Note: Descriptions are shown in the official language in which they were submitted.
WO91/04746 PCT/US90/0~44X
0 ~ -2-
ANTI-THROMBOTIC PEPTIDES AND PSEUDOPEPTIDES
This application is a continuation-in-part of
application Serial No. 415,006 filed on September 29,
1989, and issued on August 28, 1990, as U.S. Patent No.
4,952,562.
Background of the Invention
1. Field of the Invention
This invention relates to novel compounds having
anti-khrombotic activity. More particularly, the
invention relates to novel peptides and pseudopeptides
that inhibit platelet aggregation and thrombus formation
in mammalian blood thereby being useful in the prevention
and treatment of thrombosis associated with certain
disease states, such as, myocardial infarction, stroke,
peripheral arterial disease and disseminated intravascular
coagulation.
2. Description of the Prior Art
Haemostasis, the biochemistry of blood coagulation,
2S is an extremely complex and as yet not completely
understood phenomena whereby normal whole blood and body
tissue spontaneously arrest bleeding from injured blood
vessels. Effective haemostasis requires the combined
activity of vascular, platelet and plasma factors as well
as a controlling mechanism to prevent excessive clotting.
Defects, deficiencies, or excesses of any of these
components can lead to h~morrhagic or thrombotic
consequences.
Platelet adhesion, spreading and aggregation on
extracellular matrices are central events in thrombus
formation. These events are mediated by a family of
platelet adhesive glycoproteins, i.e., fibrinogen,
fibronectin, and von Willebrand factor. Fibrinogen is a
W09l/04746 PCT/US90/0544~
2~0A~
co-factor for platelet aggregation, fibronectin supports
platelet attachments and spreading reactions, and von
Willebrand factor is important in platelet attachment to
and spreading on subendothelial matrices. The binding
sites for fibrinogen, fibronectin and von Willebrand
factor have been located on the platelet membrane
glycoprotein complex IIb/IIIa.
Adhesive glycoprotein, like fibrinogen, do not bind
with normal resting platelets. However, when a platelet
is activated with an agonist such as thrombin or adenosine
diphosphate, the platelet changes its shape, perhaps
making the GPIIb/IIIa binding site accessible to
fibrinogen. The novel molecules described in this
invention may block the fibrinogen receptor, thus
inhibiting platelet aggregation and subsequent thrombus
formation. Pharmaceutical agents and/or compositions
possessing such an inhibiting effect may be provided for
the prophylaxis and treatment of thrombogenic diseases,
such as myocardial infarction, stroke, peripheral arterial
disease and disseminated intravascular coagulation.
It has been observed that the presence of Arg-Gly-Asp
(RGD) is necessary in fibrinogen, fibronectin and von
Willebrand factor for their interaction with the cell
surface receptor (Ruoslahti E., Pierschbacher, Cell 1986,
44, 517-18). Two other amino acid sequences also seem to
take part in the platelet attachment function of
fibrinogen, namely, the Gly-Pro-Arg sequence, and the
dodecapeptide, His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-
Asp-Val sequence, small synthetic peptides containing the
RGD or dodecapeptide have been shown to bind to the
platelet GPIIb/IIIa receptor and competitively inhibit
binding of fibrinogen, fibronectin and von Willebrand
factor as well as inhibit aggregation of activated
platelets (Plow et al. Proc. Natl. Acad. Sci. USA 1985,
82, 8057-61; Ruggeri et al. Proc. Natl. Acad. Sci. USA
1986, 5708-12; Ginsberg et al. J. Biol. Chem. 1985, 260,
WO9l/01746 ~ PCT/US90/05448
3931-36; and Gartner et al. J. Biol. Chem. 1987, 260,
11,891-94).
The present invention is directed to novel peptides
and pseudopeptides which inhibit platelet aggregation and
subsequent thrombus formation.
W09l/04746 ' PCT/~'S90/0~44#
-5- 2Q~04 ~
SUMMARY OF THE INVENTION
In accordance with the present invention, novel
peptides and pseudopeptides are provided for the
prophylaxis and/or treatment of thrombotic disease states
having the general formulae:
o
X- CH - A - CH2-B - CH - D - CH- C - oR2
( CH2 ) m ICH2 E
N - Y COOH
X- CH- A- CH- D- CH- C - ORZ
(ICH2)m ICH2 K
N -Y COOH
R1
II
X- CH- A- CHz- B- CH- F- G
( ICHz) m Cl H2
N -Y COOH
R1
III
R1 o
11
X- C -A - CH - D -CH- C - Z
R1-C-R2 CH2 K
3 0 ( CH2 ) m COOH
N--Y
R1
IV
W O 91/04746 PC~r/US90/0544X
~QS~ 4~ -6-
R1 R1 R2 0
R2_ C--( CH2 ) m--C = C--( CR1R2 ) m--A--CH--D--CH--C--Z
1 ' 2 CH2 X
( CHz ) m COOH
N--Y
R1
V
. ~
and pharmaceutically acceptable salts thereof, wherein:
X is H,
/R1
N< or
\R2
NH- C -R2;
Y is H,
alkyl,
cycloalkyl,
aralkyl,
N_Rl
C - NR1R2 or
C- R2;
A, B and D are independently
o
C--NR2,
CH20, or
CH2--NR2;
E is H,
CH3, or
CH3
CH<
CH3
WO 91/04746 PC'r/US~()/0~448
-7- 2Q~6()47
X is H,
CH3 ~
CH3
CH<
CH3
CH2--OH,
CH3CH--OH,
<~3 CH2,
~
HO ~ CH2 or
~ CH2
F is O
C or
2 0 CH2;
G is oR2,
/
N<
2S R2
CH2- ( CIH2) n
N CH2, or
(gH
R1 and R2 are independently:
H,
alkyl,
cycloalkyl,
3 5 aryl, and
aralkyl;
Z is OR1~ NH2 or NR1R2;
WO91/04746 PCT/~S90/~5448
Q66G~$~ -8-
n is 0-5;
provided that, in formula I, when X is NH2, then:
m is 3,
Y is C-NH2,
NH
CH3
E is CH < ; and
CH3
o
that at least one radical in A, B and D is not C-NH;
and provided further that, in formula III, when X is
NH2 then:
NH
Y i s C--NH2,
m is 3,
o
F is C,
2 5 G i s OH; and
o
that one radical in A and B is not C-NH.
WOgl/04746 PCT/US90/0;44X
9 2 ~ 7
DETAILED DESCRIPTION OF THE INVENTION
As employed above and throughout the disclosure, the
following terms, unless otherwise indicated, shall be
understood to have the following meanings:
"Alkyl" means, either alone or within the various
substitutents, defined hereinbefore, a hydrocarbon having
one to about 20 carbon atoms. "Lower alkyl" means alkyl
having one to about six carbon atoms, for example, methyl,
ethyl, propyl, isopropyl, butyl, isobutyl, amyl and hexyl.
Preferred lower alkyl includes methyl, ethyl and propyl.
"Aryl" means a mononuclear and polynuclear aromatic
hydrocarbon radical which can be substituted or
unsubstituted in one or more positions. Examples of aryl
groups include phenyl, naphthyl, anthranyl, phenanthranyl,
azulyl and the like which can be substituted with one or
more of the substituents. Aryl is preferably substituted
or unsubstituted phenyl or naphthyl. Aryl substituents
include hydrogen, alkyl, alkoxy, amino, halo, aryl,
eryloxy, carboalkoxy, nitro, dialkylamino,
trifluoromethyl, thioalkyl and carbamoyl.
"Aralkyl" means an alkyl group substituted by an aryl
radical. The preferred aralkyl groups are lower alkyl
groups substituted by phenyl or substituted phenyl. The
most preferred aralkyl group is benzyl.
In accordance with the present invention, novel
compounds are provided which inhibit platelet aggregation
by inhibiting fibrinogen binding to activated platelets
and other adhesive glycoproteins involved in platelet
aggregation and blood clotting. Compounds of the present
invention, as tested by methods predictive of anti-
thrombotic activity, are believed to be useful in the
prevention and treatment of thrombosis associated with
certain diseased states, such as myocardial infarction,
W091/04746 PCT/~S90/0~8
--10--
str~ eripheral arterial disease and disseminated
intravascular coagulation.
The present compounds may also be useful for the
treatment of certain diseases associated with abnormal
cell growth since they may interfere with adhesive
interactions betweèn abnormal cells and the extracellular
matrix (Journ. of Biol. Chem., Vol. 262, No. 36 1987, pp.
17703-17711; Science, Vol. 233, 1986, pp. 467-470; and
Cell, Vol. 57, S9-69, Apr. 1989).
The compounds of the present invention may be readily
prepared by standard solid phase or solution phase peptide
synthesis using starting materials and/or readily
available intermediates from chemical supply companies
such as Aldrich or Sigma, (H. Paulsen, G. Merz, V.
Weichart, "Solid-Phase Synthesis of O-Glycopeptide
Sequences", Angew. Chem. Int. Ed. Engl. 27 (1988); H.
Mergler, R. Tanner, J. Gosteli, and P. Grogg, "Peptide
Synthesis by a Combination of Solid-Phase and Solution
Methods I: A New Very Acid-Labile Anchor Group for the
Solid-Phase Synthesis of Fully Protected Fragments.
Tetrahedron letters 29, 4005 (1988); Merrifield, R.B.,
"Solid Phase Peptide Synthesis after 25 Years: The Design
and Synthesis of Antagonists of Glucagon", Makromol. Chem.
Macromol. Symp. 19, 31 (1988)).
We prefer to use the solid phase method schematically
represented as follows:
solid support -IX1- N -P Deprotect
solid support - Xl- NH Coupling
P R
solid support - X1- NH - C - Xz- N - P
P R P R
wo 9l/n4~46 2 Q ~ ~ o ~ S9~/0~448
wherein: the solid support may be, but is not limited to,
p-alkoxy benzyl resin; and - X1- N - P is an N-protected
amino acid. P R
In the synthetic process of making the desired
compound the amino acid derivatives are added one at a
time to the insoluble resin until the total sequence has
been built up on the resin. The functional groups of the
amino acid derivatives are protected by blocking groups to
prevent cross reaction during the coupling procedure.
These blocking groups include ~-tertiary butyloxycarbonyl
(BOC), benzyloxycarbonyl (CBZ), benzyl, t-butyl, 9-fluor-
enylmethyloxycarbonyl (FMOC), 2-(trimethylsilyl)ethyl, and
4-methoxy-2,3,6-trimethylbenzenesulfonyl. Upon completion
of the coupling reaction a functional group is deprotected
by standard methods to give an active ~-amino function
which, in turn, is reacted with a protected amino acid
derivative having a free ~-carboxyl function thereon.
This procedure is repeated until the desired peptide or
pseudopeptide is formed. The compound is then deprotected
and removed from the solid support by standard procedures
to obtain the final product.
Alternatively, the compounds of the present invention
may be prepared in solution, i.e., without using a solid
support. In a manner that is similar to the solid phase
synthesis the protected amino acid derivatives or analogs
are coupled by using standard procedures, then deprotected
to yield the desired final compound.
The invention will now be further explained by the
following illustrative examples:
EXAMPLE 1
L-Arainvl-L-Aspartyl-L-Valine
l.Og of N-(9-fluorenylmethyloxycarbonyl)-L-valine p-
alkoxybenzyl alcohol resin ester (containing 0.56 mmole of
WO9l/04746 ~Q~6~ 4 PCT/~S90/0544X
-12-
amino acid) is shaken with 20 ml of 20% (v/v) piperidine
in methylene chloride for 1 hour to remove the FMOC group.
The mixture is filtered and the resin washed with
methylene chloride. The deprotected resin is treated with
0.92g of N-FMOC-L-aspartic acid-B-t-butyl ester in 15 ml
of dimethylformamide in the presence of 0.43g 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDC), 0.31 ml triethylamine, and 0.30g l-hydroxybenzo-
triazole (HOBT), for 1 1/2 hours. This is filtered,
washed with methylene chloride, and the resulting resin
treated with 20% piperidine in methylene chloride as above
to remove the FMOC group. The resulting resin derivative
is then treated as above with 1.36g N-~-FMOC-N-~-(4-
methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine in the
presence of triethyl-amine, EDC, and HOBT. The FMOC group
is removed as above. The peptide is removed from the
resin by treating with 20 ml of 95% trifluoroacetic acid
for two hours. The arginine residue is deprotected by
overnight treatment with concentrated trifluoroacetic
acid. The resulting solution is diluted with 0.5% acetic
acid, washed with 3 portions of ethyl acetate, then
lyophilized to give L-arginyl-L-aspartyl-L-valine as the
ditrifluoroacetate salt; m.p. 90-95-C.
EXAMPLE 2
L-Arainylalycyl-L-As~artyl-~-Isobutylamide
A. 1.16g of 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide hydrochloride (EDC) and 0.93 ml of triethylamine
are stirred together in 50 ml of methylene chloride for 10
minutes. 2.5g N-~-(FMOC)-L-aspartic acid B-t-butyl ester,
0.60 ml isobutylamine and 0.82g hydroxybenzotriazole
(HOBT) are added and the solution stirred at room
temperature overnight. The solution is diluted with ethyl
acetate, washed twice with water and dried over magnesium
sulfate. The filtered solution is evaporated in vacuo to
give 2.2g N-~-(FMOC)-L-aspartic acid isobutyl amide B-
butyl ester.
W091/04746 2 ~ ~ 6 o ~ ~ Pcr/~s90/o~x
-13-
B. The amide obtained in 2A is dissolved in 20%
(v/v) piperidine in methylene chloride and stirred at room
temperature for 2 hours. The solution is evaporated in
vacuo and the residue dissolved in ethyl acetate and this
solution is washed with 10% sodium bicarbonate solution,
dried over sodium sulfate, filtered and evaporated to give
1.7g L-aspartic acid-~-isobutyl amide-~-t-butyl ester.
C. 0.67g N-FMOC glycine and 0.SSg of the amide
obtained in 2B are treated under the conditions of 2A to
give N-~-(FMOC)-glycyl-L-aspartic acid isobutyl amide-B-
butyl ester.
D. The product obtained in 2C is treated as in 2B
lS to remove the FMOC protecting group to give glycyl-L-
aspartic acid isobutyl amide-B-butyl ester.
E. 0.40g of the product of 2D and 0.78g N-~-t-BOC-
N-~-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine
are treated as in 2A with 0.29g EDC, 0.17g HOBT and 0.18
ml triethylamine to give N-~-80C-N-~-methoxy-2,3,6-
trimethylbenzenesulfonyl)-L-arginylglycyl-L-aspartic acid
isobutyl amide-B-butyl ester.
2S F. 0.35g of the product obtained in 2E is treated
with concentrated trifluoroacetic acid in the presence of
two drops of ethanedithiol overnight. The solution is
diluted with 0.S% acetic acid and washed with 4x100 ml of
ethyl acetate. The aqueous solution was lyophilized to
0.19g of a white solid, L-arginylglycyl-L-aspartyl-~-
isobutylamide as the ditrifluoroacetate salt; m.p. 90-
9SC.
EXAMPLE 3
3S
L-Ornithylglycyl-L-Aspartyl-Valine
A. 1.27g L-valine t-butyl ester and 2.5g N-~-FMOC-
L-aspartic acid ~-t-butyl ester are treated as in 2A in
w09l/04746 ~6~ -14- PCT/US90/~5448
the presence of 1.16g EDC, 0.93g triethylamine and 0.82g
hydroxybenzotriazole. The resulting product is then
deprotected as in 2B to give L-aspartyl-B-t-butyl ester-L-
valine-~-t-butyl ester.
S
B. l.lg of the product obtained from 3A is treated
with N-~-FMOC-glycine in the presence of 0.60g EDC, and
0.43g of triethylamine in methylene chloride as in 2A and
the resulting product deprotected in 20% piperidine in
methylene chloride as in 2B to give 0.65g glycyl-L-
aspartyl-~-t-butyl ester-L-valine-~-t-butyl ester.
C. 0.25g of the product from 3B is treated with
0.23g N-~-t-BOC-N-~-CBZ-ornithine in 5ml of methylene
chloride in the presence of 0.12g EDC, 0.80g HOBT and 0.09
ml triethylamine as in 2A to give 0.45g N-~-t-BOC-N-~-CBZ-
L-ornithyl-glycyl-L-aspartyl-B-t-butyl ester-L-valine-~-t-
butyl ester.
D. The benzyloxycarbonyl protecting group on the
product compound of 3C is removed by dissolving 0.45g of
the protected compound in 20 ml of cyclohexene and adding
0.10g 10% palladium on carbon and heating at reflux, under
nitrogen, for 2 hours. The resulting solution is
filtered, evaporated, and chromatographed on silica gel in
chloroform/methanol/water 90:10:3 to give 0.25g N-~-t-BOC-
L-ornithyl-glycyl-L-aspartyl-~-t-butyl ester-L-valine-t-
butyl ester.
~. 0.23g of the product obtained in 3D is dissolved
in 5 ml trifluoroacetic acid with 3 drops of ethanedithiol
added. The solution is stirred for 7 hours, evaporated,
and the residue partitioned between ethyl acetate and 0.5M
acetic acid. The aqueous portion was separated and
lyophilized and the resulting solid purified by HPLC to
give L-ornithyl-glycyl-L-aspartyl-L-valine as the
di~rifluor~acetate salt; m.p. 122-25C.
W09l/04746 ~CT/US90/0~44~
-15- 2~604 ~
EXAMPLE 4
L-Arain~lsarcosvl-L-As~artyl-L-Valine
5N-~-FMOC-sarcosine was substituted for N-~-FMOC-
glycine and the resulting product was treated with
piperidine in methylene chloride as in Example 1 to remove
the FMOC group. The corresponding product was obtained.
Treating this product with the arginine derivative of
Example 1, cleaving the resulting peptide from the resin
and deprotecting as in Example 1 gave L-arginylsarcosyl-L-
aspartyl-L-valine as the ditrifluoroacetate salt; m.p.
145C (dec.).
EXAMPLE 5
L-Arainylalvcyl-L-As~artvl-L-(N-Methvl)Valine
A. lg of p-alkoxybenzylalcohol resin (0.5-1 mmole/g
of resin), 0.706g of N-FMOC-N-methyl-L-valine, 0.382g EDC,
0.270g HOBT, and 0.28 ml triethylamine are combined in 15
ml of dimethylformamide and shaken for 2 hours. The
mixture is filtered and the resin washed with DMF. The
resin is treated as above for a second time, then shaken
with 0.28 ml glacial acetic acid, 0.955g EDC, and 0.7 ml
triethylamine in DMF and deprotection effected with 20%
piperidine in methylene chloride as in lA. This gives N-
Methyl-L-valine-p-alkoxybenzyl resin ester.
B. L-aspartic acid, glycine and L-arginine are
coupled and deprotected, sequentially, as in the previous
examples and the peptide removed from the resin to give L-
arginylglycyl-L-aspartyl-L-(N-methyl)valine as~the
ditrifluoroacetate salt which decomposes at 153C.
w09l/04746 PCT/~SgO/0~8
~ -16-
~6~ EXAMPLE 6
L-Arginylalycyl-L-Aspartyl Glycine
Starting with N-FMOC-glycine-p-alkoxy benzyl resin
ester, sequentially coupling L-aspartic acid, glycine and
arginine, deprotecting and removing the peptide as in the
above examples, L-arginylglycyl-L-aspartyl glycine was
obtained as the ditrifluoroacetate salt; m.p. 85-90C.
EXAMPLE 7
N-(L-Arainyl-2-Aminoethyl)-L-Aspartvl-L-"aline
A. 1.18g EDC and 0.86 ml of triethylamine are
combined in 20 ml of methylene chloride and stirred for 10
minutes. 2.0g N-~-CBZ-L-aspartic acid B-t-butyl ester,
0.83g HOBT, 1.30g L-valine-t-butyl ester and 0.86 ml
triethylamine were added and the solution stirred
overnight. The solution is diluted with ethyl acetate and
washed with 10% citric acid solution, 10% sodium carbonate
solution, water, then dried over sodium sulfate,
evaporated to give l.9g N-~-CBZ-L-aspartyl-t-butyl ester-
L-valine-t-butyl ester.
B. 2.2g of N-~-CBZ-glycine methyl ester is
dissolved in 50 ml of anhydrous toluene and cooled to -
78C, under nitrogen. To this is added 13 ml of 1.5M
diisobutyl aluminum hydride in toluene over a period of 1
hour. The solution is stirred for an additional hour at -
78'C, then quenched by addition of 50 ml 5% hydrochloric
acid solution. The solution is extracted with ethyl
acetate which is washed with water and dried over sodium
sulfate, evaporated to give 1.55g N-~-CBZ-2-
aminoacetaldehyde.
C. ~he product from 7A is deprotected as in 3D to
give L-aspartyl-t-butyl ester-L-valine-t-butyl ester.
WO9l/04746 PCT/US90/05448
-17- 2 0 6 ~ O ~ ~
D. 1.55g of the aldehyde from 7B, 3.4g of the
product from 7C, 1.64g sodium acetate, 1.23g sodium
cyanoborohydride and lg of 3 angstrom molecular sieves are
stirred together in 100 ml methanol for 3 days. The
solution is filtered and 5 ml of 5% hydrochloric acid is
added. The solution is diluted with water and adjusted to
pH 9 with 10% sodium carbonate, then extracted with water,
and dried over sodium sulfate. The solution is evaporated
and the residue purified by flash chromatography in ethyl
acetate/hexane, 1:1, to give l.lg N-CBZ-aminoethyl-L-
aspartyl-B-t-butyl ester-L-valine-t-butyl ester.
E. The CBZ group is removed from the product of 7D
as in 3D to give N-aminoethyl-L-aspartyl-t-butyl ester-L-
valine-t-butyl ester.
F. The product from 7E is coupled with N-~-t-BOC-N-
~-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine as
in 2D and the resulting product deprotected as in 2E to
give N-(L-arginyl-2-aminoethyl)-L-aspartyl-L-valine as the
tritrifluoroacetate salt; m.p. 91-5C.
EXAMPLE 8
L-ArainylglYcvl-L-Aspartic Acid ~-BenzYl Ester
A. lg of N-t-BOC-L-aspartic acid ~-benzyl ester is
treated with 0.366g of 2-(trimethylsilyl)ethanol in the
presence of 0.S92g EDC, 0.419g HOBT and 0.43 ml
triethylamine in 20 ml of methylene chloride for 2 hours.
The product is isolated as in 2A to give N-t-BOC-L-
aspartic acid ~-benzyl ester-~-2-(trimethylsilyl)ethyl
ester.
B. The product of 8A is deprotected by treating
with 10 ml of trifluoroacetic acid in 30 ml of methylene
chloride for 2 hours at room temperature. The mixture was
cooled to 0C and 20 ml of saturated sodium carbonate
solution is added dropwise. The layers are separated and
WO91/04746 ~ 18- PCT/US~0/0 ~ ~
the organic layer dried over magnesium sulfate, filtered,
evaporated to give L-aspartic acid-~-benzyl ester-~-2-
(trimethylsilyl)ethyl ester.
C. The product of 8B and N-t-BOC glycine are
coupled in a manner similar to that described in the
previous examples to give BOC-glycyl-L-aspartic acid-~-
benzyl ester-B-2-(trimethylsilyl)ethyl ester.
D. The BOC group is removed from the product of 8C
as in 8B to give glycyl-L-aspartic acid-~-benzyl ester-~-
2-(trimethylsilyl)ethyl ester.
E. The product from 8D is coupled to N-~-BOC-N-~-
(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine as
in 2D to give N-~-BOC-N-~-(4-methoxy-2,3,6-trimethyl-
benzenesulfonyl)-L-arginyl-glycylaspartic acid ~-benzyl
ester-~-2-(trimethylsilyl)ethyl ester.
F. 0.30g of the product obtained in 8E is stirred
with 5 ml of trifluoroacetic acid at room temperature for
24 hours. The reaction mixture is then stirred with 0.5 N
acetic acid and washed with ethyl acetate. The aqueous
layer is lyophilized to give L-arginylglycyl-L-aspartic
acid ~-benzyl ester ditrifluoroacetate; m.p. 85-7C.
EXAMPLE 9
N-(6-Aminohexanoyl)-L-Aspartyl-L-Valine
A. 1.0g of N-(9-fluorenylmethoxycarbonyl)-L-valine
p-alkoxybenzyl alcohol resin ester (containing
approximately 0.56 mmol of amino acid) was deprotected by
shaking with 10 ml of a solution of 20% piperdine in
dimethylformamide for 1.5 hours. The mixture was filtered
and the resin derivative washed with methylene chloride to
give L-valine p-alkoxybenzyl resin ester.
WO91/04746 ~ ~ ~S90/0~X
--19--
B. The product from Example lA was shaken with
0.92g of N-F~OC-L-aspartic acid ~-t-butyl ester, 0.30g of
1-hydroxybenzotriazole (HOBT), 0.43 g of 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and
0.32 ml of triethylamine in 10 ml of dimethylformamide for
2 hours. The mixture was filtered and the resin washed
with methylene chloride. The resin derivative was then
deprotected as in Example lA to give L-aspartyl-~-t-butyl
ester-L-valine p-alkoxybenzyl resin ester.
C. 2.00g of 6-aminohexamoic acid and 3.23g of
sodium carbonate were dissolved together in 30 ml of
water. The solution was cooled in an ice bath and 3.32g
of di-t-butyldicarbonate in 15 ml of tetrahydrofuran was
15 added. The mixture was stirred at room temperature for 5
hours, then diluted with 400 ml of water and extracted
with ether. The aqueous solution was acidified to pH 2
with hydrochloric acid and extracted with ethyl acetate.
The ethyl acetate layer was dried over magnesium sulfate,
20 filtered and evaporated n vacuo to give N-tert-butoxy-
carbonyl-6-aminohexamoic acid.
D. The product from Example lB was shaken with
0.52g of N-BOC-6-aminohexanoic acid, 0.30g of HOBT, 0.43g
of EDC and 0.32 ml of triethylamine in 10 ml of
dimethylformamide for 17 hours. The mixture was filtered
and the resin derivative washed with methylene chloride.
The peptide derivative was deprotected and cleaved from
the resin by treating with 10 ml of 95% trifluoroacetic
acid for 2 hours. The resin was filtered off and the
filtrate diluted with 50 ml of 0.5 N acetic acid. The
aqueous solution was washed with 4x25 ml of ethyl acetate,
filtered, then lyophilized to give N-(6-aminohexanoyl)-L-
aspartyl-L-valine as the trifluoroacetate salt; m.p. 75-
85C.
WO91/04746 PCT/US90/0~X
~5~ ~ -20-
EXAMPLE 10
N- ( 7-Aminoheptanovl~-L-Aspartyl-L-Valine
A. When 7-aminoheptanoic acid was substituted for
6-aminohexanoic acid and treated in a manner similar to
that in Example lC, N-tert-butoxycarbonyl-7-aminoheptanoic
acid was obtained.
B. L-aspartyl-~-t-butyl ester-L-valine p-alkoxy-
benzyl resin ester (prepared from 1.0g of N-FMOC-valine p-
alkoxybenzyl resin ester as in Examples lA and B) was
treated with 0.55g of N-BOC-7-aminoheptanoic acid, with
0.30g of HOBT, 0.43g of EDC and 0.32 ml of triethylamine
in 10 ml of dimethylformamide in a manner similar to that
in Example lD to give N-(7-aminoheptanoyl)-L-aspartyl-L-
valine as the trifluoroacetate salt.
EXAMPLE 11
N-(7-Guanidinoheptanoyl)-L-Aspartvl-L-Valine
A. 7-Guanidinoheptanoic acid was prepared
essentially by the method of Miller, et al, Synthesis, 777
(1986), which is incorporated herein by reference. 0.50g
of 7-aminoheptanoic acid was dissolved in a solution of
0.475g of potassium carbonate in 3.5 ml of water. 0.427g
of aminoiminomethanesulfonic acid was added portionwise
over 10 minutes and the mixture stirred at room
temperature for 24 hours. The resulting solid was
collected by filtration. The guanidine was dissolved in
diluted hydrochloric acid and the solution evaporated in
vacuo. Two portions of 2-propanol were evaporated from
the residue to give 7-guanidinoheptanoic acid
hydrochloride.
B. L-aspartyl-~-t-butyl ester-L-valine p-alkoxy-
benzylalcoholester (prepared from 1.0g of N-FMOC-L-valine
p-alkoxybenzylalcohol ester resin as in Examples lA and B)
W09l/04746 PC~/US90/0;448
-21- 2 Q ~60 ~7
was treated with 0.50g sf 7-guanidinoheptanoic a~id
hydrochloride, 0.30g of HOBT, 0.43g of EDC and 0.32 ml of
triethylamine in 10 ml of dimethylformamide in a manner
similar to that in Example lD to give N-(7 guanidino-
heptanoyl)-L-aspartyl-L-valine as the trifluoroacetate
salt; m.p. 75-80C.
EXAMPLE 12
N-(8-GuanidinooctanoYl)-L-Aspartyl-L-Valine
A. 8-guanidinooctanoic acid hydrochloride was
prepared from 8-aminooctanoic acid in a manner similar to
the process used in Example 3A.
B. 0.40g of 8-guanidinooctanoic acid hydrochloride,
L-aspartyl-~-t-butyl ester-L-valine p-alkoxybenzyl resin
ester (prepared in the same manner as in Examples lA and
B), 0.22g of HOBT, 0.32g of EDC and 0.24 ml of
triethylamine were shaken in 10ml of dimethylformamide and
treated as in Example lD to give N-(8-guanidinooctanoyl)-
L-aspartyl-L-valine as the trifluoroacetate salt.
EXAMPLE 13
If 6-guanidinohexanoic acid hydrochloride is
substituted for 7-guanidinohexanoic acid hydrochloride in
Example 3B, N-(6-guanidinohexanoyl)-L-aspartyl-L-valine
can be prepared.
EXAMPLE 14
A. If 8-aminooctanoic acid is substituted for 6-
aminohexanoic acid in Example lC, N-tert-butoxycarbonyl-8-
aminooctanoic acid can be prepared.
B. If N-BOC-8-aminooctanoic acid is substituted for
N-BOC-6-aminohexanoic acid in Example lD, N-(8-amino-
WO91/04746 ~ PCT/US90/0~8
22~
octanoyl)-L-aspartyl-L-valine can be prepared as the
trifluoroacetate salt.
EXAMPLE 15
s
8-Guanidinooct-2-Enoyl-L-AsPartyl-L-Valine
A. 4g of 6-amino-1-hexanol is dissolved in 50 ml of
10~ aqueous tetrahydrofuran and the solution cooled to
O C. 7.46g of di-tert-butyldicarbonate in 25 ml of
tetrahydrofuran is added dropwise and the resulting
mixture stirred for 3 days at room temperature. The
solvent is evaporated ln vacuo and the residue dissolved
in ethyl acetate. The ethyl acetate solution is washed
with water, dried over magnesium sulfate and evaporated in
vacuo to give 7.4g of N-tert-butoxycarbonyl-6-amino-1-
hexanol.
B. To a solution of 8.8g of pyridinium
chlorochromate in 250 ml of methylene chloride is added
8.8g of 3 Angstrom molecular sieves. A solution of 7.4g
of N-tert-butoxycarbonyl-6-amino-1-hexanol in 50 ml of
methylene chloride is added dropwise and the mixture
stirred at room temperature for 2 hours. The reaction
mixture is filtered through silica gel, washing with 40%
ethylacetate in hexane, and the filtrate evaporated in
vacuo to give 6-N-tert-butoxycarbonylaminohexanol.
C. lg of 6-N-tert-butoxycarbonylaminohexanol and
1.54g of methyl(triphenylphosphoranylidene) acetate are
combined in 25 ml of chloroform and the solution refluxed
for 2 hours. The solvent is then removed in vacuo and the
residue taken up in ether and allowed to stand in the
freezer overnight. The resulting suspension is filtered,
the filtrate evaporated and the residue flash
chromatographed in 20% ethyl acetate in hexane to give
methyl-8-N-tert-butoxycarbonylamino-2-octenoate.
wo g~/047q6 2 0 ~ 6 o ~/US90/0~8
-23-
D. A solution of 3.2g of methyl 8-N-tert-
butoxycarbonylamino-2-octenoate in 25 ml of methanol and
25 ml of 1 Normal aqueous sodium hydroxide is heated at
reflux for 2 hours. The methanol is removed in vacuo and
the aqueous solution acidified with lN hydrochloric acid.
The resulting mixture is extracted with ethyl acetate.
The organic solution is dried over magnesium sulfate and
evaporated to give 8-N-tert-butoxycarbonylamino-2-octenoic
acid.
E. 3g of 8-N-tert-butoxycarbonyl-amino-2-octenoic
acid is dissolved in 30 ml of trifluoroacetic acid and the
solution stirred at room temperature for 1 hour, then
evaporated ln vacuo to give 8-amino-2-octenoic acid as the
trifluoroacetate salt.
F. 3.lg of 8-amino-2-octenoic acid trifluoro-
acetate is added to 30 ml of water and the pH adjusted to
7 with lN sodium hydroxide solution. 1.9g of potassium
carbonate is added, then 1.75g of aminoiminomethane-
sulfonic acid is added, portionwise, over lO minutes. The
mixture was stirred for 5 hours at room temperature and
the resulting solid collected by filtration. The solid is
dissoived in diluted hydrochloric acid and the solution
evaporated and two portions of 2-propanol evaporated from
the residue to give 8-guanidino-2-octenoic acid
hydrochloride.
G. L-aspartyl-~-t-butylester-L-valine p-
alkoxybenzyl resin ester tprepared from O.6g of N-FMOC-
valine p-alkoxybenzyl resin ester as in Examples lA and B)
is treated with 0.33g of 8-guanidino-2-octenoic acid
hydrochloride in the presence of 0.184g of HOBT, 0.26g of
EDC and 0.19 ml of triethylamine in 10 ml of
dimethylformamide in a manner similar to that in Example
lD to give 8-guanidinooct-2-enoyl-L-aspartyl-L-valine as
the trifluoroacetate salt.
WO9l/04746 ~ ~ -24- PCT/US9a/0544X
By using methods analogous to that used in Examples 1
through 15, the following compounds were made:
N-(5-guanidino-2-aminopentyl)glycyl-L-aspartyl-L-
valine tritrifluoroacetate; m.p. 90-9SC:
HN ~ ~ HN ~IH~
OH
~TFA
2-~N-(5-guanidinopentanoyl)glycyl-L-aspartyl]-
1,2,3,4-tetrahydroisoquinoline:
NH o o
H2N ~ N
r~
L-arginyl-glycyl-L-N-methylaspartyl-L-valine
ditrifluoroacetate:
H2N ~ OH
NH ~2TFA
3S H2N ~NH
WO91/04746 PCT/US90/05448
-25- 2Q~6047
N-(5-guanidinopentanoyl)glycyl-L-aspartylphenethyl-
amide; m.p. 90-100C:
H2N ~lHN ` NH--~ NHJ (` N1~3
O ~OH
O
.CH,COOH
N-(5-aminopentanoyl)glycyl-L-aspartyl-L-valine
trifluoroacetate; m.p. 95-99C:
o ~
H2N ~ N ~ ~ N ~ OH
. O ~po o
OH
1FA
N-(5-guanidinopentanoyl)glycyl-L-aspartyl-L-valine
dihydrochloride; m.p. 60-70C:
o ~ OH o
H2N~,,HN ~, ~ X'
NH O O
2HCI
8-Guanidino-2,2-dimethyloctanoyl-L-aspartyl-L-valine
trifluoroacetate:
NH CH~ CH3 H
3S H N/~/\~\~N--A~,p_v~l .l'FA
;
WO91/04746 ~ PCT/US90/05448
~6~ -26-
9-Guanidino-non-2-(E)-enoyl-L-aspartyl-L-valine
trifluoroacetate:
NH 0
2N ~r --~ ~ /J\N--Asp--Val .TFA
8-Guanidino-oct-3-(E)-enoyl-L-aspartyl-L-valine
trifluoroacetate:
NH
Il ~
2N/\N/`\~/~ y/N--Asp--Val .TFA
H O
;
205-Guanidinovaleroyl-L-aspartyl-L-valine
trifluoroacetate:
25IlNq~NH o ~OH 2
~ TFA
9-Aminononanoyl-L-aspartyl-L-valine trifluoroacetate:
OH
~0
O ~
35H2N ~ NH ~
o OH
TFA
WO91/04746 -27- 2 ~ ~ 6 0 ~P~ /US90/~5448
9-Guanidinononanoyl-L-aspartyl-L-valine
trifluoroacetate:
NH2 ~ ,~OH o
TFA
11-Guanidinoundecanoyl-L-aspartyl-L-valine
trifluoroacetate:
o ~
~ ~NH~
~ O I~OH O
HN~,NH O
H2N .TFA
10-Guanidinodecanoyl-L-aspartyl-L-valine
trifluoroacetate:
O
2 5 HN
H2N~bNH ~OH
.TiFA
8-Guanidino-2(R,S)-ethyloctanoyl-L-aspartyl-L-valine
trifluoroacetate:
N~
r ~ ~
o ~ OH
.TFA
W09l/04746 ~ PCr/US90/05448
~a~Q~ -28-
Compounds of the present invention were tested for
inhibition of platelet aggregation using the following
procedures:
I. Inhibition of Radiolabeled (125I) Fibrinoaen
Bindina Assay, which is essentially based on the method
described in Proc. Natl. Acad. Sci. USA Vol. 83, pp. 5708-
S712, Aug. 1986, and is as follows.
Platelets are washed free of plasma constituents by
the albumin density-gradient technigue. In each
experimental mixture platelets in modified Tyrode's buffer
are stimulated with human ~-thrombin at 22-25DC for 10
minutes (3.125 x 10" platelets per liter and thrombin at
01 NlH units/ml). Hirudin is then added at a 25-fold
excess for 5 minutes before addition of the radiolabeled
ligand and any competing ligand. After these additions,
the final platelet count in the mixture is 1 x 10"/liter.
After incubation for an additional 30 minutes at 22-25C,
bound and free ligand are separated by centrifuging 50~1
of the mixture through 300,u1 of 20% sucrose at 12,000xg
for 4 minutes. The platelet pellet is then separated from
the rest of the mixture to determine platelet-bound
radioactivity. Nonspecific binding is measured in
mixtures containing an excess of unlabeled ligand. When
binding curves are analyzed by Scatchard analysis,
nonspecific binding is derived as a fitted parameter from
the binding isotherm by means of a computerized program.
To determine the concentration of each inhibitory compound
necessary to inhibit 50% of fibrinogen binding to
thrombin-stimulated platelets (ICso), each compound is
tested at 6 or more concentrations with 12sI-labeled
fibrinogen held at 0.1761-mol/liter (60~g/ml). The ICso is
deriYed by plotting residual fibrinogen binding against
the logarithm of the sample compound's concentration.
II. Inhibition of Fibrinoaen - Mediated Platelet
Aqareqation, which is essentially based on the method
W09l/047~6 2 0 ~ S90/0~448
-29-
described in Blood, Vol. 66, No. 4, Oct. 1985, pp. g46-
952, and is as follows.
Human Platelets were isolated from freshly drawn
whole blood and were suspended in 0.14 mol/L NaCl, 2.7
mmol/L Kll, 12 mmol/L NaHCO3, 0.42 mmol/L Na2HPO4, 0.55
mmol/L glucose, and 5 mmol/L Hepes, pH 7.35 at 2 x lO~
platelets/ml. The suspension was incubated at 37C. An
aliquot of 0.4 ml of platelet suspension was activated by
human thrombin at a final concentration of 2~g/ml of
thrombin for one minute. After one minute the reaction
was stopped by a thrombin inhibitor. Serial dilution of
the compound being tested was then added to the activated
platelet, the reaction was allowed to proceed for one
minute, followed by the addition of human fibrinogen at a
final concentration of 60~g/ml of fibrinogen. Platelet
aggregation was then recorded by an aggregometer. Rate of
aggregation was used to calculate ICso.
Representative results of platelet aggregation
inhibition are shown in Table I.
WO 9 1 /04746 PCI /US90/0:~448
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WO 91/04746 PCT/US90/05448
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WO91/04746 2 0 6 6 0 4 ~ /US90/05448
-33-
The compounds of the present invention may be orally
or parenterally administered to mammals. The compounds
may be incorporated into pharmaceutical formulations
having excipients suitable for these administrations and
which do not adversely react with the compounds, for
example, water, vegetable oils, certain alcohols and
carbohydrates, gelatin and magnesium stearate. The
pharmaceutical formulations containing an active compound
of the present invention may be made into: tablets,
capsules, elixirs, drops or suppositories for enteral
administration; and solutions, suspensions or emulsions
for parenteral administration.
In general, a compound of this invention is
administered in dosages of approximately l to 200 mg per
dosage unit or higher. The daily dosage is approximately
0.02-5 mg/kg of body weight. It is to be understood,
however, that the particular dose for each patient usually
depends on very diverse factors, such as the age, body
weight, general condition of health, sex, diet and the
like of the patient, on the time and route of
administration, on the rate of excretion, on the
combination of medicaments and on the severity of the
disease.
Having described the invention, it will be apparent
to one of ordinary skill in the art that changes and
modifications can be made thereto without departing from
the spirit and scope of the invention as set forth herein.
