Note: Descriptions are shown in the official language in which they were submitted.
CA 02268264 1999-04-OS
WO 98I16525 PCT/US97/18647
Selective Factor Xa Inhibitors
Field of the Invention
This invention relates to novel heterocyclic compounds which are potent and
highly
selective inhibitors of factor Xa or factor Xa when assembled in the
prothrombinase
complex. These compounds show selectivity for factor Xa versus other proteases
of the
coagulation (e.g. thrombin, fVIIa, flXa) or the fibrinolytic cascades {e.g.
plasminogen
activators, plasmin).
Background of the Invention
Blood coagulation protects mammalian species when the integrity of the blood
vessel
I O wall is damaged and uncontrolled loss of blood threatens survival.
Coagulation, resulting
in the clotting of blood is an important component of hemostasis. Under normal
hemostatic circumstances, there is maintained an acute balance of clot
formation and clot
removal (fibrinolysis). The blood coagulation cascade involves the conversion
of a variety
of inactive enzymes (zymogens) into active enzymes which ultimately convert
the
soluble plasma protein fibrinogen into an insoluble matrix of highly cross-
linked fibrin.
See Davie, et al. , "The Coagulation Cascade: Initiation, Maintenance and
Regulation"
Biochemistry 30:10363-10370 {I991). Blood platelets which adhere to damaged
blood
vessels are activated and incorporated into the clot and thus play a major
role in the initial
formation and stabilization of hemostatic "plugs". In certain diseases of the
cardiovascular
system. deviations from normal hemostasis push the balance of clot formation
and clot
dissolution towards life-threatening thrombus formation when thrombi occlude
blood
flow in coronary vessels (myocardial infarctions) or limb and pulmonary veins
(venous
thrombosis). Although platelets and blood coagulation are both involved in
thrombus
formation, certain components of the coagulation cascade are primarily
responsible for the
amplification or acceleration of the processes involved in platelet
aggregation and fibrin
deposition.
A key enzyme in the coagulation cascade as well as in hemostasis, is thrombin.
Thrombin is intimately involved in the process of thrombus formation, but
under normal
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circumstances can also play an anticoagulant role in hemostasis through its
ability to
convert protein C into activated protein C in a thrornbomodulin-dependent
manner.
Thrombin plays a central role in thrombosis through its ability to catalyze
the
penultimate conversion of fibrinogen into fibrin and through its potent
platelet activation
activity. Direct or indirect inhibition of thrombin activity has been the
focus of a variety
of recent anticoagulant strategies as reviewed by Claeson "Synthetic Peptides
and
Peptidomimetics as Substrates and Inhibitors of Thrombin and Other Proteases
in the
Blood Coagulation System", Blood Coal. Fibrinol. i:41 I-436 (l994). The major
classes
of anticoagulants currently used in the clinic directly or indirectly affect
thrombin (i.e.
heparins, low-molecular weight heparins and coumarins). Thrombin is generated
at the
convergence of the intrinsic and extrinsic coagulation pathways by the
prothrombinase
complex. The prothrombinase complex is formed when activated Factor X (factor
Xa)
and its non-enzymatic cofactor, factor Va assemble on phospholipid surfaces in
a Ca+2-
dependent fashion as reviewed by Mann, et al., "Surface-Dependent Reactions of
the
Vitamin K-Dependent Enzymes", Blood 76:1- I 6 ( I 990). The prothrombinase
complex
converts the zymogen prothrombin into the active procoagulant thrombin.
The location of the prothrombinase complex at the convergence of the intrinsic
and
extrinsic coagulation pathways, and the significant amplification of thrombin
generation
(393.000-fold over uncomplexed factor Xa) mediated by the complex at a limited
number
of targeted catalytic units present at vascular lesion sites, suggests that
inhibition of
thrombin generation is an ideal method to block uncontrolled procoagulant
activity.
Unlike thrombin, which acts on a variety of protein substrates as well as at a
specific
receptor, factor Xa appears to have a single physiologic substrate, namely
prothrombin.
Plasma contains an endogenous inhibitor of both the factor VIIa-tissue factor
(TF)
complex and factor Xa called tissue factor pathway inhibitor (TFPI). TFPI is a
Kunitz-
type protease inhibitor with three tandem Kunitz domains. TFPI inhibits the
TF/fVIIa
complex in a two-step mechanism which includes the initial interaction of the
second
Kunitz domain of TFPI with the active site of factor Xa, thereby inhibiting
the
2
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proteolytic activity of factor Xa. The second step involves the inhibition of
the TF/fVIIa
complex by formation of a quaternary complex TF/fVIIa/TFPI/fXa as described by
Girard, et al., "Functional Significance of the Kunitz-type Inhibitory Domains
of
Lipoprotein-associated Coagulation Inhibitor", Nature 33 8:518-520 ( 1989).
Polypeptides derived from hematophagous organisms have been reported which are
highly potent and specific inhibitors of factor Xa. U. S. Patent No. 4,588,5S7
awarded to
Gasic, describes anticoagulant activity in the saliva of the Mexican leech,
Haementeria
o~cinalis. A principal component of this saliva is shown to be the polypeptide
factor
Xa inhibitor, antistasin, by Nutt, et al., "The Amino Acid Sequence of
Antistasin, a
Potent Inhibitor of Factor Xa Reveals a Repeated Internal Structure", J. Biol.
Chem.
263:10162-10167 (1988).
Another potent and highly specific inhibitor of Factor Xa, tick anticoagulant
peptide,
has been isolated from the whole body extract of the soft tick Ornithidoros
moubata, as
reported by Waxman, et al., "Tick Anticoagulant Peptide (TAP) is a Novel
Inhibitor of
Blood Coagulation Factor Xa", Science 248:593-596 ( 1990).
Other polypeptide type inhibitors of factor Xa have been reported including
the
following citations by: Condra, et al., "Isolation and Structural
Characterization of a
Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria
ghilianii",
Thromb. Haemost. b 1:437-441 ( 1989); Blankenship, et al., "Amino Acid
Sequence of
Ghilanten: Anti-coagulant-antimetastatic Principle of the South American
Leech,
Haementeria ghilianii", Biochem. Bio h s. Res. Commun. l66:1384-l389 (1990);
Brankamp, et al., "Ghilantens: Anticoagulants, Antimetastatic Proteins from
the South
American Leech Haementeria ghilianii", J. Lab. Clin. Med. 1 l5:89-97 (1990);
Jacobs, et
al., "Isolation and Characterization of a Coagulation Factor Xa Inhibitor from
Black Fly
Salivary Glands", Thromb. Haemost. 64:235-238 ( 1990); Rigbi, et al., "Bovine
Factor Xa
Inhibiting Factor and Pharmaceutical Compositions Containing the Same",
European
Patent Application, 352,903 (1990); Cox, "Coagulation Factor X Inhibitor From
the
Hundred-pace Snake Deinagkistrodon acutus venom", Toxicon 31:1445-1457 (1993);
3
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WO 98/I6525 PCT/IJS97/18647
Cappello, et al., "Ancylostoma Factor Xa Inhibitor: Partial Purification and
its
Identification as a Major Hookworm-derived Anticoagulant In Vitro", J. Infect.
Dis.
167:1474-1477 ( 1993 ); Seymour, et al. , "Ecotin is a Potent Anticoagulant
and Reversible
Tight-binding Inhibitor of Factor Xa", Biochemistry 33:3949-3958 ( l994}.
Factor Xa inhibitory compounds which are not large polypeptide-type inhibitors
have also been reported including: Tidwell, et al., "Strategies for
Anticoagulation With
Synthetic Protease Inhibitors. Xa Inhibitors Versus Thrombin Inhibitors",
Thromb. Res.
19:339-349 ( l980); Turner, et al., "p-Amidino Esters as Irreversible
Inhibitors of Factor
IXa and Xa and Thrombin", Biochemistry 25:4929-4935 (1986); Hitomi, et al.,
"Inhibitory Effect of New Synthetic Protease Inhibitor (FUT-175) on the
Coagulation
System", Haemostasis 15:164-168 ( 1985); Sturzebecher, et al. , "Synthetic
Inhibitors of
Bovine Factor Xa and Thrombin. Comparison of Their Anticoagulant Efficiency",
Thromb. Res. 54:245-252 (1989); Kam, et al., "Mechanism Based Isocoumarin
Inhibitors
for Trypsin and Blood Coagulation Serine Proteases: New Anticoagulants",
Biochemistry
27:2547-2557 ( 1988); Hauptmann, et al., "Comparison of the Anticoagulant and
Antithrombotic Effects of Synthetic Thrombin and Factor Xa Inhibitors",
Thromb.
Haemost. 63:220-223 (I990); Miyadera, et al., Japanese Patent Application JP
6327488
(1994); Nagahara, et al., "Dibasic (Amidinoaryl)propanoic Acid Derivatives as
Novel
Blood Coagulation Factor Xa Inhibitors", J. Med. Chem. 37:1200-1207 (1994);
Vlasuk, et
~ al., "Inhibitors of Thrombosis" European Patent Application, WO 93/l5756
(1993); and
Brunck, et al., "Novel Inhibitors of Factor Xa", European Patent Application,
WO
94/13693 (l994). Al-obeidi, et al., "Factor Xa Inhibitors", WO patent
95/29l89,
discloses pentapeptide X 1-Y-I-R-X2 derivatives as factor Xa inhibitors. Said
compounds
are useful for inhibiting blood clotting in the treatment of thrombosis,
stroke, and
myocardial infarction.
WO 96/l8644 to Tamara, et al., describes aromatic heterocyclic thrombin
inhibitors.
WO 95/35313 to Semple, et al., pertains to 3-amino-2-oxo-1-piperidineacetic
derivative
thrombin inhibitors.
4
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EP 0,512,831 and US Patent No. 5,281,585, both to Duggan, et al., describe
fibrinogen
receptor antagonists.
SUMMARY OF THE INVENTION
The present invention relates to novel peptide and peptide mimetic analogs,
their
pharmaceutically acceptable isomers, salts, hydrates, solvates and prodrug
derivatives.
In another aspect, the present invention includes pharmaceutical compositions
comprising a pharmaceutically effective amount of the compounds of this
invention and a
pharmaceutically acceptable carrier. These compositions are useful as potent
and specific
inhibitors of blood coagulation in mammals.
In yet another aspect, the invention relates to methods of using these
inhibitors as
therapeutic agents for disease states in mammals which have disorders of
coagulation such
as in the treatment or prevention of unstable angina, refractory angina,
myocardial
infarction, transient ischemic attacks, thrombotic stroke, embolic stroke,
disseminated
1 S intravascular coagulation including the treatment of septic shock, deep
venous thrombosis
in the prevention of pulmonary embolism or the treatment of reocclusion or
restenosis of
reperfused coronary arteries. These compositions may optionally include
anticoagulants,
antiplatelet agents, and thrombolytic agents.
In other aspects of the invention compounds are provided which are useful as
diagnostic reagents.
In preferred embodiments, the present invention provides compounds of general
formula I:
E
I
(CH2)q o cH
2~ p
A-(Cf"12)m'W'(CH2)n
Y
R2
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Wherein:
Rl is H, C,_6alkyl, C3.bcycloalkyl, C1_3alkylaryl, C,_3alkyl-C3_gcycloalkyl or
aryl and
R2 is H, C~.~alkyl, or R' and R2 are taken together to form a carbocyclic
ring;
m is an integer from 0-3;
n is an integer from 0-6;
p is an integer from 0-4;
s is an integer from 0-2;
q is an integer from 0-2;
A is selected from the group consisting of: a five to ten membered
heterocyclic ring
system containing 1-4 heteroatoms selected from the group consisting of N, O
and S; R3;
-NR3Ra;
NRIS NRIs
/ _NR3R17
~N NR3RIS ; ;
Rla
KRIS NR's NRIs
~N~RIS~ ~ and
R17 \ / 'NR3R17
R S
where R3, R'~, R~4 and R~5 are independently selected from the group
consisting of H,
-OH, Ci_6alkyl, aryl and Ci~alkylaryl; R~6 is selected from the group
consisting of H,
-OH, C,_balkyl, aryl and C,.~aikylaryi, or can be taken together with R~'~ or
R1' to form a
5-6 membered ring; and R~~ is selected from the group consisting of H, -OH,
C,_balkyl,
aryl and Ci~alkylaryl, or can be taken together with R~5 to form a 5-6
membered ring;
W is C,_6alkyl, C3_8cycloalkyl, C,_6alkenyl, aryl, or a five to ten membered
heterocyclic ring system containing 1-4 heteroatoms selected from the group
consisting of
N, O and S;
K is selected from the group consisting of a direct link, C3_8cycloalkyl,
aryl, or a five
to ten membered heterocyclic ring system containing i-4 heteroatoms selected
from the
group consisting of N, O and S;
6
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WO 98116525 PCT/US97118647
E is selected from the group consisting of R26, -NRZ6R2~,
NR29 NR29
\ ~'NRZ6R~1
N NR26Rso
R28
NR29 NR2s NR2s
~N~R3~ ~ ~ and
/'R33 \ ~NR2gR31
R2a S
where R26, R27, Rz8 and R'-9 are independently selected from the group
consisting of H,
-OH, C,_6alkyl, aryl and C,~,alkylaryl; R3~ is selected from the group
consisting of H,
C,_6alkyl, aryl and Ci~alkylaryl, or can be taken together with R28 or R29 to
form a 5-6
membered ring; and R3' is selected from the group consisting of H, C,_balkyl,
aryl and
C,.~alkylaryl. or can be taken together with R29 to form a ~-6 membered ring;
with the
proviso that when E is R26, then K must contain at least one N atom;
Y is selected from the group consisting of H,
H3
OR~2 O _ .~ ~CH3 O
B ORt3 '-8 O CH3 , B~O H3 and Z
CH3 CH / '3
CH3
where R'2 and R'' are independently selected from the group consisting of H.
C,_3alkyl
and aryl; and G is H, -COOR'g, -CONR'8R'9, -CF3, -CF2CF3 or a group having the
formula:
N'u /
or L
V RZo
1 S where:
R2~ is selected from the group consisting of H, C i_6alkyl, CZ_6alkenyl,
Co_6alkylaryl,
Ca_6alkenylaryl, C~_6alkylheterocyclo, C2_6alkenylheterocyclo, -CF3 and -
CFZCF3;
7
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J is -S-, -SO-) -SOZ-, -O- or -NR'-, where R' is H, C,_balkyl or benryl; and
L is selected from the group consisting of:
~~Rs Ra Ra
'/
~CH2)r , ~ ~ and
~~R~ ~ ~ Rs ~ ~\'Rs
a C6_~~ heterocyclic ring system substituted by Rg and R9 and containing 1-4
heteroatoms
selected from N, S and O; where r is an integer from 0-2; R6 and R' are
independently
selected from the group consisting of H, C,_balkyl, aryl, C,_6alkylaryl, -
COOR'~,
-CONR'~R", -CN and -CF3; R8 and R9 are independently selected from the group
consisting of H, C,_balkyl, aryl, C i_6alkylaryl, C,.~alkyloxy, halogen, -NO,,
-NR'~Rt',
-NR'~COR", -O-R'~, -O-COR'~) -COOR'~, -CONR'~R", -CN, -CF3, -SO~NR'~R" and
C,_6alkyl-O-R'~; and R'~ and R'' are independently selected from the group
consisting of
H, C,_6alkyl, C,_3alkylaryl and aryl;
U is -O-, -S-, -N- or -N(H)-; and
V is -O-, -S-, -N- or -N(H)-; with the proviso that at least one of U or V is -
N- or
-N(H)-;
and a11 optical isomers thereof.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
In accordance with the present invention and as used herein, the following
terms are
defined with the following meanings, unless explicitly stated otherwise.
The term "alkyl" refers to saturated aliphatic groups including straight-
chain,
branched-chain, cyclic groups, and combinations thereof,
having the number of carbon atoms specified, or if no number is specified,
having up to
12 carbon atoms. The term "cycloalkyl" refers to a mono-, bi-, or tricyclic
aliphatic ring
having 3 to 12 carbon atoms, preferably 3 to 7 carbon atoms.
8
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The term "alkenyl" refers to unsaturated aliphatic groups including straight-
chain,
branched-chain, cyclic groups, and combinations thereof, having at least one
double bond
and having the number of carbon atoms specified.
The term "aryl" refers to an unsubstituted or substituted aromatic ring(s),
substituted
with one, two or three substituents such as, by way of example and not
limitation,
C ~ _6aikoxy, C i _6 alkyl, C i _~ alkylamino, hydroxy, halogen, cyano (-CN),
hydroxyl,
mercapto, vitro (-NOZ), thioalkoxy, carboxaldehyde, carboxyl, carboalkoxy,
carboxamide,
-NR'R", -NR'COR", -OR, -OCOR, -COOR, -CONR'R", -CF3, -SOZNR'R" and
C,_balkyl-OR; aryl, C,.balkylaryl (where the R groups can be H, C,_balkyl,
C,.3aIkylaryl
and aryl), including but not limited to carbocyclic aryl, heterocyclic aryl.
biaryl and triaryl
groups and the like, all of which may be optionally substituted. Preferred
aryl groups
include phenyl, halophenyl, C~_balkylphenyl, naphthyl, biphenyl,
phenanthrenyl,
naphthacenyl, and aromatic heterocyclics or heteroaryls, the latter of which
is an aryl
group containing one to four heteroatoms selected from the group consisting of
nitrogen,
oxygen and sulfur. Aryl groups preferably have 5-14 carbon atoms making up the
rings)
structure, while heteroaryls preferably have 1-4 heteroatoms, with the
remaining 4-10
atoms being carbon atoms.
The terms "heterocyclo" and "hetero cyclic ring system" as used herein refer
to any
saturated or unsaturated mono- or bicyclic ring system, containing from one to
four
~ heteroatoms, selected from the group consisting of nitrogen, oxygen and
sulfur. A typical
heterocyclic ring system will have five to ten members, 1-4 of which are
heteroatoms.
Typical examples of monocyclic ring systems include piperidinyl, pyrrolidinyl,
pyridinyl, piperidonyl, pyrrolidonyl and thiazolyl, while examples of bicyclic
ring
systems include benzimidazolyl, benzothiazolyl and benzoxazolyh all of which
may be
substituted.
The term "carbocyciic ring" as used herein refers to any saturated or
unsaturated ring
containing from three to six carbon atoms.
The terms "alkylaryl" and "alkenylaryl" as used herein refer to an alkyl group
or
9
CA 02268264 1999-04-08
WO 98I16525 PCT/US97/18647
alkenyl group, respectively, having the number of carbon atoms designated,
appended to
one, two, or three aryl groups. The term benzyl as used herein refers to -CH2-
C6H;.
The term "alkoxy" as used herein refers to an alkyl linked to an oxygen atom,
such as
methoxy, ethoxy, and so forth.
The terms "halogen" as used herein refer to Cl, Br, F or I substituents.
The term "direct link" as used herein refers to a bond directly linking the
substituents
on each side of the direct link. When two adj acent substituents are defined
as each being a
"direct link", it is considered to be a single bond.
Two substituents are "taken together to form a 5-6 membered ring" means that
an
ethylene or a propylene bridge, respectively, is formed between the two
substituents.
The term "pharmaceutically acceptable salts" includes salts of compounds
derived
from the combination of a compound and an organic or inorganic acid. These
compounds
are useful in both free base and salt form. In practice, the use of the salt
form amounts to
use of the base form; both acid and base addition salts are within the scope
of the present
invention.
"Pharmaceutically acceptable acid addition salt" refers to those salts which
retain the
biological effectiveness and properties of the free bases and which are not
biologically or
otherwise undesirable, formed with inorganic acids such as hydrochloric acid,
hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like,
and organic acids
such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,
malefic acid,
malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic
acid, cinnamic
acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-
toluenesulfonic acid,
salicylic acid and the like.
"Pharmaceutically acceptable base addition salts" include those derived from
inorganic
bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron,
zinc,
copper, manganese, and aluminum bases, and the Like. Particularly preferred
are the
ammonium, potassium, sodium, calcium and magnesium salts. Salts derived from
pharmaceutically acceptable organic nontoxic bases include salts of primary,
secondary,
CA 02268264 1999-04-08
WO 98I16525 PCT/US97/18647
and tertiary amines, substituted amines including naturally occurring
substituted amines,
cyclic amines and basic ion exchange resins, such as isopropylamine,
trimethylamine,
diethylamine, triethylamine, tripropylamine, ethanolamine) 2-
diethyiaminoethanol,
trimethamine, dicyclohexylamine) lysine, arginine, histidine, caffeine,
procaine,
hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylgIucamine,
theobromine, purines, piperizine, piperidine, N-ethylpiperidine, polyamine
resins and the
like. Particularly preferred organic nontoxic bases are isopropylamine,
diethylamine,
ethanolamine, trimethamine, dicyclohexylamine, choline, and caffeine.
"Biological property" for the purposes herein means an in vivo effector or
antigenic
function or activity that is directly or indirectly performed by a compound of
this
invention. Effector functions include receptor or ligand binding, any enzyme
activity or
enzyme modulatory activity, any Garner binding activity, any hormonal
activity, any
activity in promoting or inhibiting adhesion of cells to an extracellular
matrix or cell
surface molecules, or any structural role. Antigenic functions include
possession of an
epitope or antigenic site that is capable of reacting with antibodies raised
against it. The
biological properties of the compounds of the present invention can be readily
characterized by the methods described in Examples I4 and 15 and by such other
methods as are well known in the art.
In addition, the following abbreviations are used in this application:
"Boc" refers to t-butoxycarbonyl.
"BOP" refers to benzotriazol-1-yloxy-tris-(dimethylamino) phosphonium
hexafluorophosphate.
"DIEA" refers to diisopropylethylamine.
"DMF" refers to N,N-dimethylformamide.
"DMSO" refers to dimethylsulfoxide.
"Et20" refers to diethyl ether.
"EtOAc" refers to ethyl acetate.
"HOAc" refers to acetic acid.
CA 02268264 1999-04-08
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"LDA" refers to lithium diisopropylamide.
"MeOH" refers to methanol.
"MeSEt" refers to methyl ethyl sulfide.
"NaN(TMS)z" refers to sodium bis-trimethyl silyl amide.
"TFA" refers to trifluoroacetic acid.
"THF" refers to tetrahydrofuran.
"Tos" refers to p-toluenesulfonyl.
In the compounds of this invention, carbon atoms bonded to four non-identical
substituents are asymmetric. Accordingly, the compounds may exist as
diastereoisomers,
enantiomers or mixtures thereof. The syntheses described herein may employ
racemates,
enantiomers or diastereomers as starting materials or intermediates.
Diastereomeric
products resulting from such syntheses may be separated by chromatographic or
crystallization methods) or by other methods known in the art. Likewise.
enantiomeric
product mixtures may be separated using the same techniques or by other
methods
known in the art. Each of the asymmetric carbon atoms, when present in the
compounds
of this invention, may be in one of two configurations (R or S) and both are
within the
scope of the present invention. In the processes described above, the final
products may,
in some cases, contain a small amount of diastereomeric or enantiomeric
products;
however, these products do not affect their therapeutic or diagnostic
application.
In a11 of the peptides of the invention, one or more amide linkages (-CO-NH-)
may
optionally be replaced with another linkage which is an isostere such as -
CH,NH-,
-CH2S-, -CH2-O-, -CH,CH2-, -CH=CH- (cis and traps}, -COCH2-, -CH(OH)CHZ-,
-CH2S0-, and -CHZSO2-. This replacement can be made by methods known in the
art.
The following references describe preparation of peptide analogs which include
these
alternative-linking moieties: Spatola, "Peptide Backbone Modifications"
(general review)
Ve$a Data, Vol. 1, Issue 3, (March 1983); Spatola, "Chemistry and Biochemistry
of
Amino Acids, Peptides and Proteins," (general review) B. Weinstein, eds.,
Marcel
Dekker, New York, p. 267 ( 1983); Morley, Trends Pharm. Sci. (general review)
pp. 463-
12
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WO 98/16525 PCTlUS97l18647
468 (1980); Hudson, et al.) Int. J. Pept. Prot. Res. 14:177-I85 (1979) (-CHZNH-
,
-CHZCH2-); Spatola, et al., Life Sci. 3 8: I 243- I 249 ( 1986) (-CHZ-S);
Hann, J. Chem. Soc.
Perkin Trans. I pp.307-314 ( 1982) (-CH=CH-, cis and trans); Almquist, et al.,
J Med.
Chem. 23 :1392- i 398 ( 1980) (-COCH~-); Jennings-White, et al., Tetrahedron
Lett.
S 23:2533 (-COCH2-) ( 1982); Szelke, et al., European Application EP 45665;
CA:97:39405
( 1982) (-CH(OH)CH2-); Holladay, et al., Tetrahedron Lett 24:4401-4404 ( 1983)
(-CH(OH)CHZ-); and Hruby, Life Sci. 31:189-199 ( 1982) (-CHZ-S-).
Preferred Embodiments
This invention relates to a new class of peptide derivatives selected from
those of
general formula I which are potent and specific inhibitors of Xa, their
pharmaceutically
acceptable compositions thereof, and the methods of using them as therapeutic
agents for
disease states in mammals characterized by abnormal thrombosis:
E
I
(~H2)S
(CH2)q ~ ( KH )
A- CH -W- CH
( 2)m ( 2)n
~N Y
H
Wherein:
R' is H. C,.~alkyl, C3.bcycloalkyl, C,_3alkylaryl, Ci_;alkyl-C3_gcycloalkyl or
aryl and
Rz is H, C,_6alkyl, or R~ and R2 are taken together to form a carbocyclic
ring;
m is an integer from 0-3;
n is an integer from 0-6;
p is an integer from 0-4;
s is an integer from 0-2;
q is an integer from 0-2;
A is selected from the group consisting of: a five to ten membered
heterocyciic ring
13
CA 02268264 1999-04-08
WO 98/16525 PCT/US97/18647
system containing 1-4 heteroatoms selected from the group consisting of N, O
and S: R3;
-NR3R4;
NR15 NRIs
~NR3R1~
~N NR3Rls ; ;
R14
NR15 NRIS NRIs
~N~RIS ~ and
~ 14 ' ~R1~ \ ~NR3R1~
R S
where R3, R~, R ~'~ and R ~ 5 are independently selected from the group
consisting of H,
-OH, C ~ _6alkyl, aryl and C, _.~alkylaryl; R 16 is selected from the group
consisting of H,
-OH, Ci_balkyl, aryl and C,_.~alkylaryl, or can be taken together with R1'1 or
R15 to form a
S-6 membered ring; and R17 is selected from the group consisting of H, -OH,
C,_balkyl,
aryl and C,.~alkylaryl, or can be taken together with R15 to form a 5-6
membered ring;
W is C,_6alkyl, C3_8cycloalkyl, C,_balkenyl, aryl, or a five to ten membered
heterocyclic ring system containing 1-4 heteroatoms selected from the group
consisting of
N, O and S;
K is selected from the group consisting of a direct link, C3_gcycloaikyl,
aryl, or a five
to ten membered heterocyclic ring system containing 1-4 heteroatoms selected
from the
group consisting of N, O and S;
E is selected from the group consisting of R26, -NR26R2~,
N R29 N R29
/~NR2sR31
~N NR2sRso
R28
NR29 NR2s NR2s
~N~R3o ~ and
Rst \ ~NR2sRs~
R2a S
where R26, R2', R2g and R29 are independently selected from the group
consisting of H,
-OH, C,_balkyl, aryl and C,.~alkylaryi; R3~ is selected from the group
consisting of H,
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WO 98I16525 PCTlITS9?/I86A7
C,_6alkyl, aryl and C,.~alkylaryl, or can be taken together with R28 or RZ9 to
form a 3-6
membered ring; and R3 ~ is selected from the group consisting of H, C,_6alkyl,
aryl and
C,.~alkylaryl, or can be taken together with Rz9 to form a 5-6 membered ring;
with the
proviso that When E is R26, then K must contain at least one N atom;
Y is selected from the group consisting of H,
~OR12 _ ~~ _ ~ ~3 O
CH3 , B~ and
~ NCH
CH3 a
CH3
where R!2 and R~3 are independently selected from the group consisting of H,
C,_3alkyl
and aryl; and G is H, -COOR~B, -CONR~gR~9, -CF3, -CFzCF3 or a group having the
formula:
N, a ~ \
or L
~ RZ~ J
where:
R2~ is selected from the group consisting of H, C,_6alkyl, CZ_6alkenyl,
C~_6alkylaryl,
C~_6alkenylaryl, C~.6alkylheterocyclo, CZ_6alkenylheterocyclo, -CF3 and -
CF~CF3;
J is -S-, -SO-, -S02-, -O- or -NR'-, where R' is H, C,_6alkyl or benzyl; and
I 5 L is selected from the group consisting of:
Ra
(CH2)~ I ~ and
'ERs
a C6_,~ heterocyclic ring system substituted by Rg and R9 and containing 1-4
heteroatoms
selected from N, S and O; where r is an integer from 0-2; R6 and RT are
independently
selected from the group consisting of H, C,_6alkyl, aryl, C,_6alkylaryl, -
COORS~,
CA 02268264 1999-04-08
WO 98l16525 PCT/US97/I8647
-CONR'~R", -CN and -CF3; Rg and R9 are independently selected from the group
consisting of H, C,_6alkyl, aryl, C ~_6aikylaryl, C,.~alkyloxy, halogen, -NO~,
-NR'~R",
-NR'~COR~', -O-R~~, -O-COR'~, -COORS~, -CONR'~R' ~, -CN, -CF3, -SO~NR~~R" and
C,_balkyl-O-R'~; and R~~ and R' ~ are independently selected from the group
consisting of
H, C,_6alkyl, C,_3alkylaryl and aryl;
U is -O-, -S-, -N- or -N(H)-; and
V is -O-, -S-, -N- or -N(H)-; with the proviso that at least one of U or V is -
N- or
-N(H)-;
and all optical isomers thereof.
Preferred R~ substituents are H and C~_6alkyl; more preferably H.
RZ is preferably H.
The integer "m" is preferably from 0-1; more preferably 0.
The integer "n" is preferably from 1-4.
The integer "p" is preferably 3.
The integer "s" is preferably 0.
The integer "q" is preferably from 0-1.
Preferred "A" substituents are R3, -R3R'',
NR15 NR15 NRIs
and
~N NR R NR3R17 ~ ~NR R
3 16 S 3 17
~ R14
R3 is preferably H, -OH or C~_6alkyl; more preferably H, -OH or methyl.
R4 is preferably H, -OH or C,_balkyl; more preferably H, -OH or methyl.
Preferred "W" substituents are CE~alkyl, C;_bcycloalkyl, aryl, or a five to
ten
membered heterocyclic ring system containing at least one N heteroatom; more
preferably
C i~alkyl, aryl, or a five to ten membered heterocyclic ring system containing
at least one
N heteroatom.
K is preferably a direct link.
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In the "E" substituent, it is preferred that R26, Rzy R28, Rz9, R'~ and R3 ~
are
independently selected from the group consisting of H and C i_6alkyl, more
preferably H
and methyl. Particularly preferred "E" substituents are -NHS, -NHC(=NH)-NH2
and
-SC(=NH)-NHS; more preferably -NHC(=NH)-NHa and -SC(=NH)-NHS.
Preferred "Y" substituents are:
~OR~z O
-B~ or
OR~3 G
R~2 is preferably H.
R~3 is preferably H.
The "G" substituent is preferably a group having the formula:
N
\L
to J
The "J" substituent is preferably -S-, -O- or -NR'-.
R' is preferably H.
The "L" substituent is preferably:
s
Ra ~/R
or
Rs ~ .\Rs
more preferably:
R$
Rs
R6 1S preferably H.
R~ is preferably H.
R8 is preferably H, -O-R~~, -COORS~, -CONR~~RI ~ or -CF3; more preferably H.
17
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WO 98I16525 PCT/US97I18647
R9 is preferably H, -O-R~~, -COORS~, -CONR~~R' ~ or -CF3; more preferably H.
Rl~ is preferably H.
R~ ~ is preferably H.
In one embodiment of the invention, R~ and RZ are H, p=3, s=0, K is a direct
link, E is
-NHC(=NH)-NH2 and Y is -CO-G, where G is a group having the formula:
N
\ L
~J /
where J and L are as defined above. This is also illustrated as a preferred
group of
compounds defined by the general structural formula II as:
HN\ 'NH2
~N'H
(CH2)~ O N\
A-(CH2~W-(CH2 ~ N v 'N
I j
O H O
wherein:
m is an integer from 0-3;
n is an integer from 0-6;
q is an integer from 0-2;
A is selected from the group consisting of R3, -NR3R4;
NR15 NR15
~NR3R1~
~N NR3Rls ;
R14
NR15 NR15 NRIs
~N~Ris and
~ 1 ~ R17 ~ ~NR3R17
R4 S .
where R3, R'', R 14, R~ 5, R ~ 6 and R ~ ~ are independently selected from the
group consisting
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WO 98/16525 PCT/US97/18647
of H, -OH and C,_6alkyl;
W is C,_6alkyl, C3_acycloalkyl, C~_balkenyl, aryl, or a five to ten membered
heterocyclic ring system containing 1-4 heteroatoms selected from the group
consisting of
N, O and S; with the proviso that when A is R3, then W must contain at least
one N
atom;
J is -S-, -SO-, -S02-, -O- or -NR'-, where R' is H; and
L is selected from the group consisting of:
Ra
(CH2)r ~ ~ and
' ~ R9 ' 'ERs
a C6_,o heterocyclic ring system substituted by R8 and R9 and containing 1-4
heteroatoms
selected from N, S and O; where r is an integer from 0-1; R6 and R' are
independently
selected from the group consisting of H, Ci_balkyl, aryl, C,_balkylaryl, -
COORIO,
-CONRI~R~ ~, -CN and -CF3; Rg and R9 are independently selected from the group
consisting of H, C,_balkyl, aryl, C,_6alkylaryi, C,.~alkyloxy, halogen, -NOa, -
NR'~R~ ~,
-NR'~COR~ ~, -O-R~~, -O-CORf~, -COORS~, -CONR~~R~ ~, -CN, -CF3, -SOZNR~~R' ~
and
C,_balkyl-O-R'~; and R'~ and R1~ are independently selected from the group
consisting of
H, C,_6alkyl, C~.3alkylaryl and aryl;
and a11 optical isomers thereof.
A preferred embodiment of compounds of general formula II have the following
stereochemistry:
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WO 98/16525 PCT/US97/18647
HN NHZ
NH
N
A- UH2)m W- 'L
J
O H O
In yet another embodiment of the invention, R~ and RZ are H, p=3, s=0, K is a
direct
Link, E is -NHC(=NH)-NH2, and Y is -CO-G, where G is a group having the
formula:
N
\L
J
where J is -S- and L is the group:
Ra
Rs
where Rg and R9 are H. This is also illustrated as a preferred group of
compounds defined
by the general structural formula III as:
HN\ 'NH2
~NH
(CH2)~ I I I
N
A-(CH2)m W-(CH2 "
O
wherein:
m is an integer from 0-3;
n is an integer from 0-6;
q is an integer from 0-2;
(CH2)q
1
{CH2 n N
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WO 98/16525 PCT/US97/18647
A is selected from the group consisting of R3, -NR3R'~,
NH NH
/ _NH2 ;
N NH2
H
NH NH NH
~N~Rts. ~ and
' ~Rt~ ~ NH
H S 2.
where R3, R'', R' 6 and R~ ~ are independently selected from the group
consisting of H, -OH
and C i.balkyl;
W is C~_balkyl, C3_8cycloalkyl, C~_6alkenyl, aryl, or a five to ten membered
heterocyclic ring system containing I-4 heteroatoms selected from the group
consisting of
N, O and S; with the proviso that when A is R3, then W must contain at least
one N
atom;
and a11 optical isomers thereof.
A preferred embodiment of compounds of general formula III have the following
stereochemistry
HN NHZ
NH
(CH2)q
O
N
A- (CH2)rri W- (CH2 ~ '~ S
O H O
This invention also encompasses all pharmaceutically acceptable isomers,
salts,
hydrates and solvates of the compounds of formulas I, II and III. In addition,
the
compounds of formulas I, II and III can exist in various isomeric and
tautomeric forms,
and all such forms are meant to be included in the invention, along with
pharmaceutically
acceptable salts, hydrates and solvates of such isomers and tautomers.
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WO 98I16525 PCT/US97/18647
The compounds of this invention may be isolated as the free acid or base or
converted
to salts of various inorganic and organic acids and bases. Such salts are
within the scope
of this invention. Non-toxic and physiologically compatible salts are
particularly useful
although other less desirable salts may have use in the processes of isolation
and
purification.
A number of methods are useful for the preparation of the salts described
above and
are known to those skilled in the art. For example, the free acid or free base
form of a
compound of one of the formulas above can be reacted with one or more molar
equivalents of the desired acid or base in a solvent or solvent mixture in
which the salt is
insoluble, or in a solvent like water after which the solvent is removed by
evaporation,
distillation or freeze drying. Alternatively, the free acid or base form of
the product may
be passed over an ion exchange resin to form the desired salt or one salt form
of the
product may be converted to another using the same general process.
This invention also encompasses prodrug derivatives of the compounds contained
herein. The term "prodrug" refers to a pharmacologically inactive derivative
of a parent
drug molecule that requires biotransformation, either spontaneous or
enzymatic, within
the organism to release the active drug. ~ Prodrugs are variations or
derivatives of the
compounds of this invention which have groups cleavable under metabolic
conditions.
Prodrugs become the compounds of the invention which are pharmaceutically
active in
vivo, when they undergo solvolysis under physiological conditions or undergo
enzymatic
degradation. Prodrug compounds of this invention may be called single, double,
triple
etc., depending on the number of biotransformation steps required to release
the active
drug within the organism, and indicating the number of fimctionalities present
in a
precursor-type form. Prodrug forms often offer advantages of solubility,
tissue
compatibility, or delayed release in the mammalian organism (see, Bundgard,
Design of
Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985 and Silverman, The Organic
Chemistry of Drug Design and Drug Action, pp. 352-401, Academic Press, San
Diego,
CA, 1992). Prodrugs commonly known in the art include acid derivatives well
known to
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CA 02268264 1999-04-08
WO 98I16525 PCT/LTS97118647
practitioners of the art) such as, for example, esters prepared by reaction of
the parent
acids with a suitable alcohol, or amides prepared by reaction of the parent
acid compound
with an amine, or basic groups reacted to form an acylated base derivative.
Moreover, the
prodrug derivatives of this invention may be combined with other features
herein taught
S to enhance bioavailability.
The following structures are illustrative of the compounds of the present
invention
and are not intended to be limiting in any manner:
HN~NH2 HNyNHz
H NH N'H
HZN N
O N H N
N " /~ 2 ~ / N~ N
~,N S N S
O H O O H O
HN~NHz H N
NH
NH
H
HN N N HzN I ~ O N
i N N /S
l~ O H O O hi O
HNyNHz
lNH HNyNHz
Nf'H Nl H
NJLN NS> HzN~N O N
N
HN O H O g
O H O
HNyNHz
HN~NH lp
NH NH NH
HpN
'NIH I / N~N N H N ~ / N~N N
O H O O H O
HN NH2
HNyNHz
'NH NH
N N N N
HpN--~S1 N~LN /~ ~ ' N~N /N~CF3
O H O H O H O
z3
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HN~NHZ
HN'yNHp
NH N1H
N N N
N S N
S
~"~1-~3 N' H O HgC~N O H
NH
HN NHp
E"~N~NHZ
NH
O N
N~N S N~N S
N i O hl
H3C.N' .~ O H O
HN~NH2
HN~NH 1p
NH
NH
N N ~ N N NS
HzN H O S HN~ O H O
~NH ~Z
HN~NHZ HN~NH2
NH NH
H2N~~ N N HzN~ S N N
NH O H O NH O H O
HN~ NHZ HNyNH2
1NH ,NH
H2N~~~'~~~~N N N N N
NH O H H3C~NJ O H O
NH
1
HN~NH2
HN~NH2 NH
NH NH
H2N , ~ O N N %~ ~ /
N ~ N S
S H3G~N O H O
O H 1TO
NH
HNyNH2 HN N NFi~
INH
HaGN N ' % N
O H O O HZN~N O H O
NH
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WO 98116525 PCTI1JS97118647
HN~NHZ
HN~NHZ 1NH
NH
CF3
HN O N~ N~ %N~
N l \ / ~ N g
N g HN ~ , p H O
O H O
NHZ
HN~NHp HN~NH2
NH NH
N N j~ N ~l \ /
N O
HN O I-1 O H3Gt~+ O H O
HsC
HNyNHp
HN~NH2
lNH NH
H2N.~~.,,,~ N N ~ HZIV~S ~,,,'~ N N
$ NH O H O NH O H O
As mentioned above, the compounds of this invention find utility as
therapeutic
agents for disease states in mammals which have disorders of coagulation such
as in the
treatment or prevention of unstable angina, refractory angina, myocardial
infarction,
transient ischemic attacks, thrombotic stroke, embolic stroke, disseminated
intravascular
coagulation including the treatment of septic shock, deep venous thrombosis in
the
prevention of pulmonary embolism or the treatment of reocclusion or restenosis
of
reperfused coronary arteries. Further, these compounds are useful for the
treatment or
prophylaxis of those diseases which involve the production and/or action of
factor
Xa/prothrombinase complex. This includes a number of thrombotic and
prothrombotic
states in which the coagulation cascade is activated which include but are not
limited to,
deep venous thrombosis, pulmonary embolism, myocardial infarction, stroke,
thromboembolic complications of surgery and peripheral arterial occlusion.
Accordingly, a method for preventing or treating a condition in a mammal
characterized by undesired thrombosis comprises administering to the mammal a
therapeutically effective amount of a compound of this invention. In addition
to the
disease states noted above, other diseases treatable or preventable by the
administration
CA 02268264 1999-04-08
WO 98/16525 PCT/US97/18647
of compounds o~ this invention include, without limitation, occlusive coronary
thrombus
formation resulting from either thrombolytic therapy or percutaneous
transluminal
coronary angioplasty, thrombus formation in the venous vasculature,
disseminated
intravascular coagulopathy, a condition wherein there is rapid consumption of
coagulation
factors and systemic coagulation which results in the formation of life-
threatening thrombi
occurring throughout the microvasculature leading to widespread organ failure,
hemorrhagic stroke, renal dialysis, blood oxygenation, and cardiac
catheterization.
The compounds of the invention also find utility in a method for inhibiting
the
coagulation biological samples, which comprises the administration of a
compound of the
invention.
The compounds of the present invention may also be used in combination with
other
therapeutic or diagnostic agents. In certain preferred embodiments, the
compounds of
this invention may be coadministered along with other compounds typically
prescribed
for these conditions according to generally accepted medical practice such as
anticoagulant
agents, thrombolytic agents, or other antithrombotics, including platelet
aggregation
inhibitors, tissue plasminogen activators, urokinase, prourokinase,
streptokinase, heparin,
aspirin, or warfarin. The compounds of the present invention may act in a
synergistic
fashion to prevent reocciusion following a successful thrombolytic therapy
and/or reduce
the time to reperfusion. These compounds may also allow for reduced doses of
the
thrombolytic agents to be used and therefore minimize potential hemorrhagic
side-effects.
The compounds of this invention can be utilized in vivo, ordinarily in mammals
such as
primates, (e.g. humans), sheep, horses, cattle, pigs, dogs, cats, rats and
mice, or in vitro.
The biological properties of the compounds of the present invention can be
readily
characterized by methods that are wel! known in the art, for example by the in
vitro
protease activity assays and in vivo studies to evaluate antithrombotic
efficacy, and
effects on hemostasis and hematological parameters, such as are illustrated in
the
examples.
Diagnostic applications of the compounds of this invention will typically
utilize
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WO 98/16525 PCT/US97/18647
formulations in the form of solutions or suspensions. In the management of
thrombotic
disorders the compounds of this invention may be utilized in compositions such
as
tablets, capsules or elixirs for oral administration. suppositories, sterile
solutions or
suspensions or injectable administration, and the like, or incorporated into
shaped articles.
Subjects in need of treatment (typically mammalian) using the compounds of
this
invention can be administered dosages that will provide optimal efficacy. The
dose and
method of administration will vary from subject to subject and be dependent
upon such
factors as the type of mammal being treated, its sex, weight, diet, concurrent
medication,
overall clinical condition, the particular compounds employed, the specific
use for which
these compounds are employed, and other factors which those skilled in the
medical arts
will recognize.
Formulations of the compounds of this invention are prepared for storage or
administration by mixing the compound having a desired degree of purity with
physiologically acceptable carriers, excipients, stabilizers etc., and may be
provided in
sustained release or timed release formulations. Acceptable carriers or
diluents for
therapeutic use are well known in the pharmaceutical field, and are described,
for example,
in Remington's Pharmaceutical Sciences, Mack Publishing Co., (A.R. Gennaro
edit.
1985). Such materials are nontoxic to the recipients at the dosages and
concentrations
employed, and include buffers such as phosphate. citrate) acetate and other
organic acid
. salts, antioxidants such as ascorbic acid, low molecular weight (less than
about ten .
residues) peptides such as polyarginine, proteins, such as serum albumin,
gelatin, or
immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidinone, amino
acids
such as glycine, glutamic acid, aspartic acid, or arginine, monosaccharides,
disaccharides,
and other carbohydrates including cellulose or its derivatives, glucose,
mannose or
dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or
sorbitol,
counterions such as sodium and/or nonionic surfactants such as Tween,
Pluronics or
polyethyleneglycol.
Dosage formulations of the compounds of this invention to be used for
therapeutic
27
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WO 98/165Z5 PCT1US971i8647
administration must be sterile. Sterility is readily accomplished by
filtration through
sterile membranes such as 0.2 micron membranes, or by other conventional
methods.
Formulations typically will be stored in lyophilized form or as an aqueous
solution. The
pH of the preparations of this invention typically will be 3-11, more
preferably ~-9 and
most preferably 7-8. It will be understood that use of certain of the
foregoing excipients,
carriers, or stabilizers will result in the formation of cyclic polypeptide
salts. While the
preferred route of administration is by injection, other methods of
administration are also
anticipated such as orally, intravenously (bolus and/or infusion),
subcutaneously,
intramuscularly, colonically, rectally, nasally, transdermally or
intraperitoneally,
employing a variety of dosage forms such as suppositories, implanted pellets
or small
cylinders, aerosols, oral dosage formulations and topical formulations such as
ointments,
drops and dermal patches. The compounds of this invention are desirably
incorporated
into shaped articles such as implants which may employ inert materials such as
biodegradable polymers or synthetic silicones, for example, Silastic, silicone
rubber or
other polymers commercially available.
The compounds of the invention may 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 lipids, such
as
cholesterol, stearylamine or phosphatidylcholines.
The compounds of this invention may also be delivered by the use of
antibodies,
antibody fragments, growth factors, hormones, or other targeting moieties. to
which the
compound molecules are coupled. The compounds of this invention may also be
coupled
with suitable polymers as targetable drug carriers. Such polymers can include
polyvinylpyrrolidinone, pyran copolymer, polyhydroxy-propyl-methacrylamide-
phenol,
polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysine
substituted
with palmitoyl residues. Furthermore, compounds of the invention 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
28
CA 02268264 1999-04-08
WO 98I16525 PCT/US97/18647
acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,
polyacetals,
polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block
copolymers of hydrogels. Polymers and semipermeable polymer matrices may be
formed
into shaped articles, such as valves, stems, tubing, prostheses and the like.
Therapeutic compound liquid formulations generally are placed into a container
having
a sterile access port, for example, an intravenous solution bag or vial having
a stopper
pierceable by hypodermic injection needle.
Therapeutically effective dosages may be determined by either in vitro or in
vivo
methods. For each particular compound of the present invention, individual
determinations may be made to determine the optimal dosage required. The range
of
therapeutically effective dosages will be influenced by the route of
administration, the
therapeutic objectives and the condition of the patient. For injection by
hypodermic
needle, it may be assumed the dosage is delivered into the body's fluids. For
other routes
of administration, the absorption efficiency must be individually determined
for each
compound by methods well known in pharmacology. Accordingly, it may be
necessary
for the therapist to titer the dosage and modify the route of administration
as required to
obtain the optimal therapeutic effect. The determination of effective dosage
levels, that
is, the dosage levels necessary to achieve the desired result, will be readily
determined by
one skilled in the art. Typically, applications of compound are commenced at
lower
dosage levels, with dosage levels being increased until the desired effect is
achieved.
The compounds of the invention can be administered orally or parenterally in
an
effective amount within the dosage range of about 0.1 to 100 mg/kg, preferably
about 0.5
to 50 mglkg and more preferably about 1 to 20 mg/kg on a regimen in a single
or 2 to 4
divided daily doses andlor continuous infusion.
Typically, about 5 to 500 mg of a compound or mixture of compounds of this
invention, as the free acid or base form or as a pharmaceutically acceptable
salt, is
compounded with a physiologically acceptable vehicle, carrier, excipient,
binder,
preservative, stabilizer, dye, flavor etc., as called for by accepted
pharmaceutical practice.
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CA 02268264 1999-04-08
WO 98l16525 PCT/ITS97/I8647
The amount of active ingredient in these compositions is such that a suitable
dosage in the
range indicated is obtained.
Typical adjuvants which may be incorporated into tablets, capsules and the
like are
binders such as acacia, corn starch or gelatin, and excipients such as
microcrystalline
cellulose, disintegrating agents like corn starch or alginic acid, lubricants
such as
magnesium stearate, sweetening agents such as sucrose or lactose, or flavoring
agents.
When a dosage form is a capsule. in addition to the above materials it may
also contain
liquid carriers such as water, saline, or a fatty oil. Other materials of
various types may
be used as coatings or as modifiers of the physical form of the dosage unit.
Sterile
compositions for injection can be formulated according to conventional
pharmaceutical
practice. For example, dissolution or suspension of the active compound in a
vehicle such
as an oil or a synthetic fatty vehicle (ike ethyl oleate, or into a Iiposome
may be desired.
Buffers, preservatives, antioxidants and the like can be incorporated
according to accepted
pharmaceutical practice.
Preparation of the Disclosed Compounds
The compounds of the present invention may be synthesized by either solid or
liquid
phase methods described and referenced in standard textbooks, or by a
combination of
both methods. These methods are well known in the art. See, Bodanszky, "The
o Principles of Peptide Synthesis", Hafner, et al., Eds., Springer-Verlag,
Berlin, 1984. .
Starting materials used in any of these methods are commercially available
from
chemical vendors such as Aldrich, Sigma, Nova Biochemicals, Bachem
Biosciences, and
the like, or may be readily synthesized by known procedures.
Reactions are carried out in standard laboratory glassware and reaction
vessels under
reaction conditions of standard temperature and pressure, except where
otherwise
indicated.
During the synthesis of these compounds, the functional groups of the amino
acid
derivatives used in these methods are protected by blocking groups to prevent
cross
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reaction during the coupling procedure. Examples of suitable blocking groups
and their
use are described in "The Peptides: Analysis, Synthesis, Biology", Academic
Press, Vol.
3 (Gross, et al., Eds., 1981 ) and V ol. 9 ( 1987), the disclosures of which
are incorporated
herein by reference.
Two exemplary synthesis schemes are outlined directly below, and the specific
syntheses are described in the Examples. The reaction products are isolated
and purified
by conventional methods, typically by solvent extraction into a compatible
solvent. The
products may be further purified by column chromatography (reversed-phase
HPLC) or
other appropriate methods.
to Formula IV
The peptide derivatives can be prepared by coupling of protected Fragment-1
(Scheme 1 ) to protected Fragment-2 (Scheme 2). The protecting groups are
finally
removed by HF-cleavage.
Most compounds are purified by reversed-phase HPLC and characterized by ion-
spray MS spectrometry
Scheme I
7 ((//~~~'
Pre aration of Fra meat-1 - OH
P 9
BOC-N. J O
31
Fragment -1 Fragment-2
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v_
off ~goc)20 H Swern r H Wilting
HN N ~ Boc~ N~~.,
Boc~
Et ~ oEt i_DA
v II
Boc~N~ 0 Boc~N~ 0 CHp=CHCN
'CN ~-NH p
oEt H2 oEt t . aq. NaOH
Pt0 N ~ v ~ 2. Cyclizat on
Boc~ Boc' BOP
0
NaN(TMS)2
NH - ~ ~ OEt
BrCH2C00Et N o
Boc
Boc~N O
O
aq. NaOH N' ~
v _OH
Boc~ NJ O
Scheme II
Synthesis of Fragment-2 and Final Inhibitor
H 0 H 0 H 0
_ ~ I' ' ~N
Bo~N~OH .N~ .OMe ,N
BOP Boc N Boc
---~ ~ ----~' S
MeNHOMe ~ Li
NH NH S NH
HN ~ NH-Tos HN ~ NH-Tos HN ~ NH-Tos
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H O
N
H ~I
TFA ~ + N BOP
~COOH
Boc-N O
NH
HN i ' NH-Tos
HN~NH-Tos HN~NH 2
'~N'H NH
O N
N~ N~ ~ N
N S
8oc-N~ v d ~ ~ ' HN~ Y O H O
The chemical reactions described in Schemes I and II can easily be modified
and
combined with other techniques that are well known in the art to produce other
compounds within the family of formula I.
Methods of incorporating "A" substituents such as R3, -NR3R'~;
NR15 NR~~
~NR3Rt7
~N NR3R~6 ; '
Rya
NR15 NRt5 NRtS
~N~Ris. ~ and
' ~Rt7 ~ ~NR3Rt~
R S
are well known in the art. The chemistry of using the various R substituents
(H, -OH,
C~_balkyl and C~.~aIkylaryl) is also well known in the art. Methods of
incorporating "W"
substituents such as Ci_balkyl, C3_gcycloalkyl, aryl, or a five to ten
membered heterocyclic
ring system containing 1-4 heteroatoms selected from the group consisting of
N, O and S,
are also well known in the art. In addition, "A" and "W" substituents are
illustrated in
Example 13.
The "R," substituent illustrated in Fragments -l and -2 is -H. Fragments
similar to
these structures, where R~ is C,_balkyl, C3_6cycloalkyl, C,_3alkylaryl, or
aryl, can easily be
synthesized by techniques that are well known in the art. See, for example,
Duggan, et
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al., U.S. Patent No. 5,28l,585.
The "E" substituent illustrated in Fragment-2 (-NHC(=NH)-NH2) is merely
illustrative of one of the various "E" substituents encompassed by the
invention.
Fragments similar to the structure of Fragment-2, where E is -NH2, -SC{=NH)-
NH2 or
-C(=NH)-NH2, can easily be synthesized by techniques that are well known in
the art.
The "Y" substituent illustrated in Fragment-2 is -CO-G, where G is:
N
\ L
J
where: J is -S- and L is
R8
R9
and Rg and R9 are H. Fragments similar to the structure of Fragment-2, where G
is:
N
~J /
and J is -SO- or -S02- can easily be synthesized by techniques that are well
known in the
art. Fragments where J is -O- can be synthesized by techniques illustrated in
J. Am.
Chem. Soc. I 14: 1854-1863 (1992), J. Med. Chem. 38:76-85 (1995), and J. Med.
Chem.
37:3492-3502 ( 1994). Lastly, fragments where J is -NR'-, where R' is H,
C,_6alkyl or
benzyl, can be synthesized by techniques illustrated in J. Med. Chem. 37:3492-
3502
( i 994). All of these references are incorporated herein by reference.
Fragments similar to the structure of Fragment-2, where G is:
N
~J /
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and L is:
Rs
~~ R a
OH2)r
~~ R~ ~\'Rs
where R6, R', Rg and R9 are not H, can also be easily synthesized by
techniques that are
well known in the art.
Fragments similar to the structure of Fragment-2, where G is H, -COORS 8,
-CONR~gR~9, -CF3 or -CF2CF3, can easily be synthesized by techniques that are
well
known in the art.
Fragments similar to the structure of Fragment-2, where G is:
N~~
~O~ \ RZo
and U and V are the various substituents (-O-, -S-, -N-, -N(H)-) can also
easily be
synthesized by well known techniques illustrated in J. Med. Chem. 38: 1355-
137l ( 1995)
and J. Med. Chem. 37: ?421-2436 ( 1994).
- Fragments similar to the structure of Fragment-2, where "Y" is a boron-
containing
group can easily be synthesized by techniques that are well known in the art.
See for
example. J. ~. Chem. 60:37I7-3722 (l995).
The mechanisms for coupling the fragments used to synthesize the compounds of
the
instant invention, are also well known in the art.
Without further elaboration, it is believed that one skilled in the art can
utilized the
present invention to its fullest extent. Therefore, the following preferred
specific
embodiments are to be construed as merely illustrative and do not limit the
remainder of
the disclosure in any way whatsoever.
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EXAMPLE 1
Preparation of Boc-Arg(Tos)-N(Me)OMe
H O
Boc~ N ~ N'OMe
CH3
NH
HN i 'NH-Tos
To a suspension of Boc-Arg(Tos)-OH (2 g, 4.7 mmol) in DMF (20 mL) at 0~C is
added MeNHOMe.HCI ( 1 g, 10.3 rrunol), DIEA (6 mL) and BOP (2.5 g, 5.6 mmol).
The
solution is stirred at 0~C for 10 h. DMF is evaporated by vacuum. The oily
residue is
dissolved in EtOAc (200 mL) and water (20 mL). The organic layer is washed
with sat.
NaHC03, water (20 mL), 1 M HC1 ( 10 mL) and sat. NaCI (2 X 20 mL). The organic
layer is dried over MgSOa, filtered and evaporated to give a suspension. The
suspension
is filtered. washed with cold EtOAc ( 10 mL) and dried to give Boc-Arg(Tos)-
N(Me)OMe
( 1.5 g, 70 % yield). FAB-MS (M+H)+ = 472
EXAMPLE 2
Preparation of Boc-Axg(Tos)-Thiazole
H O
~~S
Boc' N ~''
N
NH
HN ~ NH-Tos
To a solution of thiazole (2.5 g, 29 mmol) in THF (25 mL) at -78~C is added n-
BuLi
( 1.6 M in hexane, 19 mL) dropwise. The mixture is stirred for 30 min. Then a
solution of
Boc-Arg(Tos)-N(Me)OMe ( 1.7 g, 3.6 mmol) in THF (50 mL) is added to the
Iithiothiazole mixture at -78~C. The solution is stirred for 2 h. 1 M HCI (30
mL) is added
to the reaction mixture and warmed to room temperature. The mixture is
extracted with
EtOAc ( 100 mL). The organic layer is washed with sat. NaCI (30 mL), dried
over
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MgSO~, filtered and evaporated. The crude oily residue is purified by flash
column over
Si02 (50% EtOAc in CHZCIZ) to give Boc-Arg(Tos)-Thiazole ( 1.5 g, 84% yield)
as a
powder. DCI -MS (M+H)+ = 496
EXAMPLE 3
Preparation of H-Arg(Tos)-Thiazole
H O
~ ~S
TFA ~ H ~N ~ '(
N
NH
HN i 'NH-Tos
To a solution of Boc-Arg(Tos)-Thiazole (300 mg, 0.6 mmol) in CH~C1, ( 10 mL)
at
0~C is added TFA { 10 mL). The solution is stirred at 0~C for 2 h. The solvent
and excess
TFA are evaporated to an oily residue which is used directly without further
purification.
FZC d RAPT F d
Preparation of N-Boc-4-piperidineethanol
off
N
Boc~
A stirred solution of 4-piperidineethanol ( 1.87 g, 14 mmol) and DMF (5 mL) at
0~C is
treated with N-t-butoxycarbonyl anhydride (3 g, 14 mmol). After 1 h, the
cooling bath is
removed and the reaction mixture stirred for an additional 20 h. The reaction
mixture is
then diluted with ether, and concentrated to furnish the title compound (2.6
g, 82%) as a
colorless oil.
EXAMPLE 5
Preparation of Ethyl 4-(N-Boc-piperidin-4-yl)-trans-crotonate
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OEt
Boc~N O
To a stirred solution of oxalyl chloride (0.43 mL, 5.0 mmol) in CH~CI~ (30 mL)
at -
78~C is added DMSO (0.S2 mL, 7.0 mmol) dropwise. After gas evolution subsided
(5
min), the alcohol of Example 4 {0.8 g, 3.5 mmol) in CHZCIz (20 mL) is added in
a stream.
After 20 min, (carbethoxymethylene) triphenyl-phosphorane ( I .4 g, 4.0 mmol)
is added.
After 2 h, the reaction mixture is diluted with petroleum ether, washed with
water, 5%
KHS04 and sat. NaCI, dried over MgSO,~, and concentrated. Flash column
chromatography (15% EtOAc in hexane) gave the title compound (0.57 g, 38%) as
a
colorless oil.
EXAMPLE 6
Preparation of Ethyl 4-(N-Boc-piperdin-4-yl)butyrate
OEt
N
Boc~ O
The olefin of Example 5 (2.6 g, 8.7 mmol) in EtOAc (50 mL) at room temperature
is
stirred under a hydrogen atmosphere ( 1 atm) in the presence of 10% Pd/C (500
mg)
overnight. The reaction mixture is then purged with argon, followed by
filtration through
a Celite pad. Concentration of the filtrate followed by flash chromatography (
10%
EtOAc in hexane) gave the title ester (2.4 g, 92%) as a crystalline solid.
EXAMPLE 7
Preparation of:
-CN
OEt
Boc~ N O
To a solution of TiCl4 (4.2 mL, 4.2 mmol, 1 M in CH2Cl2) and CH2C12 (25 mL) at
0~C
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is added titanium isopropoxide (0.42 mL, 1.4 mmol). After 15 min,
diisopropylethylamine (1 mL, 6.3 mmol) is added dropwise to form a solution.
After 10
min, the ester of Example 6 (~ mmol) in CHZCIZ (7 mL) is added, followed by
stirring at
0~C for 1 h. ~Acrylonitrile (3.3 mL, 50 mmoI) is added dropwise at 0~C to the
solution.
S After 4 h, the reaction is quenched with sat. NH.~CI ( 15 mL) at 0~C and
then the mixture
extracted with CH2CI2. The combined organic extracts are washed with sat.
NaHC03 and
sat. NaCI, dried over MgS04, and concentrated. Flash column chromatography
gives the
compound shown above, as an oil.
EXAMPLE 8
Preparation of:
~NH2 HCI
OEt
Bocf N O
A mixture of the compound of Example 7 (40 mmol), Pt02 (2.0 g), MeOH (70 mL)
and CHC13 (? mL) is shaken on the Parr apparatus under a hydrogen atmosphere
(60 psi)
at room temperature for 3 h. The reaction mixture is filtered through a Celite
pad and
concentrated to furnish the crude amine HCl as a solid.
EXAMPLE 9
Preparation of:
NH
N
Boc~ O
The crude amine.HCl of Example 8 ( 10 mmol), acetonitrile (?5 mL), and NaHC03
(3
g) is stirred at room temperature for 20 h. The heterogeneous mixture is then
filtered and
filtrate concentrated. Flash chromatography gives the compound shown above, as
a
powder.
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EXAMPLE 10
Preparation of
O
N' ~
_OEt
N
Boc~ O
To a stirred solution of the title compound of Example 9 (6 g, 22 mmol) and
THF ( 1 S
mL) at -78~C is added NaN(TMS)2 (24.S mL, 24.5 mmol, 1 M in hexane) dropwise.
After 15 min, ethyl bromoacetate (5.2 mL, 45 mmol) is added and then the
reaction
mixture warmed to 0~C for 1 h. The reaction is quenched with HOAc ( 1.0 mL)
and then
the mixture diluted with EtOAc, washed with water and sat. NaCI, dried over
MgS04,
and concentrated. Flash chromatography (40% EtOAc in hexane) gave the compound
shown above (7.6 g, 78%), as an oil.
EXAMPLE 11
Preparation of:
1
N' ~
_OH
N
Boc~ O
A solution of the compound of Example 10 (6.0 g, 15 mmol), 1 N NaOH (50 mL, 50
mmol), and MeOH (75 mL) is stirred at room temperature for 1 h. The reaction
mixture
is then acidified with 5% KHS04 and then extracted with EtOAc. The organic
portion is
washed with sat. NaCI, dried over MgSO~, and concentrated to give the compound
shown
above (5.6 g, l00%), as an oil.
EXAMPLE 12
Preparation of:
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HN ' ' NH-Tos
~NH
1 ~
N~N
,~ J 11 H I N
Boc~N~ O O
The compounds of Example 11 ( 1 mmol) and Example 3 ( 1 mmol) are dissolved in
DMF (5 mL) and cooled to 0~C. The solution is neutralized with DIEA ( 1 mL)
followed
by the addition of coupling reagent BOP (I.1 mmol). The solution is stirred
for 1-2 h and
HPLC analysis shows the completion of reaction. Solvent is removed by vacuum
at
30~C. The residue is dissolved in a mixture of EtOAc-HZO (50 mL:10 mL) and
aqueous
layer is discarded. The organic layer is washed with sat. NaHC03 (2 X 10 mL),
sat. NaCI
(2 X 10 mL), dried over MgSO.~, filtered and evaporated. The oil residue is
purified by
flash column on SiO~ to give the compound shown above, as a powder.
EXAMPLE I3
Preparation of:
HN ' ' NHp
~N'H
N~ N
II H I N
H~N~ O O
100 mg of the compound of Example 12, 1 ml of anisole and 4 drops of MeSEt are
1 S placed in HF-cleavage vessel and cooled under liquid N2. 10 ml of HF is
then condensed
and the mixture is stirred at 0~C for 1.25 h. HF is removed under vacuum to
give a gum-
like residue. The residue is triturated with 20 ml of 50% Et20-hexane and the
solvent
removed by filtration. The gum residue is dissolved in 30 ml of 30% aq. HOAc
and
filtered through the above sintered funnel. The filtrate is lyophilized to
give a powder
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which is purified by RP-HPLC to give the compound shown above, as a powder.
EXAMPLE 14 (Determination of IC;~,)
The compounds of the present invention are first dissolved in a buffer to give
solutions containing concentrations such that assay concentrations range from
0-100 ltM.
In assays for thrombin, prothrombinase and factor Xa, a synthetic chromogenic
substrate
would be added to a solution containing a test compound and the enzyme of
interest and
the residual catalytic activity of that enzyme would then be determined
spectrophotometrically.
The IC;o of a compound is determined from the substrate turnover. The IC;o is
the
concentration of test compound giving 50% inhibition of the substrate
turnover.
Preferred compounds of the invention desirably have an IC;o of less than Q00
nM in the
factor Xa assay, preferably less than 200 nM, and more preferably less than
100 nM.
Preferred compounds of the invention desirably have an IC;o of less than 4.0
~.M in the
prothrombinase assay, preferably Less than 200 nM, and more preferably Iess
than 10
nM. Preferred compounds of the invention desirably have an IC;o of greater
than 1.0 p,M
in the thrombin assay, preferably greater than 10.0 ~tM, and more preferably
greater than
100.0 ~tM.
Amidolvtic Assays for determining protease inhibition activity
a Factor Xa and thrombin assays are performed at room temperature, in 0.02 M
Tris
HCl buffer, pH 7.5, containing 0.15 M NaCI. The rates of hydrolysis of the
para-
nitroanilide substrate S-276S (Chromogenix) for factor Xa, and the substrate
Chromozym
TH (Boehringer Mannheim) for thrombin following preincubation of the enzyme
with the
test compound for 5 minutes at room temperature are determined using a Softmax
96-well
plate reader (Molecular Devices), monitored at 405 nm to measure the time
dependent
appearance of p-nitroanilide.
The prothrombinase inhibition assay is performed in a plasma free system with
modifications to the method as described by Sinha, et al., Thromb. Res.,
7S:427-436
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( 1994). The activity of the prothrombinase complex is determined by measuring
the time
course of thrombin generation using the p-nitroanilide substrate Chromozym TH.
The
assay consists of a S minute preincubation of selected compounds to be tested
as
inhibitors with the complex formed from factor Xa (0.5 nM), factor Va (2 nM),
phosphatidyl serine:phosphatidyl choline (25:75, 20 ~.M) in 20 mM Tris HCl
buffer, pH
7.5, containing 0.15 M NaCI, S mM CaCl2 and 0.1 % bovine serum albumin.
Aliquots
from the complex-test compound mixture are added to prothrombin ( 1 nM) and
Chromozym TH (0.1 mM). The rate of substrate cleavage is monitored at 405 nm
for
two minutes. Several concentrations of a given test compound are assayed in
duplicate.
A standard curve of thrombin generation by an equivalent amount of untreated
complex is
then used for determination of percent inhibition.
EXAMPLE 15
The antithrombotic efficacy of the compounds of this invention can readily be
evaluated using a series of studies in rabbits, as described below. These
studies are also
useful in evaluating a compounds effects on hemostasis and its the
hematological
parameters.
Antithrombotic Efficacy in a Rabbit Model of Venous Thrombosis
A rabbit deep vein thrombosis model as described by Hollenbach, et al.,
Thromb.
Haemost. 71:357-362 ( 1994), is used to determine the in vivo antithrombotic
activity of
the compounds of the present invention. Rabbits are anesthetized with LM.
injections of
Ketamine, Xylazine, and Acepromazine cocktail.
A standardized protocol consists of insertion of a thrombogenic cotton thread
and
copper wire apparatus into the abdominal vena cava of the anesthetized rabbit.
A non-
occlusive thrombus is allowed to develop in the central venous circulation and
inhibition
of thrombus growth is then used as a measure of the antithrombotic activity of
the
compound being evaluated. Test agents or control saline are administered
through a
marginal ear vein catheter. A femoral vein catheter is used for blood sampling
prior to and
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during steady state infusion of the compound being evaluated. Initiation of
thrombus
formation will begin immediately after advancement of the cotton thread
apparatus into
the central venous circulation. The compounds being evaluated are administered
from
time=30 minutes to time=150 minutes at which point the experiment is
terminated. The
rabbits are euthanized and the thrombus excised by surgical dissection and
characterized
by weight and histology. Blood samples are then analyzed for changes in
hematological
and coagulation parameters.
Although the invention has been described with reference to the disclosed
embodiments, those skilled in the art will readily appreciate that the
specific experiments
detailed are only illustrative of the invention. It should be understood that
various
modifications can be made without departing from the spirit of the invention.
Accordingly, the invention is limited only by the following claims.
44
