U.S. patent application number 09/960864 was filed with the patent office on 2003-02-06 for beta-sheet mimetics and methods relating to the use thereof.
This patent application is currently assigned to Molecumetics Ltd.. Invention is credited to Kahn, Michael S., Mathew, Jessymol, McMillan, Michael K., Ogbu, Cyprian O., Qabar, Maher N., Tulinsky, John E..
Application Number | 20030027819 09/960864 |
Document ID | / |
Family ID | 26724594 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030027819 |
Kind Code |
A1 |
Qabar, Maher N. ; et
al. |
February 6, 2003 |
Beta-sheet mimetics and methods relating to the use thereof
Abstract
.beta.-sheet mimetics and methods relating to the same are
disclosed. The .beta.-sheet mimetics have utility as protease and
kinase inhibitors, as well as inhibitors of transcription factors
and protein-protein binding interactions. Methods of the invention
include administration of a .beta.-sheet mimetic, or use of the
same for the manufacture of a medicament for treatment of a variety
of conditions associated with the targeted protease, kinase,
transcription factor and/or protein-protein binding
interaction.
Inventors: |
Qabar, Maher N.; (Redmond,
WA) ; McMillan, Michael K.; (Bellevue, WA) ;
Kahn, Michael S.; (Kirkland, WA) ; Tulinsky, John
E.; (Seattle, WA) ; Ogbu, Cyprian O.;
(Bellevue, WA) ; Mathew, Jessymol; (Bellevue,
WA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
Molecumetics Ltd.
Bellevue
WA
|
Family ID: |
26724594 |
Appl. No.: |
09/960864 |
Filed: |
September 21, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09960864 |
Sep 21, 2001 |
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09501052 |
Feb 9, 2000 |
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09501052 |
Feb 9, 2000 |
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09022934 |
Feb 12, 1998 |
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09501052 |
Feb 9, 2000 |
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08797915 |
Feb 10, 1997 |
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09501052 |
Feb 9, 2000 |
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08692420 |
Aug 5, 1996 |
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60047067 |
May 19, 1997 |
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Current U.S.
Class: |
514/224.2 ;
514/229.5; 514/299; 514/367; 514/373; 514/412; 514/434; 514/456;
514/469 |
Current CPC
Class: |
C07K 5/1024 20130101;
A61K 31/5025 20130101; C07K 1/047 20130101; A61K 31/428 20130101;
A61K 31/43 20130101; A61K 31/424 20130101; A61K 31/4196 20130101;
A61K 31/4035 20130101; A61K 31/407 20130101; C07K 5/021 20130101;
C07K 5/06191 20130101; C07K 5/0821 20130101; A61K 31/437 20130101;
C07K 7/06 20130101; C07K 5/06139 20130101; A61K 31/4152
20130101 |
Class at
Publication: |
514/224.2 ;
514/229.5; 514/299; 514/367; 514/373; 514/412; 514/434; 514/456;
514/469 |
International
Class: |
A61K 031/542; A61K
031/5383; A61K 031/4745; A61K 031/429; A61K 031/424 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 5, 1997 |
US |
PCT/US97/13622 |
Claims
What is claimed is:
1. A method for inhibiting a protease, comprising administering to
an animal in need thereof an effective amount of a compound having
the structure: 892and pharmaceutically acceptable salts thereof,
wherein A is selected from --C(.dbd.O)--, --(CH.sub.2).sub.0-4--,
--C(.dbd.O)(CH.sub.2).sub.1-3--, --(CH.sub.2).sub.1-2O-- and
--(CH.sub.2).sub.1-2S--; B is selected from N and CH; C is selected
from --C(.dbd.O)--, --C(.dbd.O)(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-3--, --O--, --S--, --O--(CH.sub.2).sub.1-2-- and
--S(CH.sub.2).sub.1-2--; D is selected from N and C(R.sub.4); E is
selected from 893F is an optional carbonyl moiety; R.sub.1 and
R.sub.4 are independently selected from amino acid side chain
moieties and derivatives thereof; R.sub.2 and R.sub.2' represent
one or more ring substituents individually selected from an amino
acid side chain moiety and derivatives thereof, or R.sub.2 taken
together with C or Y forms a fused substituted or unsubstituted
homocyclic or heterocyclic ring; R.sub.3 is selected from an amino
acid side chain moiety and derivatives thereof, or taken together
with C forms a bridging moiety selected from
--(CH.sub.2).sub.1-2--, --O-- and --S--; Y and Z represent the
remainder of the molecule; and any two adjacent CH groups of the
bicyclic ring may form a double bond.
2. The method of claim 1 wherein E is 894
3. The method of claim 1 wherein E is 895
4. The method of claim 1 wherein E is 896with the proviso that Z
does not contain an --NH-- moiety attached to the carbon atom
bearing the R.sub.1 substituent.
5. The method of claim 1 wherein the protease is a serine
protease.
6. The method of claim 5 wherein the serine protease is selected
from thrombin, Factor X, Factor IX, Factor VII, Factor XI,
urokinase, HCV protease, chymase, tryptase and kallikrein.
7. The method of claim 5 wherein the serine protease is
thrombin.
8. The method of claim 5 wherein the serine protease is Factor
VII
9. The method of claim 1 wherein the protease is selected from an
aspartic, cysteine and metallo protease.
10. A method for inhibiting a kinase, comprising administering to
an animal in need thereof an effective amount of a compound having
the structure: 897and pharmaceutically acceptable salts thereof,
wherein A is selected from --C(.dbd.O)--, --(CH.sub.2).sub.0-4--,
--C(.dbd.O)(CH.sub.2).sub.1-3--, --(CH.sub.2).sub.1-2O-- and
--(CH.sub.2).sub.1-2S--; B is selected from N and CH; C is selected
from --C(.dbd.O)--, --C(.dbd.O)(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-3--, --O--, --S--, --O--(CH.sub.2).sub.1-2-- and
--S(CH.sub.2).sub.1-2--; D is selected from N and C(R.sub.4); E is
selected from 898F is an optional carbonyl moiety; R.sub.1 and
R.sub.4 are independently selected from amino acid side chain
moieties and derivatives thereof; R.sub.2 and R.sub.2' represent
one or more ring substituents individually selected from an amino
acid side chain moiety and derivatives thereof, or R.sub.2 taken
together with C or Y forms a fused substituted or unsubstituted
homocyclic or heterocyclic ring; R.sub.3 is selected from an amino
acid side chain moiety and derivatives thereof, or taken together
with C forms a bridging moiety selected from
--(CH.sub.2).sub.1-2--, --O-- and --S--; Y and Z represent the
remainder of the molecule; and any two adjacent CH groups of the
bicyclic ring may form a double bond.
11. The method of claim 10 wherein E is 899
12. The method of claim 10 wherein E is 900
13. The method of claim 10 wherein E is 901with the proviso that Z
does not contain an --NH-- moiety attached to the carbon atom
bearing the R.sub.1 substituent.
14. The method of claims 10 wherein the kinase is a
serine/threonine or tyrosine kinase.
15. A method for inhibiting a transcription factor, comprising
administering to an animal in need thereof an effective amount of a
compound having the structure: 902and pharmaceutically acceptable
salts thereof, wherein A is selected from --C(.dbd.O)--,
--(CH.sub.2).sub.0-4--, --C(.dbd.O)(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.1-2O-- and --(CH.sub.2).sub.1-2S--; B is selected
from N and CH; C is selected from --C(.dbd.O)--,
--C(.dbd.O)(CH.sub.2).sub.1-3--- , --(CH.sub.2).sub.0-3--, --O--,
--S--, --O--(CH.sub.2).sub.1-2-- and --S(CH.sub.2).sub.1-2--; D is
selected from N and C(R.sub.4); E is selected from 903F is an
optional carbonyl moiety; R.sub.1 and R.sub.4 are independently
selected from amino acid side chain moieties and derivatives
thereof; R.sub.2 and R.sub.2.sup.1 represent one or more ring
substituents individually selected from an amino acid side chain
moiety and derivatives thereof, or R.sub.2 taken together with C or
Y forms a fused substituted or unsubstituted homocyclic or
heterocyclic ring; R.sub.3 is selected from an amino acid side
chain moiety and derivatives thereof, or taken together with C
forms a bridging moiety selected from --(CH.sub.2).sub.1-2--, --O--
and --S--; Y and Z represent the remainder of the molecule; and any
two adjacent CH groups of the bicyclic ring may form a double
bond.
16. The method of claim 15 wherein E is 904
17. The method of claim 15 wherein E is 905
18. The method of claim 15 wherein E is 906with the proviso that Z
does not contain an --NH-- moiety attached to the carbon atom
bearing the R.sub.1 substituent.
19. The method of claim 15 wherein the ability of the transcription
factor to bind DNA is controlled by reduction of a cysteine residue
by a cellular oxidoreductase.
20. The method of claim 15 wherein the transcription factor is
selected from NF-.kappa.B, AP-1, Myb, GRE, STAT-1 through -6, NFAT,
IRF-1 and MAF.
21. The method of claim 15 wherein the transcription factor is
NF-.kappa.B.
22. The method of claim 15 wherein the transcription factor is
AP-1.
23. The method of claim 19 wherein the cellular oxidoreductase is
ref-1.
24. The method of claim 15 wherein the warm-blooded animal has been
diagnosed with, or is at risk of developing, a condition selected
from Crohn's disease, asthma, rheumatoid arthritis,
ischemia-reperfusion injury, GVHD, ALS, Alzheimer's disease,
allograft rejection, adult T-cell leukemia, cancer and inflammatory
bowel disease.
25. A method for inhibiting protein-protein binding interactions,
comprising administering to an animal in need thereof an effective
amount of a compound having the structure: 907and pharmaceutically
acceptable salts thereof, wherein A is selected from --C(.dbd.O)--,
--(CH.sub.2).sub.0-4--, --C(.dbd.O)(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.1-2O-- and --(CH.sub.2).sub.1-2S--; B is selected
from N and CH; C is selected from --C(.dbd.O)--,
--C(.dbd.O)(CH.sub.2).sub.1-3--- , --(CH.sub.2).sub.0-3--, --O--,
--S--, --O--(CH.sub.2).sub.1-2-- and --S(CH.sub.2).sub.1-2--; D is
selected from N and C(R.sub.4); E is selected from 908F is an
optional carbonyl moiety; R.sub.1 and R.sub.4 are independently
selected from amino acid side chain moieties and derivatives
thereof; R.sub.2 and R.sub.2' represent one or more ring
substituents individually selected from an amino acid side chain
moiety and derivatives thereof, or R.sub.2 taken together with C or
Y forms a fused substituted or unsubstituted homocyclic or
heterocyclic ring; R.sub.3 is selected from an amino acid side
chain moiety and derivatives thereof, or taken together with C
forms a bridging moiety selected from --(CH.sub.2).sub.1-2--, --O--
and --S--; Y and Z represent the remainder of the molecule; and any
two adjacent CH groups of the bicyclic ring may form a double
bond.
26. The method of claim 25 wherein E is 909
27. The method of claim 25 wherein E is 910
28. The method of claim 25 wherein E is 911with the proviso that Z
does not contain an --NH-- moiety attached to the carbon atom
bearing the R.sub.1 substituent.
29. The method of claim 25 wherein the protein-protein binding
interaction is between the SH2 domain or the PDZ domain and another
protein.
Description
CROSS-REFERENCE TO PRIOR APPLICATIONS
[0001] This application claims the benefit of PCT Application No.
PCT/US97/13622, filed August 4, 1997, and U.S. Provisional
Application No. 60/047,067, filed May 19, 1997, and is a
continuation-in-part of U.S. application Ser. No. 08/797,915, filed
Feb. 10, 1997, U.S. application Ser. No. 08/692,420, filed Aug. 5,
1996, and U.S. application Ser. No. 08/725,073, filed Oct. 2, 1996;
which are continuation-in-parts of U.S. application Ser. No.
08/624,690, filed Mar. 25, 1996; which is a continuation-in-part of
U.S. application Ser. No. 08/549,006, filed Oct. 27, 1995; which is
a continuation-in-part of U.S. application Ser. No. 08/410,518,
filed Mar. 24, 1995.
TECHNICAL FIELD
[0002] This invention relates generally to .beta.-sheet mimetics
and, more specifically, to .beta.-sheet mimetics which inhibit
biologically active peptides and proteins.
BACKGROUND OF THE INVENTION
[0003] The .beta.-sheet conformation (also referred to as a
N-strand conformation) is a secondary structure present in many
polypeptides. The .beta.-sheet conformation is nearly fully
extended, with axial distances between adjacent amino acids of
approximately 3.5 .ANG.. The .beta.-sheet is stabilized by hydrogen
bonds between NH and CO groups in different polypeptides strands.
Additionally, the dipoles of the peptide bonds alternate along the
strands which imparts intrinsic stability to the .beta.-sheet. The
adjacent strands in the .beta.-sheet can run in the same direction
(i.e., a parallel .beta.-sheet) or in opposite directions (i.e., an
antiparallel .beta.-sheet). Although the two forms differ slightly
in dihedral angles, both are sterically favorable. The extended
conformation of the .beta.-sheet conformation results in the amino
acid side chains protruding on alternating faces of the
.beta.-sheet.
[0004] The importance of .beta.-sheets in peptides and proteins is
well established (e.g., Richardson, Nature 268:495-499, 1977;
Halverson et al., J. Am. Chem Soc. 113:6701-6704, 1991; Zhang, J.
Biol. Chem. 266:15591-15596, 1991; Madden et al., Nature
353:321-325, 1991). The .beta.-sheet is important in a number of
biological protein-protein recognition events, including
interactions between proteases and their substrates, protein
kinases and their substrates or inhibitors, the binding of SH2
domain containing proteins to their cognate phosphotyrosine
containing protein targets, farnesyl transferase to its protein
substrates, and MHC I and II and their antigenic peptides, and has
been implicated in many disease states.
[0005] Inhibitors that mimic the .beta.-sheet structure of
biologically active proteins or peptides would have utility in the
treatment of a wide variety of conditions. For example, Ras, the
protein product of the ras oncogene, is a membrane bound protein
involved in signal transduction regulating cell division and
growth. Mutations in the ras gene are among the most common genetic
abnormalities associated with human cancers (Barbacid, M. "ras
genes," 56:779-827, 1987). These mutations result in a growth
signal which is always "on," leading to a cancerous cell. In order
to localize to the cell membrane, Ras requires prenylation of the
cysteine within its C-terminal CaaX sequence by farnesyl
transferase (FTase). (In the sequence CaaX "a," is defined as an
amino acid with a hydrophobic side chain and "X" is another amino
acid.) This post-translational modification is crucial to its
activity. Peptidyl inhibitors of FTase with the sequence CaaX have
been shown to block or slow the growth of tumors in cell culture
and in whole animals (Kohl et al., "Selective inhibition of
ras-dependent transformation by a farnesyltransferase inhibitor,"
Science 260:1934-1937, 1993; Buss, J. E. & Marsters, Jr., J. C.
"Farnesyl transferase inhibitors: the successes and surprises of a
new class of potential cancer chemotherapeutics," Chemistry and
Biology 2:787-791, 1995).
[0006] SH2 domains, originally identified in the src subfamily of
PTKs, are noncatalytic sequences and consist of about 100 amino
acids conserved among a variety of signal transducing proteins
(Cohen et al., Cell 80:237-248, 1995). SH2 -domains function as
phosphotyrosine- binding modules and mediate critical
protein-protein associations (Pawson, Nature 573-580, 1995). In
particular, the role of SH2 domains has been clearly defined as
critical signal transducers for receptor tyrosine kinases (RTKs
such as EGF-R, PDGF, insulin receptor, etc.).
Phosphotyrosine-containing sites on autophosphorylated RTKs serve
as binding sites for SH2-proteins and thereby mediate the
activation of biochemical signaling pathways (Carpenter, G., FAESEB
J. 6:3283-3289, 1992; Sierke, S. and Koland, J., Biochem.
32:10162-10108, 1993). The SH2 domains are responsible for coupling
the activated growth-factor receptors to cellular responses which
include alterations in gene expression, cell proliferation,
cytoskeletal architecture and metabolism.
[0007] At least 20 cytosolic proteins have been identified that
contain SH2 domains and function in intracellular signaling. The
distribution of SH2 domains is not restricted to a particular
protein family, but is found in several classes of proteins,
protein kinases, lipid kinases, protein phosphatases,
phospholipases, Ras-controlling proteins and some transcription
factors. Many of the SH2-containing proteins have known enzymatic
activities while others (Grb2 and Crk) function as "linkers" and
"adapters" between cell surface receptors and downstream effector
molecules (Marengere, L., et al., Nature 369:502-505, 1994).
Examples of proteins it containing SH2 domains with enzymatic
activities that are activated in signal transduction include, but
are not limited to, the src subfamily of protein tyrosine kinases
(src (pp60.sup.c-src), abl, lck, fyn, fgr and others),
phospholipase-C-.gamma. (PLC-.gamma.), phosphatidylinositol
3-kinase (Pl-3-kinase), p21-ras GTPase activating protein (GAP) and
SH2 containing protein tyrosine phosphatases (SH-PTPase) (Songyang
et al., Cell 72:767-778, 1993). Intracellular tyrosines are
phosphorylated when surface receptors are engaged by diverse
ligands for growth factor receptors, cytokine receptors, insulin
receptor, and antigen-mediated signaling through T- or B-cell
receptors. The phosphorylation of proteins at tyrosine residues is
critical in the cellular signal transduction, neoplastic
transformation and control of the cell cycle. Due to the central
role these various SH2-proteins occupy in transmitting signals from
activated cell surface receptors into a cascade of additional
molecular interactions that ultimately define cellular responses,
inhibitors which block specific SH2-protein binding are desirable
as agents for a variety of potential therapeutic applications.
[0008] Disease areas in which tyrosine phosphorylation and
inhibition of SH2 binding represent targets for drug development
include the following:
[0009] Cancer:
[0010] SH2 domains which mediate signaling are clearly significant
elements in the regulation of oncogene and protooncogene tyrosine
kinase activity and cellular proliferation (Carpenter, Fed. Am.
Soc. Exp. Biol. J. 6:3283-3289, 1992). The SH2 domains define an
important set of substrates through which activated RTKs mediate
signaling and through which nonreceptor tyrosine kinases associate
with RTKs and are thus targets for anticancer drug development. The
ability to block interaction of the RTK with the SH2-containing
substrate using a mimetic inhibitor provides a means to abrogate
signaling and thereby eliminate oncogenic activity. The biological
significance is also illustrated by the v-crk oncogene, a protein
composed almost entirely of SH domains, which is able to bring
about cellular transformation by interacting with phosphotyrosine
containing proteins. As above, the ability of inhibitors to block
v-crk binding via its SH2 domain to other proteins would be
expected to be effective as an anticancer agent.
[0011] Immune Regulation:
[0012] Regulation of many immune responses is mediated through
receptors that transmit signals through tyrosine kinases containing
SH2 domains. T-cell activation via the antigen specific T-cell
receptor (TCR) initiates a signal transduction cascade leading to
lymphokine secretion and cell proliferation. One of the earliest
biochemical responses following TCR activation is an increase in
tyrosine kinase activity. In particular, T-cell activation and
proliferation is controlled through T-cell receptor mediated
activation of p56.sup.lck and p59.sup.fyn tyrosine kinases, as well
as ZAP-70 and Syk (Weiss and Litman, Cell 76:263-274, 1994) which
contain SH2 domains. Additional evidence indicates that several
src-family kinases (lck, blk, fyn) participate in signal
transduction pathways leading from B-cell antigen receptors and
hence may serve to integrate stimuli received from several
independent receptor structures. Thus, inhibitors that block
interactions of these SH2 domain kinases with their cognate
receptors could serve as immunosuppressive agents with utility in
autoimmune diseases, transplant rejection or as anti-inflammatory
agents as well as anticancer drugs in cases of lymphocytic
leukemias.
[0013] Additionally, non-transmembrane PTPase containing SH2
domains are known and nomenclature refers to them as SH-PTP1 and
SH-PTP2 (Neel, Cell Biology 4:419-432, 1993) SH-PTP1 is identical
to PTP1C, HCP or SHP and SH-PTP2 is also known as PTP1D or PTP2C.
SH-PTP1 is expressed at high levels in hematopoietic cells of all
lineages and all stages of differentiation. Since the SH-PTP1 gene
was identified as responsible for the motheaten (me) mouse
phenotype, this provides a basis for predicting the effects of
inhibitors that would block its interaction with its cellular
substates. Thus, inhibition of SH-PTP1 function would be expected
to result in impaired T-cell responses to mitogenic stimulation,
decreased NK cell function, and depletion of B-cell precursors with
potential therapeutic applications as described above.
[0014] Diabetes: In Type 2 (non-insulin dependent) diabetes,
tyrosine phosphatases (SH-PTP2) counter-balance the effect of
activated insulin-receptor kinases and may represent important drug
targets. In vitro experiments show that injection of PTPase blocks
insulin stimulated-phosphorylation of tyrosyl residues on
endogenous proteins. Thus, inhibitors could serve to modulate
insulin action in diabetes.
[0015] Neural Regeneration:
[0016] Glial growth factors are ligands that are specific
activators of erb-B2 receptor tyrosine kinase (p185.sup.erbB2) to
promote tyrosine phosphorylation and mitogenic responses of Schwann
cells. Consequently, regulation of tyrosine phosphorylation by
altering activity in Schwann cells following nerve injury could be
an important therapeutic strategy. Inhibitors of erb-B2 signaling
activity could have a significant role in treatment of tumors of
glial cell origin.
[0017] Another class of .beta.-sheet mimetics are inhibitors of
protein kinases, which include the protein tyrosine kinases and
serine/threonine kinases.
[0018] A wide variety of cellular substrates for polypeptide growth
factor receptors that possess intrinsic tyrosine kinase activity
have now been characterized. Although there is a tremendous
diversity among the numerous members of the receptors
tyrosine-kinases (RTK) family, the signaling mechanisms used by
these receptors share many common features. Biochemical and
molecular genetic studies have shown that binding of the ligand to
the extracellular domain of the RTK rapidly activates the intrinsic
tyrosine kinase catalytic activity of the intracellular domain. The
increased activity results in tyrosine-specific phosphorylation of
a number of intracellular substrates which contain a common
sequence motif. Consequently, this causes activation of numerous
downstream signaling molecules and a cascade of intracellular
pathways that regulate phospholipid metabolism, arachidonate
metabolism, protein phosphorylation (involving other protein
kinases), calcium mobilization and transcriptional regulation. The
growth-factor-dependent tyrosine kinase activity of the RTK
cytoplasmic domain is the primary mechanism for generation of
intracellular signals that initiate multiple cellular responses.
Thus, inhibitors which would serve as alternate substrates or
inhibitors of tyrosine kinase activity have the potential to block
this signaling.
[0019] Many of the RTK subfamilies are recognizable on the basis of
architectural similarities in the catalytic domain as well as
distinctive motifs in the extracellular ligand binding regions.
Based upon these structural considerations, a nomenclature defining
several subfamilies of RTKs, each containing several members, has
been developed (Hanks, Curr. Opin. Struc. Biol. 1:369-383, 1991;
Ullrich, A., and Schlessinger, J. Cell 61:203-212, 1990). Examples
of receptor subfamilies referred to on the basis of their
prototypic members include: EGF-receptor, insulin receptor,
platelet-derived growth factor (PDGF-receptor), fibroblast growth
factor receptors (FGFRs), TRK receptor and EPH/ECK receptors.
Members in each of these subfamilies represent molecular targets
for the development of mimetic inhibitors that would block tyrosine
kinase activity and prevent intracellular signal transduction.
Several therapeutic areas in which these targets have value are
identified below.
[0020] Cancer:
[0021] In addition to mediating normal cellular growth, members of
the EGFR family of RTKs are frequently overexpressed in a variety
of aggressive epithelial carcinomas and this is thought to directly
contribute to malignant tumor development. A number of studies have
shown that the EGFR is frequently amplified in certain types of
tumors, including glioblastomas, squamous carcinomas, and brain
tumors (Wong et al., Proc. Natl. Acad Sci USA 84:6899-6903, 1987).
Additionally, HER2/p185.sup.erbB2 (alternatively referred to as
"neu" in the rat), HER3/p160.sup.erbB3 HER4/p180.sup.erba4
(Plowman, G. et al., Proc. Natl. Acad. Sci. USA 90:1746-1750 (1993)
are three RTKs which have extensive amino acid sequence homology to
the EGFR. HER2/p185.sup.erbB2 is frequently amplified and
overexpressed in human breast tumors and ovarian carcinomas (Wong
et al., Proc. Natl. Acad. Sci. USA 84:6899-6903, 1987), and this
amplification is correlated with poor patient prognosis.
Simultaneous overexpression of p185.sup.neu and the EGFR
synergistically transforms rodent fibroblasts and this condition is
often observed in human cancers. Finally, HER3 expression is
amplified in a variety of human adenocarcinomas. Several inhibitors
are known which demonstrate inhibitory activity in vitro against
the EGFR and block EGF-dependent cell proliferation which indicates
therapeutic potential of compounds with this activity. In addition,
in human chronic myelogenous leukemia, enhanced tyrosine kinase
activity underlies the disease as a consequence of activation of
the cellular c-abl protooncogene. Inhibitors would function as
anticancer agents.
[0022] Angiogenesis:
[0023] Currently, there are at least seven FGFR members which
mediate a diverse array of biological responses, including the
capacity to induce angiogenesis. In addition, a group of RTKs with
seven lgLs has been proposed to represent a separate subfamily. Its
known members, FLT1, FLK1 and FLT4 show a similarity of structure
and expression. These receptors mediate the actions of Vascular
Endothelial Growth Factor (VEGF). Several lines of evidence
indicate that this subfamily of growth factor receptors play an
important role in the formation of blood vessels. Since blood
vessel formation is a process reactivated by tumors in order to
supply oxygen to these cells, .beta.-strand mimetics that inhibit
these growth factors' kinase activities could serve to suppress
tumor growth through inhibition of angiogenesis.
[0024] Restenosis:
[0025] The PDGF receptor is of great interest as a target for
inhibition in the cardiovascular field since it is believed to play
a significant role in restenosis after coronary balloon
angioplasties and also in atherosclerosis. The release of PDGF by
platelets at damaged surfaces of blood vessels results in
stimulation of PDGF receptors on vascular smooth muscle cells, and
eventual neointimal thickening. A mimetic inhibitor of kinase
activity would prevent proliferation and lead to greater successful
outcomes from this surgical procedure.
[0026] Many components of signal transduction pathways involve
phosphorylation of serine/threonine (ser/thr) residues of protein
substrates. Some of these substrates are themselves protein kinases
whose activity is modulated by phosphorylation. Two prominent
ser/thr-specific protein kinases play a central role in signal
transduction: cyclic AMP-dependent protein kinase A (PKA) and the
protein kinase C (PKC family). Numerous other serine/threonine
specific kinases, including the family of mitogen-activated protein
(MAP) kinases serve as important signal transduction proteins which
are activated in either growth-factor receptor or cytokine receptor
signaling. Other protein ser/thr kinases important for
intracellular signaling are Calcium-dependent protein kinase
(CaM-kinase II) and the c-raf-protooncogene.
[0027] PKC plays a crucial role in cell-surface signal transduction
for controlling a variety of physiological processes (Nishizuka,
Nature 334:661-665, 1988) and represents a large family of
isoenzymes which differ in their structure and expression in
different tissues, as well as their substrate specificity (Hug and
Sarre, Biochem J. 291:329-343, 1993) Molecular cloning has
demonstrated at least 8 isoenzymes. Due to this diversity and
differential expression, activation of individual isoenzymes
produces differing cell-specific responses: stimulation of growth,
inhibition of differentiation, or induction of differentiation. Due
to its ability to stimulate cellular proliferation, it represents a
target for anticancer drug development (Powis, Trends in Pharm.
Sci. 12:188-194, 1991). Overexpression of PKC isoenzymes in
mammalian cells is correlated with enhanced expression of early
protooncogenes such as c-jun, c-fos, c-myc and one overexpressing
cell line gives rise to tumors in nude mice.
[0028] Therapeutic applications within the area of immune
regulation are evident since activation of T-cells by antigens
involves activation of PKC. Activated PKC subsequently activates a
branch of the signal cascade that is necessary for transcriptional
activation of NF-.kappa.B, production of IL-2, and ultimately,
T-cell proliferation. Inhibitors that block signaling through this
branch pathway have been shown to prevent T-cell activation. Thus,
mimetics that would function as inhibitors of PKC in T-cells would
block signaling and serve as possible immunosuppressants useful in
transplant rejection or as anticancer agents for lymphocytic
leukemias. Activators of PKC cause edema and inflammation in mouse
skin (Hennings et al., Carcinogenesis 8:1342-1346, 1987) and thus
inhibitors are also expected to serve as potent anti-inflammatory
compounds. Such anti-inflammatory activates would find use in
asthma, arthritis and other inflammatory mediated processes. In
addition, staurosporine and its analogs, UCN01 and CGP4125, which
have been characterized as potent PKC inhibitors in vitro, have
anti-tumor activity in animal models (Powis, Trends in Pharm. Sci.
12:188-194, 1991), and related compounds are being considered for
clinical trials.
[0029] With regard to protease inhibition, Cathepsin B is a
lysosomal cysteine protease normally involved in proenzyme
processing and protein turnover. Elevated levels of activity have
been implicated in tumor metastasis (Sloane, B. F. et al.,
"Cathepsin B and its endogenous inhibitors: the role in tumor
malignancy," Cancer Metastasis Rev. 9:333-352, 1990), rheumatoid
arthritis (Werb, Z. "Proteinases and matrix degradation," in
Textbook of Rheumatology, Keller, W. N.; Harris, W. D.; Ruddy, S.;
Sledge, C. S., Eds., 1989, W.B. Saunder Co., Philadelphia, Pa., pp.
300-321), and muscular dystrophy (Katunuma N. & Kominami E.,
"Abnormal expression of lysosomal cysteine proteinases in muscle
wasting diseases," Rev. Physiol. Biochem. Pharmacol. 108:1-20,
1987).
[0030] Calpains are cytosolic or membrane bound Ca++-activated
proteases which are responsible for degradation of cytoskeletal
proteins in response to changing calcium levels within the cell.
They contribute to tissue degradation in arthritis and muscular
dystrophy (see Wang K. K. & Yuen P. W., "Calpain inhibition: an
overview of its therapeutic potential," Trends Pharmacol. Sci.
15:412-419, 1994).
[0031] Interleukin Converting Enzyme (ICE) cleaves pro-IL-1 beta to
IL-1 beta, a key mediator of inflammation, and therefore inhibitors
of ICE may prove useful in the treatment of arthritis (see, e.g.,
Miller B. E. et al., "Inhibition of mature IL-1 beta production in
murine macrophages and a murine model of inflammation by WIN 67694,
an inhibitor of IL-1 beta converting enzyme," J. Immunol.
154:1331-1338, 1995). ICE or ICE-like proteases may also function
in apoptosis (programmed cell death) and therefore play roles in
cancer, AIDS, Alzheimer's disease, and other diseases in which
disregulated apoptosis is involved (see Barr, P. J.; Tomei, L. D.,
"Apoptosis and its Role in Human Disease," Biotechnol. 12:487-493,
1994).
[0032] HIV protease plays a key role in the life cycle of HIV, the
AIDS virus. In the final steps of viral maturation it cleaves
polyprotein precursors to the functional enzymes and structural
proteins of the virion core. HIV protease inhibitors were quickly
identified as an excellent therapeutic target for AIDS (see Huff,
J. R., "HIV protease: a novel chemotherapeutic target for AIDS," J.
Med. Chem. 34:2305-2314) and have already proven useful in its
treatment as evidenced by the recent FDA approval of ritonavir,
Crixivan, and saquinavir.
[0033] Hepatitis C virus (HCV) is the major cause of non-A and
non-B hepatitis in the world today. It is estimated to infect up to
50 million people. Currently there is no satisfactory treatment
available to halt the progression of this debilitating disease.
During the life cycle of the virus, a polyprotein of about 3000
amino acids is produced and is proteolytically cleaved by host and
viral proteases to produce the mature viral gene products. A serine
proteinase located within the HCV NS3 protein cleaves at four
specific sites to produce non-structural proteins considered
essential for viral replication. Hence, inhibitors of HCV protease
are attractive targets for drug design, and could be of great
therapeutic benefit. (Neddermann et al., Biol. Chem. 378:469-476,
1997.)
[0034] Angiotensin converting enzyme (ACE) is part of the
renin-angiotensin system which plays a central role in the
regulation of blood pressure. ACE cleaves angiotensin I to the
octapeptide angiotensin II, a potent pressor agent due to its
vasoconstrictor activity. Inhibition of ACE has proved
therapeutically useful in the treatment of hypertension (Williams,
G. H., "Converting-enzyme inhibitors in the treatment of
hypertension," N. Engl. J. Med. 319:1517-1525, 1989.
[0035] Collagenases cleave collagen, the major constituent of the
extracellular matrix (e.g., connective tissue, skin, blood
vessels). Elevated collagenase activity contributes to arthritis
(Krane S. M. et al., "Mechanisms of matrix degradation in
rheumatoid arthritis," Ann. N.Y. Acad. Sci. 580:340-354, 1990.),
tumor metastasis (Flug M. & Kopf-Maier P., "The basement
membrane and its involvement in carcinoma cell invasion," Acta
Anat. Basel 152:69-84, 1995), and other diseases involving the
degradation of connective tissue.
[0036] Trypsin-like serine proteases form a large and highly
selective family of enzymes involved in hemostasis/coagulation
(Davie, E. W. and K. Fujikawa, "Basic mechanisms in blood
coagulation," Ann. Rev. 799-829, 1975) and complement activation
(Muller-Eberhard, H. J., "Complement," Ann. Rev. Biochem.
44:697-724, 1975). Sequencing of these proteases has shown the
presence of a homologous trypsin-like core with amino acid
insertions that modify specificity and which are generally
responsible for interactions with other macromolecular components
(Magnusson et al., "Proteolysis and Physiological Regulation,"
Miami Winter Symposia 11:203-239, 1976).
[0037] Thrombin, a trypsin-like serine protease, acts to provide
limited proteolysis, both in the generation of fibrin from
fibrinogen and the activation of the platelet receptor, and thus
plays a critical role in thrombosis and hemostasis (Mann, K. G.,
"The assembly of blood clotting complexes on membranes," Trends
Biochem. Sci. 12:229-233, 1987). Thrombin exhibits remarkable
specificity in the removal of fibrinopeptides A and B of fibrinogen
through the selective cleavage of only two Arg-Gly bonds of the
one-hundred and eighty-one Arg- or Lys-Xaa sequences in fibrinogen
(Blomback, H., Blood Clotting Enzymology, Seeger, W. H. (ed.),
Academic Press, New York, 1967, pp. 143-215).
[0038] Many significant disease states are related to abnormal
hemostasis, including acute coronary syndromes. Aspirin and heparin
are widely used in the treatment of patients with acute coronary
syndromes. However, these agents have several intrinsic
limitations. For example, thrombosis complicating the rupture of
atherosclerotic plaque tends to be a thrombin-mediated,
platelet-dependent process that is relatively resistant to
inhibition by aspirin and heparin (Fuster et al., "The pathogenesis
of coronary artery disease and the acute coronary syndromes," N.
Engl. J. Med. 326:242-50, 1992).
[0039] Thrombin inhibitors prevent thrombus formation at sites of
vascular injury in vivo. Furthermore, since thrombin is also a
potent growth factor which initiates smooth muscle cell
proliferation at sites of mechanical injury in the coronary artery,
inhibitors block this proliferative smooth muscle cell response and
reduce restenosis. Thrombin inhibitors would also reduce the
inflammatory response in vascular wall cells (Harker et al., Am. J.
Cardiol. 75:12B-16B, 1995).
[0040] Furthermore, at least two well-defined transcription
factors, nuclear factor (NF) .kappa.B and activator protein (AP)
-1, are regulated by the intracellular reduction-oxidation (redox)
state. The regulation of gene expression by the redox state holds
promising therapeutic implications. For example, binding sites of
the redox-regulated transcription factors NF-.kappa.B and AP-1 are
located in the promoter region of a large variety of genes that are
directly involved in the pathogenesis of diseases, such as AIDS,
cancer, atherosclerosis and diabetic complications (Sen and Packer,
FASEB Journal 10:709-720, 1996). More specifically, the binding of
transcription factors such NF-.kappa.B and AP-1 to consensus sites
on DNA is driven by oxidant-antioxidant homeostasis, especially by
the thiol-disulfide balance.
[0041] In the case of NF-.kappa.B, a physiologically relevant thiol
that plays a crucial role in the regulation of NF-.kappa.B function
is reduced thioredoxin or a reduced thioredoxin-like protein.
Thioredoxin is an important protein oxidoreductase with antioxidant
functions. Thioredoxin has been found to upregulate DNA binding of
activated NF-.kappa.B and thus augments gene expression (Schenk et
al., Proc. Natl. Acad. Sci. USA 91:1672-1676, 1994). Thioredoxin
has been implicated in reducing activated cytosolic NF-.kappa.B
(specifically reduction of cys-62), which may thus contribute to
its nuclear translocation and DNA binding (Hayashi et at., J. Blol.
Chem. 268:11380-11388, 1993).
[0042] DNA binding activity of Fos and Jun in the AP-1 complex has
also been found to be regulated by the redox state (Abate et al.,
Science 249:1157-1162, 1990). Each protein contains a single
conserved cysteine (flanked by lysine and arginine) in its DNA
binding domain. This thiol does not appear to be part of a
disulfide bond and may exist as a sulfenic or sulfinic acid in its
oxidized form. Ref-1, a bifunctional nuclear protein also
possessing endonuclease DNA repair activity, stimulates AP-1 DNA
binding by reduction of this regulatory cysteine. A Fos mutant in
which the critical cysteine was replaced with serine elicited a
three-fold increase in AP-1 DNA binding activity and was no longer
subject to redox control (Okuno et al., Oncogene 8:695-701, 1993).
Hence, since at least four members of the fos family, 3 of the jun
family, and at least 4 of the ATF/CREB family of transcription
factors all contain this conserved cysteine, redox control of
transcription factors appears widespread.
[0043] As mentioned above, the regulation of transcription factors
such as NF-.kappa.B and AP-1 have important therapeutic
implications. For example, AP-1 is an important mediator of tumor
production (Yoshioka et al., Proc. Natl. Acad. Sci. USA
92:4972-4976, 1995). Thus, compounds that repress AP-1
transcriptional activity have utility in the treatment of cancer.
Furthermore, due to its direct role in regulating responses to
inflammatory cytokines and endotoxins, the activation of
NF-.kappa.B plays an important role in the development of chronic
diseases such as rheumatoid arthritis and acute conditions such as
septic shock. Autoimmune diseases, such as systemic lupus
erythromatus (SLE), and Alzheimer's disease are also believed
involved in activation of NF-.kappa.B. Similarly, NF-.kappa.B plays
an important role in the activation of HIV gene expression. Further
conditions which are believed to involve NF-.kappa.B include the
flu, atherosclerosis, oncogenesis and ataxia telangiectasia
(AT).
[0044] Proteins containing PDZ domains constitute an additional
potential target for b-sheet mimetics. These domains of 80-100
amino acid residues mediate protein-protein interactions by binding
to a consensus X-Ser/Thr-X-Val sequence at the very carboxyl
terminus of proteins. There are also examples of protein
interactions via PDZ domains that are internal (or non C-terminal).
The crystal structure of liganded and unliganded PDZ domains have
been determined and show a six b-strand and two a-helix structure
that binds the consensus recognition polypeptide sequence through a
b-sheet conformation. Hence, screening of appropriate b-sheet
mimetics should prove a valid strategy for targeting PDZ
domain-containing proteins. The targets of PDZ domain-containing
proteins are varied but important in signal transduction. PSD-95, a
membrane associated guanylate kinase contains three PDZ domains,
two of which target the Shaker-type K.sup.+ channel and the
N-methyl-D-aspartate (NMDA) receptor resulting in their clustering
that is required for their function. PTPL1/FAP1, a protein tyrosine
phosphatase, has five PDZ domains, two of which interact with Fas,
a transmembrane protein of the tumor necrosis factor receptor
family, that mediates apoptosis in many cell types. Hence,
compounds targeting proteins containing the PDZ domains may prove
useful as anticancer agents.
[0045] Tryptase, a trypsin-like serine protease found exclusively
in mast cells, has attracted much interest due to its potential
role as a mediator of inflammation. For example, in the lung
tryptase is released along with other mediators of inflammation in
response to binding of an inhaled antigen to cell-surface IgE
receptors (Ishizaka and Ishizaka, Prog. Allergy 34:188-235, 1984).
Tryptase has also been shown to cleave vasoactive intestinal
peptide in vitro (Caughey et al., J. Pharmacol. Exp. Ther.
244:133-137, 1988; Tam and Caughey, Am. J. Respir. Cell Mol. Biol.
3:27-32, 1990). These results suggest that tryptase may increase
bronchoconstriction via proteolysis of bronchodilating peptides in
asthma patients. Consistent with this hypothesis is the recent
finding that synthetic tryptase inhibitors blocked airway responses
in allergic sheep (Clark et al., Am. J. Respir. Crit. Care Med.
152:2076-2083, 1995).
[0046] Tryptase activates extracellular matrix- degrading proteins
prostromelysin (pro-MMP-3) and procollagenase (pro-MMP-1) via
MMP-3, suggesting a role for the enzyme in tissue remodeling and
inflammation (Gruber et al., J. Clin. Invest. 84:8154-8158, 1989)
and therefore possibly in rheumatoid arthritis. Additionally,
prostromelysin, when activated, has been shown to degrade the
extracellular matrix around atherosclerotic plaques. Since
abnormally high levels of tryptase-containing mast cells have been
found in coronary atheromas, tryptase may play a role in
atheromatous rupture (release of the thrombus), the final event of
coronary atherosclerosis (Kaartinen et al., Circulation
90:1669-1678, 1994).
[0047] Other activities of tryptase include the following. Tryptase
cleaves fibrinogen but is not inactivated in the presence of
endogenous proteinase inhibitors (Schwartz et al., J. Immunol.
135:2762-2767, 1985; Ren et al., J. Immunol. 159:3540-3548, 1997),
and may function as a local anticoagulant. It has been demonstrated
to be a potent mitogen for fibroblasts and may be involved in
pulmonary fibrosis and interstitial lung disease (Ruoss et al., J.
Clin. Invest. 88:493-499, 1991). Tryptase may also be responsible
for the activation of PAR-2 (proteinase activated receptor-2) on
endothelial cells and keratinocytes (Molino et al., J. Biol. Chem.
272:4043-4049, 1997).
[0048] Given the central role of mast cells in allergic and
inflammatory responses, inhibition of tryptase may result in
significant therapeutic effects. Inhibitors of tryptase may be
useful for preventing or treating asthma, pulmonary fibrosis and
interstitial pneumonia, nephritis, hepatic fibrosis, hepatitis,
hepatic cirrhosis, scleroderma, psoriasis, atopic dermatitis,
chronic rheumatoid arthritis, influenza, Crohn's disease,
ulcerative colitis, inflammatory bowel disease, nasal allergy, and
atherosclerosis.
[0049] Chymase is a chymotrypsin-like protease that is also
released from mast cells. It has been demonstrated to cleave
angiotensin-I (ang-I) to angiotensin-II (ang-II) with greater
efficiency and selectivity than angiotensin-I converting enzyme
(ACE) (Okunishi et al., J. Hypertension 2: 227-284, 1984; Urata et
al., Circ. Res. 66: 883-890, 1990). In heart tissue chymase has
been shown to be a major source of ang-II production from ang-I
(Dell'Italia et al., Am. J. Physiol. (Heart Circ. Physiol. 38)
269:H2065-H2073, 1996). In addition, increased chymase activity has
been demonstrated in balloon-injury induced hypertrophied vessels
in dogs (Shiota et al., FEBS Lett. 323:239-242, 1993). Such
evidence suggests that inhibition of chymase may be therapeutic for
hypertension, ischaemic heart disease, and congestive heart
failure.
[0050] Urokinase-type plasminogen activator (uPA) is a trypsin-like
serine proteinase which converts plasminogen to plasmin as part of
the fibrinolytic system. It has long been used for thrombolysis in
acute massive pulmonary embolism. Other research has shown that uPA
is also a key initiator of the extra-cellular proteolytic cascade
involved in cellular invasiveness (Mullins and Rohlich, Biochim.
Biophys. Acta 695: 177-214, 1983; Testa and Quigly, Cancer Metast.
Rev. 9:353-367, 1990). In addition uPA binds to uPA receptor (uPAR)
through its growth factor domain and further modulates the activity
of other proteins involved in cell migration. Overexpression of uPA
appears to play a part in cancer invasiveness and metastasis; high
levels of uPA, PAI-1 (plasminogen activator inhibitor-1), and uPAR
correlate with poor patient prognosis. A variety of research in
various model systems demonstrates that inhibitors of uPA decrease
tumor cell invasiveness and metastasis (Testa and Quigly, ibid;
Andreasen et al., Int J Cancer 72:1-22, 1997). Hence, inhibition of
uPA may be useful in the treatment of breast cancer, prostate
cancer, ovarian cancer, human renal cell cancer, gastric cancer,
and lung cancer. Recent evidence indicates that inhibitors of uPA
may also be useful in the prevention of restenosis (Loskutoff,
Circulation 96:2772-2774, 1997).
[0051] In view of the important biological role played by the
.beta.-sheet, there is a need in the art for compounds which can
stabilize the intrinsic .beta.-sheet structure of a naturally
occurring or synthetic peptide, protein or molecule. There is also
a need in the art for making stable .beta.-sheet structures, as
well as the use of such stabilized structures to effect or modify
biological recognition events which involve .beta.-sheet
structures. The present invention fulfills these needs and provides
further related advantages.
SUMMARY OF THE INVENTION
[0052] Briefly stated, the present invention is directed to
.beta.-sheet mimetics and the use thereof, including use for the
manufacture of a medicament for achieving therapeutic effects in a
warm-blooded animal through one or more of protease inhibition,
kinase inhibition, regulation of a transcription factor and/or by
inhibiting protein-protein binding interactions. The therapeutic
effects result from administering to the warm-blooded animal a
therapeutically effective amount of a .beta.-sheet mimetic
including a bicyclic ring system, wherein the .beta.-sheet mimetic
has the general structure (I) (including pharmaceutically
acceptable salts thereof): 1
[0053] wherein
[0054] A is selected from --C(.dbd.O)--, --(CH.sub.2).sub.0-4--,
--C(.dbd.O)(CH.sub.2).sub.1-3--, --(CH.sub.2).sub.1-2O-- and
--(CH.sub.2).sub.1-2S--;
[0055] B is selected from N and CH;
[0056] C is selected from --C(.dbd.O)--,
--C(.dbd.O)(CH.sub.2).sub.1-3--, --(CH.sub.2).sub.0-3--, --O--,
--S--, --O--(CH.sub.2).sub.1-2-- and --S(CH.sub.2).sub.1-2--;
[0057] D is selected from N and C(R.sub.4);
[0058] E is selected from 2
[0059] F is an optional carbonyl moiety;
[0060] R.sub.1 and R.sub.4 are independently selected from amino
acid side chain moieties and derivatives thereof;
[0061] R.sub.2 and R.sub.2' represent one or more ring substituents
individually selected from an amino acid side chain moiety and
derivatives thereof, or R.sub.2 taken together with C or Y forms a
fused substituted or unsubstituted homocyclic or heteocyclic
ring;
[0062] R.sub.3 is selected from an amino acid side chain moiety and
derivatives thereof, or taken together with C forms a bridging
moiety selected from --(CH.sub.2).sub.1-2--, --O-- and --S--;
[0063] Y and Z represent the remainder of the molecule; and
[0064] any two adjacent CH groups of the bicyclic ring may form a
double bond.
[0065] In one embodiment where F (i.e., the optional carbonyl
moiety) is present and E is --N(Z)--, the compounds of this
invention include the following structure (II): 3
[0066] wherein A, B, C, D, R.sub.2, R.sub.2', R.sub.3, Y and Z are
as defined above with regard to structure (I).
[0067] In a preferred aspect of this embodiment, A is either
--C(.dbd.O)-- or --(CH.sub.2)-- and C is --(CH.sub.2).sub.2--, as
represented by the following structures (IIa) and (IIb) 4
[0068] In this embodiment; the six-member ring may be saturated or
unsaturated (including aromatic). For example, when B and D of
structures (IIa) and (IIb) are both --CH-- (and thus constitute
adjacent CH groups that may form a double bond), compounds of this
invention include the following aromatic structures (IIc) and
(IId): 5
[0069] Similarly, the following unsaturated compounds having
structures (IIe) and (IIf) are also representative of the compounds
of structures (IIa) and (IIb): 6
[0070] In another embodiment where F is present and E is
--C(R.sub.1)(NHZ)--, the compounds of this invention include the
following structure (III): 7
[0071] wherein A, B, C, D, R.sub.1, R.sub.2, R.sub.2', R.sub.3, Y
and Z are as defined above with regard to structure (I).
[0072] In a preferred aspect of this embodiment, A is either
--C(.dbd.O)-- or --(CH.sub.2)-- and C is --(CH.sub.2).sub.2--, as
represented by the following structures (IIIa) and (IIIb) 8
[0073] In this embodiment, the six-member ring may be saturated or
unsaturated (including aromatic). For example, when B and D of
structures (IIIa) and (IIIb) are both --CH-- (and thus constitute
adjacent CH groups that may form a double bond), compounds of this
invention include the following aromatic structures (IIIc) and
(IIId): 9
[0074] Similarly, the following unsaturated compounds having
structures (IIIe) and (IIf) are also representative of the
compounds of structures (IIIa) and (IIIb): 10
[0075] In a further embodiment when F is present and E is
--C(R.sub.1)(Z)--, the compounds of this invention include the
following structure (IV): 11
[0076] wherein A, B, C, D, R.sub.1, R.sub.2, R.sub.2', R.sub.3, Y
and Z are as defined above with regard to structure (I), and with
the proviso that Z does not contain an --NH-- moiety attached to
the carbon atom having the R.sub.1 substituent (and thus distinct
from the compounds of structure (III) above).
[0077] In a preferred aspect of this embodiment, A is either
--(C.dbd.O)-- or --(CH.sub.2)-- and C is --(CH.sub.2).sub.2--, as
represented by the following structures (IVa) and (IVb): 12
[0078] In this embodiment, the six-member ring may be saturated or
unsaturated (including aromatic). For example, when B and D of
structures (IVa) and (IVb) are both --CH-- (and thus constitute
adjacent CH groups that may form a double bond), compounds of this
invention include the following aromatic structures (IVc) and
(IVd): 13
[0079] Similarly, the following unsaturated compounds having
structures (IVe) and (IVf) are also representative of the compounds
of structures (IVa) and (IVb): 14
[0080] In a further embodiment where F is not present and E is
either --N(Z)--, --C(R.sub.1)(NHZ)-- or --C(R.sub.1)(Z)--, the
compounds of this invention include the following structures (V),
(VI) and (VII): 15
[0081] wherein A, B, C, D, R.sub.1, R.sub.2, R.sub.2', R.sub.3, Y
and Z are as defined above with regard to structure (I).
[0082] In still a further embodiment where R.sub.3 taken together
with C forms a bridging moiety, compounds of this invention include
the following structure (VIII): 16
[0083] where X is a bridging moiety selected from
--(CH.sub.2).sub.1-2--, --O-- and --S--, and A, B, C, D, E, F,
R.sub.2, R.sub.2', Y and Z are as defined above with regard to
structure (I).
[0084] In one aspect of this embodiment where F is present, A is
--C(.dbd.O)--, C is --(CH.sub.2).sub.2-- and E is either --N(Z)--
or --C(R.sub.1)(NHZ)--, compounds of this invention include those
of the following structures (VIIIa) and (VIIIb): 17
[0085] In yet a further embodiment where F is present, R.sub.2
taken together with C forms a substituted or unsubstituted,
homocyclic or heterocyclic fused ring as represented by structures
(IX) and (X): 18
[0086] wherein A, B, C, D, E, R.sub.2, R.sub.2', R.sub.3 and Y are
as defined above, and R' is one or more optional ring
substituents.
[0087] In one aspect of structure (IX), R.sub.2 and C taken
together form a fused five-, six-, seven- or eight-membered ring as
represented by structures (IXa) and (IXb): 19
[0088] wherein A, B, D, E, R.sub.2, R.sub.2', R.sub.3, R' and Y are
as defined above.
[0089] In one aspect of structure (X), R.sub.2 and C taken together
form a fused five-, six-, seven- or eight-membered ring as
represented by structures (Xa) and (Xb) 20
[0090] wherein A, B, C, D, E, Y, R.sub.2, R.sub.2', R.sub.3 and R'
are as defined above, and X is selected from --C(.dbd.O)--, --NH--,
--NR'--, --O-- and --S--.
[0091] In still a further embodiment where F is present, R.sub.2
taken together with Y forms a substituted or unsubstituted,
homocyclic or heterocyclic fused ring as represented by structure
(XI): 21
[0092] wherein A, B. C, D, E, R.sub.2, R.sub.2' and R.sub.3 are as
defined above.
[0093] In one aspect of this embodiment, R.sub.2 and Y taken
together form a fused five-, six-, seven- or eight-membered ring as
represented by structures (XIa) and (XIb): 22
[0094] wherein A, B, C, D, E, R.sub.2, R.sub.2' and R' are as
defined above, R' is an optional substituent, and X is selected
from --NH--, --NR'--, --O-- and --S--.
[0095] These and other aspects of this invention will become
apparent upon reference to the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0096] FIG. 1 is a plot showing the effect of various
concentrations of structure (20b) on platelet deposition in a
vascular graft.
[0097] FIG. 2 is a plot showing the effect of various
concentrations of structure (39) on platelet deposition in a
vascular graft.
[0098] FIG. 3 is a plot showing the effect of various
concentrations of structure (29b) on platelet deposition in a
vascular graft.
[0099] FIGS. 4A and 4B illustrate the bioavailability of structure
(20b) via both IV and PO administration.
[0100] FIGS. 5A and 5B illustrate the ability of structures (221-5)
and (221-6) to serve as antithrombotic agents.
DETAILED DESCRIPTION OF THE INVENTION
[0101] As mentioned above, the .beta.-sheet is an important
structural component for many biological recognition events. The
.beta.-sheet mimetics of this invention serve to impart and/or
stabilize the .beta.-sheet structure of a natural or synthetic
peptide, protein or molecule, particularly with regard to
conformational stability. In addition, the .beta.-sheet mimetics of
this invention are more resistant to proteolytic breakdown, thus
rendering a peptide, protein or molecule containing the same more
resistant to degradation. The .beta.-sheet mimetic may be
positioned at either the C-terminus or N-terminus of the protein,
peptide or molecule, or it may be located within the protein,
peptide or molecule itself, and more than one .beta.-sheet mimetic
of the present invention may be incorporated in a protein, peptide
or molecule.
[0102] The .beta.-sheet mimetics of this invention are generally
represented by structure (I) above, as well as the more specific
embodiments represented by structures (II) through (XI). The
.beta.-sheet mimetics of this invention may be constructed to mimic
the three-dimensional conformation of a .beta.-sheet comprised of
naturally occurring L-amino acids, as well as the structure of a
.beta.-sheet comprised of one or more D-amino acids. Thus, all
stereoconformations of the .beta.-sheet mimetics of structure (I)
are within the scope of this invention.
[0103] For example, .beta.-sheet mimetics of structure (II) include
the following structures (II') and (II"): 23
[0104] Similarly, .beta.-sheet mimetics of structure (III) include
the following structures (III') through (III""): 24
[0105] The .beta.-sheet mimetics of structure (IV) include these
same stereoconfirmations, but with the "Z-NH" moiety of structures
(III') through (III"") replaced with a "Z" moiety.
[0106] As used herein, the term "an amino acid side chain moiety"
as used to define the R.sub.1, R.sub.2, R.sub.2', R.sub.3, and
R.sub.4 moieties represents any amino acid side chain moiety
present in naturally occurring proteins, including (but not limited
to) the naturally occurring amino acid side chain moieties
identified in Table 1 below. Other naturally occurring side chain
moieties of this invention include (but are not limited to) the
side chain moieties of phenylglycine, 3,5-dibromotyrosine,
3,5-diiodotyrosine, hydroxylysine, naphthylalanine, thienylalanine,
.gamma.-carboxyglutamate, phosphotyrosine, phosphoserine and
glycosylated amino acids such as glycosylated serine, asparagine
and threonine.
1 TABLE 1 Amino Acid Side Chin Moiety Amino Acid --H Glycine
--CH.sub.3 Alanine --CH(CH.sub.3).sub.2 Valine
--CH.sub.2CH(CH.sub.3).sub.2 Leucine --CH(CH.sub.3)CH.sub.2CH.sub.3
Isoleucine --(CH.sub.2).sub.4NH.sub.3.sup.+ Lysine
--(CH.sub.2).sub.3NHC(NH.- sub.2)NH.sub.2.sup.+ Arginine 25
Histidine --CH.sub.2COO.sup.- Aspartic acid
--CH.sub.2CH.sub.2COO.sup.- Glutamic acid --CH.sub.2CONH.sub.2
Asparagine --CH.sub.2CH.sub.2CONH.sub.2 Glutamine 26 Phenylalanine
27 Tyrosine 28 Tryptophan --CH.sub.2SH Cysteine
--CH.sub.2CH.sub.2SCH.sub.3 Methionine --CH.sub.2OH Serine
--CH(OH)CH.sub.3 Threonine
[0107] In addition to naturally occurring amino acid side chain
moieties, the amino acid side chain moieties of the present
invention also include various derivatives thereof. As used herein,
a "derivative" of an amino acid side chain moiety includes all
modifications and/or variations to naturally occurring amino acid
side chain moieties. For example, the amino acid side chain
moieties of alanine, valine, leucine, isoleucine, phenylglycine and
phenylalanine may generally be classified as lower chain alkyl,
aryl or aralkyl moieties. Derivatives of amino acid side chain
moieties include other straight chain or branched, cyclic or
noncyclic, substituted or unsubstituted, saturated or unsaturated
lower chain alkyl, aryl or aralkyl moieties.
[0108] As used herein, "lower chain alkyl moieties" contain from
1-12 carbon atoms, "lower chain aryl moieties" contain from 6-12
carbon atoms, and "lower chain aralkyl moieties" contain from 7-12
carbon atoms. Thus, in one embodiment, the amino acid side chain
derivative is selected from a C.sub.1-12 alkyl, a C.sub.6-12 aryl
and a C.sub.1-12 aralkyl, and in a more preferred embodiment, from
a C.sub.1-7 alkyl, a C.sub.6-10 aryl and a C.sub.7-11 aralkyl.
[0109] Amino acid side chain derivatives of this invention further
include substituted derivatives of lower chain alkyl, aryl and
aralkyl moieties, wherein the substituent is selected from (but are
not limited to) one or more of the following chemical moieties:
--OH, --OR, --COOH, --COOR, --CONH.sub.2, --NH.sub.2, --NHR, --NRR,
--SH, --SR, --SO.sub.2R, --SO.sub.2H, --SOR and halogen (including
F, Cl, Br and I), wherein each occurrence of R is independently
selected from a lower chain alkyl, aryl or aralkyl moiety.
Moreover, cyclic lower chain alkyl, aryl and aralkyl moieties of
this invention include naphthalene, as well as heterocyclic
compounds such as thiophene, pyrrole, furan, imidazole, oxazole,
thiazole, pyrazole, 3-pyrroline, pyrrolidine, pyridine, pyrimidine,
purine, quinoline, isoquinoline and carbazole. Amino acid side
chain derivatives further include heteroalkyl derivatives of the
alkyl portion of the lower chain alkyl and aralkyl moieties,
including (but not limited to) alkyl and aralkyl phosphonates and
silanes.
[0110] As used in the context of this invention, the term
"remainder of the molecule" (as represented by Y and Z) may be any
chemical moiety, including (but not limited to) amino acid side
chain moieties and derivatives thereof as defined above. For
example, when the .beta.-sheet mimetic is located within the length
of a peptide or protein, Y and Z may represent amino acids of the
peptide or protein. Alternatively, if two or more .beta.-sheet
mimetics are linked, the Y moiety of a first .beta.-sheet mimetic
may represent a second .beta.-sheet mimetic while, conversely, the
Z moiety of the second .beta.-sheet mimetic represents the first
.beta.-sheet mimetic.
[0111] When the .beta.-sheet mimetic is located at the end of a
peptide or protein, or when the .beta.-sheet mimetic is not
associated with a peptide or protein, Y and/or Z may represent a
suitable terminating moiety. For example, representative
terminating moieties for the Z moiety include (but are not limited
to) --H, --OH, --R, --C(.dbd.O)R and --SO.sub.2R (where R is
selected from a lower chain alkyl moiety, a lower chain aryl moiety
and a lower chain aralkyl moiety), or may be a suitable protecting
group for protein synthesis, such as BOC, FMOC and CBZ (i.e.,
tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl and
benzyloxycarbonyl, respectively).
[0112] Similarly, representative terminating moieties for the Y
moiety include (but are not limited to) --H, --OH, --R,
--SO.sub.2R, --SOR, --SO.sub.2NHR, --CF.sub.3, --C.sub.2F.sub.5,
--NHOH, --NHNHR, --C(.dbd.O)H, --C(.dbd.O)R, --C(.dbd.O)CF.sub.3,
--C(.dbd.O)OR, --C(.dbd.O)CH.sub.2OR, --C(.dbd.O)NHR, --CH.sub.2X',
--C(.dbd.O)CH.sub.2X', --C(.dbd.O)C(.dbd.O)NRR,
--C(.dbd.O)CHN.sub.2, 29
[0113] --C(.dbd.O)CH.dbd.CHC(.dbd.O)OH,
--C(.dbd.O)CH.dbd.CHC(.dbd.O)R, --C(.dbd.O)CH.dbd.CHC(.dbd.O)OR,
--C(.dbd.O)CH.dbd.CHC(.dbd.O)NRR, --CH(OH)CH.dbd.CHC(.dbd.O)OH,
--CH(OH)CH.dbd.CHC(.dbd.O)R, --CH(OH)CH.dbd.CHC(.dbd.O)OR,
--CH(OH)CH.dbd.CHC(.dbd.O)NRR, --CH.dbd.CHSO.sub.2R and
--SO.sub.2CH.dbd.CHR (where X' is Cl, F, Br or I, and each
occurrence of R is independently selected from a lower chain alkyl
moiety, a lower chain aryl moiety and a lower chain aralkyl
moiety), or a heterocyclic moiety, such as pyridine, pyran,
thiophan, pyrrole, furan, thiophene, thiazole, benzthiazole,
oxazole, benzoxazole, imidazole and benzimidazole.
[0114] More specifically, suitable Z and Y terminating moieties of
this invention include the following groups:
2 Z Moieties Y Moieties R.sup.6--X-- --X--R.sup.6 R.sup.7--X--
--X--R.sup.7 (R.sup.8).sub.2N--X-- --X--N(R.sup.8).sub.2
R.sup.9--O--X-- --X--O--R.sup.9 R.sup.10--S(O).sub.p--X--
--X--S(O).sub.pR.sup.10
[0115] wherein
[0116] X is optionally present and selected from a straight chain
or branched, cyclic or noncyclic, saturated or nonsaturated
C.sub.1-2 alkyl optionally substituted with one or more
substituents selected from halogen, .dbd.O, OR, ONRR, C(O)R,
C(O)OR, CN, OC(O)R, C(O)NRR, C(O)NROR, NH.sub.2, NO.sub.2, NHOR,
C(NR), NHR, C(NR)NHR, NHC(NR)NHR, P(OR).sub.3 and SiRRR;
[0117] R.sup.6 is selected from H, CN, NO.sub.2 SIRRR and
P(OR).sub.3;
[0118] R.sup.7 is selected from C.sub.5-14 aryl, C.sub.4-13
heteroaryl, C.sub.3-14 cycloalkyl, C.sub.5-14 cycloalkylene, and
C.sub.2-13 heterocycloalkyl, each of which may be optionally
substituted with one or more substituents selected from X, halogen,
OR, ONRR, C(O)R, C(O)OR, CN, OC(O)R, NR.sub.2, C(O)NRR, C(O)NROR,
NRC(O)R, C(NR)NHR, NHC(NR)NHR, NO.sub.2 SO.sub.2R, SO.sub.2NRR,
SiRRR, OP(OR).sub.3, CH.sub.2P(OR).sub.3 and
CF.sub.2P(OR).sub.3;
[0119] R.sup.8 is, at each occurrence, independently selected from
H, X, R.sup.7, halogen, OR, .dbd.C.dbd.O, C(O)R, C(O)OR, CN,
OC(O)R, NR.sub.2, C(O)NRR, NRC(O)R, C(NR)NHR, NO.sub.2, SO.sub.2R,
SO.sub.2NRR, SiRRR and P(OR).sub.3, or, taken together, may form a
saturated or unsaturated C.sub.2-14 cycloalkyl optionally
substituted with one or more substituents selected from .dbd.O, X,
R.sup.7, halogen, OR, .dbd.C.dbd.O, C(O)R, C(O)OR, CN, OC(O)R,
NR.sub.2, C(O)NRR, NRC(O)R, C(NR)NHR, NO.sub.2 SO.sub.2R,
SO.sub.2NRR, SiRRR and P(OR).sub.3;
[0120] R.sup.9 is selected from H, X, R.sup.7, halogen C(O)R,
C(O)OR, CN, NR.sub.2 C(O)NRR, NRC(O)R, C(NR)NHR, NO.sub.2,
SO.sub.2R, SO.sub.2NRR, SIRRR and P(OR).sub.3, where p=0-2;
[0121] R.sup.10 is selected from H, X, R.sub.1, halogen, OR, C(O)R,
C(O)OR, CN, NR.sub.2 and C(O)NRR; and
[0122] each occurrence of R in the above definitions of R.sup.6
through R.sup.10 is independently selected from H, C.sub.1-6 alkyl,
C.sub.3-14 cycloalkyl, C.sub.5-14 cycloalkylene, C.sub.6-14 aryl,
C.sub.4-13 heteroaryl and C.sub.2-13 heterocycloalkyl, or, when two
R groups are present, taken together form a saturated or
unsaturated C.sub.2-8 cycloalkyl.
[0123] In the context of protease inhibitors, the Y terminating
moiety further includes the following group:
--N({C(R.sup.11).sub.2C(O)--T.sub.m}.sub.n--W.sub.k--R.sup.12).sub.2
[0124] wherein
[0125] m=0-1;
[0126] n0-20;
[0127] k=0-1;
[0128] R.sup.11 is, at each occurrence, independently selected from
an amino acid side chain moiety and derivative thereof;
[0129] T is, at each occurrence, independently selected from
C.dbd.O, C(O)--N(R.sup.12) and N(R.sup.12);
[0130] W is, at each occurrence, independently selected from a
C.sub.2-14 heterocycle; and
[0131] R.sup.12 is, at each occurrence, independently selected from
H, X, X--R.sup.5, X--R.sup.7, X--N(R.sup.8).sub.2, X--O--R.sup.9,
X--S(O)R.sup.10 and P(OR).sub.3, or, taken together, may form a
saturated or unsaturated C.sub.2-14 cycloalkyl optionally
substituted with one or more substituents selected from halogen,
.dbd.O, OR, ONRR, C(O)R, C(O)OR, CN, OC(O)R, C(O)NRR, C(O)NROR,
NH.sub.2, NO.sub.2, NHOR, C(NR)NHR, NHC(NR)NHR, P(OR).sub.3 and
SiRRR; and
[0132] R, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, X and p are
as defined immediately above;
[0133] with the proviso that when n=0 each of R.sup.12 are not both
hydrogen.
[0134] In the context of structure (I) above, any two adjacent CH
groups of the bicyclic ring may form a double bond. Such double
bonds may be present in isolation or conjugation with one or more
additional double bonds, including aromatic ring systems. For
example, representative isolated double bonds includes compounds of
structures (IIe), (IIf), (IIIe), (IIIf), (IVe), (IVf), (VIIIa) and
(VIIIb) above. Representative aromatic compounds resulting from
conjugated double bonds are depicted by structures (IIc), (IId),
(IIIc), (IIId), (IVc) and (IVd) above.
[0135] Within a specific embodiment of this invention, .beta.-sheet
mimetics are disclosed having structure (II) above, wherein A is
--C(.dbd.O)--, B is N, C is --(CH.sub.2).sub.2-- or
--C(.dbd.O)CH.sub.2--, D is N, and the optional carbonyl moiety F
is present, as represented by the following structures (IIg), (IIh)
and (IIh') 30
[0136] Similarly, when B and D are both CH, representative
.beta.-sheet mimetics of this invention include compounds of the
following structures (IIi), (IIj) and (IIj') 31
[0137] Within another specific embodiment of this invention,
.beta.-sheet mimetics are disclosed having structure (III) above.
In one aspect of this embodiment, D is N and the compound has the
following structure (IIIi): 32
[0138] wherein A is selected from --C(.dbd.O)--,
--(CH.sub.2).sub.0-4-- and --C(.dbd.0)(CH.sub.2).sub.1-3--; B is
selected from N and CH; C is selected from --C(.dbd.O)-- and
--(CH.sub.2).sub.0-3--;and the bicyclic ring system is saturated
(i.e., contains no double bonds between adjacent CH groups of the
bicyclic ring system).
[0139] In this embodiment where B is CH and R.sub.3 is hydrogen,
compounds are disclosed having the following structures (IIIj),
(IIIk) and (IIIl): 33
[0140] In an embodiment of structure (IIIi) where B is N and
R.sub.3 is hydrogen, compounds are disclosed having the following
structures (IIIm), (IIIn) and (IIIo): 34
[0141] In preferred embodiments of this aspect of the invention,
compounds are disclosed having the following structures (IIIp),
(IIIq), (IIIr) and (IIIr') 35
[0142] In another embodiment of structure (IIIi) above, compounds
are disclosed having the following structure (IIIs): 36
[0143] wherein A is selected from --(CH.sub.2).sub.0-4--,
--(CH.sub.2).sub.1-2O-- and --(CH.sub.2).sub.1-2S--; C is selected
from --(CH.sub.2).sub.0-3--, --O--, --S--, --O(CH.sub.2).sub.1-2--
and --S(CH.sub.2).sub.1-2--; and the bicyclic ring system is
saturated.
[0144] In an embodiment of structure (IIIs) where A is
--(CH.sub.2).sub.0-4--, compounds are disclosed having the
following structure (IIIf): 37
[0145] In an embodiment of structure (IIIs) where A is
--(CH.sub.2).sub.1-2O-- or --(CH.sub.2).sub.1-2S--, compounds are
disclosed having the following structures (IIIu) and (IIIv): 38
[0146] In an embodiment of structure (IIIs) where C is
--(CH.sub.2).sub.1-3--, compounds are disclosed having the
following structure (IIIw): 39
[0147] where A is selected from --(CH.sub.2).sub.1-4--,
--(CH.sub.2).sub.1-2O----and --(CH.sub.2).sub.1-2S--.
[0148] In an embodiment of structure (IIIs) where C is --O-- or
--S--, compounds are disclosed having the following structures
(IIIx) and (IIIy): 40
[0149] In an embodiment of structure (IIIs) where C is
--O(CH.sub.2).sub.1-2-- or --S(CH.sub.2).sub.1-2--, compounds are
disclosed having the following structures (IIIz) and (IIIza) 41
[0150] Within a further embodiment of this invention, .beta.-sheet
mimetics are disclosed having strucutre (IV) above. In one aspect
of this embodiment, A is --C(.dbd.O)--, B is CH or N, C is
--(CH.sub.2).sub.2-- or --C(.dbd.O)CH.sub.2--, D is N and the
optional carbonyl moiety is present, as represented by the
following structures (IVg), (IVg'), (IVh) and (IVh'): 42
[0151] In embodiments of this invention where F is not present,
compounds having structures (V), (VI) and (VII) are disclosed. With
respect to compounds of structure (V), when A is --C(.dbd.O)--, B
and D are both CH or N, and C is --(CH.sub.2).sub.2--,
representative compounds of this invention include the following
structures (Va), (Vb) and (Vc): 43
[0152] Similarly, in structure (VI), when A is --C(.dbd.O)--, B and
D are both CH or N, and C is --(CH.sub.2).sub.2--, representative
compounds of this invention include the following structures (VIa),
(VIb) and (VIc): 44
[0153] As for structure (VII), when A is --C(.dbd.O)--, B and D are
both CH or N, and C is --(CH.sub.2).sub.2--, representative
compounds of this invention include the following structures
(VIIa), (VIIb) and (VIIc): 45
[0154] With regard to compounds of structure (VIII), in one
embodiment B and D of structures (VIIIa) and (VIIIb) are both CH or
N and X is --S--, --O-- or --(CH.sub.2).sub.2--, yielding compounds
of structures (VIIIc), (VIIId), (VIIIe) and (VIIIf): 46
[0155] In an embodiment of structure (IX), wherein A is
--C(.dbd.O)--, B and D are both N, E is --N(Z)--,
--C(R.sub.1)(NHZ)-- or --C(R.sub.1)(Z)--, and F is present,
compounds of this invention include structures (IXc) through (IXh).
47
[0156] In an embodiment of structure (X), where A is --C(.dbd.O)--,
B is N, D is N and E is Z--N, compounds of this invention include
structures (Xc) and (Xd): 48
[0157] In an embodiment of structure (XI), where A is
--C(.dbd.O)--, B is N, C is --CH.sub.2C(.dbd.O)--, D is N and E is
Z-N, compounds of this invention include structure (XIc): 49
[0158] The .beta.-sheet mimetics of this invention may be
synthesized by one skilled in the art by known organic synthesis
techniques. For example, the various embodiments of structure (I)
may be synthesized according to the following reaction schemes.
[0159] Representative compounds of structure (III) can be
synthesized by the following reaction schemes (where n=0-4, p=0-3
and m=0-2): 50 51 52
[0160] In addition, representative compounds of structure (IIIl)
having structure (IIIl"') may be synthesized by the following
reaction scheme, and when A of structure (IIIl) is
--C(.dbd.O)(CH.sub.2).sub.1-3--, a related compound (designated
(IIIi') below) can be synthesized by the following reaction scheme:
53 54
[0161] Representative compounds of structure (IIIi) wherein R.sub.3
is an amino acid side chain moiety or derivative thereof may also
be prepared according to the above scheme (4). 55 56 57 58 59 60 61
62 63 64
[0162] According to the definition of structure (I) above, the
bicyclic ring system may contain adjacent CH groups (i.e., the
bicyclic ring system may be formed, at least in part, by a
--CH--CH-- group). Compounds wherein such a --CH--CH-- group is
replaced with a --C.dbd.C-- are also included within the scope of
structure (I) (i.e., any two adjacent CH groups of the bicyclic
ring may together form a double bond).
[0163] Reaction Schemes (15), (16) and (17) illustrate further
synthetic methodology for preparing representative compounds of
structure (III). 65 66 67
[0164] Representative compounds of structure (IV) may be prepared
by the following reaction schemes (18) through (21). 68 69
[0165] Alternatively, structures (IVc) and (IVd) may be made by
reaction scheme (19-1). 70 71 72
[0166] Alternatively, structure (IVf) may be made by the following
reaction scheme (21-1). 73
[0167] Representative compounds of structure (VIII) may be
synthesized either from urazoles or pyrazolidine diones by reaction
schemes (22) and (23). 74 75
[0168] Alternatively, the pyrazolidine dione starting material may
be synthesized by the following reaction scheme: 76
[0169] Representative compounds of structure (II) may be
synthesized by the following reaction scheme (24): 77
[0170] Further representative compounds of structure (II) may be
made by the following reaction scheme (25): 78
[0171] Further representative compounds of structure (III) may be
made by the following reaction scheme (26): 79
[0172] Compounds of structures (V), (VI) and (VII) may be made by
the same general techniques as disclosed above for compounds of
structures (II), (III) and (IV), with the exception that the
respective precursor intermediate does not contain a carbonyl
moiety at position F.
[0173] Further, compounds of structure (IX) may be prepared
according to reaction scheme (27): 80
[0174] Representative compounds of structure (IIe) may be made by
the following reaction scheme (28): 81
[0175] Representative compounds of structure (X) may be made by the
following reaction scheme (29): 82
[0176] Representative compounds of structure (XIc) may be made by
the following reaction scheme (30): 83
[0177] Representative compounds of structure (XId) may be made by
the following reaction scheme (31): 84
[0178] Representative compounds of structure (IIh') may be made by
the following reaction schemes (32) and (33): 85 86
[0179] In one embodiment of .beta.-sheet mimetics of this
invention, Y groups have the structure: 87
[0180] where a preferred stereochemistry is: 88
[0181] Preferred R.sub.4 groups are organoamine moieties having
from about 2 to about 10 carbon atoms and at least one nitrogen
atom. Suitable organoamine moieties have the chemical formula
C.sub.2-10H.sub.4-20N.sub.- 1-6O.sub.0-2; and preferably have the
chemical formula C.sub.3-7H.sub.7-14N.sub.1-4O.sub.0-1. Exemplary
organoamine moieties of the invention are (wherein R is selected
from hydrogen, halogen (e.g., fluorine), lower chain alkyl (e.g.,
methyl), and hydroxy lower chain alkyl (e.g., hydroxymethyl); and X
is selected from CH.sub.2, NH, S and O): 89
[0182] In the above structure, R.sub.5 is selected from (a) alkyl
of 1 to about 12 carbon atoms, optionally substituted with 1-4 of
halide, C.sub.1-5alkoxy and nitro, (b)
--C(.dbd.O)NH--C.sub.1-5alkyl, wherein the alkyl group is
optionally substituted with halide or C.sub.1,alkoxy, (c)
--C(.dbd.O)NH--C.sub.1-10aralkyl where the aryl group may be
optionally substituted with up to five groups independently
selected from nitro, halide, --NH--(C.dbd.O)C.sub.1-5alkyl,
--NH--(C.dbd.O)C.sub.6-10aryl, C.sub.1-5alkyl and C.sub.1-5alkoxy,
and (d) monocyclic and bicyclic heteroaryl of 4 to about 11 ring
atoms, where the ring atoms are selected from carbon and the
heteroatoms oxygen, nitrogen and sulfur, and where the heteroaryl
ring may be optionally substituted with up to about 4 of halide,
C.sub.1-5alkyl, C.sub.1-5alkoxy, --C(.dbd.O)NHC.sub.1-5alkyl,
--C(.dbd.O)NHC.sub.6-10aryl, amino, --C(.dbd.O)OC.sub.1-5alkyl and
--C(.dbd.O)OC.sub.6-10aryl. 90
[0183] wherein R.sub.6 is hydrogen, nitro, halide,
NH--C(.dbd.O)--C.sub.1-- 5alkyl, NH--C(.dbd.O)--C.sub.6-10aryl,
C.sub.1-C.sub.5alkyl and C.sub.1-C.sub.5 alkoxy;
[0184] wherein X is halide; 91
[0185] wherein E is --O--, --NH-- or --S-- and R.sub.7 and R.sub.8
are independently selected from hydrogen, C.sub.1-5alkyl,
--C(.dbd.O)OC.sub.1-5alkyl, --C(.dbd.O)OC.sub.6-10aryl,
--C(.dbd.O)NHC.sub.1-5alkyl and --C(.dbd.O)NHC.sub.6-10aryl; and
92
[0186] wherein E and R.sub.6 are as defined previously.
[0187] The .beta.-sheet mimetics of the present invention may be
used in standard peptide synthesis protocols, including automated
solid phase peptide synthesis. Peptide synthesis is a stepwise
process where a peptide is formed by elongation of the peptide
chain through the stepwise addition of single amino acids. Amino
acids are linked to the peptide chain through the formation of a
peptide (amide) bond. The peptide link is formed by coupling the
amino group of the peptide to the carboxylic acid group of the
amino acid. The peptide is thus synthesized from the carboxyl
terminus to the amino terminus. The individual steps of amino acid
addition are repeated until a peptide (or protein) of desired
length and amino acid sequence is synthesized.
[0188] To accomplish peptide (or protein or molecule) synthesis as
described above, the amino group of the amino acid to be added to
the peptide should not interfere with peptide bond formation
between the amino acid and the peptide (i.e., the coupling of the
amino acid's carboxyl group to the amino group of the peptide). To
prevent such interference, the amino groups of the amino acids used
in peptide synthesis are protected with suitable protecting groups.
Typical amino protecting groups include, for example, BOC and FMOC
groups. Accordingly, in one embodiment of the present invention,
the .beta.-sheet mimetics of the present invention bear a free
carboxylic acid group and a protected amino group, and are thus
suitable for incorporation into a peptide by standard synthetic
techniques.
[0189] The .beta.-sheet mimetics of this invention may be
synthesized on solid support, typically via a suitable linker. The
.beta.-sheet mimetics may then be cleaved from the solid support
by, for example, aminolysis, and screened as competitive substrates
against appropriate agents, such as the chromogenic substrate BAPNA
(benzyoylarginine paranitroanalide) (see Eichler and Houghten,
Biochemistry 32:11035-11041, 1993) (incorporated herein by
reference). Alternatively, by employing a suitable linker moiety,
such screening may be performed while the .beta.-sheet mimetics are
still attached to the solid support.
[0190] Once a substrate is selected by the above kinetic analysis,
the .beta.-sheet mimetic may be converted into an inhibitor by
modifications to the C-terminal--that is, by modification to the Y
moiety. For example, the terminal Y moiety may be replaced with
--CH.sub.2Cl, --CF.sub.3, --H, or --C(O)NHR. Appropriate R moieties
may be selected using a library of substrates, or using a library
of inhibitors generated using a modification of the procedure of
Wasserman and Ho (J. Org. Chem. 59:4364-4366, 1994) (incorporated
herein by reference).
[0191] Libraries of compounds containing .beta.-strand templates
may be constructed to determine the optimal sequence for substrate
recognition or binding. Representative strategies to use such
libraries are discussed below.
[0192] A representative .beta.-sheet mimetic substrate library may
be constructed as follows. It should be understood that the
following is exemplary of methodology that may be used to prepare a
.beta.-sheet mimetic substrate library, and that other libraries
may be prepared in an analogous manner.
[0193] In a first step, a library of the following type: 93
[0194] R.sub.1, R.sub.3, R=amino acid side chain moieities or
derivatives thereof; Y=H, Ac, SO.sub.2R; and the circled "P"
represents a solid support.
[0195] may be constructed on a solid support (PEGA resin, Meldal,
M. Tetrahedron Lett. 33:3077-80, 1992; controlled pore glass, Singh
et al., J. Med. Chem. 38:217-19, 1995). The solid support may then
be placed in a dialysis bag (Bednarski et al., J. Am. Chem. Soc.
109:1283-5, 1987) with the enzyme (e.g., a protease) in an
appropriate buffer. The bag is then placed in a beaker with bulk
buffer. The enzymatic reaction is monitored as a function of time
by HPLC and materials cleaved from the polymer are analyzed by
MS/MS. This strategy provides information concerning the best
substrates for a particular target.
[0196] The synthesis of the .beta.-sheet mimetic is illustrated by
the retrosynthetic procedure shown next: 94
[0197] The complexity of the library generated by this technique is
(R.sub.1)(R.sub.3)(R)(Y). Assuming R.sub.1, R.sub.3 and R are
selected from naturally occurring amino acid side chains moieties,
n is constant, and Y is H, Ac or --SO.sub.2R as defined above, a
library having on the order of 24,000 members [(20)(20)(20)(3)] is
generated.
[0198] After screening the library against a specific target (e.g.,
enzyme), the library may then recovered and screened with a second
target, and so on.
[0199] In addition, a library of inhibitors can be constructed and
screened in a standard chromogenic assay. For example, the library
may be constructed as follows, where the following example is
merely representative of the inhibitor libraries that may be
prepared in an analogous manner to the specific example provided
below. 95
[0200] inhibitors of serine or cysteinyl proteases
[0201] (See Wasserman et al., J. Org. Chem. 59:4364-6, 1994.)
[0202] A further alternative strategy is to link the library
through the sidechain R group as shown below. 96
[0203] A library of aspartic protease inhibitors may be constructed
having the following exemplary structure, and then cleaved from the
resin and screened: 97
[0204] Similarly, for metalloproteases, a library having the
exemplary structure shown below may be constructed and then cleaved
from the resin to provide a library of hydroxamic acids: 98
[0205] The activity of the .beta.-sheet mimetics of this invention
may be further illustrated by reference to Table 2 which lists a
number of biologically active peptides. In particular, the peptides
of Table 2 are known to have biological activity as substrates or
inhibitors.
3TABLE 2 Biologically Active Peptides Protease Inhibitors: (a)
(D)FPR (Thrombin) Enzyme 40:144-48, 1988 (b) (D)IEGR (Factor X)
Handbook of Synthetic Substrates for the Coagulation and
Fibronlytic Systems, H. C. Hemker, pp. 1-175, 1983, Martinus
Nijhoff publishers, The Hague. Protein Kinase Substrates and
Inhibitors: (c) LRRASLG (Serine Kinase) Biochem. Biophys. Res.
Commun. 61:559, 1974 (d) LPYA (Tyrosine Kinase) J. Bio. Chem.
263:5024, 1988 (e) PKI (Serine Kinase) Science 253:1414-20, 1991
CAAX Inhibitors: (f) (H)--CVIM--(OH) Proc. Natl. Acad. Sci. USA
88:732-36, 1991 (g) (H)--CVFM--(OH) Bioorg. Med. Chem. Letters
4:887-92, 1994 (h) (H)--CIT-(homoserine lactone) Science
260:1934-37, 1993 SH2 Peptide Analogs: (i) .sup.PYZPZS.sup.PYZPZS
(IRS 1 analogue) Biochemistry 33:9376-81, 1994 (j)
EPQ.sup.PYEEIPIYL (Src SH.sub.2 binding motif) Cell 72:767-68, 1993
Class MHC I Peptides: (k) TYQRTRALV (Influenza nucleoprotein) J.
Exp. Med. 175:481-87, 1991 (1) RGYVYQGL (VSV) Ann. Rev. Imm.
21:211-44, 1993 .sup.PY = phosphorylated Y Z = norleucine
[0206] More generally, the .beta.-sheet mimetics of this invention
can be synthesized to mimic any number of biologically active
peptides by appropriate choice of the R.sub.2, R.sub.2', R.sub.3,
F, Y and Z moieties (as well as the A, B, C, D and E moieties of
structure (I) itself). This is further illustrated by Table 3 which
discloses various modifications which may be made to the
.beta.-sheet mimetics of structure. (I) to yield biologically
active compounds. In Table 3, R.sub.2 and R.sub.3 are independently
chosen from among the atoms or groups shown under the
"R.sub.2/R.sub.3" column.
4TABLE 3 Modifications to Structure (I) to Yield Biological Active
Compounds (I) 99 R.sub.1 R.sub.2/R.sub.3 Y Z I. PROTEASE INHIBITORS
A. Serine 1. Thrombin C.sub.6-C.sub.10 aromatic (e.g., phenyl,
benzyl, naphthyl), C.sub.1-C.sub.10aliphatic or cycloaliphatic,
substituted C.sub.6-C.sub.10aromatic, --SiR.sub.3, --CO.sub.2H,
--CO.sub.2R hydrogen 100 hydrogen, alkyl, aryl, 101 102103 104 R =
aliphatic 105 106 107 108 X = CH.sub.2, NH, S, O R = H, CH.sub.3
109 110 111 112 {circle over (2)} = CH.sub.2Cl CF.sub.3 113 114 115
X = O, S, NH R = CO.sub.2H, CO.sub.2R, SO.sub.2R, COCF.sub.3 116 X
= O, S, NH R = CO.sub.2H, SO.sub.2R, CO.sub.2R 117 R = CO.sub.2H,
CO.sub.2 SO.sub.2R, COCF.sub.3 2. Elastase
C.sub.1-C.sub.10aliphatic hydrogen or C.sub.1-C.sub.10 C.sub.10
heterocyclic 118 acyl {circle over (1)} = --CH.sub.3
--CH(CH.sub.3).sub.2 or 119 aromatic or aliphatic 3. Factor X
C.sub.1-C.sub.10aliphatic carboxylic hydrogen 120 D(Ile) Acyl
Dansyl aromatic carboxylic 121 C.sub.1-C.sub.10 acidic heterocyclic
122 123 124 125 126 X = CH.sub.2, NH 127 128 {circle over (2)} =
--CH.sub.2Cl --CF.sub.3 129 130 131 X = O, S, NH R = CO.sub.2H,
CO.sub.2R, SO.sub.2R, COCF.sub.3 132 X = O, S, NH R = CO.sub.2H,
SO.sub.2R, CO.sub.2R 133 R = CO.sub.2H, CO.sub.2 SO.sub.2R,
COCF.sub.3 {circle over (3)} = aliphatic cycloaliphatic peptide B.
Aspartic 1. HIV1 C.sub.1-C.sub.10 aliphatic or arginine
C.sub.1-C.sub.10aliphatic or 134 acyl 135 {circle over (1)} =
C.sub.1-C.sub.10 aliphatic arginine 136 {circle over (1)} =
C.sub.1-C.sub.10 aliphatic C.sub.1-C.sub.10 aromatic {circle over
(2)} = amino acid C.sub.1-C.sub.10 alkyl C.sub.1-C.sub.10 aryl acyl
hydrogen C. Cysteins 1. Cathepsin B C.sub.6-C.sub.10 aromatic
C.sub.1-C.sub.10 aliphatic hydrogen C.sub.1-C.sub.10 basic aromatic
hydrophobic 137 benzyl acyl 138 --CH.sub.2OAc --CH.sub.2N.sub.2+
--H 139 {circle over (2)} = C1-C10 aliphatic 2. Calpain
C.sub.6-C.sub.10 aromatic, C.sub.1-C.sub.10 aliphatic, hydrophobic
C.sub.1-C.sub.10aliphatic 140 benzyl acyl {circle over (1)} =
C.sub.1-C.sub.10 aromatic, hydrophobic {circle over (2)} =
--CH.sub.2F --CH.sub.2N.sub.2 --CH.sub.2OAc --H 3. ICE
C.sub.1-C.sub.10 aliphatic hydrogen 141 dihydro- cinnamic,
aromatic, aliphatic, acetyl {circle over (1)} = --H --CH.sub.2F
--CH.sub.2N.sub.2.sup.+ 142 --CH.sub.2OAc 143 {circle over (2)} =
C1-C10 aliphatic C1-C10 aromatic D. Metallo 1. ACE
C.sub.1-C.sub.10aliphatic indoyl C.sub.1-C.sub.10aromatic --OH 144
{circle over (1)} = C.sub.1-C.sub.10 alkyl C.sub.1-C.sub.10 aryl 2.
Collagenase C.sub.1-C.sub.10 alkyl hydrogen
C.sub.1-C.sub.10aromatic, C.sub.1-C.sub.10 aliphatic, 145 hydroxyl
C.sub.1-C.sub.10 basic {circle over (1)} = alkyl 146 {circle over
(1)} = hydrogen C.sub.1-C.sub.10 alkyl or 147 C.sub.6-C.sub.10
C.sub.1-C.sub.10 alkyl --NHOH hydroxyl aromatic C.sub.1-C.sub.10
aliphatic 148 1 = hydrogen C.sub.1-C.sub.10 alkyl, or 149 II.
KINASE INHIBITORS A. Serine/ amino acid amino acid side Serine,
amino acid Threonine side chin chain Threonine B. Tyrosine amino
acid side amino acid side Tyrosine amino acid chain chain C.
Histidine amino acid side amino acid side Histidine amino acid
chain chain III. MHC II INHIBITORS A. Class I 1. HIV gp120 hydrogen
hydrogen 150 151 a. Class II 1. HA (306-18) hydrogen 152
-YVKQNTLKLAT hydrogen 2. HSP 65(3-13) Cl--hydrophobic hydrogen
-YDEEARR -TK
[0207] When the .beta.-sheet mimetics of this invention are
substituted for one or more amino acids of a biologically active
peptide, the structure of the resulting .beta.-sheet modified
peptide (prior to cleavage from the solid support, such as PAM) may
be represented by the following diagram, where AA.sub.1 through
AA.sub.3 represent the same or different amino acids: 153
[0208] The precise .beta.-sheet mimetic may be chosen by any of a
variety of techniques, including computer modeling, randomization
techniques and/or by utilizing natural substrate selection assays.
The .beta.-sheet mimetic may also be generated by synthesizing a
library of .beta.-sheet mimetics, and screening such library
members to identify active members as disclosed above.
[0209] Once the optimized .beta.-sheet mimetic is chosen,
modification may then be made to the various amino acids attached
thereto. A series of .beta.-sheet modified peptides having a
variety of amino acid substitutions are then cleaved from the solid
support and assayed to identify a preferred substrate. It should be
understood that the generation of such substrates may involve the
synthesis and screening of a number of .beta.-sheet modified
peptides, wherein each .beta.-sheet modified peptide has a variety
of amino acid substitutions in combination with a variety of
different .beta.-sheet mimetics. In addition, it should also be
recognized that, following cleavage of the .beta.-sheet modified
peptide from the solid support, the Z moiety is AA.sub.3 and the Y
moiety is AA.sub.2 and AA.sub.1 in the above diagram. (While this
diagram is presented for illustration, additional or fewer amino
acids may be linked to the .beta.-sheet mimetic--that is, AA.sub.3
may be absent or additional amino acids my be joined thereto; and
AA.sub.2 and/or AA.sub.1 may be omitted or additional amino acids
may be joined thereto).
[0210] Once a preferred substrate is identified by the procedures
disclosed above, the substrate may be readily converted to an
inhibitor by known techniques. For example, the C-terminal amino
acid (in this case AA.sub.1) may be modified by addition of a
number of moieties known to impart inhibitor activity to a
substrate, including (but not limited to) --CF.sub.3 (a known
reversible serine protease inhibitor), --CH.sub.2Cl (a known
irreversible serine protease inhibitor), --CHN.sub.2 and
--CH.sub.2S(CH.sub.3).sub.2.sup.+ (known cysteinyl protease
inhibitors), --NHOH (a known metalloprotease inhibitor), 154
[0211] (a known cysteinyl protease inhibitor), and 155
[0212] (a known aspartyl protease inhibitor).
[0213] While the utility of the .beta.-sheet mimetics of this
invention have been disclosed with regard to certain embodiments,
it will be understood that a wide variety and type of compounds can
be made which includes the .beta.-sheet mimetics of the present
invention. For example, a .beta.-sheet mimetic of this invention
may be substituted for two or more amino acids of a peptide or
protein. In addition to improving and/or modifying the .beta.-sheet
structure of a peptide or protein, especially with regard to
conformational stability, the .beta.-sheet mimetics of this
invention also serve to inhibit proteolytic breakdown. This results
in the added advantage of peptides or proteins which are less prone
to proteolytic breakdown due to incorporation of the .beta.-sheet
mimetics of this invention.
[0214] More specifically, the .beta.-sheet mimetics of this
invention have broad utility in naturally occurring or synthetic
peptides, proteins and molecules. For example, peptides, proteins
and molecules. For example, the .beta.-sheet mimetics disclosed
herein have activity as inhibitors of kinases and proteases, as
well as having utility as MHC II inhibitors. For example, the
.beta.-sheet mimetics of this invention have activity as inhibitors
of the large family of trypsin-like serine proteases, including
those preferring arginine or lysine as a P' substituent. These
enzymes are involved in hemostasis and include (but are not limited
to) Factor VIIa, Factor IXa, Factor Xa, Factor XIa, thrombin,
kallikrein, urokinase (which is also involved in cancer metastasis)
and plasmin. A related enzyme, tryptase, is involved in
inflammatory responses. Thus, the ability to selectively inhibit
these enzymes has wide utility in therapeutic applications
involving cardiovascular disease, inflammatory diseases, and
oncology.
[0215] For example, compounds of the following structures represent
further embodiments of this invention in the context of Factor VIIa
and thrombin inhibitors.
[0216] Factor VIIa Inhibitors:
5 156 157 Z G 158 159 160 161 162 X and/or X' = halogen,
--SO.sub.2NH.sub.2, --C(.dbd.O)NH.sub.2, --CH.sub.2NAc, --NO.sub.2
163 164 R = --SO.sub.2NH.sub.2, --SO.sub.2CH.sub.3, --SO.sub.2Ar,
--CH.sub.2aryl, --CH.sub.2heteroaryl, --C(.dbd.O)CH.sub.2aryl,
--C(.dbd.O) CH.sub.2heteroaryl R' = ring substituent Y 165 R' 166
167 168 R" 169 170 171 172 173 174 175 176 R''' = alkyl, aryl
[0217] Thrombin Inhibitors:
6 177 178 179 R.sub.2 --H, --CH3 Y 180 181 182 183 184 Z 185 186
187 188 R = R' or R .noteq. R' 189 X = substituent m = 0-4
[0218] In another aspect, the present invention encompasses
pharmaceutical compositions prepared for storage or administration
which comprise a therapeutically effective amount of a .beta.-sheet
mimetic or compound of the present invention in a pharmaceutically
acceptable carrier. Anticoagulant therapy is indicated for the
treatment and prevention of a variety of thrombotic conditions,
particularly coronary artery and cerebrovascular disease. Those
experienced in this field are readily aware of the circumstances
requiring anticoagulant therapy.
[0219] The "therapeutically effective amount" of a compound of the
present invention will depend on the route of administration, the
type of warm-blooded animal being treated, and the physical
characteristics of the specific animal under consideration. These
factors and their relationship to determining this amount are well
known to skilled practitioners in the medical arts. This amount and
the method of administration can be tailored to achieve optimal
efficacy but will depend on such factors as weight, diet,
concurrent medication and other factors which as noted those
skilled in the medical arts will recognize.
[0220] The "therapeutically effective amount" of the compound of
the present invention can range broadly depending upon the desired
affects and the therapeutic indication. Typically, dosages will be
between about 0.01 mg/kg and 100 mg/kg body weight, preferably
between about 0.01 and 10 mg/kg, body weight.
[0221] "Pharmaceutically acceptable carriers" for therapeutic use
are well known in the pharmaceutical art, and are described, for
example, in Remingtons Pharmaceutical Sciences, Mack Publishing Co.
(A. R. Gennaro edit. 1985). For example, sterile saline and
phosphate-buffered saline at physiological pH may be used.
Preservatives, stabilizers, dyes and even flavoring agents may be
provided in the pharmaceutical composition. For example, sodium
benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be
added as preservatives. In addition, antioxidants and suspending
agents may be used.
[0222] Thrombin inhibition is useful not only in the anticoagulant
therapy of individuals having thrombotic conditions, but is useful
whenever inhibition of blood coagulation is required such as to
prevent coagulation of stored whole blood and to prevent
coagulation in other biological samples for testing or storage.
Thus, the thrombin inhibitors can be added to or contacted with any
medium containing or suspected of containing thrombin and in which
it is desired that blood coagulation be inhibited (e.g., when
contacting the mammal's blood with material selected from the group
consisting of vascular grafts, stems, orthopedic prosthesis,
cardiac prosthesis, and extracorporeal circulation systems).
[0223] The thrombin inhibitors can be co-administered with suitable
anti-coagulation agents or thrombolytic agents such as plasminogen
activators or streptokinase to achieve synergistic effects in the
treatment of various vascular pathologies. For example, thrombin
inhibitors enhance the efficiency of tissue plasminogen
activator-mediated thrombolytic reperfusion. Thrombin inhibitors
may be administered first following thrombus formation, and tissue
plasminogen activator or other plasminogen activator is
administered thereafter. They may also be combined with heparin,
aspirin, or warfarin.
[0224] The thrombin inhibitors of the invention can be administered
in such oral forms as tablets, capsules (each of which includes
sustained release or timed release formulations), pills, powders,
granules, elixers, tinctures, suspensions, syrups, and emulsions.
Likewise, they may be administered in intravenous (bolus or
infusion), intraperitoneal, subcutaneous, or intramuscular form,
all using forms well known to those of ordinary skill in the
pharmaceutical arts. An effective but non-toxic amount of the
compound desired can be employed as an anti-aggregation agent or
treating ocular build up of fibrin. The compounds may be
administered intraocularly or topically as well as orally or
parenterally.
[0225] The thrombin inhibitors can be administered in the form of a
depot injection or implant preparation which may be formulated in
such a manner as to permit a sustained release of the active
ingredient. The active ingredient can be compressed into pellets or
small cylinders and implanted subcutaneously or intramuscularly as
depot injections or implants. Implants may employ inert materials
such as biodegradable polymers or synthetic silicones, for example,
Silastic, silicone rubber or other polymers manufactured by the
Dow-Corning Corporation.
[0226] The thrombin inhibitors can also be administered in the form
of liposome delivery systems, such as small unilamellar vesicles,
large unilamellar vesicles and multilamellar vesicles. Liposomes
can be formed from a variety of phospholipids, such as cholesterol,
stearylamine or phosphatidylcholines.
[0227] The thrombin inhibitors may also be delivered by the use of
monoclonal antibodies as individual carriers to which the compound
molecules are coupled. The thrombin inhibitors may also be coupled
with soluble polymers as targetable drug carriers. Such polymers
can include polyvinlypyrrolidone, pyran copolymer,
polyhydroxy-propyl-methacrylamide-- phenol,
polyhydroxyethyl-aspartarnide-phenol, or polyethyleneoxide-polylys-
ine substituted with palmitoyl residues. Furthermore, the thrombin
inhibitors may be coupled to a class of biodegradable polymers
useful in achieving controlled release of a drug, for example,
polylactic acid, polyglycolic acid, copolymers of polylactic and
polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric
acid, polyorthoesters, polyacetals, polydibydropyrans,
polycyanoacrylates and cross linked or amphipathic block copolymers
of hydrogels.
[0228] The dose and method of administration can be tailored to
achieve optimal efficacy but will depend on such factors as weight,
diet, concurrent medication and other factors which those skilled
in the medical arts will recognize. When administration is to be
parenteral, such as intravenous on a daily basis, injectable
pharmaceutical compositions can be prepared in conventional forms,
either as liquid solutions or suspensions, solid forms suitable for
solution or suspension in liquid prior to injection, or as
emulsions.
[0229] Tablets suitable for oral administration of active compounds
of the invention can be prepared as follows:
7 Amount-mg Active Compound 25.0 50.0 100.0 Microcrystalline 37.25
100.0 200.0 cellulose Modified food corn 37.25 4.25 8.5 starch
Magnesium stearate 0.50 0.75 1.5
[0230] All of the active compound, cellulose, and a portion of the
corn starch are mixed and granulated to 10% corn starch paste. The
resulting granulation is sieved, dried and blended with the
remainder of the corn starch and the magnesium stearate. The
resulting granulation is then compressed into tablets containing
25.0, 50.0, and 100.0 mg, respectively, of active ingredient per
tablet.
[0231] An intravenous dosage form of the above-indicated active
compounds may be prepared as follows:
8 Active Compound 0.5-10.0 mg Sodium Citrate 5-50 mg Citric Acid
1-15 mg Sodium Chloride 1-8 mg Water for q.s. to 1 ml Injection
(USP)
[0232] Utilizing the above quantities, the active compound is
dissolved at room temperature in a previously prepared solution of
sodium chloride, citric acid, and sodium citrate in Water for
Injection (USP, see page 1636 of United States
Pharmacopoeia/National Formulary for 1995, published by United
States Pharmacopoeia Convention, Inc., Rockville, Md., copyright
1994).
[0233] Compounds of the present invention when made and selected as
disclosed are useful as potent inhibitors of thrombin in vitro and
in vivo. As such, these compounds are useful as in vitro diagnostic
reagents to prevent the clotting of blood and as in vivo
pharmaceutical agents to prevent thrombosis in mammals suspected of
having a condition characterized by abnormal thrombosis.
[0234] The compounds of the present invention are useful as in
vitro diagnostic reagents for inhibiting clotting in blood drawing
tubes. The use of stoppered test tubes having a vacuum therein as a
means to draw blood obtained by venipuncture into the tube is well
known in the medical arts (Kasten, B. L., "Specimen Collection,"
Laboratory Test Handbook, 2nd Edition, Lexi-Comp Inc., Cleveland
pp. 16-17, Edits. Jacobs, D. S. et al. 1990). Such vacuum tubes may
be free of clot-inhibiting additives, in which case, they are
useful for the isolation of mammalian serum from the blood they may
alternatively contain clot-inhibiting additives (such as heparin
salts, EDTA salts, citrate salts or oxalate salts), in which case,
they are useful for the isolation of mammalian plasma from the
blood. The compounds of the present invention are potent inhibitors
of factor Xa or thrombin, and as such, can be incorporated into
blood collection tubes to prevent clotting of the mammalian blood
drawn into them.
[0235] The compounds of the present invention may be used alone, in
combination of other compounds of the present invention, or in
combination with other known inhibitors of clotting, in the blood
collection tubes. The amount to be added to such tubes is that
amount sufficient to inhibit the formation of a clot when mammalian
blood is drawn into the tube. The addition of the compounds to such
tubes may be accomplished by methods well known in the art, such as
by introduction of a liquid composition thereof, as a solid
composition thereof, or liquid composition which is lyophilized to
a solid. The compounds of the present invention are added to blood
collection tubes in such amounts that, when combined with 2 to 10
mL of mammalian blood, the concentration of such compounds will be
sufficient to inhibit clot formation. Typically, the required
concentration will be about 1 to 10,000 nM, with 10 to 1000 nM
being preferred.
[0236] With respect to regulation of transcription factors, the
compounds of this invention regulate transcription factors whose
ability to bind to DNA is controlled by reduction of a cysteine
residue by a cellular oxidoreductase. In one embodiment, the
transcription factor is NF-.kappa.B. In this embodiment, the
compounds of this invention have activity as mediators of immune
and/or inflammatory responses, or serve to control cell growth. In
another embodiment, the transcription factor is AP-1, and the
cellular oxidoreductase is Ref-1. In this embodiment, the compounds
of this invention have activity as anti-inflammatory and/or
anticancer agents. In yet further embodiments, the transcription
factor is selected from Myb and glucocorticoid receptor. Other
transcription factors that may be regulated within the context of
this invention also include: those of the NFkB family, such as
Rel-A, c-Rel, Rel-B, p50 and p52; those of the AP-1 family, such as
Fos, FosB, Fra-1, Fra-2, Jun, JunB and JunD; ATF; CREB; STAT-1, -2,
-3, -4, -5 and -6; NFAT-1, -2 and -4; MAF; Thyroid Factor; IRF;
Oct-1 and -2; NF-Y; Egr-1; and USF-43.
[0237] In the practice of the methods of this invention, a
therapeutically effective amount of a compound of this invention is
administered to a warm-blooded animal in need thereof. For example,
the compounds of this invention may be administered to a
warm-blooded animal that has been diagnosed with, or is at risk of
developing, a condition selected from Chrohns disease, asthma,
rheumatoid arthritis, ischemia, reperfusion injury, graft versus
host disease (GVHD), amyotrophic lateral sclerosis (ALS),
Alzheimer's disease, allograft rejection and adult T-cell
leukemia.
[0238] The following examples are offered by way of illustration,
not limitation.
EXAMPLES
Example 1
Synthesis of Representative .beta.-Sheet Mimetic
[0239] This example illustrates the synthesis of a representative
.beta.-sheet mimetic of this invention.
[0240] Synthesis of Structure (1) 190
[0241] Phenylalanine benzaldimine, structure (1), was synthesized
as follows. To a mixture of L-phenylalanine methyl ester
hydrochloride (7.19 g, 33.3 mmol) and benzaldehyde (3.4 ml, 33.5
mmol) stirred in CH.sub.2Cl.sub.2 (150 ml) at room temperature was
added triethylamine (7.0 ml, 50 mmol). Anhydrous magnesium sulfate
(2 g) was added to the resulting solution and the mixture was
stirred for 14 h then filtered through a 1 inch pad of Celite with
CH.sub.2Cl.sub.2. The filtrate was concentrated under reduced
pressure to ca. one half of its initial volume then diluted with an
equal volume of hexanes. The mixture was extracted twice with
saturated aqueous NaHCO.sub.3, H.sub.2O and brine then dried over
anhydrous Na.sub.2SO.sub.4 and filtered. Concentration of the
filtrate under vacuum yielded 8.32 g (93% yield) of colorless oil.
.sup.1H NMR analysis indicated nearly pure (>95%) phenylalanine
benzaldimine. The crude product was used without further
purification.
[0242] Synthesis of Structure (2): 191
[0243] .alpha.-Allylphenylalanine benzaldimine, structure (2), was
synthesized as follows. To a solution of diisopropylamine (4.3 ml,
33 mmol) stirred in THF (150 ml) at -78.degree. C. was added
dropwise a solution of n-butyllithium (13 ml of a 2.5 M hexane
solution, 33 mmol). The resulting solution was stirred for 20 min.
then a solution of phenylalanine benzaldimine (7.97 g, 29.8 mmol)
in THF (30 ml) was slowly added. The resulting dark red-orange
solution was stirred for 15 min. then allyl bromide (3.1 ml, 36
mmol) was added. The pale yellow solution was stirred for 30 min.
at -78.degree. C. then allowed to warm to room temperature and
stirred an additional 1 h. Saturated aqueous ammonium chloride was
added and the mixture was poured into ethyl acetate. The organic
phase was separated and washed with water and brine then dried over
anhydrous sodium sulfate and filtered. Concentration of the
filtrate under vacuum yielded 8.54 g of a viscous yellow oil.
Purification by column chromatography yielded 7.93 g (87%) of
.alpha.-allylphenylalanine benzaldimine as a viscous colorless
oil.
[0244] Synthesis of Structure (3): 192
[0245] .alpha.-Allylphenylalanine hydrochloride, structure (3), was
synthesized as follows. To a solution of .alpha.-allylphenylalanine
benzaldimine (5.94 g, 19.3 mmol) stirred in methanol (50 ml) was
added 5% aqueous hydrochloric acid (10 ml). The solution was
stirred at room temperature for 2 h then concentrated under vacuum
to an orange-brown caramel. The crude product was dissolved in
CHCl.sub.3 (10 ml) and the solution was heated to boiling. Hexanes
(.about.150 ml) were added and the slightly cloudy mixture was
allowed to cool. The liquid was decanted away from the crystallized
solid then the solid was rinsed with hexanes and collected. Removal
of residual solvents under vacuum yielded 3.56 g (72%) of pure
.alpha.-allylphenylalanine hydrochloride as a white crystalline
solid. .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.86 (3H, br s),
7.32-7.26 (5H, m), 6.06 (1H, dddd, J=17.5, 10.5, 7.6, 7.3 Hz), 5.33
(1H, d, J=17.5 Hz), 5.30 (1H, d, J=10.5 Hz), 3.70 (3H, s), 3.41
(1H, d, J=14.1 Hz), 3.35 (1H, d, J=14.1 Hz), 2.98 (1H, dd, J=14.5,
7.3 Hz), 2.88 (1H, dd, J=14.5, 7.6 Hz).
[0246] Synthesis of Structure (4) 193
[0247] N-tert-butyloxycarbonyl-.alpha.-allylphenylalanine,
structure (4) was synthesized as follows. To a solution of D,L
.alpha.-allylphenylalani- ne hydrochloride (565 mg, 2.21 mmol)
stirred in a mixture of THF (15 ml) and water (5 ml) was added
di-tert-butyl dicarbonate followed by careful addition of solid
sodium bicarbonate in small portions. The resulting two phase
mixture was vigorously stirred at room temperature for 2 days then
diluted with ethyl acetate. The organic phase was separated and
washed with water and brine then dried over anhydrous sodium
sulfate and filtered. Concentration of the filtrate under vacuum
yielded a colorless oil that was purified by column chromatography
(5 to 10% EtOAc in hexanes gradient elution) to yield 596 mg (86%)
of N-tert-butyloxycarbonyl-.alpha- .-allylphenylalanine.
[0248] TLC R.sub.f=0.70 (silica, 20% EtOAc in hexanes); .sup.1H NMR
(500 MHz, CDCl.sub.3) .delta. 7.26-7.21 (3H, m), 7.05 (2H, d, J=6.1
Hz), 5.64 (1H, dddd, J=14.8, 7.6, 7.2, 7.2 Hz), 5.33 (1H, br s),
5.12-5.08 (2H, m), 3.75 (3H, s), 3.61 (1H, d, J=13.5 Hz), 3.21 (1H,
dd, J=13.7, 7.2 Hz), 3.11 (1H, d, J=13.5 Hz), 2.59 (1H, dd, J=13.7,
7.6 Hz), 1.47 (9H, s)
[0249] Synthesis of Structure (5): 194
[0250] An aldehyde of structure (5) was synthesized as follows.
Ozone was bubbled through a solution of 2.10 g (6.57 mmol) of the
structure (4) olefin stirred at -78.degree. C. in a mixture of
CH.sub.2Cl.sub.2 (50 ml) and methanol (15 ml) until the solution
was distinctly blue in color. The solution was stirred an
additional 15 min. then dimethyl sulfide was slowly added. The
resulting colorless solution was stirred at -78.degree. C. for 10
min. then allowed to warm to room temperature and stirred for 6 h.
The solution was concentrated under vacuum to 2.72 g of viscous
pale yellow oil which was purified by column chromatography (10 to
20% EtOAc in hexanes gradient elution) to yield 1.63 g of pure
aldehyde as a viscous colorless oil.
[0251] TLC R.sub.f=0.3 (silica, 20% EtOAc in hexanes); .sup.1H NMR
(500 MHz, CDCl.sub.3) .delta. 9.69 (1H, br s), 7.30-7.25 (3H, m,),
7.02 (2H, m,), 5.56 (1H, br s), 3.87 (1H, d, J=17.7 Hz,), 3.75 (3H,
s,), 3.63 (1H, d, J=13.2 Hz), 3.08 (1H. d, J=17.7 Hz), 2.98 (1H, d,
J=13.2 Hz,), 1.46 (9H, s,).
[0252] Synthesis of Structure (6): 195
[0253] A hydrazone of structure (6) was synthesized as follows. To
a solution of the aldehyde of structure (5) (1.62 g, 5.03 mmol)
stirred in THF (50 ml) at room temperature was added hydrazine
hydrate (0.32 ml, 6.5 mmol). The resulting solution was stirred at
room temperature for 10 min. then heated to reflux for 3 days. The
solution was allowed to cool to room temperature then concentrated
under vacuum to 1.59 g (105% crude yield) of colorless foam. The
crude hydrazone product, structure (6), was used without
purification.
[0254] TLC R.sub.f=0.7 (50% EtOAc in hexanes); .sup.1H NMR (500
MHz, CDCl.sub.3) .delta. 8.55 (1H, br s), 7.32-7.26 (3H, m), 7.17
(1H, br s), 7.09 (2H, m), 5.55 (1H, br s), 3.45 (1H, d, J=17.7 Hz),
3.29 (1H, d, J=13.5 Hz), 2.90 (1H, d, J=13.5 Hz), 2.88 (1H, dd,
J=17.7, 1.3 Hz), 1.46 (9H, s); MS (CI+, NH.sub.3) m/z 304.1
(M+H.sup.+).
[0255] Synthesis of Structure (7): 196
[0256] A cyclic hydrazide of structure (7) was synthesized as
follows. The crude hydrazone of structure (6) (55 mg, 0.18 mmol)
and platinum oxide (5 mg, 0.02 mmol) were taken up in methanol and
the flask was fitted with a three-way stopcock attached to a rubber
balloon. The flask was flushed with hydrogen gas three times, the
balloon was inflated with hydrogen, and the mixture was stirred
vigorously under a hydrogen atmosphere for 17 hours. The mixture
was filtered through Celite with ethyl acetate and the filtrate was
concentrated under vacuum to a white form. Purification of the
white foam by flash chromatography yielded 44 mg of the pure cyclic
hydrazide of structure (7) (80%).
[0257] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.34-7.28 (3H. m),
7.21 (2H, m), 6.95 (1H, br s), 5.29 (1H, br s), 3.91 (1H, br s),
3.35 (1H, d, J=12.9 Hz), 3.00 (1H, ddd, J=13.9, 5.3, 5.0 Hz), 2.96
(1H, d, J=12.9 Hz), 2.67 (1H, br m), 2.38 (1H, br m), 2.30 (1H,
ddd, J=13.9, 5.4, 5.0 Hz), 1.45 (9H, s); MS (CI+, NH.sub.3) m/z
306.2 (M+H.sup.+).
[0258] Synthesis of Structure (8): 197
[0259] Structure (8) was synthesized as follows. To a solution of
the cyclic hydrazide of structure (7) (4.07 g, 13.32 mmol) stirred
in ethyl acrylate (200 ml) at 90.degree. C. was added formaldehyde
(1.2 mL of a 37% aqueous solution). The mixture was heated to
reflux for 15 h then allowed to cool to room temperature and
concentrated under vacuum to a white foam. The products were
separated by column chromatography (5% then 10% acetone/chloroform)
to yield 0.851 g of the least polar diastereomer of the bicyclic
ester, structure (8b), and a more polar diastereomer (8a). The
impure fractions were subjected to a second chromatography to
afford more pure structure (8b), 25% combined yield.
[0260] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.27-7.21 (3H, m),
7.09 (2H, d, J=6.5 Hz), 5.59 (1H, br s), 4.52 (1H, dd, J=9.1, 3.4
Hz), 4.21 (2H, m)), 3.40 (1H, d, J=12.5 Hz), 3.32 (1H, d, J=12.5
Hz), 3.10 (2H, m), 2.79 (1H, br m), 2.66 (1H, br m),2.79 (1H, br
m), 2.66 (1H, br m), 2.54 (1H, br m), 2.46 (1H, m), 2.18 (1H, m),
1.44 (9H, s), 1.28 (3H, t, J=7.0 Hz); MS (CI+, NH.sub.3) 418.4
(M+H.sup.+) 198
[0261] Synthesis of Structure (9b) 199
[0262] Structure (9b) was synthesized as follows. To a solution of
the least polar ethyl ester (i.e., structure (8b)) (31 mg, 0.074
mmol) stirred in THF (1 ml) was added aqueous lithium hydroxide (1
M, 0.15 ml). The resulting mixture was stirred at room temperature
for 2 h then the reaction was quenched with 5% aqueous citric acid.
The mixture was extracted with ethyl acetate (2.times.) then the
combined extracts were washed with water and brine. The organic
layer was dried over anhydrous sodium sulfate, filtered and
concentrated under vacuum to a colorless glass. The crude acid,
structure (9b), was used in subsequent experiments without further
purification.
[0263] Synthesis of Structure (10b): 200
[0264] Structure (10b) was synthesized as follows. The crude acid
of structure (9b) (30 mg, 0.074 mmol), HArg(PMC)PNA (41 mg, 0.074
mmol), and HOBt (15 mg, 0.098 mmol) were dissolved in THF (1 ml)
then diisopropylethylamine (0.026 ml, 0.15 mmol) was added followed
by EDC (16 mg, 0.084 mmol). The resulting mixture was stirred at
room temperature for 4 h then diluted with ethyl acetate and
extracted with 5% aqueous citric acid, saturated aqueous sodium
bicarbonate, water and brine. The organic layer was dried over
anhydrous sodium sulfate, filtered and concentrated under vacuum to
54 mg of pale yellow glass. The products were separated by column
chromatography to yield 33 mg (50%) of a mixture of diastereomers
of the coupled (i.e., protected) product, structure (lob). MS (CI+,
NH.sub.3) m/z 566.6 (M+H.sup.+)
[0265] Synthesis of Structure (11b): 201
[0266] A .beta.-sheet mimetic of structure (11b) was synthesized as
follows. A solution of 0.25 ml of H.sub.2O, 0.125 ml of
1,2-ethanedithiol and 360 mg of phenol in 5 ml of TFA was prepared
and the protected product of structure (lob) (33 mg, 0.035 mmol)
was dissolved in 2 ml of this solution. The resulting solution was
stirred at room temperature for 3 h then concentrated under reduced
pressure. Ether was added to the concentrate and the resulting
precipitate was collected by centrifugation. The precipitate was
triturated with ether and centrifuged two more times then dried in
a vacuum desiccator for 14 h. The crude product (14 mg) was
purified by HPLC chromatography to yield the .beta.-sheet mimetic
of structure (11b) MS (CI+, NH.sub.3) m/z 954.8 (M+Na.sup.+).
[0267] Synthesis of Structure (12b): 202
[0268] Structure (12b) was synthesized as follows. To a solution of
the crude acid of structure (9b) (24 mg, 0.062 mmol) and
N-methylmorpholine (0.008 ml), stirred in THF (1 ml) at -50.degree.
C. was added isobutyl chloroformate. The resulting cloudy mixture
was stirred for 10 min. then 0.016 ml (0.14 mmol) of
N-methylmorpholine was added followed by a solution of
HArg(Mtr)CH.sub.2Cl (50 mg, 0.068 mmol) in THF (0.5 ml). The
mixture was kept at -50.degree. C. for 20 min. then was allowed to
warm to room temperature during 1 h. The mixture was diluted with
ethyl acetate and extracted with 5% aqueous citric acid, saturated
aqueous sodium bicarbonate and brine. The organic layer was dried
over anhydrous sodium sulfate, filtered and concentrated under
vacuum to yield 49 mg of colorless glass, structure (12).
Separation by column chromatography yielded 12 mg of a less polar
diastereomer and 16 mg of a more polar diastereomer.
[0269] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.93 (1H. br s),
7.39-7.31 (3H, m), 7.16 (2H, d, J=6.9 Hz), 6.52 (1H, s), 6.30 (1H,
br s), 5.27 (1H, s), 4.74 (1H, dd, J=9.1, 6.9 Hz), 4.42 (1H, br d,
J=6.8 Hz), 4.33 (1H, d, J=6.8 Hz), 3.82 (3H, s), 3.28 (1H, d,
J=13.3 Hz), 3.26-3.12 (4H, m)), 2.98 (1H, d, J=13.3 Hz), 2.69 (3H,
s), 2.60 (3H, s), 2.59-2.33 (4H, m),r 2.25-2.10 (3H, m), 2.11 (3H,
s), 1.77 (1H, br m), 1.70-1.55 (3H, br m), 1.32 (9H, s).
[0270] Synthesis of Structure (13b): 203
[0271] A .beta.-sheet mimetic of structure (13b) was synthesized as
follows. The more polar diastereomer of structure (12b) (16 mg,
0.021 mmol) was dissolved in 95% TFA/H.sub.2O (1 ml) and the
resulting solution was stirred at room temperature for 6 h then
concentrated under vacuum to 11 mg of crude material. The crude
product was triturated with ether and the precipitate was washed
twice with ether then dried under high vacuum for 14 h. .sup.1H NMR
analysis indicated a 1:1 mixture of fully deprotected product and
product containing the Mtr protecting group. The mixture was
dissolved in 95% TFA/H.sub.2O and stirred for 2 days and the
product was recovered as above. Purification of the product by HPLC
yielded 5 mg of the pure compound of structure (13b). MS (EI+) m/z
477.9 (M.sup.+)
Example 2
Synthesis of Representative .beta.-Sheet Mimetic
[0272] This example illustrates the synthesis of a further
representative .beta.-sheet mimetic of this invention.
[0273] Synthesis of Structure (14): 204
[0274] N,O-Dimethyl hydroxamate, structure (14), was synthesized as
follows. To a mixture of
Boc-Ng-4-methoxy-2,3,6-trimethylbenzenesulfonyl-- L-arginine (8.26
g, 14.38 mmol), N,O-dimethylhydroxylamine hydrochloride (2.78 g,
28.5 mmol) and 1-hydroxybenzotriazole hydrate (2.45 g, 16.0 mmol)
stirred in THF (150 ml) at ambient temperature was added
N,N-diisopropylethylamine (7.5 ml, 43 mmol) followed by solid EDC
(3.01 g, 15.7 mmol). The resulting solution was stirred for 16 h
then diluted with ethyl acetate (200 ml) and extracted sequentially
with 5% aqueous citric acid, saturated aqueous sodium bicarbonate,
water and brine. The organic solution was dried over anhydrous
sodium sulfate and filtered. Concentration of the filtrate under
vacuum yielded 7.412 g of white foam.
[0275] .sup.1H NMR (500 Mhz, CDCl.sub.3): .delta. 6.52 (1H, s),
6.17 (1H, br s), 5.49 (1H, d, J=8.8 Hz), 4.64 (1H, br t), 3.82 (3H,
s), 3.72 (3H, s), 3.36 (1H, br m), 3.18 (3H, s), 3.17 (1H, br m),
2.69 (3H, s), 2.61 (3H, s), 2.12 (3H, 2), 1.85-1.55 (5H, m), 1.41
(9H, s); MS (FB+): m/z 530.5 (M+H.sup.+).
[0276] Synthesis of Structure (15): 205
[0277] Structure (15) was synthesized as follows. To a solution of
the arginine amide (7.412 g, 13.99 mmol) stirred in dichloromethane
(150 ml) at room temperature was added N,N-diisopropylethylamine
(2.9 ml, 17 mmol) followed by di-tert-butyldicarbonate (3.5 ml,
15.4 mmol) and N,N-dimethylaminopyridine (0.175 g, 1.43 mmol). The
resulting solution was stirred for 1.5 h then poured into water.
The aqueous layer was separated and extracted with two 100 ml
portions of dichloromethane. The combine extracts were shaken with
brine then dried over anhydrous sodium sulfate and filtered.
Concentration of the filtrate under vacuum yielded a white foam
that was purified by flash chromatography to yield 8.372 g of white
foam.
[0278] .sup.1H NMR (500 MHz, CDCl.sub.3): .delta. 9.79 (1H, s),
8.30 (1H, t, J=4.96), 6.54 (1H, s), 5.18 (1H, d, J=9.16 Hz), 4.64
(1H, m), 3.83 (3H, s), 3.74 (3H, s), 3.28 (2H, dd, J=12.6, 6.9 Hz),
3.18 (3H, s), 2.70 (3H, s), 2.62 (3H, s), 2.14 (3H, s), 1.73-1.50
(5H. m), 1.48 (9H, s), 1.42 (9H, s); MS (FB+): m/z 630.6
(M+H.sup.+).
[0279] Synthesis of Structure (16): 206
[0280] The arginal, structure (16), was synthesized as follows. To
a solution of the arginine amide structure (15) stirred in toluene
at -78.degree. C. under a dry argon atmosphere was added a solution
of diisobutylaluminum hydride in toluene (1.0 M, 7.3ml) dropwise
over a period of 15 minutes. The resulting solution was stirred for
30 minutes then a second portion of diisobutylaluminum hydride (3.5
ml) was added and stirring was continued for 15 minutes. Methanol
(3 ml) was added dropwise and the solution was stirred at
-78.degree. C. for 10 minutes then allowed to warm to room
temperature. The mixture was diluted with ethyl acetate (100 ml)
and stirred vigorously with 50 ml of saturated aqueous potassium
sodium tartrate for 2.5 h. The aqueous phase was separated and
extracted with ethyl acetate (2.times.100 ml). The extracts were
combined with the original organic solution and shaken with brine
then dried over anhydrous sodium sulfate and filtered.
Concentration of the filtrate under vacuum yielded a white foam
that was separated by flash chromatography to yield 1.617 g of the
aldehyde as a white foam.
[0281] .sup.1H NMR (500 MHz, CDCl.sub.3): .delta. 9.82 (1H, s),
9.47 (1H, s), 8.35 (1H, br t), 6.55 (1H, 8), 5.07 (1H, d, J=6.9
Hz), 4.18 (1H, br m), 3.84 (3H, s), 3.25 (2H, m), 2.70 (3H, s),
2.62 (3H, s), 2.14 (3H, s), 1.89 (1H, m), 1.63-1.55 (4H, m), 1.49
(9H, s), 1.44 (9H, s); MS (FB+) m/z 571.6 (M+H.sup.+).
[0282] Synthesis of Structure (17): 207
[0283] Hydroxybenzothiazole, structure (17), was synthesized as
follows. To a solution of benzothiazole (1.55 ml, 14 mmol) stirred
in anhydrous diethyl ether (60 ml) at -78.degree. C. under a dry
argon atmosphere was added a solution of n-butyllithium (2.5 M in
hexane, 5.6 ml, 14 mmol) dropwise over a period of 10 minutes. The
resulting orange solution was stirred for 45 minutes then a
solution of the arginal structure (16) (1.609 g, 2.819 mmol) in
diethyl ether (5 ml) was slowly added. The solution was stirred for
1.5 h then saturated aqueous ammonium chloride solution was added
and the mixture was allowed to warm to room temperature. The
mixture was extracted with ethyl acetate (3.times.100 ml) and the
combined extracts were extracted with water and brine then dried
over anhydrous sodium sulfate and filtered. Concentration of the
filtrate under vacuum yielded a yellow oil that was purified by
flash chromatography (30% then 40% ethyl acetate/hexanes eluent) to
yield 1.22 g of the hydroxybenzothiazoles (ca. 2:1 mixture of
diastereomers) as a white foam.
[0284] The mixture of hydroxybenzothiazoles (1.003 g, 1.414 mmol)
was stirred in CH.sub.2Cl.sub.2 (12 ml) at room temperature and
trifluoroacetic acid (3 ml) was added. The resulting solution was
stirred for 1.5 h then concentrated under reduced pressure to yield
1.22 g of the benzothiazolylarginol trifluoroacetic acid salt as a
yellow foam.
[0285] MS (EI+): m/z 506.2 (M+H.sup.+).
[0286] Synthesis of Structure (18b): 208
[0287] The bicyclic compound, structure (18b) was synthesized as
follows. The bicyclic acid of structure (9b) from Example 1 (151
mg, 0.387 mmol) and HOBt hydrate (71 mg, 0.46 mmol) were dissolved
in THF (5 ml) and diisopropylethylamine (0.34 ml, 1.9 mmol) was
added followed by EDC (89 mg, 0.46 mmol). After stirring for ten
minutes a solution of the benzothiazolylarginol trifluoroacetic
acid salt (structure (17) 273 mg, 0.372 mmol) in THF (1 ml) was
added along with a THF (0.5 ml) rinse. The mixture was stirred at
room temperature for 15 h then diluted with ethyl acetate and
extracted sequentially with 5% aqueous citric acid, saturated
aqueous sodium bicarbonate, water and brine. The organic solution
was dried over anhydrous sodium sulfate, filtered and concentrated
under vacuum to 297 mg of a yellow glass. .sup.1H NMR analysis
indicated a mixture of four diastereomeric amides which included
structure (18b).
[0288] MS (ES+): m/z 877 (M.sup.+).
[0289] Synthesis of Structure (19b): 209
[0290] Structure (19b) was synthesized as follows. The crude
hydroxybenzothiazole (247 mg, 0.282 mmol) was dissolved in
CH.sub.2Cl.sub.2 (5 ml) and Dess-Martin periodinane (241 mg, 0.588
mmol) was added. The mixture was stirred at room temperature for 6
h then diluted with ethyl acetate and stirred vigorously with 10%
aqueous sodium thiosulfate for 10 minutes. The organic solution was
separated and extracted with saturated aqueous sodium bicarbonate,
water and brine then dried over anhydrous sodium sulfate and
filtered. Concentration of the filtrate under vacuum yielded 252 mg
of yellow glass. .sup.1H NMR analysis indicated a mixture of two
diastereomeric ketobenzothiazoles which included structure
(19b).
[0291] Synthesis of Structure (20b): 210
[0292] The ketobenzothiazole, structure (20), was synthesized as
follows. Ketobenzothiazole (19) (41 mg, 0.047 mmol) was dissolved
in 95% aqueous trifluoroacetic (0.95 ml) acid and thioanisole (0.05
ml) was added. The resulting dark solution was stirred for 30 hours
at room temperature then concentrated under vacuum to a dark brown
gum. The gum was triturated with diethyl ether and centrifuged. The
solution was removed and the solid remaining was triturated and
collected as above two more times. The yellow solid was dried in a
vacuum desiccator for 2 hours then purified by HPLC (Vydac reverse
phase C-4 column (22.times.250 mm ID). Mobile phase: A=0.05% TFA in
water; B=0.05% TFA in acetonitrile. The flow rate was 10.0 mL/min.
The gradient used was 8% B to 22% B over 25 min, and isochratic at
22% thereafter. The peak of interest (structure (20b)) eluted at 42
minutes) to give 2.5 mg of the deprotected product, structure
(20b).
[0293] MS (ES+): 563.5 (M+H.sup.+).
Example 3
Activity of a Representative .beta.-Sheet Mimetic as a Proteolytic
Substrate
[0294] This example illustrates the ability of a representative
.beta.-sheet mimetic of this invention to selectively serve as a
substrate for thrombin and Factor VII. The .beta.-sheet mimetic of
structure (11b) above was synthesized according the procedures
disclosed in Example 1, and used in this experiment without further
modification.
[0295] Both the thrombin and Factor VII assays of this experiment
were carried out at 37.degree. C. using a Hitachi UV/Vis
spectrophotometer (model U-3000). Structure (11b) was dissolved in
deionized water. The concentration was determined from the
absorbance at 342 nm. Extinction coefficient of 8270 liters/mol/cm
was employed. The rate of structure (11b) hydrolysis was determined
from the change in absorbance at 405 nm using an extinction
coefficient for p-nitroaniline of 9920 liters/mol/cm for reaction
buffers. Initial velocities were calculated from the initial linear
portion of the reaction progress curve. Kinetic parameters were
determined by unweighted nonlinear least-squares fitting of the
simple Michaelis-Menten equation to the experimental data using
GraFit (Version 3.0, Erithacus Software Limited).
[0296] For the thrombin assay, experiments were performed in pH 8.4
Tris buffer (Tris, 0.05M; NaCl, 0.15M). 6.4 NIH units of bovine
thrombin (from Sigma) were dissolved into 10 ml of the assay buffer
to yield 10 nM thrombin solution. In a UV cuvette, 130 to 148 .mu.l
of the buffer and 100 .mu.l of the thrombin solutions were added,
preincubated at 37.degree. C. for 2 minutes, and finally 2 to 20
microliters (to make the final volume at 250 .mu.l) of 0.24 mM
structure (11b) solution was added to initiate the reaction. The
first two minutes of the reactions were recorded for initial
velocity determination. Eight structure (11b) concentration points
were collected to obtain the kinetic parameters. k.sub.cat and
K.sub.M were calculated to be 50 s.sup.-1 and 3 .mu.M,
respectively. k.sub.cat/K.sub.M was found to be 1.67.times.10.sup.7
M.sup.-1 s.sup.-1.
[0297] For the Factor VII assay, pH 8.0 Tris buffer (0.05 M Tris, 5
mM CaCl.sub.2, 0.15 M NaCl, 0.1% TWEEN 20, 0.1% BSA) was used. 10
.mu.l of 20 .mu.M human Factor VIIa (FVIIa) and 22 .mu.M of human
tissue factor (TF) was brought to assay buffer to make 160 nM FVIIa
and TF solutions, respectively. 40 to 48 .mu.l of buffer, 25 .mu.l
of FVIIa and 25 .mu.l TF solution were added to a cuvette, and
incubated at 37.degree. C. for 5 minutes, then 2 to 10 .mu.l of 2.4
mM structure (11b) solution was added to the cuvette to initiate
reaction (final volume was 100 ml). The initial 3 minutes reaction
progress curves were recorded. Five structure (11b) concentration
points were collected. The initial rates were linear least-square
fitted against the concentrations of structure (11b) with GraFit.
The k.sub.cat/K.sub.M was calculated from the slope and found to be
17,500 M.sup.-1s.sup.-1.
[0298] In both the thrombin and Factor VII assay of this
experiment, (D)FPR-PNA was run as a control. Activity of structure
(11b) compared to the control was 0.76 and 1.38 for thrombin and
Factor VII, respectively (Factor VII:
K.sub.cat/K.sub.M=1.27.times.10.sup.4 M.sup.-1 S.sup.-1; thrombin:
K.sub.cat/K.sub.M=2.20.times.10.sup.7 M.sup.-1 S.sup.-1).
Example 4
Activity of a Representative .beta.-Sheet Mimetic as a Protease
Inhibitor
[0299] This example illustrates the ability of a representative
.beta.-sheet mimetic of this invention to function as a protease
inhibitor for thrombin, Factor VII, Factor X, urokinase, tissue
plasminogen activator (t-PA), protein C, plasmin and trypsin. The
.beta.-sheet mimetic of structure (13b) above was synthesized
according to the procedures disclosed in Example 1, and used in
this experiment.
[0300] All inhibition assays of this experiment were performed at
room temperature in 96 well microplates using a Bio-Rad microplate
reader (Model 3550). 0.29 mg of structure (13b) was dissolved into
200 ml of 0.02 N hydrochloric acid deionized water solution. This
solution (2.05 mM) served as the stock solution for all the
inhibition assays. The hydrolysis of chromogenic substrates was
monitored at 405 nm. The reaction progress curves were recorded by
reading the plates typically 90 times with 30 seconds to 2 minute
intervals. The initial rate were determined by unweighted nonlinear
least-squares fitting to a first order reaction in GraFit. The
determined initial velocities were then nonlinear least-square
fitted against the concentrations of structure (13b) using GraFit
to obtain IC.sub.50. Typically, eight structure (13b) concentration
points were employed for IC.sub.50 determination.
[0301] For the thrombin assay, N-p-tosyl-Gly-Pro-Arg-pNA (from
Sigma) was used at 0.5 mM concentration in 16 DMSO (v/v) pH 8.4
Tris buffer as substrate. From structure (13b) stock solution two
steps of dilution were made. First, 1:2000 dilution into 0.02 N
hydrochloride solution, then 1:100 dilution into pH 8.4 Tris
buffer. The final dilution of structure (13b) served as the first
point (10 nM). Seven sequential dilutions were made from the first
point with a dilution factor of 2. Into each reaction well, 100
.mu.l of 10 .mu.M thrombin solution and 50 .mu.l of structure (13b)
solution was added. The mixture of the enzyme and inhibitor was
incubated for 20 minutes, then 100 .mu.l of 0.5 mM substrate
solution was added to initiate the reaction. The IC.sub.50 of
structure (13b) against thrombin was found to be 1.2.+-.0.2 nM.
[0302] In the Factor VII assay, S-2288 (from Pharmacia),
D-Ile-Pro-Arg-pNA was used at 20 .mu.M in deionized water as
substrate. From the stock of structure (13b), a 1:100 dilution was
made into pH 8.0 Tris buffer. This dilution served as the first
point of the inhibitor (20 .mu.M). From this concentration point 6
more sequential dilutions were made with a dilution factor of 2. 50
.mu.l of 16 nM FVIIa and TF complex solution and 40 .mu.l of the
inhibitor solutions were added into each well, the mixtures were
incubated for 20 minutes before 10 .mu.l of 20 mM S-2288 was added.
IC.sub.50 of structure (13b) against factor VII was found to be
140.+-.3 nM.
[0303] In the Factor X assay, buffer and substrate are the same as
used for thrombin assay. A 1:100 dilution was made into pH 8.4 Tris
buffer to serve as the first point. Seven dilutions with a dilution
factor of 2 were made. The assay protocol is the same as for
thrombin except 25 nM of bovine factor Xa (from Sigma) in pH 8.4
Tris buffer was used instead of thrombin. IC.sub.50 of structure
(13b) against factor X was found to be 385.+-.17 nM.
[0304] In the urokinase assay, buffer was pH 8.8 0.05 M Tris and
0.05 M NaCl in deionized water. S-2444 (from Sigma),
pyroGlu-Gly-Arg-pNA at 0.5 mM in water was utilized as substrate.
The same dilution procedure was used as for Factor VII and Factor
X. Assay protocol is the same as for thrombin except 18.5 nM of
human urokinase (from Sigma) was utilized. IC.sub.50 was found to
be 927.+-.138 nM.
[0305] Tissue Plasminogen Activator (t-PA):
[0306] Buffer, substrate and the dilution scheme of structure (13b)
were the same as utilized for Factor VII assay.
[0307] Activated Protein C (aPC):
[0308] Buffer was the same as used in thrombin assay. 1.25 mM
S-2366 in the assay buffer was utilized as substrate. Dilutions of
structure (13b) were the same as in urokinase assay.
[0309] Plasmin:
[0310] Buffer (see thrombin assay); S-2551 (from Pharmacia),
D-Val-Leu-Lys-pNA at 1.25 mM in assay buffer was utilized as
substrate. For dilutions of structure (13b) (see urokinase
assay).
[0311] In the trypsin assay, pH 7.8 Tris (0.10 M Tris and 0.02 M
CaCl.sub.2) was utilized as the buffer. BAPNA (from Sigma) was used
at 1 mg/ml in 1% DMSO (v/v) deionized water solution as substrate.
The same dilutions of structure (13b) were made as for Factor VII
assay. 40 .mu.l of 50 .mu.g/ml bovine trypsin (from Sigma) and 20
.mu.l of structure (13b) solution were added to a reaction well,
the mixture was incubated for 5 minutes before 40 .mu.l of 1 mg/ml
BAPNA was added to initiate the reaction. The IC.sub.50 of
structure (13b) against trypsin was found to be 160.+-.8 nM.
[0312] In the above assays, (D)FPR-CH.sub.2Cl ("PPACK") was run as
a control. Activity of structure (13b) compared to the control was
enhanced (see Table 4).
9 TABLE 4 IC.sub.50 (nM) Enzymes PPACK Structure (13b) Thrombin 1.5
1.2 Factor VII 200 140 Factor X 165 385 Protein C 281 528 Plasmin
699 978 Trypsin 212 16 Urokinase 508 927 t-PA 106 632
[0313] With respect to prothrombin time (PT), this was determined
by incubating (30 minutes at 37.degree. C.) 100 .mu.l of control
plasma (from Sigma) with 1-5 .mu.l of buffer (0.05 M Tris, 0.15 M
NaCl, pH=8.4) or test compound (i.e., PPACK or structure (13b)) in
buffer. Then 200 .mu.l of prewarmed (at 37.degree. C. for .about.10
minutes) thromboplastin with calcium (from Sigma) was rapidly added
into the plasma sample. The time required to form clot was manually
recorded with a stop watch (see Table 5), and was found to be
comparable with PPACK.
10 TABLE 5 PT (second) Concentration PPACK Structure (13b) 0
(Control) 13 13 1 pM -- 13 10 pM -- 17 50 pM -- 18 100 pM -- 23 200
pM -- 24 500 pM 15 27 1 nM 18 30 10 nM 22 31 20 nM 25 -- 30 nM --
31 40 nM 28 -- 50 nM -- 30 60 nM 30 -- 80 nM 31 33
Example 5
Activity of a Representative .beta.-Sheet Mimetic as a Protease
Inhibitor
[0314] This example illustrates the ability of a further
representative .beta.-sheet mimetic of this invention to function
as an inhibitor for thrombin, Factor VII, Factor X, urokinase,
Tissue Plasminogen Activator, Activated Protein C, plasmin,
tryptase and trypsin. The .beta.-sheet mimetic of structure (20b)
above was synthesized according to the procedures disclosed in
Example 2, and used in this experiment.
[0315] All inhibition assays were performed at room temperature in
96 well microplates using Bio-Rad microplate reader (Model 3550). A
1 mM solution of structure (20b) in water served as the stock
solution for all the inhibition assays. The hydrolysis of
chromogenic substrates was monitored at 405 nm. The reaction
progress curves were recorded by reading the plates, typically 60
times with 30 second to 2 minute intervals. Initial rates were
determined by unweighted nonlinear least-squares fitting to a first
order reaction in GraFit (Erithacus Software Limited, London,
England). The determined initial velocities were then nonlinear
least-square fitted against the concentrations of structure (20b)
using GraFit to obtain Ki. The general format of these assays are:
100 ml of a substrate solution and 100 ml of structure (20b)
solution were added in a microplate well, then 50 ml of enzyme
solution was added to initiate the reaction. Typically, eight
structure (20b) concentration points were employed for Ki
determination. The values of Ki of structure (20b) against nine
serine proteases are tabulated in Table 6.
[0316] Thrombin:
[0317] N-p-tosyl-Gly-Pro-Arg-pNA (from Sigma) was used at 0.5 mM
concentration in 1% DMSO (v/v) pH8.0 tris buffer (tris, 50 mM,
TWEEN 20, 0.16, BSA, 0.1%, NaCl, 0.15 M, CaCl.sub.2, 5 mM) as
substrate. From structure (20b) stock solution two steps of
dilution were made, first, 1:100 dilution in water, then 1:50
dilution in the pH8.0 tris buffer to serve as the first point (200
nM). Seven sequential dilutions were made from the first point for
the assay.
[0318] Factor VII:
[0319] S-2288 (from Pharmacia), D-Ile-Pro-Arg-pNA was used at 2.05
mM in the pH 8.0 tris buffer (see thrombin assay). From the stock
of structure (20b), a 1:100 dilution was made in the tris buffer.
From this concentration point seven more sequential dilutions were
made for the assay.
[0320] Factor X:
[0321] Buffer and substrate were the same as used for thrombin
assay. A 1:100 dilution was made in the pH8.0 tris buffer to serve
as the first point. Seven more dilutions from the first were made
for the assay.
[0322] Urokinase:
[0323] Buffer, 50 mM tris, 50 mM NaCl, pH=8.8. S-2444 (from Sigma),
pyroGlu-Gly-Arg-pNA at 0.25 mM in buffer was utilized as substrate.
1:10 dilution in buffer was made from the stock of structure (20b)
as the first point, then seven more dilutions from the first point
were made for the assay.
[0324] Tissue Plasminogen Activator (t-PA):
[0325] Buffer, substrate and the dilution scheme of structure (20b)
were the same as utilized for Factor VII assay.
[0326] Activated Protein C (aPC):
[0327] Buffer was the same as used in thrombin assay. 1.25 mM
S-2366 in the assay buffer was utilized as substrate. Dilutions of
structure (20b) were the same as in urokinase assay.
[0328] Plasmin:
[0329] Buffer (see thrombin assay); S-2251 (from Pharmacia),
D-Val-Leu-Lys-pNA at 1.25 mM in assay buffer was utilized as
substrate. For dilutions of structure (20b) (see urokinase
assay).
[0330] Tryptase:
[0331] 0.1 M tris, 0.2 M NaCl, 0.1 mg/ml heparin, pH=8.0 was
utilized as buffer. 0.5 mM S-2366 (from Pharmacia),
L-pyroGlu-Pro-Arg-pNA in buffer was used as substrate. From the 1
mM stock of structure (20b), 10 mM solution was made in water, then
1 mM solution was made in buffer from the 10 mM solution to serve
as the first concentration point. From this point seven more
dilutions were made for the assay.
[0332] Trypsin:
[0333] Buffer, substrate and the dilution scheme of structure (20b)
were the same as used for thrombin.
11 TABLE 6 K.sub.i (nM) Assay Structure Enzyme Source Conc. (nM)
(20b) thrombin bovine plasma 2 0.66 factor VII human 4 270 factor X
bovine plasma 8 966 urokinase human kidney 3.7 600 t-PA human 10
495 APC human plasma 1 3320 plasmin bovine plasma 4 415 tryptase
human lung 2 12.4 trypsin bovine 5 0.64 pancreas
[0334] As illustrated by the data presented in Table 6 above,
structure (20b) functioned as a good thrombin inhibitor, with good
specificity against fibrinolytic enzymes.
Example 6
Synthesis of Representative .beta.-Sheet Mimetic
[0335] This example illustrates the synthesis of a representative
.beta.-sheet mimetic of this invention having the following
structure (21): 211
[0336] Structure (21) was synthesized as follows. A solution of 48
mg (0.859 mmol) N.sup.a-FMOC-N.sup.e-Cbz-a-ethanal-Lys-Ome
[synthesized from N.sup.e-Cbz-Lys-OMe by the same method used for
the preparation of structure (5) from Phe-OMe], 15.9 mg (0.0859
mmol) Cys-OEt.HCl, and 13.2 .mu.L (0.0945 mmol) TEA were in 0.43 mL
CH.sub.2Cl.sub.2 were stirred under Ar for 2 hr at room
temperature. Bis(bis(trimethylsilyl)amino)tin(I- I) (39.8 .mu.L)
was added and the reaction stirred overnight. The reaction solution
was diluted with 10 mL EtOAc and washed with 6 mL each 10% citrate,
water, and brine. The organic layer was dried over
Na.sub.2SO.sub.4, filtered, and concentrated. The resulting residue
was purified by flash chromatography on silica gel using 40%
EtOAc/hexanes to give, after drying in vacuo, 12.9 mg of colorless
oil (23%) as a mixture of diastereomers by .sup.1H NMR
(CDCl.sub.3). MS ES(+) m/z 658.2 (MH.sup.+, 30), 675.3 (M+Na.sup.+,
100), 696.1 (M+K.sup.+, 45).
Example 7
Synthesis of Representative .beta.-Sheet Mimetic
[0337] This example illustrates the synthesis of a further
representative .beta.-sheet mimetic of this invention.
[0338] Synthesis of Structure (22) 212
[0339] Structure (22) was synthesized as follows. To a stirred
solution of Cbz-Glu(OBn)-OH (5 g, 13.5 mmol) with DMAP (270 mg) and
methanol (3 ml) in dichloromethane (100 ml) was added EDCI (3 g) at
0C. After stirring at 0.degree. C. for 3 h, the solution was
stirred at room temperature (rt) overnight. After concentration,
the residue was taken up into EtOAc (100 ml) and 1N HCl (100 ml).
The aqueous phase was separated and extracted with EtOAc (100 ml).
The combined organic extracts were washed with sat. NaHCO.sub.3
(100 ml), brine (100 ml), dried (MgSO.sub.4), passed through a
short pad of silica gel, and concentrated to provide 4.95 g an oil
(95%). The product was pure enough to use for the next reaction
without any further purification. .sup.1H NMR (CDCl.sub.3) .delta.
2.00 (m, 1H), 2.25 (m, 1H), 2.50 (m, 2H), 3.74 (s, 3H, OCH.sub.3),
4.42 (m, 1H, CHNH), 5.10 and 5.11 (two s, 4H, CH.sub.2Ph), 5.40 (d,
1H, NH), 7.35 (s, 10H, phenyls); MS CI(isobutane) m/z 386
(M+H.sup.+).
[0340] Synthesis of Structure (23): 213
[0341] Structure (23) was synthesized as follows: To a stirred
solution of L-Glu-OH (4.41 g, 30 mmol) with triethylamine (8.4 ml,
60 mmol) in 1,4-dioxane (40 ml) and H.sub.2O (20 ml) was added
Boc.sub.2O (7 g, 32 mmol) at rt. After stirring for 1.5 h, the
solution was acidified with 6N HCl (pH 2), and extracted with EtOAc
(3.times.100 ml). The combined organic extracts were washed with
H.sub.2O (100 ml), brine (50 ml), dried (Na.sub.2SO.sub.4), and
concentrated to provide an oil (9.5 g). Without further
purification, the oil was used in the next reaction.
[0342] A mixture of above oil (9.5 g) with paraformaldehyde (5 g)
and p-TsOH-H.sub.2O (400 mg) in 1,2-dichloroethane (200 ml) was
heated at reflux with a Dean-Stark condenser, which was filled with
molecular sieve 4A, for 6 h. After addition of EtOAc (100 ml) and
sat. NaHCO.sub.3 (50 ml), the solution was extracted with sat.
NaHCO.sub.3 (3.times.50 ml). The combined aqueous extracts were
acidified with 6N HCl (pH 2), and extracted with EtOAc (3.times.100
ml). The combined organic extracts were washed with brine (100 ml),
dried (Na.sub.2SO.sub.4), and concentrated to provide an oil. The
crude oil was purified by flash chromatography (hexane:EtOAc=80:20
to 70:30 to 60:40) to provide an oil (4.04 g, 52%) which solidified
slowly upon standing. .sup.1H NMR (CDCl.sub.3) .delta. 1.49 (s, 9H,
C(CH.sub.3).sub.3), 2.18 (m, 1H, --CH.sub.2CH.sub.2), 2.29 (m, 1H,
CH.sub.2CH.sub.2), 2.52 (m, 2H, --CH.sub.2CH.sub.2--), 4.33 (m, 1H,
NHCHCH.sub.2), 5.16 (d, 1H, J=4.5 Hz, NCH.sub.2O), 5.50 (br, 1H,
NCH.sub.2O); .sup.13C NMR (CDCl.sub.3) .delta. 25.85, 28.29, 29.33,
54.16, 79.10, 82.69, 152.47, 172.37, 178.13; MS (ES+) m/z 260
(M+H.sup.+), 282 (M+Na.sup.+), 298 (M+K.sup.+).
[0343] Synthesis of Structure (24): 214
[0344] Structure (24) was synthesized as follows. To a stirred
solution of 1,1,1,3,3,3-hexamethyldisilazane (2.1 ml, 10 mmol) in
THF (10 ml) was added n-BuLi (4 ml of 2.5M in hexane, 10 mmol) at
0.degree. C. The resulting solution was stirred at the same
temperature for 30 min. After cooling to -78.degree. C., to this
stirred solution was added a solution of carboxylic acid (23) (1.02
g, 3.94 mmol) in THF (10 ml) followed by rinsings of the addition
syringe with 5 ml THF. The resulting solution was stirred at
-78.degree. C. for 1 h, and PhCH.sub.2Br (0.46 ml, 3.9 mmol) was
added. After stirring at -30.degree. C. for 3 h, to this solution
was added 1N HCl (50 ml) and the resulting solution was extracted
with EtOAc (100 ml). The organic extract was washed with brine (50
ml), dried (Na.sub.2SO.sub.4), and concentrated to provide an oil.
The crude product was purified by flash chromatography
(hexane:EtOAc .dbd.80:20 to 60:40 to 50:50) to provide a foamy
solid (1.35 g, 98%): .sup.1H NMR (CDCl.sub.3) .delta. 1.55 and 1.63
(two s, 9H, ratio 1.5:1 by rotamer, OC(CH.sub.3).sub.3), 2.2-2.4
(m, 3H, --CH.sub.2CH.sub.2--), 2.6-2.9 (set of m, 1H,
--CH.sub.2CH.sub.2--), 3.04 (d, 1H, J=13.5 Hz, --CH.sub.2Ph), 3.33
and 3.58 (two d, 1H, J=13 Hz, ratio 2:1, --CH.sub.2Ph), 4.03 (two
d, 1H, J=4 Hz, A of ABq, --NCH.sub.2O--), 4.96 (two d, 1H, J=4 Hz,
B of ABq, --NCH.sub.2O--); MS (ES-) m/z 348 (M-H.sup.+)
[0345] Synthesis of Structure (25): 215
[0346] Synthesis of structure (25) was carried out as follows. To a
stirred solution of carboxylic acid (24) (1.05 g, 3.0 mmol) in dry
THF (5 ml) was added 1,1'-carbonyldiimidazole (500 mg, 3.1 mmol) at
rt. The resulting solution was stirred at rt for 30 min. The
solution of acyl imidazole was used for the next reaction without
purification.
[0347] Meanwhile, to a stirred solution of
1,1,1,3,3,3-hexamethyldisilazan- e (1.6 ml, 7.5 mmol) in THF (5 ml)
was added n-BuLi (3 ml of 2.5 M solution in hexane, 7.5 mmol) at
0.degree. C. After stirring at the same temperature for 30 min, the
solution was cooled to -78.degree. C. To the stirred solution was
added a solution of Cbz-Glu(OBn)-OMe (1.16 g, 3 mmol) in THF (5 ml)
followed by rinsings of the addition syringe with 2 ml THF. The
resulting solution was stirred at the same temperature for 15 min.
To this stirred solution was added the above acyl imidazole in 3 ml
THF. After stirring 30 min. at -78.degree. C., to this solution was
added sat. NH.sub.4Cl (50 ml) and extracted with EtOAc (2.times.75
ml). The combined organic extracts were washed with sat.
NaHCO.sub.3 (50 ml), brine (50 ml), dried (Na.sub.2SO.sub.4),
passed through a short pad of silica gel, and concentrated to
provide an oil. The crude product was purified by flash
chromatography (hexane: EtOAc=90:10 to 80:20 to 70:30 to 60:40) to
provide an oil (1.48 g, 69%): MS (ES+) m/z 734.4
(M+NH.sub.4.sup.+).
[0348] Synthesis of Structure (26a) 216
[0349] Structure (26a) was synthesized as follows. A stirred
solution of above starting keto ester (25) (530 mg, 0.7 mmol) in
EtOH/AcOH (10/1 ml) was treated with 10% Pd/C (ca. 100 mg) under 20
atm pressure of H.sub.2 for 2 days. After filtration through a
short pad of Celite, the filtrate was concentrated and dissolved in
EtOAc (50 ml). The solution was washed with 1N HCl (30 ml), sat.
NaHCO.sub.3 (30 ml), brine (30 ml), dried (Na.sub.2SO.sub.4), and
concentrated to provide an oil. The crude product was purified by
flash chromatography (hexane: EtOAc=80:20 to 60:40 to 50:50 to
20:80 to 0:100) to provide a foamy solid (95 mg, 34%). TLC (EtOAc)
R.sub.f 0.68; NMR (CDCl.sub.3) .delta. 1.38 (two s, 9H,
OC(CH.sub.3).sub.3), 1.63 (s, 1H), 1.75 (m, 2H), 2.05 (m, 5H),
2.1-2.3 (set of m, 1H), 3.00 (d, 1H, J=14 Hz, CH.sub.2Ph), 3.21 (d,
1H, J=13.5 Hz, CH.sub.2Ph), 3.74 (collapsed two s, 4H, OCH.sub.3
and NCH), 4.53 (d, 1H, J=9.5 Hz), 5.01 (br, 1H, NH); MS (ES+) m/z
403 (M+H+), 425 (M+Na.sup.+). Stereochemistry was assigned by 2D
NMR.
[0350] Synthesis of Structure (27a) 217
[0351] Structure (27a) was synthesized as follows. To a solution of
28 mg (0.070 mmol) of the bicyclic ester (26a) stirred in 1 ml THF
at room temperature was added 0.14 ml 1.0 M aqueous lithium
hydroxide solution. The mixture was stirred vigorously for 20 h
then quenched with 5% aqueous citric acid (1 ml). The mixture was
extracted with ethyl acetate (3.times.25 ml) then the combined
extracts were washed with water and brine and dried over anhydrous
sodium sulfate. Filtration and concentration of the filtrate under
vacuum gave 26 mg of white foam, used without further
purification.
[0352] Synthesis of Structure (28a) 218
[0353] Structure (28a) was synthesized as follows. The bicyclic
acid (27a) (26 mg, 0.067 mmol), benzothiazolylarginol
trifluoroacetic acid salt (structure (17) 61 mg, 0.083 mmol) EDC
(21 mg, 0.11 mmol) and HOBt hydrate (16 mg, 0.10 mmol) were
dissolved in THF (5 ml) and diisopropylethylamine (0.34 ml, 1.9
mmol) was added. The mixture was stirred at room temperature for 15
h then diluted with ethyl acetate and extracted sequentially with
5% aqueous citric acid, saturated aqueous sodium bicarbonate, water
and brine. The organic solution was dried over anhydrous sodium
sulfate, filtered and concentrated under vacuum to 60 mg of a
yellow glass. .sup.1H NMR analysis indicated a mixture of four
diastereomeric amides. MS (ES+): m/z 898 (M+Na.sup.+).
[0354] Synthesis of Structure (29a): 219
[0355] A .beta.-sheet mimetic of structure (29a) was synthesized as
follows. The crude hydroxybenzothiazole (28a) (60 mg, 0.068 mmol)
was dissolved in CH.sub.2Cl.sub.2 (2 ml) and Dess-Martin
periodinane (58 mg, 0.14 mmol) was added. The mixture was stirred
at room temperature for 6 h then diluted with ethyl acetate and
stirred vigorously with 10% aqueous sodium thiosulfate for 10
minutes. The organic solution was separated and extracted with
saturated aqueous sodium bicarbonate, water and brine then dried
over anhydrous sodium sulfate and filtered. Concentration of the
filtrate under vacuum yielded 42 mg of yellow glass. .sup.1H NMR
analysis indicated a mixture of two diastereomeric
ketobenzothiazoles.
[0356] The ketobenzothiazole (42 mg, 0.048 mmol) was dissolved in
959 aqueous trifluoroacetic (0.95 ml) acid and thioanisole (0.05
ml) was added. The resulting dark solution was stirred for 18 hours
at room temperature then concentrated under vacuum to a dark brown
gum. The gum was triturated with diethyl ether and centrifuged. The
solution was removed and the solid remaining was triturated and
collected as above two more times. The yellow solid was dried in a
vacuum desiccator for 2 hours then purified by HPLC to give 1.4 mg
of the deprotected product. MS (ES+): 562.4 (M+H.sup.+). HPLC:
(t.sub.R=21.17 min.).
[0357] Synthesis of Structure (26b): 220
[0358] Structure (26b) was synthesized as follows. A stirred
solution of above starting keto ester (25) (615 mg, 0.86 mmol) in
MeOH/AcOH (10/1 ml) was treated with 10% Pd/c (ca. 60 mg) under 20
atm pressure of H.sub.2 for 3 days. After filtration through a
short pad of Celite, the filtrate was concentrated to provide an
oil. The crude product was purified by flash chromatography
(hexane: EtOAc=80:20 to 60:40 to 50:50 to 0:100) to collect the
more polar fraction (50 mg). Rf 0.12 (hexane: EtOAc=60:40); MS
(ES+) m/z 433 (M+H.sup.+).
[0359] Above oil was treated with p-TsOH.H.sub.2O (5 mg) in
1,2-dichloroethane (10 ml) at reflux temperature for 2 days. After
concentration, the oily product was purified by preparative TLC
(hexane: EtOAc=80:20 to 60:40) to give an oil (10 mg). TLC Rf 0.36
(hexane: EtOAc .dbd.60:40); .sup.1H NMR (CDCl.sub.3) .delta. 1.43
(s, 9H), 1.66 (m, 3H), 1.89 (m, 3H), 2.14 (m, 1H), 2.75 (m, 1H),
2.98 (m, 1H, CHN), 3.72 (s, 3H, Me), 4.30 (m, 1H), 5.59 (d, 1H,
NH), 7.1-7.3 (m, SH, phenyl); MS CI(NH.sub.3) 403.2 (M+H.sup.+).
Stereochemistry was assigned by 2D NMR.
[0360] Synthesis of Structure (28b): 221
[0361] Structure (28b) was synthesized as follows. To a solution of
12 mg (0.030 mmol) of the bicyclic ester (26b) stirred in THF 1 ml
at room temperature was added 0.060 ml 1.0 M aqueous lithium
hydroxide solution. The mixture was stirred vigorously for 25 h
then quenched with 5% aqueous citric acid (1 ml). The mixture was
extracted with ethyl acetate (3.times.25 ml) then the combined
extracts were washed with water and brine and dried over anhydrous
sodium sulfate. Filtration and concentration of the filtrate under
vacuum gave 19 mg of white foam.
[0362] The foam, benzothiazolylarginol trifluoroacetic acid salt
(30 mg, 0.041 mmol) EDC (10 mg, 0.052 mmol) and HOBt hydrate (9 mg,
0.059 mmol) were dissolved in THF (2 ml) and diisopropylethylamine
(0.026 ml, 0.15 mmol) was added. The mixture was stirred at room
temperature for 30 h then diluted with ethyl acetate and extracted
sequentially with 5% aqueous citric acid, saturated aqueous sodium
bicarbonate, water and brine. The organic solution was dried over
anhydrous sodium sulfate, filtered and concentrated under vacuum to
28 mg of a yellow glass. .sup.1H NMR analysis indicated a mixture
of four diastereomeric amides. MS (ES+): m/z 898 (M+Na.sup.+).
[0363] Synthesis of Structure (29b) 222
[0364] Structure (29b) was synthesized as follows. The crude
hydroxybenzothiazole (28b) (28 mg) was dissolved in
CH.sub.2Cl.sub.2 (2 ml) and Dess-Martin periodinane (29 mg, 0.071
mmol) was added. The mixture was stirred at room temperature for 18
h then diluted with ethyl acetate and stirred vigorously with 10%
aqueous sodium thiosulfate for 10 minutes. The organic solution was
separated and extracted with saturated aqueous sodium bicarbonate,
water and brine then dried over anhydrous sodium sulfate and
filtered. Concentration of the filtrate under vacuum yielded 32 mg
of yellow glass. .sup.1H NMR analysis indicated a mixture of two
diastereomeric ketobenzothiazoles.
[0365] The ketobenzothiazole (32 mg) was dissolved in 95% aqueous
trifluoroacetic (0.95 ml) acid and thioanisole (0.05 ml) was added.
The resulting dark solution was stirred for 20 hours at room
temperature then concentrated under vacuum to a dark brown gum. The
gum was triturated with diethyl ether and centrifuged. The solution
was removed and the remaining solid was triturated and collected as
above two more times. The yellow solid was dried in a vacuum
desiccator for 2 hours then purified by HPLC to give 1.3 mg of the
deprotected product. MS (FB+): 562.36 (M+H.sup.+); HPLC:
t.sub.R=21.51 min. (Gradient 0 to 90% 0.1% TFA in CH.sub.3CN/0.1%
TFA in H.sub.2O over 40 min.).
Example 8
Activity of Representative .beta.-Sheet Mimetic as a Protease
Inhibitor
[0366] This example illustrates the ability of a further
representative .beta.-sheet mimetic of this invention to function
as an inhibitor for thrombin, Factor VII, Factor X, Factor XI, and
trypsin. The .beta.-sheet mimetics of structures (29a) and (29b)
above were synthesized according to the procedures disclosed in
Example 7, and used in this experiment.
[0367] The proteinase inhibitor assays were performed as described
in Example 5 except as described below for Factor XI. The results
are presented in Table 7.
[0368] Factor XI. The same buffer was utilized in this assay as in
the thrombin assay. 1 mM S-2366 (from Pharmacia),
L-pyroGlu-Pro-Arg-pNA, solution in water was used as substrate.
From a 1 mM stock solution of structure (29a) or (29b) in water, a
1:10 dilution was made in buffer. From this 100 .mu.M solution,
seven serial 1:5 dilutions were made in buffer for assay.
12 TABLE 7 K.sub.i (nM) Enzymes Structure (29a) Structure (29b)
Thrombin 10.4 0.085 Trypsin 0.54 0.20 Factor VII 1800 -- Factor X
4600 17 Factor XI 391 --
Example 9
Activities of Representative .beta.-Sheet Mimetics as a Protease
Inhibitor
[0369] This example illustrates the ability of further
representative .beta.-sheet mimetics of this invention to function
as an inhibitor for thrombin, Factor VII, Factor X, Factor XI,
tryptase, aPC, plasmin, tPA, urokinase and trypsin. The
.beta.-sheet mimetics of structures (20) and (29b) above were
synthesized according to the procedures disclosed in Examples 2 and
7, respectively, and used in this experiment.
[0370] The proteinase inhibitor assays were performed as described
in Example 5 except as described in Example 8 for Factor XI.
The-results are presented in Table 8.
13TABLE 8 Structure (20b) Structure (29b) 223 224 Ki (nM)
Selectivity* Ki (nM) Selectivity* Thrombin 0.65 1 0.085 1 Trypsin
0.62 0.95 0.23 2.7 Factor VII 270 415 200 2353 Factor X 222 342
19.3 227 Factor XI 27.0 42 75.3 886 Tryptase 12.3 18.9 9.0 106 aPC
3320 5108 1250 14706 Plasmin 415 638 251 2953 tPA 495 762 92.9 1093
Urokinase 600 923 335 3941 *selectivity is the ratio of Ki of an
enzyme to the Ki of thrombin
Example 10
Synthesis of Representative .beta.-Sheet Mimetics
[0371] This example illustrates the synthesis of a further
representative .beta.-sheet mimetic of this invention.
[0372] Synthesis of Structure (30): 225
[0373] Structure (30) was synthesized as follows. n-Butyllithium
(700 .mu.L, 1.75 mmol, 2.5M in hexanes) was added over 5 min to a
solution of tris(methylthio)methane (256 .mu.L, 1.95 mmol) in THF
(1 ml) at -78.degree. C. The mixture was stirred for 40 min then
treated with a solution of bis-Boc-argininal (structure (16) from
Example 2) (100 mg, 1.75 mmol) in 2 ml THF, dropwise, over a period
of 5 min. After stirring for 1.5 h, the reaction was quenched with
saturated NH.sub.4Cl solution and allowed to warm to room
temperature. The layers were separated and the aqueous layer
extracted with EtOAc (3.times.), washed with brine (1.times.),
dried (Na.sub.2SO.sub.4) and concentrated. Purification by flash
chromatography (EtOAc:Hexane 1:4) yielded 93 mg (73%) of the
orthothiomethyl ester (structure (30)) and 8 mg of recovered
aldehyde (structure (16)). .sup.1H NMR (500 MHz, CDCl.sub.3.)
.delta. 9.80 (s, 1H), 8.32 (t, J=5.0 Hz, 1H), 6.54 (s, 1H), 5.23
(d, J=9.0 Hz, 1H), 4.0 (m, 1H), 3.84 (s, 3H), 3.64 (br s, 1H), 3.38
(br s, 1H), 3.31 (m, 2H), 2.70 (s, 3H), 2.62 (s, 3H), 2.19 (s, 9H),
2.14 (s, 3H), 1.68-1.50 (m, 4H), 1.49 (s, 9H), 1.43 (s, 9H).
[0374] Synthesis of Structure (31): 226
[0375] Structure (31) was synthesized as follows. A mixture of 77
mg (0.11 mmol) of the orthothiomethyl ester (structure (30)), 117
mg (0.43 mmol) of mercuric chloride, and 39 mg (0.18 mmol) of
mercuric oxide in 2.5 ml of 12:1 methanol/water was stirred at rt
for 4 h. The mixture was filtered through Celite and the residue
washed with EtOAc (3.times.). The filtrate was diluted with water
and extracted with EtOAc (3.times.). The organic layer was washed
twice with 75% NH.sub.4OAc/NH.sub.4Cl, then with NH.sub.4Cl and
dried (Na.sub.2SO.sub.4). The solvent was removed in vacuo and the
residue purified by flash chromatography (EtOAc/Hex, 1:3) to give
48 mg (72%) of the two diastereomers of structure (31) in a 1:2.7
ratio. .sup.1H NMR (500 MHz, CDCl.sub.3) (major diastereomer)
.delta. 9.80 (s, 1H), 8.33 (t, J=5.0 Hz, 1H), 6.54 (s, 1H), 4.66
(d, J=10.5 Hz, 1H), 4.08 (dd, J=5.0, 2.0 Hz, 1H), 3.97 (m, 1H),
3.84 (s, 3H), 3.77 (s, 3H), 3.30 (m, 2H), 3.06 (d, J=5.0 Hz, 1H),
2.70 (s, 3H), 2.63 (s, 3H), 2.14 (s, 3H), 1.68-1.50 (m, 4H), 1.49
(s, 9H), 1.40 (s, 9H); MS (ES+) m/z 631.5 (M+H.sup.+).
[0376] Synthesis of Structure (32): 227
[0377] Structure (32) was synthesized as follows. A solution of 32
mg of the methyl ester (structure (31)) (0.051 mmol) in THF/water
(4 ml, 1:3) was treated with 5 mg (0.119 mmol) of LiOH.H.sub.2O.
After stirring for 45 min, the reaction was diluted with 5% citric
acid and extracted with ethyl acetate (3.times.). The combined
extracts were washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated to give 30 mg (96%) of structure (32) as a white
solid. The product was used without further purification. .sup.1H
NMR 500 MHz, CDCl.sub.3) .delta. 9.80 (br s, 1H), 8.29 (br s, 1H),
6.54 (s, 1H), 5.62 (br s, 1H), 4.08 (m, 1H), 3.82 (s, 3H), 3.27 (br
s, 3H), 2.69 (s, 3H), 2.62 (s, 3H), 2.13 (s, 3H), 1.65-1.50 (m,
4H), 1.48 (s, 9H), 1.37 (s, 9H); MS (ES-) m/z 615.5
(M-H.sup.+).
[0378] Synthesis of Structure (33): 228
[0379] Structure (33) was synthesized as follows. To a solution of
the compound of structure (32) (29 mg, 0.047 mmol), HOBt (8 mg,
0.056 mmol) and EDC (11 mg, 0.056 mmol) in THF (5 ml),
phenethylamine (7 ml, 0.056 mmol) was added followed by
diisopropylethylamine (12 .mu.L, 0.071 mmol). The reaction mixture
was stirred at rt overnight and diluted with 5% citric acid. The
organic layer was separated and the aqueous phase extracted with
EtOAc (3.times.). The combined extracts were washed with a
saturated solution of NaHCO.sub.3, brine, dried over
Na.sub.2SO.sub.4, and filtered. After concentration the crude
product was purified by chromatography (EtOAc/Hex, 1:1) to give 26
mg (77%) of structure (33) over two steps. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 9.84 (s, 1H), 8.34 (t, J=5 Hz, 1H), 7.28 (m,
3H), 7.21 (m, 2H), 7.04 (m, 1H), 6.55 (s, 1H), 5.16 (d, J=8.5 Hz,
1H), 4.56 (d, J=5 Hz, 1H), 4.11 (dd, J=5.0, 3.0 Hz, 1H), 3.98 (m,
1H), 3.84 (s, 3H), 3.66 (m, 1H), 3.51 (m, 2H), 3.17 (m, 1H), 2.81
(t, J=7.5 Hz, 2H), 2.71 (s, 3H), 2.65 (s, 3H), 2.14 (s, 3H),
1.68-1.52 (m, 4H), 1.49 (s, 9H), 1.39 (s, 9H); MS (FAB+) m/z 720.6
(M+H.sup.+) (FAB-) m/z 718.5 (M-H.sup.+).
[0380] Synthesis of Structure (34): 229
[0381] Structure (34) was synthesized as follows. To a solution of
phenethylamide (structure (33), 25 mg, 0.035 mmol) in THF (5 ml)
was added 18 mg of p-toluenesulfonic acid monohydrate (0.093 mmol).
The reaction mixture was stirred at rt overnight to give a baseline
spot by TLC. The solution was concentrated in vacuo, and the
residue washed twice with ether removing excess pTsOH to give
structure (34) as a yellowish-white solid, which was used without
further purification. .sup.1H NMR (500 MHz, CDCl.sub.3) was
consistent with the expected product, however, individual peak
assignment was difficult due to broadening. MS (ES+) m/z 520.4
(M+H.sup.+).
[0382] Structure (34) was reacted with structure (9a) of Example 1
(in an analogous manner to the procedure described in Example 2 for
the synthesis of structure (18)), followed by oxidation and
deprotection (in an analogous manner as described with respect to
the oxidation and deprotection of structures (18) and (19),
respectively) to provide structure (35) as identified in Table 9
below.
Example 11
Synthesis of Representative .beta.-Sheet Mimetics
[0383] This example illustrates the synthesis of a further
representative .beta.-sheet mimetic of this invention.
[0384] Synthesis of Structure (36): 230
[0385] Structure (36) was synthesized in an analogous fashion to
compound (34) starting with benzylamine and structure (32). .sup.1H
NMR (500 MHz, CDCl.sub.3 ) was consistent with the expected
product, however, individual peak assignment was difficult due to
broadening. MS (FAB+) m/z 506.4 (M+H.sup.+).
[0386] Structure (36) was reacted with structure (9a) of Example 1
(in an analogous manner to the procedure described in Example 2 for
the synthesis of structure (18)), followed by oxidation and
deprotection (in an analogous manner as described with respect to
the oxidation and deprotection of structures (18) and (19),
respectively) to provide structure (37) as identified in Table 9
below.
Example 12
Synthesis of Representative .beta.-Sheet Mimetics
[0387] This example illustrates the synthesis of a further
representative .beta.-sheet mimetic of this invention.
[0388] Synthesis of Structure (38): 231
[0389] Structure (38) was synthesized in an analogous fashion to
structure (34) starting with p-chlorophenethylamine and structure
(32). .sup.1H NMR (500 MHz, CDCl.sub.3) was consistent with the
expected product, individual peak assignment was difficult due to
broadening. MS (ES+) m/z 554.5 (M+H.sup.+).
[0390] Structure (38) was reacted with structure (9a) of Example 1
(in an analogous manner to the procedure described in Example 2 for
the synthesis of structure (18)), followed by oxidation and
deprotection (in an analogous manner as described with respect to
the oxidation and deprotection of structures (18) and (19),
respectively) to provide structure (39) as identified in Table 9
below.
Example 13
Synthesis of Representative .beta.-Sheet Mimetics
[0391] This example illustrates the synthesis of a further
representative .beta.-sheet mimetic of this invention.
[0392] Synthesis of Structure (40): 232
[0393] Structure (40) was synthesized in an analogous fashion to
compound (34) using p-methoxyphenethylamine and structure (32).
.sup.1H NMR (500 MHz, CDCl.sub.3) was consistent with the expected
product, however, individual assignment was difficult due to
broadening. MS (ES+) m/z 550.5 (M+H.sup.+).
[0394] Structure (40) was reacted with structure (9a) of Example 1
(in an analogous manner to the procedure described in Example 2 for
the synthesis of structure (18)), followed by oxidation and
deprotection (in an analogous manner as described with respect to
the oxidation and deprotection of structures (18) and (19),
respectively) to provide structure (41) as identified in Table 9
below.
Example 14
Synthesis of Representative .beta.-Sheet Mimetics
[0395] This example illustrates the synthesis of a further
representative .beta.-sheet mimetic of this invention.
[0396] Synthesis of Structure (42): 233
[0397] Structure (42) was prepared as follows. In a 10 ml
round-bottomed flask were added CH.sub.2Cl.sub.2 (10 ml), methyl
2,3-dimethylaminopropio- nate dihydrochloride (19.9 mg, 0.103 mmol,
1.5 eq), and diisopropylethylamine (53 ml, 0.304 mmol, 4.4 eq).
This suspension was stirred magnetically at room temperature for 1
h at which time was added the compound of structure (30) (50 mg,
0.068 mmol, 1 eq), mercury(II)chloride (82.4 mg, 0.304 mmol, 4.4
eq), and mercury(II)oxide (25.7 mg, 0.120 mmol, 1.7 eq). The
resulting yellow suspension was stirred for 16.5 h during which
time the suspension turned gray. The reaction was diluted with
CH.sub.2Cl.sub.2 (50 ml), washed with saturated aqueous NH.sub.4Cl
(5 ml), saturated aqueous NaCl (5 ml) and dried over
Na.sub.2SO.sub.4. The cloudy suspension was filtered and the
solvent removed in vacuo. The white solid was purified on
preparative thin-layer chromatography. to produce the imidazoline
structure (42) (25.3 mg, 52% yield) as a clear amorphous solid.:
R.sub.f 0.11 (10% MeOH/CHCl.sub.3); .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 9.82 (s, 0.6H, N'H, mixture of tautomers), 9.78
(s, 0.4H, N"H), 8.35 (dd, J=4.3, 11 Hz, .sup.1H, N-5), 6.54 (s, 1H,
ArH), 5.08 (d, J=11 Hz, 1H, CHOH), 4.52 (m, 1H, imidazoline
CH.sub.2), 4.38 (d, J=21 Hz, 1H), 3.8-4.0 (m, 2H), 3.86 (s, 3H,
CO.sub.2CH.sub.3), 3.767 (s, 3H, ArOCH.sub.3), 3.5-3.7 (m, 2H, C-5
CH.sub.2), 3.16-3.27 (m, C-5 CH.sub.2), 2.70 (s, 3H, ArCH.sub.3),
2.63 (s, 3H, ArCH.sub.3), 2.14 (s, 3H, ArCH.sub.3), 1.5-1.7 (m, 4H,
C-3 and C-4 CH2), 1.49 (s, 9H, Boc), 1.46 (s, 9H, Boc); IR (film)
1725.56, 1685.68, 1618.36, 1585.45, 1207.09, 1148.85 cm.sup.-1; MS
(ES+) m/e 699.4 (M+H.sup.+).
[0398] Synthesis of Structure (43): 234
[0399] Structure (43) was synthesized as follows. In a 25 ml
round-bottomed flask was placed the compound of structure (42) (230
mg, 0.33 mmol), CHCl.sub.3 (5 ml) and MnO.sub.2 (500 mg, 5.75
mmol,; 17.4 eq). After stirring for 5 h the suspension was filtered
and the solid washed with methanol. The solvent was removed in
vacuo and the residue was dissolved in ethyl acetate (5 ml) and
methanol (1 ml) and a fresh portion of MnO.sub.2 (500 mg) was
introduced and the reaction stirred for 15 h at room temperature.
The solid was filtered and the solvent removed in vacuo. The
residue was purified via column chromatography on silica gel,
eluting with 1:1 ethyl acetate:hexane, then pure ethyl acetate,
then 1:9 methanol:ethyl acetate to obtain the desired product
(structure (43), 190 mg, 83% yield) as an amorphous solid.: R.sub.f
0.64 (70:30-ethyl acetate:hexane); .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 10.70 (bs, 1H, imidazole NH), 9.70 (s, 1H),
8.28 (s, 1H), 7.84 (s, 1H), 6.54 (s, 1H, ArH), 5.35 (m, 1H, aH),
5.25 (s, 1H, BocNH), 3.926 (s, 3H), 3.840 (s, 3H), 3.15-3.40 (m,
2H), 2.682 (s, 3H), 2.133 (s, 3H), 1.52-1.70 (m, 4H), 1.470 (s,
9H), 1.424 (s, 9H); IR (film) 1724.68, 1619.03, 1277.72, 1151.93,
1120.61 cm.sup.-1; MS (ES+) m/e 695.2 (M+H.sup.+, 22), 717.2
(M+Na.sup.+, 100).
[0400] Synthesis of Structure (44): 235
[0401] Structure (44) was synthesized by the same method used to
construct structure (33) to structure (34). The product was used in
the coupling without further purification.
[0402] Structure (44) was reacted with structure (9a) of Example 1
(in an analogous manner to the procedure described in Example 2 for
the synthesis of structure (18)), followed by deprotection (in an
analogous manner as described with respect to the deprotection of
structure (19) respectively) to provide structure (45) as
identified in Table 9 below. In the preparation of structure (45),
the coupling step was performed with the carbonyl compound of
structure (44), rather than with the analogous hydroxy
compound.
Example 15
Synthesis of Representative .beta.-Sheet Mimetics
[0403] This example illustrates the synthesis of a further
representative .beta.-sheet mimetic of this invention.
[0404] Synthesis of Structure (46): 236
[0405] Structure (46) was synthesized in an analogous fashion to
structure (17) starting from structure (16) and thiazole. This
compound was used in the coupling step without further
purification.
[0406] Structure. (46) was reacted with structure (9a) of Example 1
(in an analogous manner to the procedure described in Example 2 for
the synthesis of structure (18)), followed by oxidation and
deprotection (in an analogous manner as described with respect to
the oxidation and deprotection of structures (18) and (19),
respectively) to provide structure (47) as identified in Table 9
below.
Example 16
[0407] Synthesis of Representative .beta.-Sheet Mimetics
[0408] This example illustrates the synthesis of a further
representative .beta.-sheet mimetic of this invention.
[0409] Synthesis of Structure (48): 237
[0410] To a solution of .beta.-Boc-.beta.-Fmoc-2,3-diaminopropionic
acid (818 mg, 1.92 mmol) stirred in THF (5 ml) at -25.degree. C.
was added 4-methylmorpholine (0.23 ml, 2.1 mmol) followed by
isobutylchioroformate (0.25 ml, 1.9 mmol). The resulting suspension
was stirred for 5 minutes and then filtered with the aid of 5 ml of
THF. The filtrate was cooled in an ice/water bath then sodium
borohydride (152 mg, 0.40 mmol) dissolved in water (2.5 ml) was
added dropwise. The mixture was stirred for 15 minutes then water
(50 ml) was added and the mixture was extracted with
CH.sub.2Cl.sub.2 (3.times.50 ml). The combined extracts were washed
with brine, dried over anhydrous sodium sulfate and filtered.
Concentration of the filtrate under vacuum yielded a pale yellow
solid that was purified by flash chromatography (50% ethyl
acetate/hexanes eluent) to give 596 mg of the alcohol as a white
solid.
[0411] The alcohol (224 mg, 0.543 mmol) was. dissolved in methylene
chloride and Dess-Martin periodinane (262 mg, 0.64 mmol) was added.
The mixture was stirred at room temperature for 1 h then diluted
with ethyl acetate (so ml) and extracted sequentially with 10%
aqueous Na.sub.2S.sub.2O.sub.3, saturated aqueous NaHCO.sub.3, and
brine. The organic solution was dried over anhydrous sodium
sulfate, filtered and concentrated under vacuum to a white solid.
Purification of the solid by flash chromatography yielded 169 mg of
the aldehyde structure (48) as a white solid.
[0412] Synthesis of Structure (49): 238
[0413] Structure (49) was synthesized in an analogous fashion to
structure (17) starting from structure (48) and benzothiazole. This
compound was used as a 1:1 mixture of diastereomers in the coupling
step (described below) without further purification. MS (EI+): m/z
446.4 (M+H.sup.+).
[0414] Synthesis of Structure (50): 239
[0415] Structure (49) and bicyclic acid structure (9a) (27 mg,
0.069 mmol) and HOBt hydrate (71 mg, 0.46 mmol) were dissolved in
THF (1 ml) and diisopropylethylamine (0.0.059 ml, 0.34 mmol) was
added followed by EDC (19 mg, 0.099 mmol). The mixture was stirred
at room temperature for 20 h then diluted with ethyl acetate and
extracted sequentially with 5% aqueous citric acid, saturated
aqueous sodium bicarbonate, water and brine. The organic solution
was dried over anhydrous sodium sulfate, filtered and concentrated
under vacuum to 61 mg of a yellow foam. .sup.1H NMR analysis
indicated a mixture of diastereomeric amides.
[0416] The foam was dissolved in CH.sub.3CN and diethylamine was
added. The solution was stirred at room temperature for 30 minutes
then concentrated under vacuum to a yellow foam. The foam was
rinsed with hexanes and dissolved in DMF (0.5 ml). In a separate
flask, carbonyldiimidazole (16 mg, 0.99 mmol) and guanidine
hydrochloride (10 mg, 0.10 mmol) were dissolved in DMF (1 ml) and
diisopropylethylamine (0.035 ml, 0.20 mmol) was added followed by
DMAP (1 mg). The solution was stirred for 1.5 h at room temperature
then the solution of amine was added and stirring was continued for
16 h. The solution was concentrated under vacuum then water was
added to the residue and the mixture was extracted with ethyl
acetate (3.times.25 ml). The combined extracts were washed with
brine, dried over anhydrous sodium sulfate and filtered.
Concentration of the filtrate under vacuum yielded 58 mg of
structure (50) as a yellow foam. MS (ES+): m/z 680.6
(M+H.sup.+).
[0417] Structure (50) was oxidized to provide the corresponding
ketone of structure (51).
Example 17
Activities of Representative .beta.-Sheet Mimetics as a Protease
Inhibitor
[0418] This example illustrates the ability of further
representative .beta.-sheet mimetics of this invention to function
as an inhibitor for thrombin, Factor VII, Factor X, Factor XI,
tryptase, aPC, plasmin, tPA, urokinase thrombin thrombomodulin
complex and trypsin. The .beta.-sheet mimetics of the structures
listed in Table 9 had the inhibition activities shown in Table
10.
[0419] The proteinase inhibitor assays were performed as described
in Example 9. The assay for thrombin- thrombomodulin complex was
conducted as for thrombin except that prior to the addition of
inhibitor and substrate, thrombin was preincubated with 4 nM
thrombomodulin for 20 minutes at room temperature.
14TABLE 9 Structures, Synthetic Precursors, and Physical Data for
Various Serine Protease Inhibitors 240 Struc- ture Number
B.sup..delta. R.sub.4 R.sub.5 241 M.S. (ES+) HPLC* R.T. (min) (47)
N 242 243 (46) 513.5 (M + H.sup.+) 15.9 (20b) N 244 245 (17) 563.5
(M + H.sup.+) 17.9 (37) N 246 247 (36) 563.6 (M + H.sup.+) 16.9
(39) N 248 249 (38) 611.3 (M + H.sup.+) 19.8 (29a).sup..epsilon. CH
250 251 (17) 562.4 (M + H.sup.+) 21.2 (35) N 252 253 (34) 577.4 (M
+ H.sup.+) 18.1 (45) N 254 255 (44) 554.2 (M + H.sup.+) 15.7 (51) N
256 257 (49) 578.3 (M + H.sup.+) 22.3 (29b) CH 258 259 (17) FAB
562.4 (M + H.sup.+) 21.5 (41) N 260 261 (40) 607.4 (M + H.sup.+)
18.2 (13) N 262 263 Arg(Mtr) --CH.sub.2Cl 477.9 (M + H.sup.+) 14.9
.sup..delta.The stereochemistry of the template for B = CH is (3R,
6R, 9S) except where noted (see footnote .epsilon.).
.sup..epsilon.Template stereochemistry is (3S, 6R, 9S). *HPLC was
performed on a reverse phase C-18 column 5 using a gradient of
0-90% acetonitrile/water, 0.1% TFA.
[0420]
15TABLE 10 Ki (M) Inhibition Activity of Various Compounds Against
Serine Proteases Structure Factor Factor Number Thrombin VII Factor
X XI Urokinase T.T.C..sup.a aPC.sup.b Plasmin tPA.sup.c Trypsin
Tryptase 35 7.10E-11 1.64E-08 3.45E-07.sup.e 2.70E-11 37 7.32E-11
7.73E-11 29.sup.b 8.50E-11 2.00E-07 1.93E-08 7.53E-08 3.35E-07
8.80E-11 1.25E-06 2.51E-07 9.29E-08 2.30E-10 9.00E-09 39 3.10E-10
41 4.50E-10 20.sup.b 6.50E-10 2.70E-07 2.22E-07 2.70E-08 6.00E-07
3.32E-06 4.15E-07 4.95E-07 6.20E-10 1.24E-08 47 2.40E-09 9.68E-07
1.50E-06.sup.e 1.90E-09 45 5.40E-09 2.96E-05 3.80E-05 1.24E-06
6.90E-09 2.56E-05 2.38E-05 1.72E-05 5.24E-08 1.65E-06 51 7.25E-09
4.26E-06 5.70E-05 1.73E-06 3.79E-08 29.sup.a 1.04E-08 1.77E-06
4.65E-06.sup.a 3.91E-07 5.40E-10 13.sup.d 1.20E-09 1.40E-07
3.86E-07.sup.e 9.27E-07 5.28E-07 9.78E-07 6.32E.07 1.60E-07
.sup.aThrombin thrombomodulin complex, .sup.bactivated Protein C,
.sup.ctissue Plasminogen Activator, .sup.dIC50, .sup.ebovine
plasma
Example 18
Effect of Representative .beta.-Sheet Mimetics on Platelet
Deposition in a Vascular Graft
[0421] The effect of compounds of the invention on platelet
deposition in a vascular graft, was measured according to the
procedure of Hanson et al. "Interruption of acute
platelet-dependent thrombosis by synthetic antithrombin
D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone" Proc. Natl.
Acad. Sci., USA 85:3148-3188, (1988), except that the compound was
introduced proximal to the shunt as described in Kelly et al.,
Proc. Natl. Acad. Sci., USA 89:6040-6044 (1992). The results are
shown in FIGS. 1, 2 and 3 for structures (20b), (39) and (29b),
respectively.
Example 19
Synthesis of Representative .beta.-Sheet Mimetics
[0422] This example illustrates the synthesis of a further
representative .beta.-sheet mimetic of this invention having the
structure shown below. 264
[0423] Structure (52) may be synthesized employing the following
intermediate (53) in place of intermediate (16) in Example 2:
265
[0424] Intermediate (53) may be synthesized by the following
reaction scheme: 266
[0425] Alternatively, intermediate (53) may be synthesized by the
following reaction scheme: 267
Example 20
Representative .beta.-Sheet Mimetics Which Bind to MHC I and MHC
II
[0426] The following structures (54), (55) and (56) were
synthesized by the techniques disclosed herein.
[0427] The ability of structures (54) and (55) to bind to MHC I
molecules can be demonstrated essentially as described by Elliot et
al. (Nature 351:402-406, 1991). Similarly, the ability of structure
(56) to bind to MHC II molecules can be demonstrated by the
procedure of Kwok et al. (J. Immunol. 155:2468-2476, 1995). 268 269
270
Example 21
Representative .beta.-Sheet Mimetics Which Bind the SH2 Domain
[0428] The following structure (57) was synthesized, and structure
(58) may be synthesized, by the techniques disclosed herein.
271
[0429] MS ES(-) 104.3 (M-H.sup.+);HPLC R.sub.t 17.28' (0-90%
acetonitrile/H.sub.2O, 0.1% TFA). 272
[0430] The ability of structure (58) to bind to the SH2 domain of
STAT6, or of structure (57) to bind the SH2 domain of the protein
tyrosine phosphatase SH-PTP1 can be demonstrated by the procedures
disclosed by Payne et al. (PNAS 90:4902-4906, 1993). Libraries of
SH2 binding mimetics may be screened by the procedure of Songyang
et al. (Cell 72:767-778, 1993).
Example 22
Representative .beta.-Sheet Mimetics Which Bind Protein Kinases
[0431] The following structure (59) may be synthesized by the
techniques disclosed herein. 273
[0432] The ability of structure (59) to act as a substrate or
inhibitor of protein kinases may be demonstrated by the procedure
of Songyang et al. (Current Biology 4:973-982, 1994).
Example 23
Synthesis of Representative .beta.-Sheet Mimetics
[0433] This example illustrates the synthesis of representative
.beta.-sheet mimetics of this invention having the following
structure (60) through (63), wherein B is N or CH: 274
[0434] Synthesis of Structure (60): 275
[0435] Synthesis of Structure (61): 276
[0436] Alternative Synthesis of Structure (61): 277
[0437] Synthesis of Structure (62) 278
[0438] Alternative Synthesis of Structure (62): 279
[0439] Synthesis of Structure (63): 280
Example 24
Bioavailability of Representative .beta.-Sheet Mimetics
[0440] This example illustrates the bioavailability of the compound
of structure (20b) as synthesized in Example 2 above, and having
the biological activity reported in Example 9 above.
[0441] Specifically, a pharmacodynamic and pharmacokinetic study of
structure (20b) was conducted in male Sprague Dawley rats. Rats
were administered a saline solution of structure (20b) at 4 mg/kg
intravenously (IV) or 10 mg/kg orally (PO). Groups of rats (n
.dbd.3 or 4) were sacrificed and exsanguinated at 0.25, 0.5, 1, 2,
4 and 8 hours following dosing. Efficacy parameters, aPTT and TT,
were measured for each plasma sample. Concentrations of structure
(20b) in plasma were determined by a trypsin inhibition assay. The
results of this experiment are presented in FIGS. 4A and 4B for
dosing of 4 mg/kg IV and 10 mg/kg PO, respectively. The data
presented in FIGS. 4A and 4B illustrate in vivo efficacy of
structure (20b) via both IV and PO administration.
Non-compartmental pharmacokinetic analysis of mean structure (20b)
concentration values demonstrate terminal halflives of 7.5 hr (IV)
and 4.5 hr (PO). The bioavailability of orally administered
structure (20b) is approximately 27%.
Example 25
Synthesis of Representative .beta.-Sheet Mimetics
[0442] This example illustrates the synthesis of a further
representative .beta.-sheet mimetics of this invention having the
structure shown below.
[0443] Synthesis of Structure (64) 281
[0444] Structure (64) was synthesized as follows. A 150 ml round
bottom flask was charged with 5.19 grams (24.7 mmol) of
1,2,3-benzene tricarboxylic acid, 75 ml of toluene, and 3.3 ML
(24.7 mmol) of triethyl amine. The reaction was heated at reflux
for 3 hours with the azeotropic removal water. At this time 2.07 ml
of aniline was added, and the reaction again refluxed for six hours
with the azeotropic removal of water. Upon cooling the reaction
solution a crystalline product formed and was filtered off (4.68
g). The solution was then extracted with NaHCO.sub.3 and ethyl
acetate, and the bicarbonate layer acidified and reextracted with a
second EtOAc wash. The organic layer was dried over NaSO.sub.4,
filtered, and the solvent removed to give an additional 1.24 grams
of product. The total yield was 5.92 g (82%). .sup.1H NMR
(CDCl.sub.3) .delta. 7.41, (d, 2H, J=10 Hz), 7.48 (t, 1H, J=10 Hz),
7.55 (t, 2H, J=10 Hz), 7.98 (t, 1H, J=10 Hz ), 8.20 (d, 1H, J=10
Hz), 8.70 (d, 1H, J=10 Hz); MS (ES-) 266 (M-H.sup.+).
[0445] Synthesis of Structure (65) 282
[0446] Structure (65) was synthesized as follows. The imide-acid of
structure (64) (53.4 mg, 0.2 mmol) in THF (2 ml) was cooled to
-40.degree. C. and treated with 24.2 .mu.l (0.22 mmol) of NMM and
28.2 .mu.l IBCF (0.22 mmol). The reaction was stirred for 3 minutes
and then 0.69 ml (0.69 mmol) of a 1 M solution of diazomethane in
ether was added. The temperature was slowly raised to -20 degrees,
and the reaction stirred for 2 h at this temperature. The reaction
was warmed to 0.degree. C. and stirred for 3 h more.
[0447] The reaction was diluted with EtOAc (30 ml) and the organic
phase washed with 5% citric acid, NaHCO.sub.3, and saturated NaCl.
It was then dried over Na.sub.2SO.sub.4 and concentrated to give
62.4 mg of residue. This crude product was dissolved in THF, cooled
to -40.degree. C., and treated with 74 ul of a 4 M solution of HCl
in dioxane. The reaction was warmed to -20.degree. C. and stirred
for 1 h. Subsequently the reaction was stirred for 2 h at 0.degree.
C. TLC of the reaction mixture at this point showed disappearance
of the starting diazoketone. The solvent was removed, and the
product purified by preparative TLC (EtOAc/hexanes, 7/3) to give
22.6 mg (38%) of pure chloromethylketone. .sup.1H NMR (CDCl.sub.3)
.delta. 4.93 (s, 2H), 7.35-7.60 (m, 5H), 7.9 (m, 2H), 8.12 (dd, 1H,
J=9, 1.8 Hz); MS (EI); 299.1 (M.sup.+), 264.0 (M.sup.+-Cl), 250.2
(M.sup.+-CH.sub.2Cl).
[0448] Synthesis of Structure (66): 283
[0449] Structure (66) was synthesized as follows. To a stirred
suspension of 910 mg (5.14 mmol) of 4-phenyl urazole in 50 ml of
methylene chloride, was added 1.654 g (5.14 mmol) of iodobenzene
diacetate. A deep red color developed, and with stirring, all
material went into solution. After stirring for 15 minutes at room
temperature, 560 mg of 90% pure 2,4-penatdienoic acid was added and
the color gradually faded as a white solid formed. After fifteen
minutes an additional 70 mg of pentadienoic acid was added. After
stirring for 2 h at room temperature, the methylene chloride was
removed under reduced pressure. Ether was added (25 ml) and the
resulting suspension was cooled to -20.degree. C. and solid
material (1.41 g, 100%) filtered off. The product could be
recrystallized from EtOAc/cyclohexane. .sup.1H NMR (CDCl.sub.3)
.delta. 4.04, (d, 1H, J=20 Hz), 4.40 (d, 1H, J=20 Hz), 5.17 (s,
1H), 6.13 (m, 2H) 7.4-7.5 (m, SH); MS (ES-): 271.9 (M-H.sup.+),
228.1 (M--CO.sub.2H).
[0450] Synthesis of Structure (67): 284
[0451] Structure (67) was synthesized as follows. The Diels-Alder
adduct of structure (66) (432 mg, 1.57 mmol) was mixed with 150 mg
10% Pd/C in 50 ml MeOH. The reaction was stirred overnight under a
hydrogen atmosphere (hydrogen balloon). After 18 h, an aliquot (1
ml) was removed and the solvent evaporated under reduced pressure.
.sup.1H NMR of the residue showed greater than 95% conversion to
the saturated product. The reaction mixture was filtered through
celite, and the solvent removed via rotary evaporator, to give 424
mg of crystalline product. .sup.1H NMR (CDCl.sub.3) .delta. 1.72
(m, 1H), 1.91 (m, 1H), 2.02 (m, 1H), 2.31 (m, 1H) 3.18 (m, 1H);
4.18 (d, 1H, J=10 Hz), 4.88 (d, 1H, J=12 Hz), 7.35-7.5 (m, 5H); MS
(ES-) 274 (M-H.sup.+).
[0452] Synthesis of Structure (68) 285
[0453] Structure (68) was synthesized as follows. To a solution of
450 mg (1.64 mmol) of (67) in 40 ml of methylene chloride was added
142 .mu.L of oxalyl chloride (1.64 mmol) and a drop of DMF. The
reaction was stirred at room temperature overnight under Ar. The
methylene chloride was removed via rotary evaporator and 30 ml of
THF added. This solution was cooled to -20 degrees and 2 ml of a 1
M solution of diazomethane in ether added. This was stirred 4 h,
while gradually warming to room temperature. The reaction was then
cooled to -78 degrees, and 500 uL of 4 M HCl in dioxane added. The
reaction was again stirred under Ar while gradually warming to room
temperature. Solvents were removed under reduced pressure to give a
mixture (by .sup.1H NMR analysis) of chloromethylketone and methyl
ester. This was chromatographed on silica gel (EtOAc) to give 185
mg (36%) of chloromethylketone. .sup.1H NMR (CDCl.sub.3) .delta.
1.62 (m, 1H), 1.86 (m, 1H), 2.08 (m, 1H), 2.39 (m, 1H), 3.26 (m,
1H), 3.97 (m, 1H), 4. 20 ({fraction (1/2)} of AB quartet, 1H, J=15
Hz), 4.26 ({fraction (1/2)} of AB quartet, 1H, J=15 Hz), 4.94 (m,
1H), 7.35-7.55 (m, 5H); MS (ES+): 308 (M +H+), 330
(M+Na.sup.+).
[0454] Synthesis of Structure (69): 286
[0455] Structure (69) was synthesized as follows. To 4-phenyl
urazole (1.179 g, 6.65 mmol) in 60 ml methylene chloride 2.14 g of
iodobenzene diacetate (6.64 mmol) was added and the reaction
mixture stirred at room temperature. A deep red color developed as
all the solids gradually dissolved. After about 15 minutes, 640 mg
of sorbinal (6.66 mmol) in 10 ml methylene chloride was added to
the reaction flask, and the red color slowly faded. After two
hours, the methylene chloride was removed under reduced pressure.
Ether (30 ml) was added to the resulting residue, and cooled to -20
degrees overnight. The solid material (1.55 g, 86% yield) formed
was collected on filter paper. .sup.1H NMR (CDCl.sub.3) 1.54 (d,
3H, J=7.5 Hz), 4.57 (m, 1H), 4.90 (m, 1H) 5.86 (m, 1H), 6.09 (m,
1H), 7.38 (m, 1H), 7.50 (t, 2H), 7.58, (m, 2H), 9.6 (s, 1H); MS
(CI, NH.sub.3): 272 (M +H+), 289 (M+NH.sub.4.sup.+).
[0456] Synthesis of Structure (70): 287
[0457] To 0.78 grams (3.0 mmol) of the acid of structure (64) in a
100 ml round-bottomed flask was added 20 ml THF and the reaction
mixture was cooled to -20 C. 4-Methyl morpholine (0.34 ml, 3.0
mmol) was added and was followed by the addition of 0.42 ml (3.3
mmol) isobutylchloroformate. The resultant suspension was stirred
for 5 min, and then a suspension of 0.34 grams (9.0 mmol) of
sodiumborohydride in 0.9 ml water was added rapidly. After 4-5 min,
40 ml of water were added and the suspension was extracted with 125
ml of ethylacetate. The EtOAc layer was then washed with water and
brine and dried over MgSO.sub.4. Filtration and solvent evaporation
provided the crude alcohol.
[0458] The crude alcohol was dissolved in 40 ml dichloromethane and
2.0 grams (4.7 mmol) of Dess-Martin periodinane reagent were added
at room temperature. The reaction was stirred for 2 h, diluted with
40 ml dichloromethane and washed with 3 x 20 ml 1:1 (by volume)
solution of 10% sodiumbicarbonate and 10% sodiumthiosulfate,
1.times.40 ml water, 1.times.40 ml brine and dried over magnesium
sulfate. Filtration, solvent evaporation, and flash chromatography
using 30% EtOAc/hexanes afforded the pure aldehyde (0.5 g, 67% 2
steps). .sup.1H NMR (CDCl.sub.3, 500 Mhz) d 11.09 (s, 1H), 8.33
(dd, 1H, J=8, 1 Hz), 8.20 (dd, 1H, J=8, 1 Hz), 7.93 (t, 1H, J=8
Hz), 7.54 (m, 2H), 7.45 (m, 3H).
[0459] Synthesis of Structure (71) 288
[0460] To 3 ml tetrahydrofuran in a 25 ml round-bottomed flask was
added 0.066 ml (0.69 mmol) of methyl propiolate and the solution
was cooled to -78.degree. C. n-Butyl lithium (0.28 ml, 0.69 mmol)
was added dropwise and the reaction allowed to stir for 7-10 min at
which point a 3 ml dichloromethane solution of 0.15 g (0.6 mmol) of
the aldehyde of structure (70) was rapidly added. The reaction was
stirred at -78.degree. C. for 35-45 min then it was quenched with
1.5 ml of saturated ammonium chloride solution. The organic
solvents were removed under reduced pressure and the aqueous layer
was extracted with 24 ml of EtOAc which in turn was washed with
brine. The organic layer was dried over sodium sulfate, filtered,
and the solvent evaporated under reduced pressure to afford the
crude product. Preparative TLC purification using 40% EtOAc/hexanes
afforded product (107 mg, 47%). .sup.1H NMR (CDCl.sub.3, 500 MHz) d
7.98 (dd, 1H, J=7.0, 1.0 Hz), 7.88 (dd, 1H, J=7.5, 1 Hz), 7.83 (d,
1H, J=7.0 Hz), 7.54 (m, 2H), 7.45 (m, 3H), 6.01 (d, 1H, J=9 Hz),
5.02 (d, 1H, J=9 Hz), 3.78 (s, 3H). MS (EI) 335 (M+), 275.
Example 26
Activity of Representative .beta.-Sheet Mimetics
[0461] In this example, the compounds of Example 25 were assayed
for inhibition of TNF induced V-CAM expression in human umbilical
vein entothelial cells (HUVEC). Upon stimulation with inflammatory
cytokines, HUVEC express cell surface adhesion molecules, including
E-selectin, V-CAM, and I-CAM. Proteasome antagonists inhibit
TNF.alpha. induced expression of these adhesion molecules, thereby
providing a mechanism for regulating leucocyte adhesion and the
inflammatory response.
[0462] More specifically, compounds (65), (68), (69) and (71) were
assayed by the procedures set forth by Deisher, Kaushansky and
Harlan ("Inhibitors of Topoisomerase II Prevent Cytokine-Induced
Expression of Vascular Cell Adhesion Molecule-1, While Augmenting
the Expression of Endothelial Leukocyte Adhesion Molecule-1 on
Human Umbilical Vein Endothelial Cells," Cell Adhesion Commun.
1:133-42, 1993) (incorporated herein by reference), with the
exception that tetramethyl benzidine was used in place of
o-phenylenediamine-peroxide.
[0463] The results of this experiment are as follows: compound
(65), 9.6.+-.0.1 .mu.M; compound (68), 14.2.+-.0.8 .mu.M; compound
(69), 32.4.+-.1.7 .mu.M; and compound (71) 4.9.+-.0.18 .mu.M.
Example 27
Synthesis of Representative Linkers Used in the Solid Phase
Synthesis of .beta.-Sheet Mimetics
[0464] This example illustrates the synthesis of linkers used in
the solid-phase synthesis of .beta.-sheet mimetics. 289
[0465] Synthesis of Structure (72): 290
[0466] In a 500 mL round-bottomed flask were placed
tris(methylthio)methyl arginol (30) (10.70 g, 14.8 mmol) and
CH.sub.2Cl.sub.2 (20 mL) with magnetic stirring. In a 125 mL
Erlenmeyer flask were placed cysteine methyl ester hydrochloride
(3.81 g, 22.2 mmol), CH.sub.2Cl.sub.2 (50 mL), and
diisopropylethylamine (8.5 mL, 6.3 g, 48.7 mmol). This mixture was
stirred until the cysteine methyl ester had dissolved (25 min), and
the solution appeared as a faintly cloudy suspension of
diisopropylethylamine hydrochloride. This suspension was added to
the flask containing the arginol and additional CH.sub.2Cl.sub.2
(100 mL) was added to the reaction. HgCl.sub.2 (17.7 g, 65.1 mmol)
and HgO (5.46 g, 25.2 mmol) were added to the reaction mixture and
the suspension was stirred rapidly enough so that the mercury salts
remained suspended. The flask was lightly capped and stirred at
room temperature for 22 h, by which time the starting material had
been consumed. The yellow solution was quenched with saturated
ammonium chloride and diluted with CH.sub.2Cl.sub.2. The layers
were separated and the aqueous layer extracted 2.times. with
CH.sub.2Cl.sub.2. The combined organic layers were dried over
Na.sub.2SO.sub.4/MgSO.sub.4 and filtered through a pad of silica
gel. The solvents were removed in vacuo and the residue purified
two successive times on silica gel, the first time eluting with 7:3
ethyl acetate/hexane, and the second time eluting with 1:1 ethyl
acetate/hexane, then 7:3 ethyl acetate/hexane. The combined
purifications afforded 7.97 g (75% yield) of the
N.sub..alpha.,N.sub.G-bisBoc-N.sub.G'-- Mtr-1-[(4-carboxymethyl)
thiazolin-2-yl] arginol as a pale yellow foam.: .sup.1H NMR (500
MHz, CDCl.sub.3) .delta. 9.81 (s; 1H, N.sub.G--H), 8.30 (t, J=5.5
Hz, 1H, N.sub.d--H), 6.54 (s, 1H, ArH), 5.12 (t, J=8.5 Hz, 1H,
CHOH), 4.95 (d, J=8.0 Hz, 1H, BocNH), 4.45 (bs, 1H, NCHCO2Me), 3.83
(s, 3H, ArOCH.sub.3), 3.80 (s, 3H, CO.sub.2CH.sub.3), 3.64 (dd,
J=11.5, 9.0 Hz, 1H, CH.sub.2S), 3.58 (t, J=8.5 Hz, 1H, CH.sub.2S),
3.37-3.31 (m, 1H, CH.sub.2-guanidine), 3.31-3.25 (m, 1H,
CH.sub.2-guanidine), 2.70 (s, 3H, ArCH.sub.3), 2.63 (s, 3H,
ArCH.sub.3), 2.14 (s, 3H, ArCH.sub.3), 1.54-1.70 (m, 4H,
C.sub..beta.H and C.sub..gamma. H), 1.49 (s, 9H, N.sub.G Boc), 1.40
(s, 9H, N.sub..alpha. Boc), C.sub..alpha.H not observed.
[0467] Synthesis of Structure (73): 291
[0468] A 300 mL round-bottomed flask was charged with chloroform
(20 mL) and arginol (72) (7.97 g, 11.1 mmol), and equipped for
magnetic stirring. Manganese(IV)dioxide (9.65 g, 111 mmol, 10 eq.)
was added, and the flask was stoppered. Additional chloroform (10
mL) was added, and the suspension was vigorously stirred for 8 h at
room temperature after which time it was filtered through silica
gel, rinsing with ethyl acetate. The solvent was removed in vacuo
and the residue was purified by column chromatography on silica
gel, (45:55 EtOAc/hexane) to give
N.sub..alpha.,N.sub.G-bisBoc-N.sub.G'-Mtr-1-[(4'-carboxymethyl)
thiazol-2-yl] arginol (4.83 g, 61% yield) as a pale yellow
amorphous solid, and 1.89 g (24%) of recovered starting material.:
.sup.1H NMR (500 MHz, CDCl.sub.3) d 9.84 (s, 1H, N.sub.G--H), 8.36
(bs, 1H, N.sub..delta.--H), 8.15 (s, 1H, SCH.dbd.C), 6.54 (s, 1H,
ArH), 5.10 (d, J=8.5 Hz, 1H, BocN.sub..alpha.H), 3.95 (s, 3H,
ArOCH.sub.3), 3.95-3.87 (m, 1H, C.sub.aH), 3.83 (s, 3H,
CO.sub.2CH.sub.3), 3.43-3.33 (m, 1H, CH.sub.2-guanidine), 3.33-3.25
(m, 1H, CH.sub.2-guanidine), 2.70 (s, 3H, ArCH.sub.3), 2.63 (s, 3H,
ArCH.sub.3), 2.14 (s, 3H, ArCH.sub.3), 1.80-1.55 (m, 4H,
C.sub..beta.H and C.sub..gamma. H), 1.50 (s, 9H, N.sub.GBoc), 1.35
(s, 9H, N.sub..alpha.Boc); IR (neat) 3328, 1727, 1619, 1566, 1278,
1242, 1152, 1121 cm.sup.-1; MS (ES+) m/z 714 (M+H.sup.+, 100) 736
(M+Na.sup..alpha., 9), 716 (35), 715 (45).
[0469] Synthesis of Structure (74): 292
[0470] To a 25 mL conical flask containing H.sub.20 (1 mL) was
added 2.0N LiOH (0.25 mL, 0.50 mmol, 1.5 eq.) and
N.sub..alpha.,N.sub.G-bisBoc-N.sub-
.G'-Mtr-1-[(4'-carboxymethyl)thiazol-2-yl] arginol (238 mg, 0.33
mmol) as a solution in THF (1 mL). A second portion of THF (1 mL)
was used to rinse the flask containing the arginol and added to the
reaction. The homogeneous mixture was magnetically stirred at room
temperature for 6.5 h at which time 5% HCl (0.34 mL, 0.55 mmol) and
ethyl acetate (10 mL) were added. The organic layer was separated
and the aqueous layer extracted with 2.times.10 mL ethyl acetate.
The combined organic layers were washed with saturated NaCl and
dried over Na.sub.2SO.sub.4. The solvent was removed to afford 212
mg (92% yield) of
N.sub..alpha.,N.sub.G-bisBoc-N.sub.G'-Mtr-1-[(4'-carboxylic
acid)thiazol-2-yl] arginol as a pale yellow foam.: .sup.1H NMR (500
MHz, CDCl.sub.3) .delta. 9.84 (s, 1H, N.sub.G--H), 8.39 (t, J=5.0
Hz, 1H, N.sub..delta.--H), 8.22 (s, 1H, SCH.dbd.C), 6.54 (s, 1H,
ArH), 5.11 (d, J=8.0 Hz, 1H, BocN.sub..alpha.H), 4.02-3.95 (m, 1H,
C.sub..alpha.H), 3.95 (s, 3H, ArOCH.sub.3), 3.45-3.36 (m, 1H,
CH.sub.2-guanidine), 3.36-3.27 (m, 1H, CH.sub.2-guanidine), 2.69
(s, 3H, ArCH.sub.3), 2.63 (s, 3H, ArCH.sub.3), 2.14 (s, 3H,
ArCH.sub.3), 1.83-1.62 (m, 4H, C.sub..beta.H and C.sub..gamma. H),
1.50 (s, 9H, N.sub.GBoc), 1.34 (s, 9H, N.sub..alpha.Boc); MS (ES+)
ml/z 700.3 (M+H.sup.+, 100), 722.3 (M+Na.sup.+, 10), 702.3 (20),
701.3 (38).
[0471] Synthesis of Structure (75): 293
[0472] A 250 mL round-bottomed flask equipped for magnetic stirring
was charged with CH.sub.2Cl.sub.2 (10 mL), the acid (74) (3.40 g,
4.86 mmol), and trifluoroacetic acid (2 mL). After 1.5 h the
reaction was incomplete. Additional trifluoroacetic acid (5 mL) was
added and the solution was stirred 4 h more. The solvent was
removed in vacuo and the residue taken up in THF (50 mL). Saturated
NaHCO.sub.3 solution (50 mL) was added (pH-7-8) followed by
9-fluorenylmethyl-N-succinimidyl carbonate (1.97 g, 5.83 mmol, 1.2
eq.) in THF (20 mL). After 16 h stirring at room temperature,
starting material was still present and the pH=7.0. A 2 M
Na.sub.2CO.sub.3 solution (.about.3 mL) was added (pH=8.5) followed
by a second portion of FmocONSu (328 mg, 0.97 mmol, 0.2 eq.). The
solution was stirred for 2 h more at room temperature. The reaction
mixture was washed 2.times.100 mL hexane. Ethyl acetate (100 mL)
was added and the reaction mixture acidified to pH=0 with 6 N HCl.
The organic layer was separated and the aqueous layer extracted
2.times.100 mL ethyl acetate. The combined organic layers were
washed with saturated NaCl and dried over Na.sub.2SO.sub.4. The
solvent was removed to afford the crude Fmoc acid as a brown foam.
This foam was dissolved in a minimum of ethyl acetate and pipetted
into ethyl ether (250 mL). The precipitate was centrifuged and
collected. The supernatant was concentrated and dropped into ethyl
ether (50 mL). The white precipitate was centrifuged and the
combined precipitates dried in vacuo to afford 3.42 g (98% yield)
of the N.sub..alpha.-Fmoc-N.sub.G'-Mtr-1-[(4'-carboxylic
acid)thiazol-2-yl] arginol as a white powder.: .sup.1H NMR (500
MHz, CDCl.sub.3) .delta. 8.07 (s, 1H, SCH.dbd.C), 7.70 (d, J=7.0
Hz, 2H, Fmoc ArH), 7.46 (dd, J=5.0, 7.5 Hz, 2H, Fmoc ArH), 7.34
(dd, J=4.0, 7.5 Hz, 2H, Fmoc ArH), 7.23 (d, J=7.5 Hz, 2H, Fmoc
ArH), 6.49 (s, 1H, ArH), 4.94 (s, 1H, FmocN.sub.aH), 4.32-4.23 (m,
2H, FmocCH.sub.2), 4.07 (t, J=5.5 Hz, 1H, FmocCH), 4.02-3.95 (m,
1H, C.sub.aH), 3.78 (s, 3H, ArOCH.sub.3), 3.27-3.17 (m, 1H,
CH.sub.2-guanidine), 3.17-3.10 (m, 1H, CH.sub.2-guanidine), 2.64
(s, 3H, ArCH.sub.3), 2.57 (s, 3H, ArCH.sub.3), 2.08 (s, 3H,
ArCH.sub.3), 1.74-1.49 (m, 4H, C.sub.bH and C.sub.g H); MS (ES+)
m/z 722.3 (M+H.sup.+, 85), 736.3 (M+Na.sup.+, 21), 723.2 (35).
[0473] Synthesis of Structure (76): 294
[0474] The arginol ester derivative (42) (1.35 g, 1.93 mmol) was
dissolved in 70 mL of EtOAc at room temperature. To the solution
was added manganese (IV) dioxide (5 g, 89.2 mmol) and the
suspension was stirred vigorously for 5 h at room temperature after
which time it was filtered through silica gel. The solvent was
removed and the residue was purified by flash chromatography
(30hexane/EtOAc) to give the desired alcohol (76) (0.23 g, 18%) and
the ketone (0.153 g, 11.5%). .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 9.80 (s, 1H, N.sub.G--H), 8.34 (bs, 1H, N.sub.a--H), 7.65
(s, 1H, NCH.dbd.C), 6.54 (s, 1H, ArH), 5.20 (b, 1H,
BocN.sub..alpha.H), 4.89 (s, 1H, CHOH), 4.15 (b, 1H,
c.sub..alpha.H), 3.84 (s, 3H, ArOCH.sub.3), 3.83 (s, 3H,
CO.sub.2CH.sub.3), 3.70-3.6 (b, 1H, CH.sub.2-guanidine), 3.25-3.15
(m, 1H, CH.sub.2-guanidine), 2.70 (s, 3H, ArCH.sub.3), 2.63 (s, 3H,
ArCH.sub.3), 2.14 (s, 3H, ArCH.sub.3), 1.80-1.55 (m, 4H,
C.sub..beta.H and C.sub..gamma. H), 1.50 (s, 9H, N.sub.GBoc), 1.35
(s, 9H, N.sub..alpha.Boc); MS (ES+) m/e 697 (M+H.sup.+, 100).
[0475] Synthesis of Structure (77): 295
[0476] The ester (76) (70 mg, 0.1 mmol) was dissolved in a mixture
of THF (10 mL) and water (10 mL). To the solution was added LiOH
(18 mg, 4.3 mmol) and the solution was heated to reflux for 7 h.
The resulting solution was evaporated. The residue was dissolved in
water and extracted with ether. The aqueous layer was evaporated.
The resulting residue was dissolved in MeOH, and Dowex resin
(50W.times.8, H.sup.+ form) was added to acidify the solution. The
resin was filtered off, and the filtrate was evaporated to furnish
the acid (35 mg, 60%). .sup.1H NMR (500 MHz, CD.sub.3OD) .delta.
7.7(s, 1H, NCH.dbd.C), 6.70 (s, 1H, ArH), 4.3 (m, 1H,
C.sub..alpha.H), 3.87 (s, 3H, ArOCH.sub.3), 3.34 (mt, 1H,
CH.sub.2-guanidine), 3.3-3.2 (m, 1H, CH.sub.2-guanidine), 2.69 (s,
3H, ArCH.sub.3), 2.61 (s, 3H, ArCH.sub.3), 2.14 (s, 3H,
ArCH.sub.3), 1.73-1.62 (m, 4H, C.sub..beta.H and C.sub..gamma.H),
1.34 (s, 9H, N.sub..alpha.Boc); MS (ES+) m/z 583.3 (M+H.sup.+,
100).
[0477] Synthesis of Structure (78) 296
[0478] To 4-(chloroethyl)benzoic acid (8.0 g, 0.046 mol) in
CH.sub.3CN/DMF (80 mL:80 mL) were added NaN.sub.3 (6.0 g, 0.092
mol), tetra-n-butylammonium azide (cat.), tetra-n-butylammonium
iodide (cat.) and the reaction was heated at gentle reflux for 7-9
h at which point the reaction mixture transformed into one solid
block. Water (350 mL) and EtOAc (500 mL) were added and the aqueous
layer was extracted with EtOAc (2.times.400 mL). The organic layer
was washed with H.sub.2O (250 mL), brine (300 mL) and dried over
Na.sub.2SO.sub.4. Filtration and solvent evaporation afforded a
yellowish solid (9.4 g) which was pure enough to use in the next
step. IR (CDCl3) v.sup.-1 2111.
[0479] Synthesis of Structure (79): 297
[0480] To a solution of (78) (9.4 g, 0.053 mol) in THF/DME (175
mL:60 mL) was added tripherylphosphine (15.2 g, 0.058 mol) and the
reaction was stirred for 10 m. H.sub.2O (1.2 mL) was added and the
reaction was vigorously stirred at rt for 22-24 h at which point
the solution turned into a thick suspension. The off-white solid
was filtered and washed with THF (3.times.40 mL) to afford, after
drying, 16.4 g of pure iminophosphorane. MS (ES+) (M+H.sup.+)
426.1.
[0481] Synthesis of Structure (80) 298
[0482] The iminophosphorane (79) was suspended in THF/H.sub.20 (320
mL : 190 mL) and 2N HCl (64 mL) was added and the reaction was
heated at reflux for 5 h. Concentrated HCl (11 mL) was added and
reflux continued for an additional 20 h. The solvents were removed
under vacuo and the resultant off-white solid was dried under high
vacuum for 2 h (18.0 g) and used in the next step without further
purification.
[0483] Synthesis of Structure (81): 299
[0484] To a suspension of 4-(aminoethyl)benzoic acid .HCl (80) (9.0
g, 0.019 mol, theoretical) in CH.sub.3CN (320 mL) was added TEA
(7.7 mL, 0.053 mol) and the suspension was cooled to 0.degree. C.
Fmoc-ONSu (9.3 g, 0.026 mol) was added in one portion and the
reaction was allowed to warm to rt over 1 h and stirred an
additional 1 h. The solvent was removed under reduced pressure and
the residue was dissolved in EtOAc (1200 mL), washed with 10%
citric acid (220 mL) and brine (220 mL) and dried over
Na.sub.2SO.sub.4. Filtration and solvent evaporation afforded the
crude product which was purified by flash chromatography using 8%
MeOH/CHCl.sub.3 to afford pure product (2.4 g). .sup.1H NMR
(CDCl.sub.3) .delta. 2.81 (t, 2H, J=7.0 Hz), 3.36 (m, 2H), 4.15 (t,
1H, J=6.5 Hz), 4.36 (d, 2H, J=7.0 Hz), 5.243 (br s, 1H), 7.18 (d,
2H, J=8.0 Hz), 7.26 (m, 2H), 7.35 (t, 2H, J=7.5 Hz), 7.52 (d, 2H,
J=7.5 Hz), 7.71(d, 2H, J=7.5 Hz), 7.92 (d, 2H, J=8.0 Hz). MS (ES+)
(M+H.sup.+) 387.7. 300
[0485] Synthesis of Structure (82): 301
[0486] To 2.20 -g (8.4 mmol) of 4-iodo-methylbenzoate under
nitrogen was added 1.95 g (12.26 mmol) of Boc-propargyl amine, 0.33
g (1.26 mmol) of triphenylphosphine, 0.08 g (0.42 mmol) of
copper(I) iodide, 2.11 mL (15.1 mmol) of triethylamine, and 250 mL
of DMF. The solution was stirred and degassed with nitrogen for 15
min followed by the addition of 0.10 g (0.42 mmol) of palladium(II)
acetate and stirring at room temperature for 18 h. The solution was
diluted with EtOAc and washed with 5% citric acid (4.times.), brine
(2.times.) and dried over MgSO.sub.4. Purification by column
chromatography (silica gel, 9:1 hexanes/EtOAc) afforded ester (82)
(2.37 g, 98%) as an orange solid: .sup.1H NMR (CDCl.sub.3, 500 MHz)
.delta. 1.47 (s, 9H), 3.91 (s, 3H), 4.17 (m, 2H), 4.80 (broad s,
1H), 7.46 (d, J=8.5 Hz, 2H), 7.97 (d, J=8.5 Hz, 2H).
[0487] Synthesis of Structure (83): 302
[0488] To 2.86 g (9.88 mmol) of alkyne (82) under 1 atm of H.sub.2
was added 40 mL of anhydrous diethyl ether and a catalytic amount
of platinum(IV) oxide. The reaction was monitored by TLC and
complete after 13 h. The mixture was filtered through a pad of
Celite, washed with diethyl ether and the solvent was removed in
vacuo to give ester (83) (2.72 g 94%)as an orange oil: .sup.1H NMR
(CDCl.sub.3, 500 MHz) .delta. 1.44 (s, 9H), 1.82 (m, 2H), 2.69 (m,
2H), 3.15 (m, 2H), 3.89 (s, 3H), 4.55 (broad s, 1H), 7.23 (d, J=8.0
Hz, 2H), 7.94 (d, J=8.0 Hz, 2H); MS (ES+) m/z 294 (M+H.sup.+).
[0489] Synthesis of Structure (84): 303
[0490] To 2.72 g (9.27 mmol) of ester (83) was added 1.17 g (27.18
mmol) of lithium hydroxide monohydrate, 50 mL of THF and 50 mL of
H.sub.2O. The solution was stirred at room temperature for 16 h and
quenched with 5% citric acid. The reaction was extracted with EtOAc
(4.times.) and the combined extracts were washed with brine and
dried over MgSO.sub.4. Removal of the solvent afforded acid (84)
(2.38 g, 92%) as a pale yellow solid: .sup.1H NMR (CD.sub.3OD, 500
MHz) .delta. 1.44 (s, 9H), 1.80 (m, 2H), 2.70(m, 2H), 3.07 (m, 2H),
7.30 (d, J=8.0 Hz), 7.92 (d, J=8.0 Hz).
[0491] Synthesis of Structure (85): 304
[0492] To 2.38 g of acid (84) was added 20 mL of dichloromethane
and 20 mL of TFA. The solution was stirred for 2 h at room
temperature and the solvent removed in vacuo to give amino acid
(85) (3.57 g) as a pale orange solid: .sup.1H NMR (CD.sub.3OD, 500
MHz) .delta. 1.98 (m, 2H), 2.79 (m, 2H), 2.95 (m, 2H), 7.35 (d,
J=8.0 Hz), 7.97 (d, J=8.0 Hz).
[0493] Synthesis of Structure (86): 305
[0494] To 3.57 g (12.20 mmol)of amino acid (85) was added 70 mL of
1,4 dioxane, 70 of H.sub.2O, 1.29 g (12.20 mmol), and 4.93 9 (14.6
mmol). of N-(9-fluorenylmethoxycarbonyloxy)succinimide. The cloudy
mixture was stirred for 48 h, diluted with a large volume of EtOAc
and washed with saturated ammonium chloride. The mixture was
extracted with EtOAc (3.times.) and the combined organics were
washed with saturated bicarbonate, brine and dried over sodium
sulfate. Removal of the solvent in vacuo gave a pale yellow solid
which was washed with ether to afford acid (86) (2.85 g 58%; 83%
based on (75) as a white powder: .sup.1H NMR (CD.sub.3OD, 500 MHz)
.delta. 1.81 (m, 2H), 2.68 (m, 2H), 3.12 (m, 2H), 4.37 (m, 2H),7.30
(m, 4H), 7.38 (m, 2H), 7.65 (d, J=8.0 Hz, 2H), 7.79 (d, J=7.5 Hz,
2H), 7.92 (d, J=8.0 Hz, 2H); MS (ES+) m/z 402 (M+H.sup.+). 306
[0495] Synthesis of Structure (87): 307
[0496] A solution of cyanomethyl triphenylphosphonium chloride
(CMTPP) (8.2 g, 24 mmol) was prepared in 75 mL of dichloromethane
and stirred for 10 m. With the addition of Fmoc-Lys(Boc) (10 g,
21.3 mmol), 1-(dimethylaminopropyl)-3-ethyl carbodiimide
hydrochloride (EDCI) (4.9 g, 25.6 mmol) and 4-Dimethylaminopyridine
(DMAP) (2.2 mmol), the reaction vessel was sealed and stirred for
twelve hours at room temperature. The solvent was concentrated in
vacuo to an oil which was dissolved in 300 mL of ethyl acetate and
100 mL 1N HCl with stirring. The layers were separated and the
organic phase was extracted 2.times.50 mL of brine. The ethyl
acetate was dried over magnesium sulfate and concentrated to a
solid. This material was used without further purification. MS
(ES+) 752 (M+H.sup.+).
[0497] Synthesis of Structure (88): 308
[0498] The compound of structure (87) (16 g, 21.3 mmol) was
dissolved in 100 mL of MeOH and cooled to -78.degree. C. Ozone was
bubbled through the reaction solution with a gas dispersion tube
for 3 h. The product was isolated by removal of MeOH under reduced
pressure and was purified on a silica gel column (200 g dry weight)
equilibrated in a mobile phase of ethyl acetate/hexane (3:7). The
product was eluted with ethyl acetate/hexane (4:6), and gave after
drying 5.1 g (47% for the two steps). MS (ES+) 511 (M+H.sup.+).
[0499] Synthesis of Structure (89): 309
[0500] The keto ester (88) (5.1 g, 9.8 mmol) was dissolved in 100
mL of THF. After the addition of tetramethylammonium borohydride
(1.4 g, 11.8 mmol) to the solution, the vessel was sealed and
stirred for 4 h. The reaction was incomplete at this point and more
borohydride (0.21 g, 2.4 mmol) was added and stirring was continued
for an additional h. The reaction mixture was concentrated to an
oil in vacuo and applied to a silica gel column (150 g dry weight)
equilibrated and eluted with ethyl acetate/hexane (4:6) to give 2.7
g (53%) of product. MS (ES+) 513 (M+H.sup.+).
[0501] Synthesis of Structure (90): 310
[0502] The hydroxy ester (89) (2.7 g, 5.3 mmol) was dissolved in
100 ML of THF and cooled to 0.degree.-5.degree. C. 0.2N LiOH (66.5
mL, 13.3 mmol) was added to the chilled solution and stirred for
thirty minutes. The reaction was incomplete at that time and more
0.2N LiOH (10.4 mL, 2.1 mmol) was added. The reaction was stirred
of another thirty minutes and then quenched with 300 mL of ethyl
acetate/0.2N HCl (2:1). The aqueous phase was separated, washed
with 100 mL of ethyl acetate and the combined organic extracts were
dried over magnesium sulfate and filtered. The filtrate was
evaporated in vacuo to an oil and dried to a solid (2.0 g, 78%)
CDCl.sub.3 .delta. 1.2-1.8 (m,15H), 3.1 (m,2H), 4.1-4.5 (m,5H), 4.6
(m,1H), 5.4 (m,1H), 7.2 (m,2H), 7.4 (m,2H), 7.6 (m,2H), 7.8 (m,2H);
MS (ES+) Sol (M+H.sup.+).
[0503] Synthesis of Structure (91):
[0504] Structure (91) was synthesized by standard procedures as
shown in the following scheme. 311
Example 28
Synthesis of Representative Components for the Solid Phase
Synthesis of .beta.-Sheet Mimetics
[0505] Urazole Synthesis
[0506] The following syntheses are representative of the procedures
used to prepare the urazole components used in the solid phase
synthesis of .beta.-sheet mimetics of this invention.
[0507] Synthesis of Structure (92): 312
[0508] Structure (92) was synthesized by a minor modification of
the method of Cookson and Gupte (Org. Syntheses, Vol. VI (1988),
936). 2-n-Butylaniline (12.0 mL, 76.6 mmol) in 160 mL of EtOAc was
added via addition funnel to 324 mL of 20% phosgene in toluene at
rt over 30 mn. The solution was refluxed 30 mL and the solvent
removed by distillation. The residual oil was dissolved in 75 mL of
chloroform and was added via addition funnel over 15 min to a
suspension of methyl hydrazinocarboxylate (6.90 g, 76.6 mmol) in
toluene at rt. The mixture was refluxed for 1.5 h during which time
all the solids dissolved. Upon cooling to rt, a precipitate formed
and was collected by vacuum filtration. It was washed with toluene
and dried in vacuo to give 18.17 g of off-white powder (89%). The
product was used in the next step without further purification. TLC
(CH.sub.2Cl.sub.2/MeOH, 95/5) R.sub.f 0.12; .sup.1H NMR
(CD.sub.3OD) .delta. 0.94 (t, 3H, J=7.4 Hz), 1.39 (m, 2H), 1.56 (m,
2H), 2.61 (m, 2H), 3.74 (s, 3H), 7.09-7.21 (m, 4H); MS (ES+) m/z
265.8 (M+H.sup.+, 100).
[0509] Synthesis of Structure (93): 313
[0510] The compound of structure (92) (18.03 g, 68.0 mmol) was
suspended in 190 mL of 4 N KOH and heated to reflux for 2 hours.
Upon cooling, the now clear pink solution was extracted with ether
(6.times.) and acidified with concentrated HCl. The precipitate was
collected by vacuum filtration, washed with water and EtOAc, and
dried in vacuo overnight to yield 14.00 g of white solid (88%). [If
necessary, urazoles may be recrystallized from MeOH or another
suitable solvent system.] TLC (CH.sub.2Cl.sub.2/MeOH/AcOH, 94/4/2)
R.sub.f 0.63; Purity* by UV: .sup.397%; .sup.1H NMR (CD.sub.3OD)
.delta. 0.89 (t, 3H, J=7.3 Hz), 1.32 (m, 2H), 1.51 (m, 2H),
7.18-7.42 (m, 4H); MS (ES-) m/z 232 (M-H.sup.+). Note: urazoles
generally give poor mass spectra. *A rough check of purity may be
obtained by measuring the UV absorbance of the triazoline derived
from oxidation of the urazole as follows. Urazole (5-10 mg) and
bis(trifluoroacetoxy)iodobenzene (40 mg) are dissolved in DMF to 5
mL in a volumetric flask. The absorbance of this pink solution is
measured at 520 nm (.epsilon..apprxeq.177) in a cuvette with a 1 cm
path length against a DMF blank. Under these conditions, the purity
of the parent urazole is obtained by the following equation:
Purity=2.82(A)(MW)/(m), where A is the absorbance, MW is the
molecular weight of the urazole, and m is the weight in mg of the
sample urazole.
[0511] Synthesis of Structure (94): 314
[0512] 4-(Fluoromethyl)-benzylamine (4.1 mL, 28.5 mmol) was added
to a stirring solution of methyl hydrazinocarboxylate (2.56 g, 28.5
mmol) and 1,1'-carbonyldiimidazole (4.62 g, 28.5 mmol) in THF (25
mL). The solution was stirred at room temperature for 18 hours. A
white precipitate formed that was collected by vacuum filtration,
washed with cold THF, and dried in vacuo to yield 3.22 g of (94)
(39%). .sup.1H NMR (DMSO-d.sub.6) .delta. 3.57 (s, 3H), 4.26 (d,
2H, J=6.0 Hz), 7.44 (d, 2H, J=8.0 Hz), 7.64 (d, 2H, J=8.0 Hz); MS
(ES+) m/z 292 (M+H.sup.+, 100).
[0513] Synthesis of Structure (95): 315
[0514] The compound of structure (94) (3.22 g, 11.0 mmol) was
suspended in 20 mL of 4 N KOH and heated to reflux for 3 hours.
Upon cooling the solution was acidified with concentrated HCl. A
white precipitate formed and was collected by vacuum filtration,
washed with cold water, and dried in vacuo overnight to yield 2.45
g of white solid (86%). Purity by UV: 383%; .sup.1H NMR (DMSO)
.delta. 4.62 (s, 2H), 7.46 (d, 2H, J=8.0 Hz), 7.70 (d, 2H, J=8.0
Hz), 10.29 (bs, 2H); MS (ES-) m/z 258 (M-H.sup.+, 100).
[0515] Diene Synthesis
[0516] The following syntheses are representative of the procedures
used to prepare the diene components used in the solid phase
synthesis of .beta.-sheet mimetics of this invention.
[0517] Synthesis of Structure (95): 316
[0518] A solution of methacrolein (7.01 g, 100 mmol) and methyl
(triphenylphosphoranilidene)acetate (35.11 g, 105 mmol) in 150 mL
of dry dichloromethane was refluxed for 2 h under a nitrogen
atmosphere. The solvent was evaporated under reduced pressure, and
the product was purified by chromatography on a short silica gel
column (EtOAc-hexanes, 1:9). After evaporation of the
product-containing fractions, compound (95) was obtained as a clear
oil (8.71 g, 69%). TLC (EtOAc-hexanes, 1:4) R.sub.f 0.59 .sup.1H
NMR (CDCl.sub.3) .delta. 1.89 (s, 3H), 3.766 (s, 3H), 5.33-5.37 (m,
2H), 5.87 (d, J=16 Hz, 1H), 7.37 (d, J=16 Hz, 1H).
[0519] Synthesis of Structure (96): 317
[0520] Compound (96) was synthesized by a modification of the
procedure of K. Sato et al. (J. Org. Chem. 32:177, 1967). To a
suspension of NaH (60% in mineral oil, 0.40 g, 10 mmol) in 25 mL of
dry THF, cooled to 0.degree. under a nitrogen atmosphere, triethyl
phosphonocrotonate (2.50 g, 10 mmol) was added dropwise with
stirring. After the addition, the solution was stirred at 0.degree.
C. for 1.5 h. To the brown-red solution, maintained at 0.degree.
C., 3,3-dimethylbutyraldehyde (1.00 g, 10 mmol) was added dropwise.
The solution was allowed to warm up to room temperature, and
stirred for 1 h at room temperature. The mixture was diluted with
ethyl acetate (75 mL) and water (75 mL), and the two layers were
separated. The organic layer was washed with water (2.times.50 mL)
and brine (75 mL), and dried over sodium sulfate. The solvent was
removed under reduced pressure, and flash chromatography on silica
(EtOAc/hexanes, 1:9) yielded 1.00 g (51%) of (96) as a pale yellow
solid. TLC (EtOAc/hexanes, 1:9) R.sub.f 0.60 .sup.1H NMR
(CDCl.sub.3) .delta. 0.90 (s, 9H), 1.29 (t, J=7 Hz, 3H), 2.04 (d,
J=6 Hz, 2H), 4.19 (q, J=7 Hz, 2H), 5.80 (d, J=15.5 Hz, 1H),
6.13-6.17 (m, 2H), 7.24-7.39 (m, 2H).
[0521] Synthesis of Structure (97): 318
[0522] A solution of methyl 7,7-dimethyl-2,4-octadienoate (96)
(0.99 g, 5 mmol) and sodium hydroxide (0.60 g, 15 mmol) in methanol
(15 mL) and water (5 mL) was refluxed for 30 min. After cooling to
room temperature, the solvent was removed in vacuo, and the residue
was dissolved in water (30 mL). The resulting solution was
acidified with conc. HCl to pH 2, and the precipitate was collected
by filtration, washed with water (10 mL), and dried in vacuo to
yield 0.84 (99%) of the acid as a white solid. .sup.1H NMR
(CDCl.sub.3) .delta. 0.92 (s, 3H), 2.07 (d, J=6.5 Hz, 2H), 5.80 (d,
J=15.5 Hz, 1H), 6.19-6.23 (m, 2H), 7.34-7.40 (m, 2H).
Example 29
Solid Phase Synthesis of Representative .beta.-Sheet Mimetics
[0523] This example illustrates the solid phase synthesis of
representative .beta.-sheet mimetics (100) through (227) (Tables
11-15). The compounds of this example were synthesized according to
the following reaction scheme: 319
[0524] General Procedure:
[0525] The synthesis of .beta.-strand mimetics was initiated by
deprotection of Fmoc PAL resin using 25% piperidine in DMF.
Following extensive washing with DMF, the resin was treated with
the acid fluoride of N-Fmoc-4-aminomethylbenzoic acid, or (81), or
(86) and Hunigs' base in DMF until the Kaiser test was negative.
Alternatively, the Fmoc-protected thiazole-(75) or imidazole-based
(77) linkers were coupled to the resin using BOP, HOBt and DIEA. In
some instances Fmoc-Leu or another amino acid was attached to the
resin prior to the thiazole-(75) or imidazole-based (77) linkers
via the same methodology. In the case of structures (217)-(221),
the isocyanate (91) was coupled to Wang resin overnight in the
presence of catalytic HCl in dichloromethane. Deprotection of all
Fmoc-protected linkers was effected by treatment with 25%
piperidine in DMF, and deprotection the Boc-protected linker (77)
was effected by TMS-Cl (1 M) and phenol (3 M) in dichloromethane
for 30 min. The lysinol derivative (90) was coupled to resin-bound
linkers N-Fmoc-4-aminomethylbenzoic acid, (81), or (86) using
PyBOP, HOBt and Hunigs' base in DMF until a negative Kaiser test
was achieved. Treatment of the resin with 25% piperidine in DMF
then cleaved the FMOC group. Following washing with DMF a dienoic
acid was coupled to the resin-bound linkers using PyBOP, HOBt and
Hunigs' base in DMF until the result of a Kaiser test was negative.
The cycloaddition was performed by pretreatment of a solution of a
pyrazolidinedione (not shown) or urazole in DMF with a solution of
[bis(trifluoroacetoxy)iodo]benzene in DMF. The polymer-supported
diene was treated with the resulting solution for 2-16 hours. The
resin was then washed with DMF and CH.sub.2Cl.sub.2. Oxidation to
the ketoamide was effected by treatment of the resin with a
solution of Dess-Martin periodinane in DMSO for 60 min. The resin
was washed with CH.sub.2Cl.sub.2 and the product was cleaved from
the resin by treatment of the resin with 95:5 TFA:H.sub.2O for 1-12
h. The supernatant was collected and the resin was washed with
additional TFA. The combined filtrates were concentrated in vacuo.
The residue was precipitated with diethyl ether and the ether was
decanted. The resulting solid was reconstituted in 1:1
CH.sub.3CN:H.sub.2O and lyophilized. Compounds (100) through (227)
in Tables 11-15 each gave the expected (M+H.sup.+) peak when
submitted to LCMS (ES+). The compounds were assayed for inhibition
of coagulation enzymes as mixtures of diastereomers.
[0526] All of the compounds listed in Tables 11-15 had Ki<100 nM
as thrombin inhibitors, or had activity as Factor VIIa inhibitors
(Table 15). The compounds noted with an "*" in Tables 11-15 had a
Ki<10 nM as thrombin inhibitors and represent preferred
embodiments.
16TABLE 11 320 Cpd. No. R1 R3 R4 R6 100* 321 322 323 101* 324 325
326 102* 327 328 329 103* 330 331 332 104* 333 334 335 105* 336 337
338 106* 339 340 341 107* 342 343 344 108* 345 346 347 109* 348 349
350 110* 351 352 353 111* 354 355 356 112* 357 358 359 113* 360 361
362 114* 363 364 365 115* 366 367 368 116* 369 370 371 117* 372 373
374 118* 375 376 377 119* 378 379 380 120* 381 382 383 121* 384 385
386 122* 387 388 389 390 123* 391 392 393 394 124* 395 396 397 398
125* 399 400 401 402 126* 403 404 405 127* 406 407 408 128* 409 410
411 129* 412 413 414 130* 415 416 417 131* 418 419 420 132* 421 422
423 133* 424 425 426 134* 427 428 429 135* 430 431 432 136* 433 434
435 137* 436 437 438 138* 439 440 441 139* 442 443 444 140* 445 446
447 141* 448 449 450 142* 451 452 453 143* 454 455 456 144* 457 458
459 145* 460 461 462 146* 463 464 465 147 466 467 468 148 469 470
471 149 472 473 474 150 475 476 477 151 478 479 480 152 481 482 483
153 484 485 486 154 487 488 489 155 490 491 492 156 493 494 495 157
496 497 498 158 499 500 501 159 502 503 504 160 505 506 507 161 508
509 510 162 511 512 513 514 163 515 516 517 164 518 519 520 165 521
522 523 166 524 525 526 167 527 528 529 168 530 531 532 169 533 534
535 170 536 537 538 171 539 540 541 172 542 543 544 173 545 546 547
174 548 549 550 175 551 552 553 176 554 555 556 177 557 558 559 178
560 561 562 179 563 564 565 180 566 567 568 181 569 570 571 182 572
573 574 183 575 576 577 184 578 579 580 185 581 582 583 186 584 585
586
[0527]
17TABLE 12 587 Cpd. No. R1 R2 R3 R4 R5 R6 MS ES* (M + H.sup.+) 187*
588 589 590 591 592 694 188* 593 594 595 596 597 626 189* 598 599
600 601 602 605 190* 603 604 605 606 607 486 191 608 609 610 611
612 656 192 613 614 615 616 617 619 193 618 619 620 621 622 647 194
623 624 625 626 627 557 195 628 629 630 631 632 635 196 633 634 635
636 637 590
[0528]
18TABLE 13 638 MS (ES+) Cpd. No. R1 R3 R4 R6 (M + H.sup.+) 197 639
640 641 584 198* 642 643 644 659 199 645 646 647 648 848 200 649
650 651 814 201* 652 653 654 642 202 655 656 657 567 203 658 659
660 534 204 661 662 663 587 205 664 665 666 812 206 667 668 669 656
207 670 671 672 673 664 208 674 675 676 630 209 677 678 679 588 210
680 681 682 552 211* 683 684 685 576 212 686 687 688 705 213 689
690 691 648 214 692 693 694 724 215* 695 696 697 666 216 698 699
700 780
[0529]
19TABLE 14 701 MS (ES+) Cpd. No. R1 R3 R4 R7 (M + H.sup.+) 217* 702
703 704 503 218 705 706 707 426 219* 708 709 710 441 220 711 712
713 503 221 714 715 716 455 221-1 717 718 719 545 221-2 720 721 722
456 221-3 723 724 725 545 221-4 726 727 728 581 221-5* 729 730 731
490 221-6* 732 733 734 518 221-7 735 736 737 486 221-8 738 739 740
465 221-9 741 742 743 441 221-10 744 745 746 432 221-11 747 748 749
471 221-12* 750 751 752 454 221-13* 753 754 755 503 221-14* 756 757
477 221-15* 758 759 489 221-16 760 761 762 506 221-17* 763 764 765
514 221-18* 766 767 768 530 221-19* 769 X.sub.7--NH.sub.2 491
221-20* 770 X.sub.7--NH.sub.2 507 221-21 771 772 503
[0530]
20TABLE 15 773 MS (ES+) Cpd. No. R1 R3 R4 R5 R6 (M + H.sup.+) 222*
774 775 776 777 640 223* 778 779 780 781 730 224* 782 783 784 785
575 225* 786 787 788 789 697 226* 790 791 792 793 694 227 794 795
796 797 626 227-1 798 799 800 801 647 227-2 802 803 804 805 613
227-3 806 807 808 809 599 227-4 810 811 812 813 654 227-5 814 815
816 817 599 227-6 818 819 820 821 615 227-7 822 823 824 825 660
227-8 826 827 828 829 691 227-9 830 831 832 833 638 227-10 834 835
836 837 624 227-11 838 839 840 841 786 227-12 842 843 844 845 653
227-13 846 847 848 849 633 227-14 850 851 852 853 573 227-15 854
855 856 857 733 227-16 858 859 860 861 654 227-17 862 863 864 865
711 227-18 866 867 868 869 669 227-19 870 871 872 873 724 227-20
874 875 876 877 725 227-30 878 879 880 881 705
Example 30
Synthesis of Representative .beta.-Sheet Mimetics
[0531] This example further illustrates the synthesis of
representative .beta.-sheet mimetics of this invention. 882
[0532] Synthesis of Structure (228): 883
[0533] Methyl-2,4-dioxo-pentanoate (14.4 g, 0.10 mol) and 10.6 g of
trimethyl orthoacetate were dissolved in 100 mL of methanol
followed by the addition of 300 .mu.L of acetyl chloride. This
solution was then stirred at room temperature for 6 h. An aliquot
was then taken and the solvent removed using a rotary evaporator.
.sup.1H NMR analysis of the residue suggested a complete conversion
to the methyl enol ether. The reaction solution was evaporated in
vacuo. By .sup.1H NMR, purity was 90%, and the material was used
for the next step without purification.
[0534] 2-Methoxy-4-oxo-2-pentenone (1.58 g, 10 mmol) and 1.63 g of
t-butyldimethylsilyl chloride (11 mmol) were dissolved in 15 mL of
DMF. Triethylamine (1.553 mL, 12 mmol) was added and the reaction
stirred overnight under argon at rt. The next morning 50 mL of
hexane was added and the reaction was extracted with cold
NaHCO.sub.3 solution. The hexane layer was dried over
Na.sub.2SO.sub.4 and hexane removed under vacuum to give 2.01 g of
the diene as an oil (78%), which was used without further
purification. NMR (CDCl.sub.3) .delta. 0.16 (s, 6H), 0.94 (s, 9H),
3.53 (s, 3H),3.73 (s, 3H),4.32 (bs, 1H), 4.6 (bs,1H), 6.21
(s,1H).
[0535] Synthesis of Structure(229): 884
[0536] To a mixture of 4-phenyl urazole (177 mg, 1 mmol) and
iodobenzene diacetate (322 mg, 1 mmol) in CH.sub.2Cl.sub.2 (5 mL)
was added a solution of the diene (228) (269 mg, 1.05 mmol) in
CH.sub.2Cl.sub.2. The reaction mixture was stirred 30 min, and then
cooled to 0.degree. C. BF.sub.3.OEt.sub.2 (141 mg, 1 mmol) was
added dropwise and the reaction stirred for 30 min, diluted with
CH.sub.2Cl.sub.2 (50 mL), washed with NaHCO.sub.3 solution
(2.times.15 mL), water (15 mL) and brine, dried and evaporated.
Crude product was purified by column chromatography on silica gel
(EtOAc/hexane, 1:3, v/v) to afford pure product (97 mg, 32%).
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.53-7.42 (m, 5H),6.30
(s, 1H), 4.47 (s, 2H), 3.97 (s, 3H); MS (EI, 12 eV) 301 (M.sup.+,
100), 273.4, 246.3, 154.4, 119.5.
Example 31
Synthesis of Representative .beta.-Sheet Mimetics
[0537] This example further illustrates the synthesis of
representative .beta.-sheet mimetics of this invention.
[0538] Synthesis of Structure (24) 885
[0539] To a 250 mL flame-dried round bottom flask was added 130 mL
of dry THF. The flask was cooled to -78.degree. C. under an argon
atmosphere, and 10 mL of 2.5 M n-BuLi were added followed by 5.3 mL
of hexamethyldisilazane. This solution was stirred at -78.degree.
C. for 30 min., and then 2.2 mL of methyl propiolate were added.
After stirring at -78.degree. C. for 50 min., 2.5 mL (22 mmol) of
hexadienal were added. The reaction was then slowly warmed to
-30.degree. C. over a period of 4 h. After an hour at -30.degree.
C., it was quenched by addition of aqueous tartaric acid solution.
The reaction mixture was then partitioned between EtOAc and water,
and the aqueous layer was washed with additional ethyl acetate. The
combined organic layers were then washed with saturated sodium
chloride, dried over sodium sulfate, and concentrated to give about
4.1 g of a reddish oil. Flash chromatography via silica gel (20%
ethyl acetate/80% hexane) gave 3.1 g of a yellowish oil (78%).
.sup.1H NRM (CDCl.sub.3) .delta. 1.78 (d, 3H, J=9), 3.79, (s, 3H),
5.01 (bs, 1H), 5.63 (dd, 1H, J=9, 16), 5.84 (m, 1H), 6.06 (m, 1H),
6.38 (dd, 1H, J=16, 9).
[0540] Synthesis of Structure (25) 886
[0541] A 500 mL roundbottom flask was charged with phenyl urazole
(4.91 g) and 150 mL of methylene chloride. Iodobenzene diacetate
(8.94 g) was added to the flask and the reaction stirred for 10
min. as a deep red color developed. A solution of 5.0 g of compound
(230) dissolved in 50 mL of methylene chloride was then added, and
the reaction instantaneously decolorized. The reaction was stirred
at room temperature for 3 additional hours. The solvent was removed
on rotary evaporator and the residue placed under high vacuum
overnight. The residue was purified via flash chromatography on
silica gel (40% EtOAc/hexane) to give 8.3 g of a 60/40
diastereomeric mixture of epimeric alcohols (84%). .sup.1H NMR
(CDCl.sub.3) (isomer 1): .delta. 1.474 (d, 3H, J=7), 3.773 (s, 3H),
4.66 (m, 2H), 4.83 (s, 1H), 5.73 (d, 1H, J=10), 6.19 (bd, 1H,
J=10), 7.4=7.56 (m, 5H); (isomer 2): .delta. 1.53 (d, 3H, J=7),
3.77 (s, 3H), 4.64 (m, 1H, 4.72 (m, 1H), 5.18 (bs, 1H), 6.1 (s, 2H)
7.36-7.56 (m, 5H); MS (ES+): 356 (M+1), 378 (M+Na).
[0542] Synthesis of Structure (26) 887
[0543] A solution of 1.0 g of (231) as a diastereomeric mixture of
acetylene alcohols was dissolved in 40 mL of MeOH and cooled to
0.degree. C. in an ice bath. To the reaction mixture 80 mg (3
equivalents of hydride) of powdered sodium borohydride was added
with stirring. After an hour at 0.degree. C., the reaction was
warmed to room temperature and stirred for an additional hour. It
was quenched by addition of 100 mL EtOAc and 60 mL of water. The
layers were separated in a separatory funnel, and the aqueous phase
extracted twice with additional EtOAc. The combined organic phases
were then washed with saturated sodium chloride and dried over
sodium sulfate. The organic solvent was removed by rotary
evaporator and the residue purified by flash chromatography (40/60
EtOAc/hexanes) to give 630 mg of a mixture of diastereomeric
alcohols (63%). .sup.1H NMR (CDCl.sub.3) isomer 1: .delta. 1.39 (d,
3H, J=11), 3.78 (s, 3H), 4.68 (m, 1H), 4.71 (m, 1H), 4.75 (m, 1H),
5.81 (d, 1H, J=10), 6.16 (dm, 1H, J=10), 6.26 (d, 1H, J=9), 7.01
(d, J=9), 7.01 (d, J=9), 7.35=7.5 (m, 5H). Isomer 2: 1.43 (d, 3H,
j=10), 3.72 (s, 3H), 4.5 (m, 2H), 5.53 (d, 1H, J=12), 5.86 (m, 2H),
6.12 (d, 1H, J=10), 6.89 (d, 1H, J=10), 7.35-7.5 (m, 5H). MS (ES+)
358 (M+1).
[0544] Synthesis of Structure (27) 888
[0545] To a solution of 357 mg of compound (231) as a
diastereomeric mixture in 50 mL of methylene chloride was added 424
mg of powdered Dess-Martin reagent. The reaction stirred at room
temperature for 6 h. It was then stirred for five minutes with a
sodium thiosulfate solution and extracted with aqueous bicarbonate
solution. The organic phase was washed with saturated sodium
chloride and dried over anhydrous sodium sulfate. The methylene
chloride was removed by rotary evaporation to give 348 mg of a
solid residue (97%). .delta. 1.61 (d, 3H, J=9 Hz), 3.82 (s, 3H),
4.52 (bm, 1H), 5.16 (s, 1H), 5.93 (bd, 1H, J=10 Hz), 6.01 (bd, 1H,
J=10 Hz), 6.88 (d, 1H, J=15 Hz), 7.29 (d, 1H, J=15 Hz), 7.35-7.55
(m, 5H); MS (EI) 355 (M.degree. ).
[0546] Synthesis of Structure (28) 889
[0547] A 100 mL roundbottom flask was charged with 357 mg of
compound (232) as an isomeric mixture of alcohols and 25 mL of THF.
The reaction solution was cooled to 0.degree. C., the reaction was
allowed to warm up to room temperature, and stirred for an
additional hour. It was then extracted with 40 mL of EtOAc and 30
mL of water. The aqueous phase was acidified with 1 mmol of
tartaric acid, and reextracted with 40 mL of fresh EtOAc. The
organic phase was dried over anhydrous NaSO.sub.4, filtered and the
solvent removed via rotary evaporator to give 328 mg of a solid
residue. .sup.1H NMR (CDCl.sub.3) isomer 1: .delta. 1.37 (d, 3H,
J=6.5), 4.61 (m, 1H), 4.65 (m, 1H), 4.68 (m, 1H), 5.77 (d, 1H,
J=11), 6.12 (d, 1H, J=1l), 6.23 (d, 1H, J=15), 7.083 (d, 1H, J=15),
7.35-7.54 (m, 5H); isomer 2: 1.47 (d, 3H, J=6.5), 4.5 (m, 1H), 4.58
(m, 1H), 4.96 (m, 1H), 5.9 (m, 2H), 6.12 (d, 1H, J=16), 6.98 (d,
1H, J=16) 7.35=7.54 (m, 5H).
Example 32
[0548] In this example, compounds (231) and (233) of Example 31
were assayed for their ability to block insulin disulfide reduction
by thioredoxin. Thioredoxin has been shown to up-regulate NF-kB for
DNA binding by reduction of a disulfide bond involving Cys62 of the
p50 subunit of NF-kB. Thioredoxin is also known to reduce the
disulfide bonds in insulin 10.sup.4 times faster than low molecular
weight thiols (Holmgren, J. Biol. Chem. 254:9627-9632, 1979)
(incorporated herein by reference). Therefore, if an inhibitor of
NF-kB activation is acting via inhibition of thioredoxin, it should
also be able to block reduction of insulin by thioredoxin. The
following assay measures spectrophotometrically the increasing
turbidity of insulin at 650 nm as its disulfide bonds are reduced
in the presence of thioredoxin.
[0549] A slight modification of the method of Holmgren was used. on
a 96 well microtiter plate solutions of thioredoxin in 0.1 M
potassium phosphate pH 6.5 buffer were preactivated for 15 minutes
in the presence of 0.33 mM dithiothreitol (DTT) and 2 mM EDTA.
Solutions of substrate and inhibitor were added to a final
concentration of 8 .mu.M thioredoxin, 0.13 mM insulin, and 0-100
.mu.M of either compound (231) or (233). The turbidity of the
solutions was measured at 650 nM over the course of 60 minutes on a
Spectra Max 250 absorbance plate reader (Molecular Devices). The
results demonstrate that turbidity decreases with increasing
concentration of compounds (231) or (233).
[0550] As a negative control, inhibitor in the presence of DTT and
EDTA, but without thioredoxin present did not display turbidity
(DTT did not reduce thioredoxin over the time period examined). As
a positive control, the structurally related natural products
parthenolide and santonin were tested in the above assay in place
of the inhibitors. Parthenolide, which contains an unsaturated
exomethylene lactone and is known to inhibit NF-kB activation in a
concentration dependent fashion (Bork et al., FEBS Lett. 402:
85-90, 1997), similarly blocked thioredoxin-induced turbidity of
insulin. Santonin, which contains a saturated lactone group and
does not inhibit NF-kB activation, did not block
thioredoxin-induced turbidity of insulin. Taken together, these
results are evidence that compounds (231) and (233) prevent NF-kB
activation by inhibition of thioredoxin.
Example 33
Activity of a Representative .beta.-Sheet Mimetic as a Protease
Inhibitor
[0551] 890
[0552] This example further illustrates the activity of a
.beta.-sheet mimetic of structure (234) (prepared by methods
disclosed in reaction scheme 20) as an inhibitor of the
metalloproteinases leucine aminopeptidase M and thermolysin. The
method is a modification of that of Spungin-Bialik et al., FEBS
Lett. (1996) 380, 79-82.
[0553] The following protocol was used: A buffer solution
containing 50 mM Tris-Cl, 100 mM NaCl, 1 mM CaCl.sub.2, 0.005%
Triton X-100 (pH=7.5) is prepared. A second buffer solution, 40 mM
in EDTA, is prepared from the first. A 750 .mu.M solution of
substrate, Suc-Ala-Ala-Phe-pNA, is prepared in water from a 50 mM
stock solution DMSO. A 15 nM solution of thermolysin is prepared by
diluting with buffer a 200 .mu.M thermolysin stock solution in 20%
glycerol/H.sub.2O. Dilute the commercially available solution of
Leucine Aminopeptidase M (Sigma, 2.6 mg/ml stock in H.sub.2O) down
to 50 .mu.g/ml with buffer. The inhibitor in 50% EtOH/H.sub.2O was
diluted with water to 3.times. the desired concentration levels.
Add 50 .mu.l of enzyme, substrate, and inhibitor per well (96 well
microtiter plate) to the desired number of microtiter strips. This
will yield final concentrations of 5 nM for thermolysin and 250
.mu.M for the substrate. The wells should then be incubated at rt
for 20 minutes. After 20 minutes, add the EDTA in buffer solution
to all wells at 50 .mu.l per well. and add simultaneously to the
wells at 50 .mu.l per well. This will yield a final concentration
of 10 .mu.g/ml. The plate should be read 100.times. at 405 nm with
21 second intervals. K.sub.i values were calculated as before
(Example 5). The values of K.sub.i obtained for compound (234) were
6 and 11 .mu.M for thermolysin and leucine aminopeptidase M,
respectively. These results demonstrate that a .beta.-sheet mimetic
of this invention can function as a metalloproteinase
inhibitor.
Example 34
Activity of a Representative .beta.-Sheet Mimetic as a Protease
Inhibitor
[0554] 891
[0555] This example further illustrates the activity of a
.beta.-sheet mimetic of structure (235) (prepared by methods
disclosed in reaction scheme 15) as an inhibitor of the cysteine
proteinase, papain. The assay method is a modification of that of
Mellor et al., Biochem. J. (1993) 290, 289.
[0556] The assay was conducted in a microtiter plate as in Example
4. The following protocol was used: Prepare a buffer containing
0.05 M sodium citrate, 0.15 M NaCl, 2 mM DTT, 1 mM EDTA (pH=6.5). A
2 mM stock solution of substrate (Ac-Phe-Gly-pNA) is diluted to 200
.mu.M in buffer. A 5 mM stock solution (in 50% EtOH/H2O) of the
inhibitor is diluted to 500 .mu.M in buffer, and six serial 1:5
dilutions are made. Aliquots of 100 .mu.L each of buffer,
substrate, and inhibitor (at the appropriate concentrations) are
added per well to an eight well microtiter strip. A 1.0 mM stock
solution of papain is diluted to 200 .mu.M in buffer and incubated
for 5 min prior to addition of a 100 .mu.L aliquot to the assay
wells. The plate should be read 100.times. at 405 nm with 21 second
intervals. IC.sub.50 values were calculated as before (Example 4)
Compound (234) exhibited an IC.sub.50 value of 8 .mu.M. This result
demonstrates that a .beta.-sheet mimetic of this invention can
function as a cysteine proteinase inhibitor.
Example 35
Activity of Representative .beta.-Sheet Mimetics as Antithrombotic
Agents
[0557] This example illustrates the activity of .beta.-sheet
mimetics of structures (221-14) and (221-21) of Table 14 as
antithrombotic agents. Rats (Splague Dawley) were fasted overnight
and used under pentobarbital anesthesia. A polyethylene tube
containing a 5 cm silk thread was placed between the right cartid
artery and the left junglar vein. Thirty minutes after oral
administration of 100 mg.kg of one of the above compounds
(dissolved with 50% propylene glycol at 20 mg/ml, and orally
administered 5 ml/kg), or one minute after intravenous
administration of the Argatroban (0.3 mg/kg), blood was circulated
through the tube for seven minutes. At the end of circulation, the
tube was removed and the thrombus wet weight and thrombus protein
content were measured. Blood was also withdrawn from the abdominal
artery and APTT was measured.
[0558] The results of these experiments are presented in FIGS. 5A
and 5B, which plot thrombus protein (.mu.g/thrombus) for each of a
negative control (without added compound), compound 221-14 (FIG.
5A) or 221-21 (FIG. 5B) and Argatroban (a positive control). These
results illustrate that both of the tested compounds significantly
inhibit thrombus formation.
[0559] From the foregoing, it will be understood that, although
specific embodiments of this invention have been described herein
for purposes of illustration, various modifications may be made
without departing from the spirit and scope of the invention.
Accordingly, the invention is not limited except by the appended
claims.
* * * * *