Analogs of hirudin having anti-platelet activity

This invention relates to peptide derivatives which are useful anticoagulant agents.

FIELD OF THE INVENTION 
This invention relates to peptide analogs having medical use as as 
anticoagulant and antiplatelet agents. 
BACKGROUND OF INVENTION 
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 intravascular coagulation. Prophylactic 
administration of anticoagulants is believed to prevent a recurrence 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. Arterial thrombosis, particularly in 
arteries supplying the heart muscle and brain, is a leading cause of 
death. 
Antiplatelet agents are useful therapeutic agents in the pharmacological 
treatment of those platelet associated thromboembolic diseases that are 
primarily arterial in origin. For example, antiplatelet agents can be used 
to prevent reoccurence myocardial infarction and strokes. 
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. 
Applicant has previously discovered a specific region of hirudin that is 
responsible, at least in part, for its anticoagulant activity. This region 
has been chemically synthesized and certain of its analogs appear 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 fibrinogen to the recognition site 
of thrombin, a prerequisite to fibrin production and clot formation. 
Several reports have described the ability of the oligopeptide Arg-Gly-Asp 
and related peptides to inhibit the platelet-dependent thrombus formation. 
Y. Cadroy, et al., J. Clin. Invest. 84, 939-944 (1989). Applicant has 
discovered several means of incorporating both the antiplatelet 
Arg-Gly-Asp fragment and the previously noted Hirudin C-terminal fragment 
antithrombin analogs into a single entity having both actions. 
SUMMARY OF THE INVENTION 
Peptide derivatives of the formula 
EQU X---A.sub.1 A.sub.2 A.sub.3 A.sub.4 A.sub.5 A.sub.6 A.sub.7 A.sub.8 A.sub.9 
A.sub.10 A.sub.11 --Y (1) 
wherein 
X is an amino terminal residue selected from aminomethylbenzoyl, 
guanidinomethylbenzoyl, guanidinomethylcyclohexylcarbonyl, 
aminomethylcyclohexylcaronyl, hydrogen, alkyl substituted guanidino or 
alkyl substituted amino; 
A.sub.1 is Gly or a bond; 
A.sub.2 is Asp; 
A.sub.3 is Phe, SubPhe, .beta.-(2- and 3-thienyl)alanine, .beta.-(2-and 
3-furanyl)alanine, .beta.-(2-, 3-, and 4-pyridyl)alanine, 
.beta.-(benzothienyl-2- and 3-yl)alanine, .beta.-(1- and 
2-naphthyl)alanine, Tyr or Trp; 
A.sub.4 is Glu, Asp, Ser(OSO.sub.3 H), Ser(OPO.sub.3 H), hSer(OSO.sub.3 H), 
cysteic acid or homocysteic acid; 
A.sub.5 is any amino acid; 
A.sub.6 is Ile, Val, Leu, Nle, or Phe; 
A.sub.7 is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4-carboxylate, Sar, 
NMePgl or D-Ala; 
A.sub.8 is any amino acid; 
A.sub.9 is any amino acid; 
A.sub.10 is a lipophilic amino acid selected from Tyr, Trp, Phe, Leu, Nle, 
Ile, Val, Cha and Pro or is a dipeptide containing at least one of these 
lipophilic amino acids; 
A.sub.11 is a bond or is a peptide fragment containing from one to five 
residues of any amino acid; and 
Y is a carboxy terminal residue selected from OH, C.sub.1 -C.sub.6 alkoxy, 
amino, mono- or di-(C.sub.1 -C.sub.4) alkyl substituted amino, or 
benzylamino; 
and the pharmaceutically acceptable salts thereof are useful anticoagulant 
agents. 
DETAILED DESCRIPTION OF THE INVENTION 
The following common abbreviations of the amino acids are used throughout 
this specification: 
Gly--glycine 
Ala--alanine 
Val--valine 
Leu--leucine 
Ile--isoleucine 
Cha--cyclohexylalanine 
Orn--ornithine 
Pro--proline 
Phe--phenylalanine 
Trp--tryptophan 
Met--methionine 
Ser--serine 
Thr--threonine 
Cys--cysteine 
Tyr--tyrosine 
Asn--asparagine 
Gln--glutamine 
Asp--aspartic acid 
Glu--glutaminc acid 
Lys--lysine 
Hly--homolysine 
Arg--arginine 
Har--homoarginine 
His--histidine 
Nle--norleucine 
Hyp--hydroxyproline 
Glt--glutaryl 
Mal--maleyl 
Npa--.beta.-(2-naphthyl)alanine 
3,4-dehydroPro--3,4-dehydroproline 
Tyr(SO.sub.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 
pClPhe--para-chloro-phenylalanine 
pNO.sub.2 Phe--para-nitro-phenylalanine 
Tyr(Me)--O'-methyl-4-tyrosine 
5GP--5-guanidinopentyl 
5AP--5-aminopentyl 
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, isopro- pyl, 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 moieties per group, for example, acetyl, benzoyl 
succinyl, maleyl, and glutaryl. 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 "nonprotein" .alpha.-amino acids commonly 
utilized by those in the peptide chemistry arts when preparing synthetic 
analogs of naturally occurring peptides. The naturally occurring amino 
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" .alpha.-amino 
acids are norleucine, norvaline, alloisoleucine, homoarginine, 
thiaproline, dehydroproline, hydroxyproline (Hyp), homoserine, 
Ser(OSO.sub.3 H), hSer(OSO.sub.3 H), cysteic acid, homocysteic acid, 
cyclohexylglycine (Chg), .alpha.-amino-n-butyric acid (Aba), 
cyclohexylalanine (Cha), aminophenylbutyric acid (Pba), phenylalanines 
substituted at the ortho, meta, or paraposition of the phenyl moiety with 
one or two of the following, a (C.sub.1 -C.sub.4) alkyl, (C.sub.1 
-C.sub.4) alkoxy, halogen, or nitro groups or substituted with a 
methylenedioxy group, .beta.-2- and 3-thienylalanine, .beta.-2- and 
3-furanylalanine, .beta.-2-, 3-, and 4-pyridylalanine, 
.beta.-(benzothienyl-2- and 3-yl)alanine, .beta.-(1- and 
2-naphthyl)alanine, O-alkylated derivates 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 "any amino acid" is also intended to encompass those 
naturally occurring and non-protein .alpha.-amino acids of the formulae 
##STR1## 
wherein p, q, and r are each independently an integer of from 1 to 5 and 
wherein R is a hydrogen or a (C.sub.1 -C.sub.4)alkyl group. 
The term "lipophilic amino 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, referred to herein, are of the L-configuration, including 
those amino acids depicted by formulae 2, 3, and 4. For example, any of 
the amino acids of the A.sub.1 or A.sub.10 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. As 
is also customary when using the three-letter code for the amino acids, a 
three-letter code beginning with an upper case letter indicates the 
L-configuration and a three-letter code beginning with a lower-case letter 
indicates the D-configuration. 
The polypeptides of formula 1 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, malonic, 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 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, dibenzylamine, 
1-ethenamine, N,N'-dibenzylethylenediamine, dihydroabietylamine, 
N-(lower)alkylpiperidine, and any other suitable amine. 
As with any generic group of chemical compounds, certain groups are 
preferred. Applicants prefer those peptide derivatives of formula 1 
wherein 
X is aminomethylbenzoyl, guanidinomethylbenzoyl, 
guanidinomethylcyclohexylcarbonyl, aminomethylcyclohexylcarbonyl, or alkyl 
substituted amino. 
Also preferred are those formula 1 compounds wherein 
A.sub.1 is Gly or a bond; 
A.sub.2 is Asp; 
A.sub.3 is Phe, Trp, or Tyr(OCH.sub.3); 
A.sub.4 is Glu, or Asp; 
A.sub.5 is Pro; 
A.sub.6 is Ile or Leu; 
A.sub.7 is Pro; 
A.sub.8 is Glu or Asp; 
A.sub.9 is Glu or Phe; 
A.sub.10 is Ala-Cha, Trp or Phe; 
A.sub.11 is Glu or glu; and 
Y is OH or NH.sub.2. 
Especially preferred are those peptide derivatives of formula 1 wherein 
X is an amino terminal residue selected from aminomethylbenzoyl, 
guanidinomethylbenzoyl, guanidinomethylcyclohexylcarbonyl, 
aminomethylcyclohexylcaronyl, hydrogen, alkyl substituted guanidino or 
alkyl substituted amino; 
A.sub.1 is Gly or a bond; 
A.sub.2 is Asp; 
A.sub.3 is Phe, Trp, or Tyr(OCH.sub.3); 
A.sub.4 is Glu; 
A.sub.5 is Pro; 
A.sub.6 is Ile; 
A.sub.7 is Pro; 
A.sub.8 is Glu; 
A.sub.9 is Glu; 
A.sub.10 is Ala-Cha; 
A.sub.11 is glu; and 
Y is OH. 
The proteins of this invention can be prepared by a variety of procedures 
readily known to those skilled in the art. Such procedures include the 
solid phase sequential and block synthesis, gene cloning and combinations 
of these techniques. The solid phase sequential procedure can be performed 
using established automated methods such as by use of an automated peptide 
sythesizer. In this procedure an .alpha.-amino protected amino acid is 
bound to a resin support. The resin support employed can be any suitable 
resin conventionally employed in the art for the solid phase preparation 
of polypeptides, preferably polystyrene which has been cross-linked with 
from 0.5 to about 3 percent divinyl benzene, which has been either 
chloromethylated or hydroxymethylated to provide sites for ester formation 
with the initially introduced .alpha.-amino protected amino acid. 
An example of a hydroxymethyl resin is described by Bodanszky, et al., 
Chem. Ind. (London) 38, 1597-98 (1966). A chloromethylated resin is 
commercially available from Bio Rad Laboratories, Richmond, Calif., and 
the preparation of such a resin is described by Stewart et al., "Solid 
Phase Peptide Synthesis" (Freeman & Co., San Francisco 1969), Chapter 1, 
pp. 1-6. The protected amino acid can be bound to the resin by the 
procedure of Gisin, Helv. Chem Acta, 56, 1476 (1973). Many resin bound, 
protected amino acids are commercially available. As an example, to 
prepare a polypeptide of this invention wherein the carboxy terminal end 
is a Thr residue, a tert-butyloxycarbonyl (Boc) protected Thr bound to a 
benzylated, hydroxymethylated phenylacetamidomethyl (PAM) resin can be 
used and is commercially available. 
Following the coupling of the .alpha.-amino protected amino acid to the 
resin support, the protecting group is removed using any suitable 
procedure such as by using trifluoroacetic acid in methylene chloride, 
trifluoroacetic acid alone, or HCl in dioxane. The deprotection is carried 
out at a temperature of between 0.degree. C. and room temperature. Other 
standard cleaving reagents and conditions for removal of specific 
.alpha.-amino protecting groups may be used. After removal of the 
.alpha.-amino protecting group the other amino protected amino acids are 
coupled step-wise in the desired order. Alternatively, multiple amino acid 
groups may be coupled by the solution method prior to coupling with the 
resin supported amino acid sequence. 
The .alpha.-amino protecting group employed with each amino acid introduced 
into the polypeptide sequence may be any such protecting group known to 
the art. Among the classes of .alpha.-amino protecting groups contemplated 
are (1) acyl type protecting groups such as: formyl, trifluoroacetyl, 
phthalyl, toluenesulfonyl (tosyl), benzenesulfonyl, nitrophenylsulfenyl, 
tritylsulfenyl, o-nitrophenoxyacetyl and .alpha.-chlorobutyryl; (2) 
aromatic urethan type protecting groups such as benzyloxycarbonyl and 
substituted benzyloxycarbonyl, such as p-chlorobenzyloxycarbonyl, 
p-nitrobenzyl- carbonyl, p-bromobenzyloxycarbonyl, 
p-methoxybenzyloxycarbonyl, 1-(p-biphenylyl)-l-methylethoxycarbonyl, 
.alpha.,.alpha.-dimethyl-3,5dimethoxybenzyloxycarbonyl and 
benzhydryloxycarbonyl; (3) aliphatic urethan protecting groups such as 
tertbutyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, 
isopropyloxycarbonyl, ethoxycarbonyl and allyloxycarbonyl; (4) cycloalkyl 
urethan type protecting groups such as cyclopentyloxycarbonyl, 
adamantyloxycarbonyl and cyclohexyloxycarbonyl; (5) thio urethan type 
protecting groups such as phenylthiocarbonyl; (6) alkyl type protecting 
groups such as triphenylmethyl (trityl) and benzyl; and (7) trialkylsilane 
groups such as trimethylsilane. The preferred .alpha.-amino protecting 
group is tert-butyloxycarbonyl. 
The selection of an appropriate coupling reagent is within the skill of the 
art. A particularly suitable coupling reagent where the amino acid to be 
added is Gln, Asn or Arg is N,N'-diisopropylcarbodiimide and 
1-hydroxybenzotriazole. The use of these reagents prevents nitrile and 
lactam formation. Other coupling agents are (1) carbodiimides (e.g., 
N,N'-dicyclohexylcarbodiimide and 
N-ethyl-N'-(y-dimethylaminopropylcarbodiimide); (2) cyanamides (e.g., 
N,N-dibenzylcyanamide); (3) ketenimines; (4) isoxazolium salts (e.g., 
N-ethyl-5-phenyl-isoxazolium-3'-sulfonate; (5) monocyclic nitrogen 
containing heterocyclic amides of aromatic character containing one 
through four nitrogens in the ring such as imidazolides, pyrazolides, and 
1,2,4-triazolides. Specific heterocyclic amides that are useful include 
N,N'-carbonyldiimidazole and N,N-carbonyl-di-1,2,4-triazole; (6) 
alkoxylated acetylene (e.g., ethoxyacetylene); (7) reagents which form a 
mixed anhydride with the carboxyl moiety of the amino acid (e.g., 
ethylchloroformate and isobutylchloroformate) or the symmetrical anhydride 
of the amino acid to be coupled (e.g., Boc-Ala-O-Ala-Boc) and (8) nitrogen 
containing heterocyclic compounds having a hydroxy group on one ring 
nitrogen (e.g., N-hydroxyphthalimide, N-hydroxysuccinimide and 
1-hydroxybenzotriazole). Other activating reagents and their use in 
peptide coupling are described by Kapoor, J. Pharm. Sci., 59, pp. 1-27 
(1970). Applicants prefer the use of the symmetrical anhydride as a 
coupling reagent for all amino acids except Arg, Asn and Gln. 
Each protected amino acid or amino acid sequence is introduced into the 
solid phase reactor in about a four-fold excess and the coupling is 
carried out in a medium of dimethylformamide: methylene chloride (1:1) or 
in dimethylformamide alone or preferably methylene chloride alone. In 
cases where incomplete coupling occurs, the coupling procedure is repeated 
before removal of the .alpha.-amino protecting group, prior to the 
coupling of the next amino acid in the solid phase reactor. The success of 
the coupling reaction at each stage of the synthesis is monitored by the 
ninhydrin reaction as described by E. Kaiser et al, Analyt. Biochem. 34, 
595 (1970). 
After the desired amino acid sequence has been obtained, the peptide is 
removed from the resin. This can be done by hydrolysis such as by 
treatment of the resin bound polypeptide with a solution of dimethyl 
sulfide, p-cresol and thiocresol in dilute aqueous hydrofluoric acid. 
As is known in the art of solid phase peptide synthesis many of the amino 
acids bear functionalities requiring protection during the chain 
preparation. The use and selection of the appropriate protecting group is 
within the ability of those skilled in the art and will depend upon the 
amino acid to be protected and the presence of other protected amino acid 
residues on the peptide. The selection of such a side chain protecting 
group is critical in that it must be one which is not removed by cleavage 
during cleavage of the protecting group of the .alpha.-amino moiety. For 
example, suitable side chain protecting groups for lysine are 
benzyloxycarbonyl and substituted benzyloxycarbonyl, said substituent 
being selected from halo (e.g., chloro, bromo, fluoro) and nitro (e.g., 
2-chlorobenzyloxycarbonyl, p-nitrobenzyloxy-carbonyl, 
3,4-dichlorobenzyloxycarbonyl), tosyl, t-amyloxycarbonyl, 
t-butyloxycarbonyl and diisopropylmethoxycarbonyl. The alcoholic hydroxyl 
group of threonine and serine can be protected with an acetyl, benzoyl, 
tert-butyl, trityl, benzyl, 2,6-dichlorobenzyl or benzyloxycarbonyl group. 
The preferred protecting group is benzyl. 
These groups can be removed by procedures well known in the art. Typically 
protecting group removal is done after the peptide chain synthesis is 
complete but the protecting groups can be removed at any other appropriate 
time. 
The antiplatelet dose of a peptide analog of this invention is from 0.2 
mg/kg to 250 mg/kg of patient body weight per day depending on the 
patient, the severity of the thromobotic condition to be treated and the 
peptide analog selected. The suitable dose for a particular patient can be 
readily determined. Preferably from 1 to 4 daily doses would be 
administered typically with from 5 mg to 100 mg of active compound per 
dose. 
Antiplatelet therapy is indicated for the prevention of recurrence of 
myocardial infarction and stroke as well as other disease conditions 
associated with platelet aggregation. Those experienced in this field are 
readily aware of the circumstances requiring anticoagulant and 
antiplatelet therapy. The term "patient" used herein is taken to mean 
mammals such as primates, including humans, sheep, horses, cattle, pigs, 
dogs, cats, rats and mice. 
Although some of the peptide derivatives may survive passage through the 
gut following oral administration, applicants prefer non-oral 
administration, for example, subcutaneous, intravenous, intramuscular or 
intraperitoneal; administration by depot injection; by implant 
preparation; or by application to the mucous membranes, such as, that of 
the nose, throat and bronchial tubes, for example, in an aerosol can 
contain a peptide derivative of this invention in a spray or dry powder 
form. 
For parenteral administration the compounds may be administered as 
injectable dosages of a solution or suspension of the compound in a 
physiologically acceptable diluent with a pharmaceutical carrier which can 
be a sterile liquid such as water and oils with or without the addition of 
a surfactant and other pharmaceutically acceptable adjuvants. Illustrative 
of oils which can be employed in these preparations are those of 
petroleum, animal, vegetable, or synthetic origin, for example, peanut 
oil, soybean oil, and mineral oil. In general, water, saline, aqueous 
dextrose and related sugar solutions, ethanol and glycols such as 
propylene glycol or polyethylene glycol are preferred liquid carriers, 
particularly for injectable solutions. 
The compounds can be administered in the form of a depot injection or 
implant preparation which may be formulated in such a manner as to permit 
a sustained release of the active ingredient. The active ingredient can be 
compressed into pellets or small cylinders and implanted subcutaneously or 
intramuscularly as depot injections or implants. Implants may employ inert 
materials such as biodegradable polymers or synthetic silicones, for 
example, Silastic, silicone rubber manufactured by the Dow-Corning 
Corporation.