CA2027314A1 - Fibrinogen receptor antagonists - Google Patents
Fibrinogen receptor antagonistsInfo
- Publication number
- CA2027314A1 CA2027314A1 CA002027314A CA2027314A CA2027314A1 CA 2027314 A1 CA2027314 A1 CA 2027314A1 CA 002027314 A CA002027314 A CA 002027314A CA 2027314 A CA2027314 A CA 2027314A CA 2027314 A1 CA2027314 A1 CA 2027314A1
- Authority
- CA
- Canada
- Prior art keywords
- amino acid
- substituted
- gly
- asp
- cys
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/75—Fibrinogen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
Abstract
TITLE OF THE INVENTION:
FIBRINOGEN RECEPTOR ANTAGONISTS
ABSTRACT OF THE DISCLOSURE:
A fibrinogen receptor antagonist of the formula wherein XX represents a synthetic alpha-amino acid containing a phenyl or C3-C8 cycloalkyl group, and ZZ
represents a sequence of 1, 2, 3 or 4 amino acids.
FIBRINOGEN RECEPTOR ANTAGONISTS
ABSTRACT OF THE DISCLOSURE:
A fibrinogen receptor antagonist of the formula wherein XX represents a synthetic alpha-amino acid containing a phenyl or C3-C8 cycloalkyl group, and ZZ
represents a sequence of 1, 2, 3 or 4 amino acids.
Description
3 1 ~
ll/RP6 TITLE OF T~E INVENTION:
FIBRINOGEN RECEPTOR ANTAGONISTS
BACKGROUND OF T~E INVENTION
This invention relates to compounds for inhibiting the binding of fibrinogen to blood platelets, and for inhibiting the aggregation of blood platelets.
Fibrinogen is a glycoprotein, present in blood plasma, which participates in platelet aggregation and fibrin formation. Platelets are cell-~ike anucleated fragments, found in the blood of all mammalæ, which participate in blood coagulation.
Interaction of fibrinogen with a receptor on the 2s platelet membrane glycoprotein complex IIb/IIIa is known to be ess2ntial for normal platelet function.
ll/RP6 TITLE OF T~E INVENTION:
FIBRINOGEN RECEPTOR ANTAGONISTS
BACKGROUND OF T~E INVENTION
This invention relates to compounds for inhibiting the binding of fibrinogen to blood platelets, and for inhibiting the aggregation of blood platelets.
Fibrinogen is a glycoprotein, present in blood plasma, which participates in platelet aggregation and fibrin formation. Platelets are cell-~ike anucleated fragments, found in the blood of all mammalæ, which participate in blood coagulation.
Interaction of fibrinogen with a receptor on the 2s platelet membrane glycoprotein complex IIb/IIIa is known to be ess2ntial for normal platelet function.
2~ `3~
ll/RP6 - 2 - 18016 Zimmerman et al., U.S. Patent No. 4,683,2~
describes peptides having utility in the study of fibrinogen-platelet, platelet-platelet, and cell-cell interactions. The peptides are described as having utility where it i8 desirable to retard or prevent formation of a thrombus or clot in the blood. The general formula for the peptides is:
H2N-(Ch)-Arg-Gly-A8p-(Cx)-H
where Ch and Cx are sequences of amino acids.
Pier chbacher et al., ~.S. Patent No.
4,589,881, describeæ the Requence of an 11.5 kDal polypeptide fragment of fibronectin which em~odies the cell-attachment-promoting activity of fibronectin. A specifically described fragment is:
H-Tyr-Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro-Ile-Ser-Ile-Asn-Tyr-Arg-Thr-Glu-Ile-Asp-Lys-Pro-Ser-Gln-Met-OH
Ruoslahti et al., U.S. Patent No. 4,614.517, describes tetrapeptides which alter cell-attachment activity of cells to various substrates. The peptides are stated to "consist essentially of" the following sequence:
X-Arg-Gly-Asp-Ser-Y
wherein g is H or one or more amino acids and Y is OH
or one or amino acids. Fi~ure 1 lists the polypeptides 2 ~
ll/RP6 - 3 - 18016 that were synthesized by Ruoslahti et al. ,,in ~determining the smalle~t peptide exhibiting cell attachment activity".
Ruoslahti et al., U.S. Patent No. 4,578,079, describes similar tetrapeptides having Ser substituted with Thr or Cys.
Pierschbacher et al., ~E~ Natl~ Acad. Sci. USA, Vol. 81, pp.5985-5988, October 1984 describe variants of the cell recognition site of fibronectin that retain attachment-promoting activity. They assayed the cell attachment-promoting activitieæ of a number of structures closely resembling the Arg-Gly-Asp-Ser peptide, and found "that the arginine, glycine, and aspartate reæidues cannot be replaced even with closely related amino acids, but that several amino acids can replace serine without loss of activity."
Ruoslahti et al., Science, Vol. 238, pp. 491-497, October 23, 1987, discuss cell adhesion proteins.
They specifically state that "[e]lucidation of the amino acit sequence of the cell-attachment domain in fibronectin and it~ duplication with synthetic peptides establish the seguence Arg-Gly-Asp (RGD) as the essential structure recognized by cells in fibronectin".
Cheresh, Proc. Natl. Acad. Sci. ~SA, Vol. 84, pp.
6471-6475, September 1~87, describes the Arg-Gly-Asp-directed adhesion receptor involved in attachment to fibrinogen and von Willebrand Factor.
i 3 1 ~
ll/~P6 - 4 - 1~016 Adams et al., U. S. Patent No. 4,857,508,~describes tetrapeptides which inhibit platelet aggregation and the formation of a thrombus. The tetrapeptides have the formula:
, ~-Gly-Asp-Y
wherein X can be ~2NC(=N~)NH(C~2~nCH(Z)COOH or Ac-Arg, wherein Z = H, N~2, or NH-Acyl and n=1-4, and wherein Y can be Tyr-NH2, Phe-NH2 or a group of a specifically defined formula.
Applicants have discovered fibrinogen receptor antagonistæ which do not contain the amino acid seguence Arg-Gly-Asp which is taught in the art as specifically required for binding to platelet membrane glycoprotein complex IIb/IIIa.
~MMA~Y QF T~E INVENTION
Compounds of the present invention inhibit binding of fibrinogen to the platelet membrane glycoprotein complex IIb/IIIa receptor and contain an amino acid sequence:
XX-Gly-Asp wherein XX is a synthetic alpha amino acid containing either a phenyl or C3-Cg cycloalkyl group. These compound~ are surpriæing in view of the prior art ll/RP6 - 5 - 18016 which teaches that the ~equence Arg-Gly-As,p is required in order to achieve binding to the IIb/IIIa receptor.
The present invention is a fibrinogen receptor antagonist having the following structure:
Z Z
\ XX-Gly-~s p /
wherein XX represents a synthetic a-amino acid as defined below and Z~ represent~ a sequence of 1, 2, 3, or 4 amino acids as defined below.
XX shares an amide bond with Gly and an amide bond with ZZ, and is defined as having a side chain X
NH
2s ll (CH2)n- AA ~(C~2)n - N C NHR (i) H
or _ (CH2)n- M --(CH2)n NXR (ii) 3~
ll/RP6 - 6 - .~18016 .
wherein: n i~ 0,1,2,3 or 4;
n' is 0,1,2,3 or 4;
AA i8 di6ubstitutet phenyl, either not substituted with additional groups or substituted with Cl-4 al~yl, al~oxy or hydro y ; C3-C8 cycloalkyl, preferably cyclohe~yl, either not substituted with additional group8 or substituted with Cl-4 alkyl, alkoxy or hydroxy; and R is ~, Cl-6 al~yl, ~ubstituted or unsubstituted aryl, substituted or unsubstituted arylmethyl or substituted or unsubstituted cycloal~yl.
Preferably, the side chain of ~X is defined by (ii) wherein n is 1, n' is 1, M is unsub~titute~ phenyl and R is E.More preferably, the ~ide chain is ~H2 ~{:H2-NH2 where xx i p-aminomethylphenylalanine.
Also preferred iB the side chain of gX defined by (ii) wherein n i8 1, n' i8 O, R is ~ and M is unsubstituted cyclohe y l. More preferably, the side chain i~
CH2 ~ NH2 ll/RP6 - 7 - 18016 Preferred compounds o~ the invention are those having selectivity over integrin receptors. The preferred compounds include those wherein XX i~ a synthetic alpha-amino acid containing an amino group side chain, as represented above by (il).
ZZ is defined as follows:
A ~ ~, _ y, $ E' ' ~ ' -'-'` ( ) --~ (1 ) wherein:
A' is H, acylamido, acylaminoacylamido, acylamino-N-methylamino-acylamido;
R' and R'l are independently H, methyl, 2s ethyl or a lower alkyl group having 1 to 5 carbons;
~ ' is S-S, CH2-S, S-CH2, CH2CH2, C~2, C~2C~2C~2- C~2-S-S- C~2-S-S-CH2, S-S-CH2; and 2~h ~3:L~
ll/RP6 ~ 18016 ~ ' i8 ~, C00~, CONH2, CON8R2, coNR3~, CH20H,C02R2,CH3 wherein R2 is an alkyl gr~up having 1 to 4 carbon atoms, R3R4 i~ an al~yl group having 1 to 4 carbon atoms or NR3R4 i8 a secondary amino acid, or o //N--NH
N ¦ ;
or æz i~ N=~
A ~ X
B C ~t~-6~h~
wherein:
A' is as defined above;
R' and R'l are as defined above;
X' - Y' is as defined above;
B' is a D- or L- a-amino acid;
C' i8 a D- or L- secondary ~-amino acid, preferably selectet ~rom proline, ~- methylproline, ~,~- dimethylproline, ~-hydro~yproline, anhydroproline, thioproline, ~ -ll/RP6 - 9 ~ ..18016 methylthioproline, ~ dimethylthioproline, pipecolic acid, azetidine carbo y lic acid,~nd an N-methyl amino acid, or a D- or L- primary a-amino acid; and E~ i~ as defined abo~e;
or ZZ i8 R~ ' R R
N _ RS- (~
(xX-Cl wherein:
A' is as defined above;
R' and R~l are aæ defined above;
~' - Y' are a~ defined above;
E' is ag defined above;
F' iB an L-amino acid, preferably selected from tryptophan, phenylalanine, leucine, valine, isoleucine, a-naphthylalanine, ~-naphthylalanine, methionine, tyro~ine, arg inine, lysine, homoarg inine, ornithine, hi6tidine, substituted tryptophan, substituted phenylalanine or ~ubstituted tyrosine;
and R5 is H or methyl;
__ _ ___ 3 ~ ~
ll/RP6 - 10 - 18016 or :~Z iS
R~t ' ~ It _~X--y ~ ~E
~C' I 5C
wherein A' is as defined above;
R' and R'l are as defined above;
X'-Y~ is as defined above;
Cl is as defined above; and E~ is as defined above.
or ZZ is 2 5 ~ E
- G~x- C~-ds~ F
2 ~ 2 ~
11/RP6 ~ 18016 wherein "-A' is as defined above;
R~ and R'l are as defined above;
X~-Y~ is a3 defined above, F' is as defined above;
G' i~ a D- or L-a-amino acid, 8econdary cyclic amino acid, or N-methyl amino acid;
~' i8 as defined above; and R5 is as def ined above.
~0 2s ll/RP6 - 12 - 18016 The present invention al~o is a fibrinogen receptor antagonist of the formula lo ~ 1l I~
B-Q-C-C-Gly-Asp-NH-CH
X E' wherein:
B representæ zero one or two ~ubstituted or unsubstituted amino acids;
Q represents ~, N~, N~2 or Ac-NH;
X represents the side chain of amino acid ~X
as previously defined; and I' i a ~ide chain of an amino acid previously defined by F', and E' is H, COOH, CONH2, CONHR2, CoNR3R4, CH20H, C02R2, C~3 wherein R~ i8 an alkyl group having 1 to 4 carbon atoms, R3R4 i~ an al~yl group having 1 to 4 carbon atoms or NR3R4 i8 a secondary amino acid, or ~ ~ ~'J'~
ll/RP6 - 13 - 18016 ,N-NnH
N
`N~; `
provided that when B ie zero 6ubstituted or unsubstituted amino acid~, then Q is ~, N~2 or Ac-N~, and that when B i~ one or two gub~tituted or unsubstituted amino acids, then Q i8 N~.
In a preferred embodiment of the present invention, the fibrinogen receptor antagonist has the following formula:
~ZZ
(L-AMF)-Gly-Asp wherein ZZ is:
25Ac-Cy8 Cy8-OH
Asn-Pro~ (L-AMF-Gly-Asp) r 30Ac-Cy8 Cys-OH
Asn-DiMeTzl --.... -(~-AME-Gly-Asp) i3~
ll/~P6 - 14 - 1~016 Exemplary compounds of the invention are:
r Ac-Cys-Asn-Pro-(L-AMF)-Gly-Asp-Cys-OH;
-Ac-Cys-A6n-Pro-(~-AMF)-Gly-Asp-Cys-O~;
Ac-Cys-Asn-(DiMeTzl)-(LrAMF)-Gly-Asp-Cys-O~
r Ac-Cys-Asn-(DiMeTzl)-(~-AMF)-Gly-Asp-Cys-OH
c(Aha-(L-AMF)-Gly-Asp-Trp-Pro);
c(Aha-(~-AMF)-Gly-Asp-Trp-Pro);
. .
Ac-Cy~-Asn-(DiMeTzl)(t-ACh~Ala)-Gly-Asp-Cys-OH;
Ac-Cys-(DiMeTzl)-(~-AChxAla)-Gly-Asp-Cys-OH;
. ~
Ac-Cys-Asn-(DiMeTzl)-(c-AChxAla)-Gly-Aæp-Cys-OH;
.
Ac-Cys-(DiMeTzl)-(~-AChxAla)-Gly-Asp-Cys-OH;
r Ac-Cys-Asn-(DiMeTzl)-(~-GuaChxAla)-Gly-Asp-Cys-OH;
r ~
Ac-C~s-(DiMeTzl)-(t-GuaChxAla)-Gly-Asp-Cys-O~;
, ~
Ac-Cys-Asn-(DiMeTzl)-(~-GuaChxAla)-Gly-Asp-Cys-OH;
Ac-Cys-(DiMeTzl)-(c-GuaChxAla)-Gly-Asp-Cys-OH;
Ac-Cys-Asn-~DiMeTzl)-(t-AChxGly)-Gly-Asp-Cys-OH;
Ac-Cys-Asn-(DiMeTzl)-(~-GuaChxGly)-Gly-Asp-Cys OH;
il/RP6 ~ 18016 (~2N ~ )G;y-Asp-Trp-OH;
(~-AMF) N~2 (~-AMF)-Gly-Asp-Trp-O~;
~ -AChxAla)-Gly-Asp-Trp-OH;
(~-~-AChxAla)-Gly-Asp-Trp-OH;
-AChxAla)-Gly-Asp-Trp-OH;
(k-~-AChxAla)-Gly-Asp-Trp-O~;
~ GuaChxGly)-Gly-Asp-Trp-OH;
(L-t-GuaChxGly)-Gly-Asp-Trp-OH;
(D-c-GuaChxGly)-Gly-Asp-Trp-O~;
(k-c-GuaChxGly)-Gly-Asp-Trp-o~;
~2N ~ Gly-Asp-Trp-OH;
-Ac-Cys-AME-Gly-Asp-Cys-OH;
Ac-Cys-(c-AChxAla)-Gly-Asp-Cys-O~;
r Ac-Cy~ AChxAla)-Gly-Asp-Cys-OH;
ll/RP6 - 16 - 18016 t Ac-Pen-AME-Gly-Asp-Cys-O~; i Ac-Cys-AMF-Gly-Asp-Trp-(N-MeCys)-OH;
Ac-Cys-(~-AChxAla)-Gly-Asp-Trp-(N-MeCys)-O~;
r- ~
Ac-Cys-(~-AChxAla)-Gly-Asp-Trp-(N-MeCys) O~;
Ac-Cys-(DiMeTzl)-AMF-Gly-Asp-Cys-O~;
~ ~ ~
1o Ac-Cys-(DiMeTzl)-(c-AChxAla)-Gly-Asp-Cys-OH;
Ac-Cys-(DiMeTzl)-(t-AChxAla)-Gly-Asp-Cy~-OH;
f _ ~
Ac-Cys-AMF-Gly-Asp-Trp-Pro-Cys-N~2;
c(Aha-AME-Gly-Asp-Trp-Pro);
c(Ahex-AMF-Gly-Asp-Trp-Pro);
c(Aha-(~-AChxAla)-Gly-Asp-Trp-Pro);
c(Ahex-(c-AChxAla)-Gly-Asp-Trp-Pro);
c(Aha-(t-AChxAla)-Gly-Asp-Trp-Pro);
2s c(Ahex-(t-AChxAla)-Gly-Asp-Trp-Pro);
c(Aha-(~-GuaChxGly)-Gly-Asp-Trp-Pro);
c(Ahe~ -GuaChxGly)-Gly-Asp-Trp-Pro);
c(Aha-(t-GuaChxGly)-Gly-Asp-Trp-Pro);
ll/RP6 - 17 - 18016 c(Ahex-(t-GuaChxGly)-Gly-Asp-Trp,~ro);
t - ~
Ac-Cy~-Asn-(DiMeTzl)-(~-AChxGly)-Gly-Asp-Cys-O~;
and - - ~
Ac-Cys-Asn-(DiMeTzl~-(c-GuaChxGly)-Gly-A~p-Cys-OH;
Preferred compounds are:
r Ac-Cys-Asn-Pro-(~-AME)-Gly-Asp-Cys-OH;
1 0 1 _ ~
Ac-Cys -Asn- (DiMeTzl)-(L-AME)-Gly-Asp-Cys-O~;
Ac-Cys-Asn-(DiMeTzl)-(c-AChxAla)-Gly-Asp-Cys-O~;
c(~ha-(~ AMF)-Gly-Asp-Trp-Pro) NH2 ~ Gly-Asp-Irp-O~
and Ac-Cy6-Asn-(DiMeTzl)-(~-AChxAla)-Gly-A~p-Cy~-OH.
In addition to the common three letter abbreviations used to identi~y common amino acids, applicants have used the following abbreviation designations:
- ll/RP6 - 1~ - 18016 AME aminomethyl pheyylalanine t-AChxAla trans-aminocyclohexylalanine c-AChxAla cis-aminocyclohexylalanine t-AChxGly trans-aminocyclohexylglycine c-AChxGly cis-aminocyclohexylglycine GuaChxAla guanidocyclohexylalanine GuaChxGly Guanidocyclohexylglycine DiMeTzl Dimethylthioproline Aha 7-NH2 heptanoic acid Ahex 6-N~2 hexanoic acid The invention also includes compositions, compri~ing fibrinogen receptor antagonist peptides of the present invention and one or more pharma~o-logically acceptable carriers, e.g. saline, at a pharmacologically acceptable pH, e.g. 7.4, which are ~uitable for continuou~ intravenous or oral or intravenous bolus administration for promoting inhibition of platelet aggregation.
The invention also includes methods for inhibiting platelet aggregation which comprise administering to a patient, either by continuous intravenous or oral or intravenous bolus method, an effective amount of a composition of the present invention.
pETAILED DESCRIPTION OF l~E INVENTION
Compounds of the invention are fibrinogen receptor antagonists which inhibit fibrinogen induced platelet aggregation. These compounds are prepared by solid phase syntheæis which is well known in the ~ 7 ~ -~ f~
ll/RP6 - 19 - 18016 art, or by liquid method well known in th~ art (Neurath, ~ill & Boeder, Eds~ "The Proteins~ 3rd Edition, Vol. II, Academic Press, 1976).
The compounds of the invention are specifically useful for preventing formation of blood clots by inhibiting the binding of fibrinogen to the platelet membrane glycoprotein complex IIb/IIIa receptor. Preferred compound8 have 8electivity over other integrin receptors, and thus are specifically designed for preventing thrombosis.
The procedures for synthesizing synthetic amino acids defined by XX are well know in the art.
PEPTIDES, Structure and Function, Pierce Chemical Company ~Rockford, IL) (1985), Deber et al.
Eds, Nutt et al., "Novel Conformationally Constrained Amino Acids as Lysine-9 Substitutions in Somato~tatin Analogs: pp. 441-444, describe procedures for preparing cis- and trans-4-aminocyclohexylglycine (AChxGly), ciæ- and tranæ- 4-aminocyclohexylalanine (ACh~Ala), and para-amino-methylphenylalanine (p-ANF). The procedures described by Nutt et al. are incorporated by reference.
Phenyl guanidines, benzyl guanidines, methylguanidines and N, N'-diethylguanidines are prepared from primary amines by general procedures well known in the art.
Trans-GuaChxAla, cis-GuaChxAla, trans-GuaChxGly and cis-GuaChxGly may be prepared by the following general procedure:
ll/RP6 - 20 - 18016 NH
~CH3 )~2 CH3 NEt 3 tNH .HN~3 NHa using reagent 3,5-dimethylpyrazole-1-carboxamidine nitrate, Methods of Enzymology 25b, 558 (1972).
7 ,S03 NH~ 7 6 RN=C \~NR
R-NH2 \NH3 R- NX
wherein ~6 i~ an alpha Boc-amino acid ~ide chain or the side chain of an alpha amino acid in a peptide, and R7 is alkyl, aryl, arylal~yl or cycloalkyl having 1-6 carbon~, perferably cyclohexyl.
ll/RP6 - 21 - 18016 Alkyl- or aryl- iminomethane sulfonic aci~s are prepared by oxidation of the correæponding thioureas, as described in A.E. Miller and J.J. Bischoff ~ynthe~is, pp. 777-779 (1986). Guanylation occurs in agueous K2C03, as described above. Alternatively, S the reaction may be carried out in dimethylformamide - Et3N (@ p~ 9). Reaction time i~ 24-48 hours in an aqueou~ system, and 3-20 hours in dimethylformamide.
Compound~ of the invention may be prepared using solid phase peptide synthesis, such as that described by Merrifield, J. Am. Chem. Soc.. 85, 2149 (1964), although other equivalent chemical syntheses known in the art can also be used, such as the syntheses of Houghten, ~Q. Natl. Acad. Sci., 82, 5132 (1985). Solid-pha~e synthesis is commenced from the C-terminus of the peptide by coupling a protected amino acid to a suitable resin, as generally set forth in ~.S. Patent No. 4,244,946, issued Jan. 21, 1982 to Rivier et al., the disclosure of which is hereby incorporated by reference. Solution method can be used aæ described by Neurath et al. Chapter 2, pp. 106-~53. Examples of synthesis of this general type are set forth in U.S. Patent Nos. 4,305,872 and 4,316,891.
In ~ynthesizing these polypeptides, the carboxyl terminal amino acid, having itæ alpha-amino group suitably protected, i6 covalently coupled to a chloromethylated polystyrene resin or the like. The chloromethylated polystyrene resin is composed of fine beads (20-70 microns in diameter) of a synthetic resin prepared by copolymerization of styrene with 1 to 2 percent divinylbenzene. The benzene rings in the resin are chloromethylated in a Fried~l-Crafts reaction with chloromethyl methyl ether and 6tanni~
chloride. The Friedel-Crafts reaction is continued until the resin contains 0.5 to 5 mmoles o~ chlorine per gram of ~e8in. After removal of the alpha amino 5 protecting group, as by using trifluoroacetic acid in methylene chloride, the amino protected derivative of the next amino acid in the sequence i8 added along with a condensation coupling agent such as dicyclohexylcarbodiimide. The remaining alpha-amino and ~ide-chain-protected amino acids are then coupled by condensation stepwi~e in the desired order to obtain an intermediate compound connected to the resin.
The condensation between two amino acids, or an amino acid and a peptide, or a peptide and a peptide can be carried out according to the usual condensation methods 6uch as azide method, mixed acid anhydride method, DCC (dicyclohexyl-carbodiimide) method, BOP (benzotriazole-l-yloxytris (dimethylamino) phosphonium hexafluorophosphate method, active ester method (p~nitrophenyl ester method, N-hydroxy-succinimido ester method, cyanomethyl ester method, etc.), Woodward reagent K method, carbonyldiimidazol method, oxidation-reduction method. In the case of elongating the peptide chain in the ~olid phase method, the peptide is attached to an insoluble carrier at the C-termina~ amino acid. For insoluble carrier~, tho~e which react with the carboxy group of the C-terminal amino acid to form a bond which is readily cleaved later, for example, halomethyl resin such as chloromethyl resin and bromomethyl resin, ll/~P6 - 23 - 18016 hydroxymethyl resin, aminomethyl resin, b~nzhydryl-amine re~in, t-al~yloxycarbonylhydrazide resin an p-hydroxymethylphenylacetylamidomethyl resin(PAM).
Common to chemical syn~hese~ of peptides is the protection of the reactive side-chain groups of the various amino acid moieties with suitable protecting groups at that ~ite until the group i8 ultimately removed after the chain has been completely a~sembled. Also common i~ the protection of the alpha-amino group on a amino acid or a fragment while that entity reacts at the carboxyl group followed by the ~elective removal of the alpha-amino-protecting group to allow ~ubsequent reaction to take place at that location.
Accordingly, it is common that, as a step in the æynthesiæ, an intermediate compound i3 produced which includes each of the amino acid reæidues located in the desired sequence in the peptide chain with variouæ of theæe residueæ having æide-chain protecting groups. These protecting groups are then commonly removed substantially at the æame time so as to produce the deæired resultant product following purification.
The applicable protective groupæ for protecting the alpha-and omega-æide chain amino 2s group~ are exemplified ~uch aæ benzylo~ycarbonyl (hereinafter abbreviated as Z), isonicotinyloxy-carbonyl (iNOC), O-chlorobenzyloxycarbonyl ~Z(2-Cl)], p-nitrobenzyloxycarbonyl ~Z(N02)~,p-methoxybenzyl-oxycarbonyl ~Z(OMe)],t-butoxycarbonyl (Boc), t-amyloxycarbonyl (Aoc), isobornyloxycarbonyl, adamantyloxycarbonyl, 2-(4-biphenyl)-2- propyloxy-carbonyl (Bpoc),9-fluorenylmethoxycarbonyl (Fmoc), ll/RP6 - 24 - 1801~
methylsulfonylethoxycarbonyl (Msc), triflu~roacetyl, phthalyl, formyl, 2-nitrophenylæulphenyl (NPS), diphenylphosphinothioyl (Ppt), dimethylphos-phinothioyl (Mpt) and the like.
Protective groups for carboxy group include, for example, benzyl ester (OBzl), cyclohexyl ester (Chx) 4-nitrobenzyl ester (ONb), t-butyl ester (OBut), 4-pyridylmethyl ester (OPic), and the like.
It is desirable that specific amino acids such a~
arginine, cysteine, and serine possesing a functional group other than amino and carboxyl groups are protected by a suitable protective group as occasion demands. For example, the guanidino group in arginine may be protected with nitro, p-toluene-sulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzenesulfonyl, 4-methoxy-2, 6-dimethyl-benzenesulfonyl (Mds), 1,3,5-trimethylphenylsulfonyl (Mts~, and the like. The thiol group in cysteine may be protected with benzyl, p-methoxybenzyl~
triphenylmethyl, acetylamidomethyl, ethylcarbamoyl, 4-methylbenzyl, 2,4,6-trimethylbenzyl (Tmb) etc., and the hydroxyl group in serine can be protected with benzyl, t-butyl, acetyl, tetrahydropyranyl etc.
Stewart and Young, "Solid Phase Peptide Synthesis:, Pierce Chemical Company, Roc~ford, IL
2s (1984) provides detailed information regarding procedures for preparing peptides. Protection of a-amino groups is described on pages 14-18, and side-chain blockage is described on pages 18-28. A
table of protecting groups for amine, hydroxyl and sulfhydryl functions i~ provided on pages 149-151.
These descriptions are hereby incorporated by reference.
ll/RP6 - ~5 - 18016 After the desired amino-acid seq~ence has been completed, the intermediate peptide is removed from the resin support by treatment with a reagent, such as liquid HF, which not only cleaves the peptide from the resin, but also cleaves all the remaining protecting groups from the side chain which do not interfere in the cyclization reaction. Potentially reactive Ride chains functionalities are protected with blocking groups which are stable to HF. The peptides are cyclized by any one of several kno~n procedures (see Schroder and Lubke, "The Peptides:
Methods of Peptide Synthesis" Vol. I, Academic Press, New York (1965), pp. 271-286, the contents of which are hereby incorporated by reference), e.g. ~y forming a disulfide bridge between the cysteine re~idues using iodine in AcO~, or air sxidation at pH
8 in dilute NH4 OAc buffer. The polypeptide can then be purified by gel permeation chromatography followed by preparative HPLC, as described in Rivier et al., Peptides: Structure and Biological Function (1979) pp. 125-128.
EXAMPL~ 1 Synthesis of aminomethylphenylalanine a~ a-BOC-Cbz-p-aminomethvl-D.L-phenvlalanine ~or use in synthesi~ of Ac-Cys~Pmb)-Asn-Pro-(D.L-AMF(Cbz~)-1y-As~(Bzl~-Cys-(Pmb)-opam(~) ~'l3~
ll/RP6 - 26 - 18016 NaOEt (CO2 Et) 2 r+ AcNH-cHyco2Et)2 ~ CN
To 500 ml dry EtOH (4A sieves) was added 5.9g Na (0.256m) under nitrogen, 55.67g (0.2563m) of acetamido diethylmalonate and 50 g (0.2563m) of p-cyanobenzylbromide. The mixture was heated to reflux which resulted in complete dissolution of starting materials and product. After 1 hour, the reaction ~olution waæ cooled, 1.5 liters of water was added and the precipitate was filtered to give 77.3g of crude product which was recrystallized from 450 ml of EtOH to give 70.56g of product (83% yield), mp 174-5 175~5 C; IBCHC13 ~97~ ~NH), 5.78 (ester) 6.0 (amide) 4.52~ (CN) Rf (95-5-0.5 - C~C13-MeO~-H20) = 0.75 PP~
NMR
CDC13: 1.3 (t, Ç~3CH20), 2.05 (s, ~3C), 3.75 (s, ar-~2-C), 4.3 (m, CH3~2~0) 6.5 (s, N~), 7.2 (d, arom), 7.6 (d, arom) ~.J~ ~
ll/RP6 - 27 - 18016 ~,~cO,Et);~ H2_Pd/c ~,N--~(CO,Et), N~ EtO~1~50% }~AC NH1~c To a suspen6ion of 20g (60mm) of p-CN-benzyl N-acetyl-diethylmalonate in 200 ml EtOH - 50% HOAc (8:2) under a N2 gtream wa~ added 4g of 10% Pd/C and the mixture wa~ treated with ~2 in a Parr Shaker for 70 l~ minutes after which period 96% of theoretical amount of ~2 was conæumed. The mixture was filtered through Celite, the filtrate was evaporated ~n vacuo to drynes~
~o give a solid residue which was triturated with EtOAc, filtered and dried to give 21.45g ~90.2% yield) of product.
1~ in CHC13 show~ no CN at 4.5 ~.
(95-5-0.5 CHC13-MeOH-conc.NH40H) = 0.3 (ninhydrin +) ppm ~MB C~2C @3.65 singlet, ~N @3.9 singlet ~l2N~CO~Et)~ 6N HCl ~N~COOH
A ~olution of 21g (53 mm) of N-acetyl diethyl ester p-aminomethylbenzyl aminomalonate in 100 ml of 6N ~Cl was refluxed for 24 hrs. The reaction solution was evaporated ~n vacuo to give 16.6g of product aæ ~olid.
Rf (60-30-4-6, CHC13-MeO$-$2O-N$4OH) = 0.15 ~o OOH CuCl~. 2H~O CbZ~ COOH
G~N--J~o~ N~, To all of p-aminomethylphenylalanine (53 mm) (prepared above) in 200 ml H2O was added 4.92g of CuC12.2~2O. The mixture waæ adjusted to pH of 9 with NaOH. To the reaction mixture waæ added 18.26g 2~ 3~
ll/RP6 - 29 - 1~016 (58 mm) of the N-benzyloxycarbonylogy-t-nol~bornene 2,3-dicarbo~imide reagent and the reaction mixture waæ kept at 5C for 18 hr~. The ~olid was filtered, washed with ~2 and EtOAc, and redi~solved in HOAc and ~Cl to o~tain a pH of 0.5. ~pon ~tanding, 9.5g of product as the zwitterion precipitated. The filtrate wae treated with ~2S, fil~ered through a pad of celite and pyridine was added to the filtrate to pH 6. The flocculent precipitate was filtered to give a second crop of product (1.5g). Total yield was llg (58% yield). Anal. calcd. for C18H20 N2 4 calcd. fd N = 8.53 7.99 C ~65.84 66.65 H = 6.14 6.13 NMR in D20 and NaOD evidenced product to have the Cbz group on the NH2CH2 and not the a-NH2, CbZNH--~OOH ~ ~30C-ON--CbzN~ OOH
E~oc A suspension of 7.0g (21.3mm) of omega-Cbz -p-aminomethyl-DL-phenylalanine in 70 ml H20 and 35 ml of T~F was treated with 9.27 ml (63.9mm) of NEt3 and 5.51 g (22.36mm) of Boc-ON (Aldrich) for 24 hrs during which time all ætarting material went into solution. To the reaction solution was added lSO ml 3 ~ ~
ll/RP6 - 30 - 18016 of ethyl ether, the H20 layer was separat~ and the ether layer was washed two times with H20; the combined H20 layers were back-washed once with ether and acidified with citric acid to give a gummy solid. The aqueou~ supernatant wa~ decanted, the gummy solid was extracted into EtOAc, the EtOAc solution was dried over MgS04, filtered and evaporated to a foamy residue (8.73g). The crude product was crystallized from EtOAc-pet Et20 to give 7.22g (79.3% yield), m.p. 133-133.5C.
TLC Rf = 0.35 (80-20-2, CHC13-MeO~-N~40H) NMR CD30D : 1.4(Boc), 2.9, 3.15 (m, ~ 2)~
4.25(8, C~2N), 4.3 (m, a-H), 5.1(s,C~2-Cbæ) 7.2 ,7.3(arom, Cbz, ~ ) ~XAMPL~L.2 Svntheæis of Ac-Cys(Pmb)-Asn-Pro-rD.L-AMF~Cbz~l-Gly-Asp(Bzl)-Cys~Pmb)-OPam ~
.~nd ultimately Ac-Cys-Asn-Pro-~D.L-AMF)-Gly-Asp-Cys-O~
PM~3 Starting with Boc-Cys-O-Pam-resin, the alpha-amino Boc protecting group (tert-butylcarbonyl~
ll/RP6 - 31 - 18016 is removed (while the Cy6 æide-chain remains protected by p-methylbenzyl) using trifluoroacetic acid and methylene chloride, and the a-deprotected cysteine neutralized with diisopropylethyl amine.
Boc-protected A8p (benzyl) (Asp (Bzl)) is then coupled to cysteine mediated by dicyclohexyl-carbodiimide, and deprotected with trifluoroacetic acid and methylene chloride. Asp is then neutralized with diisopropylethylamine. Following thi 8 stepwise procedure of coupling with dicyclohe~ylcarbodiimide, lo deprotection with trifluoroacetic acid and methylene chloride, and neutralization with diisopropyl-ethylamine, Boc-protected Gly, AMF, Pro, Asn, Cys residues are coupled in succession. AME is--additionally protected by Cbz, (AMF (Cbz)), and the final Cys residue ls again additionally protected by p-methylbenzyl. The f inal Cys is then acetylated with acetic anhydride.
Following acetylation, the following peptide-resin iæ ~ormed:
PMB Cbz Bzl PMB
Acetyl-Cy6-Asn-Pro-(D,L-AMF)-Gly-Asp-Cys-0-Pam ~
Cleavage of the peptide from the reein i8 achieved using ~F/anisole (9:1 (v/v)) to form:
H
Acetyl-Cy~-Asn-Pro-(D,L-AMF)-Gly-Asp-Cys-0~.
A cyclic 6tructure is formed by formation of a disul~ide bridge between the cysteine residues.
The peptide i8 dissolved in 50-80% Ac0~:~20 at room temperature, and the ~olution stirred during rapid ll/RP6 - 32 - 18016 addition of a solution of iodine in AcOH ~o a final concentration of 2.25 mg/ml of iodine. After 1-2 hours reaction time, excess I2 and AcO~ are removed by rotary evaporation under vacuum and the aqueous solution containing the cyclized pep~ide is purified using preparative HPLC in 0.1% TFA H20-C~3CN gradient at which stage the D- and L- diastereomers are separated by conventional means. The final TFA salt product iB converted to ~OAc salt by passing through an ion exchange column BioRad AG3-~4A (acetate cycle). The fini6hed peptide i8:
\
Acetyl-Cys-Asn-Pro-(k-AMF)-Gly-Asp-Cys-O~.
As an alternative to forming the disulfide bridge by iodine oxidation, the free SH peptide is dis~olved in 1-5% HOAc at a concentration of approximately 2 mg/ml and the solution adjusted to appro~imately pH 7-8.5 with concentrated NH40H. Cyclization is accomplished under brisk stirsing (preferably with a small piece of copper wire added to accelerate the reaction) during a period of 1-4 hours at 25. The reaction mixture is then concentrated as before and product purified by preparative HPLC.
Synthesis of Ac-Cys-Asn-(Di~Tzl~-~L-AMF~-Gly-Asp-Cys-Q~
.
ll/RP6 - 33 - 18016 The same procedure for synthesizi~g the cyclic peptide of Example 2 is followed, except that Pro is replaced with DiMeTzl.
Thera~eutic Utility Compounds of the invention may be administered to patients where prevention of thrombosis by inhibiting binding of fibrinogen to the platelet membrane glycoprotein complex IIb/IIIa receptor is desired. They are useful in surgery on peripheral arteries (arterial grafts, carotid endarterectomy) and in cardiovascular surgery where manipulation of arteries and organs, and/or the interaction of platelets with artificial su~faces, leads to platelet aggregation and consumption. The lS aggregated platelets may form thrombi and thromboemboli. Polypeptides of the invention may be administered to these surgical patients to prevent the formation of thrombi and thromboemboli.
Extracorporeal circulation is routinely used for cardiovascular ~urgery in order to oxygenate blood. Platelets adhere to surfaces of the extracorporeal circuit. Adhesion is dependent on the interaction between GPIIb/IIIa on the platelet membranes and fibrino~en adsorbed to the surface of the circuit. (Gluszko et al., Amer. J. Physiol 1987l 252:H, pp 615-621). Platelets released from artificial surfaces show impaired hemostatic function. Polypeptides of the invention may be administered to prevent adhesion.
Other applications of these polypeptides include prevention of platelet thrombosis, ll/RP6 - 34 - 18016 thromboembolism and reocclusion during an~;after thrombolytic therapy and prevention of platelet thrombosis, thromboembolism and reocclusion after angioplasty of coronary and other arteries and after coronary artery bypass procedures. Polypeptides of the invention may al60 be u~ed to prevent myocardial infarction.
These polypeptides may be administered by any convenient means which will result in its delivery into the blood ~tream in substantial amount including continuous intravenous or bolus injection or oral methods. Compositions of the invention include peptides of the invention and pharmacologically acceptable carriers, e.g. ~aline, at a p~ level e.g. 7.4, suitable for achieving inhibition of platelet aggregation. They may be combined with thrombolytic agents such as plasminogen activatoræ or streptokinase in order to inhibit platelet aggregation. They may also be combined with anticoagulants ~uch as heparin, aspirin or warfarin.
Intravenou~ administration is presently contemplated as the preferred administration route. They are soluble in water, and may therefore be effectively administered in ~olution.
In one exemplary application, a suitable amount of peptide is intravenously admini~tered to a heart attack victim undergoing angioplasty.
Administrat~on occurs during or several minutes prior to angioplasty, and is in an amount sufficient to inhibit platelet aggregation, e.g. an amount which achieves a steady state plasma concentration of between about 0.05-30 ~M per kilo, preferably between ~ ~w~
ll/RP6 - 35 - 1~016 about 0.3-3 ~M per kilo. When thiæ amoun~ is achieved, an infusion of between about 1-100 ~M per kilo per min., preferably between about 10-30 ~M per kilo per min. i~ maintained to inhibit platelet aggregation.. Should the patient need to undergo bypass surgery, administration may be stopped immediately and will not cause complicatione during surgery that would be caused by other materials such as aspirin or monoclonal antibodies, the effects of which last hours after cessation of administration.
The present invention also includes a pharmaceutical composition comprising peptideæ of the present invention and tissue type plasminogen activator or streptokinase. The invention al~o includes a method for promoting thrombolysis and preventing reocclusion is a patient which compri~es administering to the patient an effective amount of compositions of the invention.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. Thus, the specific examples described above should not be interpreted as limiting the scope of the present invention.
ll/RP6 - 2 - 18016 Zimmerman et al., U.S. Patent No. 4,683,2~
describes peptides having utility in the study of fibrinogen-platelet, platelet-platelet, and cell-cell interactions. The peptides are described as having utility where it i8 desirable to retard or prevent formation of a thrombus or clot in the blood. The general formula for the peptides is:
H2N-(Ch)-Arg-Gly-A8p-(Cx)-H
where Ch and Cx are sequences of amino acids.
Pier chbacher et al., ~.S. Patent No.
4,589,881, describeæ the Requence of an 11.5 kDal polypeptide fragment of fibronectin which em~odies the cell-attachment-promoting activity of fibronectin. A specifically described fragment is:
H-Tyr-Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro-Ile-Ser-Ile-Asn-Tyr-Arg-Thr-Glu-Ile-Asp-Lys-Pro-Ser-Gln-Met-OH
Ruoslahti et al., U.S. Patent No. 4,614.517, describes tetrapeptides which alter cell-attachment activity of cells to various substrates. The peptides are stated to "consist essentially of" the following sequence:
X-Arg-Gly-Asp-Ser-Y
wherein g is H or one or more amino acids and Y is OH
or one or amino acids. Fi~ure 1 lists the polypeptides 2 ~
ll/RP6 - 3 - 18016 that were synthesized by Ruoslahti et al. ,,in ~determining the smalle~t peptide exhibiting cell attachment activity".
Ruoslahti et al., U.S. Patent No. 4,578,079, describes similar tetrapeptides having Ser substituted with Thr or Cys.
Pierschbacher et al., ~E~ Natl~ Acad. Sci. USA, Vol. 81, pp.5985-5988, October 1984 describe variants of the cell recognition site of fibronectin that retain attachment-promoting activity. They assayed the cell attachment-promoting activitieæ of a number of structures closely resembling the Arg-Gly-Asp-Ser peptide, and found "that the arginine, glycine, and aspartate reæidues cannot be replaced even with closely related amino acids, but that several amino acids can replace serine without loss of activity."
Ruoslahti et al., Science, Vol. 238, pp. 491-497, October 23, 1987, discuss cell adhesion proteins.
They specifically state that "[e]lucidation of the amino acit sequence of the cell-attachment domain in fibronectin and it~ duplication with synthetic peptides establish the seguence Arg-Gly-Asp (RGD) as the essential structure recognized by cells in fibronectin".
Cheresh, Proc. Natl. Acad. Sci. ~SA, Vol. 84, pp.
6471-6475, September 1~87, describes the Arg-Gly-Asp-directed adhesion receptor involved in attachment to fibrinogen and von Willebrand Factor.
i 3 1 ~
ll/~P6 - 4 - 1~016 Adams et al., U. S. Patent No. 4,857,508,~describes tetrapeptides which inhibit platelet aggregation and the formation of a thrombus. The tetrapeptides have the formula:
, ~-Gly-Asp-Y
wherein X can be ~2NC(=N~)NH(C~2~nCH(Z)COOH or Ac-Arg, wherein Z = H, N~2, or NH-Acyl and n=1-4, and wherein Y can be Tyr-NH2, Phe-NH2 or a group of a specifically defined formula.
Applicants have discovered fibrinogen receptor antagonistæ which do not contain the amino acid seguence Arg-Gly-Asp which is taught in the art as specifically required for binding to platelet membrane glycoprotein complex IIb/IIIa.
~MMA~Y QF T~E INVENTION
Compounds of the present invention inhibit binding of fibrinogen to the platelet membrane glycoprotein complex IIb/IIIa receptor and contain an amino acid sequence:
XX-Gly-Asp wherein XX is a synthetic alpha amino acid containing either a phenyl or C3-Cg cycloalkyl group. These compound~ are surpriæing in view of the prior art ll/RP6 - 5 - 18016 which teaches that the ~equence Arg-Gly-As,p is required in order to achieve binding to the IIb/IIIa receptor.
The present invention is a fibrinogen receptor antagonist having the following structure:
Z Z
\ XX-Gly-~s p /
wherein XX represents a synthetic a-amino acid as defined below and Z~ represent~ a sequence of 1, 2, 3, or 4 amino acids as defined below.
XX shares an amide bond with Gly and an amide bond with ZZ, and is defined as having a side chain X
NH
2s ll (CH2)n- AA ~(C~2)n - N C NHR (i) H
or _ (CH2)n- M --(CH2)n NXR (ii) 3~
ll/RP6 - 6 - .~18016 .
wherein: n i~ 0,1,2,3 or 4;
n' is 0,1,2,3 or 4;
AA i8 di6ubstitutet phenyl, either not substituted with additional groups or substituted with Cl-4 al~yl, al~oxy or hydro y ; C3-C8 cycloalkyl, preferably cyclohe~yl, either not substituted with additional group8 or substituted with Cl-4 alkyl, alkoxy or hydroxy; and R is ~, Cl-6 al~yl, ~ubstituted or unsubstituted aryl, substituted or unsubstituted arylmethyl or substituted or unsubstituted cycloal~yl.
Preferably, the side chain of ~X is defined by (ii) wherein n is 1, n' is 1, M is unsub~titute~ phenyl and R is E.More preferably, the ~ide chain is ~H2 ~{:H2-NH2 where xx i p-aminomethylphenylalanine.
Also preferred iB the side chain of gX defined by (ii) wherein n i8 1, n' i8 O, R is ~ and M is unsubstituted cyclohe y l. More preferably, the side chain i~
CH2 ~ NH2 ll/RP6 - 7 - 18016 Preferred compounds o~ the invention are those having selectivity over integrin receptors. The preferred compounds include those wherein XX i~ a synthetic alpha-amino acid containing an amino group side chain, as represented above by (il).
ZZ is defined as follows:
A ~ ~, _ y, $ E' ' ~ ' -'-'` ( ) --~ (1 ) wherein:
A' is H, acylamido, acylaminoacylamido, acylamino-N-methylamino-acylamido;
R' and R'l are independently H, methyl, 2s ethyl or a lower alkyl group having 1 to 5 carbons;
~ ' is S-S, CH2-S, S-CH2, CH2CH2, C~2, C~2C~2C~2- C~2-S-S- C~2-S-S-CH2, S-S-CH2; and 2~h ~3:L~
ll/RP6 ~ 18016 ~ ' i8 ~, C00~, CONH2, CON8R2, coNR3~, CH20H,C02R2,CH3 wherein R2 is an alkyl gr~up having 1 to 4 carbon atoms, R3R4 i~ an al~yl group having 1 to 4 carbon atoms or NR3R4 i8 a secondary amino acid, or o //N--NH
N ¦ ;
or æz i~ N=~
A ~ X
B C ~t~-6~h~
wherein:
A' is as defined above;
R' and R'l are as defined above;
X' - Y' is as defined above;
B' is a D- or L- a-amino acid;
C' i8 a D- or L- secondary ~-amino acid, preferably selectet ~rom proline, ~- methylproline, ~,~- dimethylproline, ~-hydro~yproline, anhydroproline, thioproline, ~ -ll/RP6 - 9 ~ ..18016 methylthioproline, ~ dimethylthioproline, pipecolic acid, azetidine carbo y lic acid,~nd an N-methyl amino acid, or a D- or L- primary a-amino acid; and E~ i~ as defined abo~e;
or ZZ i8 R~ ' R R
N _ RS- (~
(xX-Cl wherein:
A' is as defined above;
R' and R~l are aæ defined above;
~' - Y' are a~ defined above;
E' is ag defined above;
F' iB an L-amino acid, preferably selected from tryptophan, phenylalanine, leucine, valine, isoleucine, a-naphthylalanine, ~-naphthylalanine, methionine, tyro~ine, arg inine, lysine, homoarg inine, ornithine, hi6tidine, substituted tryptophan, substituted phenylalanine or ~ubstituted tyrosine;
and R5 is H or methyl;
__ _ ___ 3 ~ ~
ll/RP6 - 10 - 18016 or :~Z iS
R~t ' ~ It _~X--y ~ ~E
~C' I 5C
wherein A' is as defined above;
R' and R'l are as defined above;
X'-Y~ is as defined above;
Cl is as defined above; and E~ is as defined above.
or ZZ is 2 5 ~ E
- G~x- C~-ds~ F
2 ~ 2 ~
11/RP6 ~ 18016 wherein "-A' is as defined above;
R~ and R'l are as defined above;
X~-Y~ is a3 defined above, F' is as defined above;
G' i~ a D- or L-a-amino acid, 8econdary cyclic amino acid, or N-methyl amino acid;
~' i8 as defined above; and R5 is as def ined above.
~0 2s ll/RP6 - 12 - 18016 The present invention al~o is a fibrinogen receptor antagonist of the formula lo ~ 1l I~
B-Q-C-C-Gly-Asp-NH-CH
X E' wherein:
B representæ zero one or two ~ubstituted or unsubstituted amino acids;
Q represents ~, N~, N~2 or Ac-NH;
X represents the side chain of amino acid ~X
as previously defined; and I' i a ~ide chain of an amino acid previously defined by F', and E' is H, COOH, CONH2, CONHR2, CoNR3R4, CH20H, C02R2, C~3 wherein R~ i8 an alkyl group having 1 to 4 carbon atoms, R3R4 i~ an al~yl group having 1 to 4 carbon atoms or NR3R4 i8 a secondary amino acid, or ~ ~ ~'J'~
ll/RP6 - 13 - 18016 ,N-NnH
N
`N~; `
provided that when B ie zero 6ubstituted or unsubstituted amino acid~, then Q is ~, N~2 or Ac-N~, and that when B i~ one or two gub~tituted or unsubstituted amino acids, then Q i8 N~.
In a preferred embodiment of the present invention, the fibrinogen receptor antagonist has the following formula:
~ZZ
(L-AMF)-Gly-Asp wherein ZZ is:
25Ac-Cy8 Cy8-OH
Asn-Pro~ (L-AMF-Gly-Asp) r 30Ac-Cy8 Cys-OH
Asn-DiMeTzl --.... -(~-AME-Gly-Asp) i3~
ll/~P6 - 14 - 1~016 Exemplary compounds of the invention are:
r Ac-Cys-Asn-Pro-(L-AMF)-Gly-Asp-Cys-OH;
-Ac-Cys-A6n-Pro-(~-AMF)-Gly-Asp-Cys-O~;
Ac-Cys-Asn-(DiMeTzl)-(LrAMF)-Gly-Asp-Cys-O~
r Ac-Cys-Asn-(DiMeTzl)-(~-AMF)-Gly-Asp-Cys-OH
c(Aha-(L-AMF)-Gly-Asp-Trp-Pro);
c(Aha-(~-AMF)-Gly-Asp-Trp-Pro);
. .
Ac-Cy~-Asn-(DiMeTzl)(t-ACh~Ala)-Gly-Asp-Cys-OH;
Ac-Cys-(DiMeTzl)-(~-AChxAla)-Gly-Asp-Cys-OH;
. ~
Ac-Cys-Asn-(DiMeTzl)-(c-AChxAla)-Gly-Aæp-Cys-OH;
.
Ac-Cys-(DiMeTzl)-(~-AChxAla)-Gly-Asp-Cys-OH;
r Ac-Cys-Asn-(DiMeTzl)-(~-GuaChxAla)-Gly-Asp-Cys-OH;
r ~
Ac-C~s-(DiMeTzl)-(t-GuaChxAla)-Gly-Asp-Cys-O~;
, ~
Ac-Cys-Asn-(DiMeTzl)-(~-GuaChxAla)-Gly-Asp-Cys-OH;
Ac-Cys-(DiMeTzl)-(c-GuaChxAla)-Gly-Asp-Cys-OH;
Ac-Cys-Asn-~DiMeTzl)-(t-AChxGly)-Gly-Asp-Cys-OH;
Ac-Cys-Asn-(DiMeTzl)-(~-GuaChxGly)-Gly-Asp-Cys OH;
il/RP6 ~ 18016 (~2N ~ )G;y-Asp-Trp-OH;
(~-AMF) N~2 (~-AMF)-Gly-Asp-Trp-O~;
~ -AChxAla)-Gly-Asp-Trp-OH;
(~-~-AChxAla)-Gly-Asp-Trp-OH;
-AChxAla)-Gly-Asp-Trp-OH;
(k-~-AChxAla)-Gly-Asp-Trp-O~;
~ GuaChxGly)-Gly-Asp-Trp-OH;
(L-t-GuaChxGly)-Gly-Asp-Trp-OH;
(D-c-GuaChxGly)-Gly-Asp-Trp-O~;
(k-c-GuaChxGly)-Gly-Asp-Trp-o~;
~2N ~ Gly-Asp-Trp-OH;
-Ac-Cys-AME-Gly-Asp-Cys-OH;
Ac-Cys-(c-AChxAla)-Gly-Asp-Cys-O~;
r Ac-Cy~ AChxAla)-Gly-Asp-Cys-OH;
ll/RP6 - 16 - 18016 t Ac-Pen-AME-Gly-Asp-Cys-O~; i Ac-Cys-AMF-Gly-Asp-Trp-(N-MeCys)-OH;
Ac-Cys-(~-AChxAla)-Gly-Asp-Trp-(N-MeCys)-O~;
r- ~
Ac-Cys-(~-AChxAla)-Gly-Asp-Trp-(N-MeCys) O~;
Ac-Cys-(DiMeTzl)-AMF-Gly-Asp-Cys-O~;
~ ~ ~
1o Ac-Cys-(DiMeTzl)-(c-AChxAla)-Gly-Asp-Cys-OH;
Ac-Cys-(DiMeTzl)-(t-AChxAla)-Gly-Asp-Cy~-OH;
f _ ~
Ac-Cys-AMF-Gly-Asp-Trp-Pro-Cys-N~2;
c(Aha-AME-Gly-Asp-Trp-Pro);
c(Ahex-AMF-Gly-Asp-Trp-Pro);
c(Aha-(~-AChxAla)-Gly-Asp-Trp-Pro);
c(Ahex-(c-AChxAla)-Gly-Asp-Trp-Pro);
c(Aha-(t-AChxAla)-Gly-Asp-Trp-Pro);
2s c(Ahex-(t-AChxAla)-Gly-Asp-Trp-Pro);
c(Aha-(~-GuaChxGly)-Gly-Asp-Trp-Pro);
c(Ahe~ -GuaChxGly)-Gly-Asp-Trp-Pro);
c(Aha-(t-GuaChxGly)-Gly-Asp-Trp-Pro);
ll/RP6 - 17 - 18016 c(Ahex-(t-GuaChxGly)-Gly-Asp-Trp,~ro);
t - ~
Ac-Cy~-Asn-(DiMeTzl)-(~-AChxGly)-Gly-Asp-Cys-O~;
and - - ~
Ac-Cys-Asn-(DiMeTzl~-(c-GuaChxGly)-Gly-A~p-Cys-OH;
Preferred compounds are:
r Ac-Cys-Asn-Pro-(~-AME)-Gly-Asp-Cys-OH;
1 0 1 _ ~
Ac-Cys -Asn- (DiMeTzl)-(L-AME)-Gly-Asp-Cys-O~;
Ac-Cys-Asn-(DiMeTzl)-(c-AChxAla)-Gly-Asp-Cys-O~;
c(~ha-(~ AMF)-Gly-Asp-Trp-Pro) NH2 ~ Gly-Asp-Irp-O~
and Ac-Cy6-Asn-(DiMeTzl)-(~-AChxAla)-Gly-A~p-Cy~-OH.
In addition to the common three letter abbreviations used to identi~y common amino acids, applicants have used the following abbreviation designations:
- ll/RP6 - 1~ - 18016 AME aminomethyl pheyylalanine t-AChxAla trans-aminocyclohexylalanine c-AChxAla cis-aminocyclohexylalanine t-AChxGly trans-aminocyclohexylglycine c-AChxGly cis-aminocyclohexylglycine GuaChxAla guanidocyclohexylalanine GuaChxGly Guanidocyclohexylglycine DiMeTzl Dimethylthioproline Aha 7-NH2 heptanoic acid Ahex 6-N~2 hexanoic acid The invention also includes compositions, compri~ing fibrinogen receptor antagonist peptides of the present invention and one or more pharma~o-logically acceptable carriers, e.g. saline, at a pharmacologically acceptable pH, e.g. 7.4, which are ~uitable for continuou~ intravenous or oral or intravenous bolus administration for promoting inhibition of platelet aggregation.
The invention also includes methods for inhibiting platelet aggregation which comprise administering to a patient, either by continuous intravenous or oral or intravenous bolus method, an effective amount of a composition of the present invention.
pETAILED DESCRIPTION OF l~E INVENTION
Compounds of the invention are fibrinogen receptor antagonists which inhibit fibrinogen induced platelet aggregation. These compounds are prepared by solid phase syntheæis which is well known in the ~ 7 ~ -~ f~
ll/RP6 - 19 - 18016 art, or by liquid method well known in th~ art (Neurath, ~ill & Boeder, Eds~ "The Proteins~ 3rd Edition, Vol. II, Academic Press, 1976).
The compounds of the invention are specifically useful for preventing formation of blood clots by inhibiting the binding of fibrinogen to the platelet membrane glycoprotein complex IIb/IIIa receptor. Preferred compound8 have 8electivity over other integrin receptors, and thus are specifically designed for preventing thrombosis.
The procedures for synthesizing synthetic amino acids defined by XX are well know in the art.
PEPTIDES, Structure and Function, Pierce Chemical Company ~Rockford, IL) (1985), Deber et al.
Eds, Nutt et al., "Novel Conformationally Constrained Amino Acids as Lysine-9 Substitutions in Somato~tatin Analogs: pp. 441-444, describe procedures for preparing cis- and trans-4-aminocyclohexylglycine (AChxGly), ciæ- and tranæ- 4-aminocyclohexylalanine (ACh~Ala), and para-amino-methylphenylalanine (p-ANF). The procedures described by Nutt et al. are incorporated by reference.
Phenyl guanidines, benzyl guanidines, methylguanidines and N, N'-diethylguanidines are prepared from primary amines by general procedures well known in the art.
Trans-GuaChxAla, cis-GuaChxAla, trans-GuaChxGly and cis-GuaChxGly may be prepared by the following general procedure:
ll/RP6 - 20 - 18016 NH
~CH3 )~2 CH3 NEt 3 tNH .HN~3 NHa using reagent 3,5-dimethylpyrazole-1-carboxamidine nitrate, Methods of Enzymology 25b, 558 (1972).
7 ,S03 NH~ 7 6 RN=C \~NR
R-NH2 \NH3 R- NX
wherein ~6 i~ an alpha Boc-amino acid ~ide chain or the side chain of an alpha amino acid in a peptide, and R7 is alkyl, aryl, arylal~yl or cycloalkyl having 1-6 carbon~, perferably cyclohexyl.
ll/RP6 - 21 - 18016 Alkyl- or aryl- iminomethane sulfonic aci~s are prepared by oxidation of the correæponding thioureas, as described in A.E. Miller and J.J. Bischoff ~ynthe~is, pp. 777-779 (1986). Guanylation occurs in agueous K2C03, as described above. Alternatively, S the reaction may be carried out in dimethylformamide - Et3N (@ p~ 9). Reaction time i~ 24-48 hours in an aqueou~ system, and 3-20 hours in dimethylformamide.
Compound~ of the invention may be prepared using solid phase peptide synthesis, such as that described by Merrifield, J. Am. Chem. Soc.. 85, 2149 (1964), although other equivalent chemical syntheses known in the art can also be used, such as the syntheses of Houghten, ~Q. Natl. Acad. Sci., 82, 5132 (1985). Solid-pha~e synthesis is commenced from the C-terminus of the peptide by coupling a protected amino acid to a suitable resin, as generally set forth in ~.S. Patent No. 4,244,946, issued Jan. 21, 1982 to Rivier et al., the disclosure of which is hereby incorporated by reference. Solution method can be used aæ described by Neurath et al. Chapter 2, pp. 106-~53. Examples of synthesis of this general type are set forth in U.S. Patent Nos. 4,305,872 and 4,316,891.
In ~ynthesizing these polypeptides, the carboxyl terminal amino acid, having itæ alpha-amino group suitably protected, i6 covalently coupled to a chloromethylated polystyrene resin or the like. The chloromethylated polystyrene resin is composed of fine beads (20-70 microns in diameter) of a synthetic resin prepared by copolymerization of styrene with 1 to 2 percent divinylbenzene. The benzene rings in the resin are chloromethylated in a Fried~l-Crafts reaction with chloromethyl methyl ether and 6tanni~
chloride. The Friedel-Crafts reaction is continued until the resin contains 0.5 to 5 mmoles o~ chlorine per gram of ~e8in. After removal of the alpha amino 5 protecting group, as by using trifluoroacetic acid in methylene chloride, the amino protected derivative of the next amino acid in the sequence i8 added along with a condensation coupling agent such as dicyclohexylcarbodiimide. The remaining alpha-amino and ~ide-chain-protected amino acids are then coupled by condensation stepwi~e in the desired order to obtain an intermediate compound connected to the resin.
The condensation between two amino acids, or an amino acid and a peptide, or a peptide and a peptide can be carried out according to the usual condensation methods 6uch as azide method, mixed acid anhydride method, DCC (dicyclohexyl-carbodiimide) method, BOP (benzotriazole-l-yloxytris (dimethylamino) phosphonium hexafluorophosphate method, active ester method (p~nitrophenyl ester method, N-hydroxy-succinimido ester method, cyanomethyl ester method, etc.), Woodward reagent K method, carbonyldiimidazol method, oxidation-reduction method. In the case of elongating the peptide chain in the ~olid phase method, the peptide is attached to an insoluble carrier at the C-termina~ amino acid. For insoluble carrier~, tho~e which react with the carboxy group of the C-terminal amino acid to form a bond which is readily cleaved later, for example, halomethyl resin such as chloromethyl resin and bromomethyl resin, ll/~P6 - 23 - 18016 hydroxymethyl resin, aminomethyl resin, b~nzhydryl-amine re~in, t-al~yloxycarbonylhydrazide resin an p-hydroxymethylphenylacetylamidomethyl resin(PAM).
Common to chemical syn~hese~ of peptides is the protection of the reactive side-chain groups of the various amino acid moieties with suitable protecting groups at that ~ite until the group i8 ultimately removed after the chain has been completely a~sembled. Also common i~ the protection of the alpha-amino group on a amino acid or a fragment while that entity reacts at the carboxyl group followed by the ~elective removal of the alpha-amino-protecting group to allow ~ubsequent reaction to take place at that location.
Accordingly, it is common that, as a step in the æynthesiæ, an intermediate compound i3 produced which includes each of the amino acid reæidues located in the desired sequence in the peptide chain with variouæ of theæe residueæ having æide-chain protecting groups. These protecting groups are then commonly removed substantially at the æame time so as to produce the deæired resultant product following purification.
The applicable protective groupæ for protecting the alpha-and omega-æide chain amino 2s group~ are exemplified ~uch aæ benzylo~ycarbonyl (hereinafter abbreviated as Z), isonicotinyloxy-carbonyl (iNOC), O-chlorobenzyloxycarbonyl ~Z(2-Cl)], p-nitrobenzyloxycarbonyl ~Z(N02)~,p-methoxybenzyl-oxycarbonyl ~Z(OMe)],t-butoxycarbonyl (Boc), t-amyloxycarbonyl (Aoc), isobornyloxycarbonyl, adamantyloxycarbonyl, 2-(4-biphenyl)-2- propyloxy-carbonyl (Bpoc),9-fluorenylmethoxycarbonyl (Fmoc), ll/RP6 - 24 - 1801~
methylsulfonylethoxycarbonyl (Msc), triflu~roacetyl, phthalyl, formyl, 2-nitrophenylæulphenyl (NPS), diphenylphosphinothioyl (Ppt), dimethylphos-phinothioyl (Mpt) and the like.
Protective groups for carboxy group include, for example, benzyl ester (OBzl), cyclohexyl ester (Chx) 4-nitrobenzyl ester (ONb), t-butyl ester (OBut), 4-pyridylmethyl ester (OPic), and the like.
It is desirable that specific amino acids such a~
arginine, cysteine, and serine possesing a functional group other than amino and carboxyl groups are protected by a suitable protective group as occasion demands. For example, the guanidino group in arginine may be protected with nitro, p-toluene-sulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzenesulfonyl, 4-methoxy-2, 6-dimethyl-benzenesulfonyl (Mds), 1,3,5-trimethylphenylsulfonyl (Mts~, and the like. The thiol group in cysteine may be protected with benzyl, p-methoxybenzyl~
triphenylmethyl, acetylamidomethyl, ethylcarbamoyl, 4-methylbenzyl, 2,4,6-trimethylbenzyl (Tmb) etc., and the hydroxyl group in serine can be protected with benzyl, t-butyl, acetyl, tetrahydropyranyl etc.
Stewart and Young, "Solid Phase Peptide Synthesis:, Pierce Chemical Company, Roc~ford, IL
2s (1984) provides detailed information regarding procedures for preparing peptides. Protection of a-amino groups is described on pages 14-18, and side-chain blockage is described on pages 18-28. A
table of protecting groups for amine, hydroxyl and sulfhydryl functions i~ provided on pages 149-151.
These descriptions are hereby incorporated by reference.
ll/RP6 - ~5 - 18016 After the desired amino-acid seq~ence has been completed, the intermediate peptide is removed from the resin support by treatment with a reagent, such as liquid HF, which not only cleaves the peptide from the resin, but also cleaves all the remaining protecting groups from the side chain which do not interfere in the cyclization reaction. Potentially reactive Ride chains functionalities are protected with blocking groups which are stable to HF. The peptides are cyclized by any one of several kno~n procedures (see Schroder and Lubke, "The Peptides:
Methods of Peptide Synthesis" Vol. I, Academic Press, New York (1965), pp. 271-286, the contents of which are hereby incorporated by reference), e.g. ~y forming a disulfide bridge between the cysteine re~idues using iodine in AcO~, or air sxidation at pH
8 in dilute NH4 OAc buffer. The polypeptide can then be purified by gel permeation chromatography followed by preparative HPLC, as described in Rivier et al., Peptides: Structure and Biological Function (1979) pp. 125-128.
EXAMPL~ 1 Synthesis of aminomethylphenylalanine a~ a-BOC-Cbz-p-aminomethvl-D.L-phenvlalanine ~or use in synthesi~ of Ac-Cys~Pmb)-Asn-Pro-(D.L-AMF(Cbz~)-1y-As~(Bzl~-Cys-(Pmb)-opam(~) ~'l3~
ll/RP6 - 26 - 18016 NaOEt (CO2 Et) 2 r+ AcNH-cHyco2Et)2 ~ CN
To 500 ml dry EtOH (4A sieves) was added 5.9g Na (0.256m) under nitrogen, 55.67g (0.2563m) of acetamido diethylmalonate and 50 g (0.2563m) of p-cyanobenzylbromide. The mixture was heated to reflux which resulted in complete dissolution of starting materials and product. After 1 hour, the reaction ~olution waæ cooled, 1.5 liters of water was added and the precipitate was filtered to give 77.3g of crude product which was recrystallized from 450 ml of EtOH to give 70.56g of product (83% yield), mp 174-5 175~5 C; IBCHC13 ~97~ ~NH), 5.78 (ester) 6.0 (amide) 4.52~ (CN) Rf (95-5-0.5 - C~C13-MeO~-H20) = 0.75 PP~
NMR
CDC13: 1.3 (t, Ç~3CH20), 2.05 (s, ~3C), 3.75 (s, ar-~2-C), 4.3 (m, CH3~2~0) 6.5 (s, N~), 7.2 (d, arom), 7.6 (d, arom) ~.J~ ~
ll/RP6 - 27 - 18016 ~,~cO,Et);~ H2_Pd/c ~,N--~(CO,Et), N~ EtO~1~50% }~AC NH1~c To a suspen6ion of 20g (60mm) of p-CN-benzyl N-acetyl-diethylmalonate in 200 ml EtOH - 50% HOAc (8:2) under a N2 gtream wa~ added 4g of 10% Pd/C and the mixture wa~ treated with ~2 in a Parr Shaker for 70 l~ minutes after which period 96% of theoretical amount of ~2 was conæumed. The mixture was filtered through Celite, the filtrate was evaporated ~n vacuo to drynes~
~o give a solid residue which was triturated with EtOAc, filtered and dried to give 21.45g ~90.2% yield) of product.
1~ in CHC13 show~ no CN at 4.5 ~.
(95-5-0.5 CHC13-MeOH-conc.NH40H) = 0.3 (ninhydrin +) ppm ~MB C~2C @3.65 singlet, ~N @3.9 singlet ~l2N~CO~Et)~ 6N HCl ~N~COOH
A ~olution of 21g (53 mm) of N-acetyl diethyl ester p-aminomethylbenzyl aminomalonate in 100 ml of 6N ~Cl was refluxed for 24 hrs. The reaction solution was evaporated ~n vacuo to give 16.6g of product aæ ~olid.
Rf (60-30-4-6, CHC13-MeO$-$2O-N$4OH) = 0.15 ~o OOH CuCl~. 2H~O CbZ~ COOH
G~N--J~o~ N~, To all of p-aminomethylphenylalanine (53 mm) (prepared above) in 200 ml H2O was added 4.92g of CuC12.2~2O. The mixture waæ adjusted to pH of 9 with NaOH. To the reaction mixture waæ added 18.26g 2~ 3~
ll/RP6 - 29 - 1~016 (58 mm) of the N-benzyloxycarbonylogy-t-nol~bornene 2,3-dicarbo~imide reagent and the reaction mixture waæ kept at 5C for 18 hr~. The ~olid was filtered, washed with ~2 and EtOAc, and redi~solved in HOAc and ~Cl to o~tain a pH of 0.5. ~pon ~tanding, 9.5g of product as the zwitterion precipitated. The filtrate wae treated with ~2S, fil~ered through a pad of celite and pyridine was added to the filtrate to pH 6. The flocculent precipitate was filtered to give a second crop of product (1.5g). Total yield was llg (58% yield). Anal. calcd. for C18H20 N2 4 calcd. fd N = 8.53 7.99 C ~65.84 66.65 H = 6.14 6.13 NMR in D20 and NaOD evidenced product to have the Cbz group on the NH2CH2 and not the a-NH2, CbZNH--~OOH ~ ~30C-ON--CbzN~ OOH
E~oc A suspension of 7.0g (21.3mm) of omega-Cbz -p-aminomethyl-DL-phenylalanine in 70 ml H20 and 35 ml of T~F was treated with 9.27 ml (63.9mm) of NEt3 and 5.51 g (22.36mm) of Boc-ON (Aldrich) for 24 hrs during which time all ætarting material went into solution. To the reaction solution was added lSO ml 3 ~ ~
ll/RP6 - 30 - 18016 of ethyl ether, the H20 layer was separat~ and the ether layer was washed two times with H20; the combined H20 layers were back-washed once with ether and acidified with citric acid to give a gummy solid. The aqueou~ supernatant wa~ decanted, the gummy solid was extracted into EtOAc, the EtOAc solution was dried over MgS04, filtered and evaporated to a foamy residue (8.73g). The crude product was crystallized from EtOAc-pet Et20 to give 7.22g (79.3% yield), m.p. 133-133.5C.
TLC Rf = 0.35 (80-20-2, CHC13-MeO~-N~40H) NMR CD30D : 1.4(Boc), 2.9, 3.15 (m, ~ 2)~
4.25(8, C~2N), 4.3 (m, a-H), 5.1(s,C~2-Cbæ) 7.2 ,7.3(arom, Cbz, ~ ) ~XAMPL~L.2 Svntheæis of Ac-Cys(Pmb)-Asn-Pro-rD.L-AMF~Cbz~l-Gly-Asp(Bzl)-Cys~Pmb)-OPam ~
.~nd ultimately Ac-Cys-Asn-Pro-~D.L-AMF)-Gly-Asp-Cys-O~
PM~3 Starting with Boc-Cys-O-Pam-resin, the alpha-amino Boc protecting group (tert-butylcarbonyl~
ll/RP6 - 31 - 18016 is removed (while the Cy6 æide-chain remains protected by p-methylbenzyl) using trifluoroacetic acid and methylene chloride, and the a-deprotected cysteine neutralized with diisopropylethyl amine.
Boc-protected A8p (benzyl) (Asp (Bzl)) is then coupled to cysteine mediated by dicyclohexyl-carbodiimide, and deprotected with trifluoroacetic acid and methylene chloride. Asp is then neutralized with diisopropylethylamine. Following thi 8 stepwise procedure of coupling with dicyclohe~ylcarbodiimide, lo deprotection with trifluoroacetic acid and methylene chloride, and neutralization with diisopropyl-ethylamine, Boc-protected Gly, AMF, Pro, Asn, Cys residues are coupled in succession. AME is--additionally protected by Cbz, (AMF (Cbz)), and the final Cys residue ls again additionally protected by p-methylbenzyl. The f inal Cys is then acetylated with acetic anhydride.
Following acetylation, the following peptide-resin iæ ~ormed:
PMB Cbz Bzl PMB
Acetyl-Cy6-Asn-Pro-(D,L-AMF)-Gly-Asp-Cys-0-Pam ~
Cleavage of the peptide from the reein i8 achieved using ~F/anisole (9:1 (v/v)) to form:
H
Acetyl-Cy~-Asn-Pro-(D,L-AMF)-Gly-Asp-Cys-0~.
A cyclic 6tructure is formed by formation of a disul~ide bridge between the cysteine residues.
The peptide i8 dissolved in 50-80% Ac0~:~20 at room temperature, and the ~olution stirred during rapid ll/RP6 - 32 - 18016 addition of a solution of iodine in AcOH ~o a final concentration of 2.25 mg/ml of iodine. After 1-2 hours reaction time, excess I2 and AcO~ are removed by rotary evaporation under vacuum and the aqueous solution containing the cyclized pep~ide is purified using preparative HPLC in 0.1% TFA H20-C~3CN gradient at which stage the D- and L- diastereomers are separated by conventional means. The final TFA salt product iB converted to ~OAc salt by passing through an ion exchange column BioRad AG3-~4A (acetate cycle). The fini6hed peptide i8:
\
Acetyl-Cys-Asn-Pro-(k-AMF)-Gly-Asp-Cys-O~.
As an alternative to forming the disulfide bridge by iodine oxidation, the free SH peptide is dis~olved in 1-5% HOAc at a concentration of approximately 2 mg/ml and the solution adjusted to appro~imately pH 7-8.5 with concentrated NH40H. Cyclization is accomplished under brisk stirsing (preferably with a small piece of copper wire added to accelerate the reaction) during a period of 1-4 hours at 25. The reaction mixture is then concentrated as before and product purified by preparative HPLC.
Synthesis of Ac-Cys-Asn-(Di~Tzl~-~L-AMF~-Gly-Asp-Cys-Q~
.
ll/RP6 - 33 - 18016 The same procedure for synthesizi~g the cyclic peptide of Example 2 is followed, except that Pro is replaced with DiMeTzl.
Thera~eutic Utility Compounds of the invention may be administered to patients where prevention of thrombosis by inhibiting binding of fibrinogen to the platelet membrane glycoprotein complex IIb/IIIa receptor is desired. They are useful in surgery on peripheral arteries (arterial grafts, carotid endarterectomy) and in cardiovascular surgery where manipulation of arteries and organs, and/or the interaction of platelets with artificial su~faces, leads to platelet aggregation and consumption. The lS aggregated platelets may form thrombi and thromboemboli. Polypeptides of the invention may be administered to these surgical patients to prevent the formation of thrombi and thromboemboli.
Extracorporeal circulation is routinely used for cardiovascular ~urgery in order to oxygenate blood. Platelets adhere to surfaces of the extracorporeal circuit. Adhesion is dependent on the interaction between GPIIb/IIIa on the platelet membranes and fibrino~en adsorbed to the surface of the circuit. (Gluszko et al., Amer. J. Physiol 1987l 252:H, pp 615-621). Platelets released from artificial surfaces show impaired hemostatic function. Polypeptides of the invention may be administered to prevent adhesion.
Other applications of these polypeptides include prevention of platelet thrombosis, ll/RP6 - 34 - 18016 thromboembolism and reocclusion during an~;after thrombolytic therapy and prevention of platelet thrombosis, thromboembolism and reocclusion after angioplasty of coronary and other arteries and after coronary artery bypass procedures. Polypeptides of the invention may al60 be u~ed to prevent myocardial infarction.
These polypeptides may be administered by any convenient means which will result in its delivery into the blood ~tream in substantial amount including continuous intravenous or bolus injection or oral methods. Compositions of the invention include peptides of the invention and pharmacologically acceptable carriers, e.g. ~aline, at a p~ level e.g. 7.4, suitable for achieving inhibition of platelet aggregation. They may be combined with thrombolytic agents such as plasminogen activatoræ or streptokinase in order to inhibit platelet aggregation. They may also be combined with anticoagulants ~uch as heparin, aspirin or warfarin.
Intravenou~ administration is presently contemplated as the preferred administration route. They are soluble in water, and may therefore be effectively administered in ~olution.
In one exemplary application, a suitable amount of peptide is intravenously admini~tered to a heart attack victim undergoing angioplasty.
Administrat~on occurs during or several minutes prior to angioplasty, and is in an amount sufficient to inhibit platelet aggregation, e.g. an amount which achieves a steady state plasma concentration of between about 0.05-30 ~M per kilo, preferably between ~ ~w~
ll/RP6 - 35 - 1~016 about 0.3-3 ~M per kilo. When thiæ amoun~ is achieved, an infusion of between about 1-100 ~M per kilo per min., preferably between about 10-30 ~M per kilo per min. i~ maintained to inhibit platelet aggregation.. Should the patient need to undergo bypass surgery, administration may be stopped immediately and will not cause complicatione during surgery that would be caused by other materials such as aspirin or monoclonal antibodies, the effects of which last hours after cessation of administration.
The present invention also includes a pharmaceutical composition comprising peptideæ of the present invention and tissue type plasminogen activator or streptokinase. The invention al~o includes a method for promoting thrombolysis and preventing reocclusion is a patient which compri~es administering to the patient an effective amount of compositions of the invention.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. Thus, the specific examples described above should not be interpreted as limiting the scope of the present invention.
Claims (9)
1. A fibrinogen receptor antagonist which comprises the sequence XX-Gly-Asp wherein XX represents a synthetic alpha-amino acid having a side-chain, X, containing a phenyl group or C3-C8 cycloalkyl group.
2. A fibrinogen receptor antagonist of the formula:
wherein XX represents a synthetic alpha-amino acid having a side chain, X, containing a phenyl or cyclohexyl group, and ZZ represent a sequence of 1, 2, 3 or 4 substituted or unsubstituted amino acids.
wherein XX represents a synthetic alpha-amino acid having a side chain, X, containing a phenyl or cyclohexyl group, and ZZ represent a sequence of 1, 2, 3 or 4 substituted or unsubstituted amino acids.
3. A fibrinogen receptor antagonist of the formula:
wherein B represents zero, one or two substituted or unsubstituted amino acids; Q represents H,NH,NH2, or Ac-NH; X represents an amino acid side chain containing a phenyl or C3-C8 cycloalkyl group; I' is a side chain of an L-amino acid, and E' is H, COOH, CONH2, CONHR2, CONR3R4, CH2OH, CO2R2, CH3 wherein R2 is an alkyl group having 1 to 4 carbon atoms, R3R4 is an alkyl group having 1 to 4 carbon atoms or NR3R4 is a secondary amino acid, or ;
provided that when B is zero substituted or unsubstituted amino acidæ, then Q is H,NH2 or Ac-NH, and that when B is one or two substituted or unsubstituted amino acids, then Q is NH.
wherein B represents zero, one or two substituted or unsubstituted amino acids; Q represents H,NH,NH2, or Ac-NH; X represents an amino acid side chain containing a phenyl or C3-C8 cycloalkyl group; I' is a side chain of an L-amino acid, and E' is H, COOH, CONH2, CONHR2, CONR3R4, CH2OH, CO2R2, CH3 wherein R2 is an alkyl group having 1 to 4 carbon atoms, R3R4 is an alkyl group having 1 to 4 carbon atoms or NR3R4 is a secondary amino acid, or ;
provided that when B is zero substituted or unsubstituted amino acidæ, then Q is H,NH2 or Ac-NH, and that when B is one or two substituted or unsubstituted amino acids, then Q is NH.
4. A compound of claim 1, claim 2, or claim 3 wherein X is defined as (i) or -(CH2)n-AA-(CH2)n'-NHR (ii) ll/RP6 - 38 - 18016 wherein: n iæ 0,1,2,3 or 4;
n' is 0,1,2,3 or 4;
AA is disubstituted phenyl, either not substituted with additional groups or substituted with C1-4 alkyl, alkoxy or hydroxy; C3-C8 cycloalkyl, either not substituted with additional groups or substituted with C1-4 alkyl, alkoxy or hydroxy; and R is H, C1-6 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylmethyl, or substituted or unsubstituted cycloalkyl.
n' is 0,1,2,3 or 4;
AA is disubstituted phenyl, either not substituted with additional groups or substituted with C1-4 alkyl, alkoxy or hydroxy; C3-C8 cycloalkyl, either not substituted with additional groups or substituted with C1-4 alkyl, alkoxy or hydroxy; and R is H, C1-6 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylmethyl, or substituted or unsubstituted cycloalkyl.
5. A compound of claim 2 wherein ZZ is 1, 2, 3 or 4 amino acids according to formulas I, II, III, IV or V:
(I) (II) (D) (III) (IV) (V) wherein A' is H, acylamido, acylaminoacylamido, acylamino-N-methylaminoacylamido;
R' and R'1 are independently H, methyl, ethyl or a iower alkyl group having 1 to 5 carbons;
X'-Y' is S-S, CH2-S, S-CH2, CH2CH2, CH2,CH2CH2CH2- CH2-S-S- CH2-S-S-CH2- S-S-CH2; and E' is H, COOH, CONH2, CONHR2, CONR3R4, CH2OH,CO2R2,CH3 wherein R2 is an alkyl group having 1 to 4 carbon atoms, R3R4 is an alkyl group having 1 to 4 carbon atoms or NR3R4 is a secondary amino acid.
or B' is a D- or L- a-amino acid;
C' is a D- or L- secondary .alpha.-amino acid or a D- or L- primary .alpha.-amino acid; --F' is an L- amino acid ;
G' is a D- or L- .alpha.-amino acid, secondary cyclic amino acid, or N-methyl amino acid; and R5 is H or methyl.
(I) (II) (D) (III) (IV) (V) wherein A' is H, acylamido, acylaminoacylamido, acylamino-N-methylaminoacylamido;
R' and R'1 are independently H, methyl, ethyl or a iower alkyl group having 1 to 5 carbons;
X'-Y' is S-S, CH2-S, S-CH2, CH2CH2, CH2,CH2CH2CH2- CH2-S-S- CH2-S-S-CH2- S-S-CH2; and E' is H, COOH, CONH2, CONHR2, CONR3R4, CH2OH,CO2R2,CH3 wherein R2 is an alkyl group having 1 to 4 carbon atoms, R3R4 is an alkyl group having 1 to 4 carbon atoms or NR3R4 is a secondary amino acid.
or B' is a D- or L- a-amino acid;
C' is a D- or L- secondary .alpha.-amino acid or a D- or L- primary .alpha.-amino acid; --F' is an L- amino acid ;
G' is a D- or L- .alpha.-amino acid, secondary cyclic amino acid, or N-methyl amino acid; and R5 is H or methyl.
6. A compound of claim 5 wherein:
C' is selected from the group consisting of proline, .beta.-methylproline, .beta.,.beta.- dimethylproline, .gamma.-hydroxyproline, anhydroproline, thioproline, .beta.-methylthioproline, .beta.,.beta. - dimethylthioproline, pipecolic acid, azetidine carboxylic acid and an N-methyl amino acid; and F' is selected from the group consisting of tryptophan, phenylalanine, leucine, valine, isoleucine, alpha-naphthylalanine, .beta.-naphthylalanine, methionine, tyrosine, arginine, lysine, homoarginine, ornithine, histidine, substituted tryptophan, substituted phenylalanine or substituted tyrosine.
C' is selected from the group consisting of proline, .beta.-methylproline, .beta.,.beta.- dimethylproline, .gamma.-hydroxyproline, anhydroproline, thioproline, .beta.-methylthioproline, .beta.,.beta. - dimethylthioproline, pipecolic acid, azetidine carboxylic acid and an N-methyl amino acid; and F' is selected from the group consisting of tryptophan, phenylalanine, leucine, valine, isoleucine, alpha-naphthylalanine, .beta.-naphthylalanine, methionine, tyrosine, arginine, lysine, homoarginine, ornithine, histidine, substituted tryptophan, substituted phenylalanine or substituted tyrosine.
7. A compound of claim 4 whlch is
8. A compound of claim 4 which is
9. A composition for inhibiting fibrinogen-dependent platelet aggregation in a mammal comprising a peptide of claim 1 and a pharmaceutically acceptable carrier.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US42104989A | 1989-10-13 | 1989-10-13 | |
| US421,049 | 1989-10-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2027314A1 true CA2027314A1 (en) | 1991-04-14 |
Family
ID=23668978
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002027314A Abandoned CA2027314A1 (en) | 1989-10-13 | 1990-10-11 | Fibrinogen receptor antagonists |
Country Status (17)
| Country | Link |
|---|---|
| US (1) | US5374622A (en) |
| EP (1) | EP0422937B1 (en) |
| JP (1) | JP2559289B2 (en) |
| KR (1) | KR910007958A (en) |
| AT (1) | ATE102219T1 (en) |
| AU (1) | AU644380B2 (en) |
| CA (1) | CA2027314A1 (en) |
| DE (1) | DE69007004T2 (en) |
| DK (1) | DK0422937T3 (en) |
| ES (1) | ES2062400T3 (en) |
| FI (1) | FI905023A0 (en) |
| IE (1) | IE66253B1 (en) |
| IL (1) | IL95918A0 (en) |
| NO (1) | NO904430L (en) |
| NZ (1) | NZ235564A (en) |
| PT (1) | PT95581A (en) |
| ZA (1) | ZA908159B (en) |
Families Citing this family (31)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5851839A (en) * | 1989-06-16 | 1998-12-22 | Cor Therapeutics, Inc. | Platelet aggregation inhibitors |
| US5686568A (en) * | 1989-06-16 | 1997-11-11 | Cor Therapeutics, Inc. | Platelet aggregration inhibitors |
| US5643872A (en) * | 1989-10-23 | 1997-07-01 | Smithkline Beecham Corporation | Cyclic anti-aggregatory peptides |
| DK0570493T3 (en) * | 1991-02-08 | 2000-06-26 | Diatide Inc | Technetium-99m-labeled polypeptides for imaging. |
| US6019958A (en) * | 1991-02-08 | 2000-02-01 | Diatide, Inc. | Technetium-99m labeled peptides for imaging inflammation |
| US5830856A (en) * | 1991-02-08 | 1998-11-03 | Diatide, Inc. | Radiolabeled compounds for thrombus imaging |
| US5736122A (en) * | 1991-02-08 | 1998-04-07 | Diatide, Inc. | Technetium-99m labeled peptides for thrombus imaging |
| US5561220A (en) * | 1991-02-08 | 1996-10-01 | Diatech, Inc. | Technetium-99m labeled peptides for imaging inflammation |
| US6107459A (en) * | 1991-02-08 | 2000-08-22 | Diatide, Inc. | Technetium-99m labeled peptides for diagnostic imaging |
| US5645815A (en) * | 1991-02-08 | 1997-07-08 | Diatide, Inc. | Radiolabled compounds for thrombus imaging |
| IL103252A (en) | 1991-09-30 | 1997-03-18 | Du Pont Merck Pharma | CYCLIC COMPOUNDS USEFUL AS INHIBITORS OF PLATELET GLYCOPROTEIN IIb/IIIa AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM |
| US5635477A (en) * | 1991-09-30 | 1997-06-03 | The Dupont Merck Pharmaceutical Company | Cyclic compounds useful as inhibitors of platelet glycoprotein IIB/IIIA |
| US5674863A (en) * | 1991-10-18 | 1997-10-07 | Genentech, Inc. | Nonpeptidyl integrin inhibitors having specificity for the GPIIb IIIa receptor |
| US5250679A (en) * | 1991-10-18 | 1993-10-05 | Genentech, Inc. | Nonpeptidyl platelet aggregation inhibitors having specificity for the GPIIb III.sub. receptor |
| WO1993008818A1 (en) * | 1991-11-07 | 1993-05-13 | The University Of Southern California | Compositions and methods for preventing adhesion formation |
| US5968476A (en) * | 1992-05-21 | 1999-10-19 | Diatide, Inc. | Technetium-99m labeled peptides for thrombus imaging |
| US5338725A (en) * | 1992-06-30 | 1994-08-16 | The Research Foundation Of The State University Of New York | Anti-aggregatory agents for platelets |
| JPH08502261A (en) * | 1992-10-02 | 1996-03-12 | ダイアテク，インコーポレイテッド | Multivalent antithrombotic agent |
| EP0672059A1 (en) * | 1992-11-18 | 1995-09-20 | The Du Pont Merck Pharmaceutical Company | CYCLIC COMPOUNDS LINKED BY A HETEROCYCLIC RING USEFUL AS INHIBITORS OF PLATELET GLYCOPROTEIN IIb/IIIa |
| NZ263455A (en) * | 1993-03-29 | 1997-10-24 | Du Pont Merck Pharma | Synthesis of platelet glycoprotein iib/iiia inhibitors |
| SG49788A1 (en) * | 1993-04-08 | 1998-06-15 | Diatide Inc | Radiolabeled compounds for thrombus imaging |
| US5646000A (en) * | 1994-09-29 | 1997-07-08 | Merck & Co., Inc. | Compounds and methods for identifying activated platelets |
| US5858972A (en) * | 1996-01-11 | 1999-01-12 | La Jolla Cancer Research Foundation | Antithrombotic agents and methods of use |
| US6623981B2 (en) * | 1998-01-27 | 2003-09-23 | Bristol-Myers Squibb Company | Detection of patients at risk for developing integrin antagonist/agonist mediated disease states |
| ID26082A (en) | 1998-03-19 | 2000-11-23 | Briston Myers Squib Company | DWIFASA CONTROLLED REMOVAL DELIVERY SYSTEM FOR HIGH SOLUTION PHARMACY SUBSTANCES AND ITS METHODS |
| TR200100139T2 (en) | 1998-04-16 | 2001-06-21 | Texas Biotechnology Corporation | N, N-two substituted amides that inhibit the binding of integrins to their receptors |
| US6808698B1 (en) | 1999-03-26 | 2004-10-26 | Bristol-Myers Squibb Pharma Company | Method for localization of blood clots |
| US6685914B1 (en) | 1999-09-13 | 2004-02-03 | Bristol-Myers Squibb Pharma Company | Macrocyclic chelants for metallopharmaceuticals |
| US7317104B2 (en) | 2003-06-13 | 2008-01-08 | Bristol-Myers Squibb Pharma Company | Chelants and macrocyclic metal complex radiopharmaceuticals thereof |
| US7319149B2 (en) | 2003-06-13 | 2008-01-15 | Bristol-Myers Squibb Pharma Company | Chelants and macrocyclic metal complex radiopharmaceuticals thereof |
| RU2016125229A (en) | 2010-07-09 | 2018-12-04 | БиЭйчВи ФАРМА, ИНК. | COMBINED DELIVERY SYSTEM WITH IMMEDIATE / SLOW-DELIVERY FOR MEDICINES WITH A SHORT HALF-HOUR, INCLUDING REMOGLYPHLOSIN |
Family Cites Families (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5921656A (en) * | 1982-07-28 | 1984-02-03 | Takeda Chem Ind Ltd | Polypeptide derivative and preparation thereof |
| GB8305985D0 (en) * | 1983-03-04 | 1983-04-07 | Szelke M | Enzyme inhibition |
| US4988621A (en) * | 1985-05-24 | 1991-01-29 | La Jolla Cancer Research Foundation | Peptides in cell detachment and aggregation |
| CA1311583C (en) * | 1986-12-15 | 1992-12-15 | Gerard Armand Marguerie De Rotrou | Peptide derivatives and their application, in particular in therapy |
| US4857508A (en) * | 1987-12-03 | 1989-08-15 | Monsanto Company | Novel platelet-aggregation inhibitor peptide derivatives |
| EP0394326B1 (en) * | 1987-12-10 | 1996-07-31 | La Jolla Cancer Research Foundation | Methods for the production of conformationally stabilized cell adhesion peptides |
| ZW6189A1 (en) * | 1988-05-09 | 1990-05-09 | Smithkline Beckman Corp | Anti-aggregatory peptides |
| JP2945680B2 (en) * | 1988-09-09 | 1999-09-06 | 旭硝子株式会社 | Peptide derivatives and their uses |
| CA2008116C (en) * | 1989-02-23 | 2001-11-20 | Thomas Weller | Glycine derivatives |
| US4952562A (en) * | 1989-09-29 | 1990-08-28 | Rorer Pharmaceutical Corporation | Anti-thrombotic peptides and pseudopeptides |
| NZ235563A (en) * | 1989-10-13 | 1993-04-28 | Merck & Co Inc | Fibrinogen receptor antagonist and pharmaceutical composition |
| CA2027936C (en) * | 1989-10-23 | 2001-07-24 | Smithkline Beecham Corporation | Cyclic anti-aggregatory peptides |
-
1990
- 1990-10-04 NZ NZ235564A patent/NZ235564A/en unknown
- 1990-10-08 IL IL95918A patent/IL95918A0/en unknown
- 1990-10-10 IE IE362990A patent/IE66253B1/en not_active IP Right Cessation
- 1990-10-11 AT AT90311148T patent/ATE102219T1/en not_active IP Right Cessation
- 1990-10-11 ES ES90311148T patent/ES2062400T3/en not_active Expired - Lifetime
- 1990-10-11 EP EP90311148A patent/EP0422937B1/en not_active Expired - Lifetime
- 1990-10-11 DK DK90311148.2T patent/DK0422937T3/en active
- 1990-10-11 CA CA002027314A patent/CA2027314A1/en not_active Abandoned
- 1990-10-11 DE DE69007004T patent/DE69007004T2/en not_active Expired - Fee Related
- 1990-10-12 JP JP2272498A patent/JP2559289B2/en not_active Expired - Fee Related
- 1990-10-12 NO NO90904430A patent/NO904430L/en unknown
- 1990-10-12 AU AU64568/90A patent/AU644380B2/en not_active Ceased
- 1990-10-12 FI FI905023A patent/FI905023A0/en not_active Application Discontinuation
- 1990-10-12 KR KR1019900016166A patent/KR910007958A/en not_active Application Discontinuation
- 1990-10-12 PT PT95581A patent/PT95581A/en not_active Application Discontinuation
- 1990-10-12 ZA ZA908159A patent/ZA908159B/en unknown
-
1993
- 1993-01-28 US US08/010,234 patent/US5374622A/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| IL95918A0 (en) | 1991-07-18 |
| ES2062400T3 (en) | 1994-12-16 |
| NO904430D0 (en) | 1990-10-12 |
| EP0422937B1 (en) | 1994-03-02 |
| DE69007004D1 (en) | 1994-04-07 |
| DE69007004T2 (en) | 1994-10-06 |
| PT95581A (en) | 1991-09-13 |
| JPH03206097A (en) | 1991-09-09 |
| IE66253B1 (en) | 1995-12-27 |
| US5374622A (en) | 1994-12-20 |
| ATE102219T1 (en) | 1994-03-15 |
| AU6456890A (en) | 1991-04-18 |
| NZ235564A (en) | 1993-10-26 |
| ZA908159B (en) | 1991-07-31 |
| EP0422937A1 (en) | 1991-04-17 |
| NO904430L (en) | 1991-04-15 |
| FI905023A0 (en) | 1990-10-12 |
| IE903629A1 (en) | 1991-04-24 |
| JP2559289B2 (en) | 1996-12-04 |
| DK0422937T3 (en) | 1994-03-28 |
| AU644380B2 (en) | 1993-12-09 |
| KR910007958A (en) | 1991-05-30 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |