U.S. patent application number 10/679478 was filed with the patent office on 2006-07-27 for cell adhesion inhibitors.
This patent application is currently assigned to Biogen, Inc., a Massachusetts corporation. Invention is credited to Steven P. Adams, Alfredo C. Castro, Julio Herman Cuervo, Charles E. Hammond, Wen-Cherng Lee, Yu-Sheng Liao, Ko-Chung Lin, Juswinder Singh, Craig N. Zimmerman.
Application Number | 20060166866 10/679478 |
Document ID | / |
Family ID | 23484763 |
Filed Date | 2006-07-27 |
United States Patent
Application |
20060166866 |
Kind Code |
A1 |
Adams; Steven P. ; et
al. |
July 27, 2006 |
Cell adhesion inhibitors
Abstract
The present invention relates to novel compounds that are useful
for inhibition and prevention of cell adhesion and cell
adhesion-mediated pathologies. This invention also relates to
pharmaceutical formulations comprising these compounds and methods
of using them for inhibition and prevention of cell adhesion and
cell adhesion-mediated pathologies. The compounds and
pharmaceutical compositions of this invention can be used as
therapeutic or prophylactic agents. They are particularly
well-suited for treatment of many inflammatory and autoimmune
diseases.
Inventors: |
Adams; Steven P.; (Andover,
MA) ; Lin; Ko-Chung; (Lexington, MA) ; Lee;
Wen-Cherng; (Lexington, MA) ; Castro; Alfredo C.;
(Woburn, MA) ; Zimmerman; Craig N.; (Somerville,
MA) ; Hammond; Charles E.; (Burlington, MA) ;
Liao; Yu-Sheng; (Lexington, MA) ; Cuervo; Julio
Herman; (Arlington, MA) ; Singh; Juswinder;
(Malden, MA) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Biogen, Inc., a Massachusetts
corporation
|
Family ID: |
23484763 |
Appl. No.: |
10/679478 |
Filed: |
October 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10002341 |
Oct 23, 2001 |
6630512 |
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10679478 |
Oct 7, 2003 |
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08875321 |
Sep 19, 1997 |
6376538 |
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PCT/US96/01349 |
Jan 18, 1996 |
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10002341 |
Oct 23, 2001 |
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08376372 |
Jan 23, 1995 |
6306840 |
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08875321 |
Sep 19, 1997 |
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Current U.S.
Class: |
514/283 ;
514/1.3; 514/1.7; 514/12.2; 514/16.6; 514/17.9; 514/19.1; 514/6.9;
562/15; 562/450 |
Current CPC
Class: |
A61P 37/04 20180101;
A61P 37/06 20180101; A61P 11/00 20180101; C07C 271/22 20130101;
C07D 209/42 20130101; A61P 25/28 20180101; C07D 215/48 20130101;
A61P 3/10 20180101; A61P 37/00 20180101; C07D 213/55 20130101; A61P
43/00 20180101; A61K 38/00 20130101; A61P 25/00 20180101; A61P 3/00
20180101; C07D 405/12 20130101; C07C 323/60 20130101; C07C 311/06
20130101; C07C 255/19 20130101; C07C 275/42 20130101; C07C 275/28
20130101; C07C 311/21 20130101; C07C 275/54 20130101; C07C 2601/14
20170501; A61P 17/06 20180101; C07D 317/60 20130101; C07C 2601/02
20170501; C07C 317/50 20130101; A61P 19/02 20180101; A61P 11/06
20180101; C07D 277/48 20130101; A61P 3/08 20180101; A61P 29/00
20180101; C07D 213/75 20130101; A61P 1/00 20180101; C07C 237/22
20130101; C07K 7/06 20130101 |
Class at
Publication: |
514/007 ;
514/019; 562/450; 562/015 |
International
Class: |
A61K 38/04 20060101
A61K038/04; C07K 5/04 20060101 C07K005/04 |
Claims
1. A cell adhesion inhibitory compound of formula (I): ##STR38## or
a pharmaceutically acceptable derivative thereof, wherein: X is
--CO.sub.2H; Y is selected from the group consisting of --CO--,
--CH.sub.2--, --SO.sub.2-- and --PO.sub.2--; R.sub.1 is selected
from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkyl-substituted alkyl,
cycloalkenyl-substituted cycloalkyl, alkoxy, alkenoxy, alkynoxy,
alkylamino, alkenylamino, alkynylamino, N-alkylurea-substituted
alkyl, alkylcarbonylamino-substituted alkyl, and
aminocarbonyl-substituted alkyl; R.sub.2 is selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, and
cycloalkenyl; R.sub.3 is selected from the group consisting of
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
hydroxy-substituted alkyl, alkoxy-substituted alkyl,
amino-substituted alkyl, thiol-substituted alkyl,
alkylsulfonyl-substituted alkyl, (hydroxy-substituted
alkylthio)-substituted alkyl, thioalkoxy-substituted alkyl,
acylamino-substituted alkyl, alkylsulfonylamino-substituted alkyl,
[N-(alkyl, alkenyl or alkynyl)-or N,N-[dialkyl, dialkenyl,
dialkynyl or (alkyl,alkenyl)-amino]carbonyl-substituted alkyl,
carboxyl-substituted alkyl, dialkylamino-substituted
acylaminoalkyl, and and amino acid side chains selected from
arginine, asparagine, glutamine, S-methyl cysteine, methionine and
corresponding sulfoxide and sulfone derivatives thereof, glycine,
leucine, isoleucine, allo-isoleucine, tert-leucine, norleucine,
alanine, ornithine, glutamine, valine, threonine, serine, aspartic
acid, beta-cyanoalanine, and allothreonine; R.sub.4 is selected
from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl,
cycloalkenyl, alkynyl, amido, aminocarbonyl, mono- or
dialkylaminocarbonyl, mono- or diacylaminocarbonyl, aliphatic acyl,
alkyl optionally substituted with substitutents selected from the
group consisting of amino, carboxy, hydroxy, mercapto, mono- or
dialkylamino, mono- or diacylamino, alkoxy, alkenoxy, thioalkoxy,
thioalkenoxy, and thioalkynoxy; and n is 0, 1 or 2.
2. The compound according to claim 1, wherein R.sub.4 is selected
from the group consisting of alkyl, cycloalkyl, alkyenyl,
cycloalkenyl, and alkynyl.
3. The compound according to claim 1, wherein R.sub.1 is selected
from the group consisting of cyanomethyl, cyclohexylmethyl, methyl,
n-hexyl, t-butoxy, t-butylamino, 5-(N'-t-butylurea)pentyl,
2,2-dimethylpropyl, and hydroxyethylthiomethyl.
4. The compound according to claim 1, wherein R.sub.1 is selected
from the group consisting of cyanomethyl, cyclohexylmethyl, methyl,
n-hexyl, t-butoxy, t-butylamino, 5-(N'-t-butylurea)pentyl, and
2,2-dimethylpropyl.
5. The compound according to claim 1, wherein R.sub.2 is hydrogen
or methyl.
6. The compound according to claim 5, wherein R.sub.2 is
hydrogen.
7. The compound according to claim 1, wherein R.sub.3 is selected
from the group consisting of 2-(methylsulfonyl)-ethyl,
3-(hydroxy-propylthio)-methyl, 4-(methylsulfonylamino)-butyl,
4-acetylaminobutyl, aminomethyl, butyl, hydroxymethyl, isobutyl,
methyl, methylthiomethyl, propyl, N,N-(methylpropargyl)-amino,
2-(methylthio)-ethyl, 2-(N,N-dimethylamino)-ethyl, 4-amino-butyl,
t-butoxy-carbonylaminomethyl, sec-butyl, t-butyl,
N,N-dimethyl-aminocarbonylmethyl, 1,1-ethano, 1-hydroxyethyl,
1-methoxyethyl, carbonylmethyl, 2-methylsulfinylethyl, asparagine
side-chain, 4-(methylurea)butyl, 4-methylsulfonylaminobutyl,
hydroxymethylthiomethyl, 2-methylsulfonylethyl,
4-propionylaminobutyl, 4-ethoxycarbonylaminobutyl,
methoxycarbonylaminobutyl, carbomethoxymethylthiomethyl,
4-t-butylureabutyl, carboxymethylthiomethyl,
dimethylamidomethylthiomethyl, acetylaminopropyl,
3-methylureapropyl, 4-trifluoroacetylaminobutyl,
dimethylaminomethylthiomethyl, dimethylaminoethylthiomethyl, and
4-(dimethylaminoacetylamino)butyl.
8. The compound according to claim 7, wherein R.sub.3 is selected
from the group consisting of 2-(methylsulfonyl)-ethyl,
3-(hydroxypropylthio)-methyl, 4-(methylsulfonylamino)-butyl,
4-acetylaminobutyl, aminomethyl, butyl, hydroxymethyl, isobutyl,
methyl, methylthiomethyl, propyl, N,N-(methylpropargyl)-amino,
2-(methylthio)-ethyl, 2-(N,N-dimethylamino)-ethyl, 4-amino-butyl,
t-butoxy-carbonylaminomethyl, sec-butyl, t-butyl,
N,N-dimethyl-aminocarbonylmethyl, 1,1-ethano, 1-hydroxyethyl,
1-methoxyethyl, carbonylmethyl, 2-methylsulfinylethyl, and
asparagine side chain.
9. The compound according to claim 7, wherein R.sub.3 is selected
from the group consisting of 2-(methylsulfonyl)-ethyl,
3-(hydroxypropylthio)-methyl, 4-(methylsulfonylamino)-butyl,
4-acetylaminobutyl, isobutyl, 2-(methylthio)-ethyl, and
4-(ethoxycarbonylamino)butyl.
10. The compound according to claim 9, wherein R.sub.3 is selected
from the group consisting of 2-(methylsulfonyl)-ethyl,
3-(hydroxypropylthio)-methyl, 4-(methylsulfonylamino)-butyl,
4-acetylaminobutyl, isobutyl, and 2-(methylthio)-ethyl.
11. The compound according to claim 1, wherein R.sub.4 is selected
from the group consisting of methyl, 4-methylsulfonylamino,
4-propionylamino, n-pentyl, carboxymethyl, 2-carboxyethyl, allyl,
ethynyl, 2-propenyl, 2-propynyl, and propyl.
12. The compound according to claim 11, wherein R.sub.4 is
methyl.
13. The compound according to claim 11, wherein R.sub.4 is allyl or
ethynyl.
14. The compound according to claim 1, wherein Y is --CO--,
--CH.sub.2-- or --SO.sub.2--.
15. The cell adhesion inhibitory compound according to claim 14,
wherein Y is --CO--.
16. The cell adhesion inhibitory compound according to claim 1,
wherein n is 1.
17. A pharmaceutical composition comprising a compound according to
claim 1 in an amount effective for prevention, inhibition or
suppression of VLA-4 mediated cell adhesion and a pharmaceutically
acceptable carrier.
18. The pharmaceutical composition according to claim 17, further
comprising an agent selected from the group consisting of
corticosteriods, bronchodilators, antiasthmatics,
antiinflammatories, antirheumatics, immunosuppressants,
antimetabolites, immunonodulators, antipsoriatics and
antidiabetics.
19. A method of preventing, inhibiting or suppressing cell adhesion
in a mammal comprising the step of administering to said mammal the
pharmaceutical composition according to claim 17.
20. The method according to claim 19, wherein said method is used
for preventing, inhibiting or suppressing cell adhesion-associated
inflammation.
21. The method according to claim 20, wherein said method is used
for preventing, inhibiting or suppressing cell adhesion-associated
immune or autoimmune response.
22. The method according to claim 19, wherein said method is used
to treat or prevent a disease selected from the group consisting of
asthma, arthritis, psoriasis, transplantation rejection, multiple
sclerosis, diabetes and inflammatory bowel disease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation-in-part of U.S. patent application
Ser. No. 08/376,372, filed Jan. 23, 1995, now pending.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to novel compounds that are
useful for inhibition and prevention of cell adhesion and cell
adhesion-mediated pathologies. This invention also relates to
pharmaceutical formulations comprising these compounds and methods
of using them for inhibition and prevention of cell adhesion and
cell adhesion-mediated pathologies. The compounds and
pharmaceutical compositions of this invention can be used as
therapeutic or prophylactic agents. They are particularly
well-suited for treatment of many inflammatory and autoimmune
diseases.
BACKGROUND OF THE INVENTION
[0003] Cell adhesion is a process by which cells associate with
each other, migrate towards a specific target or localize within
the extra-cellular matrix. As such, cell adhesion constitutes one
of the fundamental mechanisms underlying numerous biological
phenomena. For example, cell adhesion is responsible for the
adhesion of hemoatopoietic cells to endothelial cells and the
subsequent migration of those hemopoietic cells out of blood
vessels and to the site of injury. As such, cell adhesion plays a
role in pathologies such as inflammation and immune reactions in
mammals.
[0004] Investigations into the molecular basis for cell adhesion
have revealed that various cell-surface
macromolecules--collectively known as cell adhesion molecules or
receptors--mediate cell-cell and cell-matrix interactions. For
example, proteins of the superfamily called "integrins" are the key
mediators in adhesive interactions between hematopoietic cells and
their microenvironment (M. E. Hemler, "VLA Proteins in the Integrin
Family: Structures, Functions, and Their Role on Leukocytes.", Ann.
Rev. Immunol., 8, p. 365 (1990)). Integrins are non-covalent
heterodimeric complexes consisting of two subunits called .alpha.
and .beta.. There are at least 12 different a subunits
(.alpha.1-.alpha.6, .alpha.-L, .alpha.-M, .alpha.-X, .alpha.-IIB,
.alpha.-V and .alpha.-E) and at least 9 different
.beta.(.beta.1-.beta.9) subunits. Based on the type of its .alpha.
and .beta. subunit components, each integrin molecule is
categorized into a subfamily.
[0005] .alpha.4.beta.1 integrin, also known as very late antigen-4
("VLA-4"), CD49d/CD29, is a leukocyte cell surface receptor that
participates in a wide variety of both cell-cell and cell-matrix
adhesive interactions (M. E. Hemler, Ann. Rev. Immunol., 8, p. 365
(1990)). It serves as a receptor for the cytokine-inducible
endothelial cell surface protein, vascular cell adhesion molecule-1
("VCAM-1"), as well as to the extracellular matrix protein
fibronectin. ("FN") (Ruegg et al., J. Cell Biol., 177, p. 179
(1991); Wayner et al., J. Cell Biol., 105, p. 1873 (1987); Kramer
et al., J. Biol. Chem., 264, p. 4684 (1989); Gehlsen et al.
Science, 24, p. 1228 (1988)). Anti-VLA4 monoclonal antibodies
("mAb's") have been shown to inhibit VLA4-dependent adhesive
interactions both in vitro and in vivo (Ferguson et al. Proc. Natl.
Acad. Sci., 88, p. 8072 (1991); Ferguson et al., J. Immunol., 150,
p. 1172 (1993)). Results of in vivo experiments suggest that this
inhibition of VLA-4-dependent cell adhesion may prevent or inhibit
several inflammatory and autoimmune pathologies (R. L. Lobb et al.,
"The Pathophysiologic Role of .alpha.-4 Integrins In Vivo", J.
Clin. Invest., 94, pp. 1722-28 (1994)).
[0006] In order to identify the minimum active amino acid sequence
necessary to bind VLA-4, Komoriya et al. ("The Minimal Essential
Sequence for a Major Cell Type-Specific Adhesion Site (CS1) Within
the Alternatively Spliced Type III Connecting Segment Domain of
Fibronectin Is Leucine-Aspartic Acid-Valine", J. Biol. Chem., 266
(23), pp. 15075-79 (1991)) synthesized a variety of overlapping
peptides based on the amino acid sequence of the CS-1 region (the
VLA-4 binding domain) of a particular species of fibronectin. They
identified an 8-amino acid peptide, Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr
[SEQ ID NO: 1], as well as two smaller overlapping pentapeptides,
Glu-Ile-Leu-Asp-Val [SEQ ID NO: 2] and Leu-Asp-Val-Pro-Ser [SEQ ID
NO: 3), that possessed inhibitory activity against FN-dependent
cell adhesion. These results suggested the tripeptide Leu-Asp-Val
as a minimum sequence for cell-adhesion activity. It was later
shown that Leu-Asp-Val binds only to lymphocytes that express an
actived form of VLA-4, thus bringing into question the utility of
such a peptide in vivo (E. A. Wayner et al., "Activation-Dependent
Recognition by Hematopoietic Cells of the LDV Sequence in the V
Region of Fibronectin", J. Cell. Biol., 116(2), pp. 489-497
(1992)). However, certain larger peptides containing the LDV
sequence were subsequently shown to be active L vivo [T. A.
Ferguson et al., "Two Integrin Binding Peptides Abrogate
T-cell-Mediated Immune Responses In Vivo," Proc. Natl. Acad. Sci.
USA, 88, pp. 8072-76 (1991); and S. M. Wahl et al., "Synthetic
Fibronectin Peptides Suppress Arthritis in Rats by Interrupting
Leukocyte Adhesion and Recruitment," J. Clin. Invest., 94, pp.
655-62 (1994)].
[0007] A cyclic pentapeptide, ##STR1## (wherein TPro denotes
4-thioproline), which can inhibit both VLA-4 and VLA-5 adhesion to
FN has also been described (D. M. Nowlin et al. "A Novel Cyclic
Pentapeptide Inhibits .alpha.4.beta.1 and .alpha.5.beta.1
Integrin-mediated Cell Adhesion", J. Biol. Chem., 268(27), pp.
20352-59 (1993); and PCT publication PCT/US91/04862). This peptide
was based on the tripeptide sequence Arg-Gly-Asp from FN which had
been known as a common motif in the recognition site for several
extracellular-matrix proteins.
[0008] Despite these advances, there remains a need for small,
specific inhibitors of VLA-4-dependent cell adhesion. Ideally, such
inhibitors would be semi-peptidic or non-peptidic so that they may
be orally administered. Such compounds would provide useful agents
for treatment, prevention or suppression of various pathologies
mediated by cell adhesion and VLA-4 binding.
SUMMARY OF THE INVENTION
[0009] The present invention solves this problem by providing novel
non-peptidic compounds that specifically inhibit the binding of
ligands to VLA-4. These compounds are useful for inhibition,
prevention and-suppression of VLA-4-mediated cell adhesion and
pathologies associated with that adhesion, such as inflammation and
immune reactions. The compounds of this invention may be used alone
or in combination with other therapeutic or prophylactic agents to
inhibit, prevent or suppress cell adhesion. This invention also
provides pharmaceutical formulations containing these
VLA-4-mediated cell adhesion inhibitors and methods of using the
compounds and compositions of the invention for inhibition of cell
adhesion.
[0010] According-to one embodiment of this invention, these novel
compounds, compositions and methods are advantageously used to
treat inflammatory and immune diseases. The present invention also
provides methods for preparing the compounds of this invention and
intermediates useful in those methods.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
[0011] As used herein, the term "alkyl", alone or in combination,
refers to a straight-chain or branched-chain alkyl radical
containing from 1 to 10, preferably from 1 to 6 and more preferably
from 1 to 4, carbon atoms. Examples of such radicals include, but
are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, decyl and
the like.
[0012] The term "alkenyl", alone or in combination, refers to a
straight-chain or branched-chain alkenyl radical containing from 2
to 10, preferably from 2 to 6 and more preferably from 2 to 4,
carbon atoms. Examples of such radicals include, but are not
limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- and
Z-butenyl, E- and Z-isobutenyl, E- and Z-pentenyl, decenyl and the
like.
[0013] The term "alkynyl", alone or in combination, refers to a
straight-chain or branched-chain alkynyl radical containing from 2
to 10, preferably from 2 to 6 and more preferably from 2 to 4,
carbon atoms. Examples of such radicals include, but are not
limited to, ethynyl (acetylenyl), propynyl, propargyl, butynyl,
hexynyl, decynyl and the like.
[0014] The term "cycloalkyl", alone or in combination, refers to a
cyclic alkyl radical containing from 3 to 8, preferably from 3 to
6, carbon atoms. Examples of such cycloalkyl radicals include, but
are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl and the like.
[0015] The term "cycloalkenyl", alone or in combination, refers to
a cyclic carbocycle containing from 4 to 8, preferably 5 or 6,
carbon atoms and one or more double bonds. Examples of such
cycloalkenyl radicals include, but are not limited to,
cyclopentenyl, cyclohexenyl, cyclopentadienyl and the like.
[0016] The term "aryl" refers to a carbocyclic aromatic group
selected from the group consisting of phenyl, naphthyl, indenyl,
indanyl, azulenyl, fluorenyl, and anthracenyl; or a heterocyclic
aromatic group selected from the group consisting of furyl,
thienyl, pyridyl, pyrrolyl, oxazolyly, thiazolyl, imidazolyl,
pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl,
1,2,3-oxadiazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl,
pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazinyl,
1,3,5-trithianyl, indolizinyl, indolyl, isoindolyl, 3H-indolyl,
indolinyl, benzo[b]furanyl, 2,3-dihydrobenzofuranyl,
benzo[b]thiophenyl, 1H-indazolyl, benzimidazolyl, benzthiazolyl,
purinyl, 4H-quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl,
phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl,
pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, and
phenoxazinyl.
[0017] "Aryl" groups, as defined in this application may
independently contain one to four substituents which are
independently selected from the group consisting of hydrogen,
halogen, hydroxyl, amino, nitro, trifluoromethyl, trifluoromethoxy,
alkyl, alkenyl, alkynyl, cyano, carboxy, carboalkoxy,
Ar'-substituted alkyl, Ar'-substituted alkenyl or alkynyl,
1,2-dioxymethylene, 1,2-dioxyethylene, alkoxy, alkenoxy or
alkynoxy, Ar'-substituted alkoxy, Ar'-substituted alkenoxy or
alkynoxy, alkylamino, alkenylamino or alkynylamino, Ar'-substituted
alkylamino, Ar'-substituted alkenylamino or alkynylamino,
Ar'-substituted carbonyloxy, alkylcarbonyloxy, aliphatic or
aromatic acyl, Ar'-substituted acyl, Ar'-substituted
alkylcarbonyloxy, Ar'-substituted carbonylamino, Ar'-substituted
amino, Ar'-substituted oxyl Ar'-substituted carbonyl,
alkylcarbonylamino, Ar'-substituted alkylcarbonylamino,
alkoxy-carbonylamino, Ar'-substituted alkoxycarbonyl-amino,
Ar'-oxycarbonylamino, alkylsulfonylamino, mono- or
bis-(Ar'-sulfonyl)amino, Ar'-substituted alkyl-sulfonylamino,
morpholinocarbonylamino, thiomorpholinocarbonylamino, N-alkyl
guanidino, N--Ar' guanidino, N--N-(Ar',alkyl) guanidino,
N,N-(Ar',Ar')guanidino, N,N-dialkyl guanidino, N,N,N-trialkyl
guanidino, N-alkyl urea, N,N-dialkyl urea, N--Ar' urea,
N,N-(Ar',alkyl) urea and N,N--(Ar').sub.2 urea; acylcarbonylamino;
Ar'-substituted aryl; aromatic acyl-substituted aromatic or
aliphatic acyl; Ar'-substituted heterocyclyl; Ar'-substituted
cycloalkyl or cycloalkenyl; heterocyclylalkoxy; N,N--(Ar',
hydroxyl) urea; Ar'-substituted cycloalkyl and cycloalkenyl;
Ar'-substituted biaryl; Ar'-substituted aminocarbonylamino;
Ar'-mercapto-substituted alkyl; Ar'-amino-substituted aryl;
Ar'-oxysubstituted alkyl; Ar'-substituted aminocycloalkyl and
cycloalkenyl; aralkylaminosulfonyl; aralkoxyalkyl;
N--Ar'-substituted thiourea; N-aralkoxyurea; N-hydroxylurea;
N-alkenylurea; N,N-(alkyl, hydroxyl)urea; heterocyclyl;
thioaryloxy-substituted aryl; N,N-(aryl,alkyl)hydrazino;
Ar'-substituted sulfonylheterocyclyl; aralkyl-substituted
heterocyclyl; cycloalkyl and cycloakenyl-substituted heterocyclyl;
cycloalkyl-fused aryl; aryloxy-substituted alkyl;
heterocyclylamino; Ar'-substituted arylaminosulfonyl;
thioaryl-substituted thioxy; and Ar'-substituted alkenoyl;
aliphatic or aromatic acylaminocarbonyl; aliphatic or aromatic
acyl-substituted alkenyl; Ar'-substituted aminocarbonyloxy;
Ar',Ar'-disubstituted aryl; aliphatic or aromatic acyl-substituted
acyl; benzofused-heterocyclylcarbonylamino; Ar'-substituted
hydrazino; Ar'-substituted aminosulfonyl; Ar'-substituted
alkylimino; Ar'-substituted heterocyclyl; Ar',Ar'-disubstituted
acylamino; Ar'-substituted cycloalkenonylamino; heterocyclylalkoxy;
N,N--Ar', hydroxylurea; N,N'-Ar', hydroxylurea;
heterocyclylcarbonylamino; Ar'-substituted
aminocarbonylheterocyclyl; Ar'-substituted aminocarbonyl;
Ar'-substituted carbonylamino; Ar'-substituted aminosulfonylamino;
Ar'-substituted mercaptoalkyl; Ar'-amino substituted biaryl;
araIkylaminoalkoxy; alkyl- and aryloxy-substituted alkoxy;
heterocyclylcarbonyl; Ar'-substituted sulfonylalkyl; Ar'-amino
carbocyclyl; aralkylsulfonyl; aryl-substituted alkenyl;
heterocyclylalkylamino; heterocyclylalkylaminocarbonyl;
Ar'-substituted sulfonylaminoalkyl; Ar'-substituted cycloalkyl;
thioaryloxyalkyl; thioaryloxymercapto; cycloalkylcarbonylalkyl;
cycloalkyl-substituted amino; Ar'-substituted arylamino;
aryloxycarbonylalkyl; phosphorodiamidyl acid or ester;
aryloxydimethylsiloxy; 1,3-indandionylcarbonylalkyl;
1,3-indandionylcarbonyl; oxamidyl; heterocyclylalkylidenyl;
formamidinyl; benzalizinyl; benzalhydrazino; arylsulfonylurea;
benzilylamino;
4-(N-2-carboxyalkyl-1-(1,3-benzodioxol-5-yl)-amino-N-leucinylalkylamidyla-
rylurea); Ar'-carbamoyloxy and alkyl- and aryloxy-substituted urea;
wherein "Ar'" is a carbocyclic or heterocyclic aryl group as
defined above having one to three substituents selected from the
group consisting of hydrogen, halogen, hydroxyl, amino, nitro,
trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl,
1,2-dioxymethylene, 1,2-dioxyethylene, alkoxy, alkenoxy, alkynoxy,
alkylamino, alkenylamino or alkynylamino, alkylcarbonyloxy,
aliphatic or aromatic acyl, alkylcarbonylamino,
alkoxycarbonylamino, alkylsulfonylamino, N-alkyl or N,N-dialkyl
urea.
[0018] The term "alkoxy", alone or in combination, refers to an
alkyl ether radical, wherein the term "alkyl" is as defined above.
Examples of suitable alkyl ether radicals include, but are not
limited to methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy,
iso-butoxy, sec-butoxy, tert-butoxy and the like.
[0019] The term "alkenoxy", alone or in combination, refers to a
radical of formula alkenyl-O--, wherein the term "alkenyl" is as
defined above provided that the radical is not an enol ether.
Examples of suitable alkenoxy radicals include, but are not limited
to, allyloxy, E- and Z-3-methyl-2-propenoxy and the like.
[0020] The term "alkynyloxy", alone or in combination, refers to a
radical of formula alkynyl-O--, wherein the term "alkynyl" is as
defined above provided that the radical is not an ynol ether.
Examples of suitable alkynoxy radicals include, but are not limited
to, propargyloxy, 2-butynyloxy and the like.
[0021] The term "thioalkoxyl" refers to a thioether radical of
formula alkyl-S--, wherein alkyl is as defined above.
[0022] The term "alkylamino", alone or in combination, refers to a
mono- or di-alkyl-substituted amino radical (i.e., a radical of
formula alkyl-NH-- or (alkyl).sub.2-N--), wherein the term "alkyl"
is as defined above. Examples of suitable alkylamino radicals
include, but are not limited to, methylamino, ethylamino,
propylamino, isopropylamino, t-butylamino, N,N-diethylamino and the
like.
[0023] The term "alkenylamino", alone or in combination, refers to
a radical of formula alkenyl-NH-- or (alkenyl).sub.2N--, wherein
the term "alkenyl" is as defined above, provided that the radical
is not an enamine. An example of such alkenylamino radicals is the
allylamino radical.
[0024] The term "alkynylamino", alone or in combination, refers to
a radical of formula alkynyl-NH-- or (alkynyl).sub.2N--, wherein
the term "alkynyl" is as defined above, provided that the radical
is not an ynamine. An example of such alkynylamino radicals is the
propargyl amino radical.
[0025] The term "aryloxy", alone or in combination, refers to a
radical of formula aryl-O--, wherein aryl is as defined above.
Examples of aryloxy radicals include, but are not limited to,
phenoxy, naphthoxy, pyridyloxy and the like.
[0026] The term "arylamino", alone or in combination, refers to a
radical of formula aryl-NH--, wherein aryl is as defined above.
Examples of arylamino radicals include, but are not limited to,
phenylamino (anilido), naphthylamino, 2-, 3- and 4-pyridylamino and
the like.
[0027] The term "biaryl", alone or in combination, refers to a
radical of formula aryl-aryl-, wherein the term "aryl" is as
defined above.
[0028] The term "thioaryl", alone or in combination, refers to a
radical of formula aryl-S--, wherein the term "aryl" is as defined
above. An example of a thioaryl radical is the thiophenyl
radical.
[0029] The term "aryl-fused cycloalkyl", alone or in combination,
refers to a cycloalkyl radical which shares two adjacent atoms with
an aryl radical, wherein the terms "cycloalkyl" and "aryl" are as
defined above. An example of an aryl-fused cycloalkyl radical is
the benzofused cyclobutyl radical.
[0030] The term "aliphatic acyl", alone or in combination, refers
to radicals of formula alkyl-CO--, alkenyl-CO-- and alkynyl-CO--
derived from an alkane-, alkene- or alkyncarboxylic acid, wherein
the terms "alkyl", "alkenyl" and "alkynyl" are as defined above.
Examples of such aliphatic acyl radicals include, but are not
limited to, acetyl, propionyl, butyryl, valeryl, 4-methylvaleryl,
acryloyl, crotyl, propiolyl, methylpropiolyl and the like.
[0031] The term "aromatic acyl", alone or in combination, refers to
a radical of formula aryl-CO--, wherein the term "aryl" is as
defined above. Examples of suitable aromatic acyl radicals include,
but are not limited to, benzoyl, 4-halobenzoyl, 4-carboxybenzoyl,
naphthoyl, pyridylcarbonyl and the like.
[0032] The terms "morpholinocarbonyl" and "thiomorpholinocarbonyl",
alone or in combination with other terms, refer to an
N-carbonylated morpholino and an N-carbonylated thiomorpholino
radical, respectively.
[0033] The term "alkylcarbonylamino", alone or in combination,
refers to a radical of formula alkyl-CONH, wherein the term "alkyl"
is as defined above.
[0034] The term "alkoxycarbonylamino", alone or in combination,
refers to a radical of formula alkyl-OCONH--, wherein the term
"alkyl" is as defined above.
[0035] The term "alkylsulfonylamino", alone or in combination,
refers to a radical of formula alkyl-SO.sub.2NH-- wherein the term
"alkyl" is as defined above.
[0036] The term "arylsulfonylamino", alone or in combination,
refers to a radical of formula aryl-SO.sub.2NH--, wherein the term
"aryl" is as defined above.
[0037] The term "N-alkylurea", alone or in combination, refers to a
radical of formula alkyl-NH--CO--NH--, wherein the term "alkyl" is
as defined above.
[0038] The term "N-arylurea", alone or in combination, refers to a
radical of formula aryl-NH--CO--NH--, wherein the term "aryl" is as
defined above.
[0039] The term "halogen" means fluorine, chlorine, bromine and
iodine.
[0040] The term "heterocycle" (and corresponding "heterocyclyl"
radical form) unless otherwise defined herein, refers to a stable
3-7 membered monocyclic heterocyclic ring or 8-11 membered bicyclic
heterocyclic ring which is unsaturated, and which may be optionally
benzofused. Each heterocycle consists of one or more carbon atoms
and from one to four heteroatoms selected from the group consisting
of nitrogen, oxygen and sulfur. As used herein, the terms "nitrogen
and sulfur heteroatoms" include any oxidized form of nitrogen and
sulfur, and the quaternized form of any basic nitrogen. In
addition, any ring nitrogen may be optionally substituted with a
substituent R.sup.4, as defined herein for compounds of formula I.
A heterocycle may be attached at any endocyciic carbon or
heteroatom which results in the creation of a stable structure.
Preferred heterocycles include 5-7 membered monocyclic heterocycles
and 8-10 membered bicyclic heterocycles. Heterocycles may be
optionally oxo-substituted at 1-3 ring positions and may optionally
be independently substituted with 1-4 substituents selected from
the group of "aryl" substituents described above.
[0041] The term "leaving group" generally refers to groups readily
displaceable by a nucleophile, such as an amine, and alcohol or a
thiol nucleophile. Such leaving groups are well known and include
carboxylates, N-hydroxysuccinimide, N-hydroxybenzotriazole, halogen
(halides), triflates, tosylates, mesylates, alkoxy, thioalkoxy and
the like.
[0042] The terms "activated derivative of a suitably protected
.alpha.-amino acid" and "activated substituted-phenylacetic acid
derivative" refer to the corresponding acyl halides (e.g. acid
fluoride, acid chloride and acid bromide), corresponding activated
esters (e.g. nitrophenyl ester, the ester of
1-hydroxybenzotriazole, HOBT, or the ester of hydroxysuccinimide,
HOSu), and other conventional derivatives within the skill of the
art.
[0043] In view of the above definitions, other chemical terms used
throughout this application can be easily understood by those of
skill in the art. Terms may be used alone or in any combination
thereof. The preferred and more preferred chain lengths of the
radicals apply to all such combinations.
[0044] This invention provides compounds which are capable of
inhibiting VLA-4-mediated cell adhesion by inhibiting the binding
of ligands to that receptor. These compounds are represented by
formula (I): ##STR2## and
[0045] pharmaceutically acceptable derivatives thereof;
[0046] wherein:
[0047] X is selected from the group consisting of --CO.sub.2H,
--PO.sup.-.sub.3H, --SO.sub.2R.sub.5, --SO.sub.3H,
--OPO.sup.-.sub.3H, --CO.sub.2R.sub.4 and --C(O)N(R.sub.4).sub.2;
[0048] wherein R.sub.5 is selected from the group consisting of
alkyl, alkenyl, alkynyl,.cycloalkyl, cycloalkenyl, aryl,
aryl-substituted alkyl, and aryl-substituted alkenyl or
alkynyl;
[0049] Y is selected from the group consisting of --CO--,
--SO.sub.2-- and --PO.sub.2--;
[0050] R.sub.1 is selected from the group consisting of alkyl,
alkenyl, alkynyl, cycloalkyl, aryl-fused cycloalkyl, cycloalkenyl,
aryl, aryl-substituted alkyl ("aralkyl"), aryl-substituted alkenyl
or alkynyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted
cycloalkyl, biaryl, alkoxy, alkenoxy, alkynoxy, aryl-substituted
alkoxy ("aralkoxy"), aryl-substituted alkenoxy or alkynoxy,
alkylamino, alkenylamino or alkynylamino, aryl-substituted
alkylamino, aryl-substituted alkenylamino or alkynylamino, aryloxy,
arylamino, N-alkylurea-substituted alkyl, N-arylurea-substituted
alkyl, alkylcarbonylamino-substituted alkyl,
aminocarbonyl-substituted alkyl, heterocyclyl,
heterocyclyl-substituted alkyl, heterocyclyl-substituted amino,
carboxyalkyl substituted aralkyl, oxocarbocyclyl-fused aryl and
heterocyclylalkyl;
[0051] R.sub.2 is selected from the group consisting of hydrogen,
aryl, alkyl, alkenyl or alkynyl, cycloalkyl, cycloalkenyl,
aryl-substituted alkyl and wherein R.sub.2 and R.sub.3 may be taken
together with the atoms to which they are attached, to form a
heterocycle;
[0052] R.sub.3 is selected from the group consisting of alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl,
aryl-substituted alkenyl or alkynyl, hydroxy-substituted alkyl,
alkoxy-substituted alkyl, aralkoxy-substituted alkyl,
amino-substituted alkyl, (aryl-substituted
alkyloxycarbonylamino)-substituted alkyl, thiol-substituted alkyl,
alkylsulfonyl-substituted alkyl, (hydroxy-substituted
alkylthio)-substitute alkyl, thioalkoxy-substituted alkyl,
acylamino-substituted alkyl, alkylsulfonylamino-substituted alkyl,
aryl-sulfonylamino-substituted alkyl, morpholino-alkyl,
thiomorpholino-alkyl, morpholino carbonyl-substituted alkyl,
thiomorpholinocarbonyl-substituted alkyl, [N-(alkyl, alkenyl or
alkynyl)- or N,N-[dialkyl, dialkenyl, dialkynyl or
(alkyl,alkenyl)-aminolcarbonyl-substituted alkyl,
carboxyl-substituted alkyl, dialkylamino-substituted acylaminoalkyl
and amino acid side chains selected from arginine, asparagine,
glutamine, S-methyl cysteine, methionine and corresponding
sulfoxide and sulfone derivatives thereof, glycine, leucine,
isoleucine, allo-isoleucine, tert-leucine, norleucine,
phenylalanine, tyrosine, tryptophan, proline, alanine, ornithine,
histidine, glutamine, valine, threonine, serine, aspartic acid,
beta-cyanoalanine, and allothreonine, wherein R.sub.2 and R.sub.3
may be taken together with the atoms to which they are attached, to
form a heterocycle;
[0053] R.sub.4 is selected from the group consisting of aryl,
alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl and
aryl-substituted alkyl, hydrogen, heterocyclyl,
heterocyclylcarbonyl, aminocarbonyl, amido, mono- or
dialkylaminocarbonyl, mono- or diarylaminocarbonyl,
alkylarylaminocarbonyl, diarylaminocarbonyl, mono- or
diacylaminocarbonyl, aromatic or aliphatic acyl, alkyl optionally
substituted by substituents selected from the group consisting of
amino, hydroxy, mercapto, mono- or dialkylamino, mono- or
diarylamino, alkylarylamino, diarylamino, mono- or diacylamino,
alkoxy, alkenoxy, aryloxy, thioalkoxy, thioalkenoxy, thioalkynoxy,
thioaryloxy and heterocyclyl; and
[0054] n is 0, 1 or 2.
[0055] A "pharmaceutically acceptable derivative" denotes any
pharmaceutically acceptable salt, ester, or salt of such ester, of
a compound of this invention. The invention also includes any other
compound which, upon administration to a patient, is capable of
providing (directly or indirectly) a compound of this invention
(e.g. a prodrug). The invention also includes metabolites or
residues of a compound of this invention characterized by the
ability to inhibit, prevent or suppress cell adhesion and cell
adhesion-mediated pathologies.
[0056] In another preferred embodiment of this invention, R.sub.1
is selected from the group consisting of benzyloxy, cyanomethyl,
cyclohexylmethyl, methyl, n-hexyl, N-phenylamino, phenyl,
phenylcarbonyl, phenyl-methyl, t-butoxy, t-butylamino, 1-indanyl,
1-naphthyl-methyl, 1-phenylcyclopropyl, 2-(4-hydroxyl-phenyl)ethyl,
2-(benzyloxycarbonylamino)-phenylmethyl,
2-(bis(phenyl-sulfonyl)amino)-phenylmethyl,
2-(N'-phenylurea)phenyl-methyl, 2-aminophenylmethyl,
2-benzamidophenylmethyl, 2-bromo-4-hydroxy-5-methoxyphenylmethyl,
2-hydroxyphenyl-methyl, 2-naphthylmethyl, 2-phenylethyl,
2-pyridylmethyl, 2-quinolinyl, 2-[4-(N'-phenylurea)phenyl]-ethyl,
3-(benzyloxycarbonylamino)-phenylmethyl, 3-
(N'-phenylurea)-phenylmethyl, 3-(N'-phenylurea)propyl,
3-(phenylsulfon-amido)-phenylmethyl, 3-acetamidophenyl-methyl,
3-amino-phenylmethyl, 3-benzamidophenylmethyl,
3-hydroxy-4-(N'-phenylurea)-phenylmethyl, 3-hydroxyphenylmethyl,
3-indolyl, 3-methyoxy-4-(N'-phenylurea)-phenylmethyl,
3-methoxy-4-(N'-(2-methylphenyl)urea)-phenylmethyl,
3-methyl-4-(N'-phenylurea)-phenylmethyl, 3-nitrophenylmethyl,
3-phenylpropyl, 3-pyridylmethyl, 4-(2-aminobenzamido)-phenylmethyl,
4-(benzamido)phenylmethyl,
4-(benzyloxy-carbonylamino)-phenylmethyl,
4-(morpholinocarbonyl-amino)-phenylmethyl,
4-(N'-(2-chlorophenyl)urea)-phenylmethyl,
4-(N'-(2-chlorophenyl)urea)-3-methoxyphenylmethyl,
4-(N'-(2-ethylphenyl)urea)-phenylmethyl,
4-(N'-(2-isopropylphenyl)urea)-phenylmethyl,
4-(N'-(2-methoxyphenyl)urea)phenylmethyl,
4-(N'-(2-methyl-3-pyridyl)urea)-phenyl-methyl,
4-(N'-(2-nitrophenyl)urea)-phenylmethyl,
4-(N'-(2-pyridyl)urea)-phenylmethyl,
4-(N'-(2-t-butylphenyl)-urea)-phenylmethyl,
4-(N'-(2-thiazolyl)urea)-phenyl-methyl,
4-(N'-(3-chlorophenyl)urea)-phenylmethyl,
4-(N'-(3-methoxyphenyl)urea)-phenylmethyl,
4-(N'-(3-pyridyl)-urea)-phenylmethyl,
4-(N'-(4-pyridyl)urea)-phenylmethyl,
4-(N'-(3-methylphenyl)urea)-phenylmethyl,
4-(N'-(2-methylphenyl)-urea)-phenylmethyl,
4-(N'-benzylurea)phenylmethyl, 4-(N'-cyclohexylurea)-phenylmethyl,
4-(N'-ethylurea)-phenylmethyl, 4-(N'-isopropylurea)-phenylmethyl,
4-(N'-methylurea)phenyl-methyl, 4-(N'-p-toluylurea)-phenyl-methyl,
4-(N'-phenylurea)phenyl, 4-(N'-phenylurea)phenyl-amino,
4-(N'-phenylurea)phenylmethyl, 4-(N'-t-butylurea)-phenylmethyl,
4-(phenylamindcarbonylamino-methyl)-phenyl,
4-(phenylsulfonamido)-phenylmethyl,
4-(t-butoxycarbonylamino)-phenylmethyl, 4-acetamido-phenylmethyl,
4-aminophenylamino, 4-amino-phenylmethyl, 4-benzamidophenylmethyl,
4-chlorophenylmethyl, 4-hydroxy-3-nitrophenylmethyl,
4-hydroxyphenylmethyl, 4-methoxyphenylmethyl, 4-nitrophenylamino,
4-nitrophenylmethyl, 4-phenacetamidophenylmethyl,
4-phenylphenylmethyl, 4-pyridylmethyl,
4-trifluoro-methylphenylmethyl,
4-(2-(N'-methylurea)benzamido]-phenylmethyl,
4-(N-'-(2-methylphenyl)urea) phenyl-methyl,
4-(N'-phenyl-N''-methylguanidino)phenyl-methyl,
5-(N'-phenylurea)pentyl, 5-(N'-t-butylurea)-pentyl,
2,2-dimethylpropyl, 2,2-diphenylmethyl, 2,3-benzocyclobutyl,
3,4-dihydroxyphenylmethyl, 3,5-dimethoxy-4-hydroxy-phenylmethyl,
4-(1-indolecarbo-xylamino)-phenylmethyl,
6-methoxy-5-(N'-(2-methyl-phenyl)urea)-2-pyridylmethyl,
4-(1,3-benzoxazol 2-ylamino)-phenylmethyl and
4-(1,3-imdazol-2-ylamino)-phenylmethyl, 3-carboxy-1-phenylpropyl;
3-hydroxy-4-(2-methylphenyl)ureaphenylmethyl;
3-hydroxy-4-(2-chlorophenyl)ureaphenylmethyl; 6-(phenylurea)heptyl,
4-phenylurea)butyl; 2-thienylmethyl;
4-(2,6-dimethylphenylurea)phenylmethyl;
4-(2-hydroxyphenylurea)phenylmethyl;
3-butoxy-4-(2-methylphenyl)ureaphenylmethyl;
3-butoxy-4-(phenylurea)phenylmethyl;
4-(N-2-pyrazinylurea)phenylmethyl; 2-phenylethynyl;
5-phenylurea-2-pyridylmethyl;
5-(2-methylphenylurea)-2-pyridylmethyl;
4-(3-methyl-2-pyridylurea)phenylmethyl;
3-nitro-4-(phenylurea)phenylmethyl;
3-acylamino-4-(phenylurea)phenylmethyl; 4-(N,N-phenyl,
methylurea)phenylmethyl; 4-(3-hydroxyphenylurea)phenylmethyl;
4-(2-acetylaminophenylurea)phenylmethyl;
4-(2-propionylaminophenylurea)phenylmethyl;
4-(3-benzyloxy-2-pyridylurea)phenylmethyl;
4-(3-methyl-2-pyridylurea)phenylmethyl;
4-(indolylcarbonylamino)phenylmethyl;
2-(4-(phenylurea)phenyl)oxiranyl; 4-(N,N'-phenyl,
methylurea)phenylmethyl; 4-(2-dimethylaminophenylurea)phenylmethyl;
4-(2-benzimidazolylamino)phenylmethyl;
4-(2-benzoxazolylamino)phenylmethyl;
4-(2-benzthiazolylamino)phenylmethyl;
4-(tetrahydroquinolinylcarbonylamino)phenylmethyl;
1,3-dimethyl-3-(phenylurea)butyl; hydroxyethylthiomethyl;
4-(phenylurea)phenylethenyl; 3-amino-4-(phenylurea)phenylmethyl;
4-(4-hydroxyphenylurea)phenylmethyl;
4-(2-aminophenylurea)phenylmethyl;
4-((2-methylurea)phenylurea)phenyl;
4-(2-hydroxyphenylurea)-3-methoxyphenylmethyl;
4-(2-methylsulfonylmethylphenylurea)phenylmethyl;
4-(2-methylphenylurea)tetrahydro-2-pyrimidonylmethyl;
3-methoxy-4-(phenylurea)-2-pyridylmethyl;
4-(2-trifluoromethylphenylurea)phenylmethyl;
4-(3-methyl-2-pyridylurea)phenylmethyl;
4-(2,4(1H,3H)-quinazolinedionyl)phenylmethyl;
4-thioureaphenylmethyl; 4-(phenylthiourea)phenylmethyl;
4-(pyrrolidinylcarbonylamino)phenylmethyl;
4-(2-benzoxazolinonylcarbonylamino)phenylmethyl;
4-(benzyloxyurea)phenylmethyl;
4-(thiazolidinylcarbonylamino)phenylmethyl;
4-benzoylureaphenylmethyl; hydroxylureaphenylmethyl;
N',N'-methyl,hydroxylureaphenylmethyl;
4-(N'-allylurea)phenylmethyl;
4-(3-pyrrolidinylcarbonylamino)phenylmethyl;
4-(1-pyrrolylcarbonylamino)phenylmethyl;
4-(2-pyrrolylcarbonylamino)phenylmethyl;
4-(propylurea)phenylmethyl; 4-(methoxyurea)phenylmethyl;
4-(dimethylurea)phenylmethyl; 4-(2-quinazolinylamino)phenylmethyl;
4-(2-furanoylamino)phenylmethyl;
4-(2-hydroxy-6-methylphenylurea)phenylmethyl;
4-(2-pyridylcarbonylamino)phenylmethyl;
4-(3-hydroxy-2-methylphenylurea)phenylmethyl;
4-(2-fluorophenylurea)phenylmethyl;
4-(3-fluorophenylurea)phenylmethyl;
4-(4-fluorophenylurea)phenylmethyl;
4-(2-quinolinylcarbonylamino)phenylmethyl;
4-(isoquinolinylcarbonylamino)phenylmethyl;
4-(2,3-dimethylphenylurea)phenylmethyl;
4-(2,5-dimethylphenylurea)phenylmethyl;
4-(2-methyl-4-fluorophenylurea)phenylmethyl;
4-(2-methyl-3-fluorophenylurea)phenylmethyl;
3-carboxy-3-phenylpropyl;
4-(5-hydroxy-2-methylphenylurea)phenylmethyl;.
4-(4-hydroxy-2-methylphenylurea)phenylmethyl;
4-(2,4-difluorophenylurea)phenylmethyl; 3-dibenzofuranylcarbonyl;
4-(phenoxycarbonylamino)phenylmethyl; 3-phenylureapropyl;
4-(phenylaminocarbonyloxy)phenylmethyl; 4-cinnamoylphenylmethyl;
dibenzofuranylmethyl;
4-(2-methylphenylaminocarbonyloxy)phenylmethyl;
methylphenylurea)phenylamino;
4-(3-indolylcarbonylamino)phenylmethyl;
4-(phenylaminocarbonyl)phenylmethyl; 4-phenylalkynylphenylmethyl;
4-(3-pyrrolylcarbonylamino)phenylmethyl; 5-nitrobenzofuran-2-yl;
5-(2-methylphenylurea-)benzofuran-2-yl; 3-carboxy-3-phenylpropyl;
2-(3-pyridyl)-thiazol-4-yl; 2-(4-pyridyl)-thiazol-4-yl; 2-oxo- and
4-oxo-4,5,6,7-tetrahydrobenzo[b]furan-3-yl;
3-methoxy-4-(phenylcarbamoyloxy)phenylnethyl;
5-amino-benzofuran-2-yl; benzilylaminophenylmethyl and
4-(N-2-carboxyethyl-1-(1,3-benzodioxolyl-5-yl)amino-N-leucinylacetamidylp-
henylurea)phenylmethyl.
[0057] Most preferably, R.sub.1 is selected from the group
consisting of 4-hydroxyphenylmethyl,
3-methoxy-4-(N'-phenylurea)-phenylmethyl,
4-(N'-phenylurea)-phenyl-methyl,
4-(N'-(2-methylphenyl)-urea)phenylmethyl,
4-(N'-2-pyridyl)-urea)-phenylmethyl,
3-methoxy-4-(N'-(2-methylphenyl)urea)phenylmethyl,
6-methoxy-5-(N'-(2-methylphenyl)urea)-2-pyridylmethyl,
4-(N'-3-methyl-2-pyridylurea)phenylmethyl,
3-methoxy-4-(N',-3-methyl-2-pyridylurea)phenylmethyl, and
3-methoxy-4-(N'-2-pyridylurea)phenylmethyl.
[0058] In an alternate preferred embodiment, R.sub.1 is an
aryl-substituted C.sub.1-C.sub.4 alkyl group. More preferably, is a
(N--Ar'-urea)-para-substituted arylalkyl group, and most
preferably, a (N--Ar'-urea)-para-substituted phenylmethyl
group.
[0059] According to another preferred embodiment, R.sub.2 is
selected from the group consisting of hydrogen, methyl or
phenacetyl. Most preferably, R.sub.2 is hydrogen.
[0060] According to another preferred embodiment, R.sub.3 is
selected from the group consisting of 2-(methyl-sulfonyl)-ethyl,
3-(hyrdoxypropylthio)-methyl, 4-(methylsulfonylamino)-butyl,
4-acetylaminobutyl, aminomethyl, benzyl, butyl, hydroxymethyl,
isobutyl, methyl, methylthiomethyl, phenylmethyl, propyl,
4-(benzyloxycarbonylamino)-butyl, N,N-(methylpropargyl) amino,
2-(methylthio)-ethyl, 2-(morpholino-N-carbonyl)-ethyl,
2-(N-morpholino)-ethyl, 2-(N,N-dimethylamino)-ethyl, 4-amino-butyl,
4-benzyloxyphenylmethyl, 2-benzylthiomethyl,
t-butoxycarbonylaminomethyl, sec-butyl, t-butyl,
N,N-dimethylaminocarbonylmethyl, 1,1-ethano,
*4-hydroxyphenylmethyl, 1-hydroxyethyl, 1-methoxyethyl,
4-methoxyphenylmethyl, benzyloxy-methyl,
[0061] *The amino acid side chain derived from
1-amino-cyclopropylcarboxylic acid. benzylthio-methyl,
carbonylmethyl, 2-methylsulfinyl-ethyl,
morpholino-N-carbonylmethyl, thiomorpholino-N-carbonylmethyl,
2-phenylethyl, asparagine side-chain, proline side-chain,
2-thiazolyl-methyl, 4-(phenylurea)butyl; 4-(methylurea)butyl;
morpholinocarbonylmethylthiomethyl; morpholinoethylthiomethyl;
3-pyridylmethyl; 4-methylsulfonylaminobutyl;
hydroxymethylthiomethyl; 2-methylsulfonylethyl,
4-propionylaminobutyl; 4-ethoxycarbonylaminobutyl;
methoxycarbonylaminobutyl; carbomethoxymethylthiomethyl;
4-t-butylureabutyl; carboxymethylthiomethyl;
dimethylamidomethylthiomethyl; acetylaminopropyl;
3-methylureapropyl; 4-biotinoylaminobutyl; 2-thienylmethyl;
3-pyridylmethyl; 4-trifluoroacetylaminobutyl;
dimethylaminomethylthiomethyl; dimethylaminoethylthiomethyl;
4-(dimethylaminoacetylamino)butyl or in combination with R.sub.2
forms a proline, azetidine or pipecolinic ring.
[0062] Most preferably, R.sub.3 is selected from the group
consisting of isobutyl, 2-(methylthio)-ethyl,
3-(hydroxypropylthio)-methyl, 2-(methylsulfonyl)-ethyl,
4-acetylamino-butyl, 4-(methylsulfonylamino)-butyl, and
4-(ethoxycarbonylamino)butyl.
[0063] According to yet another embodiment, R.sub.4 is selected
from the group consisting of 4-carbomethoxy-phenyl,
4-carboxyphenyl, 4-fluorophenyl, 4-methoxy-phenyl, benzyl, methyl,
phenyl, phenylmethyl, phenylethyl, 4-chlorophenyl,
3,4-difluorophenyl, 3,4-dimethoxyphenyl, 2-methoxyphenyl,
3-methoxyphenyl, 4-methoxyphenyl, 2-nitrophenyl, 3-pyridyl,
4-phenoxyphenyl; 4-ethoxyphenyl; 4-nitrophenyl;
4-acetylaminophenyl; 4-methylureaphenyl; 2-fluorophenyl; naphthyl;
3-fluorophenyl; 3-nitrophenyl; hydrogen; 2-nitrophenyl;
4-cyanophenyl; 3-methoxyphenyl; 4-methylsulfonylamino;
3-cyanophenyl; 4-propionylamino; 4-aminophenyl; 3-aminophenyl;
4-trifluoromethoxyphenyl; 4-methylphenyl; 4-amino-3-nitrophenyl;
4-hydroxy-3-methoxyphenyl; 4-hexyloxyphenyl; 4-methylthiophenyl;
3-furanyl; 4-dimethylaminophenyl; 3-hydroxy-4-nitrophenyl;
n-pentyl; carboxymethyl; 2-carboxyethyl; ethynyl; 2-thienyl;
2-propenyl; 2-propynyl; methyl; and propyl. More preferably,
R.sub.4 is selected from the group consisting 4-methoxyphenyl,
3,4-dimethoxyphenyl, 4-fluorophenyl, 4-carboxyphenyl,
4-carbomethoxyphenyl, phenylethyl, phenylmethyl, allyl, ethynyl,
and 3,4-methylenedioxyphenyl.
[0064] In another preferred embodiment Y is CO, CH.sub.2 or
SO.sub.2. Most preferably, Y is CO.
[0065] According to another preferred embodiment, X in formula (I)
is COOH.
[0066] According to yet another preferred embodiment, n is 1.
[0067] Examples of some preferred compounds of this invention
wherein X is a carboxyl group and n is 1 are provided in Table 1.
TABLE-US-00001 TABLE 1 ##STR3## Bio # R.sup.1 R.sup.2 R.sup.3
R.sup.4 Y 1002 cyanomethyl H isobutyl phenyl CO 1003
cyclohexylmethyl H isobutyl phenyl CO 1004 2-pyridylmethyl H
isobutyl phenyl CO 1005 3-pyridylmethyl H isobutyl phenyl CO 1006
4-hydroxyphenylmethyl H isobutyl 1,3-benzo- CO dioxol-5-yl 1007
4-pyridylmethyl H isobutyl phenyl CO 1008 phenyl H isobutyl phenyl
CO 1009 4-phenylphenylmethyl H isobutyl phenyl CO 1010
4-chlorophenylmethyl H isobutyl phenyl CO 1011
4-trifluoromethylphenylmethyl H isobutyl phenyl CO 1013
phenylmethyl H isobutyl phenyl SO.sub.2 1014 3-indolyl H isobutyl
phenyl CO 1015 4-benzamidophenylmethyl H isobutyl phenyl CO 1016
4-aminophenylmethyl H isobutyl phenyl CO 1017 1-phenylcyclopropyl H
isobutyl phenyl CO 1018 3-acetamidophenylmethyl H isobutyl phenyl
CO 1020 3-benzamidophenylmethyl H isobutyl phenyl CO 1021
1-naphthylmethyl H isobutyl phenyl CO 1022 2-naphthylmethyl H
isobutyl phenyl CO 1023 4-phenacetamidophenylmethyl H isobutyl
phenyl CO 1024 2-aminophenylmethyl H isobutyl phenyl CO 1025
2-(bis(phenylsulfonyl)amino)- H isobutyl phenyl CO phenylmethyl
1026 2-benzamidophenylmethyl H isobutyl phenyl CO 1027
2-(benzyloxycarbonylamino)- H isobutyl phenyl CO phenylmethyl 1028
4-(2-aminobenzamido)- H isobutyl phenyl CO phenylmethyl 1029
4-[2-(N'-methylurea)-benzamido]- H isobutyl phenyl CO phenylmethyl
1030 3-aminophenylmethyl H isobutyl phenyl CO 1031
3-(benzyloxycarbonylamino)- H isobutyl phenyl CO phenylmethyl 1032
3-(phenylsulfonamido)- H isobutyl phenyl CO phenylmethyl 1036
phenylmethyl H isobutyl 1,3-benzo- CO dioxol-5-yl 1037
4-(N'-phenylurea)- H 2-thiazolyl- phenyl CO phenylmethyl methyl
1038 phenylmethyl H propyl phenyl CO 1039 phenylmethyl H butyl
phenyl CO 1040 phenylmethyl H sec-butyl phenyl CO 1041 t-butoxy H
hydroxymethyl phenyl CO 1042 t-buyoxy H phenylmethyl phenyl CO 1043
t-butoxy H 1,1-ethano phenyl CO 1044 t-butoxy methyl isobutyl
phenyl CO 1045 phenylmethyl H hydroxymethyl phenyl CO 1046
phenylmethyl H phenylmethyl phenyl CO 1047 phenylmethyl H proline
side-chain phenyl CO 1048 phenylmethyl H 1,1-ethano.sup.1 phenyl CO
1049 B phenylmethyl H asparagine phenyl CO side-chain 1050
4-(N'-phenylurea)phenylmethyl H isobutyl 1,3-benzo- CO dioxol-5-yl
1051 4-(N'-phenylurea)phenyl H isobutyl phenyl CO 1052
2-[4-(N'-phenylurea)phenyl]- H isobutyl phenyl CO thyl 1053
4-(N'-phenylurea)phenylmethyl methyl isobutyl phenyl CO 1054
3-(N'-phenylurea)phenylmethyl H isobutyl phenyl CO 1055
4-(N'-phenylurea)phenylmethyl methyl isobutyl 1,3-benzo- CO
dioxol-5-yl 1056 3-methoxy-4-(N'-phenylurea)- H isobutyl 1,3-benzo-
CO phenylmethyl dioxol-5-yl 1057 3-hydroxy-4-(N-phenylurea)- H
isobutyl 1,3-benzo- CO phenylmethyl dioxol-5-yL 1058
3-methyl-4-(N'-phenylurea)- H isobutyl 1,3-benzo- CO phenylmethyl
dioxol-5-yl 1060 4-(N'-phenylurea)phenylmethyl H isobutyl phenyl CO
1063 4-(N'-phenylurea)phenylmethyl H isobutyl benzyl CO 1064
4-(N'-methylurea)phenylmethyl H isobutyl 1,3-benzo- CO dioxol-5-yl
1065 4-(N'-isopropylurea)- H isobutyl 1,3-benzo- CO phenylmethyl
dioxol-5-yl 1066 4-(N'-phenylurea)phenylmethyl H isobutyl
1,3-benzo- CO dioxol-5-yl 1067 4-(N'-p-toluylurea)- H isobutyl
1,3-benzo- CO phenylmethyl dioxol-5-yl 1068 4-(N'-cyclohexylurea)-
H isobutyl 1,3-benzo- CO phenylmethyl dioxol-5-yl 1069
4-(N'-phenylurea)phenylmethyl H isobutyl 2-methoxy CO phenyl 1070
4-hydroxyphenylmethyl H isobutyl 2-methoxy CO phenyl 1072
4-(N'-phenylurea)phenylmethyl H isobutyl 3-methoxy CO phenyl 1073
4-(benzyloxycarbonylamino)- H isobutyl 1,3-benzo- CO phenylmethyl
dioxol-5-yl 1074 4-(phenylsulfonamido) H isobutyl 1,3-benzo- CO
phenyl-methyl dioxol-5-yl 1075 4-(benzamido)phenylmethyl H isobutyl
1,3-benzo- CO dioxol-5-yl 1076 4-(N'-t-butylurea)phenylmethyl H
isobutyl 1,3-benzo- CO dioxol-5-yl 1077
4-(N'-ethylurea)phenylmethyl H isobutyl 1,3-benzo- CO dioxol-5-yl
1078 4-(N'-(3-methaxyphenyl)urea)- H isobutyl 1,3-benz- CO
phenylmethyl dioxol-5-yl 1079 4-(N'-(2-methoxyphenyl)urea)- H
isobutyl 1,3-benzo- CO phenylmethyl dioxol-5-yl 1080
4-(N'-(3-pyridyl)urea)- H isobutyl 1,3-benzo- CO phenylmethyl
dioxol-5-yl 1081 phenylmethyl H isobutyl phenyl CO 1082
3-phenylpropyl H isobutyl phenyl CO 1083 methyl H isobutyl phenyl
CO 1084 2-(4-hydroxyphenyl)ethyl H isobutyl phenyl CO 1085
benzyloxy H isobutyl phenyl CO 1086 N-phenylamino H isobutyl phenyl
CO 1087 2-(4-hydroxyphenyl)ethyl methyl isobutyl phenyl CO 1088
4-(N'-phenylurea)phenylmethyl H isobutyl 4-methoxy CO phenyl 1089
4-(N'-phenylurea)phenylmethyl H 2-(methylthio)- 4-methoxy CO ethyl
phenyl 1090 4-(N'-phenylurea)phenylmethyl H isobutyl 1,3-benzo- CO
dioxol-5-yl 1091 4-hydroxyphenylmethyl H isobutyl phenyl CO 1092
4-methoxyphenylmethyl H isobutyl phenyl CO 1093 4-nitrophenylmethyl
H isobutyl phenyl CO 1094 n-hexyl H isobutyl phenyl CO 1096
2-hydroxyphenylmethyl H isobutyl phenyl CO 1097
3-hydroxyphenylmethyl H isobutyl phenyl CO 1098
3,4-dihydroxyphenylmethyl H isobutyl phenyl CO 1099
2,2-diphenylethyl H isobutyl phenyl CO 1100 2-bromo-4-hydroxy-5- H
isobutyl phenyl CO methoxyphenylmethyl 1101 4-(b
nzyloxycarbonylamino)- H isobutyl phenyl CO phenylmethyl 1102
2-(N'-phenylurea)phenylmethyl H isobutyl phonyl CO 1103
4-aminophenylmethyl H isobutyl phenyl CO 1104
4-(phenylsulfonamido)phenyl- H isobutyl phenyl CO methyl 1105
4-(benzamido)phenylmethyl H isobutyl phenyl CO 1106
5-(N'-phenylurea)pentyl H isobutyl phenyl CO 1107
5-(N'-t-butylurea)pentyl H isobutyl phenyl CO 1108
4-nitrophenylamino H isobutyl phenyl CO 1109 4-aminophenylamino H
isobutyl phenyl 1110 4-(N'-phenylurea)phenylamino H isobutyl phenyl
1111 3,5-dimethoxy-4-hydroxy- H isobutyl phenyl phenylmethyl 1112
4-hydroxy-3-nitrophenylmethyl H isobutyl phenyl 1113
3-nitrophenylmethyl H isobutyl phenyl 1114 phenylmethyl methyl
isobutyl phenyl CO 1115 phenylmethyl H isobutyl 4-chloro CO phenyl
1116 phenylmethyl H 1-hydroxy-ethyl phenyl CO 1117 phenylmethyl H
1-methoxy-ethyl phenyl CO 1119 phenylmethyl H methyl phenyl CO 1120
phenylmethyl methyl methyl phenyl CO 1122 phenylmethyl H 4-methoxy-
phenyl CO phenylmethyl 1123 phenylmethyl H 2-phenylethyl phenyl CO
1124 phenylmethyl H 4-benzyloxy- phenyl CO phenylmethyl 1125 ph
nylmethyl H 4-hydroxy- phenyl CO phenylmethyl 1126 phenylmethyl H
benzyloxy- phenyl CO methyl 1127 phenylmethyl H benzylthio- phenyl
CO methyl 1128 4-(N'-phenylurea)phanylmethyl H isobutyl 1,3-benzo-
CO dioxol-5-yl 1129 4-(N'-phenylurea)phenylmethyl H benzyl
1,3-benzo- CO dioxol-5-yl 1130 4-(N'-phenylurea)phenylmethyl H
benzyl phenyl CO 1131 4-(N'-phenylurea)phenylmethyl H sec-butyl
phenyl CO 1132 4-(N'-phenylurea)phenylmethyl H 4-(benzyloxy- phenyl
CO carbonylamino)- butyl 1133 4-(N'-phenylurea)phenylmethyl H
sec-butyl 1,3-benzo- CO dioxol-5-yl 1134
4-(N'-phenylurea)phenylmethyl H t-butoxy- phenyl CO carbonylamino-
methyl 1135 4-(N'-phenylurea)phenylmethyl H 2-(methylthio)- phenyl
CO ethyl 1136 4-(N'-phenylurea)phenylmethyl H 2-benzylthio- phenyl
CO methyl 1137 phenylmethyl H isobutyl 2-nitro CO phenyl 1138
4-(N'-phenylurea)phenylmethyl H aminomethyl phenyl CO 1139
4-(N'-phenylurea)phenylmethyl H 4-amino-butyl phenyl CO 1140
phenylcarbonyl H isobutyl phenyl CH.sub.2 1141 phenylcarbonyl phen-
isobutyl phenyl CH.sub.2 acyl 1142 2,3-benzocyclobutyl H isobutyl
phenyl CO 1143 4-hydroxyphenylmethyl H isobutyl benzyl CO 1144
4-hydroxyphenylmethyl H isobutyl phenyl CO 1145
4-(t-butoxycarbonylamino)- H isobutyl phenyl CO phenylmethyl 1146
4-hydroxyphenylmethyl H isobutyl 3-methoxy CO phenyl 1147
4-acetamidophenylmethyl H is butyl phenyl CO 1148
4-hydroxyphenylmethyl H isobutyl 3-pyridyl CO 1149 2-quinolinyl H
isobutyl phenyl CO 1150 2-phenylethyl H isobutyl phenyl CO 1152
2,2-dimethylpropyl H isobutyl phenyl CO 1153 benzyloxy H isobutyl
3-pyridyl CO 1154 t-butylamino H isobutyl phenyl CO 1155
phenylmethyl H t-butyl phenyl CO 1156 methyl H t-butyl phenyl CO
1157 phenylmethyl H isobutyl benzyl CO 1158 phenylmethyl H isobutyl
1,3-benzo- CO dioxol-5-yl 1159 phenylmethyl H isobutyl 2-methoxy CO
phenyl 1160 phenylmethyl H isobutyl 3-methoxy CO phenyl 1162
benzyloxy H isobutyl methyl CO 1163 4-(N'-phenylurea)phenylmethyl H
2-(methylthio)- 1,3-benzo- CO ethyl dioxol-5-yl 1164 phenylmethyl H
2-(methylthio)- 1,3-benzo- CO ethyl dioxol-5-yl 1168
4-(N'-(m-toluyl)urea)- H isobutyl 1,3-benzo- CO phenylmethyl
dioxol-5-yl 1169 4-(N'-benzylurea)- H isobutyl 1,3-benzo- CO
phenylmethyl dioxol-5-yl 1170 4-(N'-phenylurea)phenylmethyl H
morpholino-N- 1,3-benzo- CO carbonylmethyl dioxol-5-yl 1173
4-hydroxyphenylmethyl H isobutyl 4-methoxy CO phenyl 1174
4-hydroxyphenylmethyl H 2-(methylthio)- 4-methoxy CO ethyl phenyl
1175 phenylmethyl H 2-(methylthio)- 4-methoxy CO ethyl phenyl 1176
4-(N'-phanylurea)phenylmethyl H thiomorpholino-N- 1,3-benzo- CO
carbonylmethyl dioxol-5-yl 1177 4-(N'-phenylurea)phenylmethyl H
N,N-(methylprop 1,3-benzo- CO argyl)amino dioxol-5-yl
carbonyl-methyl 1178 phenylmethyl H isobutyl 4-methoxy CO phenyl
1179 4-(N'-(o-toluyl)urea)- H isobutyl 1,3-benzo- CO phenylmethyl
dioxol-5-yl 1180 4-(N'-(2-thiazolyl)urea)- H isobutyl 4-methoxy CO
phenylmethyl phenyl 1181 4-(N'-(3-chlorophenyl)urea)- H isobutyl
1,3-benzo- CO phenylmethyl dioxol-5-yl 1182 4-(N'-(4-pyridyl)urea)-
H isobutyl 1,3-benzo- CO phenylmethyl dioxol-5-yl 1185
4-(N'-(2-chlorophenyl)urea)- H isobutyl 1,3-benzo- CO
phenylmethyl dioxol-5-yl 1186 4-(N'-phenylurea)phenylmethyl H
isobutyl isobutylamino- CO carbonyl 1187 3-(N'-phenylurea)propyl H
isobutyl phenyl CO 1188 1-phenylcyclopropyl H isobutyl 1,3-benzo-
CO dioxol-5-yl 1189 1-indanyl H isobutyl phenyl CO 1190
4-(N'-(o-toluyl)urea)- H isobutyl 4-methoxy CO phenylmethyl phenyl
1191 4-(N'-phenylurea)phenylmethyl H 2-(N-morpholino)- 1,3-benzo-
CO ethyl dioxol-5-yl 1192 4-(N'-(2-methoxyphenyl)urea)- H isobutyl
4-methoxy CO phenylmethyl phenyl 1193 4-(N'-phenylurea)phenylmethyl
methyl isobutyl 4-methoxy CO phenyl 1194 4-(N'-(2-pyridyl)urea)- H
isobutyl 4-methoxy CO phenylmethyl phenyl 1195
4-(N'-phenylurea)phenylmethyl H isobutyl 3,4-difluoro- CO phenyl
1196 4-(N'-phenylurea)phenylmethyl H is butyl 3,4-dimeth CO
oxy-phenyl 1197 4-(N'-(o-toluyl)urea)- H isobutyl phenyl CO
phenylmethyl 1198 4-(morpholinocarbonylamino)- H isobutyl
1,3-benzo- CO phenylmethyl dioxol-5-yl 1199
4-(N'-phenylurea)phenylmethyl H 2-methyl- 4-methoxy CO
sulfinylethyl phenyl 1200 4-(N'-(2-ethylphenyl)urea)- H isobutyl
1,3-benzo- CO phenylmethyl dioxol-5-yl 1201
4-(N'-(2-nitrophenyl)urea)- H isobutyl 1,3-benzo- CO phenylmethyl
dioxol-5-yl 1206 4-(N'-(2-isopropylphenyl)urea)- H isobutyl
1,3-benzo- CO phenylmethyl dioxol-5-yl 1207
4-(N'-(2-isopropylphenyl)urea)- H isobutyl 4-methoxy CO
phenylmethyl phenyl 1208 4-(N'-(2-ethylphenyl)urea)- H isobutyl
4-methoxy CO phenylmethyl phenyl 1209 4-(N'-(2-t-butylphenyl)urea)-
H isobutyl 1,3-benzo- CO phenylmethyl dioxol-5-yl 1210
4-(N'-(o-toluyl)urea)- H isobutyl 1,3-benzo- CO phenylmethyl
dioxol-5-yl 1212 4-(N'-(o-toluyl)urea)- H isobutyl 3,4-dimethoxy CO
phenylmethyl phenyl 1214 4-(N'-phenylurea)phenylmethyl H
N,N-dimethyl 1,3-benzo- CO amino- dioxol-5-yl carbonylmethyl 1215
4-(N'-phenylurea)phenylmethyl H 2-(N,N-dimethyl- 1,3-benzo-
amino)-ethyl dioxol-5-yl 1216 4-(N'-phenylurea)phenylmethyl H
2-(morpholino-N- 1,3-benzo- CO carbonyl)-ethyl dioxol-5-yl 1217
4-(N'-(o-toluyl)urea)- H 4-(benzyloxy- 3,4-dimethoxy CO
phenylmethyl carbonylamino)- phenyl butyl 1218
4-(N'-(2-pyridyl)urea)- H isobutyl 3,4-dimethoxy CO phenylmethyl
phenyl 1219 4-(N'-(3-pyridyl)urea)- H isobutyl 3,4-dim thoxy CO
phenylmethyl ph nyl 1220 4-(N'-(2-methyl-3-pyridyl) H isobutyl
4-methoxy CO urea)-phenylmethyl phenyl 1221
3-methoxy-4-(N'-(o-toluyl)- H isobutyl 1,3-benzo- CO
urea)phenylmethyl dioxol-5-yl 1222 4-(N'-(2-chlorophenyl)urea)- H
isobutyl 1,3-benzo- CO 3-methoxyphenylmethyl dioxol-5-yl 1223
4-(phenylaminocarbonylamino- H isobutyl 1,3-benzo- CO
methyl)-phenyl dioxol-5-yl 1224 4-(N'-(o-toluyl)urea)- H
2-(methylthio)- 3,4-dimethoxy CO phenylmethyl ethyl phenyl 1225
4-(N'-(o-toluyl)urea)- H 4-(benzyloxy- 1,3-benzo- CO phenylmethyl
carbonylamino)- dioxol-5-yl butyl 1227 4-(N'-(o-toluyl)urea)- H
methylthiomethyl 1,3-benzo- CO phenylmethyl dioxol-5-yl 1238
4-(N'-(o-toluyl)urea)- H 2-(methylthio)- 4-methoxy CO phenylmethyl
ethyl phenyl 1245 4-(N'-(o-toluyl)urea)- H 2-(methyl-sulfonyl)-
1,3-benzo- CO phenylmethyl ethyl dioxol-5-yl 1246
4-(N'-(o-toluyl)urea)- H 3-(hyrdoxypropy- 1,3-benzo- CO
phenylmethyl thio)-methyl dioxol-5-yl 1248 4-(N'-(o-toluyl)urea)- H
isobutyl 4-fluorophenyl CO phenylmethyl 1270 4-(N'-(o-toluyl)urea)-
H 4-acetylamino- 1,3-benzo- CO phenylmethyl butyl dioxol-5-yl 1272
4-(N'-(2-methylphenyl) H 4-(methoxy 1,3-benzo- CO urea)
phenylmethyl carbonyl amino) dioxol-5-yl butyl 1282
4-(N'-(o-toluyl)urea)-pyrid-5- H isobutyl 1,3-benzo- CO yllmethyl
dioxol-5-yl 1294 4-(N'-(o-toluyl)urea)- H 4-(methylsulfonyl-
1,3-benzo- CO phenylmethyl amino)-butyl dioxol-5-yl 1311
4-(N'-(3-methyl-2-pyridyl) H 4-(methoxy 3,4-dimethoxy CO urea)
phenylmethyl carbonyl amino) phenyl butyl 1319 4-(indolylcarbonyl H
isobutyl 1,3-benzo- CO amino)phenylmethyl dioxol-5-yl 1321
4-(N'-(o-toluyl)urea)- H isobutyl 4- CO phenylmethyl carboxyphenyl
1327 4-(1-indolecarboxylamino)- H isobutyl 1,3-benzo- CO
phenylmethyl diaxol-5-yl 1336 6-methoxy-5-(N'-(o-toluyl) H isobutyl
1,3-benzo- CO urea)-2-pyridylmethyl dioxol-5-yl 1345
4-(N'-(o-toluyl)urea)- H dimethylamino 1,3-benzo- CO phenylmethyl
ethyithiomethyl dioxol-5-yl 1347 4-(N'-2-pyridyl) H 2-(methylthio)-
3,4-dimethoxy- CO urea)phenylmethyl ethyl phenyl 1358
4-(N'-phenylthiourea) H isobutyl 1,3-benzo- CO phenylmethyl
dioxol-5-yl 1360 4-(N'-(o-toluyl)urea)- H isobutyl 2,3-dihydro- CO
phenylmethyl benzofuran-5- yl 1361 4-(N'-(o-totuyl)urea)- H
methylthio 4-carbometh- CO phenylmethyl ethyl oxy phenyl 1380
4-(N'-phenyl-N''-methyl- H isobutyl 1,3-benzo- CO
guanidino)-phenylmethyl dioxol-5-yl 1382 4-(N'-(o-toluyl)urea)- H
4-(methylsulfonyl- 4-carbometh- CO phenylmethyl amino)-butyl
oxy-phenyl 1388 4-(phenylurea) H isobutyl 4-carbometh- CO
phenylmethyl oxy-phenyl 1390 4-(1,3-imidazol-2-ylamino)- H isobutyl
1,3-benzo- CO phenylmethyl dioxol-5-yl 1393 4-(N'-(2-pyridyl) H
2-(methylthio)- 1,3-benzo- CO urea) phenylmethyl ethyl dioxol-5-yl
1396 4-(1,3-benzoxazol-2-ylamino)- H isobutyl 1,3-benzo- CO
phenylmethyl dioxol-5-yl 1400 4-(N'-(2-methylphenyl)urea)- H
isobutyl phenylethyl CO phenylmethyl 1429
4-(N'-(3-methyl-2-pyridyl) H 2-(methylthio)- 1,3-benzo- CO urea)
phenylmethyl ethyl dioxol-5-yl 1444 4-(2-b nzoxazolinonyl H
isobutyl 1,3-benzo- CO carbonylamino)phenyl- dioxol-5-yl methyl
1474 4-(2-pyrrolylcarbonylamino) H isobutyl 1,3-benzo- CO
phenylmethyl dioxol-5-yl 1475 4-(N'-allylurea)phenyl- H isobutyl
1,3-benzo- CO methyl dioxol-5-yl 1490 4-(N'-(2-methylphenyl) H
isobutyl ethynyl CO urea)phenylmethyl 1515 4-(N'-(2-methylphenyl) H
isobutyl allyl CO urea)phenylmethyl 1525 4-(N'-(2-fluorophenyl) H
isobutyl 3,4-diemthoxy- CO urea)phenylmethyl phenyl 1526
4-(4-fluorophenylurea) H isobutyl 3,4-dimethoxy CO phenylmethyl
phenyl 1536 4-(N'-(2-methylphenyl) H isobutyl methyl CO
urea)phenylmethyl 1594 4-(N'-2-methylpenylurea)- H isobutyl H CO
phenylmethyl 1648 4-(2-indoylycarbonylamino) H isobutyl H CO
phenylmethyl 1655 4-(3-indolylcarbonylamino) H isobutyl 1,3-benzo-
CO phenylmethyl dioxol-5-yl 1721 4-(N'-(2-methylphenyl) H isobutyl
morpholino- CO urea)phenylmethyl methyl 1725 3-methoxy4-(N'-phenyl
urea) H 2-(methylthio)- 1,3-benzo- CO phenylmethyl ethyl
dioxol-5-yl 1726 3-methoxy-4-(N'-phenyl urea) H isobutyl
3,4-dimethoxy- CO phenylmethyl phenyl 1727 3-methoxy-4-(N'-phenyl
urea) H 2-(methylthio)- 3,4-dimethoxy- CO phenylmethyl ethyl phenyl
1728 3-methoxy-4-(N'-2-pyridyl urea) H isobutyl 3,4-dimethoxy- CO
phenylmethyl phenyl 1729 3-methoxy-4-(N'-3-methyl-2- H isobutyl
3,4-dimethoxy- CO pyridyl) ur a phenylmethyl phenyl 1730
3-methoxy-4-(N'-3-methyl-2- H 2-(methylthio)- 3,4-dimethoxy- CO
pyridyl) urea phenylmethyl ethyl phenyl 1731
3-methoxy-4-(N'-3-methyl-2- H 2-(methylthio)- 1,3-benzo- CO
pyridyl) urea phenylmethyl ethyl di xol-5-yl 1732
4-(N'-(3-methyl-2-pyridyl) H 2-(methylthio)- 3,4-dimethoxy- CO
urea) phenylmethyl ethyl phenyl
[0068] The more preferred compounds of formula (I) are: BIO-1006,
BIO-1056, BIO-1089, BIO-1179, BIO-1194, BIO-1221, BIO-1224,
BIO-1238, BIO-1245, BIO-1246, BIO-1248, BIO-1270, BIO-1282,
BIO-1294, BIO-1321, BIO-1336 BIO-1382 and BIO-1400. Even more
preferred compounds are BIO-1218, BIO-1272, BIO-1311, BIO-1319,
BIO-1345, BIO-1347, BIO-1358, BIO-1361, BIO 1388, BIO-1390,
BIO-1393, BIO-1396, BIO-1429, BIO-1444, BIO-1474, BIO-1475,
BIO-1490,- BIO-1515, BIO-1525, BIO-1526, BIO-1536, BIO-1594,
BIO-1648, BIO-1655, BIO-1721, BIO-1725, BIO-1726, BIO-1727,
BIO-1728, BIO-1729, BIO-1730, BIO-1731, and BIO-1732. Most
preferred are BIO-1218, BIO-1272, BIO-1311, BIO-1347, BIO-1393,
BIO-1429, BIO-1515, BIO-1725, BIO-1726, BIO-1727, BIO-1728,
BIO-1729, BIO-1730, BIO-1731, and BIO-1732.
[0069] Compounds of this invention may be synthesized using any
conventional technique. Preferably, these compounds are chemically
synthesized from readily available starting materials, such as
.alpha.-amino acids. Modular and convergent methods for the
synthesis of these compounds are also preferred. In a convergent
approach, for example, large sections of the final product are
brought together in the last stages of the synthesis, rather than
by incremental addition of small pieces to a growing molecular
chain.
[0070] According to one embodiment, compounds of the present
invention may be synthesized in the following manner. A protected
chiral amine is added to an .alpha.,.beta.-unsaturated ester to
produce a protected .beta.-amino acid ester. Upon suitable
deprotection, the .beta.-amino acid ester is coupled to an
appropriate activated-ester moiety. The coupled product, if
suitably functionalized, may be further reacted with yet another
activated ester moiety. This material can be further manipulated to
give the desired compounds of the invention. At each step of the
above sequence, the ester can be hydrolyzed to the corresponding
acid to give another compound of the invention.
[0071] Alternatively, the activated ester moieties mentioned above
can be attached together first, then the resulting compound can be
attached to the .beta.-amino acid ester portion. At this point the
final manipulations and/or necessary deprotection steps can be
performed.
[0072] Alternatively, under suitable conditions, the desired
functionalities can be incorporated (protected or unprotected) in
one of the activated ester moieties. That piece is then coupled
with a .beta.-amino acid ester or a moiety consisting of a
.beta.-amino ester previously coupled to an activated ester. The
resulting product can then be subjected to any deprotection steps,
if necessary, to give compounds of the invention.
[0073] Alternatively, the chiral .beta.-amino acid esters used in
the synthesis of the compounds of this invention may be synthesized
by well-known techniques, such as those described in U.S. Pat. No.
5,344,957, the disclosure of which is herein incorporated by
references.
[0074] The compounds of this invention may also be modified by
appending appropriate functionalities to enhance selective
biological properties. Such modifications are known in the art and
include those which increase biological penetration into a given
biological system (e.g., blood, lymphatic system, central nervous
system), increase oral availability, increase solubility to allow
administration by injection, alter metabolism and alter rate of
excretion.
[0075] As used throughout this application, the term "patient"
refers to mammals, including humans. And the term "cell" refers to
mammalian cells, including human cells.
[0076] Once synthesized, the activities and VLA-4 specificities of
the compounds according to this invention may be determined using
in vitro and in vivo assays.
[0077] For example, the cell adhesion inhibitory activity of these
compounds may be measured by determining the concentration of
inhibitor required to block the binding of VLA-4-expressing cells
to fibronectin- or CS1-coated plates. In this assay microtiter
wells are coated with either fibronectin (containing the CS-1
sequence) or CS-1. If CS-1 is used, it must be conjugated to a
carrier protein, such as bovine serum albumin, in order to bind to
the wells. Once the wells are coated, varying concentrations of the
test compound are then added together with appropriately labelled,
VLA-4-expressing cells. Alternatively, the test compound may be
added first and allowed to incubate with the coated wells prior to
the addition of the cells. The cells are allowed to incubate in the
wells format least 30 minutes. Following incubation, the wells are
emptied and washed. Inhibition of binding is measured by
quantitating the fluorescence or radioactivity bound to the plate
for each of the various concentrations of test compound, as well as
for controls containing no test compound.
[0078] VLA-4-expressing cells that may be utilized in this assay
include Ramos cells, Jurkat cells, A375 melanoma cells, as well as
human peripheral blood lymophocytes (PBLs). The cells used in this
assay may be fluorescently or radioactively labelled.
[0079] A direct binding assay may also be employed to quantitate
the inhibitory activity of the compounds of this invention. In this
assay, a VCAM-IgG fusion protein containing the first two
immunoglobin domains of VCAM (D1D2) attached above the hinge region
of an IgG1 molecule ("VCAM 2D-IgG"), is conjugated to a marker
enzyme, such as alkaline phosphatase ("AP"). The synthesis of this
VCAM-IgG fusion is described in PCT publication WO 90/13300, the
disclosure of which is herein incorporated by reference. The
conjugation of that fusion to a marker enzyme is achieved by
cross-linking methods well-known in the art.
[0080] The VCAM-IgG enzyme conjugate is then placed in the wells of
a muti-well filtration plate, such as that contained in the
Millipore Multiscreen Assay System (Millipore Corp., Bedford,
Mass.). Varying concentrations of the test inhibitory compound are
then added to the wells followed by addition of VLA-4-expressing
cells. The cells, compound and VCAM-IgG enzyme conjugate are-mixed
together and allowed to incubate at room temperature.
[0081] Following incubation, the wells are vacuum drained, leaving
behind the cells and any bound VCAM. Quantitation of bound VCAM is
determined by adding an appropriate calorimetric substrate for the
enzyme conjugated to VCAM-IgG and determining the amount of
reaction product. Decreased reaction product indicates increased
cell adhesion inhibitory activity.
[0082] In order to assess the VLA-4 inhibitory specificity of the
compounds of this invention, assays for other major groups of
integrins, i.e., .beta.2 and .beta.3, as well as other .beta.1
integrins, such as VLA-5, VLA-6 and .alpha.4.beta.7 are performed.
These assays may be similar to the adhesion inhibition and direct
binding assays described above, substituting the appropriate
integrin-expressing cell and corresponding ligand. For example,
polymorphonuclear cells (PMNs) express .beta.2 integrins on their
surface and bind to ICAM. .beta.3 integrins are involved in
platelet aggregation and inhibition may be measured in a standard
platelet aggregation assay. VLA-5 binds specifically to Arg-Gly-Asp
sequences, while VLA-6 binds to laminin. .alpha.4.beta.7 is a
recently discovered homologue of VLA-4, which also binds
fibronectin and VCAM. Specificity with respect to .alpha.4.beta.7
is determined in a binding assay that utilizes the above-described
VCAM-IgG-enzyme marker conjugate and a cell line that expresses
.alpha.4.beta.7, but not VLA-4, such as RPMI-8866 cells.
[0083] Once VLA-4-specific inhibitors are identified, they may be
further characterized in in vivo assays. One such assay tests the
inhibition of contact hypersensitivity in an animal, such as
described by P. L. Chisholm et al., "Monoclonal Antibodies to the
Integrin .alpha.-4 Subunit Inhibit the Murine Contact
Hypersensitivity Response", Eur. J. Immunol., 23, pp. 682-688
(1993) and in "Current Protocols in Immunology", J. E. Coligan, et
al., Eds., John Wiley & Sons, New York, 1, pp. 4.2.1-4.2.5
(1991), the disclosures of which is herein incorporated by
reference. In this assay, the skin of the animal is sensitized by
exposure to an irritant, such as dinitrofluorobenzene, followed by
light physical irritation, such as scratching the skin lightly with
a sharp edge. Following a recovery period, the animals are
re-sensitized following the same procedure. Several days after
sensitization, one ear of the animal is exposed to the chemical
irritant, while the other ear is treated with a non-irritant
control solution. Shortly after-treating the ears, the animals are
given various doses of the VLA-4 inhibitor by subcutaneous
injection. In vivo inhibition of cell adhesion-associated
inflammation is assessed by measuring the ear swelling response of
the animal in the treated versus untreated ear. Swellling is
measured using calipers or other suitable instrument to measure ear
thickness. In this manner, one may identify those inhibitors of
this invention which are best suited for inhibiting
inflammation.
[0084] Another in vivo assay that may be employed to test the
inhibitors of this invention is the sheep asthma assay. This assay
is performed essentially as described in W. M. Abraham et al.,
".alpha.-Integrins Mediate Antigeninduced Late Bronchial Responses
and Prolonged Airway Hyperresponsiveness in Sheep," J. Clin.
Invest., 93, pp. 776-87 (1994), the disclosure of which is herein
incorporated by reference. This assay measures inhibition of
Ascaris antigen-induced late phase airway responses and airway
hyperresponsiveness in asthmatic sheep.
[0085] The compounds of the present invention may be used in the
form of pharmaceutically acceptable salts derived from inorganic or
organic acids and bases. Included among such acid salts are the
following: acetate, adipate, alginate, aspartate, benzoate,
benzenesulfonate, bisulfate, butyrate, citrate, camphorate,
camphorsulfonate, cyclopentanepropionate, digIuconate,
dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,
glycerophosphate, hemisulfate, heptanoate, hexanoate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,
lactate, maleate, methanesulfonate, 2-naphthalenesulfonate,
nicotinate, oxalate, pamoate, pectinate, persulfate,
3-phenyl-propionate, picrate, pivalate, propionate, succinate,
tartrate, thiocyanate, tosylate and undecanoate. Base salts include
ammonium salts, alkali metal salts, such as sodium and potassium
salts, alkaline earth metal salts, such as calcium and magnesium
salts, salts with organic bases, such as dicyclohexylamine salts,
N-methyl-D-glucamine, and salts with amino acids such as arginine,
lysine, and so forth. Also, the basic nitrogen-containing groups
can be quaternized with such agents as lower alkyl halides, such as
methyl, ethyl, propyl, and butyl chloride, bromides and iodides;
dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl
sulfates, long chain halides such as decyl, lauryl, myristyl and
stearyl chlorides, bromides and iodides, aralkyl halides, such as
benzyl and phenethyl bromides and others. Water or oil-soluble or
dispersible products are thereby obtained.
[0086] The compounds of the present invention may be formulated
into pharmaceutical compositions that may be administered orally,
parenterally, by inhalation spray, topically, rectally, nasally,
buccally, vaginally or via an implanted reservoir. The term
"parenteral" as used herein includes subcutaneous, intravenous,
intramuscular, intra-articular, intra-synovial, intrasternal,
intrathecal, intrahepatic, intralesional and intracranial injection
or infusion techniques.
[0087] The pharmaceutical compositions of this invention comprise
any of the compounds of the present invention, or pharmaceutically
acceptable salts thereof, together with any pharmaceutically
acceptable carrier. The term "carrier" as used herein includes
acceptable adjuvants and vehicles. Pharmaceutically acceptable
carriers that may be used in the pharmaceutical compositions of
this invention include, but are not limited to, ion exchangers,
alumina, aluminum stearate, lecithin, serum proteins, such as human
serum albumin, buffer substances such as phosphates, glycine,
sorbic acid, potassium sorbate,. partial glyceride mixtures of
saturated vegetable fatty acids, water, salts or electrolytes, such
as protamine sulfate, disodium hydrogen phosphate, potassium
hydrogen phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based
substances, polyethylene glycol, sodium carboxymethylcellulose,
polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,
polyethylene glycol and wool fat.
[0088] According to this invention, the pharmaceutical compositions
may be in the form of a sterile injectable preparation, for example
a sterile injectable aqueous or oleaginous suspension. This
suspension may be formulated according to techniques known in the
art using suitable dispersing or wetting agents and suspending
agents. The sterile injectable preparation may also be a sterile
injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium-chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed including
synthetic mono- or di-glycerides. Fatty acids, such as oleic acid
and its glyceride derivatives are useful in the preparation of
injectables, as do natural pharmaceutically-acceptable oils, such
as olive oil or castor oil, especially in their polyoxyethylated
versions. These oil solutions or suspensions may also contain a
long-chain alcohol diluent or dispersant, such as Ph. Helv or
similar alcohol.
[0089] The pharmaceutical compositions of this invention may be
orally administered in any orally acceptable dosage form including,
but not limited to, capsules, tablets, aqueous suspensions or
solutions. In the case of tablets for oral use, carriers which are
commonly used include lactose and corn starch. Lubricating agents,
such as magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose
and dried corn starch. When aqueous suspensions are required for
oral use, the active ingredient is combined with emulsifying and
suspending agents. If desired, certain sweetening, flavoring or
coloring agents may also be added.
[0090] Alternatively, the pharmaceutical compositions of this
invention may be administered in the form of suppositories for
rectal administration. These can be prepared by mixing the agent
with a suitable non-irritating excipient which is solid at room
temperature but liquid at the rectal temperature and therefore will
melt in the rectum to release the drug. Such materials include
cocoa butter, beeswax and polyethylene glycols.
[0091] The pharmaceutical compositions of this invention may also
be administered topically, especially when the target of treatment
includes areas or organs readily accessible by topical application,
including diseases of the eye, the skin, or the lower intestinal
tract. Suitable topical formulations are readily prepared for each
of these areas or organs.
[0092] Topical application for the lower intestinal tract can be
effected in a rectal suppository formulation (see-above) or in a
suitable enema formulation. Topically-transdermal patches may also
be used.
[0093] For topical applications, the pharmaceutical compositions
may be formulated in a suitable ointment containing the active
component suspended or dissolved in one or more carriers. Carriers
for topical administration of the compounds of this invention
include, but are not limited to, mineral oil, liquid petrolatum,
white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutical compositions can be formulated in
a suitable lotion or cream containing the active components
suspended or dissolved in one or more pharmaceutically acceptable
carriers. Suitable carriers include, but are not limited to,
mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters
wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and
water.
[0094] For ophthalmic use, the pharmaceutical compositions may be
formulated as micronized suspensions in isotonic, pH adjusted
sterile saline, or, preferably, as solutions in isotonic, pH
adjusted sterile saline, either with our without a preservative
such as benzylalkonium chloride. Alternatively, for ophthalmic
uses.sub.r the pharmaceutical compositions may be formulated in an
ointment such as petrolatum.
[0095] The pharmaceutical compositions of this invention may also
be administered by nasal aerosol or inhalation through the use of a
nebulizer, a dry-powder inhaler or a metered dose inhaler. Such
compositions are prepared according to techniques well-known in the
art of pharmaceutical formulation and may be prepared. as solutions
in saline, employing benzyl alcohol or other suitable
preservatives, absorption promoters to enhance bioavailability,
fluorocarbons, and/or other conventional solubilizing or dispersing
agents.
[0096] The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary
depending upon the host treated, and the particular mode of
administration. It should be understood, however, that a specific
dosage and treatment regimen for any particular patient will depend
upon a variety of factors, including the activity of the specific
compound employed, the age, body weight, general health, sex, diet,
time of administration, rate of excretion, drug combination, and
the judgment of the treating physician and the severity of the
particular disease being treated. The amount of active ingredient
may also depend upon the therapeutic or prophylactic agent, if any,
with which the ingredient is co-administered.
[0097] The dosage and dose rate of the compounds of this invention
effective to prevent, suppress or inhibit cell adhesion will depend
on a variety of factors, such as the nature of the inhibitor, the
size of the patient, the goal of the treatment, the nature of the
pathology to be treated, the specific pharmaceutical composition
used, and the judgment of the treating physician. Dosage levels of
between about 0.001 and about 100 mg/kg body weight per day,
preferably between about 0.1 and about 10 mg/kg body weight per day
of the active ingredient compound are useful.
[0098] According to another embodiment compositions containing a
compound of this invention may also comprise an additional agent
selected from the group consisting of corticosteroids,
bronchodilators, antiasthmatics (mast cell stabilizers),
anti-inflammatories, antirheumatics, immunosuppressants,
antimetabolites, immunonodulators, antipsoriatics and
antidiabetics. Specific compounds within each of these classes may
be selected from any of those listed under the appropriate group
headings in "Comprehensive Medicinal Chemistry," Pergamon Press,
Oxford, England, pp. 970-986 (1990), the disclosure of which is
herein incorporated by reference. Also included within this group
are compounds such as theophylline, sulfasalazine and
aminosalicylates (antiinflammatories); cyclosporin, FK-506, and
rapamycin (immunosuppressants); cyclophosphamide and methotrexate
(antimetabolites); and interferons (immunomodulators).
[0099] According to other embodiments, the invention provides
methods for preventing, inhibiting or suppressing cell
adhesion-associated inflammation and cell adhesion-associated
immune or autoimmune responses. VLA4-associated cell adhesion plays
a central role in a variety of inflammation, immune and autoimmune
diseases. Thus, inhibition of cell adhesion by the compounds of
this invention may be utilized in methods of treating or preventing
inflammatory, immune and autoimmune diseases. Preferaby the
diseases to be treated with the methods of this invention are
selected from asthma, arthritis, psoriasis, transplantation
rejection, multiple sclerosis, diabetes and inflammatory bowel
disease.
[0100] These methods may employ the compounds of this invention in
a monotherapy or in combination with an anti-inflammatory or
immunosuppressive agent. Such combination therapies include
administration of the agents in a single dosage form or in multiple
dosage forms administered at the same time or at different
times.
[0101] In order that this invention may be more fully understood,
the following examples are set forth. These examples are for the
purpose of illustration only and are not to be construed as
limiting the scope of the invention in any way.
Procedure A--Synthesis of Cinnamate Esters
[0102] Method A: To a cinnamic acid or substituted cinnamic acid
(1.0 mmol) in CH.sub.2Cl.sub.2 (10 ml) was added. (COCl).sub.2 (1.5
mmol) slowly. The reaction mixture was stirred at r.t. for 4 h and
the solvent was removed in vacuo to afford the acid chloride.
Methanol or t-butyl alcohol (5 ml) was added to quantitatively
provide the methyl or t-butyl ester after removal of the
solvents.
[0103] Method B: To an appropriate aldehyde (1.0 mmol) in THF (10
ml) was added t-butoxycarbonyl methylene triphenylphosphorane (1.0
mmol, Aldrich) and the resulting mixture was stirred at room
temperature for 16 h. The reaction mixture was diluted with
petroleum ether (10 ml) and was filtered through a pad of celite.
The filtrate was collected and concentrated in vacuo to afford the
desired product. ##STR4##
[0104] Method A; Yield: 95%; (CDCl.sub.3' 300 MHz, ppm): 7.57 (d,
1H, J=16 Hz), 7.47 (m, 2 H), 7.34 (m, 3H), 6.35 (d, 1H, J=16 Hz),
1.52 (s, 9H); ##STR5##
[0105] Method B; Yield: 90%; (CDCl.sub.3' 300 MHz, ppm): 7.48 (d,
1H), 7.28-7.18 (m, 5H), 5.69 (d, 2H), 3.44 (d, 2H), 1.42 (s, 9H);
##STR6##
[0106] Method A; Yield: 94%; (CDCl.sub.3' 300 MHz, ppm): 7.95 (d,
1H, J=16 Hz), 7.49 (d, 1H), 7.42 (t, 1H), 6.94 (dd, 2H), 6.51 (d,
2H), J=16 Hz), 3.86 (s, 3H), 3.76 (s, 3H); ##STR7##
[0107] Method A; Yield: 92%; (CDCl.sub.3' 300 MHz, ppm): 7.52 (d,
1H, J=15.9 Hz), 7.28 (t, 1H), 7.09 (d, 1H), 7.02 (br, s, 1H), 6.89
(d, 1H), 6.34 (d, 1H, J=15.9 Hz) 3.82 (s, 3H), 1.54 (s, 9 H);
##STR8##
[0108] Method A: Yield: 98%; (CDCl.sub.3' 300 MHz, ppm): 7.64 (d,
1H, J=16 Hz), 7.29 (t, 1H), 7.10 (d, 1H), 7.06 (br, s, 1H), 6.94
(d, 1H, J=16 Hz), 3.82 (s, 3H), 3.80 (s, 3H); ##STR9##
[0109] Method B; Yield: 88%; (CDCl.sub.3' 300 MHz, ppm): 8.62
(br,s, 1H), 8.51 (m, 1H), 7.72 (d, 1H), 7.48 (d, 1H, J=15.9 Hz),
7.22 (m, 1H), 6.36 (d, 1H, J=15.9 Hz), 1.49 (s, 9H); ##STR10##
[0110] Method B; Yield: 90%; (CDCl.sub.3' 300 MHz, ppm) 8.60 (br,
s, 1H), 7.66 (t, 1H), 7.55 (d, 1H, J=15.9 Hz), 7.36 (d, 1H), 7.21
(m, 1H), 6.78 (d, 1H, J=15.9 Hz), 1.52 (s, 9 H); ##STR11##
[0111] Method A; Yield: 91%; (CDCl.sub.3' 300 MHz, ppm): 7.52 (d,
1H, J=15.9 Hz), 7.44 (d, 1H, J=8.0 Hz), 6.85 (d, 1H, J=8.0 Hz),
6.21 (d, 1H, J=15.9 Hz), 3.81 (s, 3H), 1.52 (s, 9H); ##STR12##
[0112] Method A; Yield: 90%; (CDCl.sub.3' 300 MHz, ppm): 7.61 (d,
1H, J=16 Hz), 7.42 (d, 2H, J=7.9Hz), 6.86 (d, 1H, J=7.9 Hz), 6.28
(d, 1H, J=16 Hz), 3.78. (s, 3H), 3.74 (s, 3H); ##STR13##
[0113] Method B; Yield: 91%; (CDCl.sub.3' 300 MHz, ppm) 7.56 (d,
1H, J=16 Hz), 7.46 (t, 2H), 7.02 (t, 2H), 6.26 (d, 2H, J=16 Hz),
1.54 (s, 9H); ##STR14##
[0114] Method A; Yield: 89%; (CDCl.sub.3' 300 MHz, ppm): 7.47 (d,
1H, J=15.9 Hz), 7.01 (d, 1H, J=8.3 Hz), 6.98 (br, S, 1H), 6.78 (d,
1H, J=8.3 Hz), 3.84 (s, 6H), 1.48 (s, 9H); ##STR15##
[0115] Method A; Yield: 91%; (CDCl.sub.3' 300 MHz, ppm): 7.61 (d,
1H, J=15.9 Hz, 7.07 (d, 1H,; J=8.3 Hz), 7.02 (br, s, 1H), 6.83 (d,
1H, J=8.3 Hz), 6.28 (d, 1H, J=15.9 Hz), 3.88 (s, 3H), 3.76 (s, 3H);
##STR16##
[0116] Method A; Yield: 92%; (CDCl.sub.3' 300 MHz, ppm): 7.46 (d,
1H, J=16.1 Hz), 6.99 (s, 1H), 6.97 (d, 1H), 6.76 (d, 1H), 6.18 (d,
1H, J=16.1 Hz), 5.96 (s, 2H), 1.50 (s, 9H); ##STR17##
[0117] Method A; Yield: 88%; (CDCl.sub.3' 300 MHz, ppm): 7.55 (d,
1H, J=15.9 Hz), 6.98-6.75 (m, 2H), 6.22 (d, 1H, J=15.9 Hz), 5.96
(s, 2H), 3.75 (s, 3H); ##STR18##
[0118] Method B; Yield: 89%; (CDCl.sub.3' 300 MHz, ppm): 7.45 (d,
1H, J=15.8 Hz), 6.99 (s, 1H), 6.98 (d, 1H), 6.80 (d, 1H), 6.18 (d,
1H, J=15.8 Hz), 4.21 (br,s, 4H), 1.49 s, 9H); ##STR19##
[0119] Method B; Yield: 88%. ##STR20##
[0120] Method B; Yield: 93%; .sup.1HNMR (CDCl.sub.3): .delta. 8.00
(2H, d, J=5.5 Hz), 7.53 (2H, d, J=5.5 Hz), 7.58 (1H, d, J=10, 7
Hz), 6.42 (1H, d, J=10.7 Hz), 3.90 (3H, s), 1.51 (9H, s).
Procedure B--Synthesis of .beta.-Amino Acids
[0121] A 2 L round bottom flask, equipped with a magnetic stir bar,
was charged with 1000 mL of MeOH and the flask tared with its
contents. Anhydrous HCl (11 g, 0.29 mol) was bubbled in from a
cylinder. To this solution was added a cinnamic acid (0.29 mol)
neat in one portion. The resulting mixture was heated at reflux
until the reaction was judged complete by TLC analysis. The
reaction was cooled to RT, then refrigerated overnight. The
crystalline product was collected by suction filtration on a medium
frit and the cake washed with cold MeOH. The solid was dried on the
filter to give a white or nearly white product. Precursor to
.beta.-3: Yield: 94%; TLC (3:1 hexane/EtOAc; UV): R.sub.f=0.48;
mp=134-136.degree. C.; .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.58 (d,
1H, J=15.9 Hz), 7.00-6.97 (m, 3H), 6.79 (d, 1H, J=7.9 Hz), 6.24 (d,
1H, J=15.9 Hz), 5.98 (s, 2H), 3.77 (s, 3H); MS (FAB): 206.
[0122] Precursor to 9-5: Yield: 84%; TLC (3:1 hexane/EtOAc; UV):
R.sub.f=0.48; mp=89-91.degree. C.; .sup.1H NMR (CDCl.sub.3, 300
MHz) 7.63 (d, 1H, J=15.9 Hz), 7.46 (d, 2H, J=8.7 Hz), 6.89 (d, 2H,
J=8.7 Hz), 6.29 (d, 1H, J=15.9 Hz), 3.82 (s, 3H), 3.77 (s, 3H); MS
(FAB): 192.
Michael addition of (R)-(+)-N-benzyl-1-phenylethylamine to methyl
4-methoxy-cinnamate
[0123] A 1 L 3-neck round bottom flask, equipped with a stopper,
thermometer, and 250,mL addition funnel with an Ar inlet was
charged with (R)-(+)-N-benzyl-1-phenylethylamine hydrochloride
(0.132 mol, 32.6 g, 1.1 eq based on cinnamate) and the apparatus
flushed with Ar 30 min. The salt was suspended in dry THF (200 mL)
and the mixture cooled to an internal temperature of -70.degree. C.
with a dry ice/acetone bath. To the suspension. was added n-BuLi
(2.5 M in hexanes, 0.257 mol, 103 mL, 1.95 eq based on amine
hydrochloride) from the addition funnel at such a rate that the
internal temperature did not exceed -65.degree. C. The addition
required 90 min. After completing the addition, the reaction was
stirred at -70.degree. C. for 1 hr. A solution of methyl
4-methoxycinnamate (0.120 mol, 23 g, 1 eq).in THF (125 mL) was
added from the addition funnel over 90 min at such a rate that the
internal temperature did not exceed -65.degree. C. After completing
the addition, the reaction was stirred at -70.degree. C. 2 hrs. TLC
analysis indicated complete reaction. The reaction was quenched
cold by the addition of 5% citric acid (250 mL) and stirred
overnight at RT. In a 2 L separatory funnel, the layers were
separated and the organic washed with 5% citric acid (1.times.125
mL). The combined aqueous were extracted with EtOAc (1.times.200
mL). The combined organics were then washed with 5% NaHCO.sub.3
(1.times.150 mL) and brine (1.times.150 mL) and dried (MgSO.sub.4).
Filtration and evaporation to constant weight provided crude
product (50.04 g, 103% of theory) as a viscous oil which solidified
on standing. Pure material was obtained by triturating and stirring
crude product with heptane (1.5-2 mL/g, 75-100 mL total volume) at
RT overnight. The solids were collected by suction filtration on a
medium frit and the cake washed by flooding with cold heptane
(2.times.50 mL). The solids were dried on the filter to give pure
product (28.93 g, 60% yield) as a white powder. TLC (4:1
hexane/EtOAc): Rf=0.50 (I.sub.2, UV); mp=87-88.degree. C.; .sup.1H
NMR (CDCl.sub.3, 300 MHz): 1.20 (d, 3H, J=6.9 Hz), 2.51 (dd, 1H,
J=9.4, 14.8 Hz), 2.66 (dd, 1H, J=5.7, 14.8 Hz), 3.45 (s, 3H), 3.67
(ABq, 2H, J=14.7 Hz), 3.79 (s, 3H), 3.98 (q, 1H, J=6.8 Hz), 4.37
(dd, 1H, J=5.7, 9.3 Hz), 6.86 (d, 2H, J=8.6 Hz), 7.16-7.33 (m, 10
H), 7.40 (d, 2H, J=7.3 Hz); MS (FAB): 404
Hydrogenolysis of Benzyl Groups
[0124] The above adduct (0.071 mol, 28 g) was suspended in MeOH
(300 mL) and treated with formic acid (96%, 0.179 mol, 8.25 g, 6.8
mL, 2.5 eq) neat in one portion with stirring. To this suspension
was added Degussa type E101 NE/W 10% Pd/C (50% wet, 0.00179 mol,
3.81 g, 0.025 eq) in one portion. The resulting mixture was heated
at reflux for 1-2 hr until judged complete by TLC analysis. The
mixture was cooled to RT, then filtered on a pad of Celite, washing
the flask and pad with MeOH (150 mL). The combined filtrates were
evaporated to give crude product (15.42 g, 102% of theory) as an
oil. The crude product was dissolved in i-PrOH (250 mL) and heated
to a gentle reflux. D-tartaric acid (0.071 mol, 10.76 g, 1 eq) was
added as a solid in one portion. Heating was continued for 15 min,
during which time the salt precipitated as a fine white solid. The
mixture was cooled to RT, then refrigerated overnight. The
crystalline salt was collected by suction filtration on a medium
frit, washing with cold i-PrOH (50-75 mL), and dried on the filter
to give product (23 g, 79%). The above salt was converted to the
free base by dissolving in a minimum volume of H.sub.2O (125 mL)
and treating the solution with solid NaHCO.sub.3 until the aqueous
was saturated. This was extracted with EtOAc (3.times.100 mL). The
combined organics were washed with brine (1.times.100 mL) and dried
(MgSO.sub.4). Filtration and evaporation provided pure product
(11.75 g, 78%) as a nearly colorless oil which solidified on
cooling.
[0125] TLC (9:1 CHCl.sub.3/MeOH): R.sub.f=0.30 (I.sub.2, UV); HPLC
(reverse phase; MeCN/H.sub.2O/TFA gradient): 96% pure, R.sub.t=17.9
min;
[0126] .sup.1H NMR (CDCl.sub.3, 300 MHz): 1.87 (br s, 2H), 2.62 (d,
2H, J=6.9 Hz), 3.64 (s, 3H), 3.76 (s, 3H), 4.35 (t, 1H, J=6.9 Hz),
6.84 (d, 2H, J=8.6 Hz), 7.25 (d, 2H, J=8.6 Hz); MS (FAB): 210.
##STR21##
[0127] .sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm): 7.41-7.28 (m, 5H),
4.18 (q, 2H), 2.65 (d, 2H), 2.12 (br, 2H), 1.16 (t, 3H).
##STR22##
[0128] .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 6.81 (d, 1H, J=1.6
Hz), 6.72 (d, 1H, J=7.9 Hz), 6.66 (d, 1H, J=7.9 Hz), 5.85 (s, 2H),
4.22 (1H, dd, J=7.5 Hz and 7.3 Hz), 2.47 (2H, dd, J=7.5 Hz and 5.6
Hz), 2.21 (s, 2H), 1.35 (9H, s). ##STR23##
[0129] .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 6.82 (d, 1H, J=1.6
Hz), 6.76 (d, 1H, J=7.9 Hz), 6.73 (d, 1H, J=7.9 Hz), 5.89 (s, 2H),
4.29 (1H, dd, J=6.9 Hz and 6.8 Hz), 3.63 (3H, s), 2.57 (d, 2H,
J=6.9 Hz), 1.75 (s, 2H); ##STR24##
[0130] .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 6.79-6.78 (m, 3H),
4.32 (t, 1H, J=6.7 Hz), 3.75 (s, 3H), 3.72 (s, 3H), 2.52 (d, 2H,
J=6.8 Hz), 1.82 (br, 2H), 1.42 (s, 9H).; ##STR25##
[0131] .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.20 (d, J=8.6 Hz),
6.80 (d, 2H, J=8.6 Hz), 4.30 (t, 1H, 6.8 Hz), 3.71 (s, 3H), 3.60
(s, 3H), 2.57 (d, 2H, J=6.8 Hz), 1.91 (s, 2H); ##STR26##
[0132] .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.24 (d, J=8.4 Hz),
6.82 (d, 2H, J=8.4 Hz), 4.26 (t, 1H, 6.8 Hz), 3.66 (s, 3H), 2.47
(d, 2H, J=6.6 Hz), 1.41 (s, 9H); ##STR27##
[0133] .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.21 (dd, 1H, J=8.2
Hz and 8.1 Hz), 6.95-6.93 (m, 2H), 6.78 (d, 1H, 6.8 Hz), 4.34 (t,
1H, J=6.7 Hz), 3.79 (s, 3H), 2.54 (d, 2H, J=6.9 Hz), 1.74 (s, 2H),
1.40 (s, 9H); ##STR28##
[0134] .sup.1H NMR (CDCl.sub.3' 300 MHz, ppm): 7.34-7.08 (m, 2H),
6.82-6.68 (m, 2H), 4.45 (m, 1H), 3.65 (s, 3H), 3.49 (s, 3H), 2.58
(d, 2H), 1.68 (br s, 2H). ##STR29##
[0135] .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.28-7.25 (m, 2H),
7.01 (d, 1H), 4.31 (t, 1H), 2.50 (d, 2H), 2.01 (br, 2H), 1.41 (s,
9H); ##STR30##
[0136] .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 6.84 (s, 1H),
6.79-6.76 (m, 1H), 4.24-4.19 (m, 1H), 4.19 (s, 4H), 2.50 (d, 2H),
1.63 (br, 2H), 1.41 (s, 9H); ##STR31##
[0137] .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 3.34-3.05 (m, 1H),
2.65-2.58 (m, 2H), 1.65 (d, 2H); ##STR32##
[0138] .sup.1H NMR (CDCl.sub.3, 300MHz, ppm) 3.34-3.05 (m, 1H),
2.65-7.15 (m, 3H), 3.42-3.15 (m, 1H), 2.71 (dd, 1H, J=5.5 Hz and
13.3 Hz), 2.54 (dd, 1H, J=8.1 Hz and 13.3 Hz), 2.36 (dd, 1H, J=4.2
Hz and 15.7 Hz), 2.20 (dd, J=8.6 and 15.7 Hz), 1.42 (s, 9H).
##STR33##
[0139] 1M TMSCl in CH.sub.2Cl.sub.2 (33 ml, 33 mmol) was added to a
mixture of (R)-.alpha.-methylbenzylamine (3.4 g, 28 mmol) and
Et.sub.3N (4 g, 40 mmol) in THF (10 ml) was added and the mixture
was allowed to stir for 1 h at room temperature. After the solid
was removed by filtration, the solution was concentrated to afford
a liquid. This silylamine (2.4 g, 12.5 mmol) was dissolved in THF
(35 ml) and was cooled to -78.degree. C. To this cooled solution
was added n-BuLi (7.8 ml of 1.6 M solution in hexanes, 12.5 mmol)
slowly. After stirring for 0.5 h at the temperature, to the
reaction mixture was added a solution of t-butyl
trans-3-(3-pyridyl)acrylate (2.56 g, 12.4 mmol) in THF (10 ml). The
stirring was continued for another 1/2 h and the mixture was
quenched with sat. NH.sub.4Cl (20 ml) and was allowed to warm up to
room temperature and extracted with ether. The combined ether
layers were dried (K.sub.2CO.sub.3) and concentrated to afford an
oil. This oil (500 mg) was dissolved in ethanol (1.5 ml), t-butanol
(15 ml), ammonium formate (1.5 g) and 10% Pd/C (1.2 g) were added.
The resulting mixture was heated to reflux for 3 h followed by acid
and base workup to afford the desired amine .beta.-13 (300 mg).
FAB-MS=223. ##STR34##
[0140] .sup.1HNMR (CDCl.sub.3): .delta. 7.97(2H, d, J=5.4 Hz), 7.41
(2H, d, J=5.4 Hz), 4.40 (1H, t, J=4.5 Hz), 3.88 (3H,s), 2.55 (2H,
d, J=4.5 Hz), 1.71 (2H, br), 1.39 (9H, s).
General Procedure for Synthesis of M-1, M-2 and M-3
[0141] To a solution of the commercially available amino acid (1.5
mmoles) in CH.sub.2Cl.sub.2 (4 ml) and MeOH (1 ml) cooled to
0.degree. C., was added thionyl chloride (0.125 ml, 1.65 mmol). The
reaction was warmed to 40.degree. C. for 2 h, and concentrated to
dryness in vacuo to afford the desired amino ester HCl salt.
##STR35##
[0142] 89% yield; .sup.1HNMR (DMSO-d.sup.6' 300 MHz, ppm):
9.00-8.75 (3H, bm), 7.71 (2H, d, J=7.3 Hz), 7.58 (2H, d, J=7.3 Hz)
4.71 (1 H, bs), 3.64 (3 H,s), 3.40-3.06 (2H, m); ##STR36##
[0143] 85% yield as a tan solid. .sup.1HNMR (CDCl.sub.3' 300 MHz,
ppm): 7.55-7.05 (6 H, bm), 3.66 (3H, s), 3.65-3.45 (2H, bm),
3.10-2.77 (5H, bm), 2.17-1.95 (2H, bm); ##STR37##
[0144] 84% yield as a pale tan solid; .sup.1HNMR (CDCl.sub.3' 300
MHz, ppm): 8.1-7.8 (4H, bm), 7.65-7.45 (3H, bm), 5.45 (1H, br),
3.80-3.30 (2H, bm), 3.55 (3H, s).
Procedure C--Synthesis of Coupled Amino Acids
[0145] To a solution of ethyl 3-amino-3-phenyl-1-propanoate (or
other S-amino acid ester prepared by Procedure B) (0.50 g, 5.25
mmol) in CH.sub.2Cl.sub.2 (5 ml) was added BocLeuOSu (1.5 g, 4.67
mmol) (CbzLeuOSu is used for the Cbz protected analog) with cooling
and Et.sub.3N (5 drops). The mixture was stirred at room
temperature for 1 h. The reaction mixture was diluted with
CH.sub.2Cl.sub.2 (10 ml) and washed with 5% citric acid
(5ml.times.2), 5% NaHCO.sub.3 (5 ml) and sat. NaCl (5 ml). The
organic layer was dried (Na.sub.2SO.sub.4) and concentrated to
afford 1.26 g (66%). as a white solid.
Procedure D--Synthesis of Deprotected Amino Acids
[0146] To a stirred solution of the product of Procedure C (a
Boc-Leu-S-amino acid ester) (41.5 mg, 0.102 mmol) at 0-5.degree. C.
in 2 mL of CH.sub.2Cl.sub.2 was added 4 mL of TFA. The mixture was
allowed to come to room temperature with continued stirring for 1
hour. The reaction was concentrated in vacuo, redissolved in
CH.sub.2Cl.sub.2, concentrated two more times and placed under high
vacuum to remove final traces of TFA. HPLC showed complete
conversion to two new peaks of shorter retention time. The residue
can taken up in DMF and TEA added with stirring until basic to
litmus in preparation for further reaction.
[0147] A Cbz group is removed using the following method:
[0148] The product from Procedure C (where t-butyl
3-amino-3-phenyl-1-propanoate and CbzLeuOSu were used) (110 mg,
0.23 mmol) in MeOH with a catalytic amount of 10% palladium on
charcoal was stirred overnight under hydrogen at 40 psi. The
reaction was filtered through Celite.RTM. and concentrated in vacuo
yielding the free base Leu BOC .beta.-amino acid (87 mg,
quantitative) as a clear oil. .sup.1H NMR: (CDCl.sub.3, 300 MHz,
ppm), 7.30 (m, 5H), 5.33 (dd, 1H, J=6, 8.82 Hz), 4.00 (m, 1H) 2.77
(dd, 1H J=9, 15 Hz), 2.90 (dd, 1H, J=6, 15 Hz), 1.69 (m, 2H), 1.45
(m, 1H), 1.29 (s, 9H), 0.90 (d, 6H, J=6 Hz).
EXAMPLE 1
Synthesis of BIO-1002
[0149] A. A stirred solution of cyanoacetic acid (13 mg, 0.15
mmol), EDC (30 mg, 0.16 mmol), and HOBt (30 mg, 0.20 mmol) in DMF
(0.5 mL) was treated with-a solution of the amine prepared in
Procedure D (52 mg, 0.105 mmol) and diisopropylethylamine (0.30 mL,
1.7 mmol) in DMF (1.0 mL) at room temperature. After the solution
was stirred for over 18 h, the reaction was partitioned in ethyl
acetate (15 mL) and 60% sat. NaHCO.sub.3 (10 mL). The organic phase
was washed with 60% sat. aq. NaHCO.sub.3 (2.times.10 mL), H.sub.2O
(5 mL), 5% citric acid (3.times.10 mL), H.sub.2O (5 mL), and sat.
aq. NaCl (10 mL). The organic phase was dried (MgSO.sub.4) and
concentrated in vacuo to afford BIO1002-OEt (27 mg, 69%) as a foam:
.sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.58 (d, 1H), 7.45 (d, 1H),
7.40-7.20 (m, 5H), 5.28 (m, 1H), 4.46 (m, 1H), 4.05 (m, 2H), 3.23
(m, 2H), 2.79 (m, 2H), 1.78-1.53 (m, 3H), 1.23 (m, 3H), 0.90 (m,
6H).
[0150] B. A stirred solution of BIO1002-OEt (27 mg, 0.072 mmol) in
methanol (3 mL) was treated with aq. LiOH (1.0 M, 0.25 mL, 0.25
mmol) at room temperature for 22 h. The reaction was acidified with
trifluoroacetic acid then concentrated in vacuo. The crude products
were purified by HPLC to give BIO-1002A (2.5 mg, 10%) and BIO-1002B
(4.4 mg, 18%) as white solids:
[0151] BIO1002A: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 8.08 (d,
1H), 7.87 (d, 1H), 7.30-7.16 (m, 5H), 5.25 (m, 1H), 4.37 (m, 1H),
3.36 (s, 2H), 2.75 (m, 2H), 1.70-1.45 (m, 3H), 0.90 (m, 6H); HPLC
(Gradient A), 16.7 min; MS, m/z 346 BIO1002B: .sup.1H NMR
(CDCl.sub.3, 300 MHz, ppm) 8.00-7.70 (m, 2H), 7.40-7.20 (m, 5H),
5.28 (m, 1H), 4.39 (m, 1H), 3.45 (s, 2H), 2.78 (m, 2H), 1.65-1.40
(m, 3H), 0.90 (m, 6H); HPLC (Gradient A), 20.6 min; MS, m/z
346.
EXAMPLE 2
Synthesis of BIO-1003
[0152] A. The procedure as described Example 1A was performed
utilizing cyclohexylacetic acid (22 mg, 0.15 mmol), EDC (30 mg,
0.16 mmol), and HOBt (30 mg, 0.20 mmol), amine from Procedure D (52
mg, 0.105 mmol) and diisopropylethylamine (0.30 mL, 1.7 mmol) in
DMF (1.0 mL) to afford BIO1003-OEt (32 mg, 71%) as a foam: .sup.1H
NMR (CDCl.sub.3, 300 MHz, ppm) 7.42-7.18 (m, 6H), 6.08 (m, 1H),
5.36 (m, 1H), 4.50 (m, 1H), 4.05 (m, 2H), 2.81 (m, 2H), 2.11-0.80
(m, 25H).
[0153] B. The procedure as described in Example 1B was performed
utilizing BIO1003-OEt (32 mg, 0.074 mmol) and aq. LiOH (1.0 M, 0.25
mL, 0.25 mmol) in MeOH (3.0 mL) to give BIO-1003A (3.5 mg, 11%) and
BIO-1003B (5.3 mg, 18%) as white solids:
[0154] BIO-1003A: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.35-7.16
(m, 5H), 5.23 (m, 1H), 4.38 (m, 1H), 2.28 (d, 2H), 2.03 (m, 2H),
1.75-0.80 (m, 22H); HPLC (Gradient A), 34.1 min and 35.3 min (4:1);
MS, m/z 403.
[0155] BIO-1003B: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.35-7.16
(m, 5H), 5.23 (m, 1H), 4.38 (m, 1H), 2.28 (m, 2H), 2.03 (m, 2H),
1.75-0.80 (m, 22H); HPLC (Gradient A), 34.1 min and 35.3 min
(1:10); MS, m/z 403.
EXAMPLE 3
Synthesis of BIO-1014
[0156] A. Methyl 3-amino-3-phenyl-1-propanoate was coupled with
BocLeuOSu by the method described in Procedure C. This material was
subjected to the conditions used in Procedure D1 to give the
desired TFA-amine salt.
[0157] B. The procedure as described in Example 1A was performed
utilizing indole-3-carboxylic acid (19 mg, 0.12 mmol), EDC (26 mg,
0.14 mmol), HOBt (26 mg, 0.17 mmol), amine from Example 3A (44 mg,
0.11 mmol) and diisopropylethylamine (0.10 mL, 0.56 mmol) in
CH.sub.2Cl.sub.2 (5.0 mL) to afford BIO1014-OMe (25 mg, 52%) as a
foam.
[0158] C. The same procedure as described in Example 1B was
performed utilizing BIO1014-OMe (25 mg, 0.057 mmol) and aq. LiOH
(1.0 M, 0.115 mL., 0.115 mmol) in MeOH (5 mL) to give BIO-1014A
(5.1 mg, 21%) and BIO-1014B (4.7 mg, 20e) as white solids:
[0159] BIO-1014A: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 8.52 (d,
1H), 8.13 (d, 1H), 8.10 (d, 1H), 7.81 (d, 1H), 7.46-7.03 (m, 9H),
5.20 (m, 1H), 4.58 (m, 1H), 2.69 (m, 2H), 1.75-1.45 (m, 3H), 0.90
(m, 6H); HPLC (Gradient A), 28.1 min; MS, m/z 422.
[0160] BIO-1014B: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 8.55 (d,
1H), 8.18 (d, 1H), 8.13 (d, 1H), 7.79 (d, 1H), 7.46-7.03 (m, 9H),
5.20 (m, 1H), 4.58 (m, 1H), 2.70 (m, 2H), 1.55-1.40 (m, 3H), 0.90
(m, 6H); HPLC (Gradient A), 29.5 min; MS, m/z 422.
EXAMPLE 4
Synthesis of BIO-1017
[0161] A. The procedure as described in Example 1A was performed
utilizing 1-phenyl-1-cyclopropane-carboxylic acid (21 mg, 0.13
mmol), EDC (26 mg, 0.14 mmol), HOBt (26 mg, 0.17 mmol), amine from
Example 3A (44 mg, 0.11 mmol) and diisopropylethylamine (0.10 mL,
0.56 mmol) in CH.sub.2Cl.sub.2 (5.0 mL) to afford BIO1017-OMe (39
mg, 68%) as a foam.
[0162] B. The procedure as described in Example 1B was performed
utilizing BIO1017-OMe (39 mg, 0.089 mmol) and aq. LiOH (1.0 M, 0.27
mL, 0.27 mmol) in MeOH (2 mL) to give BIO-1017A (10.3 mg, 27%) and
BIO-1017B (12.2 mg, 32%) as white solids:
[0163] BIO-1017A: .sup.1H NMR (CD.sub.3SOCD.sub.3, 300 MHz, ppm)
8.46 (d, 1H), 7.40-7.20 (m, 10H), 6.30 (d, 1H), 5.09 (m, 1H), 4.33
(m, 1H), 2.62 (m, 2H), 1.50-1.20 (m, 5H), 0.98 (m, 2H), 0.82 (m,
6H); HPLC (Gradient A), 33.9 min; MS, m/z 423.
[0164] BIO-1017B: .sup.1H NMR (CD.sub.3SOCD.sub.3, 300 MHz, ppm)
8.55 (d, 1H), 7.48-7.15 (m, 10H), 6.30 (d, 1H), 5.08 (m, 1H), 4.35
(m, 1H), 2.63 (m, 2H), 1.48-1.15 (m, 5H), 1.10-0.88 (m, 2H),
0.85-0.64 (m, 6H); HPLC (Gradient A), 33.9 min and 34.5 min (1:9);
MS, m/z 423.
EXAMPLE 5
Synthesis of BIO-1022
[0165] A. The procedure as described in Example 1A was performed
utilizing 2-naphthylacetic acid (20 mg, 0.11 mmol), EDC (25 mg,
0.13 mmol), HOBt (25 mg, 0.16 mmol), amine from Example 3A (42 mg,
0.10 mmol) and diisopropylethylamine (0.10 mL, 0.56 mmol) in DMF
(2.0 mL) to afford BIO1022-OMe (36 mg, 70%) as a foam.
[0166] B. The procedure as described in Example 1B was performed
utilizing BIO1022-OMe (36 mg, 0.078 mmol) and aq. LiOH (1.0 M, 0.50
mL, 0.50 mmol) in MeOH (3 mL) to give BIO-1022A (1.7 mg, 4.8%) and
BIO-1022B (6.8 mg, 19%) as white solids:
[0167] BIO-1022A: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.90-7.17
(m, 12H), 5.30 (t, 1H), 4.45 (m, 1H), 2.79 (m, 2H), 1.68-1.33 (m,
3H), 0.87 (d, 6H); HPLC (Gradient A), 25.7 min; MS, m/z 447.
[0168] BIO-1022B: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.90-7.17
(m, 12H), 5.35 (t, 1H), 4.49 (m, 1H), 2.79 (d, 2H), 1.58-1.33 (m,
3H), 0.82 (m, 6H); HPLC (Gradient A), 25.7 min and 26.4 min (1:9);
MS, m/z 447.
EXAMPLE 6
Synthesis of BIO-1029
[0169] A. t-Butyl 3-amino-3-phenyl-1-propanoate was coupled with
BocLeuOSu using the method described in Procedure C. This material
was subjected to the conditions of Procedure D2 to give the desired
amine salt.
[0170] B. The procedure as described in Example 1A was performed
utilizing 4-(2-aminobenzamido)-phenylacetic acid (18 mg, 0.067
mmol), EDC (13 mg, 0.067 mmol), and HOBt (13 mg, 0.085 mmol), amine
from Example 6A (18 mg, 0.054 mmol) and diisopropylethylamine
(0.048 mL, 0.27 mmol) in DMF (0.5 mL) to afford
NH.sub.2-BIO1029-OtBu (32 mg, 100%) as an oil:
[0171] .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.65-7.43 (m, 4H),
7.40-7.10 (m, 9H), 6.72 (m, 2H), 6.49 (d, 1H), 5.28 (m, 1H), 4.45
(m, 1H), 3.52 (s, 2H), 2.68 (m, 2H), 2.00 (bs, 2H), 1.65-1.15 (m,
13H), 0.85 (m, 6H).
[0172] C. A solution of NH.sub.2-BIO1029-OtBu (16 mg, 0.027 mmol)
in trifluoroacetic acid (1 mL) was stirred at room temperature for
45 min and then-concentrated. The crude product was purified by
HPLC to afford NH.sub.2-BIO1029 (3.4 mg, 26%) as a white solid: MS,
m/z 531.
[0173] D. A solution of NH.sub.2-BIO1029 (3.4 mg, 0.0064 mmol),
methyl isocyanate (3 drops), and diisopropyl-ethylamine (1 drop) in
CH.sub.2Cl.sub.2 (0.30 mL) was stirred at room temperature for 18 h
and then concentrated in vacuo. The crude product was purified by
HPLC to afford BIO-1029 (2.6 mg, 69%) as a white solid: .sup.1H NMR
(CD.sub.3SOCD.sub.3, 300 MHz, ppm) consistent with structure; HPLC
(Gradient A), 28.2 min; MS, m/z 588.
EXAMPLE 7
Synthesis of BIO-1032
[0174] A. The procedure as described in Example 1A was performed
utilizing 3-amino-phenylacetic acid (29 mg, 0.19 mmol), EDC (44 mg,
0.23 mmol), and HOBt (44 mg, 0.29 mmol), amine from Example 6A (49
mg, 0.15 mmol) and diisopropylethylamine (0.17 mL, 0.95 mmol) in
DMF (1.0 mL) to afford NH.sub.2-BIO1032-OtBu (22 mg, 31%) as a foam
after flash chromatography (SiO.sub.2, 60% ethyl acetate-hexane):
.sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.45-7.05 (m, 7H), 6.75-6.50
(m, 3H), 5.97 (d, 1H), 5.30 (m, 1H), 4.46 (m, 1H), 3.50 (s, 2H),
2.71 (m, 2H), 1.70-1.39 (m, 3H), 1.33 (s, 9H), 0.84 (m, 6H).
[0175] B. A mixture of NH.sub.2-BIO1032-OtBu (7.0 mg, 0.015 mmol),
phenylsulfonyl chloride (1.7 .mu.L, 0.014 mmol), and
diisopropylethylamine (5.4 .mu.L, 0.030 mmol) in CH.sub.2Cl.sub.2
was stirred at room temperature for 18 h. The reaction mixture was
concentrated in vacuo and the residue diluted with ethyl acetate.
The organic solution was washed with 60% sat. aq. NaHCO.sub.3
(2.times.), H.sub.2O, 5% citric acid (3.times.), H.sub.2O, and sat.
aq. NaCl, dried (MgSO.sub.4) and concentrated. The residue (9 mg)
was stirred in trifluoroacetic acid (1 mL) at room temperature for
30 min before concentrating in vacuo. The resulting crude product
was purified by HPLC to afford BIO-1032 (3.9 mg, 47%) as a white
solid: .sup.1H NMR (CD.sub.3SOCD.sub.3, 300 MHz, ppm) 8.52 (d, 1H),
8.17 (d, 1H), 7.75 (d, 2H), 7.61-7.45 (m, 3H), 7.35-6.85 (m, 9H),
5.13 (m, 1H), 4.28 (m, 1H), 3.40 (m, 2H), 2.65 (bs, 2H), 1.50-1.12
(m, 3H), 0.79 (d, 3H), 0.71 (d, 3H); HPLC (Gradient B), 18.7 min;
MS, m/z 552.
EXAMPLE 8
Synthesis of BIO-1093
[0176] A. To a stirred solution of the Boc-protected amine product
of Procedure C (41.5 mg, 0.102 mmol) at 0-5.degree. C. in 2 mL of
CH.sub.2Cl.sub.2 was added 4 mL of TFA. The mixture was allowed to
come to room temperature with continued stirring for 1 hour. The
reaction was concentrated in vacuo, redissolved in
CH.sub.2Cl.sub.2, concentrated two more times and placed under high
vacuum to remove final traces of TFA. HPLC showed complete
conversion to two new peaks of shorter retention time.
[0177] B. The material from Example 8A was redissolved in 0.75 mL
DMF, cooled to 0-5.degree. C. and DIEA was added until the mixture
was basic to litmus and the ice bath was removed. This material
combined with 4-nitrophenyl acetic acid (16.5 mg, 0.091 mmol), HOBt
(20.4 mg, 0.151 mmol) and EDC (19.4mg, 0.101 mmol) under conditions
described in Example 1A to yield BIO 1093-OEt (21.4 mg, 50%) as a
clear oil.
[0178] C. A solution of BIO 1093-OEt (21.4 mg, 0.053 mmol) in 1 ml
of MeOH was stirred overnight at room temperature with 1N LiOH (130
.mu.l, 0.13 mmol). The mixture was acidified (red to litmus) with
TFA and concentrated in vacuo. Pure isomers were resolved via
preparative HPLC followed by lyophilization. Repeated dissolution
in 50/50 MeOH/CH.sub.2Cl.sub.2 and in vacuo concentration followed
by 24 hours under high vacuum provided BIO-1093 (3 mg, 13%) of each
isomer as white amorphous solids: Isomer A: .sup.1H NMR:
(CDCl.sub.3, 300 MHz, ppm), 8.09 (d 2H J=8.2 Hz), 7.38 (d, 2H,
J=8.21 Hz), 7.15 (s, 5H), 5.21 (m, 1H), 4.32 (m, 1H), 3.28 (s, 1H),
2.67 (m, 2H), 1.40 (M, 3H), 0.75 (dd, 6H J=6.9, 7.6 Hz). FAB: 442
(M+H).sup.+, 464 (M+Na).sup.+ Mw 441.43. HPLC: Gradient 1 single
peak >99% 19.5 min. Tlc: 10% MeOH/CH.sub.2Cl.sub.2 R.sub.f=0.25,
EtOAc plus 1% HOAC R.sub.f=0.35. Isomer B: .sup.1H NMR:
(CDCl.sub.3, 300 MHz, ppm), 8.0 (d, 2H, J=9.7 Hz), 7.56 (d, 1H
J=8.0 Hz), 7.73 d, 2H J=9.7 Hz), 7.07 (s, 5H), 5.15 (t, 1H, J=5.5
Hz), 4.29 (m, 1H), 3.45 (s, 2H), 2.65 (m, 2H), 1.45 (m, 3H), 0.78
(dd, 6H, J=6.9, 4.8 Hz). FAB: 442 (M+H).sup.+, 464(M+Na).sup.+, MW
441.43. HPLC: Single peak >99%, 19.3 min. Tlc: 10%
MeOH/CH.sub.2Cl.sub.2 R.sub.f=0.29, EtOAc plus 1% HOAc
R.sub.f=0.55.
EXAMPLE 9
Synthesis of RIO-1099
[0179] A. The amine from Example 3A (50.0 mg, 0.127 mmol) was,
subjected to the conditions described in Example 8B using
diphenylacetic acid (25.6 mg, 0.121 mmol), HOBt (26 mg, 0.19 mmol),
and EDC (27 mg, 0.14 mmol) in DMF to afford BIO 1099-OMe (49.2 mg,
83%) as a clear viscous oil.
[0180] B. BIO1099-OMe (49 mg, 0.1 mmol) was saponified and purified
as described in Example 8C to provide BIO-1099A (7 mg, 15%) and
BIO-1099B (5 mg, 11%) as white amorphous solids. Isomer A: .sup.1H
NMR: (CDCl.sub.3, 300 MHz, ppm), 7.95 (d, 1H 8 Hz), 7.19 (m, 15H),
6.95 (d, 1H 8 Hz), 5.25 (t, 1H, J=3.2), 4.84 (s, 1H), 4.41 (m, 1H),
2.70 (dd, 2H, J=2.5, 1.3 Hz), 1.41 (m, 3H), 0.79 (dd, 6H, (J=6 Hz).
FAB: (M+H).sup.+ 474, (M+Na).sup.+ 496 MW 472.54 HPLC: 1 peak; 100%
pure; 30.074 min. Tlc: 10% MeOH/CH.sub.2Cl.sub.2 R.sub.f=0.33;
50/50 EtOAc/Hex, 1% HOAc R.sub.f=0.45 Isomer B .sup.1H NMR:
(CDCl.sub.3, 300 MHz, ppm) 7.72 (d, 1H, 8 Hz), 7.22 (m, 15H), 5.31
(t, 1H, 1.2 Hz), 6.70 (d, 1H 8 Hz), 4.93 (s, 1H), 4.60 (m, 1H),
2.68 (s, 1H), 2.65 (m, 2H ), 1.35 (m, 3H), 0.61 (dd, 6H, J=2.5, 1.3
Hz). FAB: 473 "(M+H).sup.+, 495 (M+Na).sup.+; MW 472.54 HPLC: 1
Peak; 100%; 30.38 min. Tlc: 10% MeOH/CH.sub.2Cl.sub.2 R.sub.f=0.33,
50/50 EtOAc/Hex plus 1% HOAC R.sub.f=0.38.
EXAMPLE 10
Synthesis of BIO-1100
[0181] A. The amine salt described in Example 6A (prepared from
40.5 mg, 0.093 mmol of Boc protected material) was taken up in 1.0
mL of DMF and TEA was added with stirring until basic to
litmus.
[0182] B. The method described in Example 1A was performed using
2-bromo-5-methoxy-4-hydroxy phenyl acetic acid (23.1 mg, 0.089
mmol), HOBt (18.9 mg, 0.14 mmol), EDC (19.6 mg, 0.10 mmol) in 1.0
ml DMF and free amine prepared in Example 10A to give a white solid
(49 mg, quantitative). An aliquot was purified by preparative
reverse phase HPLC (gradient 2), lyophilized and dried-by
repeatedly dissolving in 50/50 MeOH/CH.sub.2Cl.sub.2 and
concentrated under reduced pressure to yield BIO-1100 (1.8 mg) as
an amorphous white solid. .sup.1H NMR: (CDCl.sub.3 300 MHz, ppm),
7.25 (s, 5H), 7.05 (s, 1H), 6.30 (s, 1H), 5.28 (m, 1H), 3.81 (s,
3H), 3.59 (s, 2H), 2.77 (m, 2H), 1.45 (m, 3H), 0.82 (dd, 6H J=2.5,
1.2). FAB: (M+H).sup.+521, 523; (M+Na).sup.+543, 545; MW 521.44
HPLC: Major peak at 29.1 min; >97% purity. Tlc: 10%
MeOH/CH.sub.2Cl.sub.2 R.sub.f=0.16; 50/50 EtOAc/Hex plus 1% HOAc
R.sub.f=0.28
EXAMPLE 11
Synthesis of BIO-1106
[0183] A. To a solution of 6-aminohexanoic acid (1.0 g, 7.6 mmol)
in dioxane (6 ml) and water (6 ml) containing TEA (1.7 ml, 11.25
mmol) was added BOC-ON (2.1g, 8.4 mmol, Aldrich). After stirring
for 3 h at room temperature, the reaction was diluted with water
(20 ml) and washed twice with ethyl acetate (10 ml). The aqueous
was then acidified to PH=1-2 with 1N HCl and the aqueous layer
extracted five times with ethyl acetate, dried over
Na.sub.2SO.sub.4 and concentrated to afford 1106-1 (842 mg, 51%).
.sup.1HNMR (CDCl.sub.3' 300 MHz, ppm): 4.61 (1H, bs), 3.15-2.95
(4H, bm), 2.55-2.23 (4 H, m), 1.65-1.50 (4 H), 1.46 (9 H),
1.45-1.30 (2 H, m).
[0184] B. t-Butyl 3-amino-3-phenyl-1-propanoate was coupled with
CbzLeuOSu as described in Procedure C. This material was subjected
to the conditions of Procedure D2 to give the desired free
amine.
[0185] C. N-Boc 6-aminohexanoic acid (prepared in Example 11A)
(17.3 mg, 0.075 mmol), HOBt (15.2 mg, 0.11 mmol) and EDC (17.3 mg,
0.09 mmol) were stirred in 0.5 ml of DMF at room temperature for
1.5 hours. The free amine from Example 11B (25 mg, 0.075 mmol) in
0.5 ml of DMF was added to the stirred solution of activated ester
along with two drops of TEA so that the reaction was basic to
litmus. After several hours the reaction was determined to be
incomplete by HPLC. Small portions of N-Boc-6-aminohexanoic acid,
HOBt, and EDC were then added to drive the reaction to completion.
Purification, as detailed in Example 8C, provided BIO 1106 Boc
t-butyl ester (26 mg, 63%) as a clear viscous oil. .sup.1H NMR:
(CDCl.sub.3 300 MHz, ppm), 7.40 (d, 1H, 8 Hz), 7.32-7.25 (m, 5H),
6.30 (d, 1H, J=8 Hz), 5.30 (q, 1H, J=7 Hz), 4.49 (m, 1H), 3.09 (bs,
2H), 2.79 (dd, 1H, J=8, 15 Hz), 2.69 (dd, 1H, J=7, 15 Hz), 2.20 (t,
2H J=8 Hz), 1.69-1.39 (m, 9H), 1.42 (s, 9H), 1.29 (s, 9H), 0.88 (m,
6H). HPLC: 1 peak, 100% purity at 28.3 min.
[0186] Both t-butyl protecting groups of BIO 1106 Boc t-butyl ester
were removed as described in Example 10A. The resulting residue was
stirred in 0.5 ml of DMF, made basic to litmus by the addition of
two drops of TEA, followed by phenyl isocyanate (13.6 mg, 0.3 mmol)
and stirred overnight. The reaction mixture was purified as
detailed in Example 10B resulting in BIO-1106 (3.5 mg, 29%) as a
beige amorphous solid. .sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm),
7.97 (d, 1H, 8 Hz), 7.22 (m, 11H), 6.91 (t, 1H J=8 Hz), 5.30 (m,
1H), 4.33 (m, 1H), 3.12 (m, 6H), 2.63 (m, 2H), 2.13 (t, 2H, J=6
Hz), 1.41 (bm, 9H), 0.80 (m, 6H). FAB: (M+H).sup.+ 511,
(M+Na).sup.+ 533; MW 510.59. HPLC: 1 peak; 100% at 19.4 min. Tlc:
15% MeOH/CH.sub.2Cl.sub.2 R.sub.f=0.32, 10% MeOH/EtOAc plus 1% HOAc
R.sub.f=0.31.
EXAMPLE 12
Synthesis of BIO-1142
[0187] (.+-.)-1-Benzocyclobutene carboxylic acid (16.3 mg, 0.11
mmol), HOBt (22.4 mg, 0.165 mmol), and EDC (23.7 mg, 0.121 mmol)
were stirred in 0.5 ml DMF at room temperature for 45 minutes to
give the activated ester. The product of Example 10A (15.3 mg,
0.055 mmol) was added to the activated ester and the mixture
stirred for two hours. Filtration and preparative HPLC
purification, as described in Example 10B, yielded BIO-1142 isomer
A (4.4 mg, 70%) and BIO-1142 isomer B (4.9 mg, 22%) as white
amorphous solids.
[0188] BIO-1142 isomer A: .sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm),
7.79 (d, 1H J=8 Hz), 7.31-7.05 (m, 9H), 6.81 (d, 1H J=8 Hz), 5.24
(m, 1H), 4.36 (m, 1H), 4.15 (m, 1H), 3.00-3.50 (bm, 11H), 2.70 (m,
2H), 1.43 (m, 3H), 0.70 (m, 6H). FAB: (M+H).sup.+ 409 (M+Na).sup.+
431; MW 408.46. HPLC: Major peak at 20.2 min; >99% purity. Tlc:
10% MeOH/CH.sub.2Cl.sub.2 R.sub.f=0.46, EtOAc plus 1% HOAc
R.sub.f=0.53.
[0189] BIO-1142 isomer B: .sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm),
7.92 (d, 1H, J=8 Hz), 7.31-7.05 (m, 9H), 6.91 (d, 1H, J=8 z), 5.25
(m, 1H), 4.38 (m, 1H), 4.14 (m, 1H), 3.28 (m, 2H), 2.72 (m, 2H),
1.42 (m, 3H), 0.77 (m, 6H). FAB: (M+H).sup.+ 409 (M+Na).sup.+ 431;
MW 408.46. HPLC: Major peak at 20.62 min; >96% purity. Tlc:10%
MeOH/CH.sub.2Cl.sub.2 R.sub.f=0.52; EtOAc plus 1% HOAc
R.sub.f=0.54.
EXAMPLE 13
Synthesis of BIO-1189
[0190] (.+-.)-1-indancarboxylic acid (6.2 mg, 0.038 mmol), HOBt
(7.7 mg, 0.057 mmol), and EDC (8.0 mg, 0.042 mmol) were stirred in
0.5 ml DMF at room temperature for two hours. The free amine
prepared in Example 11B was treated with TFA and this material (10
mg, 0.038 mmol) was then added and the mixture stirred overnight.
Filtration and preparative HPLC purification as described in
Example 10B yielded BIO-1189 isomer A (less than 1 mg) and isomer B
(2 mg, 12%) as white amorphous solids.
[0191] BIO-1189 isomer A: .sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm),
7.3-7.1 (m, 12H), 5.32 (m, 1H), 4.48 (m, 1H), 3.91 (t, 1H J=6.6
Hz), 3.1-2.7 (m, 3H), 2.5-2.2 (m, 1H), 1.6-1.4 (m, 3H), 0.85 (m,
6H).
[0192] FAB: (M+H).sup.+, 423 (M+Na).sup.+ 445; MW 422.5.
[0193] HPLC: Major peak 21.2 min.; >97% purity.
[0194] Tlc: 5% MeOH/CH.sub.2Cl.sub.2 R.sub.f=0.19; EtOAc plus 1%
HOAc R.sub.f=0.73.
[0195] BIO-1189 isomer B: .sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm),
7.7(d, 1H, J=8 Hz), 7.45-7.1 (m, 9H), 6.65 (d, 1H, J=8 Hz), 5.33
(m, 1H), 4.48 (m, 1H), 3.90 (t, 1H, J=6.6 Hz), 3.1-2.8 (m, 3H),
2.45-2.3 (m, 2H), 1.48 (m, 3H), 0.80 (m, 6H),
[0196] FAB: (M+H).sup.+ 423 (M+Na).sup.+ 445; MW 422.5.
[0197] HPLC: Major peak 21.5 min; >94% purity.
[0198] Tlc: 5% MeOH/CH.sub.2Cl.sub.2 R.sub.f=0.12, EtOAc plus 1%
HOAc R.sub.f=0.60.
EXAMPLE 14
Synthesis of BIO-1006
[0199] A. Amine .beta.-3 was coupled with BocLeuOSu according to
Procedure C (product recrystallized from diethyl ether) and
deprotected according to Procedure D to give the desired TFA-amine
salt.
[0200] .sup.1H-NMR (300 MHz, CDCl.sub.3) for BOC amine: 0.90 (m,
6H), 1.42 (9H), 1.55-1.75 (m, 3H), 2.8 (m, 2H), 3.61 (s, 3H), 4.05
(m, 1H), 4.83 (m, 1H), 5.26 (m, 1H), 5.92 (s, 2H), 6.68-6.78 (m,
3H), 7.06 (d, 1 H).
[0201] 1H-NMR (300 MHz, CDCl.sub.3) for TFA-amine: 0.83 (d, 3H),
0.87 (d, 3H), 1.50 (m, 1H), 1.63 (bt, 2H), 2.73-2.92 (m, 2H), 3.63
(s, 3H), 4.27 (bs, 1H), 5.26 (m, 1H), 5.95 (s, 2H), 6.66-6.78 (m,
3H), 7.58 (bs, 3H), 8.02 (d, 1 H).
[0202] B. A solution of amine-TFA salt of Example 14A (24 mg) in
CH.sub.2Cl.sub.2 was added to 4-hydroxyphenylacetic acid
succinimidyl ester (14 mg, 1.1 eq) and stirred at room temperature
for about 2 hours. The reaction mixture was washed with 5% citric
acid (2.times.), sat. aq. NaHCO.sub.3 (2.times.) and brine
(1.times.), dried (Na.sub.2SO.sub.4), filtered and concentrated to
give 28 mg of crude BIO-1006 methyl ester. .sup.1H-NMR: (300 MHz,
CDCl.sub.3) 0.82 (6 H), 1.35-1.58 (3 H), 2.62-2.82 (2 H), 3.48 (2
H), 3.57 (3 H), 4.41 (1 H), 5.70 (1 H), 5.89 (2 H), 6.08 (1 H),
6.65-6.75 (5 H), 7.04 (2 H), 7.22 (1 H).
[0203] C. Crude BIO-1006 methyl ester in MeOH was added to 1 N LiOH
and stirred at room temperature for about 1 hour. The reaction
mixture was neutralized by trifluoroacetic acid and purified by
HPLC. The clean fraction was collected and dried to give
BIO-1006.
[0204] .sup.1H-NMR (300 MHz, CDCl.sub.3): 0.73 (d, J=6 Hz, 3H),
0.80 (d, J=6 Hz, 3H), 1.35 (bt, 2 H), 1.45 (m, 1H), 2.40 (m, 2H),
3.22-3.38 (m, 2H), 4.23 (bq, 1H), 5.02 (m, 1H), 5.93 (s, 2H), 6.65
(d, J=8 Hz, 2H), 6.68-6.80 (m, 2H), 6.83 (s, 1H), 7.03 (d, J=Hz,
2H), 8.11 (bd, 1H). Mass Spec. M/z=457
EXAMPLE 15
Synthesis of BIO-1050
[0205] A. To a suspension of 4-amino phenylacetic acid (9 g, 60
mmol) and N-(benzyloxycarbonyloxy)-succinimide (15 g, 60 mmol) in
CH.sub.2Cl.sub.2 was added enough triethylamine to form a
homogeneous solution. The mixture was stirred at room temperature
for 30 min and then CH.sub.2Cl.sub.2 was removed by rotavapor. The
resulting residue was dissolved in water and acidified with 5% HCl.
The solid thus formed was filtered and washed with 5% HCl, water,
and diethyl ether to give 12 g (70%) of Cbz-aminophenylacetic acid
as a brownish powder. .sup.1H-NMR (300 MHz, DMSO-d6): 3.48 (s, 2H),
5.13 (s, 2H), 7.14 (d, 2H), 7.29-7.45 (m, 7H), 9.73 (s, 1 H).
[0206] B. The method of Example 1A was performed using
Cbz-aminophenylacetic acid from Example 15A (342 mg, 1.2 mmol) in
DMF, HOBT (275 mg, 1.8 mmol), EDC (276 mg, 1.44 mmol), and a
solution of free amine prepared in Example 14A (432 mg, 0.94 mmol)
in DMF to give the coupled product which was used without further
purification.
[0207] C. The product of Example 15B was subjected to hydrogenation
(H.sub.2, 50 psi, 10% Pd/C, MeOH/H.sub.2O, overnight). The reaction
mixture was filtered through a pad of Celite.RTM., and concentrated
to give 0.4 g (90%) of free amine as a brown powder. .sup.1H-NMR
(300 MHz, CDCl.sub.3) for (F): 0.82 (m, 6H), 1.30-1.62 (m, 3H),
2.62-2.82 (m, 2H), 3.45 (s, 2H), 3.57 (s, 3H), 4.37 (m, 1H), 5.18
(m, 1H), 5.91 (s, 2H), 6.65-6.80 (m, 5H), 7.02 (d, 2 H).
[0208] D. To a solution of free amine from Example 15C (22 mg) in
CH.sub.2Cl.sub.2 was added phenylisocyanate (8 mg, 1.5 eq) with one
drop of triethylamine. The solution was then stirred at room
temperature for 2 hours. After diluting with ethyl acetate (15 mL),
the mixture was washed with 5% citric acid (2.times.), sat. aq.
NaHCO.sub.3 (2.times.) and brine (1.times.), dried
(Na.sub.2SO.sub.4), filtered and concentrated to give the crude
phenylureamethyl ester.
[0209] E. The crude phenylureamethyl ester was dissolved in MeOH
and 1 N LiOH was added at 0.degree. C. and mixture was stirred at
room temperature for 2 h. After neutralization with trifluoroacetic
acid, the reaction mixture was purified by HPLC. The pure fraction
was collected and dried to give BIO-1050. .sup.1H-NMR: (300 MHz,
DMSO-D6): 0.76 (d, 3H), 0.80 (d, 3H), 1.30-1.50 (m, 3H), 2.52-2.72
(m, 2H), 3.28-3.50 comp, 2H), 4.30 (m, 1H), 5.06 (m, 1H), 5.97 (s,
2H), 6.70 (d, 1H), 6.79-6.87 (m, 2H), 6.95 (t, 1H), 7.13 (d, 2H),
7.25 (t, 2H), 7.85 (d, 2H), 7.43 (d, 2H), 8.12 (d, 1H), 8.40 (d,
1H), 8.60 (s, 1H), 8.66 (s, 1H). Mass Spec: M/z=575.
EXAMPLE 16
Synthesis of BIO-1068
[0210] The procedure of Example 15D was followed utilizing
cyclohexylisocyanate for phenylisocyanate. The resulting product
was hydrolyzed as described in Example 15E and the pure fraction
from HPLC purification was collected and dried to give
BIO-1068.
[0211] .sup.1H-NMR (300 MHz, DMSO-d6): 0.73 (d, J=6 Hz, 3H), 0.80
(d, J=6 Hz, 3H), 1.05-1.85 (m, 13H), 2.50-2.75 (m, 2H), 3.23-3.50
(m, 3H), 4.28 (bq, 1H), 5.05 (bq, 1H), 5.95 (bs, 2H), 6.02 (d, J=8
Hz, 1H), 6.72 (bd, 1H), 6.71 (d, J=8 Hz, 1H), 6.84 (bs, 1H), 7.08
(d, J=8 Hz, 2H), 7.25 (d, J=8 Hz, 2H), 8.07 (d, J=8 Hz, 1H), 8.20
(s, 1H), 8.40 (d, J=8 Hz, 1H).
[0212] Mass Spec. M/z=581.
EXAMPLE 17
Synthesis of BIO-1079
[0213] The procedure of Example 15D was followed utilizing
2-methoxyphenylisocyanate for phenylisocyanate. The resulting
product was hydrolyzed as described in Example 15E and the pure
fraction from HPLC purification was collected and dried to give
Bio-1079. .sup.1H-NMR (300 MHz, DMSO-d6): 0.75 (d, 3H), 0.80 (d,
3H), 1.30-1.50 (m, 3H), 2.50-2.72 (m, 2H), 3.30-3.45 (m, 2H), 3.85
(s, 3H), 4.28 (m, 1H), 5.06 (m, 1H), 5.96 (bs, 2H), 6.69-7.02 (m,
8H), 7.13 (d, 2H), 7.34 (d, 2H), 8.05-8.15 (m, 3H), 8.42 (bd, 1H),
8.87 (s, 1H), 9.13 (s, 1H). Mass Spec. M/z=605.
EXAMPLE 18
Synthesis of BIO-1082
[0214] A. Triethylamine was added to a solution of the TFA-amine
salt prepared in Procedure D (43 mg) in CH.sub.2Cl.sub.2 at
-0.degree. C. until pH reached 9.0 was reached, followed by the
addition of 4-phenylbutyryl chloride (26 mg). After stirring at
room temperature for 2 h, the reaction mixture was diluted with
ethyl acetate (20 mL) and then washed with 5% citric acid
(2.times.), sat. aq. NaHCO.sub.3 (2.times.) and brine (1.times.),
dried (Na.sub.2SO.sub.4), filtered and concentrated to give the
desired product as an ethyl ester.
[0215] B. The crude ethyl ester was dissolved in MeOH, 1 N LiOH was
added at 0.degree. C. and the mixture was stirred at room
temperature for 2 h. After neutralization with trifluoroacetic
acid, the reaction mixture was purified by HPLC. Two diastereomers
were separated and the pure fractions were collected and dried-to
give Bio-1082-A and Bio-1082-B.
[0216] Bio-1082-B .sup.1H-NMR (300 MHz, DMSO-d6): 0.79 (d, 3H),
0.83 (d, 3H), 1.29-1.37 (m, 2H), 1.47 (m, 1H), 1.70-1.83 (m, 2H),
2.08-2.17 (m, 2H), 2.48-2.58 (m, 2H), 2.67 (bt, 2H), 4.31 (m, 1H),
5.03 (m, 1H), 7.12-7.32 (m, 10H), 7.90 (d, 1H), 8.45 (d, 1 H).
Mass. Spec. M/z=425.
EXAMPLE 19
Synthesis of BIO-1148
[0217] A. Amine .beta.-13 was coupled with BocLeuOSu using the
method described in procedure C. This material was subjected to the
conditions of Procedure D1 to give the desired amine salt
1148-1.
[0218] B. To a solution of 4-hydroxyphenylacetic acid (3.0 g, 20
mmol) in DMF was added HOBT (3.7 g, 24 mmol) followed by EDC (4.2
g, 22 mmol) and the mixture was stirred at room temperature for 30
min. N-hydroxysuccinimide (2.3 g, 20 mmol) was added and stirred at
room temperature overnight. The resulting mixture was diluted with
ethyl acetate (150 ml), extracted with 5% citric acid (2.times.),
saturated NaHCO.sub.3 (2.times.) and brine (1.times.) and was dried
over anhydrous Na.sub.2SO.sub.4. Following removal of the solvent
in vacuo the product was dissolved in CH.sub.2Cl.sub.2 and
precipitated with hexanes to afford 4-hydroxyphenylacetic acid
succinimidyl ester (3.9 g, 786). .sup.1H NMR (300 MHz, DMSO-d6):
2.79 (s, 4 H), 3.93 (s, 2 H), 6.72 (d, J=8.5 Hz, 2 H), 7.12 (d,
J=8.5 Hz, 2 H), 9.41 (s, 1 H).
[0219] C. Amine salt 1148-1 was hydrolysed under MeOH/aqueous LiOH
conditions to give an acid. A solution of this acid, triethylamine,
and 4-hydroxyphenylacetic acid-OSu (prepared in Example 19B) in
CH.sub.2Cl.sub.2 was stirred at room temperature for 1 h. The
reaction mixture was purified by HPLC and the pure fraction was
collected and dried to give Bio-1148 as a mixture of two
diastereomers. .sup.1H-NMR (300 MHz, DMSO-d6): 0.70-0.90 (m, 6H),
1.29-1.63 (m, 3H), 2.73-2.85 (m, 2H), 3.17-3.40 (m, 2H), 4.15-4.30
(m, 1H), 5.12-5.28 (m, 1H), 6.58-6.68. (m, 2H), 6.94-7.06 (m, 2H),
7.54-7.67 (m, 1H), 7.93-8.16 (m, 2H), 8.53-8.75 (m, 3 H). Mass.
Spec. M/z=414.
EXAMPLE 20
Synthesis of BIO-1168
[0220] The procedure that was used in Example 15D was followed
utilizing 3-methylphenylisocyanate for phenylisocyanate. The
resulting product was hydrolyzed as described in Example 15E and
the pure fraction from HPLC purification was collected and dried to
give Bio-1168. .sup.1H-NMR (300 MHz, DMSO-d6): 0.76 (d, 3H), 0.82
(d, 3H), 1.30-1.52 (m, 3H), 2.28 (s, 3H), 2.54-2.70 (m, 2H),
3.35-3.48 (m, 2H), 4.28 (m, 1H), 5.07 (m, 1H), 5.96 (m, 2H),
6.68-6,86 (m, 4H), 7.10-7.25 (m, 4H), 7.30 (s, 1H), 7.35 (d, 2H),
8.11 (d, 1H), 8.44 (d, 1H), 8.63 (s, 1H), 8.67 (s, 1 H). Mass Spec.
M/z=589.
EXAMPLE 21
Synthesis of BIO-1179
[0221] The procedure that was used in Example 15D was followed
utilizing 2-methylphenylisocyanate for phenylisocyanate. The
resulting product was hydrolyzed as described in Example 15E and
the pure fraction from HPLC purification was collected and dried to
give Bio-1179. .sup.1H-NMR (300 MHz, DMSO-d6): 0.75 (d, 3H), 0.80
(d, 3H), 1.27-1.51 (m, 3H), 2.23 (s, 3H), 2.62 (m, 2H), 3.40 (m,
2H), 4.28 (m, 1H), 5.06 (m, 1H), 5.98 (bs, 2H), 6.71 (bd, 1H), 6.80
(d, 1H), 6.83 (bs, 1H), 6.92 (bt, 1H), 7.05-7.20 (m, 4H), 7.38 (d,
2H), 7.82 (d, 1H), 7.87 (s, 1H), 8.10 (d, 1H), 8.42 (d, 1H), 8.93
(s, 1 H). Mass Spec. M/z=589.
EXAMPLE 22
Synthesis of BIO-1195
[0222] A. Amine .beta.-9 was coupled with BocLeuOSu according to
Procedure C to give the desired product.
[0223] .sup.1H-NMR (300 MHz, CDCl.sub.3): 0.90 (m, 6H), 1.32 (s,
9H), 1.42 (s, 9H), 1.58-1.90 (m, 3H), 2.61-2.80 (m, 2H), 4.08 (m,
1H), 4.89 (bd, 1H), 5.37 (bq, 1H).sub.1 6.95-7.15 (m, 3H), 7.45
(bd, 1 H).
[0224] B. The product of Example 22A was treated with TFA as
described in Procedure D to give the corresponding TFA-amine salt
1195-2.
[0225] C. A mixture of 4-amino-phenylacetic acid (10.0 g, 66.1
mmol) and 98% phenyl isocyanate. (8.27 g, 68.0 mmol) in ethyl
acetate (100 mL) was stirred at RT for 1 h then refluxed for 1.5 h.
The mixture was allowed to cool to RT and the product was filtered,
washed with ethyl acetate, methanol, and then ether affording
phenylureaphenylacetic acid 1195-3 (17.5 g, 98%) as a white powder.
.sup.1HNMR (DMSO-d.sup.6, 300 MHz, ppm): 8.72-8.64 (m, 2H), 7.44
(d, 2H), 7.36 (d, 2H), 7.28 (d, 2H), 7.16 (d, 2H), 6.96 (t, 1H),
3.52 (s, 2H). FAB-MS=272.
[0226] D. A solution of phenylureaphenylacetic acid 1195-3, HOBT,
and EDC in DMF was stirred at room temperature for 30 min and then
the free amine prepared from the product of Example 22B and TEA
treatment was added. After stirring at room temperature overnight,
the reaction mixture was purified by HPLC and the pure fraction was
collected and dried to give Bio-1195.
[0227] .sup.1H-NMR (300 MHz, DMSO-d6): 0.71 (d, 3H), 0.78 (d, 3H),
1.25-1.46 (m, 3H), 2.56-2.72 (m, 2H), 3.26-3.41 (m, 2H), 4.21 (bq,
1H), 5.07 (bq, 1H), 6.90 (bt, 1H), 7.02-7.14 (m, 3H), 7.17-7.42 (m,
8H), 8.10 (d, 1H), 8.47 (d, 1H), 8.58 (s, 1H), 8.63 (s, 1 H). Mass
Spec. M/z=567.
EXAMPLE 23
Synthesis of BIO-1198
[0228] A. To a solution of phosgene in CH.sub.2Cl.sub.2 at
0.degree. C. was added a solution of morpholine and triethylamine
in CH.sub.2Cl.sub.2 dropwise. The reaction was then stirred at room
temperature for 30 min and concentrated in vacuo to give a white
solid. This crude product was dissolved in CH.sub.2Cl.sub.2 and
4-aminophenylacetic acid t-butyl ester was added. The mixture was
stirred at room temperature overnight, diluted with ethyl acetate
(20 mL), washed with 5% citric acid (2.times.), sat. aq NaHCO.sub.3
(2.times.) and brine (1.times.), dried (Na.sub.2SO.sub.4), filtered
and concentrated to give morpholineurea t-butyl ester 1198-1.
[0229] .sup.1H-NMR (300 MHz, CDCl.sub.3) for t-butyl ester (A):
1.40 (s, 9H), 3.38-3.46 (m, 4H), 3.60-3.70 (m, 6H), 6.67 (s, 1H),
7.13 (d, 2H), 7.27 (d, 2 H).
[0230] B. The morpholineurea t-butyl ester 1198-1 was dissolved in
CH.sub.2Cl.sub.2 and trifluoroacetic acid was added. The solution
was stirred at room temperature for 3 h. and concentrated to give
26 mg of the corresponding carboxylic acid 1198-2.
[0231] C. The method described in Example 1A was performed using
carboxylic acid 1198-2 (26 mg) dissolved in DMF, HOBT, EDC, and the
amine prepared in Example 14A to give 27 mg of crude methyl ester
1198-3.
[0232] D. A solution of crude methyl ester 1198-3 was treated as
described in Example 14C to give Bio-1198. .sup.1H-NMR (300 MHz,
DMSO-d6) for BIO 1198: 0.75 (d, 3H), 0.82 (d, 3H), 1.27-1.50 (m,
3H), 2.53-2.70 (m, 2H), 3.28-3.45 (m, 6H), 3.55-3.60 (m, 4H), 4.27
(m, 1H), 5.07 (bq, 1H), 5.96 (bs, 2H), 6.72 (bd, 1H), 6.82 (d, 1H),
6.85 (bs, 1H), 7.09 (d, 2H), 7.35 (d, 2H), 8.08 (d, 1H), 8.42 (d,
1H), 8.47 (s, 1 H). Mass Spec. M/z=569
EXAMPLE 24
Synthesis of Bio-1190
[0233] A. Amine .beta.-5 was coupled with BocLeuOSu as described in
Procedure C. This material was subjected to the conditions of
Procedure D1 to give the desired amine salt.
[0234] B. The protocol described in Example 1A was performed using
2-methylphenylureaphenylacetic acid (135 mg, 0.47 mmol) in DMF (2.5
ml), HOBt (135 mg, 0.88 mmol), EDC (0.71 mmol) and the amine salt
from Example 29A (200 mg, 0.46 mmol) (treated with Et.sub.3N until
pH 10 was reached) to-give 1190-1 (235 mg, 89%) as a white
solid.
[0235] C. To a stirred solution of 1190-1 (20 mg, 0.034 mmol) in
MeOH (3 mL) was added aqueous LiOH (3 mL of 2N). After stirring at
room temperature overnight, the reaction mixture was cooled to
0.degree. C. and acidified by adding TFA until pH=3-4 (pH paper).
The desired product was isolated and purified by LC (Vydac C18
column; gradient 8) to give 10 mg (0.017 mmol; 50%) of BIO-1190 as
a white solid. .sup.1H NMR (DMSO-d.sup.6, 300 MHz, ppm) 8.95 (s, 1
H, NH), 8.39 (d, 1 H, J=9 Hz, NH), 8.11 (d, 1 H, J=9 Hz, NH), 7.88
(s, 1 H, NH), 7.83 (d, 1 H, J=8 Hz, Ar), 7.36 (d, 2 H, J=8.4 Hz,
Ar), 7.2-7.1 (comp, 6 H, Ar), 6.92 (m, 1 H, Ar), 6.83 (d, 2 H, J=9
Hz, Ar), 5.08 (m, 1 H), 4.28 (m, 1 H), 3.70 (s, 3 H, OMe), 3.39 (d,
1 H, J=8 Hz), 3.31 (d, 1 H, J=7 Hz), 2.63 (m, 1 H), 2.23 (s, 3 H,
Me), 1.50-1.25 (comp, 3 H), 0.81 (d, 3 H, J=6 Hz), 0.75 (d, 3 H,
J=6 Hz); FABMS, m/z 575 (C.sub.32H.sub.38N.sub.4O.sub.6 of M.sup.+1
requires 575).
EXAMPLE 25
Synthesis of Bio-1197
[0236] A. Amine B-1 (0.884 g, 4.0 mmol) was coupled with BocLeuOSu
(1.32 g, 4.0 mmol) as described in Procedure C. This material was
subjected to the conditions of Procedure D1 to give the desired
amine salt (1.42 g, 85%) as a white solid.
[0237] .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm): 7.31-7.22 (m, 5H),
7.14 (d, 1H), 5.37-5.30 (m, 1H), 4.84 (m, 1H), 4.10 (m, 1H),
2.85-2.66 (m, 2H), 1.72-1.58 (m, 2H), 1.51-1.49 (m,1H), 1.48 (s,
9H), 1.29 (s, 9H), 0.91 (m, 9H).
[0238] B. The procedure of Example 1A was performed using
2-methylphenylureaphenylacetic acid (34 mg, 0.12 mmol), HOBT (20
mg, 0.14 mmol), EDC (26 mg, 0.134 mmol) and the amine salt of
Example 25A (30 mg, 0.079 mmol) in the presence of Et.sub.3N to
give 15 mg (0.028 mmol; 35%) of Bio-1197 as a white foam: FABMS,
m/z 545 (C.sub.31H.sub.36N.sub.4O.sub.5 of M.sup.+1 requires
545).
EXAMPLE 26
Synthesis of BIO-1201
[0239] A. The procedure of Example 15D was performed using the free
amine from Example 15C (40 mg, 0.086 mmol) and 2-nitrophenyl
isocyanate (28 mg, 0.172 mmol) to give 50 mg (92%) of 1201-1 as a
light yellow oil. .sup.1H NMR (DMSO-d.sup.6, 300 MHz, ppm) 8.55 (d,
1 H, NH), 8.50 (d, 1 H, NH), 8.15 (d, 1 H, NH), 8.05 (d, 1 H, NH),
7.6-6.7 (11 H, Ar), 5.85 (bs, 2 H), 5.25 (m, 1 H), 4.6 (m, 1 H),
3.8-3.55 (comp), 3.5 (s, 3 H, OMe), 2.75 (m, 2 H), 1.7-1.4 (comp, 3
H), 0.85 (m, 6 H).
[0240] B. The procedure of Example 24C was performed using 1201-1
(50 mg, 0.079 mmol) to give 17 mg (0.027 mmol; 35%) of BIO-1201 as
a light yellow solid. FABMS, m/z 620
(C.sub.31H.sub.35N.sub.5O.sub.9 of M.sup.+1 requires 620)
EXAMPLE 27
Synthesis of Bio-1217
[0241] A. Amine 0-4 (30 mg, 0.1 mmol) was coupled with
N.alpha.-t-Boc-N.epsilon.-CBZ-.sub.L-Lysine-N-Hydroxysuccinimide
(50 mg, 0.1 mmol) as described in Example 25A to give 60 mg (93%)
of 1217-1 as a white foam. .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm)
7.35-7.25 (comp, 5 H, Ar), 6.8-6.7 (comp, 3 H, Ar), 5.3-5.1 (comp,
2 H), 4.95 (m, 1 H), 4.05 (m, 1H), 3.8 (s, 3 H, OMe), 3.78 (s, 3 H,
OMe), 3.1 (m, 2 H), 2.7 (m, 2 H), 1.9-1.4 (comp), 1.35 (S, 9 H,
Bu.sup.t), 1.3 (s, 9 H, Bu.sup.t)
[0242] B. Compound 1217-1 (60 mg, 0.09 mmol) in CH.sub.2Cl.sub.2 (5
mL) was deprotected with trifluoroacetic acid (0.5 mL) as described
in Procedure D1 to give 56 mg (100%) of 1217-2 as a white foam.
[0243] .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 8.75 (bs), 7.35-7.15
(comp, Ar), 6.85-6.65 (comp, Ar), 5.4 (m), 5.2-4.9 (bs, Bn), 4.15
(m), 3.75 (bs), 3.15-2.6 (comp), 1.8 (m), 1.4-1.0 (comp).
[0244] C. The procedure of Example 1A was performed using
2-methylphenylureaphenylacetic acid (40 mg, 0.14 mmol), HOBT (23
mg, 0.167), EDC (30 mg, 0.158 mmol) and amine 1217-2 (56 mg, 0.093
mmol) was added in the presence of Et.sub.3N to give 21 mg (30%) of
BIO-1217 as a white foam. .sup.1H NMR (DMSO-d.sub.6, 300 MHz, ppm)
9.05 (m, 1H, NH), 8.4 (m, 1 H, NH), 8.1 (m, 1 H, NH), 8.0 (m, 1 H,
NH), 7.4-6.7 (comp, Ar), 5.1 (m, 1 H), 5.0 (bs, 2 H), 4.2 (m, 1 H),
3.7 (bs, 6 H, OMe), 2.9-2.6 (comp), 2.2 (s, 3 H, Me),1.6-1.1
(comp); FABMS, m/z 754 (C.sub.41H.sub.47N.sub.5O.sub.9 of M.sup.+1
requires 754).
EXAMPLE 28
Synthesis of BIO-1225
[0245] A. Amine .beta.-3 (90 mg, 0.4 mmol) coupled with
N.alpha.-t-Boc-N.epsilon.-CBZ-.sub.L-Lysine-N-Hydroxysuccinimide
(193 mg, 0.4 mmol) as described in Example 25A to give 220 mg (94%)
of 1225-1 as a white foam. .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm)
7.4-7.25 (5 H, Ar), 7.1 (m, 1 H, NH), 6.8-6.65 (3 H, Ar), 5.9 (s, 2
H), 5.25 (m, 1 H), 5.15 (m, NH), 5.05 (s, 2 H), 4.85 (m, 1 H), 4.0
(m, 1 H), 3.6 (s, 3 H, OMe), 3.15 (m, 2 H), 2.80 (m, 2 H),
1.90-1.20 (6 H), 1.4 (s, 9 H).
[0246] B. The BOC protecting group of 1225-1 (170 mg, 0.29 mmol)
was removed as described in Procedure D1 to give 100 mg (71%) of
free amine 1225-2 as a white foam.
[0247] .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 8.07 (d, 1 H, J=9
Hz), 7.4-7.2 (comp, 5 H), 6.80-6.65 (comp, 3 H), 5.90 (s, 2 H),
5.25 (m, 1 H), 5.05 (s, 2 H), 4.98 (bs, 1 H), 3.58 (s, 3 H, OMe),
3.32 (m, 1 H), 3.16 (m, 2 H), 2.27 (m, 2 H), 1.90-1.70 (comp, 3 H),
1.6-1.25 (comp, 5 H).
[0248] C. The procedure-of Example 1A was performed using
2-methylphenylureaphenylacetic acid (44 mg, 0.155 mmol), HOBT (36
mg, 0.264 mmol), EDC (47 mg, 0.248 mmol) and free amine 1225-2 (50
mg, 0.103 mmol) to give 46 mg (80%) of BIO-1225-3 as a white foam.
.sup.1H NMR (DMSO-d.sup.6, 300 MHz, ppm) .delta. 9.0-6.7 (21 H, Ar
& NH), 5.96 (s, 2 H), 5.1 (m, 2 H), 4.98 (s, 2 H), 4.2 (m, 1
H), 3.50 (s, 3 H, OMe), 3.48-3.4 (comp, 2 H), 2.88 (m, 2 H), 2.71
(m, 2 H), 2.24 (s, 3H, Me), 1.6-1.0 (comp, 6 H); FABMS, m/z 752
(C.sub.41H.sub.45N.sub.5O.sub.9 of M.sup.+1 requires 752).
[0249] D. BIO-1225-3 (25 mg, 0.033 mmol) was treated as described
in Example 24C to give 15 mg (62%) of BIO-1225 as a white solid.
FABMS, m/z 738 (C.sub.40H.sub.43N.sub.5O.sub.9 of M.sup.+1 requires
738).
EXAMPLE 29
Synthesis of BIO-1036
[0250] A. The method described in Procedure C was followed using
methyl 3-amino-5-indanyl-1-propanoate (ester M-1, preparation
described in Procedure B) (85 mg, 0.33 mmol) to give 1036-1 as a
yellow foam (96 mg, 0.22 mmol, 67%) which was used without further
purification in the next step. .sup.1HNM (CDCl.sub.3): .delta. 7.15
(3H), 6.95 (1H), 5.30 (1H), 4.95 (1H), 4.15 (1H), 3.55 (3H),
2.90-2.80 (6H), 2.05 (3H), 1.70 (2H), 1.35 (9H), 0.85 (6H).
[0251] B. Compound 1036-1 (98 mg, 0.22 mmol) was treated as
described in Procedure D to produce the corresponding amine salt.
The method described in Example 1A was performed using phenylacetic
acid and the resulting amine salt (in the presence of TEA) to give
1036-2 as a yellowish solid (75 mg, 0.17 mmol, 77%), which was used
without further purification in the next step. .sup.1HNMR
(CDCl.sub.3): .delta. 7.35-6.8 (9H), 6.25 (1H), 5.25 (1H), 4.45
(1H) 3.6 (1.5H) 3.5 (1.5H), 2.80-2.60 (6H), 2.00 (2H), 1.70-1.30
(5H), 0.85 (6H).
[0252] C. Using the general procedure above a small portion of
compound 1036-2 was hydrolyzed as described in Example 1B, purified
by HPLC and the clean fractions collected to afford Bio-1036A
(.sup..about.2 mg) m/z=437 (98% pure by HPLC) along with Bio-1036B
(.sup..about.2 mg) m/z=437 (98% pure by HPLC) as white solids.
[0253] Bio-1036A: .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta. 8.45
(1H, d, J=7.3 Hz), 8.21 (1H, d, J=7.3 Hz), 7.37-7.05 (8H, m), 5.20
(1H, m), 4.37 (1H, m), 3.57-3.43 (2H, m), 2.86 (4H, m), 2.69 (2H,
m), 2.03 (2H, m), 1.60 (1H, m), 1.49 (2H, m), 0.91 (3H, d, J=6.3
Hz), 0.84 (3H, d, J=6.3 Hz).
[0254] Bio-1036B: .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta. 8.45
(1H, d, J=8.4 Hz), 8.22 (1H, d, J=8.4 Hz), 7.40-7.00 (8H, m), 5.18
(1H, m), 4.35 (1H, m), 3.55 (2H, m), 2.85 (4H, m), 2.57 (2H, m)
2.05 (2H, m), 1.55 (1H, m), 1.40 (2H, m), 0.90 (3H, d, J=6.3 Hz),
0.75 (3H, d, J=6.3 Hz).
EXAMPLE 30
Synthesis of BIO-1137
[0255] A. The method described in Procedure C was followed using
methyl 3-amino-3-(2-nitrophenyl)-1-propanoate (ester M-3,
preparation described in Procedure B) (58 mg, 0.22 mmol) to afford
1137-1 (106 mg, 0.22 mmol, 100%) as a thick pale yellow oil.
[0256] .sup.1HNMR(CDCl.sub.3): .delta. 7.95 (1H), 7.85-7.35 (5H),
5.85 (1H), 4.95 (1H), 4.15 (1H), 3.55 (1.5H), 3.50 (1.5H), 2.90
(2H), 1.70-1.60 (2H), 1.45 (9H), 0.90 (6H).
[0257] B. Compound 1137-1 (106 mg, 0.22 mmol) was treated as
described in Example 29B to afford 1137-2 (69 mg, 0.16 mmol, 73%)
as a yellow semi-solid.
[0258] .sup.1HNMR(CDCl.sub.3): .delta. 7.90-7.15 (10H), 6.35
(0.5H), 6.20 (0.5H), 5.75 (1H), 4.45 (1H), 3.55 (1.5H), 3.50
(1.5H), 2.85 (4H), 1.70-1.30 (3H), 0.70 (6H).
[0259] C. A small portion of compound 1137-1 was hydrolyzed as
described in Example 1B, purified by HPLC and the clean fractions
isolated to afford Bio-1037A (.sup..about.1 mg) m/z=442 (97% pure
by HPLC) and Bio-1037B (.sup..about.2 mg) m/z=442 (100% pure by
HPLC).
EXAMPLE 31
Synthesis of BIO-1043
[0260] A. The commercially available N-BOC-1-aminocyclopropane
carboxylic acid (80 mg, 0.4 mmol) in DMF (3 mL) was activated at
room temperature using BOP (221 mg, 0.5 mmol). After 15 minutes the
methyl 3-amino-3-phenyl-1-propanoate HCl salt (86 mg, 0.4mmol)
(neutralized with excess Hunig's base (0.15 mL, 0.8 mmol)) was
added in DMF (1 mL). After stirring overnight at room temperature
the reaction was diluted with ethyl acetate (10 mL), washed with
60% sat. bicarbonate (2.times.10 mL), 5% citric acid (2.times.5 mL)
and brine (10 mL), dried over sodium sulfate and concentrated to
afford 1043-1 as a white foam (143 mg, 0.4 mmol, 100%). .sup.1H NMR
(CDCl.sub.3) .delta. 7.6 (1H), 7.2 (5H), 5.4-5.3 (2H), 3.55 (3H),
2.85-2.70 (2H), 1.55 (2H), 1.40 (9H), 0.9 (2H).
[0261] B. A small portion of compound 1043-1 was hydrolysed as
described in Example 1B and purified by HPLC. Collection of the
pure fractions afforded Bio-1043 (.sup..about.3 mg) m/z=349 (100%
pure by HPLC) as a white solid which was submitted for
bioassay.
[0262] .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta. 8.55-8.05 (2H,
bm), 7.5-7.15(5H, m), 5.40 (,2H, bm), 3.0-2.65 (2H, m), 1.45 (9H,
s) 1.43-1.10 (2H, m), 0.97 (1H, bm), 0.85 (1H, bm).
EXAMPLE 32
Synthesis of BIO-1115
[0263] A. The method described in Procedure C was followed using
methyl 3-amino-3-(4-chlorophenyl)-1-propanoate HCl salt (ester M-1,
preparation described in procedure B) (68 mg, 0.27 mmol) to afford
1115-1 (94 mg, 0.22 mmol, 82%) as a white foam.
[0264] .sup.1HNMR (CDCl.sub.3): 7 7.35 (1H), 7.25-7.10 (4H), 5.35
(1H), 4.95 (1H), 4.05 (1H), 3.60 (1.5H), 3.55 (1.5H), 2.80-2.65
(2H), 1.65 (2H), 1.40 (1OH), 0.80 (6H).
[0265] B. Compound 1115-1 (68 mg, 0.27 mmol) was treated as
described in Example 29B to afford crude 1115-2 (67 mg, 0.15 mmol,
68%) as a pale yellow solid.
[0266] .sup.1H NMR: .delta. 7.50 (1H), 7.40-7.00 (9H), 6.20 (1H),
5.25 (1H), 4.45 (1H), 3.60 (1.5H), 3.55 (1.5H), 2.7-2.55 (4H),
1.65-1.40 (3H), 0.80 (6H).
[0267] C. A small portion of crude 1115-1 was hydrolysed, purified
by LC and the pure fractions collected to afford Bio-1115A
(.sup..about.1 mg) m/z=431 (100% pure by HPLC) along with Bio-1115B
(.sup..about.2mg) m/z=431 (100% pure by HPLC) as white solids.
[0268] Bio-1115A: .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta. 8.46
(1H, d, J=8.2 Hz), 8.27 (1H, d, J=8.2 Hz), 7.46-7.18 (9H, m), 5.20
(1H, m), 4.35 (1H, m), 3.60-3.45 (2H, m), 2.71 (2H, d, J=7.3 Hz),
1.63 (1H, m), 1.48 (2H, m), 0.91 (3H, d, J=6.4 Hz), 0.84 (3H, d,
J=6.4 Hz).
[0269] Bio-1115B: .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.60
(1H, d, J=8 Hz), 8.26 (1H, d, J=8 Hz), 7.45-7.15 (9H, m), 5.18 (1H,
m), 4.35 (1H, m), 3.50 (2H, m), 2.70 (2H, m), 1.50(1H, m), 1.42
(2H, m), 0.85 (3H, d, J=6.3 Hz), 0.75 (3H, d, J=6.3 Hz)
EXAMPLE 33
Synthesis of BIO-1129
[0270] A. To a solution of 4-(phenylurea)phenylacetic acid (540 mg,
2.0 mmol; prepared in Example 22C) in DMF (5 mL) was added EDC (460
mg., 2.4 mmol). After storing at room temperature for 15 min,
phenylalanine t-butyl ester HCl salt (515 mg, 2.0 mmol) which was
neutralized with excess Hunig's base (0.7 mL, 4.0 mmol) was added
in DMF (3 mL). After stirring overnight the reaction was diluted
with ethyl acetate (20 mL) and washed with 60% sat. bicarbonate
(2.times.10 mL), citric acid (2.times.10 mL), brine (2.times.10
mL), dried over sodium sulfate and concentrated to afford crude
1129-1 (662 mg, 1.40 mmol, 70%) as a thick pale yellow oil.
[0271] .sup.1HNMR (CDCl.sub.3): .delta. 7.45-6.90 (16H), 6.45 (1H),
4.70 (1H), 3.4 (2H), 3.15-2.90 (2H), 1.35 (9H).
[0272] B. To crude product 1129-1 (662 mg, 1.40 mmol) was added
methylene chloride (5 mL) followed by TFA (1 mL). After stirring
overnight the reaction was concentrated to dryness and dried on a
vacuum pump. A small portion (21 mg, 0.05 mmol) was dissolved in
DMF (1 mL) and HOBt (11 mg, 0.07 mmol) was added followed by EDC
(14 mg, 0.06 mmol). After stirring for 15 min at room temperature
amine .beta.-3 (13 mg, 0.05 mmol) was added in DMF (0.5 mL) After
stirring overnight the reaction was diluted with ethyl acetate (20
mL), washed with sat. bicarbonate (2.times.10 mL), citric acid (10
mL), brine (10 mL) dried over sodium sulfate and concentrated to
afford crude 1129-2 (26 mg, 0.04 mmol, 80%) as a light tan solid.
.sup.1HNMR (CDCl.sub.3): .delta. 8.4 (1H), 7.4-6.5 (19H), 5.95
(2H), 5.7 (1H), 5.25 (1H), 4.70 (1H), 3.65-3.50 (5H), 3.10-2.65
(4H).
[0273] C. A small aliquot of crude 1129-2 was hydrolysed as
described in Example 1B and purified by HPLC to afford:
[0274] Bio-1129A (.sup..about.1.5 mg) m/z=609 (80:20 ds) (100% pure
by HPLC); and
[0275] Bio-1129B (.sup..about.2mg) m/z=609 (9:91 ds) (100% pure by
HPLC) as white solids.
[0276] Bio-1129A: .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.18
(1H, s), 8.14 (1H, s), 8.50 (1H, bd), 8.23 (1H, bd), 7.50 (2H, d,
J=8.1 Hz), 7.40-7.10 (9H, m), 7.08-6.72 (6H, m), 6.04 (2H, s), 5.15
(1H, m), 4.07 (1H, m), 3.38 (2H, m, 3.05-2.70 (2H, m), 2.62 (2H,
s).
EXAMPLE 34
Synthesis of BIO-1131
[0277] A. The method of Example 1A was performed using
phenylureaphenyl acetic acid (prepared in Example 22C) and
isoleucine methyl ester HCl salt (362 mg, 2.0 mmol) (treated with
TEA) to afford crude 1131-1 (344 mg, 1.0 mmol, 51%) as a clear
thick oil.
[0278] .sup.1HNMR (CDCl.sub.3): .delta. 7.7 (1H), 7.35-6.95 (10H),
6.60 (1H) 4.55 (1H), 3.65 (3H), 3.45 (2H), 1.90 (1H), 1.45-1.20
(3H), 0.85 (5H).
[0279] B. To a solution of crude 1131-1 (344 mg, 0.95 mmol) in
methanol (5 mL) was added 2N LiOH (2 mL). After stirring overnight
the methanol was removed, H.sub.2O (5 mL) added and the pH adjusted
to pH=1-2. The aqueous layer was extracted with ethyl acetate
(5.times.20 mL) dried over sodium sulfate and concentrated to give
1131-2 (365 mg, 0.95 mmol, 100%) as a tan solid.
[0280] .sup.1H NMR (CDCl.sub.3): .delta. 8.70 (2H), 8.30 (1H),
7.60-7.20 (8H), 7.00 (1H), 4.25 (1H), 3.55 (2H), 1.90 (1H), 1.55
(1H), 1.30 (2H), 0.85 (5H).
[0281] C. Prepared from 1131-2 (27 mg, 0.07 mmol) and amine
.beta.-3 (11 mg, 0.07 mmol) as described in Example 1A to afford
crude 1131-3 (34 mg, 89%), as a pale brownish solid. .sup.1H NMR
(CDCl.sub.3): .delta. 8.3 (2H), 7.45-6.65 (16H), 5.45 (1H),
4.45-4.30 (1H), 3.55 (2H), 3.2-2.90 (2H), 2.00-0.70 (9H).
[0282] D. A small aliquot of crude 1131-3 was hydrolysed as
described in Example 1B and purified by HPLC to afford Bio-1131A
(.sup..about.2 mg)m/z=531 (100:0ds) (100% pure by HPLC)and
Bio-1131B (.sup..about.3 mg) m/z=531 (0:100ds) (100% pure by HPLC)
as white solids.
[0283] Bio-1131A: .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta. 8.69
(1H, s), 8.63 (1H, s), 8.50 (1H, d, J=8.1 H.sub.z), 7.50 (2H, d,
J=7.8 H.sub.z), 7.44-7.22 (8H, m), 7.19 (2H, d, J=8.4 H.sub.z),
7.00 (1H, m), 5.27 (1H, m), 4.36 (1H, m), 3.52 (2H, m), 3.00 (2H,
bm), 2.71 (2H, d, J=7.3 H.sub.z), 1.70 (1H, bm), 1.44-1.26 (1H, m),
1.22-1.00 (3H, m), 0.95-0.78 (5H, m).
[0284] Bio-1131B: .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta. 8.73
(1H, s), 8.68 (1H, s), 8.60 (1H, d, J=8 Hz), 8.15 (1H, d, J=8 Hz),
7.50 (2H, d, J=7.9 Hz), 7.42 (2H, d, J=8.4 Hz), 7.37-7.23 (5H, m),
7.20 (2H, d, J=8.4 Hz), 7.00 (1H, m), 5.35 (1H, m), 4.23 (1H, m),
3.50 (2H, m), 3.05 (2H, bm), 2.71 (2H, m), 1.72 (1H, bm), 1.20 (3H,
m), 0.72-0.60 (5H, m).
EXAMPLE 35
Synthesis of BIO-1136
[0285] A. The method described in Example 1A was performed
utilizing commercially available N-BOC-S-benzyl-cysteine (25 mg,
0.08 mmol) and methyl 3-amino-3-phenyl-1-propanoate (17 mg, 0.09
mmol) to afford crude protected amine 1136-1 (42 mg, 0.08 mmol,
100%).
[0286] .sup.1H NMR (CDCl.sub.3): .delta. 7.35 (10H), 5.40-5.20
(2H), 4.20 (1H), 3.65 (1.5H), 3.55 (1.5H), 3.54 (1.5H), 3.25-2.65
(6H), 1.45-1.30 (9H).
[0287] B. The protected amine 1136-1 was treated as described in
Procedure D to give the TFA-amine salt 1136-2.
[0288] C. The method described in Example 22D was performed
utilizing free amine 1136-2 (42 mg, 0.08 mmol) (TEA treatment) to
afford crude 1136-3 which was used in the hydrolysis step without
further purification.
[0289] D. A small aliquot of crude 1136-3 was hydrolysed as
described in Example 1B and purified by HPLC to afford Bio-1136
(.sup..about.4 mg )m/z=611 (100% pure by HPLC) as a white solid.
.sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta. 9.05 (2H, bm), 8.90
(1H, br), 8.37 (1H, br), 7.50 (1H, d, J=7.7 Hz), 7.45 (1H, d, J=8.3
Hz), 7.4-7.2 (9H, m), 7.00 (1H, m) 5.25 (1H, br), 4.65 (1H, br),
3.5-3.2 (4H, m), 2.70 (2H, bm).
EXAMPLE 36
Synthesis of BIO-1176
[0290] A. To a solution of commercially available N-BOC-aspartic
acid .alpha.-benzyl ester (500 mg, 1.55.mmol) in DMF (5 mL) was
added HOEt (283 mg, 2.10 mmol) followed by EDC (343 mg, 1.80 mmol).
After stirring for 15 minutes at room temperature thiomorpholine
(500 mg, 1.54 mmol) was added followed by Hunig's base (0.7 mL, 92
mmol) and the reaction mixture stirred at room temperature
overnight. The reaction was worked up by diluting with ethyl
acetate (25 mL) and washing with 60% sat. bicarbonate (5 mL), 5%
citric acid (5 mL) and brine (5 mL). The organics were separated,
dried over sodium sulfate and concentrated to give ester 1176-1 as
a thick orange oil (421 mg, 1.03 mmol, 69%). .sup.1H NMR
(CDCl.sub.3): .delta. 7.13 (5H, m), 5.69 (1H, bd, J=9.4 Hz) 5.03
(1H, d, J=12.6 Hz), 4.42 (1H, m), 3.61 (1H, m), 3.60-3.40 (4H, m),
2.96 (1H, bm), 2.58 (1H, bm), 2.35 (4H, m) 1.22 (9H, s)
[0291] B. Ester 1176-1 (100 mg, 0.25 mmol) was treated as described
in Example 1B to afford acid 1176-2 (76 mg, 0.24 mmol, 96%) as a
clear thick oil.
[0292] .sup.1H NMR (CDCl.sub.3) .delta. 7.39-7.28 (5H, m),
7.15-6.70 (1H, br), 5.70 (1H, bs, J=6.3 Hz), 4.55 (1H, br),
4.40-3.40 (4H, m) 3.15 (1H, m), 2.80-2.52 (5H, m), 1.43 (9H,
s).
[0293] C. The method of Example 1A was performed using acid 1176-2
(32 mg, 0.10 mmol), in DMF, HOBT, EDC, and amine .beta.-3 to afford
1176-3 (36 mg, 0.07 mmol, 70%) as a thick pale yellow oil. .sup.1H
NMR (CDCl.sub.3): .delta. 7.71 (1H, br), 6.61 (3H, m) 6.00 (0.5H,
br), 5.90 (1H, s), 5.77 (0.5H, br), 5.21 (1H, m), 4.51 (1H, bm),
3.90-3.40 (4H, m), 3.39 (3H, s), 3.12-3.00 (1H, m), 2.85-2.65 (3H,
m), 2.63-2.45 (4H, m), 1.43 (4.5H, s), 1.43 (4.5H, s).
[0294] D. The protected amine 1176-3 (36 mg, 0.07 mmol) was treated
as described in Procedure D to give TFA-amine salt 1176-4 (51 mg,
0.07 mmol, 100%) as a pale yellow solid.
[0295] E. The method described in Example 22D was performed
utilizing free amine 1176-4 (42 mg, 0.08 mmol) (after TEA
treatment) to afford crude 1176-5 which was used in the hydrolysis
step without further purification. .sup.1H NMR (CDCl.sub.3):
.delta. 7.95-6.9 (13H, m), 6.61 (3H, s), 5.85 (2H, s) 5.23 (1H, m),
4.88 (1H, m), 3.89-3.60 (4H, s), 3.55 (3H, s), 3.43 (2H, br),
3.11-2.96 (2H, m), 2.71 (2H, m), 2.46 (4H, m).
[0296] F. Crude 1176-5 was hydrolyzed as described in Example 1B
and injection of a small aliquot into the HPLC afforded Bio-1176
(.sup..about.4mg) m/z=662 (>99% pure by HPLC) as a white solid.
.sup.1HNMR: (DMSO-d.sub.6) .delta. 8.69 (2H, d, J=9.8 Hz), 8.33
(1H, d, J=8.0 Hz), 8.26 (1H, d, J=8.0 Hz), 7.61 (2H, d, J=8.0 Hz),
7.43 (2H, d, J=8.0 Hz), 7.34 (2H, m), 7.21 (2H, d, J=8.0 Hz),
7.10-6.95 (4H, m), 6.11 (2H, s), 5.13 (1H, m), 4.68 (1H, m), 3.71
(4H, br), 3.56-3.18 (2H, m), 2.73-2.46 (8H, m).
EXAMPLE 37
Synthesis of BIO-1177
[0297] A. The procedure described in Example 36A was carried out
using methylpropargylamine in place of thiomorpholine to afford
crude 1177-1 (374 mg, 0.99 mmol, 66%) as a white-foam. .sup.1H NMR
(CDCl.sub.3): .delta. 7.20 (5H), 5.25 (1H), 5.10 (2H), 4.45 (1H),
4.15-3.8 (2H), 3.15-2.65 (5H), 2.2-2.15 (1H), 1.30 (9H).
[0298] B. Crude 1177-1 was treated as described in Example 1B to
afford acid 1177-2 (76 mg, 0.26 mmol, 96%) as a clear oil. .sup.1H
NMR (CDCl.sub.3): .delta. 5.35 (1H), 4.55 (1H), 4.35-3.8 (2H),
3.30-2.65 (5H), 2.4-2.25 (1H), 1.45 (9H).
[0299] C. The method of Example 1A was performed using acid 1177-2
(76 mg, 0.26 mmol), in DMF, HOBT, EDC, and amine .beta.-3 to afford
crude 1177-3 (78 mg, 0.15 mmol) as a white foam. .sup.1H NMR
(CDCl.sub.3): .delta. 7.70 (1H), 7.35 (3H), 6.65 (2H), 5.80 (1H)
5.30-5.00 (2H), 4.60(1H), 4.45-3.80 (2H), 3.60 (3H), 3.30-2.70
(5H), 2.30 (1H), 1.45 (4.5H), 1.40 (4.5H).
[0300] D. The protected amine 1177-3 (78 mg, 0.15 mmol) was treated
as described in Procedure D to afford TFA-amine salt 1177-4.
[0301] E. The-method described in Example 22D was performed
utilizing free amine 1177-4 to afford 1177-5 (52 mg, 0.08 mmol,
77%) as a tan solid. .sup.1H NMR: (CDCl.sub.3) .delta. 57.5-6.9
(14H), 6.65 (3H), 5.85 (2H) 5.25-5.00 (2H), 4.85 (1H), 4.25-3.70
(2H), 3.60 (3H), 3.55 (2H), 3.30-2.65 (5H), 2.22 (1H).
[0302] F. A small portion of 1177-5 was hydrolyzed as described in
Example 1B to afford Bio-1177 (.sup..about.2 mg) m/z=628 (100% pure
by HPLC) as a white solid.
[0303] .sup.1H NMR: (DMSO-d.sub.6) .delta. 8.64 (2H, bd), 8.27 (2H,
bm), 7.55-7.13 (7H, m), 7.11-6.75 (3H, m) 6.15 (2H, s), 5.12 (1H,
bm), 4.65 (1H, bm), 4.25 (2H, bm), 3.25 (2H, m), 3.05 (2H, br) 2.88
(1H, bm), 2.62 (2H, m).
EXAMPLE 38
Synthesis of BIO-1214
[0304] A. The procedure described in Example 36A was carried out on
the N-BOC-aspartic acid .alpha.-benzyl ester (1.60 g, 4.9 mmol)
using dimethylamine in place of thiomorpholine to afford ester
1214-1 (1.43 g, 4.1 mmol, 83%) as a thick colorless oil.
[0305] .sup.1H NMR (CDCl.sub.3): .delta. 7.32 (5H, m), 5.85 (1H,
br), 5.15 (2H, m) 4.55 (1H, br), 3.12 (1H, m), 2.94 (3H, s), 2.88
(3H, s), 2.73 (1H, m), 1.40 (9H, s).
[0306] B. Ester 1214-1 (124 mg, 0.33 mmol) was dissolved in ethyl
acetate (2 mL) and 10% Pd/C (.sup..about.50 mg) was added and the
mixture was hydrogenated under pressure (40 psi) for 2 h. The
reaction was filtered through Celite.RTM. and concentrated to
afford acid 1214-2 (95 mg, 0.33 mmol, 100%), as a colorless oil.
.sup.1H NMR: (CDCl.sub.3) .delta. 5.81 (1H, bm), 4.48 (1H, bs),
3.15 (1H, m), 3.00 (3H, s) 2.93 (3H, s), 2.59 (1H, m), 1.39 (9H,
s).
[0307] C. The method of Example 1A was performed using acid 1214-2
(28 mg, 0.10 mmol) and amine .beta.-3 (17 mg, 0.80 mmol) to afford
protected amine 1214-3 (55 mg, 0.10 mmol, 100%) as a white foam.
.sup.1H NMR: (CDCl.sub.3) .delta. 7.77 (1H, bd), 6.71 (3H, m), 6.11
(1H, bd), 5.91 (2H, s) 5.25 (1H, m), 4.51 (1H, br), 3.60 (3H, s),
3.12 (1H, m), 2.94 (3H, s), 2.90 (3H, s), 2.88-2.68 (2H, m), 2.48
(1H, m), 1.43 (9H, s).
[0308] D. The protected amine 1214-3 (55 mg, 0.10 mmol) was treated
as described in Procedure D to afford TFA-amine salt 1214-4.
[0309] E. The method described in Example 22D was performed
utilizing free amine 1214-4 to afford 1214-5 (31 mg, 0.05 mmol,
50%) as a tan solid. .sup.1H NMR (CDCl.sub.3): .delta. 7.45-6.90
(13H, m), 6.61 (3H, m), 5.85 (2H, s), 5.24 (1H, m), 4.82 (1H, m),
3.55 (3H, s), 3.47 (2H, m), 3.08-2.94 (1H, m), 2.92 (3H, s), 2.84
(3H, s), 2.77-2.50 (2H, m), 2.45 (1H, m).
[0310] F. A small portion of 1214-5 was hydrolyzed as described in
Example 1B to afford BIO-1214 (.sup..about.2 mg) m/z=604 (100%
purity by HPLC) as a white solid.
EXAMPLE 39
Synthesis of BIO-1215
[0311] A. To a solution of amide 1214-1 (prepared in Example 38A)
(671 mg, 1.9 mmol) in dry tetrahydrofuran (5 mL) cooled to
0.degree. C. was added 1 N BH.sub.3/THF solution (4.1 mL, 3.8 mmol)
dropwise. After stirring the reaction mixture for 2 h at room
temperature the reaction was quenched with methanol (2 mL) and
concentrated to dryness. Methanol (5 mL) was added and removed
three times to remove all (MeO).sub.3B formed. Drying under high
vacuum afforded amine 1215-1: (623 mg, 1.7 mmol, 90%) as a thick
colorless oil. .sup.1H NMR (CDCl.sub.3) .delta. 7.38 (5H, M), 5.48
(1H, bm), 2.65-2.35 (8H, m), 1.95 (2H, m), 1.42 (9H, s).
[0312] B. Amine 1215-1 (124 mg, 0.34 mmol) was subjected to
catalytic hydrogenation using methanol/ethyl acetate/acetic acid as
solvent and 10% Pd/C (.sup..about.50 mg). After 2 h the reaction
mixture was filtered and concentrated to give acid 1215-2 (90 mg,
0.33 mmol, 97%) as a thick colorless oil.
[0313] .sup.1H NMR (CDCl.sub.3) .delta. 5.91 (1H, br), 3.95 (1H,
br), 3.54 (1H, bm), 2.71-2.42 (8H, m), 2.15 (2H, br), 1.33 (9H,
s).
[0314] C. The method of Example 1A was performed using acid 1215-2
(55 mg, 0.12 mmol) and the amine .beta.-3 (22 mg, 0.10 mmol) to
afford protected amine 1215-3 (44 mg, 0.09 mmol, 90%) as a white
foam. .sup.1H NMR(CDCl.sub.3) .delta. 6.75 (3H, m), 6.51 (1H, bd),
5.91 (2H, s), 5.30 (1H, m), 4.37-4.12 (2H, m), 3.61 (3H, s),
2.90-2.65 (2H, m), 2.55-2.00 (10H, m), 1.42 (9H, s).
[0315] D. The protected amine 1215-3 (44 mg, 0.09 mmol) was treated
as described in Procedure D to afford TFA-amine salt 1215-4.
[0316] E. The method described in Example 22D was performed
utilizing free amine 1215-4 to afford 1215-5 (38 mg, 0.06 mmol,
70%) as a white solid.
[0317] .sup.1H NMR (CDCl.sub.3) .delta. 7.41-6.90 (13H, M), 6.71
(3H, m), 5.91 (2H, s), 5.29 (1H, m), 4.21 (1H, m), 3.61 (3H, s),
3.45 (2H, m), 2.90-2.70 (2H, m), 2.40-1.95 (10H, m).
[0318] F. A small portion of 1214-5 was hydrolyzed as described in
Example 1B to afford BIO-1215 (.sup..about.3 mg) m/z=590 (100% pure
by HPLC) as a white solid.
EXAMPLE 40
Synthesis of BIO-1227
[0319] A. The method as described in Example 1B was performed using
commercially available BOC-S-methyl-cysteine (28 mg, 0.12 mmol) and
amine 0-3 (21 mg, 0.10 mmol) to afford protected amine 1227-1 (32
mg, 0.07 mmol, 70%) as a white foam.
[0320] .sup.1H NMR (CDCl.sub.3) .delta. 7.38 (1H, bd), 6.81-6.67
(3H, m), 5.90 (2H, s), 5.40 (1H, bd), 5.37 (1H, m), 4.20 (1H, m),
3.59 (3H, s), 2.95-2.68 (4H, m), 2.10 (3H, s), 1.43 (9H, s).
[0321] B. The protected amine 1227-1 (32 mg, 0.07 mmol) was treated
as described in Procedure D to afford TFA-amine salt 1227-2.
[0322] C. The method described in Example 22D was performed
utilizing free amine 1227-2 and the 2-methylphenylureaphenylacetic
acid (28 mg, 0.10 mmol) to afford crude ester 1227-3 (29 mg, 0.047
mmol, 67%) as a light tan solid. .sup.1H NMR (CDCl.sub.3) .delta.
7.62 (1H, bd), 7.4-6.9 (12H, m), 6.80 (3H, m) 5.90 (2H, s) 5.15
(1H, m), 4.45 (1H, m), 3.63-3.45 (5H, m), 3.15-2.61 (4H, m), 2.21
(3H, s), 2.10 (3H, s).
[0323] D. A small aliquot of crude ester 1227-3 was hydrolyzed as
described in Example 1B to afford BIO-1227 (.sup..about.4 mg)
m/z=593 (>99% pure by HPLC) as a white solid. .sup.1H NMR
(DMSO-d.sub.6): .delta. 9.01 (1H, s), 8.67 (1H, d, J=7.9 Hz), 8.31
(1H, d, J=8.3 Hz), 7.97 (1H, s), 7.90 (1H, d, J=8 Hz), 7.44 (2H,d,
J=8.3 Hz), 7.23 (4H, m), 6.99 (2H, m), 6.85 (2H, m), 6.03 (2H, s),
5.16 (1H, m), 4.54 (1H, M), 3.39 (2H, m), 2.81-2.58 (4H, m) 2.30
(3H, s), 2.05 (3H, s).
EXAMPLE 41
Synthesis of BIO-1149
[0324] A. To a solution of the product from Procedure C (272 mg,
0.67 mmol) in CH.sub.2Cl.sub.2 (2.5 ml) was added TFA (2.5 ml)
slowly and the mixture was stirred at room temperature for 1 h. The
solvents were removed to give an oil. This oil was dissolved in
CH.sub.2Cl.sub.2 (2.5 ml). To this solution was added Et.sub.3N to
pH 9 and then succinimidyl 2-quinolinecarboxylic acid (170 mg, 0.63
mmol). The mixture was stirred at room temperature for 1 h followed
by usual workup (5% citric acid, 5% NaHCO.sub.3 and sat. NaCl) to
afford ester 1149-1 (200 mg, 76%) as a white solid.
[0325] B. Acid 1149-1 (200 mg, 0.43 mmol) was dissolved in methanol
(1.5 ml) and to the solution was added 1M aqueous LiOH (0.5 ml).
The mixture was stirred at room temperature for 3 h and neutralized
with 5% citric acid to pH 3 and was extracted with EtOAc (3.times.5
ml). The combined extracts were dried (Na.sub.2SO.sub.4) and
concentrated to afford 155 mg (82.5%) crude 1149. A small amount of
the crude product (30 mg) was purified by HPLC to give BIO-1149,
and the diastereomers were separated.
[0326] HPLC: Room temperature; A: 36 min; B:38 min. FAB-MS=434.
.sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm) 8.72 (m, 1H), 8.30-7.98 (m,
3H), 7.82-7.64 (m, 2H), 7.60-7.51 (m, 1H), 7.30-7.09 (m, 5H),
5.46-5.38 (m, 1H), 4.86-4.72 (m, 1H), 2.92-2.74 (m, 2H), 1.88-1.61
(m, 3H), 0.96-0.83 (m, 6H).
EXAMPLE 42
Synthesis of BIO-1152
[0327] A. To a solution of the product of Procedure D2 (33 mg, 0.1
mmol) in CH.sub.2Cl.sub.2 (0.5 ml) was added 2,2-dimethylbutyric
acid chloride (14 mg, 0.1 mmol) and Et.sub.3N (50 .mu.l). The
mixture was stirred at room temperature for 16 h. The usual workup
(5% NaHCO.sub.3, 5%. citric acid and sat. NaCl) afforded 1152-2 (37
mg, 76%) as a white solid. .sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm)
7.32-7.19 :(m,5H), 6.08 (s, 1H), 5.36-5.27 (m, 1H), 4.53-4.44 (m,
1H), 2.86-2.61 (m, 2H), 2.05 (s, 2H), 1.26. (s, 9H), 1.01 (s, 9H),
0.99-0.84 (s, 9H).
[0328] B. Ester 1152-2 was dissolved in CH.sub.2Cl.sub.2 (2.5 ml)
and TFA (2.5 ml) and stirred at room temperature for 3 h to afford
an oil. The purification of the oil by HPLC to give a pure
BIO-1152. .sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm) 8.29 (d, 1H),
7.44 (d, 1H), 7.34-7.18 (m, 5H), 5.44-5.32 (m, 1H), 4.78-4.69 (m,
1H), 3.21-3.14 (m, 2H), 2.98-2.77 (dd, 2H), 1.59-1.38 (m, 3H), 0.96
(s, 9H), 0.84 (d, 3H), 0.73 (d, 3H).
EXAMPLE 43
Synthesis of BIO-1089
[0329] A. To a solution of amine .beta.-6 (2.2 g, 8.76 mmol) in
CH.sub.2Cl.sub.2 (25 ml) was added N-BOC-methionine succinimidyl
ester (2.77 g, 8.0 mmol) and Et.sub.3N (5 drops) and the mixture
was stirred at room temperature for 1.5 h. The mixture was washed
with 5% citric acid (2.times.10 ml), 5% NaHCO.sub.3 (2.times.10 ml)
and sat. NaCl (15 ml), dried (Na.sub.2SO.sub.4) and concentrated to
give 1089-1 (3.2 g, 83%) as a white solid. .sup.1H NMR:
(CDCl.sub.3, 300 MHz, ppm) 7.27 (d, 2H), 6.81 (d, 2H), 5.31-5.20
(m, 2H), 4.38-4.28 (m, 1H), 3.72 (s, 3H), 2.82-2.64 (m, 2H), 2.12
(s, 3H), 1.44 (s, 9H), 1.30 (s, 9H).
[0330] B. To a solution of 1089-1 (3.2 g, 6.64 mmol) in EtOAc (15
ml) was added a 1M HCl-EtOAc solution (40 ml) and the mixture was
stirred at room temperature for 4.5 h. The reaction mixture was
quenched with H.sub.2O (60 ml) and the aqueous layer was collected.
It was neutralized with solid NaHCO.sub.3 to pH 8 and was extracted
with EtOAc (2.times.45 ml). The combined organic extracts were
washed with sat. NaCl (20 ml), dried (Na.sub.2SO.sub.4) and
concentrated to afford 1089-2 (1.7 g, 67%) as an oil. .sup.1H NMR:
(CDCl.sub.3, 300 MHz, ppm) 7.98 (d, 1H), 7.19 (d, 2H, J=8.3 Hz),
6.81 (d, 2H, J=8.3 Hz), 5.32-5.18 (m, 1H), 3.74 (s, 3H), 3.48-3.44
(m, 1H), 2.82-2.62 (m, 2H), 2.53 (t, 2H), 2.18-2.06 (m, 1H), 2.04
(s, 3H), 1.8-1.66 (1H), 1.31 (s, 9H).
[0331] C. The method of Example 22D was performed using 1089-2 (1.7
g, 4.45 mmol) to afford 1089-3 (2.3 g, 81.6%) as a solid. This
material was used in the next step without further
purification.
[0332] .sup.1H NMR: (DMSO-d.sup.6, 300 MHz, ppm) 8.60 (d, 2H), 8.41
(d, 1H), 8.24 (d, 1H), 7.44 (d, 2H), 7.31 (d, 2H), 7.26 (t, 2H),
7.13 (t, 2H), 6.91 (t, 1H), 6.79 (d, 2H), 5.10-5.01 (m, 1H),
4.36-4.33 (m, 1H), 3.68 (s, 3H), 3.29 (s, 2H), 2.61-2.58 (m, 2H),
1.89 (s, 3H), 1.26 (s, 9H).
[0333] D. Compound 1089-3 (2.3 g, 3.63 mmol) was dissolved in 4N
HCl-dioxane (8 ml) and the solution was stirred at room temperature
for 16 h. After the dioxane was removed, ether (15 ml) was added
and mixture was stirred for 10 min. The precipitate was collected
and was recrystallized from methanol to give pure BIO-1089 as a
pale brown solid. FAB-MS =579.
[0334] .sup.1H NMR: (DMSO-d.sup.6, 300 MHz, ppm) 8.76 (d, 2H), 8.52
(d, 1H), 7.54 (d, 2H), 7.46 (d, 2H), 7.36 (t, 1H), 7.34-7.26 (m,
4H), 7.04 (t, 1H), 6.95 (d, 2H), 5.22-5.20 (m, 1H), 4.46-4.35 (m,
1H), 3.81 (s, 3H), 3.50, (s, 2H), 3.20 (m, 2H), 2.79-2.73 (m, 2H),
2.35 (t, 2H), 2.03 (s, 3H), 1.87-1.80 (m, 2H).
EXAMPLE 44
Synthesis of BIO-1090
[0335] A. The method described in Procedure C was followed using
amine 5-10 (28 mg, 1.0 mmol) to afford 1090-1 (38 mg, 84%) as a
white solid. .sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm) 7.08 (m, 1H),
6.82 (s, 1H), 6.74-6.70 (m, 2H), 5.24-5.15 (m, 1H), 4.98-4.93 (m,
1H), 4.16-4.13 (m, 4H), 2.74-2.53 (m, 2H), 1.62-1.42 (m, 3H), 1.44
(s, 9H), 1.40 (s, 9H), 0.89 (m, 6H).
[0336] B. The white solid of 1090-1 (38 mg, 0.77 mmol) was treated
as described in Procedure D1 to give 1090-2 as an oil. This
compound was used in next step without further purification.
.sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm) 7.24-7.15 (m, 2H),
6.84-6.61 (m, 3H), 5.81-5.78 (m, 1H), 4.23 (s, 4H), 4.19-4.08 (m,
1H), 2.88-2.62 (m, 2H), 1.70-1.46 (m, 3H), 0.90-0.81 (m, 6H).
[0337] C. The method of Example 22D was performed using amine
1090-2 to afford crude 1090 (27 mg, 59%). The purification of crude
product by HPLC gave pure BIO-1090 as a white solid.
FAB-MS=603.
EXAMPLE 45
Synthesis of BIO-1194
[0338] A. To a well-stirred cold solution of methyl
p-aminophenylacetate (9.8 g, 59.4 mmol) in CH.sub.2Cl.sub.2 (200
ml) and Et.sub.3N (25 ml, 18 g, 178.2 mmol) was added COCl.sub.2
(96 ml of 1.9M solution in toluene) through an additional funnel
for 1 h. The reaction mixture was stirred at 0.degree. C. for
another 1 h. The reaction mixture was concentrated and ether: pet
ether (3:1) (125 ml) was added. The solid was filtered and the
filtrate was collected. Removal of the solvents gave crude 1194-1
as a brown liquid. The purification of crude product by
distillation (118-120.degree. C./10 mm) gave pure 1194-1 (8.5 g,
75%) as a colorless liquid.
[0339] .sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm) 7.20 (d, J=8.4 Hz),
7.02 (d, J=8.4 Hz), 3.69 (s, 3H), 3.48 (s, 2H).
[0340] B. To a solution of 1194-1 (5.73 g, 30.0 mmol) in
CH.sub.2Cl.sub.2 (60 ml) was added 2-aminopyridine (2.82 g, 30
mmol) in portions. The mixture was stirred at room temperature for
0.5 h then 35.degree. C. for 0.5 h. The resulting mixture was
diluted with pet ether (60 ml) and a white solid was formed.
Filtration of the solid gave pure 1194-2 (8.35 g, 98%) as a white
solid.
[0341] .sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm): 8.20 (s, 2H),
7.62-7.51 (m, 3H), 7.33 (d, 2H), 7.01 (d, 2H), 6.89-6.85 (m, 1H),
3.70 (s, 3H), 3.59 (s, 2H).
[0342] C. Compound 1194-2 (--5.7 g, 20.0 mmol) was dissolved in
methanol (20 ml) and to this was added 1N NaOH (40 ml). The mixture
was heated until a clear solution was formed and was stirred at
room temperature for 16 h, followed by a careful neutralization
with 1N HCl to pH 7 then with acetic acid to pH 3. The white solid
thus formed was filtered and washed with methanol (15 ml) and ether
(2.times.30 ml) to give 1194-3 (4.7 g, 87%) as a white powder.
.sup.1H NMR: (DMSO-D.sup.6, 300 MHz, ppm) 10.62 (br, s, 1H), 9.53
(br, s, 1H), 8.39 (d, 1H), 7.82 (t, 1H), 7.63-7.55 (m, 1H),
7.33-7.27 (d, 2H), 7.14-7.08 (m, 1H), 3.62 (s, 3H).
[0343] D. Standard Procedure C was followed to prepare 1194-4 by
coupling amine .beta.-6 (2.65 g, 10.56 mmol) with BocLeuOSu (3.28
g, 10 mmol) in CH.sub.2Cl.sub.2 (25 ml) and Et.sub.3N (5 drops)
then followed by deprotection (TFA/CH.sub.2Cl.sub.2) to afford
1194-4. (4.5 g, 83.6%) in two steps
[0344] 1194-4-Boc: .sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm) 7.18 (d,
2H), 6.36 (d, 2H), 5.13-5.10 (m, 1H), 4.12-4.01 (m, 1H), 3.72 (s,
3H), 2.79-2.60 (m, 2H), 1.62-1.40 (3H), 1.38 (s, 9H), 1.26 (s, 9H),
0.85-0.80 (m, 6H). 1194-4: .sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm)
7.10 (d, 2H), 6.78 (d, 2H), 5.43-5.27 (m, 1H), 4.21-4.06 (m, 1H),
3.71 (s, 3H), 2.95-2.76 (m, 1H), 2.75-2.56 (m, 1H), 1.62-1.32 (m,
6H).
[0345] E. The method of Example 1A was followed using acid 1194-3
(1.36 g, 5.0 mmol) and amine 1194-4 to afford crude BIO-1194 (2.1
g, 78%) as a white solid. The pure product (purity >97.5%) was
obtained by crystallization from methanol. .sup.1H NMR:
(CDCl.sub.3, 300 MHz, ppm) 8.03-7.97 (m, 2H), 7.59 (m, 1H), 7.51
(d, 2H), 7.18-7.07 (m, 4H), 6.27 (d, 2H), 5.24 (m, 1H), 4.39-4.36
(m, 1H), 3.61 (s, 3H), 3.43 (s, 2H), 2.69-2.66 (m, 2H), 1.54-1.33
(m, 2H), 0.86-0.75 (m, 6H).
EXAMPLE 46
Synthesis of BIO-1180
[0346] A. The method described in Example 45A was followed using
t-butyl p-aminophenylacetate to give 1180-1 in 94% yield.
FAB-MS=234. .sup.1H NMR: (CDCl.sub.3, 300 MHZ, ppm) 7.18 (d, 2H,
J=8.2 Hz), 6.98 (d, 2H, 8.2 Hz), 3.49 (s, 3H), 1.45 (s, 9H).
[0347] B. To a solution of isocyanate 1180-1 (233 mg, 1.0 mmol) in
CH.sub.2Cl.sub.2 (5 ml) was added 2-aminothiazole (100 mg, 1.0
mmol) and the mixture was heated until a clear solution was formed
and was stirred at room temperature for 1 h. Removal of the
solvents gave 1180-2 (335 mg) as a brown-yellow solid. This solid
was dissolved in CH.sub.2Cl.sub.2 (2.5 ml), and to this was added
TFA (2.5 ml). The mixture was stirred at room temperature for 1.5 h
and was concentrated to afford 1180-3 (300 mg) as a yellow-solid.
FAB-MS=278.
[0348] C. To a solution of 1180-3 (28 mg, 0.1 mmol) in DMF (0.25
ml) was added EDC (60 mg, 0.31 mmol) and DMAP (55 mg). The mixture
was stirred at room temperature for 10 min. and to this was added
amine-TFA salt .beta.-3 (23 mg, 0.051 mmol). The resulting reaction
mixture was stirred at room temperature for 16 h. The usual workup
(5% citric acid, 5% NaHCO.sub.3, sat. NaCl) drying
(Na.sub.2SO.sub.4), and concentration gave crude 1180-4 (22 mg,
72%). FAB-MS=596.
[0349] D. The crude 1180-4 was hydrolyzed as described in Example
1B to give crude Bio-1180. Purification of the crude product by
HPLC afforded pure BIO1180. HPLC: Room temperature; 26.3
min.>99% purity. FAB-MS=582. .sup.1H NMR: (DMSO-D.sup.6, 300
MHz, ppm) 9.00 (br, s, 1H), 8.52 (d, 2H, J=8.3 Hz), 8.24 (d, 2H,
J=8.3 Hz), 7.50-7.47 (m, 3H), 7.28 (d, 2H, J=8.5 Hz), 7.20 (1H, d,
J=3.5 Hz), 6.95-6.81 (m, 3H), 6.08 (d, 1H, J=1.4), 5.19-5.16 (m,
1H), 4.4-4.2 (m, 1H), 3.51 (dd, J=14.1 Hz and 23.8 Hz), 2.76-2.65
(m, 2H), 1.57-1.50 (m, 1H), 1.50-1.44 (m, 2H), 0.92 (d, 2H, J=6.3
Hz), 0.86 (d, J=6.3 Hz).
EXAMPLE 47
Synthesis of BIO-1199
[0350] To a solution of BIO-1089 (15 mg) in DMSO (1.0 ml), H.sub.2O
(2 ml) was added Oxone.RTM. (20 mg) and the mixture was stirred at
room temperature. The HPLC trace showed that Bio-1089 (Room
temperature=20 min) was disappearing and a new peak (retention
time=16.9 min) was forming. After stirring at room temperature for
16 h, the starting Bio-1089 was almost totally consumed. Bio-1199
(Room temperature=16.9 min) was isolated by HPLC and was >99%
pure. FAB-MS=595.
EXAMPLE 48
Synthesis of BIO-1207
[0351] A. Procedure C was carried out using amine .beta.-5 (220 mg,
1.053 mmol), this product was then subjected to the conditions
described in Procedure D1 to afford 1207-1 (383 mg, 88% for two
steps).
[0352] B. The method of Example 1A was followed using
p-Cbz-aminophenylacetic acid (260 mg, 0.91 mmol) and amine 1207-1
(375 mg, 0.86 mmol) (treated with Et.sub.3N) afford 1207-2 (415 mg,
82%) as a pale brown solid.
[0353] C. Compound 1207-2 (390 mg, 0.66 mmol) was deprotected as
described in Procedure D2 to afford 1207-3 (140 mg, 47%) as a pale
brown solid.
[0354] D. To a solution of 2-isopropylaniline (135 mg, 1.0 mmol) in
CH.sub.2Cl.sub.2 (2 ml) and Et.sub.3N (0.5 ml) was added COCl.sub.2
(1.6 ml of 1.9 M solution in toluene, 3.0 mmol) solution at
0.degree. C. slowly and the resulting mixture was stirred at room
temperature for 1 h and diluted with ether (15 ml). Removal of the
solid thus formed and the solvents gave 1207-4 (165 mg) as a brown
liquid. .sup.1H NMR: (CDCl.sub.3, 300 MHz, ppm) 7.87-7.64 (m, 4H),
3.83-3.74 (m, 1H), 1.81 (d, 6H).
[0355] E. To a solution of 1207-4 (12 mg, 0.074 mmol) in DMF (0.12
ml) was added 1 drop of Et.sub.3N and 1207-3 (28 mg, 0.062 mmol).
The resulting mixture was stirred for 1 h (FAB-MS=617) and was
added to methanol (2 ml) and 2M LiOH (0.25 ml). This mixture was
stirred at room temperature for 16 h and was subjected to HPLC. The
pure fractions were collected and concentrated to afford BIO-1207
as a white solid. FAB-MS=603. HPLC: Room temperature=31.2 min;
>98.5% purity.
EXAMPLE 49
Synthesis of BIO-1210
[0356] The procedure described in Example 22D was followed using
2-methylphenylureaphenylacetic acid and the free amine of the
TFA-amine salt prepared in Example 44B (65 mg). The resulting
product was subjected to HPLC. The pure fractions were collected
and concentrated to afford BIO-1210 as a white solid. FAB-MS=603.
HPLC: Room. temperature=28.6 min, >99% purity.
EXAMPLE 50
Synthesis of BIO-1224
[0357] A. Procedure C was performed using amine P-4 (48 mg, 0.2
mmol), to afford 1224-1 (82 mg, 91%) as a white solid. .sup.1H-NMR:
(CDCl.sub.3, 300 MHz, ppm) 7.49-7.39 (1H), 6.73-6.62 (m, 3H),
5.35-5.28 (m, 1H), 5.19-5.06 (m, 1H), 4.16-4.08 (m, 1H), 3.74 (s,
3H), 3.69 (s, 3H), 2.72-2.51 (m, 2H), 2.40-2.36 (m, 2H), 1.98-1.75
(m, 2H), 1.90 (s, 3H), 1.28 (s, 9H), 1.19 (s, 9H).
[0358] B. Compound 1224-1 (60 mg, 0.13 mmol) was dissolved in
CH.sub.2Cl.sub.2 (1.5 ml) and TFA (1.5 ml). The mixture was stirred
room temperature for 5 h and the solvents were removed to give
1224-2 as a TFA salt. This compound was used in the next step
without purification.
[0359] 1H NMR: (CDCl.sub.3, 300 MHz, ppm) 7.92 (br, 1H), 6.82-6.78
(m, 3H), 5.44-5.26 (m, 1H), 4.40-4.28 (m, 1H), 3.84-3.72 (m, 6H),
2.92-2.70 (m, 4H), 2.60-2.25 (m, 2H), 1.92 (s, 3H).
[0360] C. The method described in Example 22D was followed using
2-methylphenylureaphenylacetic acid (37 mg, 0.13 mmol) and amine
1224-2 (60 mg, 0.13 mmol). The resulting product was subjected to
HPLC. The pure fractions were collected and dried to afford
BIO-1224 (22 mg, 22%) as a white solid. FAB-MS=623. HPLC: Room
temperature=23.8 min, >99% purity. .sup.1H NMR: (CDCl.sub.3, 300
MHz, ppm). 7.38 (d, 1H), 6.98 (d, 2H), 6.74 (d, 2H), 6.72 (m, 2H),
6.51 (t, 1H), 6.43-6.40 (m, 1H), 6.35-6.31 (m, 1H), 4.84-4.76 (m,
1H), 4.04-3.97 (m, 1H), 3.39 (s, 6H), 3.33 (s, 2H), 2.36-2.18 (m,
2H), 1.91-1.75 (m, 2H), 1.72 (s, 3H), 1.19-0.99 (m, 2H), 0.46-0.37
(m, 6H).
EXAMPLE 51
Synthesis of Compound BIO-1056
[0361] A. A mixture of 3-methoxy-4-nitrobenzoic acid (2.01 g, 10.2
mmol) and thionyl chloride (2.3 mL, 31.5 mmol) was stirred at
80-90.degree. C. for 1.5 h. The reaction was concentrated and the
residue diluted with ether. The organic solution was washed with
sat. aq. NaHCO.sub.3 (2.times.), H.sub.2O, then sat. aq. NaCl,
dried (MgSO.sub.4) and concentrated to afford
3-methoxy-4-nitrobenzoyl chloride (1.92 g, 87%) as a white solid:
.sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.95-7.70 (m, 3H), 4.06 (s,
3H).
[0362] B. To a cold (0.degree. C.) solution of TMSCHN.sub.2 (2 M in
hexane, 1.5 mL, 3.0 mmol) and triethylamine (420 .mu.L, 3.0 mmol)
was added a solution of 3-methoxy-4-nitrobenzoyl chloride (0.52 g,
2.4 mmol) in acetonitrile (8.5 mL). The reaction was stirred at
0.degree. C. for 24 h and then concentrated. The residue was
slurried with sat. aq., NaHCO.sub.3 and the mixture extracted with
ether (3.times.). The combined ether washes were washed with water,
then sat. aq. NaCl, dried (MgSO.sub.4) and concentrated to afford
.omega.-diazo-3-methoxy-4-nitroacetophenone (0.53.g, 100%) as a
yellow foam: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.88 (d, 10 Hz,
1H), 7.61 (s, 1H), 7.27 (d, 10 Hz, 1H), 5.97 (s, 1H), 4.02 (s,
3H).
[0363] C. To a refluxing solution of
.omega.-diazo-3-methoxy-4-nitroacetophenone (7.95 g, 35.9 mmol) in
tBuOH (100 mL) was added a filtered solution of silver benzoate
(2.50 g, 10.9 mmol) in triethylamine (15 mL) dropwise over 1 h.
After refluxing for 45 min, decolorizing carbon was added and the
hot mixture filtered thru a pad of Celite. The filtrate was
concentrated and the residue diluted with ethyl acetate. The
organic solution was washed with 5% aq. NaHCO.sub.3 (2.times.),
H.sub.2O, 5% aq. citric acid, H.sub.2O, then sat. aq. NaCl, dried
(MgSO.sub.4) and concentrated to afford t-butyl
3-methoxy-4-nitrophenylacetate (8.92 g, 93%.) as a brown oil:
.sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.83 (d, 8.3 Hz, 1H), 7.03
(s, 1H), 6.93 (d, 8.3 Hz, 1H) 3.97 (s, 3H), 3.58 (s, 2H), 1.45 (s,
9H).
[0364] D. A mixture of t-butyl 3-methoxy-4-nitrophenylacetate
(0.144 g, 0.539 mmol) and 10%. Pd on carbon (0.155 g) in ethyl
acetate (8 mL) and methanol (2 mL) was stirred under H.sub.2 (40-60
psi) for 2 h. The mixture was filtered through Celite and the
filtrate concentrated to afford t-butyl
4-amino-3-methoxyphenylacetate (0.123 g, 96%) as a light yellow
oil: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 6.70 (m, 3H), 4.04 (bs,
2H), 3.84 (s, 3H), 3.42 (s, 2H), 1.43 (s, 9H).
[0365] E. To a solution of t-Butyl 4-amino-3-methoxyphenylacetate
(0.123 g, 0.52 mmol) in methylene chloride (2.0 mL) was added
phenyl isocyanate (60 .mu.L, 0.55 mmol). The reaction was stirred
for 45 min then concentrated to afford t-butyl
3-methoxy-4-phenylureidophenylacetate (0.190 g, 100%) as a pale
yellow foam: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 8.00 (d,11 Hz,
1H) 7.65-6.94 (m, 7H), 6.80 (d, 9.0 Hz, 1H,), 6.74 (s, 1H), 3.68
(s, 3H), 3.45 (s, 2H), 1.44 (s, 9H).
[0366] F. A solution of t-butyl
3-methoxy-4-phenylureidophenylacetate (0.108 g, 0.303 mmol) in
trifluoroacetic acid (5.0 mL) was stirred for 30 min. The reaction
was concentrated and the residue coevaporated with methylene
chloride (2.times.) then ether to afford
3-methoxy-4-phenylureidophenylacetic acid, (0.090 g, 99%) as a
white foam: .sup.1H NMR (CD.sub.3SOCD.sub.3, 300 MHz, ppm) 9.28
(s,1H), 8.18 (s, 1H), 8.02 (d, 7.5 Hz, 1H), 7.58-7.15 (m, 5H), 6.91
(bm, 2H), 6.77 (d, 7.5 Hz, 1H), 3.85 (s, 3H), 3.49 (s, 2H).
[0367] G. A solution of 3-methoxy-4-phenylureidophenylacetic acid
(0.33 g, 0.88 mmol), Leu-.beta.-2, prepared utilizing procedures C
and D, (0.27 g, 0.90 mmol), BOP 0.39 g, 0.90 mmol) and DIPEA (0.77
mL, 4.4 mmol) in DMF (5 mL) was stirred for 18 h. The reaction was
diluted with ethyl acetate and washed with 60% sat. aq. NaHCO.sub.3
(3.times.), H.sub.2O, 5% aq. citric acid (3.times.), H.sub.2O, then
sat. aq. NaCl, dried (MgSO.sub.4) and concentrated to afford crude
product (0.49 g). The crude product was purified by flash
chromatography (silica gel, 1:4 hexane-ethyl acetate) to give
BIO1056 t-butyl ester (0.35 g, 60%) as a white foam: .sup.1H NMR
(CDCl.sub.3, 300 MHz, ppm) 8.00 (d, 8.1 Hz, 1H), 7.55-7.20 (m, 8H),
7.05 (m, 1H), 6.70 (m, 5H), 5.89 (s, 2H), 5.18 (m, 1H), 4.50 (s,
1H), 3.63 (s, 3H), 3.47 (s, 2H), 2.67 (m, 2H), 1.68-1.40 (bm, 3H),
1.33 (s, 9H).
[0368] H. To a cold (0.degree. C.) solution of BIO1056 t-butyl
ester (0.35 g, 0.53 mmol) in methylene chloride (5.0 mL) was added
trifluoroacetic acid (5.0 mL). The reaction was allowed to warm to
RT and stirred for 1 h then concentrated to afford crude BIO1056
(0.315 g). The crude product was purified by HPLC in two portions
to give BIO1056 (0.16 g, 50%) as a white solid: .sup.1H NMR
(CD.sub.3SOCD.sub.3, 300 MHz, ppm) 9.25 (s, 1H), 8.43 (d, 8.2 Hz,
1H), 8.15 (m, 2H), 8.01 (d, 8.2 Hz, 1H), 7.50-6.55 (m, 10H), 5.97
(s, 2H), 5.08 (m, 1H), 4.31 (m, 1H), 3.85 (s, 3H), 3.41 (m, 2H),
2.64 (m, 2H), 1.55-1.22 (bm, 3H), 0.80 (m, 6H); HPLC (Gradient A),
35.2 min, (Gradient B), 19.4 min; MS, m/z 605.
EXAMPLE 52
Synthesis of Compound BIO-1221
[0369] A. To a solution of t-butyl 4-amino-3-methoxyphenylacetate
(0.024 g, 0.10 mmol) in methylene chloride (2.0 mL) was added
o-tolyl isocyanate (15 .mu.L, 0.12 mmol). The reaction was stirred
for 2 h then concentrated to afford t-Butyl
3-methoxy-4-o-tolylureidophenylacetate (0.036 g, 97%) as a tan
foam:
[0370] .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 8.05 (d, 7.9 Hz, 1H),
7.55 (d, 7.9 Hz, 1H), 7.45-7.05 (m, 5H), 6.78 (m, 2H), 3.73 (s,
3H), 3.48 (s, 2H), 2.23 (s, 3H), 1.44 (s, 9H).
[0371] B. A solution of t-butyl
3-methoxy-4-o-tolylureidophenylacetate (0.016 g, 0.043 mmol) in
trifluoroacetic acid (1.0 mL) was stirred for 1 h. The reaction was
concentrated and the residue coevaporated with methylene chloride
(2.times.) then ether to afford
3-methoxy-4-o-tolylureidophenylacetic acid (0.0135 g, 100%) as a
white residue.
[0372] C. The procedure described in Example 51G was performed
using 3-methoxy-4-o-tolylureidophenylacetic acid (0.0135 g, 0.043
mmol) and amine salt prepared from .beta.-3 utilizing procedures C
and D (0.0185 g, 0.041 mmol)to afford BIO1221 methyl ester (0.016
g, 60%) as a white foam: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm)
8.10 (d, 1H), 7.61 (d, 1H), 7.45-7.00 (m, 7H), 6.85-6.65 (m, 5H),
5.93 (s, 2H), 5.20 (m, 1H), 4.37 (m, 1H), 3.85 (s, 3H), 3.61 (s,
3H), 3.52 (s, 2H), 2.75 (m, 2H), 2.30 (s, 3H), 1.65-1.10 (bm, 3H),
0.86 (m, 6H).
[0373] D. BIO1221 methyl ester (0.016 g, 0.025 mmol) was hydrolyzed
using the method described in Example 1B to give BIO-1221 (0.0087
g, 56%) as a white powder: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm)
7.93 (d, 1H), 7.70 (d, 1H), 7.49 (d, 1H), 7.37-6.92 (m, 6H),
6.78-6.55 (m, 5H) 5.81 (s, 2H), 5.09 (m, 1H), 4.27 (m, 1H), 3.73
(s, 3H), 3.40 (s, 2H), 2.58 (m, 2H), 2.19 (s, 3H), 1.48-1.25 (bm,
3H), 0.76 (m, 6H); HPLC (Gradient A), 35.2 min; MS, m/z 619.
EXAMPLE 53
Synthesis of Compound BIO-1238
[0374] A. The procedure described in Example 43A was performed
using amine 9-5 to give 1238-1. Yield:92%. .sup.1HNMR (CDCl.sub.3,
300 MHz, ppm): 7.19 (d, 2 H, J=8.6 Hz), 6.82 (d, 2 H, J=8.6 Hz),
5.36-5.28 (m, 2 H), 4.25-4.22 (m, 1 H), 3.72 (s, 3H), 3.56 (s, 3H),
2.72-2.66 (m, 2H), 2.49-2.41 (m, 2 H), 2.1 (s, 3 H), 1.92-1.78 (m,
1H), 1.48 (s, 9 H). The Boc group was removed by
TFA/CH.sub.2Cl.sub.2 to afford TFA salt 1238-1. .sup.1HNMR
(CDCl.sub.3, 300 MHz, ppm): 7.12 (d, 2 H, J=8.5 Hz), 6.74 (d, 2 H,
J=8.5 Hz), 5.32 (m, 1 H), 4.38 (m, 1 H), 3.68 (s, 3 H), 3.51 (s, 3
H), 2.77-2.69 (m, 2 H), 2.55-2.38 (m, 1H), 2.36-2.31 (m, 1 H),
2.16-2.02 (m, 2 H), 1.91 (s, 3 H).
[0375] B. The procedure described in Example 1A was performed using
2-methylphenylureaphenylacetic acid (20 mg, 0.7 mmol) and TFA salt
1238-1 (30 mg, 0.7 mmol) to give 1238-2 (35 mg, 83 %) as a white
solid. .sup.1HNMR (DMSO-d.sup.6, 300 MHz, ppm): 7.91 (d, 1H), 7.52
(d, 2 H, J=8.5 Hz), 7.35-7.30 (m, 4 H), 7.02 (d, 1 H), 6.80 (d, 2
H, J=8.5 Hz), 5.79-5.68 (m, 1 H), 4.40-4.28 (m, 1 H), 3.71 (,s, 3
H), 3.63 (s, 3 H), 3.35-3.38 (m, 2 H), 2.49 (br, s, 2 H), 2.00 (s 3
H).
[0376] C. A solution of 1238-2 (20 mg, 0.033 mmol) in MeOH (3 mL)
and aqueous LiOH (3 mL of 2N) was stirred at room temperature
overnight, the reaction mixture was cooled to 0.degree. C. and
acidified by adding TFA. until pH=3-4 (pH paper). The desired
product was isolated and purified by LC (Vydac C18 column; gradient
8) to give 12 mg (0.017 mmol; 61%) of BIO-1238 as a white solid:
FAB-MS=595.
EXAMPLE 54
Synthesis of Compound BIO-1245
[0377] A. 1245-1 was prepared from commercially available
N-BOC-methionine sulfone (562 mg, 2.0 mmol) and amine B-3 (470 mg,
2.10 mmol) using the method described in Example 1A to afford crude
1245-1 (962 mg, 1.90 mmol, 95%) as a white foam which was used
without further purification. .sup.1HNMR(CDCl.sub.3): .delta. 7.31
(1H, d, J=8.3 Hz), 6.77-6.7(3H, m), 5.91(2H, s), 5.04(1H, d, J=7.6
Hz), 5.27 (1H, m), 4.30 (1H, br), 3.61 (3H, s), 3.15 (1H, m), 2.93
(1H, m), 2.89 (3H, s), 2.85 (2H, m), 2.22 (2H, m), 1.42 (9H,
s).
[0378] B. Compound 1245-1 (962 mg, 1.90 mmol) was treated-with 4N
HCl/dioxane as the reagent. Concentration affords the hydrochloride
salt 1245-2 as a white solid (800 mg, 1.89 mg, 1.89 mmol, 99%)
which was used without further purification.
.sup.1HNMR(CDCl.sub.3): .delta. 8.75 (1H, br), 8.20 (2H, br),
6.91-6.55 (3H, m), 5.90 (2H, bs), 5.42 (1H, br), 4.55 (1H, br) 3.60
(3H, s), 3.45-3.0 (2H, bm), 2.90 (3H, s), 2.85-2.40 (4H, bm).
[0379] C. The procedure described in Example 22D was performed
using compound 1245-2 (800 mg, 1.89 mmol) and
o-methylphenylureaphenyl acetic acid (543 mg, 1.89 mmol) to afford
crude 1245-3 (1.15 g, 1.76 mmol, 93%) as a white solid which used
without further purification.
[0380] .sup.1HNMR(DMSO=d.sub.6) .delta. 7.95 (1H,s), 7.89 (1H, d,
J=7.9 Hz), 7.43 (2H, D, J=7.9 Hz), 7.20 (4H, m), 7.00-6.78 (4H, m),
6.03 (2H,s), 5.18 (1H, m), 4.40 (1H, m), 3.58 (3H, s), 3.49 (3H,
s), 3.39 (2H, br), 2.90-2.49 (2H, m), 2.29 (3H, s), 2.00 (2H,
m)
[0381] D. Compound 1245-3 (1.1 g, 1.7 mmol) was hydrolyzed as
described in Example 1B to afford crude BIO-1245 (490 mg, 0.77
mmol, 45%)as a white solid >90% pure by HPLC. A small amount
(.sup..about.150 mg) was purified by prep HPLC to afford pure
BIO-1245(81 mg, 54% recovery) as a white solid m/z=639 (100% pure
by HPLC).
[0382] .sup.1HNMR(DMSO-d.sub.6): .delta. 8.60(0.5H, bs), 8.57 (1H,
d, J=8.1 Hz), 8.37 (1H, d, J=8.1 Hz), 8.18 (1H, s), 8.05 (0.5H, s),
7.89 (1H, d, J=8.0 Hz), 7.43 (2H, d, J=8.04 z), 7.21 (4H, m),
6.97-6.81 (4H, m), 6.03 (2H, s), 5.13 (1H,m), 4.43 (1H, m), 3.80
(1H, br), 3.49 (3H, s), 2.93 (2H, m), 2.45 (2H, m), 2.30 (3H, s),
2.01 (2H, m).
EXAMPLE 55
Synthesis of BIO-1246
[0383] A. To a suspension of L-cysteine (1.5 g, 12.4 mmol) in
methanol (8 mL) was added excess sodium methoxide (2.0 g, 37.2
mmol) followed by a catalytic amount of sodium iodide
(.sup..about.100 mg). After stirring at room temperature for 30
min. 1-bromo-2-propanol (1.7 g, 12.4 mmol) was added and the
reaction was stirred overnight. The reaction mixture was then
neutralized to pH.sup..about.7, diluted with water (20 mL) and
concentrated to remove the methanol. The solution was then diluted
with dioxane (20 mL) and triethylamine (7.0 mL, 50 mmol) was added
followed by BOCON (3.1 g, 12.4 mmol) and the reaction was stirred
at room temperature for 3 h. The reaction was worked up by diluting
with water (20 mL) and extracting with ethyl acetate (3.times.25
mL). The organic extracts were discarded and the aqueous solution
acidified to pH=1 with 1N HCl. The aqueous was extracted with ethyl
acetate (4.times.30 mL), dried over sodium sulfate and concentrated
to afford 1246-1 (2.87 g, 10.4 mmol, 83%, 2 steps) as a thick pale
yellow syrup. .sup.1HNMR(CDCl.sub.3) .delta. 5.60-5.50 (1H, br),
4.60-4.50 (1H, br), 4.44 (2H, t, J=6.3 Hz), 3.02 (2H, bm), 32.65
(2H,br) 2.03 (2H,M), 1.45 (9H, S).
[0384] B. The procedure of Example 1A was performed using 1246-1
(33 mg, 0.11 mmol) and amine 9-3 (22 mg, 0.10 mmol) to afford
1246-2 (39 mg, 0.08 mmol, 80%) as a pale yellow foam which was used
without purification in the next step. .sup.1HNMR(CDCl.sub.3):
.delta. 6.80-6.60 (3H,m), 5.91 (2H,s), 5.50 (1H,bm), 4.35 (1H,bm),
3.71, (2H,bt), 3.61 (3H, s), 3.15-2.65 (6H,m), 1.85 (2H,m), 1.46
(9H, s).
[0385] C. Compound 1246-2 (39 mg, 0.08 mmol) was treated with TFA
to give the corresponding amine-TFA salt of 1246-2 which was
subjected to the conditions described in Example 54C to give a
white solid which was directly hydrolysed as described in Example
1B to the free acid. A small aliquot was purified by HPLC. The
clean fractions were collected to afford BIO-1246(.sup..about.3 mg)
M/Z=637(100% pure by HPLC) as a white solid.
[0386] .sup.1HNMR(DMSO-d.sub.6): .delta. 9.01 (1H,s), 8.66
(1H,d,J=5.3 Hz), 8.30 (1H,d,J=5.5 Hz), 7.94(1H,s), 7.88 (1H,d,J=5.3
Hz), 7.42 (2H,d,J=5.5 Hz), 7.20-7.15 (4H,m), 7.00-6.94 (2H,m),
6.88-6.79 (2H,m), 6.02 (2H,s), 5.12 (1H,m), 4.48 (1H,m), 3.65
(2H,m), 2.90-2.45 (6H,m), 2.28 (3H,s), 1.65 (2H,m).
EXAMPLE 56
Synthesis of BIO-1248
[0387] A. A mixture of 4-fluorobenzaldehyde (2.48 g; 20 mmol),
malonic acid (2.5 g, 24 mmol) and ammonium acetate (2.16 g; 28
mmol) in ethanol (100 mL) was refluxed under argon overnight. After
cooling to room temperature, the solid precipitate was collected by
filtration and washed with ethanol (3.times.30 mL) and dried under
vacuum to give 1.0 g (27%) of white solid, which was used without
further purification.
[0388] To a suspension of the white solid (1.0 g, 9.4 mmol) in
methanol was added SOCl.sub.2 (6.01 mmol; 5.2 mL of 2 M in
CH.sub.2Cl.sub.2). The resultant solution was stirred at room
temperature overnight. After removal of excess solvent, the residue
was dissolved in EtOAc, basified with sat. NaHCO.sub.3, and dried
with Na.sub.2SO.sub.4. The organic solution was concentrated under
reduced pressure to give 900 mg (84%) of amine 1248-1 as a light
yellow oil: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.28 (m, 2 H,
Ar), 6.96 (m, 2 H, Ar), 4.46 (t, J=6.8, 1 H), 3.62 (s, 3 H, OMe),
2.58 (d, J=6.8 Hz, 2 H), 1.69 (s, 2 H, NH); TLC, 10%
MeOH/CH.sub.2Cl.sub.2, R.sub.f=0.5.
[0389] B. Amine 1248-1 (300 mg, 1.52 mmol) was coupled with
N.alpha.-t-Boc-N.epsilon.-leu-N-hydroxysuccinimide (300 mg, 1.52
mmol) using the method described in Procedure C. The resulting
adduct was deprotected with trifluoroacetic acid and, then basified
with Et.sub.3N as described in Procedure D1 to give the amine
1248-2 in 84%: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 8.20 (d,
J=7.1 Hz, 1 H), 7.24 (m, 2 H, Ar), 6.97 (m, 2 H, Ar), 5.33 (m, 1
H), 3.58 (s, 3 H, OMe), 3.38 (m, 1 H), 2.82 (m, 2 H), 1.66 (m, 2
H), 1.30 (m, 1 H), 1.22 (s, 2 H), 0.91 (m, 6 H); TLC,
10MeOH/CH.sub.2Cl.sub.2, R.sub.f=0.47 and 0.38.
[0390] C. 2-Methylphenylureaphenylacetic acid (77 mg, 0.27 mmol)
was coupled with amine 1248-2 (70 mg, 0.23 mmol) using the method
described in Example 22D to give 1248-3 in 61% yield. .sup.1H NMR
(DMSO-d.sup.6, 300 MHz, ppm) .delta. 9.15 (d, J=5.9 Hz, 1 H), 8.53
(t, J=7.5 Hz, 1 H), 8.17 (d, J=8.2 Hz, 1H), 8.0 (s, 1 H), 7.84 (d,
J=8.0 Hz, 2 H), 7.35 (m, 4 H), 7.13 (m, 6 H), 6.92 (t, J=8.2 Hz, 1
H), 5.20 (m, 1 H), 4.30 (m, 1 H), 3.52 (s, two peaks, 3 H, OMe),
3.45-3.24 (m, 2 H), 2.75 (m, 2 H), 2.24 (s, 3 H, Me), 1.57-1.33 (m,
3 H), 0.82 (m, 6 H); HPLC (gradient 1**) 21.2 min and 21.5 min
(1:24); FABMS, m/z 577 (C.sub.33H.sub.37N.sub.4O.sub.5F of
M.sup.++1 requires 577).
[0391] D. A solution of 1248-3 (22 mg, 0.038 mmol) in DMSO (1 mL)
and MeOH (2 mL) was hydrolyzed with aqueous LiOH as described in
Example 1B. The product was purified on a Vydac reverse-phase C18
column (22 mm.times.25 cm) using a linear gradient of 15 %
CH.sub.3CN/H.sub.2O (0.1% TFA) to 40% CH.sub.3CN/H.sub.2O (0.1%
TFA) with a flow rate of 10 mL/min to give BIO-1248 in 29% isolated
yield. .sup.1H NMR (DMSO-d.sup.6, 300 MHz, ppm) .delta. 8.93 (s, 1
H), 8.46 (d, J=8.3 Hz, 1 H), 8.25 (d, J=8.2 Hz,1 H), 7.87 (s, 1 H),
7.82 (d, J=8.0 Hz, 1 H), 7.33 (m, 5 H), 7.12 (m, 5 H), 6.93 (m, 1
H), 5.15 (m, 1 H), 4.28 (m, 1 H), 3.35 (m, 2 H), 2.65 (d, J=7.2 Hz,
2 H), 2.22 (s, 3 H, Me), 1.55 (m, 1 H), 1.43 (m, 2 H), 0.83 (m, 6
H); HPLC (gradient 1) 18.7 min and 19.3 min (1:24); FABMS, m/z 563
(C.sub.31H.sub.35N.sub.4O.sub.5F of M.sup.++1 requires 563).
EXAMPLE 57
Synthesis of BIO-1270
[0392] A. Amine .beta.-3 (500 mg, 2.24 mmol) was coupled with
N.alpha.-Cbz-N.epsilon.-t-Boc-L-Lys-N-hydroxysuccinimide (1.0 g,
2.1 mmol) using Procedure C to give the coupled adduct 1270-1 (1.1
g, 82%). This adduct was deprotected with trifluoroacetic acid and
was basified with Et.sub.3N as previously described in Procedure D
to give 1270-2 in 54% yield. .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm)
.delta. 7.31 (m, 6 H), 6.72 (m, 3 H), 5.90. (s, 2 H), 5.58 (d, J=9
Hz, 1 H), 5.26 (m, 1 H), 5.07 (s, 2 H), 4.15 (m, 1 H), 3.58 (s, 3
H, OMe), 2.77 (m, 2 H), 2.61 (m, 2 H), 1.79 (m, 1 H), 1.59 (m, 1
H), 1.41-1.30 (m, 6 H); TLC, 10% MeOH/CH.sub.2Cl.sub.2,
R.sub.f=0.11.
[0393] B. To a stirred solution of 1270-2 (15.5 mg, 0.032 mmol) and
pyridine (10.1 mg, 0.128 mmol) in CH.sub.2Cl.sub.2 at rt is added
acetyl chloride (7.5 mg, 0.096 mmol). After stirring for 3 hours
the reaction is concentrated and reverse phase chromatography
provided 1270-3 (16.3 mg, 95%) as a white foam.
.sup.1HNMR(CDCl.sub.3, 300 MHz, ppm) 7.32 (S, 5H), 6.70 (m, 3H)
5.91 (s, 2H), 5.82 (m, 1H), 5.55 (m, 1H), 5.25 (m, 1H), 5.09 (s,
1H), 4.13 (m, 1H), 3.60 (S, 3H), 3.28 (M, 2H), 2.9-2.4 (m, 3H),
1.94 (S, 3H), 1.9-1.76 (m, 1H) 1.70-1.58 (m, 1H), 1.52-1.42 (m,
2H), 1.36-1.22 (m, 2H).
[0394] C. Procedure D2 was performed using 1270-3 (reaction
progress was followed by HPLC) to give compound 1270-4 (14.1 mg,
quantitative yield) as a clear oil which was used as the crude
material.
[0395] D. The procedure of Example 54C was performed using 1270-4
(14.1 mg, 0.036 mmol). Purification was carried out via preperative
HPLC and provided Bio 1270-OMe (9.1 mg, 38%) as a white solid.
.sup.1HNMR(DMSO.sub.D6, 300 MHz, ppm), 8.13 (d, 1H J=10.35), 8.03
(s, 1H), 7.93 (d, 1H J=10.35), 7.83 (m, 1H), 7.49 (d, 2H J=10.35),
7.28 (m, 5H), 7.10-6.81 (m, 5H), 6.08 (s, 2H), 5.20 (dd, 1H J=9.66,
17.25), 4.33 (dd, 1H J=8.97, 15.18), 3.63 (s, 3H), 3.5 (s, 2H),
3.1-2.95 (m, 2H), 2.85-2.74 (m, 2H), 2.33 (s, 3H), 1.86 (s, 3H),
1.72-1.49 (m, 2H), 1.5-1.32 (m, 3H), 1.31-1.09 (m, 2H).
[0396] E. To Bio 1270-OMe (9.1 mg, 0.016) in 1 ml of DMSO.sub.D6
(NMR sample) was added 20 ul of 2N LiOH (0.041 mmol) and the
reaction was stirred at rt. overnight. The reaction was acidified
(red to litmus) with 3 drops of TFA and purified by preparative
HPLC This afforded BIO-1270 (6.2ng, 60%) as a white solid.
[0397] .sup.1HNMR(DMSO.sub.D6, 300 MHz, ppm), 8.5 (d, 12H J=10.35),
8.19 (d, 1H J=10.35), 7.99 (s, 1H), 7.93 (d, 1H J=10.35), 7.82 (m,
1H), 7.45 (d, 2H J=10.35). 7.28 (m, 4H), 7.05 (m, 1H), 6.98-6.89
(m, 2H) 6.86 (m, 1H), 6.09 (S, 2H), 5.66 (dd, 1H J=8.28, 16.56)
4.32 (dd, 1H J=7.59, 13.8), 3.27 (s, 2H), 2.98 (m, 2H) 2.75 (m,
2H), 2.33 (s, 3H), 1.87 (s, 3H), 1.69-1.48 (m, 2H), 1.46-1.32 (m,
3H), 1.28-1.12 (m, 2H); MS, m/z 646; HPLC (Gradient 1) 19.73 min.
100%.
[0398] Gradient 3 15% B.fwdarw.65% B 50 min.
[0399] Gradient 1 20% B.fwdarw.70% B 50 min.
EXAMPLE 58
[0400] Synthesis of BIO-1282
[0401] A. A solution of ethyl 3-pyridylacetate (1.65 g, 9.90 mmol)
in 32% peracetic acid (10 mL) was stirred at 80-90.degree. C. for 2
h. The reaction was concentrated and the residue coevaporated with
methanol (2.times.) then methylene chloride to afford ethyl
3-pyridylacetate N-oxide (1.80 g, 100%) as a white solid: .sup.1H
NMR (CDCl.sub.3, 300 MHz, ppm) 8.38 (s, 1H), 8.22 (d, 1H), 7.39 (d,
1H), 4.20 (q, 2H), 3.62 (s, 2H), 1.26 (t, 3H).
[0402] B. A solution of salicylamide (4.14 g, 30.2 mmol) and conc.
sulfuric acid (3 drops) in acetone (40 mL) was refluxed for 5 h.
The reaction was concentrated and the residue taken up in ethyl
acetate. The organic solution was washed with 1 N NaOH (2.times.),
1 N HCl (2.times.), H.sub.2O, then sat. aq. NaCl, dried
(MgSO.sub.4) and concentrated to afford
2,2-dimethyl-4-keto-1,3-benzoxazine (2.50 g, 47%) as a white solid:
.sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.92 (d, 1H), 7.60 (bs, 1H),
7.47 (m, 1H), 7.06 (m, 1H), 6.92 (d, 1H), 1.65 (s, 6H).
[0403] C. A solution of 2,2-dimethyl-4-keto-1,3-benzoxazine (1.77
g, 10.0 mmol) and PCl.sub.5 (2.09 g, 10.0 mmol) in POCl.sub.3 (3.0
mL) was stirred at RT for 1 h then at 50-60.degree. C. for 2 h. The
reaction was concentrated and the product distilled (90-95.degree.
C./2-3 mm Hg) to afford 4-chloro-2,2-dimethyl-3H-1,3-benzoxazine.
(0.496 g, 25%) as a clear oil: .sup.1H NMR (CDCl.sub.3, 300 MHz,
ppm) 7.58 (d, 1H), 7.48 (m, 1H), 6.97 (m, 1H), 6.94 (d, 1H), 1.63
(s, 6H).
[0404] D. A mixture of 4-chloro-2,2-dimethyl-3H-1,3-benzoxazine
(0.145 g, 0.741 mmol) and ethyl 3-pyridylacetate N-oxide (0.270 g,
1.49 mmol) in methylene chloride (5.0 mL) was refluxed for 20 h.
The reaction was concentrated and the residue taken up in ethyl
acetate. The organic mixture was washed with 60% sat. aq.
NaHCO.sub.3 (2.times.), H.sub.2O, sat. aq. NaCl, dried
(MgSO.sub.4), and concentrated to afford an oily residue (0.148
g).
[0405] The crude oily residue (0.148 g) in conc. HCl (10 mL) was
refluxed for 18 h. The reaction was concentrated and the residue
partitioned in H.sub.2O and methylene chloride. The aqueous
solution was washed with methylene chloride (2.times.) and then
concentrated to afford a white solid (0.105 g).
[0406] A solution of the white solid (0.105 g) in methanol (5.0 mL)
was treated with thionyl chloride (0.5 mL, 7 mmol) dropwise over 30
min. The reaction was stirred for 2 h then concentrated. The
residue was taken up in 5% aq. NH.sub.4OH and extracted with
methylene chloride (3.times.). The organic extracts were dried
(MgSO.sub.4) and concentrated to afford methyl
5-(2-aminopyridyl)acetate (0.012 g, 10% for three steps) as a white
solid: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.93 (s, 1H), 7.40
(d, 1H), 6.50 (d, 1H), 4.52 (bs, 2H), 3.70 (s, 3H), 3.49 (s, 2H);
MS, m/z 167.
[0407] E. To a solution of methyl 5-(2-aminopyridyl)acetate (0.012
g, 0.072 mmol) in methylene chloride (1.0 mL) was added o-tolyl
isocyanate (10 .mu.L, 0.081 mmol). The reaction was stirred for 1 h
then concentrated to afford a white residue (0.020 g) containing
methyl 5-(2-o-tolylureido)pyridylacetate.
[0408] F. A solution of crude methyl
5-(2-o-tolylureido)pyridylacetate (0.020 g) in methanol (1.0 mL)
was treated with 2 M LiOH (100 .mu.L, 0.20 mmol). The reaction was
stirred for 18 h then concentrated. The crude product was purified
by HPLC to afford 5-(2-o-tolylureido)pyridylacetic acid (0.013 g,
65%) as a white powder: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 8.10
(s, 1H), 7.87 (bd, 1H), 7.75 (bd, 1H), 7.21 (m, 1H), 7.08 (m, 1H),
3.62 (s, 2H); 2.38 (s, 3H); MS, m/z 286.
[0409] G. The procedure described in Example 1A was performed using
5-(2-o-tolylureido)pyridylacetic acid (0.013 g, 0.045 mmol) and the
amine prepared in Example 14A (0.022 g, 0.049 mmol) to afford
BIO1282 methyl ester (0.020 g, 60%) .sup.1H NMR (CDCl.sub.3, 300
MHz, ppm) 8.18-7.73 (m, 4H), 7.55 (d, 1H), 7.35-6.65 (m, 10H), 5.93
(s, 1H), 5.28 (m, 1H), 4.45 (m, 1H), 3.6.9-3.45 (m, 5H), 2.81 (bm,
2H), 2.20 (s, 3H), 1.54 (bm, 3H), 0.92 (m, 6H).
[0410] H. To a mixture of BIO1282 methyl ester (0.020 g, 0.033
mmol) in methanol (02.0 mL) was added 2.0 M LiOH (200 .mu.L, 0.40
mmol). The reaction was stirred for 20 h then concentrated. The
residue (containing a 4:5 mixture of BIO1282 and starting ester)
was dissolved in DMF (0.5 mL) and methanol (0.5 mL) then stirred
for an additional 28 h. The reaction was acidified with
trifluoroacetic acid and concentrated. The crude product was
purified by HPLC to give BIO-1282 (0.0056 g, 24%) as a white
powder: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 8.44 (d, 8.1 Hz,
1H), 8.26 (d, 8.3 Hz, 1H), 8.15 (s, 1H), 8.04 (d, 8.0 Hz, 1H), 7.66
(d, 8.7 Hz, 1H), 7.32-7.13 (m, 3H), 7.05-6.94 (m, 1H), 6.85-6.65
(m, 3H), 5.96 (s, 2H), 5.06 (m, 1H), 4.29 (m, 1H), 3.45 (m, 2H),
2.63 (m, 2H), 2.31 (s, 3H), 1.57-1.20 (m, 3H), 0.78 (m, 6H); HPLC
(Gradient A), 27.0 min; MS, m/z 590.
EXAMPLE 59
Synthesis of BIO-1294
[0411] A. To a stirred solution of the amine prepared in Example
57A (102 mg, 0.21 mmol) in CH.sub.2Cl.sub.2 (20) was added
CH.sub.3SO.sub.2Cl (48 mg, 32 .mu.L, 0.42 mmol) and Et.sub.3N (50
.mu.L). The resulting mixture was stirred at RT for 18 h. The
reaction mixture was diluted with CH.sub.2Cl.sub.2 (40 mL), washed
with 5% citric acid (20 mL), H.sub.2O (10 mL), Sat. NaHCO.sub.3 (20
mL), Sat. NaCl (20 mL) and dried with Na.sub.2SO.sub.4. The organic
solution was concentrated under reduced pressure to give 110 mg
(92%) of 1294-1 as a white solid: .sup.1H NMR (CDCl.sub.3, 300 MHz,
ppm) .delta. 7.30 (m, 6 H), 6.74 (m, 3 H), 5.90 (s, 2 H), 5.70 (m,
1 H), 5.25 (m, 1 H), 5.07 (s, 3 H), 4.16 (m, 1 H), 3.58 (s, 3 H,
OMe), 3.02 (m, 2 H), 2.88 (s, 3 H), 2.75 (m, 2 H), 1.76 (m, 1 H),
1.60 (m, 1 H), 1.50 (m, 2 H), 1.32 (m, 2 H); TLC, 10%
MeOH/CH.sub.2Cl.sub.2, R.sub.f=0.67.
[0412] B. To a solution of compound 1294-1 (110 mg, 0.195 mmol) was
dissolved in methanol (10 ml) was added acetic acid (0.2 ml) and
Pd(OH).sub.2 (110 mg). The resulting mixture was hydrogenated
(H.sub.2, 50 psi) at RT for 48 h. After standard work-up, 1294-2
(35 mg, 42%) was obtained as colorless oil: .sup.1H NMR
(CDCl.sub.3, 300 MHz, ppm) .delta. 8.06 (m, 1 H), 6.75 (m, 3 H),
5.92 (s, 2 H), 5.25 (m, 1 H), 5.02 (m, 1 H), 3.61 (s, 3 H), 3.35
(m, 1 H), 3.10 (m, 2 H), 2.94 (s, 3 H), 2.80 (m, 2 H), 1.87-1.30
(m, 8 H); HPLC (gradient 8) 12 min.
[0413] C. 2-Methylphenylureaphenylacetic acid (35 mg, 0.12 mmol)
was coupled with the amine 1294-2 (35 mg, 0.08 mmol) as described
in Example 1A to give compound 1294-3 in 88%. .sup.1H NMR
(CDCl.sub.3, 300 MHz, ppm) .delta. 8.50 (m, 1 H), 8.30 (s, 1 H),
8.16 (m, 1 H), 7.82 (m, 1 H), 7.40 (m, 2 H), 7.22-7.05 (m, 5 H),
7.00-6.70 (m, 5 H), 5.98 (s, 2 H), 5.11 (m, 1 H), 4.22 (m, 1 H),
3.52 (s, 3 H), 3.36. (m, 2 H), 2.91-2.62 (m, 7 H), 2.25 (s, 3 H),
1.60-1.05 (m, 6H); HPLC (gradient 8) 31 min; FABMS, m/z 696
(C.sub.34H.sub.41N.sub.5O.sub.9S of M.sup.++1 requires 696).
[0414] D. A solution of compound 1294-3 (50 mg, 0.07 mmol) in MeOH
(3 mL) was hydrolyzed with aqueous LiOH as previously described.
The product was purified on a Vydac reverse-phase C18 column (22
mm.times.25 cm) using a linear gradient of 15% CH.sub.3CN/H.sub.2O
(0.1% TFA) to 40% CH.sub.3CN/H.sub.2O (0.1% TFA) with a flow rate
of 10 mL/min to give BIO-1294 in 41% isolated yield. .sup.1H NMR
(CDCl.sub.3, 300 MHz, ppm) .delta. 8.95 (m,1 H), 8.42 (d, J=8.2 Hz,
1H), 8.08 (d, J=8.1 Hz, 1 H), 7.88 (s, 1 H), 7.83 (d, J=8.0 Hz, 2
H), 7.36 (d, J=8.2 Hz, 2 H), 7.15 (m, 4 H), 7.10-6.71 (m, 5 H),
5.97 (s, 2 H), 5.04 (m, 1 H), 4.22 (m, 1 H), 3.41-3.25 (m, 2 H),
2.83-2.80 (m, 6 H), 2.23 (s, 3 H), 1.70-1.04 (m, 6 H); HPLC
(gradient 8) 27 min; FABMS, m/z 682
(C.sub.33H.sub.39N.sub.5O.sub.9S of M.sup.++1 requires 682).
EXAMPLE 60
Synthesis of BIO-1321
[0415] A. A mixture of methyl 4-formylbenzoate (3.48 g; 20 mmol),
malonic acid (2.5 g, 24 mmol) and ammonium acetate (2.16 g; 28
mmol) in ethanol (100 mL) was refluxed under argon overnight. After
cooling to room temperature, the solid precipitate was collected by
filtration and washed with ethanol (3.times.30 mL). The white solid
was dried under vacuum overnight to give 2.8 g (63%) of 1321-1.
[0416] B. To a suspension of compound 1321-1 (1.0. g, 4.48 mmol) in
methanol (50 mL) was added SOCl.sub.2 (5.4 mmol; 2.7 mL of 2 M in
CH.sub.2Cl.sub.2). The resultant solution was stirred at room
temperature overnight. After removal of excess solvent, the residue
was dissolved in EtOAc, basified with sat. NaHCO.sub.3, and dried
with Na.sub.2SO.sub.4. The organic solution was concentrated under
reduced pressure to give 780 mg (53%) of the amine 1321-2 as a
light yellow oil: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 7.99 (m, 2
H, Ar), 7.56 (d, J=8.1 Hz , 1 H, Ar), 7.42 (d, J=8.0 Hz, 1 H, Ar),
4.46 (t, J=6.7, 1 H), 3.85 (s, 3 H, OMe), 3.65 (s, 3 H, OMe), 2.65
(d, J=6.8 Hz, 2H), 1.88 (s, 2 H, NH).
[0417] C. The amine 1321-2 (500 mg, 1.11 mmol) was coupled with
N.alpha.-t-Boc-N.epsilon.-Leucine-N-Hydroxysuccinimide (380 mg, 1.0
mmol) as described in Procedure C to give material which was
deprotected with trifluoroacetic acid and, then basified with
Et.sub.3N as described in Procedure D1 to give amine 1321-3 in 70t
yield: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 8.32 (t, J=9.1 Hz , 1
H), 8.20 (d, J=8.3 Hz, 2 H), 7.34 (m, 2 H, Ar), 5.40 (m, 1 H), 3.86
(s, 3 H, OMe), 3.58 (s, 3 H, OMe), 3.41 (m, 1 H), 2.85 (m, 2 H),
1.67 (m, 2 H), 1.53 (s, 2 H), 1.30 (m, 1 H), 0.90 (m, 6 H).
[0418] D. 2-Methylphenylureaphenylacetic acid (54 mg, 0.19 mmol)
was coupled with amine 1321-3 (70 mg, 0.23 mmol) using the method
described in Example 22D to give the 1321-4 in 87% yield: 1H NMR
(DMSO-d.sup.6, 300 MHz, ppm) .delta. 8.62 (m, 1 H), 8.18 (d, J=8.1
Hz, 1 H), 8.10 (m, 1H), 7.94-7.82 (m, 4 H), 7.48-7.34 (m, 4 H),
7.17-7.13 (m, 4 H), 6.91 (t, J=7.3 Hz, 1 H), 5.24 (m, 1 H), 4.30
(m, 1 H), 3.53 (s, two peaks, 3 H, OMe), 3.39-3.34 (m, 2 H), 3.05
(m, 2 H), 2.24 (s, 3 H, Me), 1.60-1.36 (m, 3 H), 0.83 (m, 6 H);
HPLC (gradient 8) 40 min (1:1); FABMS, m/z 617
(C.sub.33H.sub.40N.sub.4O.sub.7 of M.sup.++1 requires 617).
[0419] E. A solution of 1321-4 (70 mg, 0.11 mmol) in DMSO (1 mL)
and MeOH (2 mL) was hydrolyzed with aqueous LiOH as described in
Example 1B. The product was purified on a Vydac reverse-phase C18
column (22 mm.times.25 cm) using a linear gradient of 15 %
CH.sub.3CN/H.sub.2O (0.1% TFA) to 40 % CH.sub.3CN/H.sub.2O (0.1%
TFA) with a flow rate of 10 mL/min to give BIO-1321 (22 mg, 34%
isolated yield): .sup.1H NMR (DMSO-d.sup.6, 300 MHz, ppm) .delta.
8.95 (d, J=4.6 Hz, 1 H), 8.57 (m, 1 H), 8.13 (d, J=8.3 Hz, 1 H),
7.88-7.81 (m, 4 H), 7.44-7.32 (m, 4 H), 7.17-7.10 (m, 4 H), 6.92
(t, J=7.4 Hz, 1 H), 5.20 (m, 1 H), 4.31 (m, 1 H), 3.46-3.27 (m, 2
H), 2.70 (m, 2 H), 2.22 (s, 3 H, Me), 1.59-1.32 (m, 3 H), 0.81 (m,
6 H); HPLC (gradient 8) 27.8 min and 28.1 min (1:1); FABMS, m/z 589
(C.sub.31H.sub.36N.sub.4O.sub.7 of M.sup.++1 requires 589)
EXAMPLE 61
Synthesis of Compound 1336
[0420] A. A slurry of 2,6-dichloro-3-nitropyridine (92%, 9.9 g, 47
mmol) and K.sub.2CO.sub.3 powder (6.5 g, 47 mmol) in methanol (100
mL) was stirred for a week at RT. The reaction was filtered and
concentrated. The residue was partitioned in ethyl acetate and 60%
sat. aq. NaHCO.sub.3. The organic solution was washed with 60k sat.
aq. NaHCO.sub.3 (2.times.), H.sub.2O, then sat. aq. NaCl, dried
(MgSO.sub.4) and concentrated to afford
2-chloro-6-methoxy-5-nitropyridine and
2-chloro-6-methoxy-3-nitropyridine (8.9 g, 100%) as a light yellow
solid: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 8.31 (d, 8.3 Hz, 1H),
8.28 (d, 8.9 Hz, 1H), 7.10 (d, 8.3 Hz, 1H), 6.82 (d, 8.9 Hz, 1H),
4.15 (s, 3H), 4.06 (s, 3H).
[0421] B. A mixture of 2-chloro-6-methoxy-5-nitropyridine and
2-chloro-6-methoxy-3-nitropyridine (8.9 g, 47 mmol), t-butyl methyl
malonate (10 mL, 60 mmol), and NaH (95%, 3.1 g, 120 mmol) in THF
(250 mL) was stirred at RT for 24 h. The reaction was concentrated
and the residue treated with trifluoroacetic acid (200 mL) for 2 h.
The reaction was concentrated and the product separated by flash
chromatography (silica gel, 95:5 hexane-ethyl acetate) to afford
methyl 6-(2-methoxy-3-nitro)pyridylacetate (3.3 g, 62%) as a yellow
oil: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) 8.27 (d, 8.0 Hz, 1H),
7.04 (d, 8.0 Hz, 1H), 4.09 (s, 3H), 3.85 (s, 2H), 3.75 (s, 3H).
[0422] C. A mixture of methyl 6-(2-methoxy-3-nitro)pyridylacetate
(0.047 g, 0.21 mmol) and 10% Pd on carbon (0.063 g) in ethyl
acetate (2 mL) and ethanol (1 mL) was stirred under H.sub.2 (40-50
psi) for 6 h. The mixture was filtered thru Celite and the filtrate
concentrated to afford methyl 6-(3-amino-2-methoxy)pyridylacetate
(0.041 g, 100%) as a light yellow oil: .sup.1H NMR (CDCl.sub.3, 300
MHz, ppm) 6.82 (d, 7.6 Hz, 1H), 6.65 (d, 7.6 Hz, 1H), 3.94 (s, 3H),
3.70 (s, 3H), 3.65 (s, 2H).
[0423] D. To a solution of methyl
6-(3-amino-2-methoxy)pyridylacetate (0.078 g, 0.33 mmol) and
triethylamine (50 mL, 0.36 mmol) in methylene chloride (1.0 mL) was
added o-tolyl isocyanate (41 .mu.L, 0.36 mmol). The reaction was
stirred for 4 h then concentrated. The crude product was purified
by flash chromatography (silica gel, 3:2 hexane-ethyl acetate) to
afford the Methyl, 6-(2-methoxy-3-o-tolylureido)pyridylacetate
(0.060 g, 55%) as a white powder: .sup.1H NMR (CDCl.sub.3, 3,00
MHz, ppm) 8.33 (d, 7.9 Hz, 1H), 7.51 (d, 7.8 Hz, 1H), 7.41 (s, 1H),
7.17 (m, 2H), 7.08 (m, 2H), 6.77 (d, 7.9 Hz, 1H), 3.81 (s, 3H),
3.71 (s, 3H), 3.67 (s, 2H), 2.20 (s, 3H).
[0424] E. A solution of methyl
6-(2-methoxy-3-o-tolylureido)pyridylacetate (0.023 g, 0.070 mmol)
in 30 methanol (1.0 mL) was treated with 2 M LiOH (90 .mu.L, 0.18
mmol). The reaction was stirred for 18 h, diluted with H.sub.2O
(5.0 mL) and washed with ether (2.times.). The aqueous solution was
then acidified with 5% aq. citric acid. The product was filtered
and washed with H.sub.2O then ether to give
6-(2-Methoxy-3-o-tolylureido)pyridylacetic acid (0.014 g, 64%) as a
white solid: .sup.1H NMR (CD.sub.3OD, 300 MHz, ppm) 8.50-8.25 (m,
3H), 7.60 (bd, 1H), 7.28-7.00 (m, 3H), 4.01 (s, 3H), 3.69 (s, 2H),
2.30 (s, 3H); MS, m/z 316.
[0425] F. Procedure C was performed using amine .beta.-2. The
resulting product was subjected to the conditions described in
Procedure D1 to provide TFA-amine salt 1336-1.
[0426] G. The procedure described in Example 1A was performed using
6-(2-methoxy-3-o-tolylureido)pyridylacetic acid (0.014 g, 0.044
mmol) and amine-TFA salt 1336-1 (0.017 g, 0.045 mmol) to afford
BIO1336 t-butyl ester (0.024 g, 796) as a white foam: .sup.1H NMR
(CDCl.sub.3, 300 MHz, ppm) 8.40 (d, 7.9 Hz, 1H), 7.63 (d, 8.3 Hz,
1H), 7.50 (d, 7.9 Hz, 1H), 7.43-7.06 (m, 6H), 6.80-6.67 (m, 4H),
5.92 (s, 2H), 5.19 (m, 1H), 4.47 (m, 1H), 3.91 (s, 3H), 3.61 (s,
3H), 2.65 (m, 2H), 2.31 (s, 3H), 1.58 (m, 3H), 1.31 (s, 9H).
[0427] H. To a solution of BIO1336 t-butyl ester. (0.024 g, 0.035
mmol) in methylene chloride (3.0 mL) was added trifluoroacetic acid
(3.0 mL). The reaction was stirred for 2 h then concentrated. The
crude product was purified by HPLC to afford BIO-1336 (0.011 g,
50%) as a white powder: .sup.1H NMR (CD.sub.3SOCD.sub.3, 300 MHz,
ppm) 8.73 (s, 1H), 8.52 (s, 1H), 8.47 (d, 8.3 Hz, 1H), 8.31 (d, 7.9
Hz, 1H), 8.11 (d, 8.3 Hz, 1H), 7.81 (d, 7.9 Hz, 1H), 7.21-7.09 (m,
2H), 7.00-6.70 (m, 5H), 5.98 (s, 2H), 5.08 (m, 1H), 4.36 (m, 1H),
3.97 (s, 3H), 3.52 (m, 2H), 2.64 (m, 2H), 2.25 (s, 3H), 1.55-1.25
(m, 3H), 0.81 (m, 6H); HPLC (Gradient B), 20.0 min; MS, m/z
620.
EXAMPLE 62
Synthesis of BIO-1382
[0428] A. To methyl 6-amino-2 (S)-N-BOC-aminohexanoate
hydrochloride salt (200 mg, 0.60 mmol) in CH.sub.2Cl.sub.2 (5 ml)
and TEA (basic to litmus) is added methanesulfonyl chloride (76.2
mg, 0.67 mmol) dropwise over 2 min. at rt. Following 1 hour of
stirring the reaction is diluted with CH.sub.2Cl.sub.2 (10 ml)
partitioned 3 times with 5% citric acid (3.times.0.5 ml), water
(1.times.1 ml), brine (1.times.1 ml), and dried over MgSO.sub.4.
The organic phase was concentrated in vacuo to yield 1382-1 (230
mg, 100%) as a clear oil.
[0429] .sup.1HNMR(CDCl.sub.3, 300 MHz, ppm) 7.26 (s, 5H), 5.58 (d,
1H, J=8), 5.02 (s,2H), 4.27 (m, 1H), 3.64 (s, 3H), 3.02 (m, 2H),
2.78 (s, 3H) 1.85-1.20 (m, 6H). HPLC (Gradient 3) 24.26 min. 98%
MS, mz 373.
[0430] B. To 1382-1 (225 mg, 0.60 mmol) in 10 ml MeOH at rt with
stirring is added 2N LiOH (0.91 ml, 1.8 mmol) dropwise over 2 min.
Stirring is continued overnight. The reaction mixture is acidified
with TFA (red to litmus) and concentrated in vacuo. The clear crude
gum was taken up in EtOAc (20 ml) and worked up as described in
Example 62A yielding 1382-2 (122 mg, 57%) as a clear gum.
.sup.1HNMR(CDCl.sub.3, 300 Mz, ppm), 7.33 (s, 4H), 5.54 (d, 1H
J=7.89), 4.39 (m, 1H), 3.47 (S, 3H), 3.09 (m, 2H), 1.92-1.28 (m,
6H). HPLC (Gradient 3) 19.23 min. (100%). MS, mz 359.
[0431] C. The procedure described in Example 1A was performed using
1382-2 (48 mg, 0.13 mmol) and amine .beta.-14 (25 mg, 0.09 mmol) to
give 1382-3 (51 mg, 62%).
[0432] .sup.1HNMR(CDCl.sub.3, 300 MHz, ppm), 7.97 (d, 2H J=7.38),
7.35 (m, 7H), 5.51 (m, 1H), 5.35 (dd, 1H J=5.77, 13.50), 5.09 (s,
2H), 4.75 (m, 1H), 4.14 (m, 1H), 3.88 (s, 3H), 3.62 (s, 3H), 3.09
(m, 2H), 2.73 (m, 2H), 1.92-1.77 (m, 1H), 1.70-1.55 (m, 1H),
1.55-1.49 (m, 2H), 1.49-1.15 (m, 13H).
[0433] D. The CBZ protecting group of compound 1382-3 was removed
under catalytic hydrogenation conditions as described in Procedure
D2 to give (13.2 mg, 35%) of product 1382-4. .sup.1HNMR(CDCl.sub.3,
300 MHz, ppm). 8.23-8.12 (m, 2H), 8.02-7.82 (m, 2H), 7.49-7.38 (m,
2H), 5.50-5.31 (m, 1H), 3.86 (s, 3H), 3.57 (s, 3H), 3.20-2.65 (m,
4H), 1.89-172 (m, 1H), 1.50-1.10 (m, 14H).
[0434] E. The procedure described in Example 49 was performed using
1382-4 (15.5 mg, 0.05 mmol) to give Bio 1382 t-butyl ester (22.6
mg, 111%) as a white solid.
[0435] .sup.1HNMR(CDCl.sub.3, 300 MHz, ppm). 8.02 (d, 1H J=8.1),
7.87 (d, 2H J=8.0), 7.59 (d, 1H J=8.1), 7.29-7.19 (m, 5H),
7.11-7.02 (m, 4H), 6.92 (t, 1H J=7.19), 5.25-5.16 (m, 1H),
4.20-4.30 (m, 1H), 3.8 (s, 3H), 3.39 (s, 2H), 2.86-2.73 (m, 5H),
2.68-2.58 (m, 2H), 2.17 (s, 3H), 1.65-1.18 (m, 15H). MS, mz
752.
[0436] F. Bio 1382 t-butyl ester (27.6 mg, 0.027 mmol) is stirred
in CH.sub.2Cl.sub.2 (1 ml) at 5.degree. C. TFA (1.0 ml) is added in
one portion; the ice bath is removed and stirring is continued for
2 hours. The reaction mixture is concentrated in vacuo and
subjected to preparative HPLC purification to provide BIO-1382 (14
mg, 75%) as a white solid. .sup.1HNMR(DMSO.sub.D6, 300 MHz, , ppm)
8.71 (d, 1H J=7.82), 8.21 (d, 1H J=8.01), 8.04-7.91 (m, 3H),
7.59-7.44 (m, 3H), 7.32-7.20 (m, 3H), 7.01-6.98 (m, 2H), 5.30 (dd,
1H J=7.50, 14.93) 4.35 (m, 1H), 3.93 (s, 3H), 3.84-3.62 (m, 2H),
3.09-3.45 (m, 2H), 2.99-2.78 (m, 6H), 2.32 (s, 3H) 1.75-1.15 (m,
6H). HPLC (Gradient 3) 27.8 min. (95%). MS, mz 696.
EXAMPLE 63
Synthesis of BIO-1400
[0437] A. To 4-phenyl-1-butene (3.47 g, 3.94 mL, 26 mmol) at RT was
added chlorosulfonyl isocynate (3.54 g, 2.17 mL, 25 mmol) under
argon. The resulting mixture was stirred overnight. The reaction
mixture was added dropwise to a rapidly stirring solution of
NaHCO.sub.3 (5 g), NaHSO.sub.3 (1.5 g) and
H.sub.2O/CH.sub.2Cl.sub.2 (15 mL/10 mL) at 0.degree. C. After 1 h,
the solution was concentrated under reduced pressure and the
residue was extracted with EtOAc (2.times.50 mL). After separation,
the organic layer was washed with sat. NaCl (30 mL), dried with
Na.sub.2SO.sub.4 and concentrated under reduced pressure to give
600 mg (14%) of the beta lactam 1400-1 as a light yellow oil:
.sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) .delta. 7.30-7.13 (m, 5 H,
Ar), 6.45 (s, 1H, NH), 3.0 (ddd, J=14.8, 4.7, 1.7 Hz, 1 H), 2.64
(t, J=7.6 Hz, 2 H), 2.52 (d, J=14.8 Hz, 1H), 1.92 (m, 2 H); TLC,
50% Hex/EtOAc, R.sub.f=0.27.
[0438] B. A solution of the beta lactam 1400-1 (500 mg, 2.86 mmol),
MeOH (25 mL), and HCl (1 mL of 33%) was stirred at RT for 18 h. The
reaction mixture was, diluted with EtOAc (100 mL) and basified with
Et.sub.3N until pH=9-10 (pH paper). The resulting solution was
washed with H.sub.2O (10 mL), Sat. NaHCO.sub.3 (30 mL), Sat. NaCl
(30 mL), dried with Na.sub.2SO.sub.4, and concentrated under
reduced pressure to give 270 mg (52%) of amine 1400-2 as a yellow
oil: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) .delta. 7.28-7.15 (m, 5
H, Ar), 3.66 (s, 3 H, OMe), 2.66 (m, 2 H), 2.48 (dd, J=15.7, 4.0
Hz, 1 H), 2.29 (dd, J=15.7, 8.8 Hz, 1 H), 1.70 (m, 2 H), 1.54 (s, 2
H, NH); TLC, 10% MeOH/CH.sub.2Cl.sub.2, R.sub.f=0.35; FABMS, m/z
207 (C.sub.12H.sub.17NO.sub.2 of M.sup.++1 requires 207).
[0439] C. Free amine 1400-2 (100 mg, 0.55 mmol)) was coupled with
N.alpha.-t-Boc-N.epsilon.-leu-N-hydroxysuccinimide (163 mg, 1.52
mmol) as described in Procedure C to give material which was
deprotected with trifluoroacetic acid (0.5 mL) and then basified
with Et.sub.3N as described in Procedure D1 to give the amine
1400-3 in 95% yield: .sup.1H NMR (CDCl.sub.3, 300 MHz, ppm) .delta.
9.02 (d, J=9.0 Hz, 1 H), 7.27-7.14 (m, 5 H, Ar), 4.26 (m, 1 H),
3.64, (s, two peaks, 3 H, OMe), 3.44 (m, 1 H), 2.79 (s, 2 H), 2.62
(t, J=7.8 Hz, 1 H), 2.54 (d, J=4.9 Hz, 1 H), 1.87 (m, 2 H), 1.68
(m, 2 H), 1.36 (m, 1 H), 0.92 (m, 6 H); TLC, 10%
MeOH/CH.sub.2Cl.sub.2, R.sub.f=0.47 and 0.18; HPLC (gradient 1)
12.2 min and 13.6 min (1:1); FABMS, m/z 321
(C.sub.18H.sub.28N.sub.2O.sub.3 of M.sup.++1 requires 321).
[0440] D. 2-Methylphenylureaphenylacetic acid (64 mg, 0.24 mmol)
was coupled with free amine 1400-3 (64 mg, 0.20 mmol) as described
in Example 49 to give compound 1400-4 in 60%: .sup.1H NMR
(DMSO-d.sup.6, 300 MHz, ppm) .delta. 9.50 (d, J=6.8 Hz, 1 H),
8.26-8.17 (m, 2 H), 7.97 (d, J=6.1 Hz, 1H), 7.84 (d, J=8.0 Hz, 1
H), 7.38 (m, 4 H), 7.27-7.09 (m, 9 H), 6.91 (t, J=7.3 Hz, 1 H),
4.26 (m, 1 H), 4.03 (m, 1 H), 3.52 (s, two peaks, 3 H, OMe), 3.38
(m, 2 H), 2.57-2.40 (m, 4 H), 2.25 (s, 3 H), 1.70-1.41 (m, 5 H),
0.86 (m, 6 H); FABMS, m/z 587 (C.sub.34H.sub.42N.sub.4O.sub.5 of
M.sup.++1 requires 587).
[0441] E. Compound 1400-4 (70 mg, 0.119 mmol) in DMSO (1 mL) and
MeOH (2 mL) was hydrolyzed with aqueous LiOH as described in
Example 1B. The, product was purified on a Vydac reverse-phase C18
column (22 mm.times.25 cm) using a linear gradient of 20%
CH.sub.3CN/H.sub.2O (0.1% TFA) to 50% CH.sub.3CN/H.sub.2O (0.1%
TFA) with a flow rate of 10 mL/min to give the BIO-1400 in 22%
isolated yield: .sup.1H NMR (DMSO-d.sup.6, 300 MHz, ppm) .delta.
8.93 (m, 1 H), 8.14 (m, 1 H), 7.91-7.81 (m, 3 H), 7.34 (m, 2 H),
7.27-7.09 (m, 9 H), 6.92 (t, J=7.4 Hz, 1 H), 4.27 (m, 1 H), 4.00
(m, 1 H), 3.43 (d, J=14.2 Hz, 1 H), 3.36 (d, J=14.2 Hz, 1 H),
2.60-2.30 (m, 4 H), 2.22 (s, 3 H), 1.68-1.55 (m, 3 H), 1.45 (t,
J=6.9 Hz, 2 H), 0.86 (m, 6 H); HPLC (gradient 1) 20 min and 20.5
min (1:2.45); FABMS, m/z 573 (C.sub.33H.sub.40N.sub.4O.sub.5 of
M.sup.++1 requires 573).
[0442] Conditions for analytical HPLC:
[0443] Gradient 1: a linear gradient of 20% CH.sub.3CN/H.sub.2O
(0.1% TFA) to 70% CH.sub.3CN/H.sub.2O (0.1% TFA).
[0444] Gradient 8: a linear gradient of 15% CH.sub.3CN/H.sub.2O
(0.1% TFA) to 40% CH.sub.3CN/H.sub.2O (0.1% TFA).
EXAMPLE 64
Synthesis of BIO 1051
[0445] A. 4-Aminobenzoic acid (420 mg, 3.1 mmol) in
CH.sub.2Cl.sub.2 was treated with phenyl isocyanate (340 .mu.l, 3.1
mmol) at RT. The reaction was stirred for 20 minutes .and then
concentrated. The residue was washed with 1N HCl then excess ether
to afford the product (98 mg, 12%) as a white powder. 1H NMR:
(CDCl.sub.3, 300 MHz, ppm), 9.08 (s, 1H), 8.80 (s, 1H), 7.90 (d,
2H), 7.58 (d. 2H), 7.45 (d, 2H), 7.30 (m, 2H), 7.00 (m, 1H).
FAB:257 (M+H)+, MW 256.26.
[0446] B. A solution of the amine from Example 6A (15 mg, 0.045
mmol) and the product from Example 64A (12 mg, 0.047 mmol) in DMF
was treated with DIPEA (40 .mu.l, 0.22 mmol) and BOP (20 mg, 0.045)
at RT. After the reaction was stirred overnight it was worked up as
in Example 1A to afford BIO-1051-OtBu (18 mg, 69%) as a foam.
[0447] C. BIO-1051-OtBu (19 mg, 0.031 mmol) was treated with TFA (2
mL) at RT for 30 min and then concentrated. The crude product was
purified by HPLC to afford BIO-1051 (6.3 mg, 39%) as a white
powder: HPLC (gradient A) 19.2 min, FAB: 517 (M+H)+, MW 516.3.
EXAMPLE 65
Synthesis of BIO-1110
[0448] A. The amine from Example. 6A (49 mg, 0.15 mmol) in
CH.sub.2Cl.sub.2 was treated with TFA (10 mL) at RT. The reaction
was stirred for 3 hours and concentrated. The residue was dissolved
in DMF and neutralized with triethylamine at RT. This was followed
by addition of 4-nitrophenylphenylisocyanate (26.5 mg, 0.16 mmol)
and stirred 1 hr. at RT. Purification by HPLC resulted in 62 mg of
a beige solid. .sup.1HNMR (CDCl.sub.3, 300 MHz, ppm): 8.05 (d, 2H),
7.25 (m, 5H), 5.35 (m, 1H), 4.34 (m, 1H), 2.22 (m, 2H), 1.59 (m,
3H), 0.84 (m, 6H). FAB: 442.9 (M+H).sup.+, MW 442.41. HPLC:
(Gradient A) 21.05 min.
[0449] B. The product of Example 65A (55 mg, 0.12 mmol) was reduced
with 10% Pd/C in MeOH while stirring under 40 psi hydrogen gas. The
reaction mixture was filtered through Celite 545 and concentrated
to yield 49 mg of a beige solid. .sup.1HNMR (CDCl3, 300 MHz, ppm):
7.19 (m, 5H), 7.03 (d, 2H), 6.94 (d, 2H), 5.27 (m, 1H), 4.23 (m,
2H), 2.72 (m, 2H), 1.52 (m, 3H), 0.78 (m, 6H). FAB: 413.3
(M+H).sup.+, MW 412.45. HPLC: (Gradient A) 11.93 min.
[0450] C. The product of Example 65B (5 mg, 0.012 mmol) in DMF and
triethylamine was treated with phenylisocyanate (1.4 mg, 0.12
mmol). After stirring overnight, the material was purified by HPLC.
1HNMR (CDCl.sub.3, 300 MHz, ppm): 7.55 (d, 2H), 7.36 (m, 12H), 7.04
(m, 1H), 6.34 (d, 1H), 5.36 (m, 1H), 4.41 (m, 1H), 2.78 (m, 2H),
1.39 (m, 3H), 0.91 (m, 6H). FAB: 532 (M+H).sup.+, MW 531.36. HPLC:
(Gradient A) 20.31 min.
EXAMPLE 66
Synthesis of BIO-1527
[0451] A. To a solution of amine .beta.-3 (1 equiv.) In
CH.sub.2Cl.sub.2 was added BOC-Pro-OSu (1 equiv.) and then stirring
at rt overnight. The resulting mixture was diluted with
ethylacetate and then washed with 5% citric acid (2.times.), sat.
aq NaHCO3 (2.times.) and brine (1.times.), dried
(Na.sub.2SO.sub.4), filtered and concentrated to give crude product
as a white foam. The above crude product was dissolved in
CH.sub.2Cl.sub.2 and TFA was-added at 0.degree. C. Mixture was
stirred at room temperature for 1 hour and concentrated to give the
amine as a TFA salt.
[0452] B. To a solution of 2-methylphenylureaphenylacetic acid in
DMF was added HOBT (1.5 equiv.) and EDC (1.2 equiv.) followed by
free amine from Example 66A and then stirred at room temperature
overnight. The resulting mixture was diluted with ethylacetate and
then washed with 5% citric acid (2.times.), sat. aq NaHCO.sub.3
(2.times.) and brine (1.times.), dried (Na.sub.2SO.sub.4), filtered
and concentrated to give methyl ester. The resulting methyl ester
was dissolved in methanol and then treated with 1N LiOH (aqueous
solution). The final product (carboxylic acid) was purified by
HPLC. The pure fraction from HPLC purification was collected and
dried to give Bio-1527.
[0453] Mass Spect: 573 (M+1), 595 (M+Na).
EXAMPLE 67
Inhibition of VLA4-Dependent Adhesion to RSA-CS1
[0454] This assay was used to assess the potency of VLA4-directed
inhibitory compounds of this invention.
1. Conjugation of CS1 to BSA
[0455] We dissolved BSA-SMCC (Pierce Chemical, Rockford, Ill.;
Catalog,# 77115) in H.sub.2O at a concentration of 10 mg/mL. [SEQ
ID
NO:4]:Cys-Tyr-Asp-Glu-Leu-Pro-Gln-Leu-Val-Thr-Leu-Pro-His-Pro-Asn-Leu-His-
-GIy-Pro-Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr ("Cys-Tyr-CS1 peptide"),
which we synthesized by conventional solid phase chemistry and
purified by HPLC, was dissolved in 10 mM HEPES pH 5, 50 mM NaCl and
0.1 mM EDTA also at a concentration of 10 mg/mL. We then mixed 500
.mu.L of BSA-SMCC, 250 .mu.L of Cys-Tyr-CS1 peptide and 75 .mu.L of
1 mM HEPES pH 7.5 and allowed the conjugation reaction to proceed
for 30 minutes. We stopped the reaction by adding 1 .mu.L of
beta-mercaptoethanol. Samples were analyzed for cross-linking by
SDS-PAGE. This reaction produced multiple molecules of the
Cys-Tyr-CS1 peptide conjugate to each BSA molecule.
2. Preparation of Plates for Adhesion Assay
[0456] We coated the wells of a Linbro titertek polystyrene 96-well
flat bottom plate (Flow Laboratories, Maclean, Va.; catalog
#76-231-05) with 100 .mu.L of the above-described BSA-CS1 solution
diluted to 1 .mu.g/mL in 0.05 M NaHCO.sub.3 (15 mM NaHCO.sub.3, 35
mM Na.sub.2CO.sub.3) pH 9.2. Some wells were not coated with CS1 in
order to assess non-specific cell binding (NSB). The plate was then
incubated overnight at 4.degree. C.
[0457] Following this incubation, the contents of the wells were
removed by inverting and blotting the plate. All of the wells were
then blocked with 100 .mu.L of 1% BSA in PBS, 0.02% NaN.sub.3, for
a minimum of one hour at room temperature.
3. Preparation of Fluorescently Labelled Ramos Cells
[0458] Ramos cells are grown, maintained and labelled in RPMI 1640
culture medium containing 1% BSA. Just prior to running the assay,
we added 2',7'-bis-(2-carboxyethyl)-5 (and -6) carboxyfluorescein
acetoxymethyl ester ("BCECF-AM"; Molecular Probes Inc., Eugene,
Oreg.; catalog #B-1150) to a final concnetration of 2 .mu.M to a
culture of Ramos cells (4.times.10.sup.6 cells/mL). We incubated
the cells for 20 minutes at 37.degree. C.
[0459] Following labelling, the cells were washed twice in assay
buffer (24 mM TRIS, 137 mM NaCl, 2.7 mM KCl, pH 7.4, containing
0.1% BSA and 2 mM glucose) to remove any cations originating from
the culture medium. The cells were then resuspended in assay buffer
to 4.times.10.sup.6 cells/mL and 2 mM MnCl.sub.2 was added to
upregulate VLA4 on the surface of the cells.
4. Running the Assay
[0460] Immediately prior to running the assay, we removed the BSA
blocking solution from the 96-well plates and washed the wells with
100 .mu.L of assay buffer. We then added to each well 25 .mu.L of
test compound at 2.times. the final concentration and 25 .mu.L of
the labelled Ramos cells. Final concentrations were selected across
a range of anticipated IC50s, usually between 0.01 nM-10 .mu.M.
Each concentration of compound was tested in triplicate. The
compound and cells are allowed to incubate for 30 minutes at room
temperature.
[0461] We then emptied the contents of the plate and washed the
wells 4 times with assay buffer. Using a light microscope, we
examined the the NSB wells. If more than a few cells are bound to
those wells, we washed the plate once more to remove the excess
non-specifically bound cells.
[0462] Binding of the Ramos cells to the CS1 peptide-coated wells
was measured by adding 100 .mu.L of assay buffer to each well and
quantitating fluorescence in a Millipore Cytofluor 2300 System
platereader set at 485 nm excitation and 530 nm emission. Binding
was expressed as an IC50--the concentration of inhibitor at which
50% of control binding occurs. Percent binding is calculated by the
formula:
[(F.sub.TB-F.sub.NS)-(F.sub.I-F.sub.NS)]/[(F.sub.TB-F.sub.NS).times.100=%
binding, where F.sub.TB is total fluorescence bound to
CS1-containing wells without added inhibitor; F.sub.NS is
fluorescence bound in wells lacking CS1; and F.sub.1 is
fluorescence bound in wells containing an inhibitor of this
invention.
[0463] Other compounds according to this invention were similarly
assayed. The IC50 for each of these compounds is indicated in the
table below: TABLE-US-00002 BIO # IC.sub.50 BIO # IC.sub.50 BIO #
IC.sub.50 BIO # IC.sub.50 1002 nd 1064 B 1122 C 1185 A 1003 nd 1065
B 1123 C 1186 B 1004 C 1066 nd 1124 nd 1187 C 1005 C 1067 B 1125 nd
1188 C 1006 B 1068 B 1126 C 1189 C 1007 C 1069 A 1127 B 1190 A 1008
C 1070 B 1128 B 1191 B 1009 C 1072 A 1129 B 1192 A 1010 B 1073 B
1130 B 1193 B 1011 C 1074 B 1131 B 1194 A 1013 nd 1075 B 1132 B
1195 A 1014 C 1076 B 1133 B 1196 A 1015 B 1077 B 1134 B 1197 A 1016
C 1078 B 1135 A 1198 C 1017 C 1079 A 1136 B 1199 B 1018 C 1080 B
1137 nd 1200 B 1020 C 1081 B 1138 B 1201 B 1021 B 1082 C 1139 B
1206 A 1022 C 1083 nd 1140 nd 1207 C 1023 B 1084 nd 1141 nd 1208 B
1024 C 1085 C 1142 nd 1209 C 1025 nd 1086 B 1143 C 1210 A 1026 C
1087 C 1144 B 1212 A 1027 C 1088 A 1145 B 1214 B 1028 B 1089 A 1146
B 1215 C 1029 C 1090 A 1147 B 1216 B 1030 C 1091 B 1148 C 1217 A
1031 C 1092 C 1149 C 1218 B 1032 C 1093 C 1150 C 1219 B 1036 B 1094
C 1152 nd 1220 B 1037 B 1096 C 1153 C 1221 A 1038 C 1097 B 1154 nd
1222 A 1039 B 1098 C 1155 nd 1223 nd 1040 B 1099 C 1156 nd 1224 A
1041 nd 1100 B 1157 C 1225 nd 1042 nd 1101 C 1158 B 1227 nd 1043 nd
1102 nd 1159 C 1238 A 1044 nd 1103 C 1160 B 1245 A 1045 nd 1104 B
1162 nd 1246 A 1046 C 1105 B 1163 B 1248 A 1047 nd 1106 C 1164 B
1270 A 1048 nd 1107 C 1168 B 1282 A 1049 B nd 1108 C 1169 B 1294 A
1050 A 1109 C 1170 B 1321 A 1051 nd 1110 B 1173 B 1327 B 1052 B
1111 C 1174 B 1336 A 1053 B 1112 C 1175 B 1360 A 1054 B 1113 C 1176
B 1380 B 1055 A 1114 C 1177 B 1382 A 1056 A 1115 B 1178 B 1390 B
1057 nd 1116 nd 1179 A 1396 B 1058 nd 1117 C 1180 B 1400 A 1060 B
1119 nd 1181 B 1272 A 1063 B 1120 nd 1182 B 1311 B 1319 B 1345 A
1347 A 1358 B 1361 A 1388 A 1393 A 1429 B 1444 B 1474 B 1475 B 1490
A 1515 A 1525 B 1526 B 1536 A 1594 B 1648 B 1655 B 1721 B 1725 nd
1726 nd 1727 nd 1728 nd 1729 nd 1730 nd 1731 nd 1732 nd Table
abbreviations: A - <50 nM; B - 50 nM-10 .mu.M: C - >10 .mu.M;
nd--not determined. All compounds tested in this table demonstrated
an IC.sub.50 <1 mM
EXAMPLE 68
Direct Binding of VLA4-Presenting Cells to VCAM-IgG
[0464] We next examined the ability of the compounds of this
invention to inhibit VCAM/VLA4 binding, utilizing a
VCAM-IgG-alkaline phosphatase conjugate. To carry out this assay,
we used the Millipore Multiscreen Assay System (Millipore Corp.,
Bedford, Mass.) to wash the cells efficiently.
1. Preparation of VCAM-IgG-AP Conjugates
[0465] The construction of VCAM 2D-IgG expression vectors,
transfection of CHO cells with those constructs and purification of
the resulting expression product is described in PCT publication WO
90/13300, the disclosure of which is herein incorporated by
reference.
[0466] 1.2 ml of purified VCAM 2D-IgG (5 mg/ml in 10 mM HEPES, pH
7.5) was reacted with 44 .mu.l of Traut's reagent (2-iminothiolane,
20 mg/ml in water; Pierce Chemical, Rockford, Ill.) at room
temperature for 30 minutes. The sample was desalted on a 15 ml
Sephadex G-25 column equilibrated with 100 mM NaCl, 1.0 mM MES, pH
5.0. One ml fractions were collected and absorbance at 280 nm was
determined. The two peak fractions were pooled.
[0467] One ml of calf intestinal alkaline phosphatase (19 mg/ml;
Pierce Chemical, Rockford, Ill.) was reacted with 100 .mu.l of
sulfo-SMCC (30 mg/ml in water) and 100 .mu.l 1 M HEPES, pH 7.5 for
35 minutes at room temperature. The reaction mix was desalted on a
12 ml Sephadex G-25 column equilibrated with 150 mM NaCl, 10 mM
HEPES, pH 6.0. One ml fractions were collected and absorbance at
280 nm was determined. The two peak fractions were pooled and
stored on ice.
[0468] The alkaline phosphatase-SMCC and VCAM 2D-IgG-iminothilane
adducts were cross-linked at a molar ratio of 2:1 in Tris-HCL, pH
7.5 by incubation at room temperature for 30 minutes. Extent of
cross-linking was determined by SDS-PAGE. The cross-linked products
were stabilized by the addition of 2 mM MgCl.sub.2 and 0.25 nM
ZnCl.sub.2 and stored at 4.degree. C.
2. Binding Assay
[0469] We first blocked a 96-well filtration plate for by adding
275 .mu.L of PBS containing 0.1% Tween 20 and 2% BSA ("blocking
buffer") to each well and incubating for 1 hour at room
temperature. The plate was then placed onto a vacuum manifold and
the blocking buffer was drained through the bottom of the
filtration wells into a waste collection tray. Then we washed the
wells three times with 200-250 .mu.L of Tris-buffered saline,
containing 0.1% BSA, 2 mM glucose and 1 mM HEPES, pH 7.5 ("assay
buffer") to wash out any remaining blocking buffer. We then drained
the plates and blotted them on paper towels to remove buffer on the
underside of the plate.
[0470] We then prepared a stock solution of VCAM-IgG-AP (4 .mu.g/mL
in assay buffer) and filtered it thorugh a 0.2.mu. low protein
binding syringe filter (Gelman Sciences, Ann Arbor, Mich. # 4454).
This solution was then diluted 1:10 in assay buffer and 25 .mu.L
was added to every well of the washed plate.
[0471] We diluted the cell adhesion inhibitor being tested to
2.times. final concentration in assay buffer and added 25 .mu.L of
each dilution to triplicate wells-in the plate. Final
concentrations used ranged from 0.01 nM-10 .mu.M. Control wells for
total binding and non-specific binding recieved 25 .mu.L of assay
buffer, instead of inhibitor. Total binding wells contained cells
and VCAM-IgG-AP in assay buffer. Non-specific binding wells
contained only VCAM-IgG-AP in assay buffer.
[0472] Jurkat cells were washed once in assay buffer to remove
growth medium and resuspended at 8.times.10.sup.6/mL in assay
buffer containing 2 mM MnCl.sub.2. We added 50 .mu.l of Jurkat
cells to every well, except the non-specific binding wells, which
received 50 .mu.L of assay buffer to maintain a final assay volume
of 100 .mu.L per well. We gently mixed the contents of the wells by
tapping the sides of the plate. The plate was then allowed to
incubate undisturbed for 60 minutes at room temperature.
[0473] At the end of the 60 minute incubation, we placed the plate
on the vacuum-manifold to drain the wells. We carefully added 100
.mu.L of assay buffer containing 1 mM MnCl.sub.2 (wash buffer) to
each well so as not to disturb the cells on the bottom. The wash
buffer was removed by vacuum and the plate was washed again with
150 .mu.L of wash buffer. After draining the wash buffer again, the
underside of the plate was blotted on paper towels.
[0474] Next, we prepared a 10 mg/mL solution of
4-nitrophenylphosphate in 0.1 M glycine, 1 mM ZnCl.sub.2, pH 10.5
(substrate buffer) and added 100 .mu.L immediately added to each
well. The plate was incubated for 30 minutes at room temperature to
allow the calorimetric reaction to proceed. We stopped the reaction
by adding 100 .mu.L of 3 N NaOH to each well.
[0475] The contents of the 96-well filtration plate was then
transferred directly into a 96-well flat bottom plate using the
vacuum manifold. The plate was read at a wavelength of 405 nm to
determine the amount of VCAM conjugate bound to the cells. Percent
binding is calculated by the formula:
[(A.sub.TB-A.sub.NS)-(A.sub.I-A.sub.NS)]/[(A.sub.TB-A.sub.NS).t-
imes.100=% binding, where A.sub.TB is the absorbance at 405 nm of
CS1-containing wells without added inhibitor; A.sub.NS is the
absorbance at 405 nm in wells lacking CS1; and A, is absorbance at
405 nm in wells containing an inhibitor of this invention
[0476] We assayed other compounds of this invention in the same
assay. The IC50 values are comparable to those derived from the CS1
binding assay described in the previous example, although certain
compounds demonstrated up to 10-fold greater binding in this assay
than in the previous assay.
EXAMPLE 69
Inhibition of Mouse Contact Hypersensitivity
[0477] We anesthetized 20-g female Balb/c mice (Jackson
Laboratories, Bar Harbor, Me.) with sodium pentobarbital (90 mg/kg,
i.p.). A 3 cm.sup.2 patch of abdominal skin was then exposed by
closely shaving the fur. The skin was then scrubbed with 70%
ethanol, followed by application of 25 .mu.L of 0.5% DNFB in 4:1
v/v acetone:olive oil onto the bare abdominal skin. We then lightly
scratched the skin with the applying pipet tip to encourage mild
inflammation. Twenty four hours after the initial sensitization we
again sensitized the mouse with 25 .mu.L of 0.5% DNFB at same
abdominal skin location, again followed by light scratching with
the pipet tip. The second sensitization was performed while
restraining the unanesthetized mouse.
[0478] On Day 5 (120 hours after the initial sensitization), we
anesthetized the mice with 90:10 mg/kg ketamine:xylazine, i.p. and
applied a sub-irritant dose of 10 .mu.L of 0.2% DNFB to the dorsal
surface of the left ear. The right ear received a similar
application of the 4:1 v/v acetone:olive oil vehicle.
[0479] Four hours after challenging the immune response, we
administered various concentrations of the inhibitors of this
invention to the mice in 100 .mu.L 0.5% sodium phosphate buffer, pH
8.8, and 3% v/v DMSO by subcutaneous (s.c.) injection. Less soluble
inhibitors occasionally required up to 30% DMSO addition the
highest concentrations tested. Groups of 8 mice were used for each
treatment tested. Positive (PS2 anti-mouse VLA-4 antibody, 8 mg/kg,
i.v.), and negative control (phosphate-buffered physiological
saline, PBS, 100 .mu.L i.v.; DMSO in PBS, 100 .mu.L s.c.).groups
were routinely tested for comparison as part of the assay of test
compounds.
[0480] Twenty four hours after challenge mice were again
anesthetized with ketamine:xylazine and the ear thickness of both
ears measured with an engineer's micrometer to an accuracy of
10.sup.-4 inches. The ear swelling response for each mouse was the
difference between its control- and DNFB-challenged ear thickness.
Typical uninhibited ear swelling responses were
65-75.times.10.sup.-4 in. Inhibition of the ear swelling response
was judged by comparison of treated groups with their negative
control group. Percent inhibition was calculated as: [ ( mean
.times. .times. .times. negative .times. .times. control .times.
.times. group .times. .times. ear .times. .times. swelling ) - (
mean .times. .times. test .times. .times. .times. group .times.
.times. .times. ear .times. .times. swelling ) mean .times. .times.
negative .times. .times. control .times. .times. group .times.
.times. ear .times. .times. swelling ] 100 ##EQU1## Statistical
significance of the difference among treatment groups was evaluated
using one-way analysis of variance followed by computation of the
Tukey-Kramer Honestly Significant Difference (JMP, SAS Institute)
using p<0.05.
[0481] The inhibitors of this invention cause a. statistically
significant reduction in the ear swelling response of DNFB-treated
mice as compared to uninhibited control animals.
EXAMPLE 70
Inhibition of Ascaris Antigen-Induced Late Phase Airway Sensitivity
in Allergic Sheep
[0482] Sheep which had previously been shown to develop both early
and late bronchial responses to Ascaris suum antigen were used in
this study. The protocol used for the experiment was that described
in W. M. Abraham et al., J. Clin. Invest., 93, pp. 776-87 (1994),
except that the VLA-4 inhibitors of this invention were
administered to the animals was dissolved in 3-4 ml of 50% aqueous
ethanol and delivered by aerosol spray.
[0483] The results showed that ail of the VLA-4 inhibitors of this
invention inhibited the airway responses associated with
administration of Ascaris suum antigen.
[0484] While we have hereinbefore presented a number of embodiments
of this invention, it is apparent that our basic construction can
be altered to provide other compounds and methods which utilize the
compounds of this invention. Therefore, it will be appreciated that
the scope of this invention is to be defined by the claims appended
hereto rather than the specific embodiments which have been
presented hereinbefore by way of example.
Sequence CWU 1
1
5 1 8 PRT Artificial Sequence Synthetically generated peptide 1 Glu
Ile Leu Asp Val Pro Ser Thr 1 5 2 5 PRT Artificial Sequence
Synthetically generated peptide 2 Glu Ile Leu Asp Val 1 5 3 5 PRT
Artificial Sequence Synthetically generated peptide 3 Leu Asp Val
Pro Ser 1 5 4 27 PRT Artificial Sequence Synthetically generated
peptide 4 Cys Tyr Asp Glu Leu Pro Gln Leu Val Thr Leu Pro His Pro
Asn Leu 1 5 10 15 His Gly Pro Glu Ile Leu Asp Val Pro Ser Thr 20 25
5 5 PRT Artificial Sequence Synthetically generated peptide 5 Arg
Cys Asp Xaa Cys 1 5
* * * * *