Peptide derivatives

New peptide derivatives represented by the following general formula EQU R.sub.1 -Pro-Hyp-R.sub.2 wherein R.sub.1 is a hydrophobic radical, R.sub.2 is a hydrophilic radical, Pro is prolyl and Hyp is hydroxyprolyl show more powerful inhibitory action against platelet agglutination than conventional drugs and have potential use as anti-agglutination agent. The peptide derivative can be prepared by condensing a peptide containing a hydrophobic radical with a carboxylic acid containing a hydrophilic radical. The peptide derivative can be used in the form of parenteral injections, tablets, capsules or powder, the typical daily dose being in the range from 1 mg to 500 mg for adults.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to new peptides useful as a medicine. This invention 
also relates to processes for preparing new peptides. 
2. Description of the Prior Art 
An active peptide, Pro-Hyp-Gly-Ala-Gly, (hereinafter referred to as 
compound P), is known from Japanese Patent Application (OPI) No. 165356/82 
(the term "OPI" as used herein means an "unexamined published 
application"). This compound has potent anti-arrhythmic and 
platelet-agglutination preventing activity. 
Compound P showed low toxicity and was expected as a remedy against 
ischemic cardiac insufficiencies. However, its effective dose for oral 
administration is rather large compared with conventional drugs, and its 
action does not last long as is apparent from its half-life in the blood 
as short as about ten minutes. Therefore, improvement has been desired. 
SUMMARY OF THE INVENTION 
The object of this invention is to provide new peptide derivatives having 
higher physiological activity, cell membrane permeability, absorption 
speed and metabolic turnover compared with compound P. 
Another object of this invention is to provide processes for producing new 
peptide derivatives. 
As a result of extensive studies it has now been found that these objects 
can be achieved by production of compounds having a general structure of 
(Hydrophobic group)-Pro-Hyp-(Hydrophilic group). This invention was 
accomplished based on these findings. 
Thus this invention relates to new peptide derivatives represented by 
general formula (I) 
EQU R.sub.1 -Pro-Hyp-R.sub.2 (I) 
wherein R.sub.1 represents a hydrophobic radical, R.sub.2 represents a 
hydrophilic radical, Pro stands for prolyl and Hyp stands for 
hydroxyprolyl. 
In another aspect, this invention relates to a process for producing new 
peptide derivatives represented by general formula (I) 
EQU R.sub.1 -Pro-Hyp-R.sub.2 (I) 
wherein R.sub.1 represents a hydrophobic radical, R.sub.2 represents a 
hydrophilic radical, Pro stands for prolyl and Hyp stands for 
hydroxyprolyl, by condensing a peptide containing a hydrophobic radical 
with a carboxylic acid containing a hydrophilic radical.

DETAILED DESCRIPTION OF THE INVENTION 
The hydrophobic radical may be the residue of a hydrophobic amino acid, or 
linear or branched 1C to 10C alkyl, 6C to 10C aryl, or 7C to 10C aralkyl 
having a linear or branched alkyl moiety. These hydrophobic radicals may 
also contain, in the main and/or side chains, keto, carboxyl, hydroxyl, 
ether, ester, acid amide or amino acid residues. 
As typical examples of hydrophobic amino acids there may be mentioned, 
among others, phenylalanine, tryptophan, leucine and isoleucine. The most 
preferred hydrophobic radicals are those represented by the following 
general formula 
##STR1## 
wherein X represents --CO--, --CH(COOH)-- or --CH.sub.2 --; R.sub.3 
represents hydrogen or hydroxyl; and n is an integer from 1 to 8, 
preferably from 1 to 4. 
As examples of the hydrophilic radical may be mentioned linear or branched 
1C to 16C alkyl, 6C to 16C aryl and 7C to 16C aralkyl having a linear or 
branched alkyl moiety, containing, at the terminal thereof, carboxyl, 
sufonic acid, sulfate, amino, quaternary ammonium, phosphate, hydroxyl, 
acid amide, thiol, nitrile, thiocyanate, nitro or imino group, or halogen 
atom. In addition, some peptide residues or peptide derivatives may also 
be used as the hydrophilic radical. In the latter case, suitable peptide 
is one composed of 2 to 6 amino acids (preferably glycine (Gly) or alanine 
(Ala)), for example, a peptide represented by formula (III). 
EQU Gly-Ala-Gly (III) 
The compounds (I) of this invention can be prepared, for example, by 
condensing a peptide containing a hydrophobic radical with a carboxylic 
acid containing a hydrophilic radical. 
Of the compounds of formula (I), those in which R.sub.1 is the radical 
represented by formula (II) and R.sub.2 is the residue of compound (III), 
namely, a compound represented by formula 
##STR2## 
wherein R.sub.3, X and n have the same meanings as defined above, can be 
synthesized by condensing a peptide containing a hydrophilic radical 
represented by 
EQU (Pro).sub.m -Hyp-Gly-Ala-Gly (V) 
wherein m is 0 or 1, with a carboxylic acid containing a hydrophobic 
radical represented by a formula 
##STR3## 
wherein Y represents 
##STR4## 
when n is 1, or 
##STR5## 
when n is 0. 
As the amino-protective group may be used, for example, t-butoxycarbonyl 
(BOC), benzyloxycarbonyl (hereinafter abbreviated as Z) or tosyl group. 
The compound of formula (V) and the compound of formula (VI) each is used 
in a concentration of preferably from 0.1 to 1.0M, more preferably 0.1 to 
0.5M. 
Any known condensing agents may be used, but the preferred is a 
carbodiimide such as N,N'-dicyclohexylcarbodiimide (DCC). The condensing 
agent is preferably used in an amount of 1 to 3 mol equivalents, more 
preferably 1.1 to 1.3 mol equivalents. 
The proportion of the condensing agent, the compound of formula (V) and the 
compound of formula (VI) is preferably 1-3/1/1-3, more preferably 
1.1-1.3/1/1.1-1.3 by mol equivalent. 
Preferable solvents include ethyl acetate, acetonitrile, dichloromethane, 
chloroform, tetrahydrofuran and N,N-dimethylformamide. 
The condensation reaction is preferably carried out at a temperature in the 
range from -10.degree. to 100.degree. C., most preferably in the range 
from 0.degree. to 50.degree. C., for 2 to 20 hours. 
More particularly, a compound of formula (I) in which R.sub.1 is a radical 
represented by 
##STR6## 
and R.sub.2 is the residue of compound (III), namely, a compound of 
##STR7## 
can be synthesized by condensation between compound P and a carboxylic 
acid represented by the formula (VIII) 
##STR8## 
Compound P is a pentapeptide which may be prepared by known methods as 
detailed below (the liquid phase method and the solid phase method). 
A compound of formula (I) in which R.sub.1 is a radical represented by 
##STR9## 
and R.sub.2 is the residue of comound (III), namely, a compound 
represented by formula (IX) or (X) 
##STR10## 
can be synthesized by reaction of the compound of formula (XI) 
EQU Hyp-Gly-Ala-Gly (XI) 
with a compound of formula (XII) or (XIII), respectively. 
##STR11## 
The reaction conditions are the same as described above. 
The N-substituted proline derivatives (compounds (XII) and (XIII)) may be 
easily obtained by reductive amination from proline and a corresponding 
aldehyde or .alpha.-ketocarboxylic acid, as described in Fieser & Fieser, 
Reagents for Organic Synthesis, A Wiley-Interscience Publication, Vol. IV, 
p. 448-449 (1974). 
The compounds of formula (I) thus formed can be isolated and purified by 
gel filtration, ion-exchange chromatography, high performance liquid 
chromatography (HPLC) using a reverse column, silica gel chromatography, 
or by combinations thereof. Either of the solid phase method and the 
liquid phase method (as described in S. Aonuma et al., Chem. Pharm. Bull., 
28, 3332-3339 and 3340-3346 (1980), ibid., 32, 219-227 (1984), J. Pharm. 
Dyn., 5, 40-48 (1982) and Yakugaku Zasshi, 103, 662-666 (1983)) shown 
below may be used for the synthesis of compound P and compounds of (XI) 
and (I). 
SOLID PHASE METHOD 
##STR12## 
R: Chlorinated resin DCC: Dicyclohexylcarbodiimide 
TEA: Triethylamine, 
BOC: tert-Butoxy carbonyl 
TFA: Trifluoroacetic acid 
Bzl: Benzyl 
LIQUID PHASE METHOD 
##STR13## 
Su: Succinimide AcOEt: Ethyl acetate 
Tos: 
##STR14## 
Bzl: Benzyl 
In actual practice, it is preferable that compounds (P) and (VIII) are used 
for the succeeding steps with their protective groups left unreleased, as 
shown in the Examples given below. 
The peptide derivatives of this invention represented by the general 
formula (I) show more potent inhibitory action against platelet 
agglutination than conventional drugs and are useful as anti-agglutination 
agent. 
In actual application, said peptide derivatives may be used in the form of 
parenteral injections, tablets, capsules or powder. 
Typical daily dose of said peptide derivatives is in the range from 1 mg to 
500 mg for adults. 
The following Examples and Test Examples further illustrate this invention 
but are not intended to limit its scope. 
EXAMPLE 1 
Synthesis of 
##STR15## 
(1) Preparation of N-tert-butyloxycarbonyl-L-alanyl-glycine benzyl ester 
To a solution of 12.9 g of glycine benzyl ester tosylate in 200 ml 
acetonitrile cooled to 0.degree. C., was added dropwise triethylamine (5.3 
ml), followed by addition of 12 g of N-tert-butyloxycarbonylalanine 
N-hydroxysuccinimide ester (prepared by reaction of 
N-tert-butyloxycarbonylalanine with N-hydroxysuccinimide in the presence 
of dicyclohexylcarbodiimide). The resulting solution was stirred at 
0.degree. C. for one hour and then at room temperature for 18 hours. At 
the end of reaction, the solvent was distilled off under reduced pressure, 
the residue was extracted with ethyl acetate, and the extract was washed 
with 5% acetic acid, water and saturated aqueous solution of sodium 
chloride in that order, giving 16 g of crude product. Purification of this 
crude product by silica gel chromatography (chloroform:methanol=40:1) 
afforded 12.6 g of pure N-tert-butyloxycarbonyl-L-alanyl-glycine benzyl 
ester. 
.sup.1 H NMR (CDCl.sub.3), .delta.: 1.35 (d, J=7 Hz, 3H), 1.42 (s, 9H), 
4.02 (d, J=5 Hz, 2H), 3.95-4.35 (m, 1H), 5.12 (s, 2H), 5.45 (d, J=7 Hz), 
7.12 (t, J=6 Hz, 1H), 7.28 (s, 5H). 
IR, .nu..sub.max.sup.KBr : 3310, 1738, 1682, 1655 cm.sup.-1. 
(2) Preparation of N-tert-butyloxycarbonyl-glycyl-L-alanyl-glycine benzyl 
ester 
A solution of N-tert-butyloxycarbonyl-L-alanyl-glycine benzyl ester (12.6 
g) in 80 ml of anhydrous ethyl acetate was added dropwise over a period of 
15 minutes to 130 ml of 4N HCl solution in anhydrous ethyl acetate 
previously cooled to 0.degree. C., and the mixture was stirred at room 
temperature for 30 minutes. At the end of reaction, the solvent and excess 
hydrogen chloride were distilled off under reduced pressure, giving white 
solid. 
This solid was dissolved in 200 ml acetonitrile, the solution was cooled to 
0.degree. C., and 5.3 ml of triethylamine was added dropwise to the 
solution, followed by addition of 11.2 g of 
N-tert-butyloxycarbonyl-glycine N-hydroxysuccinimide ester (prepared by 
reaction of N-tert-butyloxycarbonyl-glycine with N-hydroxysuccinimide in 
the presence of dicyclohexylcarbodiimide). The resulting solution was 
stirred at 0.degree. C. for 20 minutes and then at room temperature for 18 
hours. At the end of reaction, 10 ml of 5% acetic acid was added, the 
solvent was distilled off under reduced pressure, the residue was 
extracted with ethyl acetate, and the extract was washed with 1% acetic 
acid, water and saturated aqueous solution of sodium chloride in that 
order, given 15 g of crude product. Purification of this crude product by 
silica gel chromatography (chloroform:methanol=40:1) afforded 10.6 g of 
pure product. 
EQU BOC-Gly-Ala-GlyOBzl 
.sup.1 H NMR (DMSO-d.sub.6 +CDCl.sub.3), .delta.: 1.26 (d, J=7 Hz, 3H), 
1.40 (s, 9H), 3.62 (d, J=5 Hz, 2H), 3.90 (d, J=5 Hz, 2H), 4.41 (t, J=7 Hz, 
1H), 5.09 (s, 2H), 6.60 (t, J=5 Hz, 1H), 7.30 (s, 5H), 7.79 (d, J=7 Hz, 
1H), 8.18 (t, J=5 Hz, 1H). 
IR, .nu..sub.max.sup.KBr : 3300, 1745, 1648 cm.sup.-1. 
(3) Preparation of 
N-tert-butyloxycarbonyl-O-benzyl-L-hydroxyprolyl-glycyl-L-alanyl-glycine 
benzyl ester 
A solution of N-tert-butyloxycarbonyl-glycyl-L-alanyl-glycine benzyl ester 
(9.95 g) in 80 ml of anhydrous ethyl acetate was added dropwise over a 
period of 15 minutes to 120 ml of 4.3N HCl solution in anhydrous ethyl 
acetate previously cooled to 0.degree. C., and the mixture was stirred at 
0.degree. C. for 10 minutes and then at room temperature for 15 minutes. 
At the end of reaction, the solvent and excess hydrogen chloride was 
distilled off under reduced pressure, giving white solid. 
This solid was suspended in 200 ml acetonitrile, the suspension was cooled 
to 6.degree. C., and 3.5 ml of triethylamine was added dropwise to the 
suspension, followed by addition of 11.6 g of 
N-tert-butyloxycarbonyl-O-benzyl-L-hydroxyproline N-hydroxysuccinimide 
ester (prepared by reaction of 
N-tert-butyloxycarbonyl-O-benzyl-L-hydroxyproline with 
N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide). 
Acetonitrile (100 ml) was further added, and the resulting mixture was 
stirred at 0.degree. C. for 20 minutes and then at room temperature for 18 
hours. At the end of reaction, 10 ml of 5% acetic acid was added, the 
solvent was distilled off under reduced pressure, the residue was 
extracted with ethyl acetate, and the extract was washed with 0.1% acetic 
acid, water and saturated aqueous solution of sodium chloride in that 
order, giving 17 g of crude product. Purification of this crude product by 
silica gel chromatography (chloroform:methanol=20:1) afforded 14 g of pure 
product. 
EQU BOC-Hyp(OBzl)-Gly-Ala-GlyOBzl 
.sup.1 H NMR (CDCl.sub.3), .delta.: 1.32-1.44 (m, 12H), 2.0-2; 0.5 (m, 2H), 
3.4-4.75 (m, 9H), 4.43 (s, 2H), 5.05 (s, 2H), 7.05-7.8 (m, 13H). 
IR, .nu..sub.max.sup.KBr : 3290, 1752, 1700, 1665, 1638 cm.sup.-1. 
(4) Preparation of 
N-tert-butyloxycarbonyl-L-prolyl-O-benzyl-L-hydroxyprolyl-glycyl-L-alanyl- 
glycine benzyl ester 
A solution of 
N-tert-butyloxycarbonyl-O-benzyl-L-hydroxyprolyl-glycyl-L-alanyl-glycine 
benzyl ester (14.0 g) in 80 ml of anhydrous ethyl acetate was added 
dropwise over a period of 15 minutes to 100 ml of 5.2N HCl solution in 
anhydrous ethyl acetate previously cooled to 0.degree. C., and the mixture 
was stirred at 0.degree. C. for 10 minutes and then at room temperature 
for 10 minutes. At the end of reaction, the solvent and excess hydrogen 
chloride were distilled off under reduced pressure, giving white solid. 
This solid was suspended in 200 ml acetonitrile, the suspension was cooled 
to 0.degree. C., and 3.3 ml of triethylamine was added dropwise to the 
suspension, followed by addition of 8.2 g of 
N-tert-butyloxycarbonyl-L-proline N-hydroxysuccinimide ester (prepared by 
reaction of N-tert-butyloxycarbonyl-L-proline with N-hydroxysuccinimide in 
the presence of dicyclohexylcarbodiimide). The resulting mixture was 
stirred at 0.degree. C. for one hour and then at room temperature for 18 
hours. At the end of reaction, 10 ml of 5% acetic acid was added, the 
solvent was distilled off under reduced pressure, the residue was 
extracted with ethyl acetate, and the extract was washed with 0.1% acetic 
acid, water and saturated aqueous solution of sodium chloride in that 
order, giving 17 g of crude product. Purification of this crude product by 
silica gel chromatography (chloroform:methanol=20:1) afforded 3.5 g of 
pure product. 
EQU BOC-Pro-Hyp(OBzl)-Gly-Ala-Gly-OBzl 
.sup.1 H NMR (CDCl.sub.3), .delta.: 1.40 (d, J=7 Hz, 3H), 1.43 (s, 9H), 
1.6-2.7 (m, 6H), 3.2-4.8 (m, 12H), 4.52 (s, 2H), 5.12 (s, 2H), 7.1-7.8 (m, 
13H). 
IR, .nu..sub.max.sup.KBr : 3310, 1752, 1670 cm.sup.-1. 
(5) Preparation of 
N-3-(4-benzyloxyphenyl)propionyl-L-prolyl-O-benzyl-L-hydroxyprolyl-glycyl- 
L-alanyl-glycine benzyl ester 
L-Prolyl-L-hydroxyprolyl-glycyl-L-alanyl-glycine benzyl ester hydrochloride 
(1.81 g) (prepared in the synthesis of 
L-prolyl-L-hydroxyprolyl-glycyl-L-alanyl-glycine benzyl ester) was 
suspended in dichloromethane, the suspension was cooled to 0.degree. C., 
triethylamine (0.42 ml), 3-(4-benzyloxyphenyl)propionic acid (*1) (1.12 g) 
and dicyclohexylcarbodiimide (0.90 g) were added to the suspension, and 
the mixture was stirred at 0.degree. C. for 90 minutes. The reaction 
mixture was filtered, the filtrate was concentrated, and the crude product 
thus obtained (3.7 g) was purified by silica gel chromatography 
(chloroform:methanol=20:1), giving 1.77 g of pure product. 
.sup.1 H NMR (CDCl.sub.3), .delta.: 1.41 (d, J=7 Hz, 3H), 1.7-4.2 (m, 19H), 
4.2-4.7 (m, 3H), 4.45 (s, 2H), 4.95 (s, 2H), 5.06 (s, 2H), 6.77 (d, J=9 
Hz, 2H), 7.08 (d, J=9 Hz, 2H), 7.1-7.8 (m, 18H). 
IR, .nu..sub.max.sup.KBr : 3310, 1750, 1650 (bs)cm.sup.-1. 
(*1) Preparation of 3-(4-benzyloxyphenyl)propionic acid: 
(1) 3-(4-Hydroxyphenyl)propionic acid (5 g), potassium carbonate (16 g) and 
tetrahydrofuran (50 ml) were placed in a three-necked flask, 8.5 ml of 
benzyl bromide was added dropwise through a syringe under stirring, and 
the mixture was heated under reflux overnight. After filtering off the 
solid matters, the solvent was distilled off, and the residue was purified 
by column chromatography on silica gel (50 g), affording 9.25 g (89.3%) of 
pure benzyl 3-(4-benzyloxyphenyl)propionate. 
(2) Benzyl 3-(4-benzyloxyphenyl)propionate (9.25 g) and a solution of 2.8 g 
potassium hydroxide in 95% ethanol (125 ml) were placed in a flask, and 
the mixture was heated under reflux for 30 minutes. At the end of 
reaction, the solvent was distilled off, ethyl acetate was added to the 
residue, and the resulting mixture was transferred to a separating funnel. 
After washing with 1N HCl and water, the ethyl acetate layer was collected 
and dried over anhydrous sodium sulfate, the solvent was distilled off 
from the dried solution, and the residue was recrystallized from ethyl 
acetate/hexane, affording 5 g (72.7%) of pure 
3-(4-benzyloxyphenyl)propionic acid. 
.sup.1 H NMR (CDCl.sub.3), .delta.: 2.4-3.1 (m, 4H), 4.98 (s, 2H), 6.82 (d, 
J=9 Hz, 2H), 7.08 (d, J=9 Hz, 2H), 7.30 (s, 5H). 
IR, .nu..sub.max.sup.KBr : 1690, 1612, 1582 cm.sup.-1. 
(6) Preparation of 
N-3-(4-hydroxyphenyl)propionyl-L-prolyl-L-hydroxyprolyl-glycyl-L-alanyl-gl 
ycine 
To a solution of 
N-3-(4-benzyloxyphenyl)propionyl-L-prolyl-O-benzyl-L-hydroxyprolyl-glycyl- 
L-alanyl-glycine benzyl ester (122 mg) in 5 ml methanol was added 61 mg of 
10% palladium/carbon, and the mixture was stirred at room temperature for 
90 minutes under normal pressure of hydrogen. At the end of reaction, the 
catalyst was filtered off, and the filtrate was concentrated, giving 69 mg 
of crude product. Purification by silica gel chromatography 
(chloroform:methanol=2:1) afforded 37 mg of pure product. 
.sup.1 H NMR (MeOH-d.sub.4 +CDCl.sub.3), .delta.: 1.40 (d, J=7 Hz, 3H), 
1.6-2.4 (m, 6H), 2.4-3.1 (m, 4H), 3.2-4.8 (m, 11H), 6.62 (d, J=8 Hz, 2H), 
6.94 (d, J=8 Hz, 2H). 
IR, .nu..sub.max.sup.KBr : 3300, 1630 cm.sup.-1. 
EXAMPLE 2 
Synthesis of 
##STR16## 
BOC-Gly (22 g) and triethylamine (17.5 ml) were dissolved in a mixture of 
ethanol (133 ml) and chloroform (67 ml), a resin (50 g) was put in this 
solution, and the mixture was stirred at room temperature for one hour and 
then heated under reflux at 90.degree. C. for 48 hours. The resin thus 
treated was collected by filtration, washed with ethanol, acetic acid, 
water and methanol in that order, and dried at a temperature below 
30.degree. C. under reduced pressure. The BOC-Gly-Resin (55.7 g; 20.3 mmol 
Gly) thus obtained was placed in a solid-phase reactor (700 ml capacity), 
and amino-acid residues were successively introduced to the resin by 
repeating the cycle of protective-group removal, neutralization and 
coupling as shown below. 
(1) Washing thrice with 350 ml of dichloromethane for three minutes; (2) 
releasing the protective groups by treating twice with 350 ml of 5% 
trifluoroacetic acid/dichloromethane (for three minutes and 20 minutes 
each); (3) washing eight times with 350 ml of dichloromethane for three 
minutes; (4) neutralization by treating twice with 350 ml of 10% 
triethylamine/dichloromethane for three minutes and ten minutes each; (5) 
washing five times with 350 ml of dichloromethane; (6) adding 60.8 mmol of 
BOC-amino-acid and 250 ml of dichloromethane and mixing for ten minutes; 
(7) adding 60.81 mmol of dicyclohexylcarbodiimide and 100 ml of 
dichloromethane and continuing reaction for two hours; and (8) washing 
five times with 350 ml of dichloromethane. 
BOC-Ala, BOC-Gly, BOC-Hyp(OBzl), BOC-Pro and 3-phenylpropionic acid were 
introduced by repeating the cycle of operations described above, and the 
resulting resin was vacuum-dried at a temperature below 30.degree. C. The 
dry resin was placed in an HF-reactor, anisole (10 ml) and anhydrous 
hydrofluoric acid (100 ml) were added, and the reaction was carried out at 
0.degree. C. for one hour. Excess hydrofluoric acid was distilled off, the 
residue was thoroughly washed with methanol, the resin was filtered off, 
and the filtrate was concentrated, leaving 8 g of oily product. 
Purification by silica gel column chromatography (chloroform:methanol=4:1) 
gave 4.3 g of pure product. 
.sup.1 H NMR (MeOH-d.sub.4 +CDCl.sub.3), .delta.: 1.41 (d, J=7 Hz, 3H), 
1.5-2.4 (m, 6H), 2.5-3.1 (m, 4H), 3.2-4.7 (m, 11H), 6.8 (s, 5H). 
IR, .nu..sub.max.sup.KBr : 3300, 1635 cm.sup.-1. 
EXAMPLE 3 
Compound (ii) in Table 1 was prepared in the same manner as Example 1 
except that N-3-(4-benzyloxyphenyl)propyl-L-proline N-hydroxysuccinimide 
ester was used in step (4). 
EXAMPLE 4 
Compound (ii-a) in Table 1 was prepared in the same manner as Example 2 
except that N-3-phenylpropyl-L-proline was used in place of BOC-Pro. 
TABLE 1 
__________________________________________________________________________ 
Compound No. 
R.sub.1 ProHypR.sub.2 IR 
__________________________________________________________________________ 
i (Example 1) 
##STR17## ProHypGlyAlaGly 3300 (br) 1630 cm.sup.-1 
i-a (Example 2) 
##STR18## ProHypGlyAlaGly 3300 (br) 1635 cm.sup.-1 
ii 
##STR19## ProHypGlyAlaGly 3250 (br) 1615 cm.sup.-1 
ii-a 
##STR20## ProHypGlyAlaGly 3250 (br) 1620 
__________________________________________________________________________ 
cm.sup.-1 
TEST EXAMPLE 1 
Platelet Agglutination Inhibitory Action 
Blood samples were taken from the abdominal artery of rats by means of a 
syringe containing 3.8% solution of sodium citrate, and the mixture 
(sodium citrate:blood=1:9) was centrifuged for ten minutes, giving 
platelet-rich plasma (PRP). 
PRP thus collected was placed in cuvettes and kept in an incubator held at 
37.degree. C. for two minutes. Solutions of the compound obtained in 
Example 1 with different concentrations (diluted with a solution of 1.5 mM 
EDTA in a mixture of Tris-HCl buffer and sodium chloride solution (1:5)) 
were added, the resulting mixture in each cuvette was incubated for three 
minutes, and Ca ionophore A-23187 (final concentration: 4 .mu.M), 
arachidonic acid (1 mM) or thrombin (0.8 U/ml) was added to measure the 
degree of agglutination. A solution of heparin sodium was used in place of 
sodium citrate when arachidonic acid was employed as agglutination 
inducer. The result of test is summarized in FIGS. 1, 2, and 3. As can be 
seen from these FIGS., HP (the compound of this invention) showed about 
ten times stronger effects than compound P (control). 
TEST EXAMPLE 2 
Acute Toxicity 
The compounds listed in Table 1 were orally, parenterally and intravenously 
administered to mice (three head for each group) at a dose of 1 g/kg. No 
mortality or abnormality was observed at all. 
While the invention has been described in detail and with reference to 
specific embodiments thereof, it will be apparent to one skilled in the 
art that various changes and modifications can be made therein without 
departing from the spirit and scope thereof.