysis by UV fluorescence was
misleading). The reaction was poured into a mixture of ice (2000 g)
and concentrated sulfuric acid (50 mL) and was stirred for
overnight. The precipitated solids were collected by filtration and
the filtrate was extracted with diethyl ether (2.times.200 mL). The
combined extracts were washed with brine, dried (MgSO.sub.4) and
filtered. Evaporation of the solvent under reduced pressure gave
the crude aldehyde, which was combined with the above solid to
afford 71.31 g (93%) of light yellow colored aldehyde suitable for
use in the next step.
Example 13.
[0237] 2-chloro-6-methylbenzoic acid. 109
[0238] A 1000 mL, three-necked, round bottomed flask equipped with
a magnetic stirrer, thermometer, addition funnel, and argon inlet
was charged with 2-chloro-6-methylbenzaldehyde (71.31 g , 461 mmol,
crude obtained from the above experiment) and 750 mL of
acetonitrile. To this suspension, a solution of monobasic sodium
phosphate (115 mmol, 15.9 g) in water 240 mL) was added followed by
hydrogen peroxide (50 mL, 30%) at room temperature. Then, a
solution of sodium chlorite (73.5 g, 811 mmol) in water (700 mL)
was added dropwise at 0.degree. C. while maintaining the
temperature below 3 .degree. C. After the addition was complete,
the yellow suspension was stirred for 15 h at 0.degree. C. to room
temperature. TLC analysis of the mixture indicated the absence of
starting material. A solution of sodium bisulfite (73 g, 701 mmol)
in water (200 mL) was added dropwise at 0.degree. C. until the
yellow color disappeared (KI-paper positive). Cooling was
maintained throughout to control the exothermic reaction. The
solvent was removed under vacuum to afford a colorless solid. The
solid was collected by filtration and the filtrate was extracted
with diethyl ether (200 mL). The above solid was dissolved in the
combined diethyl ether extracts which were then washed with 10%
NaOH solution (2.times.200 mL). The combined aqueous washings were
acidified with 10% HCl to pH 1. The resulting colorless precipitate
was collected by filtration and air-dried to afford 54.88 g (65%,
overall in two steps) of 2-chloro-6-methylbenzoic acid as a
colorless solid.
Example 14.
[0239]
N-[(2-chloro-6-methylphenyl)carbonyl]-4-nitro-L-phenylalanine
methyl ester. 110
[0240] To a solution of 4-nitro-L-phenylalanine methyl ester
hydrochloride salt (7.44 mmol, 1.94 g), 2-chloro-6-methylbenzoic
acid (8.2 mmol, 1.4 g) and HBTU (8.2 mmol, 3.11 g) in DMF (27 mL)
was added diisopropylethylamine (18.6 mmol, 3.24 mL) at room
temperature. The clear solution was stirred for 48 h at room
temperature and was diluted with 100 mL of ethyl acetate. The
solution was washed in turn with 0.5N hydrochloric acid (2.times.50
mL), saturated sodium bicarbonate solution (2 x 50 mL), brine (100
mL) then was dried (MgSO.sub.4) and filtered. Concentration of the
solution to dryness gave 2.67 g (95%) of
N-[(2-chloro-6-methylphenyl)carbonyl]-4-nitro-L-phenylalanine
methyl ester as a colorless solid, mp 120-123.degree. C. HRMS: Obs.
mass, 376.4274. Calcd. mass, 376.4238 (M+H).
Example 15.
[0241]
N-[(2-chloro-6-methylphenyl)thioxomethyl]-4-nitro-L-phenylalanine
methyl ester. 111
[0242] To a mixture of
N-[(2-chloro-6-methylphenyl)carbonyl]-4-nitro-L-phe- nylalanine
methyl ester (9.66 mmol, 3.64 g) and Lawesson's reagent (6.0 mmol,
2.46 g, 0.62 equiv.) was added toluene (15 mL, which had been
stored over 4 A molecular sieves) at room temperature. The
suspension was heated to 90-100.degree. C. and was stirred for 24
h. Examination of the resulting clear solution by TLC failed to
detect the presence of starting material. The reaction mixture was
diluted with ethyl acetate (50 mL) and washed with water (50 mL),
saturated sodium bicarbonate solution (50 mL), and brine (50 mL).
The organic extract was dried (MgSO.sub.4) filtered and evaporated
under reduced pressure. The crude compound was purified by careful
silica gel column chromatography (hexane:ethyl acetate, 4:1 to 2:1)
to obtain 1.52 g (40%) of
N-[(2-chloro-6-methylphenyl)thioxomethyl]-- 4-nitro-L-phenylalanine
methyl ester as a yellow solid, mp 150-153.degree. C. (triturated
from diethyl ether/ hexane 3:1 ratio). HRMS: Obs. mass, 393.0685.
Calcd. mass, 393.0677 (M+H).
Example 16.
[0243]
4-amino-N-[(2-chloro-6-methylphenyl)thioxomethyl]-L-phenylalanine
methyl ester. 112
[0244] To a mixture of
N-[(2-chloro-6-methylphenyl)thioxomethyl]-4-nitro-L- -phenylalanine
methyl ester (3.86 mmol, 1.52 g), zinc dust (.about.325 mesh, 39.0
mmol, 2.55 g, 10 equiv.) and ammonium chloride (58.0 mmol, 3.09 g,
15 equiv.) was added methanol (50 mL) and water (25 mL) at room
temperature. After addition of water, an exothermic reaction ensued
and the temperature rose to between 45 and 50 .degree. C. After the
suspension was stirred for 2 h at a bath temperature of
50-60.degree. C., 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 filtrate was concentrated under vacuum to
remove methanol and the product was extracted into ethyl acetate
(2.times.50 mL). The combined extracts were washed with brine (50
mL), dried (MgSO.sub.4), filtered and concentrated in vacuo to give
1.3 g (92%) of 4-amino-N-[(2-chloro-6-methylphenyl)thiox-
omethyl]-L-phenylalanine methyl ester as an amorphous yellow solid,
which was used directly for next step. HRMS: Obs. mass, 363.0932.
Calcd. mass, 363.0934 (M+H).
Example 17.
[0245]
N-[(2-chloro-6-methylphenyl)thioxomethyl]-4-[[(2,6-dichlorophenyl)c-
arbonyl]amino]-L-phenylalanine methyl ester. 113
[0246] To a solution of
4-amino-N-[(2-chloro-6-methylphenyl)thioxomethyl]-- L-phenylalanine
methyl ester (3.57 mmol, 1.296 g) and 2,6-dichlorobenzoyl chloride
(3.75 mmol, 0.785 g) in dichloromethane (20 mL) was added
diisopropylethylamine (5.35 mmol, 0.93 mL) at room temperature. The
solution was stirred for 15 h at which time TLC analysis of the
mixture indicated the absence of starting material. After the
addition of water (30 mL), the layers were separated and the
aqueous phase was extracted with dichloromethane (20 mL). The
combined extracts were washed with brine (50 mL), dried
(MgSO.sub.4) and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography ( hexane:ethyl
acetate, 4:1 to 1:1) to obtain 1.91 g (83%) of
N-[(2-chloro-6-methylphenyl)thioxomethyl]-4-[[(2,6-dichlorophenyl)carbony-
l]amino]-L-phenylalanine methyl ester as an amorphous colorless
solid. HRMS: Obs. mass, 535.0399. Calcd. mass, 535.0416 (M+H).
Example 18.
[0247]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-chloro-6-methylphenyl-
)thioxomethyl]-L-phenylalanine. 114
[0248] To a suspension of
N-[(2-chloro-6-methylphenyl)thioxomethyl]-4-[[(2-
,6-dichlorophenyl)carbonyl]amino]-L-phenylalanine methyl ester
(2.89 mmol, 1.55 g) in ethanol (8 mL) was added aqueous 1.ON sodium
hydroxide (5 mL) at room temperature. The mixture was heated to
50-55.degree. C. and the resulting clear solution was stirred for
3-4 h. TLC analysis of the mixture indicated the absence of
starting material. The mixture was concentrated to remove ethanol,
then was diluted with 15 mL of water and extracted with 25 mL of
diethyl ether to remove any neutral impurities. The aqueous layer
was acidified with 1N HCl and the precipitated colorless solid was
extracted into ethyl acetate (2.times.30 mL). The combined extracts
were washed with brine, dried (MgSO.sub.4) and concentrated in
vacuo to afford 1.45 g (96%) of N-[(2-chloro-6-methylphen-
yl)thioxomethyl]-4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalanine
as an amorphous colorless solid. HRMS: Obs. mass, 521.0241. Calcd.
mass, 521.0260 (M+H).
Example 19.
[0249]
N-[(2-chloro-6-methylphenyl)thioxomethyl]-4-[[(2,6-dichlorophenyl)c-
arbonyl]amino]-L-phenylalanine sodium salt. 115
[0250]
N-[(2-chloro-6-methylphenyl)thioxomethyl]-4-[[(2,6-dichlorophenyl)c-
arbonyl]amino]-L-phenylalanine (2.77 mmol, 1.45 g) was dissolved in
water (10 mL) containing 1.5 equivalents of aqueous 1 ON sodium
hydroxide (4.2 mL) at room temperature. The solution was loaded
into a reverse phase column size of 8 inches length with 1.5 inches
diameter containing C-18 silica gel and eluted with water to remove
excess base. The product was eluted with 5-20% methanol in water.
After the appropriate fractions were combined and concentrated, the
residue was dissolved in 50 mL water and lyophilized to afford 1.3
g of the sodium salt as a colorless amorphous solid. HRMS: Obs.
mass, 543.0076. Calcd. mass, 543.0079 (M+H).
Example 20.
[0251] 2-ethyl-6-methylbenzoic acid. 116
[0252] A 250 mL pressure bottle was charged with
2-ethyl-6-methyliodobenze- ne (30.07 mmol, 7.4 g), Pd(OAc).sub.2
(1.43 mmol, 334 mg) and dppp (1.43 mmol, 620 mg). The flask was
closed with a septum and evacuated three times with argon.
Acetonitrile (96 mL), triethylamine (189 mmol, 19.0 g, 26.25 mL)
and water (19.1 mL) were added in succession by the aid of syringe
and the rubber septum was replaced with a teflon lined cap
connected to a carbon monoxide source. The flask was now
pressurized with carbon monoxide (40 psi) and the excess pressure
was released. This process was repeated three times and finally the
mixture was stirred for 5 min under 40 psi carbon monoxide
pressure. The flask was then disconnected from the carbon monoxide
cylinder and immersed in a preheated oil bath (83-85 .degree. C.).
The reaction mixture turned black within 1 h and was stirred for
another 14 h at this temperature, then was cooled to room
temperature and the pressure was released. The resulting mixture
was diluted with diethyl ether (200 mL) and 1 ON NaOH (20 mL). The
formed sodium was extracted into water (2.times.100 mL). The
combined water extracts were acidified with 1.ON HCl and the
mixture was extracted with dichloromethane (3.times.100 mL). The
combined dichloromethane extracts were washed with brine, dried
(MgSO.sub.4), filtered and the volatiles were removed under vacuum
to provide 3.58 g (72.5%) of 2-ethyl-6-methylbenzoic acid as a
viscous brown oil which slowly solidified overnight. HR MS: Obs.
mass, 164.0833. Calcd. mass, 164.0837 (M+).
Example 21.
[0253] N-[(2-ethyl-6-methylphenyl)carbonyl]-4-nitro-L-phenylalanine
methyl ester. 117
[0254] Using the procedure described in example 14,
N-[(2-ethyl-6-methylphenyl)carbonyl]-4-nitro-L-phenylalanine methyl
ester was prepared in 72% yield as a colorless solid, mp
119-121.degree. C. HR MS: Obs. mass, 371.1610. Calcd. mass,
371.1607 (M+H).
Example 22.
[0255]
N-[(2-ethyl-6-methylphenyl)thioxomethyl]-4-nitro-L-phenylalanine
methyl ester. 118
[0256] Using the procedure described in example 15,
N-[(2-ethyl-6-methylphenyl)thioxomethyl]-4-nitro-L-phenylalanine
methyl ester was prepared in 47% yield as an amorphous colorless
solid. HR MS: Obs. mass, 387.1383. Calcd. mass, 387.1378 (M+H).
Example 23.
[0257] 4-amino-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-
L-phenylalanine methyl ester. 119
[0258] Using the general procedure described in example
16,4-amino-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalanine
methyl ester was prepared in 94% yield as an amorphous colorless
solid. HR MS: Obs. mass, 357.1640. Calcd. mass, 357.1638 (M+H).
Example 24.
[0259]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-ethyl-6-methylphenyl)-
thioxomethyl]-L-phenylalanine methyl ester. 120
[0260] Using the procedure described in example 17,
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-ethyl-6-methylphenyl)thioxo-
methyl]-L-phenylalanine methyl ester was prepared in 70% yield as
an amorphous colorless solid. HR MS: Obs. mass, 529.1094. Calcd.
mass, 529.1119 (M+H).
Example 25.
[0261]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-ethyl-6-methylphenyl)-
thioxomethyl]-L-phenylalanine. 121
[0262] Using the procedure described in example 18,
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-ethyl-6-methylphenyl)thioxo-
methyl]-L-phenylalanine was prepared in 77% yield as an amorphous
colorless solid. HR MS: Obs. mass, 515.0942. Calcd. mass, 515.0963
(M+H).
Example 26.
[0263]
N-[(2-ethyl-6-methylphenyl)thioxomethyl]-4-[[(2R)-2-(Fmoc-amino)-1--
oxo-3-(pyridin-3-yl)propyl]amino]-L-phenylalanine methyl ester.
122
[0264] Using the procedure described in example 1,
N-[(2-ethyl-6-methylphe-
nyl)thioxomethyl]-4-[[(2R)-2-(Fmoc-amino)-l-oxo-3-(pyridin-3-yl)propyl]ami-
no]-L-phenylalanine methyl ester was prepared in 72% yield as an
amorphous colorless solid. HR MS: Obs. mass, 727.2973. Calcd. mass,
727.2954 (M+H).
Example 27.
[0265]
4-[[(2R)-2-amino-1-oxo-3-(pyridin-3-yl)propyl]amino]-N-[(2-ethyl-6--
methylphenyl)thioxomethyl]-L-phenylalanine methyl ester. 123
[0266]
N-[(2-ethyl-6-methylphenyl)thioxomethyl]-4-[[(2R)-2-(Fmoc-amino)-1--
oxo-3-(pyridin-3-yl)propyl]amino]-L-phenylalanine methyl ester from
example 26 (0.308 mmol, 224 mg) was treated with 25% piperidine in
NMP (3 mL) and the solution was stirred at room temperature. Within
1 h, TLC analysis of the mixture indicated the absence of starting
material. The mixture was diluted with hexane (25 mL) and the
formed layers were separated. The bottom yellow layer was washed
with hexane, then was diluted with water and extracted with ethyl
acetate and THF (2:1, 3 x 25 mL). The combined extracts were washed
with water (50 mL), brine (50 mL), dried (MgSO.sub.4), filtered and
the solvents were removed under vacuum. The resulting residue was
dried under high vacuum to afford 126 mg (81%) of
4-[[(2R)-2-amino-1-oxo-3-(pyridin-3-yl)propyl]amino]-N-[(2-ethyl-6-met-
hylphenyl)thioxomethyl]-L-phenylalanine methyl ester as an
amorphous colorless solid. HR MS: Obs. mass, 505.2270. Calcd. mass,
505.2274 (M+H).
Example 28.
[0267] 4-[(2S,4R)-3-acetyl-5-oxo-2-phenyl-4-[(pyridin-3-yl)methyl]
imidazolidin- 1
-yl]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylala- nine
methyl ester. 124
[0268] To a solution of
4-[[(2R)-2-amino-1-oxo-3-(pyridin-3-yl)propyl]amin-
o]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalanine methyl
ester (0.224 mmol, 113 mg) in dichloromethane (0.75 mL) and
trimethyl orthoformate (0.75 mL), was added benzaldehyde (0.25
mmol, 27.5 mg). After the resulting light yellow solution was
stirred for 3 days at room temperature, it was heated to 90.degree.
C. (oil bath temperature) and excess acetic anhydride (2.0 mmol,
0.21 mL) was introduced via a syringe. The solution was stirred at
110-120.degree. C. (oil bath temperature) for 6 h, then was cooled
to room temperature and the solvents were removed in vacuo. The
crude residue was purified by RP-HPLC to obtain 95 mg (67%) of
4-[(2S,4R)-3-acetyl-5-oxo-2-phenyl-4-[(pyridin-3-yl)methyl]imidazolidin-1-
-yl]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalanine
methyl ester as an amorphous colorless solid. HR MS: Obs. mass,
635.2672. Calcd. mass, 635.2692 (M+H). Another isomer was formed in
very minor amount by HPLC (<5%) and not attempted to isolate
it.
Example 29.
[0269]
4-[(2S,4R)-3-acetyl-5-oxo-2-phenyl-4-[(pyridin-3-yl)methyl]imidazol-
idin-1-yl]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalanine.
125
[0270] The hydrolysis of
4-[(2S,4R)-3-acetyl-5-oxo-2-phenyl-4-[(pyridin-3--
yl)methyl]imidazolidin-1-yl]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-ph-
enylalanine methyl ester was carried out using the general
procedure described in example 18. The obtained crude product was
purified by RP-HPLC, using a 5-95% acetonitrile-water gradient over
30 min and the appropriate fraction was collected. The acetonitrile
was removed under vacuum and the product was extracted into ethyl
acetate:THF (3:1) (2.times.25 mL). The combined extracts were
washed with brine, dried (MgSO.sub.4), filtered and the solvents
were removed under reduced pressure. The resulting residue was
dried under high vacuum to obtain
4-[(2S,4R)-3-acetyl-5-oxo-2-phenyl-4-[(pyridin-3-yl)methyl]imidazolinidin-
-1-yl]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalanine in
30% yield as an amorphous colorless solid. HR MS: Obs. mass,
621.2520. Calcd. mass, 621.2535 (M+H).
Example 30.
[0271] N-[(2-fluorophenyl)carbonyl]-4-nitro-L-phenylalanine methyl
ester. 126
[0272] Using the general procedure described in example 14,
starting with 4-nitro-L-phenylalanine methyl ester hydrochloride
salt and 2-fluorobenzoic acid,
N-[(2-fluorophenyl)carbonyl]-4-nitro-L-phenylalanin- e methyl ester
was prepared in 99% yield as a colorless solid, mp 137-139.degree.
C. HR MS: Obs. mass, 346.0977. Calcd. mass, 346.0980, M+.
Example 31.
[0273] N-[(2-fluorophenyl)thioxomethyl]-4-nitro-L-phenylalanine
methyl ester. 127
[0274] Using the general procedure described in example 15,
starting with N-[(2-fluorophenyl)carbonyl]-4-nitro-L-phenylalanine
methyl ester,
N-[(2-fluorophenyl)thioxomethyl]-4-nitro-L-phenylalanine methyl
ester was prepared in 99% yield as an amorphous colorless solid. HR
MS: Obs. mass, 363.0816. Calcd. mass, 363.0815 (M+H).
Example 32.
[0275] 4-amino-N-[(2-fluorophenyl)thioxomethyl]-L-phenylalanine
methyl ester. 128
[0276] Using the general procedure described in example 16,
starting with
N-[(2-fluorophenyl)thioxomethyl]-4-nitro-L-phenylalanine methyl
ester, 4-amino-N-[(2-fluorophenyl)thioxomethyl]-L-phenylalanine
methyl ester was prepared in 87% yield as an amorphous colorless
solid. HR MS: Obs. mass, 332.1042. Calcd. mass, 332.1046,
(M+H).
Example 33.
[0277]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-fluorophenyl)thioxome-
thyl]-L-phenylalanine methyl ester. 129
[0278] Using the general procedure described in example 17,
starting with
4-amino-N-[(2-fluorophenyl)thioxomethyl]-L-phenylalanine methyl
ester and 2,6-dichlorobenzoyl chloride,
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[-
(2-fluorophenyl)thioxomethyl]-L-phenylalanine methyl ester was
prepared in 74% yield as an amorphous colorless solid. HR MS: Obs.
mass, 505.0561. Calcd. mass, 505.0555, (M+H).
Example 34.
[0279]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-fluorophenyl)thioxome-
thyl]-L-phenylalanine. 130
[0280] Using the general procedure described in example 18,
starting with
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-fluorophenyl)thioxomethyl]--
L-phenylalanine methyl ester,
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(-
2-fluorophenyl)thioxomethyl]-L-phenylalanine was prepared in 89%
yield as an amorphous colorless solid. HR MS: Obs. mass, 491.0407.
Calcd. mass, 491.0399 (M+H).
Example 35.
[0281]
4-nitro-N-[[(2-(trifluoromethyl)phenyl]carbonyl]-L-phenylalanine
methyl ester. 131
[0282] Using the general procedure described in example 14,
starting with 4-nitro-L-phenylalanine methyl ester hydrochloride
salt and 2-trifluoromethylbenzoic acid,
4-nitro-N-[[(2-(trifluoromethyl)phenyl]car- bonyl]-L-phenylalanine
methyl ester was prepared in 69% yield as a colorless solid, mp
152-154.degree. C. HR MS: Obs. mass, 397.1017. Calcd. mass,
397.1011 (M+H).
Example 36.
[0283]
4-nitro-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalanine
methyl ester 132
[0284] Using the general procedure described in example 15,
starting with
4-nitro-N-[[(2-(trifluoromethyl)phenyl]carbonyl]-L-phenylalanine
methyl ester,
4-nitro-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalanin-
e methyl ester was prepared in 67% yield as an amorphous colorless
solid. HR MS: Obs. mass, 412.0752. Calcd. mass, 412.0757 (M+H).
Example 37.
[0285]
4-amino-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalanine
methyl ester. 133
[0286] Using the general procedure described in example 16,
starting with
4-nitro-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalanine
methyl ester,
4-amino-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-pheny-
lalanine methyl ester was prepared in 98% yield as an amorphous
colorless solid. HR MS: Obs. mass, 382.1072. Calcd. mass, 382.1078,
(M+H).
Example 38.
[0287]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[2-(trifluoromethyl)
phenyl]thioxomethyl]-L-phenylalanine methyl ester. 134
[0288] Using the general procedure described in example 17,
starting with
4-amino-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalanine
methyl ester and 2,6-dichlorobenzoyl chloride,
4-[[(2,6-dichlorophenyl)ca-
rbonyl]amino]-N-[[2-(trifluoromethyl)phenyl]-thioxomethyl]-L-phenylalanine
methyl ester was prepared in 98% yield as an amorphous colorless
solid. HR MS: Obs. mass, 555.0511. Calcd. mass, 555.0524,
(M+H).
Example 39.
[0289]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[2-(trifluoromethyl)
phenyl]thioxomethyl]-L-phenylalanine. 135
[0290] Using the general procedure described in example 18,
starting with
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[2-(trifluoromethyl)
phenyl]thioxomethyl]-L-phenylalanine methyl ester,
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[2-(trifluoromethyl)phenyl]thi-
oxomethyl]-L-phenylalanine was prepared in 99% yield as an
amorphous colorless solid. HR MS: Obs. mass, 541.0358. Calcd. mass,
541.0367, (M+H).
Example 40.
[0291] 1-(4-bromobutyl)cyclopentanecarboxylic acid methyl ester..
136
[0292] To a solution of diisopropylamine (150 mmol, 21 mL) in THF
(100 mL) at -10.degree. C. was added dropwise a 2.5M solution of
n-butyl lithium in hexanes (145 mmol, 58 mL) while maintaining the
temperature below 0 .degree. C. After the addition was complete,
the solution was stirred for 30 min at 0.degree. C., then it was
cooled to -70.degree. C. using a dry ice/acetone bath. A solution
of methyl cyclopentanecarboxylate (100 mmol, 13.1 g) in THF (20 mL)
was added dropwise at -70.degree. C. maintaining the reaction
temperature between -60 to -70.degree. C. The mixture was then
stirred for 1 h at -50 to -60.degree. C. and 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. The cooling bath was removed and the reaction was
allowed to equilibrate to room temperature and stirred overnight.
The reaction mixture then was poured into a saturated solution of
ammonium chloride (200 mL) and the mixture was extracted with
diethyl ether (2.times.100 mL). The combined extracts were washed
with brine (150 mL), dried (MgSO.sub.4), filtered and the solution
was concentrated under reduced pressure. The resulting residue was
distilled at 120-133.degree. C./2.5 mm Hg to obtain 12.8 g (48%) of
1-(4-bromobutyl)cyclopentanecarboxylic acid methyl ester as a
colorless oil. HR MS: Obs. mass, 262.0565. Calcd. mass, 262.0568,
(M+).
Example 41.
[0293] 1-[4-(methylthio)butyl]cyclopentanecarboxylic acid methyl
ester. 137
[0294] To a solution of 1-(4-bromobutyl)cyclopentanecarboxylic acid
methyl ester (38 mmol, 10 g) in DMF (100 mL) was added sodium
thiomethoxide (72.6 mmol, 5.09 g). After the addition, an
exothermic reaction ensued and the mixture turned to a light brown
cloudy solution. The mixture was stirred for 15 h at room
temperature, then was poured into water (200 mL) and extracted with
diethyl ether (2.times.150 mL). The combined extracts were washed
with brine (150 mL), dried (MgSO.sub.4), filtered and the solution
was concentrated in vacuo. The residual material was purified by
silica gel column chromatography to afford 4.43 g (51%) of methyl
1-[4-(methylthio)butyl]cyclopentanecarboxylic acid methyl ester as
a colorless oil. HR MS: Obs. mass, 230.1343. Calcd. mass, 230.1341,
(M+).
Example 42.
[0295] 1-[4-(methylsulfonyl)butyl]cyclopentanecarboxylic acid
methyl ester. 138
[0296] To a solution of
1-[4-(methylthio)butyl]cyclopentanecarboxylic acid methyl ester
(19.2 mmol, 4.43 g) in acetic acid (20 mL) was added 30% hydrogen
peroxide (10 mL) and stirred mixture was heated to 70.degree. C.
for 15 h. TLC analysis 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 diethyl ether (3.times.100 mL). The combined extracts were
washed with brine (200 mL), dried (MgSO.sub.4), filtered and the
solvent was removed under reduced pressure. The residual material
was purified by silica gel column chromatography to afford 4.94 g
(98%) of 1-[4-(methylsulfonyl)butyl]cyclo- pentanecarboxylic acid
methyl ester as a colorless oil. LR MS (C12H22O4S): 263 (M+H).
Example 43.
[0297] 1-[4-(methylsulfonyl)butyl]cyclopentane carboxylic acid
139
[0298] To a solution of
1-[4-(methylsulfonyl)butyl]cyclopentanecarboxylic 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) and the mixture was
heated to 50-55.degree. C. After 15 h, 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 diethyl 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,
dried (MgSO.sub.4), filtered and the solution was concentrated in
vacuo. The residual material was dried under high vacuum to afford
4.31 g (92%) of 1-[4-(methylsulfonyl)butyl]cyclopen- tane
carboxylic acid as a low melting colorless solid. LR MS (C11H2O4S):
249 (M+H).
Example 44.
[0299] 1-[4-(methylthio)butyl]cyclopentanecarboxylic acid. 140
[0300] To a solution of
1-[4-(methylthio)butyl]cyclopentanecarboxylic acid methyl ester
(18.8 mmol, 4.94 g) in a mixture of THF (38 mL) and methanol (3 8
mL) was added 1 N sodium hydroxide (38 mL)and the mixture was
heated to 50-55.degree. C. for 15 h. TLC analysis of the reaction
mixture did not detect the presence of starting ester and the
reaction was cooled to room temperature,. The volatiles were
removed under reduced pressure and the residue was diluted with
water (100 mL) and was extracted with diethyl ether (2.times.50 mL)
to remove any neutral impurities. Then the separated aqueous layer
was acidified with 1 N hydrochloric acid and the mixture was
extracted with ethyl acetate (2.times.75 mL). The combined ethyl
acetate extracts were washed with brine, dried (MgSO.sub.4),
filtered, then concentrated in vacuo and the residue was dried
under high vacuum to afford 4.31 g (92%) of
1-[4-(methylthio)butyl]cyclopentanecarbo- xylic acid as a low
melting colorless solid. HR MS: Obs. mass, 216.1181. Calcd. mass,
216.1184, M+.
Example 45.
[0301]
N-[[1-[4-(methylthio)butyl]cyclopentyl]carbonyl]-4-nitro-L-phenylal-
anine methyl ester. 141
[0302] To a suspension of 4-nitro-L-phenylalanine methyl ester
hydrochloride salt (181.84 mmol, 47.41 g),
1-[4-(methylthio)butyl]cyclope- ntanecarboxylic acid (177.17 mmol,
38.33 g) in DMF (470 mL) were added HBTU (177.17 mmol, 67.2 g) and
diisopropylethylamine (443 mmol, 77 mL) at room temperature. The
clear solution was stirred for 15 h at room temperature at which
time TLC analysis of the mixture indicated the absence of starting
materials. The reaction mixture was diluted with 600 mL of ethyl
acetate then was washed in turn with 0.5N hydrochloric acid
(2.times.250 mL), saturated sodium bicarbonate solution
(2.times.250 mL) and brine (300 mL). The dried (MgSO.sub.4) organic
layer was filtered and evaporated to dryness under reduced
pressure. The crude product which was purified by silica gel column
chromatography to afford 58.5 g (78%) of
N-[[1-[4-(methylthio)butyl]cyclopentyl]carbonyl]-4-nitro-L-phenylalanine
methyl ester as an amorphous colorless solid. HRMS: Obs. mass,
423.1940. Calcd. mass, 423.1953 (M+H).
Example 46.
[0303] 4-nitro-N- [[1
-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-L-ph- enylalanine
methyl ester. 142
[0304] To a solution of
N-[[1-[4-(methylthio)butyl]cyclopentyl]carbonyl]-4-
-nitro-L-phenylalanine methyl ester (138.4 mmol, 58.5 g) in
dichloromethane (1.2 L) was added m-chloroperbenzoic acid (415
mmol, 71.7 g) at -5.degree. C. (ice-salt bath). The suspension was
stirred for 30 min at 0.degree. C. and allowed to warm to room
temperature. After 5 h, analysis of the reaction by TLC indicated
that the starting material was gone. The precipitated solid was
removed by filtration and the filtrate was concentrated under
vacuum to afford a colorless residue. The residue was dissolved in
ethyl acetate (600 mL) and was washed with saturated sodium
bicarbonate solution (3.times.300 mL). TLC analysis showed the
presence of m-chloroperbenzoic acid. Accordingly, the ethyl acetate
layer was washed in turn with saturated sodium bisulfite solution
(20 g in 150 mL of water), saturated sodium bicarbonate solution
(200 mL) and brine (300 mL). The dried (MgSO.sub.4) ethyl acetate
layer was filtered and evaporated to dryness to give a crude
product which was dissolved in ethyl acetate. Diethyl ether and
hexane were added to precipitate an oily residue. Some of the
solvent was removed under reduced pressure to obtain a white
suspension. The suspension was further diluted with diethyl ether
and the resulting solid was collected by filtration and was washed
with hexane. The colorless low melting solid was dried to furnish
53.9 g (86%) of
N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-4-nitro-L-phenyla-
lanine methyl ester, mp 40-44.degree. C. HRMS: Obs. mass, 455.1854.
Calcd. mass, 455.1852 (M+H).
Example 47.
[0305]
N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-4-nitro-L--
phenylalanine methyl ester. 143
[0306] To a solution of
N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]carbony-
l]-4-nitro-L-phenylalanine methyl ester (33 mmol, 15 g) in toluene
(100 mL, stored over 4 .ANG. molecular sieves) and freshly
distilled THF (50 mL) was added Lawesson's reagent (33 mmol, 13.35
g, 1.0 equiv.) at room temperature. The solution was heated to
60-65.degree. C. and was stirred for 48 h at which time TLC
analysis of the mixture indicated the absence of starting material.
The reaction mixture was cooled to room temperature and was poured
into saturated sodium bicarbonate solution (200 mL) and extracted
with ethyl acetate (3.times.150 mL). An oil formed in the aqueous
layer, which was separated, diluted with water and extracted with
ethyl acetate (2.times.50 mL). The combined ethyl acetate extracts
were washed with saturated sodium bicarbonate solution (200 mL),
with brine (300 mL), dried (MgSO.sub.4), filtered and the solution
was concentrated in vacuo. The residual light brown syrup was
purified by silica gel column chromatography (hexane:ethyl acetate,
1:1) to obtain 6.87 g (44%) of
N-[[l-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-4-nitro-L-phe-
nylalanine methyl ester as a fluffy yellow solid. HRMS: Obs. mass,
493.1438. Calcd. mass, 493.1443 (M+Na).
Example 48.
[0307]
4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl)-L--
phenylalanine methyl ester. 144
[0308] The poorly soluble N-[[l
-[4-(methylsulfonyl)butyl]cyclopentyl]thio-
xomethyl]-4-nitro-L-phenylalanine methyl ester (19.3 mmol, 9.07 g)
was dissolved in methanol (150 mL) and THF (20 mL) by gentle
heating with a heat gun. To this solution, zinc dust (.about.325
mesh, 193 mmol, 12.62 g, 10 equiv.) and ammonium chloride (289.5
mmol, 15.5 g, 15 equiv.) were added followed by water (75 mL) at
room temperature. After the addition of water, an exothermic
reaction ensued and the temperature rose to 45 to 50.degree. C. The
suspension was stirred for 1 h, at which time TLC analysis of the
mixture indicated the absence of starting material. The reaction
mixture was filtered and the filter cake was washed with methanol
(200 mL) and THF (100 mL). The volatiles were removed under vacuum
and the organic residue was extracted into ethyl acetate
(2.times.200 mL). The combined extracts were washed brine (250 mL)
then were dried (MgSO.sub.4), filtered and evaporated to dryness to
give 8.37 g (98%) of
4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethy-
l)-L-phenylalanine methyl ester as a colorless gum which was used
directly for next step. HRMS: Obs. mass, 441.1884. Calcd. mass,
441.1882 (M+H).
Example 49.
[0309]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)-b-
utyl]cyclopentyl]thioxomethyl]-L-phenylalanine methyl ester.
145
[0310] To a solution of
4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl-
]thioxomethyl]-L-phenylalanine methyl ester (19.0 mmol, 8.37 g) and
2,6-dichlorobenzoyl chloride (21 mmol, 4.4 g) in dichloromethane
(90 mL) was added diisopropylethylamine (32.3 mmol, 5.6 mL) at room
temperature. The solution was stirred for 15 h at which time TLC
analysis of the mixture indicated the absence of starting material.
Then, it was diluted with water (100 mL) and the two layers were
separated. The aqueous phase was extracted with dichloromethane
(100 mL) and the combined extracts were washed with brine (200 mL).
The dried (MgSO.sub.4) solution was concentrated under vacuum and
the residue was purified by silica gel column chromatography
eluting with (hexane:ethyl acetate:dichloromethane, 1:1:1) to
obtain 11.54 g (99%) of 4-[[(2,6-dichlorophenyl)carbonyl]amino]-
-N-[[1
-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanine
methyl ester as a colorless solid, mp 200-202.degree. C. HRMS: Obs.
mass, 613.1367. Calcd. mass, 613.1363 (M+H).
Example 50.
[0311]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)bu-
tyl]cyclopentyl]thioxomethyl] -L-phenylalanine. 146
[0312] To a solution of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(-
methylsulfonyl)butyl] cyclopentyl]thioxomethyl]-L-phenylalanine
methyl ester (25.86 mmol, 15.87 g) in ethanol (75 mL) was added
aqueous 1.ON sodium hydroxide (60 mL) at 50.degree. C. The mixture
was heated to 50-55.degree. C. and the resulting clear light brown
solution was stirred for 22 h at which time TLC analysis of the
mixture indicated the absence of starting material. The mixture was
diluted with water and allowed to cool to room temperature and was
filtered to remove a small amount of solids. The filtrate was
concentrated and the residual aqueous solution was washed with
diethyl ether (2.times.75 mL). The basic aqueous layer was
acidified with 3.0N HCl to form a cloudy suspension and was
extracted with ethyl acetate (3.times.100 mL). The combined ethyl
acetate extracts were washed with brine (200 mL) then the dried
(MgSO.sub.4) solution was filtered and evaporated to dryness. The
residue was taken up in dichloromethane and diluted with diethyl
ether:hexane (1: 1) to obtain a solid which was collected by
filtration. Trituration of the solid with hot ethyl acetate (100
mL) resulted in a suspension that was then diluted with diethyl
ether (.about.50 mL) and the solid was collected by filtration. The
above Trituration was repeated to afford 10.89 g (70%) of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1
-[4-(methylsulfonyl)butyl]c-
yclopentyl]thioxomethyl]-L-phenylalanine as a colorless solid. HR
MS: Obs. mass, 599.1193. Calcd. mass, 599.1208 (M+H).
Example 51.
[0313]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)-b-
utyl]cyclopentyl]thioxomethyl]-L-phenylalanine sodium salt. 147
[0314] A suspension of
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(m-
ethylsulfonyl)butyl]-cyclopentyl]thioxomethyl]-L-phenylalanine
(16.49 mmol, 9.89 g) in water (100 mL) was treated with aqueous
1.ON sodium hydroxide (16.4 mmol, 16.4 mL) at room temperature. The
mixture was heated to 40-45.degree. C. and some acetonitrile (15
mL) was added to give an essentially clear solution containing a
small amount of suspended solid. The solution was filtered and the
filtrate was lyophilized to afford 10.1 g of sodium salt as a
colorless solid. HRMS: Obs. mass, 621.1023. Calcd. mass, 621.1027
(M+H).
Example 52.
[0315]
4-[[(2,4-dimethylpyridin-3-yl)carbonyl]amino]-N-[[1-[4-(methylsulfo-
nyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanine methyl ester.
148
[0316] To an ice cold solution of 2,4-dimethyl-3-pyridinecarboxylic
acid (0.3 mmol, 45 mg) in dichloromethane (2 mL) containing one
drop of DMF, was added oxalyl chloride (0.39 mmol, 49.5 mg) at
0.degree. C. The reaction mixture was stirred for 30 min at this
temperature, was allowed to warm to room temperature and was
stirred for an additional 2 h. The solution was concentrated and
the residue was dried under high vacuum. To a mixture of above acid
chloride and 4-amino-N-[[1-[4-(methylsulfonyl)but- yl]
cyclopentyl]thioxomethyl]-L-phenylalanine methyl ester (0.2 mmol,
88 mg) in dichloromethane (3 mL) was added diisopropylethylamine (1
mmol, 0.175 mL) at room temperature. After the solution had stirred
for 15 h, TLC analysis of the mixture did not detect any starting
material remaining. The solution was diluted with water (20 mL) and
dichloromethane (20 mL) and the layers were separated. The aqueous
phase was extracted with dichloromethane (10 mL) and the combined
organic extracts were washed with brine (300 mL). The dried
(MgSO.sub.4) solution was filtered and evaporated to dryness and
the residual material was purified by RP-HPLC to obtain 74 mg (65%)
of 4-[[(2,4-dimethylpyridin-3-y-
l)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-
-L-phenylalanine methyl ester as an amorphous colorless solid.
HRMS: Obs. mass, 574.2389. Calcd. mass, 574.2409 (M+H).
Example 53.
[0317]
4-[[(2,4-dimethylpyridin-3-yl)carbonyl]amino]-N-[[1-[4-(methylsulfo-
nyl)butyl]-cyclopentyl]thioxomethyl]-L-phenylalanine
trifluoroacetic acid salt. 149
[0318] To a solution of
4-[[(2,4-dimethylpyridin-3-yl)carbonyl]amino]-N-[[-
1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanine
methyl ester (0.118 mmol, 68 mg) in ethanol (4 mL) was added
aqueous 1.ON sodium hydroxide (3 mL) at room temperature and the
stirred mixture was heated to 45-50.degree. C. After 3 h TLC
analysis of the clear solution indicated that the starting material
had been consumed. The mixture was concentrated and the crude
residue was purified by RP-HPLC to afford 54.5 mg (82%) of
4-[[(2,4-dimethylpyridin-3-yl)carbonyl]amino]-N-[[1
-[4-(methylsulfonyl)butyl]-cyclopentyl]thioxomethyl]-L-phenylalanine
trifluoroacetic acid salt as an amorphous colorless solid. HR MS:
Obs. mass, 560.2240. Calcd. mass, 560.2253 (M+H).
Example 54.
[0319] 1-(4-bromobutyl)cyclobutanecarboxylic acid ethyl ester..
150
[0320] Using the general procedure described in example 40,
starting with cyclobutanecarboxylic acid ethyl ester, 1
-(4-bromobutyl)cyclobutanecarbo- xylic acid ethyl ester was
prepared in 58% yield as a colorless oil. HR MS: Obs. mass,
263.0563. Calcd. mass, 263.0568, M+.
Example 55.
[0321] 1-[4-(methylthio)butyl]cyclobutanecarboxylic acid ethyl
ester. 151
[0322] Using the general procedure described in example 41,
starting with 1-(4-bromobutyl)cyclobutanecarboxylic acid ethyl
ester, 1-[4-(methylthio)butyl] cyclobutanecarboxylic acid ethyl
ester was prepared in 87% yield as a colorless oil. HR MS: Obs.
mass, 230.1339. Calcd. mass, 230.1340, M+.
Example 56.
[0323] ethyl 1-[4-(methylsulfonyl)butyl]cyclobutane carboxylic acid
ethyl ester. 152
[0324] Using the general procedure described in example 46,
starting with 1-[4-(methylthio)butyl]cyclobutanecarboxylic acid
ethyl ester, 1-[4-(methylsulfonyl)-butyl]cyclobutanecarboxylic acid
ethyl ester was prepared in 92% yield as a colorless oil. HR MS:
Obs. mass, 262.1231. Calcd. mass, 262.1238, M+.
Example 57.
[0325] 1-[4-(methylsulfonyl)butyl]cyclobutanecarboxylic acid.
153
[0326] Using the general procedure described in example 43,
starting with 1-[4-(methylsulfonyl)butyl] cyclobutanecarboxylic
acid ethyl ester, 1-[4-(methylsulfonyl)butyl] cyclobutanecarboxylic
acid was prepared in 92% yield as a low melting colorless solid. HR
MS: Obs. mass, 234.0921. Calcd. mass, 234.0918, (M+).
Example 58.
[0327]
N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]carbonyl]-4-nitro-L-pheny-
lalanine methyl ester. 154
[0328] Using the general procedure described in example 45,
starting with 1-[4-(methylsulfonyl)butyl] cyclobutanecarboxylic
acid and 4-nitro-L-phenylalanine methyl ester hydrochloride salt,
N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]carbonyl]-4-nitro-L-phenylalani-
ne methyl ester was prepared in 89% yield as a yellow gum. HR MS:
Obs. mass, 441.1700. Calcd. mass, 441.1696 (M+H).
Example 59.
[0329]
N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl)-4-nitro-L-p-
henylalanine methyl ester. 155
[0330] Using the general procedure described in example 47,
starting with
4-nitro-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]carbonyl]-L-phenylalani-
ne methyl ester,
N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl)-4-
-nitro-L-phenylalanine methyl ester was prepared in 80% yield as a
colorless solid, mp 150-152.degree. C. HR MS: Obs. mass, 457.1464.
Calcd. mass, 457.1467, (M+H).
Example 60.
[0331]
4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl)-L-p-
henylalanine methyl ester. 156
[0332] Using the general procedure described in example 48,
starting with
N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl)-4-nitro-L-phenyla-
lanine methyl ester,
4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thi-
oxomethyl)-L-phenylalanine methyl ester was prepared in 94% yield
as a hygroscopic solid. HR MS: Obs. mass, 427.1720. Calcd. mass,
427.1725, (M+H).
Example 61.
[0333]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)bu-
tyl]cyclobutyl]thioxomethyl]-L-phenylalanine methyl ester. 157
[0334] Using the procedure described in example 49, starting with
4-amino-N-[[1-[4-(methylsulfonyl)-butyl]cyclobutyl]thioxomethyl)-L-phenyl-
alanine methyl ester,
4-[[(2,6-dichlorophenyl)-carbonyl]amino]-N-[[1
-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine
methyl ester was obtained in 92% yield as an amorphous colorless
solid. HR MS: Obs. mass, 599.1207. Calcd. mass, 599.1208 (M+H).
Example 62.
[0335]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)-b-
utyl]cyclobutyl]thioxomethyl]-L-phenylalanine. 158
[0336] Using the general procedure described in example 50,
starting with 4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[l
-[4-(methylsulfonyl)butyl]c-
yclobutyl]thioxomethyl]-L-phenylalanine methyl ester,
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cy-
clobutyl]thioxomethyl]-L-phenylalanine was prepared in 99% yield as
an amorphous colorless solid. HR MS: Obs. mass, 585.1038. Calcd.
mass, 585.1051 (M+H).
Example 63.
[0337]
N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-4-[[[4-(tri-
fluoromethyl) pyrimidin-5-yl]carbonyl]amino]- L-phenylalanine
methyl ester. 159
[0338] Using the general procedure described in example 45,
starting with 4-(trifluoromethyl)pyrimidine-5-carboxylic acid and
4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl)-L-phenyla-
lanine methyl ester,
N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethy- l]-
4-[[[2-(trifluoromethyl)
pyrimidin-5-yl]carbonyl]amino]-L-phenylalanin- e methyl ester was
prepared in 32% yield as an amorphous colorless solid. HR MS: Obs.
mass, 601.1766. Calcd. mass, 601.1766 (M+H).
Example 64.
[0339] N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-
4-[[[2-(trifluoromethyl)
pyrimidin-5-yl]carbonyl]amino]-L-phenylalanine. 160
[0340] Using the general procedure described in example 50,
starting with
N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-4-[[[2-(trifluoro-
methyl) pyrimidin-5-yl]carbonyl]amino]-L-phenylalanine methyl
ester,
N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-4-[[[2-(trifluoro-
methyl) pyrimidin-5-yl]carbonyl]amino]-L-phenylalanine was prepared
in 22% yield as an amorphous colorless solid. HR MS: Obs. mass,
587.1619. Calcd. mass, 587.1609 (M+H).
Example 65.
[0341] Preparation 1-(3-bromopropyl)cyclobutanecarboxylic acid
ethyl ester. 161
[0342] Using the general procedure described in example 40,
starting with cyclobutanecarboxylic acid ethyl ester and
1,3-dibromopropane, 1-(3-bromopropyl)cyclobutanecarboxylic acid
ethyl ester was prepared in 33% yield as a colorless oil. HR MS:
Obs. mass, 248.0416. Calcd. mass, 248.0412 (M+).
Example 66.
[0343] 1-[3-(methylthio)propyl]cyclobutanecarboxylic acid ethyl
ester and 1-[3-(methylthio)propyl]cyclobutanecarboxylic acid.
162
[0344] Using the general procedure described in example 41,
starting with 1-(3-bromopropyl)cyclobutanecarboxylic acid ethyl
ester, 1-[3-(methylthio)propyl]cyclobutanecarboxylic acid ethyl
ester was prepared in 58% yield as a colorless oil. HR MS: Obs.
mass, 216.1182. Calcd. mass, 216.1184 (M+). Also,
1-[3-(methylthio)propyl]cyclobutanecarb- oxylic acid was obtained
in 16% yield as a colorless oil. HR MS: Obs. mass, 188.0872. Calcd.
mass, 188.0871 (M+).
Example 67.
[0345]
N-[[1-[3-(methylthio)propyl]cyclobutyl]carbonyl]-4-nitro-L-phenylal-
anine methyl ester. 163
[0346] Using the general procedure described in example 45,
starting with 4-nitro-L-phenylalanine methyl ester hydrochloride
salt and 1-[3-(methylthio)propyl]cyclobutanecarboxylic acid,
N-[[1-[3-(methylthio)propyl]cyclobutyl]carbonyl]-4-nitro-L-phenylalanine
methyl ester was prepared in 92% yield as an yellow viscous oil. HR
MS: Obs. mass, 395.1638. Calcd. mass, 395.1640 (M+H).
Example 68.
[0347]
N-[[1-[3-(methylthio)propyl]cyclobutyl]thioxomethyl]-4-nitro-L-phen-
ylalanine methyl ester. 164
[0348] Using the general procedure described in example 47,
starting with
4-nitro-N-[[1-[3-(methylthio)propyl]cyclobutyl]carbonyl]-4-nitro-L-phenyl-
alanine methyl ester,
N-[[i-[3-(methylthio)propyl]cyclobutyl]thioxomethyl]-
-4-nitro-L-phenylalanine methyl ester was prepared in 95% yield as
a colorless viscous oil. HR MS: Obs. mass, 411.1408. Calcd. mass,
411.1412 (M+H).
Example 69.
[0349]
4-amino-N-[[1-[3-(methylthio)propyl]cyclobutyl]thioxomethyl]-L-phen-
ylalanine methyl ester. 165
[0350] Using the general procedure described in example 48,
starting with N-[[1-[3-(methylthio)
propyl]cyclobutyl]thioxomethyl]-4-nitro-L-phenylala- nine methyl
ester, 4-amino-N-[[1-[3-(methylthio)propyl]cyclobutyl]thioxome-
thyl]-L-phenylalanine methyl ester was prepared in 97% yield as an
hygroscopic yellow solid. HR MS: Obs. mass, 381.1660. Calcd. mass,
381.1671 (M+H).
Example 70.
[0351] 4-[(2,6-dichlorophenylcarbonyl)amino]-N-[[1-[3-(methylthio)
propyl]cyclobutyl]thioxomethyl]-L-phenylalanine methyl ester.
166
[0352] Using the general procedure described in example 49,
starting with
4-(amino)-N-[[1-[3-(methylthio)propyl]cyclobutyl]thioxomethyl]-L-phenylal-
anine methyl ester and 2,6-dichlorobenzoyl chloride,
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-(methylthio)propyl]cyclo-
butyl]thioxomethyl]-L-phenylalanine methyl ester was prepared in
83% yield as a colorless solid, mp 184-186.degree. C. HRMS: Obs.
mass, 553.1139. Calcd. mass, 553.1153 (M+H).
Example 71.
[0353]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[l-[3-(methylthio)propyl-
]cyclobutyl]-thioxomethyl]-L-phenylalanine. 167
[0354] Using the general procedure described in example 50,
starting with 4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[l
-[3-(methylthio)propyl]cycl- obutyl]thioxomethyl]-L-phenylalanine
methyl ester, 4-[[(2,6-dichlorophenyl-
)carbonyl]amino]-N-[[1-[3-(methylthio)propyl]cyclobutyl]thioxomethyl]-L-ph-
enylalanine was prepared in 97% yield as a colorless solid, mp
186-188.degree. C. HR MS: Obs. mass, 539.0986. Calcd. mass,
539.0996 (M+H).
Example 72.
[0355] 1-[3-(methylsulfonyl)propyl]cyclobutanecarboxylic acid ethyl
ester. 168
[0356] Using the general procedure described in example 46,
starting with 1-[3-(methylthio)propyl]cyclobutanecarboxylic acid
ethyl ester, 1-[3-(methylsulfonyl)propyl]cyclobutanecarboxylic acid
ethyl ester was prepared in 87% yield as a colorless oil. HR MS:
Obs. mass, 248.1084. Calcd. mass, 248.1082 (M+).
Example 73.
[0357] 1-[3-(methylsulfonyl)propyl]cyclobutanecarboxylic acid.
169
[0358] Using the general procedure described in example 43,
starting with 1-[3-(methylsulfonyl)propyl]cyclobutanecarboxylic
acid ethyl ester, 1-[3-(methylsulfonyl)propyl]cyclobutanecarboxylic
acid was prepared in 76% yield as a colorless solid, mp
113-116.degree. C. HR MS: Obs. mass, 220.0770. Calcd. mass,
220.0769 (M+).
Example 74.
[0359] N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]carbonyl]-
4-nitro-L-phenylalanine methyl ester. 170
[0360] Using the general procedure described in example 45,
starting from 4-nitro-L-phenylalanine methyl ester and
1-[3-(methylsulfonyl)propyl]cycl- obutane carboxylic acid,
N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]carbon-
yl]-4-nitro-L-phenylalanine methyl ester was prepared in 76% yield
as a colorless amorphous solid. HR MS: Obs. mass, 427.1526. Calcd.
mass, 427.1539 (M+H).
Example 75.
[0361]
N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-4-nitro-L--
phenylalanine methyl ester. 171
[0362] Using the general procedure described in example 47,
starting with
N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]carbonyl]-4-nitro-L-phenylalan-
ine methyl ester, N-[[1 -[3
-(methylsulfonyl)propyl]cyclobutyl]thioxomethy-
l]-4-nitro-L-phenylalanine methyl ester was prepared in 88% yield
as an yellow sticky solid. HR MS: Obs. mass, 443.1309. Calcd. mass,
443.1310 (M+H).
Example 76.
[0363]
4-amino-N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-L--
phenylalanine methyl ester. 172
[0364] Using the general procedure described in example 48,
starting with
N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-4-nitro-L-phenyl-
alanine methyl ester,
4-amino-N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]t-
hioxomethyl]-L-phenylalanine methyl ester was prepared in 97% yield
as an hygroscopic yellow solid. HR MS: Obs. mass, 413.1556. Calcd.
mass, 413.1570 (M+H).
Example 77.
[0365]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-(methylsulfonyl)-p-
ropyl]cyclobutyl]thioxomethyl]-L-phenylalanine methyl ester.
173
[0366] Using the general procedure described in example 49,
starting with
4-amino-N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-L-phenyl-
alanine methyl ester and 2,6-dichlorobenzoyl chloride,
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-(methylsulfonyl)propyl]c-
yclobutyl]thioxomethyl]-L-phenylalanine methyl ester was prepared
in 82% yield as a colorless amorphous solid. HR MS: Obs. mass,
585.1056. Calcd. mass, 585.1051 (M+H).
Example 78.
[0367]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-(methylsulfonyl)
propyl]cyclobutyl]thioxomethyl] -L-phenylalanine. 174
[0368] Using the general procedure described in example 50,
starting with
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-(methylsulfonyl)propyl]c-
yclobutyl]thioxomethyl]-L-phenylalanine methyl ester,
4-[(2,6-dichlorophenylcarbonyl)amino]-N-[[1-[3-(methylsulfonyl)propyl]cyc-
lobutyl]thioxomethyl]-L-phenylalanine was prepared in 87% yield as
an amorphous colorless solid. HR MS: Obs. mass, 571.0894. Calcd.
mass, 571.0895 (M+H).
Example 79.
[0369] 2-chloro-5-(trifluoromethyl)phenol triflate. 175
[0370] To a solution of 2-chloro-5-(trifluoromethyl)phenol (24.4
mmol, 4.8 g) in dichloromethane (160 mL) was added DMAP (54.0 mmol,
6.7 g) at -70.degree. C. followed by triflic anhydride (36.6 mmol,
10.32 g, 6.16 mL) at -70.degree. C. After the addition was
complete, the suspension was stirred for 30 min at this temperature
and then warmed to room temperature. After another 3 h, when
starting material could not be detected by TLC of the reaction
mixture indicated the absence of starting material, the stirred
mixture was diluted with H.sub.2O (100 mL) and the two layers were
separated. The aqueous layer was extracted with dichloromethane
(100 mL)and then the combined dichloromethane extracts were washed
with brine. The dried (MgSO.sub.4) solution was filtered and
evaporated to dryness to give a colorless residue which was
purified by silica gel column chromatography (hexane:diethyl ether
4: 1) to obtain 6.8 g (85%) of 2-chloro-5-(trifluoromethyl)phenol
triflate as a colorless oil .HR MS: Obs. mass, 327.9388. Calcd.
mass, 327.9392 (M+).
Example 80.
[0371] 2-chloro-5-(trifluoromethyl)benzoic acid. 176
[0372] A 250 mL pressure bottle was charged with
2-chloro-5-(trifluorometh- yl)phenol triflate (20.6 mmol, 6.76 g),
Pd(OAc).sub.2 (1.71 mmol, 384 mg) and dppp (1.71 mmol, 701 mg). The
flask was closed with a septum and evacuated three times with
argon. Acetonitrile (114 mL), triethylamine (225.3 mmol, 30.7 mL)
and water (22.2 mL) were added in succession with the aid of a
syringe. The rubber septum was replaced with a teflon lined lid.
The flask was pressurized with carbon monoxide (40 psi) and the gas
was released. This process was repeated three times and finally the
reaction was stirred for 5 min under pressure. The flask was then
disconnected from the gas cylinder, immersed in a preheated oil
bath (83-85.degree. C.) and stirred for 2 h. The flask was
re-pressurized with carbon monoxide and stirred for another 1 h.
After the reaction mixture was cooled to room temperature, the
pressure was released and it diluted with diethyl ether (250 mL)
and 25 mL of 1.0 N NaOH. The sodium salt was extracted into water
(2.times.100 mL). The combined aqueous extracts were acidified with
1.0 N HCl and extracted with diethyl ether (3.times.100 mL). The
combined diethyl ether extracts were washed with brine, dried
(MgSO.sub.4), filtered and the solution was concentrated in vacuo
to furnish a light yellow solid. The solid was dissolved in diethyl
ether (100 mL) and extracted with 1.0 N NaOH solution (2.times.50
mL). The combined aqueous layers were acidified and extracted with
diethyl ether (2.times.100 mL). After the combined organic extracts
were washed with brine (100 mL), the dried (MgSO.sub.4) solution
was filtered and evaporated to dryness to give 1.6 g (35%) of
2-chloro-5-(trifluoromethyl)- benzoic acid obtained as a colorless
solid, mp 82-83.5.degree. C. HR MS: Obs. mass, 223.9852. Calcd.
mass, 223.9851 (M+).
Example 81.
[0373]
4-[[[(2-chloro-5-(trifluoromethyl)phenyl]carbonyl]amino]-N-[[1
-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine
methyl ester. 177
[0374] Using the general procedure described in example 52,
starting with 4-amino-N-[[1-[4-(methylsulfonyl)
butyl]cyclobutyl]thioxomethyl]-L-phenyl- alanine methyl ester and
2-chloro-5-(trifluoromethyl)benzoic acid,
4-[[[(2-chloro-5-(trifluoromethyl)
phenyl]carbonyl]amino]-N-[[1-[4-(methy- lsulfonyl)butyl]
cyclobutyl]thioxomethyl]-L-phenylalanine methyl ester was prepared
in 97% yield as a colorless amorphous solid. HR MS: Obs. mass,
633.1477. Calcd. mass, 633.1471 (M+H).
Example 82.
[0375]
4-[[[(2-chloro-5-(trifluoromethyl)phenyl]carbonyl]amino]-N-[[1
-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine.
178
[0376] Using the general procedure described in example 50,
starting with 4-[[[(2-chloro-5-(trifluoromethyl)
phenyl]carbonyl]amino]-N-[[1
-[4-(methylsulfonyl)butyl]-cyclobutyl]thioxomethyl]
-L-phenylalanine methyl ester,
4-[[[(2-chloro-5-(trifluoromethyl)phenyl]carbonyl]amino]-N-- [[l
-[4-(methylsulfonyl) butyl]cyclobutyl]thioxomethyl]-L-phenylalanine
was prepared in 75% yield as an amorphous colorless solid. HR MS:
Obs. mass, 619.1315. Calcd. mass, 619.1318 (M+H).
Example 83.
[0377]
4-[[[(2,6-dimethyl-4-(trifluoromethyl)pyridin-3-yl]carbonyl]amino]--
N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine
methyl ester. 179
[0378] To suspension of
2,6-dimethyl-4-(trifluoromethyl)pyridine-3-carboxy- lic acid (0.84
mmol, 184 mg) in dichloromethane (10 mL) containing DMF (3 drops)
was added dropwise oxalyl chloride (1.14 mmol, 146 mg, 0.1 mL) at
0.degree. C. for 2-3 min. After the addition was complete, the
reaction was stirred for 30 min at 0.degree. C. and then allowed to
warm to room temperature. The clear solution was stirred for
another 2 h at room temperature, then the solvent was removed under
reduced pressure and the residue was dried under high vacuum for 1
h. To a mixture of
4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenyla-
lanine methyl ester (0.7 mmol, 298 mg) and amberlyst A-21 (1.4
mmol, 900 mg) in ethyl acetate (10 mL, stored over 4 .ANG.
molecular sieves) in a 4-necked sonicator flask was added a
solution of the above prepared acid chloride in ethyl acetate (6
mL) at room temperature. The mixture was subjected for sonication
for 30 min and then was partitioned between water (100 mL) and
ethyl acetate (100 mL). The separated aqueous layer was extracted
with ethyl acetate (50 mL) and the combined extracts were washed
brine (100 mL. The dried (MgSO.sub.4) ethyl acetate layer was
filtered and evaporated to dryness in vacuo and the residue was
purified by RP-HPLC to obtain 139 mg (32%) of
4-[[[(2,6-dimethyl-4-(trifluoromethy-
l)pyridin-3-yl]carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]t-
hioxomethyl]-L-phenylalanine methyl ester as an amorphous colorless
solid. HR MS: Obs. mass, 628.2122. Calcd. mass, 628.2127 (M+H).
Example 84.
[0379]
4-[[[(2,6-dimethyl-4-(trifluoromethyl)pyridin-3-yl]carbonyl]amino]--
N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine.
180
[0380] To a suspension of
4-[[[(2,6-dimethyl-4-(trifluoromethyl)pyridin-3--
yl]carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-
-L-phenylalanine methyl ester (0.2 mmol, 125 mg) in ethanol (7 mL)
was added a 1.ON solution of sodium. hydroxide (5.0 mL) at room
temperature. Within few minutes the reaction mixture become a clear
solution and it was heated to 45-50.degree. C. and stirred for 4
hr, at which time TLC analysis of the mixture indicated the absence
of starting material. After the solution was cooled to room
temperature, the ethanol was removed in vacuo and the residue was
purified by RP-HPLC to obtain 67.5 mg (55%) of
4-[[[(2,6-dimethyl-4-(trifluoromethyl)pyridin-3-yl]carbonyl]amino]-N-[[1
-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine
methyl ester as an amorphous colorless solid. HR MS: Obs. mass,
614.1970. Calcd. mass, 614.1970 (M+H).
Example 85.
[0381]
4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-bromophenyl)thioxomet-
hyl]-L-phenylalanine. 181
[0382] Using the method described in examples 35 to 39, and
starting with 2-bromobenzoic acid and 4-nitro-L-phenylalanine
methyl ester hydrochloride salt, the title compound was prepared.
HRMS Obs. mass, 550.9593. Calcd mass, 550.9598 (M+H).
Example 86.
[0383]
4-[(2S,4R)-3-acetyl-5-oxo-2-phenyl-4-(phenylmethyl)imidazolidin-1-y-
l]-N-[[4-(methylsulfonyl)butyl]cyclopentyl]
thioxomethyl]-L-phenylalanine and
4-[(2R,4R)-3-acetyl-5-oxo-2-phenyl-4-(phenylmethyl)imidazolidin- 1
-yl]-N-[[4-(methylsulfonyl)butyl]
cyclopentyl]thioxomethyl]-L-phenylalani- ne. 182
[0384] Using the procedure described in examples 26 to 29, the
title compounds were prepared. The isomers were separated by
chromatography at the methyl ester stage. For the 2S,4R isomer,
HRMS, Obs. mass, 650.2670. Calcd mass, 650.2665 (M+Na). For the
2R,4R isomer, HRMS, obs. mass, 650.2679. Calcd. mass, 650.2665
(M+Na).
[0385] Assays:
[0386] 1. VLA-4 VCAM-1 Screening Assay
[0387] 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 is 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 are 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
are 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 are 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 is added to the VCAM-coated plates,
followed by 10 .mu.l of Ramos cell-derived VLA-4. These plates are
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 is 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 are washed four
times with VCAM Assay Buffer (200 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), is 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 is 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 are then read in a
UV/Vis spectrophotometer at 650 nM. Results are calculated as %
inhibition of total binding (i.e., VLA-4+VCAM-1 in the absence of
test compound). Selected data for compounds of this invention are
shown in the table below:
[0388] 2. Ramos (VLA-4) / VCAM-1 Cell-Based Screening Assay
Protocol
[0389] Materials:
[0390] Soluble recombinant human VCAM-l (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 .mu.g/mL leupeptin. Calcein-AM was purchased
from Molecular Probes Inc.
[0391] Methods:
[0392] 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. 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 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 incubation 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.
[0393] 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) fluorescence measurement system (excitation=485 nm,
emission=530 nm). The results are shown in the following table:
[0394] 3. MadCAM RPMI 8866 Cell Based Assay
[0395] MadCAM binding activity was quantified using an RPMI 8866
cell-based assay. RPMI 8866 cells bearing cell surface MadCAM were
labelled with fluorescent dye (calcein AM) and allowed to bind
MadCAM in the presence or absence of test compounds. A reduction in
fluoresence intensity associated with adherent cells (% inhibition)
reflects competitive inhibition of MadCAM mediated cell adhesion by
the test compound. Initially, this entailed coating 96 well plates
(Nunc F96 Maxisorp) with 25 ng/well of MadCAM (100 .mu.l/well in
coating buffer: 10 mM carbonate/bicarbonate buffer, 0.8 g/L sodium
carbonate, 1.55 g/L sodium bicarbonate, adjusted to pH 9.6 with 1N
HCL), sealing and wrapping each plate and refrigerating the plates
for at least 24 hrs. The MadCAM-coated plates were subsequently
washed twice with 0.05% Tween-20 in PBS, and then blocked for at
least 1 hr. at room temperature with of blocking buffer (1% nonfat
dry milk in PBS) to reduce non-specific binding. Following the
incubation with blocking buffer, plates were washed with PBS, hand
blotted, and the remaining liquid aspirated.
[0396] RPMI 8866 cells (2.times.10.sup.6 cells/ml.times.10 ml. per
plate.times.number of plates) were transferred to a 50 ml
centrifuge tube filled with PBS and spun at 200.times.g for 8
minutes, after which the PBS is poured off and the pellet
resuspended to 10.times.10.sup.6 cells/ml in PBS. Calcein (diluted
with 200 .mu.l DMSO from a 5 mg/ml frozen stock) was added to the
cells at 5 .mu.l/ml PBS. After incubation at 37 degrees C for 30
min. in the dark, the cells were washed in PBS and resuspended at
2.times.106 cells/ml in cell buffer (RPMI 1640 medium (no
additives)).
[0397] Test compounds were dissolved in 100% DMSO and then diluted
1:25 in binding buffer (1.5 mM CaCl.sub.2, 0.5 mM MnCl.sub.2 in 50
mM TRIS-HCl, adjusted to pH 7.5 with NaOH). Remaining dilutions
were into dilution buffer (4% DMSO in binding buffer--2% DMSO final
when diluted 1:2 in wells). A series of dilutions were performed
for each compound tested. 129 .mu.l of binding buffer was placed in
the first row of wells in the MadCAM-coated plates. 100 .mu.l/well
of dilution buffer was added to the remaining wells, followed by
5.4 .mu.l of each test compound in the appropriate dilution (in
triplicate). 100 .mu.l of cells (200,000 cells/well) were added.
Control wells contained 100 .mu.l dilution buffer +100 .mu.l cell
buffer, and 100 .mu.l dilution buffer +100 .mu.l cell buffer.
Plates were allowed to incubate for 45 min at room temperature,
after which 150 .mu.l/well PBS was added. Plates were inverted to
remove non-adherent cells, blotted and 200 .mu.l/well PBS added.
Plates were again inverted, blotted and the remaining buffer gently
aspirated. 100 .mu.l PBS was added to each well. The plates were
then read for fluorescence intensity on a fluorescence measurement
system (excitation=485 nm, emission=530 nm, sensitivity=2). A
linear regression analysis was performed to obtain the IC.sub.50 of
each compound. The results are shown in the following table:
2 ELISA IC.sub.50 nM Ramos Example (VLA/VCAM) IC.sub.50 RPMI
IC.sub.50 3 4.0 66.5 10 1.5 33 11 5.6 42.5 18 0.47 60 25 6.0 101 29
220 34 4,180 39 784 50 30 53 148 62 8 8.7 64 87 71 926 78 341 82 84
5.5 83
Example 4.
[0398] Acute airway inflammation in the atopic primate.
[0399] Airway inflammation in the monkey was determined using a
modification of the protocol described by Turner et al. (Turner et
al., 1994). Adult male cynomolgus monkeys (Macaca fascicularis,
Hazelton Labs, Denver, Pa.) weighing between 3.6 - 5.8 kg were used
in these studies. All animals exhibited positive skin and airway
responses to Ascaris suum antigen and had at least a 3-fold
increase in the sensitivity to methacholine (MCh) when subjected to
an aerosol of ascaris extract.
[0400] On the day of each experiment the animals were anesthetized
with ketamine hydrochloride, 12 mg/kg, and xylazine, 0.5 mg/kg,
intubated with a cuffed endotracheal tube (3 mm, Mallinckrodt
Medical, St. Louis, Mo.), then seated in an upright position in a
specially designed Plexiglass chair (Plas-Labs, Lansing, Mich.).
The endotracheal tube was connected to a heated Fleisch
pneumotachograph. Airflow was measured via a Validyn differential
pressure transducer (DP 45-24) that was attached to the
pneumotachograph. Transpulmonary pressure was measured via a second
Validyne transducer (DP 45-24) connected between a sidearm of the
tracheal cannula and a 18-gauge intrapleural needle inserted in the
intercostal space located below the left nipple. Recordings of
pressure and flow and the calculation of R.sub.L were made using
the Modular Instruments data acquisition system as described above.
Baseline R.sub.L was measured for all animals on the day of each
experiment and had an average value of about 0.04
cmH.sub.2O/ml/sec.
[0401] Protocol
[0402] Airway inflammation was induced by exposing the animal to an
aerosol of A. Suum extract for 60 sec. The aerosol was delivered
via a nebulizer (De Vilbiss Model 5000, Healt Care Inc., Somerset,
Pa.) that was attached to the endotracheal tube. The concentration
of extract was predetermined for each animal (500 to 50,000 PNU)
and caused at least a doubling in the airway resistance. At 24 hour
after the antigen challenge, the animals were anesthetized as
described previously and placed on a stainless steel table. Airway
inflammation was assessed by inserting a pediatric bronchoscope
into the airway lumen down to about the 4 or 5.sup.th generation
bronchi and gently ravaging with 3.times.2 ml aliquots of sterile
Hanks Balanced Salt Solution. The recovered lavage fluid then was
analyzed for the total cell and differential cell counts using
standard hematological techniques.
[0403] Drug Treatment
[0404] The animals received drug or a vehicle, p.o., administered 2
hours prior to antigen challenge. The compound of example 1 caused
a significant decrease in the number and percent of inflammatory
cells present in the lavage fluid relative to vehicle treated
control animals.
* * * * *