AU633128B2 - Anticoagulant peptides - Google Patents

Description

1 3 128 AUSTRALIA Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: o 0 e., Complete Specification Lodged: Accepted: Published: Priority Related Art: APPLICANT'S REF.: Patent of Addition of 10729/88 Name(s) of Applicant(s): S* MERRELL DOW PHARMACEUTICALS INC Address(es) of Applicant(s): 2110 East Galbraith Road Cincinnati, Ohio UNITED STATES OF AMERICA Actual Inventor(s): John L KRSTENANSKY and Simon J T MAO Address for Service is: PHILLP'S, ORMONDE AND FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne, Australia, 3000 Complete Specification for the invention entitled: ANTICOAGULANT PEPTIDES The following statement is a full description of this invention, including the best method of performing it known to applicant(s): P19/3/84 -2- ANTICOAGULANT PEPTIDES FIELD OF THE INVENTION The present application is a Patent of Addition to Australian Patent Application No 10729/88, the entire disclosure of which is incorporated herein by reference.

This invention relates to novel peptides which are useful anticoagulant agents.

BACKGROUND OF INVENTION o o 0 Anticoagulants are useful therapeutic agents in the pharmacological treatment of, for example, acute deep venous thrombosis, pulmonary embolism, acute arterial embolization of the extremities, myocardial infarction, and disseminated S intravascular coagulation. Proplylactic administration of anticoagulants is believed to prevent a recurrance of embolism in patients with rheumatic or arteriosclerotic heart disease and to prevent certain thromboembolic complications of surgery. Administration of anticoagulants has also been indicated in the treatment of coronary artery and cerebrovascular disease. Artrial thrombosis, particularly in arteries supplying the heart muscle and brain, is a leading cause of death.

Hirudin is a 65 residue polypeptide isolated from the salivary glands of leeches. It is an anticoagulant agent, which is a thrombin specific inhibitor. Although quite potent, clinical use of hirudin isolated from leech extracts seems unlikely because of its limited quantity, expense and allergic reactions which commonly follow administration of any foreign protein of this size.

In Australian Patent Application No 10729/88, applicants described their discovery of a specific region of hirudin that is responsible, at least in part, for its anticoagulant activity. This region was chemically synthesized and certain of its analogs appeared to bind to the recognition site of thrombin but not the enzymatic cleavage site which is spatially separate. Binding of the synthetic peptides competitively prevents binding of the

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-3fibrinogen to the recognition site of thrombin, a prerequisite to fibrin production and clot formation. The peptides of Australian Patent Application No 10729/88 possessed significant anticoagulant activity and their unusual ability to bind only to the recognition site without binding to the cleavage site of thrombin may allow for a scientifically interesting and therapeutically significant adjunct to anticoagulant therapy.

Australian Patent Application No 10729/88 describes peptide derivatives of the formula X-A -A 2-A 3-A-A -A -A -A -A-A -A -Y 1 2 3 4 5 6 7 8 9 10 wherein X is an amino terminal residue selected from S hydrogen, one or two alkyl groups of from 1 to 6 carbon atoms, one or two acyl groups of from 2 to 10 carbon atoms, carbobenzyloxy or t-butyloxy carbonyl; t I wherein A1 is a bond or is a peptide containing Sfrom 1 to 11 residues of any amino acid; 'wherein A2 is Phe, SubPhe, and 3-thienyl)alanine, B-(2-and 3-furanyl)alanine, and 4-pyridyl)alanine, B-(benzothienyl-2- and 3-yl)alanine, B-(1- Sand 2-naphthyl)alanine, Tyr or Trp; wherein A is Glu or Asp; wherein A 4 is any amino acid; wherein A 5 is Ile, Val, Leu, Nle or Phe wherein A 6 is Pro, Hyp, 3,4-dehydroPro, thiazolidine- 4-carboxylate, Sar, NMePgl or D-Ala; |4 wherein A7 is any amino acid; wherein A 8 is any amino acid; wherein A9 is a lipophilic amino acid selected from Tyr, Tyr(SO 3 Trp, Phe, Leu, Nle, Ile, Val, Cha and Pro or is a dipeptide containing at least one of these lipophilic amino acids; wherein AI0 is a bond or is a peptide fragment containing from one to five residues of any amino acid; and wherein Y is a carboxy terminal residue selected from OH, C1-C 6 alkoxy, amino, mono- or di-(C 1

-C

4 alkyl substituted amino, or benzylamino; are useful anticoagulant agents.

Further peptides which possess significant anticoagulant activity have now been synthesised. A number

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-4of these peptides contain fragments very different from thcse described as preferred in Australian Patent Application No 10729/88.

According to the present invention there is provided a peptide selected from any one of the following: Ac-Thr-Pro-Lys--Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe--Glu -Glu-I le-Pro-Glu-Glu-Tyr-Leu-Gln-OH Ac-Thr-Pro--Asn-Pro-Glu-Ser-His--Asn-Asn-Gly-Asp--Phe-Glu -Glu-I le-Pro-Glu-Glu-Tyr-leu-Gln-OH Suc-Tyr-Glu-Pro-I le-Pro-Glu-Ala-Cha-Asn-OH Suc-Tyr-Glu--Pro-I le-Pro--Glu-Glu-Tyr-Cha-Gln-OH Suc-Tyr-Glu-Pro-I J-e-Pro-Glu-Glu-Pro-Cha-D-Glu-OH o di-l-PoIePoGl-l-r-CaDGuO o o Suc-Tha-Glu-Pro-Ile-Pro-Glu-Glu-ro-Cha-D-Glu-OH Su-ydluPoIei Cl-Gl-l-haDGuO Sucl-Npa-Glu-Pro--Ile-Pro-Glu-Glu--Ala-Cha-D-Glu-OH *000 Suc-Tyr-Glu-Pro-I le-Pro-Glu-Glu-Ala-Cha-D-Glu-OH Fma-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala-Cha-D-Glu-OH DETAILED DESCRIPTION OF THE INVENTION The following common abbreviations of the amino acids are used throughout this specification and the claims: Gly glycine Ala alanine Val valine Leu Leucine ~LC. Ile isoleucine Pro proline Phe phenylalanine 1'Trp tryptophan Met methionine Ser serine Thr threonine Cys cysteine Tyr tyrosine Asn asparagine Gln glutarnine Asp aspartic acid

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11 oo 4 0 o 05a 0 Glu glutamic acid Lys lysine Arg arginine His histidine Nle norleucine Hyp hydroxyproline 3,4-dehydroPro 3,4-dehydroproline Tyr(SO 3 H) tyrosine sulfate Pgl phenylglycine NMePgl N-methyl-phenylglycine Sar sarcocine (N-methylglycine) pSubPhe para substituted phenylalanine SubPhe ortho, meta, or para, mono- or di-substituted phenylalanine DAla D-alanine Ac acetyl Suc succinyl pClePhe para-chloro-phenylalanine pNO 2 Phe para-nitro-phenylalanine Cha cyclohexylalanine Orn ornithine Glt glutaryl Mal maleyl Npa B- (2-naphthvl) alanine _8 -C2 yi en/ja bi%'>rue An' alkyl group and the alkyl portion of an alkoxy group is taken to include straight, branched, or cyclic alkyl groups, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, sec-pentyl, cyclopentyl, hexyl, isohexyl, cyclohexyl and cyclopentylmethyl. An acyl group of from 2 to 10 carbon atoms is taken to include straight, branched, cyclic, saturated and unsaturated acyl groups having 1 or 2 carbonyl moities per group, for example, acetyl, benzoyl, maleyl, glutaryl and succinyl. A halogen group is a fluoro, chloro, bromo or iodo group.

The term "any amino acid" as used herein includes the naturally occurring amino acids as well as other "non-protein" e-amino acids commonly utilized by those in the peptide chemistry arts when preparing synthetic analogs ,f naturally occurring peptides. The naturally occurring 00.4 9 0 00110 0400

II

I

40450 -6amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, ornithine, and lysine. Examples of "non-protein" a-amino acids are norleucine, norvaline, alloisoleucine, homoarginine, thiaproline, dehydroproline, hydroxyproline (Hyp), homoserine, cyclohexylglycine (Chg), a a-amino-n-butyric acid (Aba), cyclohexylalanine (Cha), aminophenylbutyric acid (Pba), phenylalanines substituted at Sthe ortho, meta, or paraposition of the phenyl moiety with one or two of the following, a (C 1

-C

4 )alkyl, (C 1

-C

4 alkoxy, halogen, or nitro groups or substituted with a Smethylenedioxy group, 3-2- and 3-thienylal- anine, 3-2- and S3-furanylalanine, and 4-pyridylalanine, i-(benzothienyl-2- and 3-yl)alanine, and 2-naphthyl)alanine, O-alkylated derivatives of serine, threonine, or tyrosine, S-alkylated cysteine, the O-sulfate ester of tyrosine, 3,5- diiodotyrosine and the D-isomers of the naturally occurring amino acids.

The term "lipophilic amii acid" includes Tyr, Phe, Leu, Nle, Ile, Val, His and Pro.

The natural amino acids with the exception of glycine, contain a chiral carbon atom. Unless otherwise specifically indicated, the optically active amino acids, I referred to herein, are of the L-configuration. For example, Sany of the amino acids of the Al or A10 group can be of the D- or L-configuration. As is customary, the structure of peptides written out herein is such that the amino terminal end is on the left side of the chain and the carboxy terminal end is on the right side of the chain.

The polypeptides of formula I can form pharmaceutically acceptable salts with any non-toxic, organic or inorganic acid. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulphuric and phosphoric acid and acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate.

Illustrative organic acids which form suitable salts include the mono, di and tricarboxylic acids. Illustrative of such acids are, for example, acetic, glycolic, lactic, pyruvic,

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-7malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic: cinnamic, salicylic, 2-phenoxybenzoic and sulfonic acids such as methane sulfonic acid and 2-hydroxyethane sulfonic acid. Salts of the carboxy *eq# 0 0 *0 S S 000 0 0 o 0 0 #4 0 .4 0 0 .44, 0 04 0 9 terminal amino acid moiety include the non-toxic carboxylic acid salts formed with any suitable inorganic or organic bases. Illustratively, these salts include those of alkali metals, as for example, sodium and potassium; alkaline earth metals, such as calcium and magnesium; light metals of Group IIIA including aluminum; and organic primary, secondary and tertiary amines, as for example, trialkylamines, including triethylamine, procaine, dibenzy'lamine, 1-ethenamine, N,N'-dibenzylethylenediamine, dihydroabietylamine, N-(lower) alkylpiperidine, and any other suitable amine.

EXAMPLES

This invention is illustrated by the following examples. The peptides of the following examples 1-12 were prepared in the same manner as described in Australian Patent Application No 10729/88.

EXAMPLE 1 Ac-Thr-Pro-Lys-Pro-Gln-Ser-Hi s-Asn-Asp-Gly-Asp-Phe--Glu-Glu-I le- Pro-Glu-Glu-Tyr-Leu-Gln-OH 0 00 0.0 0* 90 S 0 tO I I EXAMPLE 2 Ac-Thr-Pro-Asn-Pro-Glu-Ser-Hi s-Asn-Asn-Gly--Asp-Phe--Glu-Glu-I le- Pro-Glu-Glu-Tyr-Leu-Gln-OH EXAMPLE 3 Suc-Tyr--Glu-Pro-I le-Pro-Glu-Al a-Cha--Asn-OH EXAMPLE 4 Suc-Tyr-Gluj-Pro-I le-Pro-Glu-Glu-Tyr-Cha-Gln-OH EXAMPLE Suc-Tyr-Glu-Pro-I le-Pro-Glu-Glu-Pro-Cha-D-Glu-OH EXAMPLE 6 Suc-Tha-Glu-Pro-I le-Pro-Glu-Glu-Pro-Cha-D-Glui-OH EXAMPLE 7 Suc-Npa-Glu-Pro-Il e-Pro-Glu-Glu-Ala-Cha-D-Glu-OH EXAMPLE 8 Suc-Tyr-Glu-Pro-I le-Pro-Glu-Glu-Ala-Cha-D-Glu-OH

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a a a a a *1 SO S Amino Acids Analysis (6N HCI Hydrolysis: 24 Hrs at 1060C) Exampl His Asx Ser Glx Pro Ala Gly Ile Leu Tyr Phe Lys 1+ 1.01(l) 2.99(3) 0.81(l) 5.93(6) 3.02(3) 1.04(l) 0.98(l) 1.05(1) 0.96(1) 2 1.02(l) 4.05(4) 0.92(1) 6.01(6) 3.14(3) 1.05(l) 0.98(l) 1.031(l) 0.84(1) 3 0.98(1) 2.06(2) 2.03(2) 0.98(l) 0.95(1)0.5l 4 4.09(4) 2.00(2) 0.93(i)1.82 4.06(4) 2.97(3) 0.96(1) 1.00(1) 6 4.07(4) 2.98(3) 0.96(1)

(D

(D

(D

(n 0

CD

CD

0 0 H H H C *(Me)Tyr coelutes but not quantitated.

*Cha present, not calculated +ThrO.99(l) ++ThrO.95(1) k*(Orn standard used to quantitate) *64*Tha present, not calculated Si-9 -9- Physical Characteristics

HPLC

Example tr(mn) TLCI TLCII TLC III FAB-MS No. (14- (Rf) (Rf) (Rf) (M H) 28 dient) 1 13.70 2513.9 1297 S2 14.03 2501 3 15.83 1185 S4 18.02 1420 16.85 1345.6 6 19.08 1377* S(M Na EXAMPLE 12 4 ;This example illustrates the effectiveness of the Speptides of Examples 1-11 in reducing blood coagulation.

Human plasma was collected in EDTA (final j concentration from a healthy female volunteer who had fasted for 12 hours. The plasma was immediately sterilized by passing it through a 0.2 p filter membrane (Gelman) then aliquoted into 1 ml portions and stored at 20 0 C. In all i assays, unsulfated Na-acetylhirudin45_-5 was included as a control. A bovine thrombin solution (50 pl; 0.2 pmol; Sigma) was added to the wells of a 96 well micotiter plate (Falcon) containing 50 pl of a solution of the peptide to be tested. After a minute of agitation and a 10 minute incubation at 24 0 C, 100 pl of 1:10 diluted human plasma in 0.12 M sodium chloride, 0.01 M sodium phosphate, 0.01% sodium azide, 0.1% bovine serum albumin (pH 7.4) was added. The mixture was agitated for 10 seconds and the turbidity

(A

405 of the solution was measured at 5 minute intervals by an autoreader (Bio-Tek Model EL 309).

Reported is the ability of a 5pM concentration of peptide to delay two-fold the amount of fibrin-clot present

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I at 15 minutes in the control well. =Delayed clot formation but less than twofold. =Delayed clot formation but more than twofold).

Fibrin-clot inhibition by the examples in the present application: Examples 3, 5, 6.

Examples 1, 2, 4, 7-11.

3 04 o 4

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Claims (10)

1. A peptide derivative which is Ac-Thr-Pro-Lys-Pro- Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-I le-Pro-Glu-Glu-Tyr-Leu -Gin-OH.

2. A peptide derivative which is Ac-Thr--Pro-Asn-Pro- Glu-Ser-His-Asn-Asn-Gly-Asp-Phe-Glu-Glu-I le-Pro-Glu-Glu-Tyr-Leu I q -Gin-OH.

3. A peptide derivative Pro-Glu-Ala-Cha-Asn-OH.

4. A peptide derivative Pro-Glu-Glu-Tyr-Cha-Gln-OH. A peptide derivative Pro-Glu-Glu-Pro-Cha--D-Glu-OH.

6. A peptide derivative Pro-Glu-Glu-Pro-Cha-D-Glu-OH.

7. A peptide derivative Pro-Glu-Glu-Ala-Cha-D-Glu-OH.

8. A peptide derivative Pro-Glu-Glu-Al a-Cha-D-Glu-OH.

9. A peptide derivative Pro-Glu-Glu-Ala-Cha-D-Glu-OH. A peptide derivative Pro-Glu-Glu-Ala-Cha-D-Glu-OH.

11. A peptide derivative Pro-Glu-Glu-Al a-Cha-D-G lu-OH.

12. A method of reducing in need thereof which which is Suc-Tyr-Glu-Pro-Ile- which is Suc-Tyr-Glu-Pro-Ile- which is SUC-Tyr-Glu-Pro-Ile- which is Suc-Tha-Glu-Pro-Ile- which is Suc-Npa-Glu-Pro-Ile- which is Suc-Tyr-Glu-Pro-Ile- which is Mal-Tyr-Glu-Pro-Ile- which is Glt-Tyr-Glu-Pro-Ile- which is Fum-Tyr-Glu-Pro-Ile- blood coagulation in a patient comprises administering an I I II ~1 ''it tE t anticoagulant effective amount of a peptide derivative of one of claims 1-11 and a pharmaceutically acceptable carrier. DATED: 8 August 1990 PHILLIPS ORMONDE FITZPATRICK Patent Attorneys for: MERRELL DOW PHARMACEUTICALS INC. 1435U DMW