Alkylamides of carboxyalkanoyl peptides and method for preparation thereof

A method is described for the preparation of novel alkylamides of carboxyalkanoyl peptides of the formula ##STR1## wherein R is an aralky or an alkyl group of 1-5 carbon atoms, A is a residue of peptidically bound proline or alanine, B is a straight bond or a residue of peptidically bound proline or alanine and X is a CH.dbd.CH group or a methylene group of 1-3 carbon atoms. The described compounds are capable of inhibiting elastase.

This invention relates to a process for the preparation of alkylamides of 
carboxyalkanoyl peptides and to the peptides so produced. 
The alkylamides of dipeptides are known to be effective inhibitors of 
elastase (see Belgian Pat. Nos. 855,851 and 856,064). Additionally, the 
dipeptides evidence characteristics which are akin to amino acid 
composition optimums for elastolytic substances, for example 
alanyl-alanine or alanyl-proline. Previous studies concerning the 
optimization of a substrate for pancreatic elastase revealed the presence 
of an electrostatic bond between P.sub.4 -S.sub.4 and P.sub.5 -S.sub.5 
(see I. Schechter et al, Biochem. Biophys. Res. Commun. 32, 898 (1968) was 
of great significance with regard to the interaction between substrate and 
enzyme, the bond being viewed as related to a primary interaction (see 
Eur. J. Biochem. 69, 1 (1976), FEBS Lett. 40, 353 (1974). 
The earlier work also led to the surprising discovery that the carboxy 
group in the N-terminal part of the inhibitor as compared with the 
hydrophobic residue, such as an acetyl, substantially enhances inhibition 
capacity in the same manner as it enhances the constants of elastolytic 
hydrolysis of substrates of this type. It is further known that the 
properties of an elastolytic inhibitor are dependent upon the length of 
the peptide chain as well as at the substrate (eur. J. Biochem. 69, 1, 
1976), higher inhibition capacity being present with a tripeptide than 
with a dipeptide. Experiments with vitro inhibition constants (Ki) for the 
alkylamides of 3-carboxypropionyl and 4-carboxybutyryl di and tripeptides 
were determined using pancreatic elastase and chromogenic substrates of 
p-nitroanilides of succinyl or glutaryl tetraalanines, the results of 
which are set forth in Table 1, below. Inhibition constant of the 
inhibitors described herein were also determined using human elastase, 
that is, human duodenal elastase after stimulation with cholecystokinine 
(E.sub.1) or lyophilized human pancreated fluid obtained by cannulation of 
the pancreatic duct outlet (E.sub.2), and they were then compared with 
swine's pancreatic elastase (E.sub.3), the results of which are set forth 
in Table 2, below. 
TABLE 1 
__________________________________________________________________________ 
Inhibition constants (Ki) of alkylamides of acylated di- and 
tripeptides. 
Ki (.mu.mol) 
Inhibitor Suc--(Ala).sub.4 --NAn 
Glt--(Ala).sub.4 --NAn 
__________________________________________________________________________ 
Suc--(ala).sub.2 --NH--Et 
95.2 166 
Glt--(Ala).sub.2 --NH--Et 
68.1 109 
Suc--(Ala).sub.2 --NH--EtPh 
1456 666 
Glt--(Ala).sub.2 --NH--EtPh 
982 770 
Suc--(Ala).sub.3 --NH--Me 
510 680 
Glt--(Ala).sub.3 --NH--Me 
280 320 
Suc--(Ala).sub.3 --NH--Et 
20 29 
Glt--(Ala)--.sub.3 --NH--Et 
8.8 8.4 
Mal--(Ala).sub.3 --NH--Et 
35 30 
Ac--(Ala).sub.3 --NH--Et 
28 46 
Suc--(Ala).sub.3 --NH--Pr 
6.8 4.5 
Glt--(Ala).sub.3 --NH--Pr 
2.5 2.5 
Suc--(Ala).sub.3 --NH--iBu 
816 1474 
Glt--(Ala).sub.3 --NH--iBu 
668 794 
Suc--Ala--Pro--NH--Et 
111 80 
Suc--Ala--Pro--NH--iBu 
2042 5747 
Suc--Ala--Ala--Pro--NH--Et 
2.5 4.7 
Glt--Ala--Ala--Pro--NH-- Et 
1.6 2.7 
__________________________________________________________________________ 
TABLE 2 
__________________________________________________________________________ 
Inhibition constants (Ki) of alkylamides of acylated tripeptides. 
Ki (.mu.mol) 
Suc--(Ala).sub.4 --NAn 
Glt--(Ala).sub.4 --NAn 
E.sub.1 
E.sub.2 
E.sub.3 
E.sub.1 
E.sub.2 
E.sub.3 
__________________________________________________________________________ 
Ac--(Ala).sub.3 --NH--Et 
238 69 28 123 117 46 
Mal--(Ala).sub.3 --NH--Et 
87 69 35 73 117 30 
Suc--(Ala).sub.3 --NH--Et 
76 29 20 89 47 29 
Glt--(Ala).sub.3 --NH--Et 
15 7.4 8.8 
19 19 8.4 
Suc--Ala--Ala--Pro--NH--Et 
14 23 2.5 
19 17 4.7 
Glt--Ala--Ala--Pro--NH--Et 
10 2.9 1.6 
12 13.8 2.7 
Suc--(Ala).sub.3 --NH--Pr 
12 13 6.8 
21 14 4.5 
Glt--(Ala).sub.3 --NH--Pr 
11 3.8 2.5 
7.2 4.5 2.5 
__________________________________________________________________________ 
Suc = succinyl; 
Glt = glutaryl; 
NAn = nitroanilide; 
Et = ethyl, 
Pr = propyl; 
Mal = 3carboxyacryolyl i.e. maleyl. 
In vivo experimentation with experimental pancreatitis in rats, induced by 
the nitroorgan application of sodium desoxycholate revealed that lower 
level of serum amylase with the simultaneous application of 20 mg 
intraperitoneally of Glt-(Ala).sub.3 -NH-Et, Glt-Ala-Ala-Pro-NH-Et and 
especially Glt-Ala-Ala-Pro-Nh-Pr in a dose of 20 mg per rat was found to 
suppress the development of oedema induced by subcutaneous injection of 
elastase in the rats paws by 20 to 40%. 
When compared with known alkanoyl derivatives (dipeptides), See Biochem. 
Chem. 8, 299 (1979) a significant enhancement in the inhibition of 
elastolytic hydrolysis is evidenced, so suggesting the significance of the 
character of N-terminal substituents. 
The alkylamides of carboxyalkanoyl peptides described herein are of the 
formula 
##STR2## 
wherein 
R is a straight or branched chain alkyl group of 1-5 carbon atoms or an 
aralkyl group, 
A is a residue of peptidically bound proline or alanine, 
B is a residue of peptidically bound proline or alanine or excluded, 
X is selected from the group consisting of (CH.sub.2).sub.n and CH.dbd.CH 
groups, n being an integer from 1-3. 
The described peptides are obtained by reacting a compound of the formula 
EQU A--NH--R (2) 
wherein R and A are as described above with a compound of the formula 
EQU X--B--Ala (3) 
wherein B is as defined above and Y is a readily removed protecting group 
sucb as a benzyloxycarbonyl group. Following removal of the protecting 
group the resultant intermediate is reacted with a reactive derivative of 
dicarboxylic acid of the formula 
##STR3## 
wherein X is as defined above with its anhydride, hemihalogenide, 
hemi-ester or hemi-amide. 
The synthesis of these biologically active peptides is effected, from a 
mechanical standpoint, by fragment condensation in solution, by stepwise 
aminoacidic building or by synthesis with a solid carrier. 
Protecting groups employed in the practice of the present invention may be 
selected from among urethanes (benzyloxycarbonyl), groups which are 
unstable in weakly acidic media such as tert-butyloxycarbonyl or 
o-nitrobenzene sulphenyl, or groups capable of being reduced with metals 
or electrolytically, such as 2 halogene-ethyloxycarbonyl. 
Condensation reactions may be effected by the azide method, the 
carbondiimide method and the method of mixed anhydrides or other methods 
employed for preparing peptides.

Several examples of the practice of the present invention are set forth 
below. These examples are merely for purposes of exposition and are not to 
be construed as limiting. 
EXAMPLE 1 
Benzyloxycarbonyl Alanine Ethylamide 
14 ml. of N-ethyl piperidine was added to a solution of 23.3 grams of 
benzyloxycarbonyl alanine in 100 ml. of tetrahydrofuran and, after cooling 
to -10 degrees C., 14 ml. of sec. butyl chlorformate was added. Next, the 
mixture was stirred for 8 minutes and cooled to -6 degrees C. Then 30 ml. 
of a 4.1 solution of ethylamine in tetrahydrofuran was added. After 30 
minutes of stirring at 0 degrees C. and 2 hours of stirring at room 
temperature, the reaction solution was evaporated, gradually shaken with a 
5% solution of sodium hydrogen carbonate, with water, dried with anhydrous 
sodium carbonate, and evaporated. The evaporation residue was crystallized 
from ethyl acetate (30 ml.) and petroleum ether. 18.1 g of a product 
having a melting point of 116 to 117 degrees C. was obtained. 
Alanine Ethylamide Hydrobromide 
10 grams of benzyloxycarbonyl alanine ethylamide was treated with 40 ml. of 
36% hydrogen bromide in glacial acetic acid. Then, 150 ml. of ether was 
poured over the mixture and, after 1 hour, a crystalline product 
separated. The product was filtered off, thoroughly washed with ether and 
dried in a desiccator over phosphorus pentoxide and sodium hydroxide. 7.7 
g of the hydrobromide, having a melting point of 224 to 226 degrees C., 
was obtained. 
R.sub.f 0.26/S.sub.1, 0.58S.sub.2. 
S.sub.1 : n-butyl alcohol--acetic acid--water (4:1:1). 
S.sub.2 : n-butyl alcohol--acetic acid--pyridine--water (15:3:10:6). 
Benzyloxycarbonylalanyl-alanyl Alanine Ethylamide 
To a solution of 3.08 grams of benzyloxycarbonylalanyl alanine hydrazide in 
100 ml. of tetrahydrofuran there was added 4 ml. of an azeotropic 
hydrochloric acid solution comprising 690 mg. of sodium nitrate in 2.8 ml. 
of water, the addition being effected at a temperature of -12 degrees C. 
After 8 minutes of stirring and cooling to -10 degrees C., the reaction 
solution was diluted with 200 ml. of pre-cooled ethyl acetate. Then, an 
organic phase was extracted with a solution of sodium hydrogen carbonate 
in brine (3%) at -15 degrees C., dried with sodium sulfate and added to a 
pre-cooled solution (-10 degrees C.) of alanine ethylamide in 40 ml. of 
dimethylformamide prepared from 1.97 grams of N-ethylpiperidine. After 12 
hours at a temperature of 0 degrees C., the solution was evaporated and 
the solid evaporation residue crystallized from a mixture of 50 ml. of 
2-propyl alcohol and 50 ml. of dimethylformamide by the addition of 250 
ml. of water. 2.71 g of a product having a melting point of 271 to 273 
degrees C. was obtained. 
[.alpha.].sub.D.sup.20 -8.3.degree. (c 0.3; dimethylformamide). 
For C.sub.19 H.sub.28 N.sub.4 O.sub.5 (392.5): calculated: 58.15% C, 7.19% 
H, 14.28% N; found: 57.97% C, 7.27% H, 13.93% N. 
Alanyl-alanyl Alanine Ethylamide 
1 gram of Benzyloxycarbonylalanyl-alanyl-alanine ethylamide was treated 
with 3 ml. of 36% hydrogen bromide in acetic acid and after 1 hour, the 
original solution was treated with 30 ml. of ether. Separated hydrobromide 
was filtered off, dried for two hours in a desiccator over phosphorus 
pentoxide and sodium hydroxide, and then dissolved in 20 ml. of 90% 
aqueous methyl alcohol and deionized at Zerolit FF in OH-cyclus in methyl 
alcohol. Methanolic eluate was then evaporated and the evaporation residue 
dried by azeotropic distillation from a mixture of methyl alcohol and 
benzene. The evaporation residue was crystallized from 10 ml. of methyl 
alcohol (10 ml.) and 30 ml. of ether. 495 mg of a product having a melting 
point of 259 to 260 degrees C. was obtained. 
[.alpha.].sub.D.sup.20 -67.1.degree. (c 0.3; methyl alcohol). 
R.sub.f 0.14/S.sub.1 ; 0.58/S.sub.2. 
For C.sub.11 H.sub.22 N.sub.4 O.sub.3 (258.3): calculated: 51.15% C, 8.58% 
H, 21.69% N; found: 50.82% C, 8.63% H, 21.37% N. 
An analytical sample of alanyl-alanyl alanine ethylamide hydrobromide was 
crystallized from methyl alcohol and ether; melting point 284 to 289 
degrees C. 
For C.sub.11 H.sub.22 N.sub.4 O.sub.6.HBr (339.2): calculated: 38.95% C, 
6.83% H, 16.52% N; found: 38.70% C, 6.91% H, 16.18N. 
3-Carboxypropionylalanyl-alanyl Alanine Ethylamide 
300 mg. of succinic anhydride was added to a solution comprising 260 mg. of 
alanyl-alanyl alanine ethylamide in 5 ml. of dimethylformamide and 30 ml. 
of tetrahydrofuran. The reaction solution was heated for 2 hours under a 
reflux condenser. Then, a crystalline product separated and was filtered 
off, washed with tetrahydrofuran and ether. 180 mg. of a product having a 
melting point of 285 to 287 degrees C. was obtained by crystallization 
from water (13 ml.). 
For C.sub.15 H.sub.26 N.sub.4 O.sub.6 (359.2) [.alpha.].sub.D.sup.20 
-61.8.degree. (c 0.3; methyl alcohol): calculated: 50.16% C, 7.30% H, 
15.60% N; found: 50.10% C, 7.40% H, 15.61% N. 
4-Carboxybutyrylalanyl-alanyl Alanine Ethylamide 
This compound was prepared by a process analogous to that employed for the 
3-carboxypropionyl derivative by acylation with glutaric anhydride in a 
65% yield and having a melting point of 278 to 280 degrees C. 
[.alpha.].sub.D.sup.20 -86.0.degree. (c 0.3; 50% methyl alcohol). 
For C.sub.16 H.sub.28 N.sub.4 O.sub.5 (372.4): calculated: 51.60% C, 7.58% 
H, 15.04% N; found: 51.24% C, 7.68% H, 14.90% N. 
EXAMPLE 2 
Benzyloxycarbonyl Alanine Phenylethylamide 
This compound was prepared by a process analogous to that employed for the 
corresponding ethylamide described in EXample 1 in a yield of 87% and 
having a melting point of 125 to 126 degrees C. 
For CC.sub.19 H.sub.22 N.sub.2 O.sub.3 (326.3): [.alpha.].sub.D.sup.20 
-2.7.degree. (c 0.3; diemthylformamide): calculated: 69.94% C, 6.80% H, 
8.59% N; found: 70.32% C, 7.01% H, 8.70% N. 
Benzyloxycarbonylalanyl Alanine Phenylethylamide 
5.5 grams of N, N-dicyclohexyl carbodiimide was added to 4.9 grams of the 
solution of alanine phenylethylamide liberated from the corresponding 
benzyloxycarbonyl derivative released by 36% hydrogen bromide in acetic 
acid (R.sub.f 0.44/S.sub.1 ; 0.58/S.sub.2) and 5.6 grams of 
benzyloxycarbonyl alanine in 200 ml. of dimethylformamide cooled to -5 
degrees C. After 1 hour of stirring at 0 degrees of C. and 3 hours at room 
temperature, the separated N, N-dicyclohexylurea was filtered off, washed 
with dimethylformamide and the filtrate evaporated under vacuum. A 
crystalline evaporation residue was rubbed gradually with 1M hydrochloric 
acid, water, a 5% solution of sodium hydrogen carbonate, water, and 
crystallized from a mixture of 150 ml. of ethyl alcohol and 100 ml. of 
water. 7.75 g of a product having a melting point of 211 to 212 degrees C. 
was obtained. 
[.alpha.].sub.D.sup.20 -1.95.degree.; (c 0.3; dimethylformamide). 
For C.sub.22 H.sub.27 N.sub.3 O.sub.4 (3.97.5): calculated: 66.55% C, 6.85% 
H, 10.57% N; found: 66.89% C, 6.99% H, 10.74% N. 
Alanyl Alanine Phenylethylamide 
This compound was prepared by a process analogous to that employed for 
alanyl-alanyl alanine ethylamide in Example 1 in a 93% yield having a 
melting point of 107 to 110 degrees C. R.sub.f 0.31/S.sub.1 ; 
0.63/S.sub.2. 
[.alpha.].sub.D.sup.20 -0.44.9.degree. (c 0.3; methyl alcohol). 
For C.sub.14 H.sub.21 N.sub.3 O.sub.2 (263.4): calculated: 63.85% C, 8.04% 
H, 15.96% N; found: 63.38% C, 8.14% H, 16.20% N. 
3-Carboxypropionylalanyl Alanine Phenylethylamide 
This compound was prepared by a process analogous to that employed for 
3-carboxypropionylalanyl-alanyl alanine ethylamide in Example 1 in a 63% 
yield having a melting point of 209 to 211 degrees C. 
[.alpha.].sub.D.sup.20 -45.3.degree. (c 0.3; 50% methyl alcohol). 
For C.sub.18 H.sub.25 N.sub.3 O.sub.5 (364.2): calculated: 59.37% C, 6.92% 
H, 11.54% N; found: 59.22% C, 6.85% H, 11.82% N. 
4-Carboxybutyrylalanyl Alanine Phenylethylamide 
This compound was prepared by a process analogous to that employed for 
4-carboxybutyrylalanyl-alanyl alanine ethylamide in Example 1 in a 57% 
yield having a melting point of 208 to 209 degrees C. 
[.alpha.].sub.D.sup.20 -61.4.degree. (c 0.3; methyl alcohol). 
For C.sub.19 H.sub.27 N.sub.3 O.sub.4 (377.5): calculated: 60.46% C, 7.21% 
H, 11.13% N; found: 59.58% C, 7.19% H, 11.25% N. 
EXAMPLE 3 
Proline Ethylamide 
40 ml. of 36% hydrogen bromide in acetic acid was added to 11 grams of a 
solution of benzyloxycarbonyl proline ethylamide in 10 ml. of acetic acid. 
After 1 hour 300 ml. of ether was added to the reaction solution, so 
resulting in the separation of a non-crystalline hydrobromide. The 
hydrobromide was dried for 2 hours in desiccator over phosphorus pentoxide 
and sodium hydroxide, and then suspended in 60 ml. of a saturated 
amoniacal solution of chloroform at 0 degrees C. The resultant suspension 
was permitted to stand at 3 degrees C. for 20 minutes and then filtered, 
the filtrate being evaporated. 5.7 g of a non-crystalline product was 
obtained. R.sub.f 0.25/S.sub.1 ; 0.56/S.sub.2. 
Benzyloxycarbonylalanyl Proline Ethylamide 
9.0 grams of N, N-dicyclohexylcarbodiimide was added to a solution of 5.6 
grams of proline ethylamide and 9.0 grams of benzyloxycarbonyl alanine in 
a mixture of 5 ml. of dimethylformamide and 60 ml. of tetrahydrofuran 
cooled to -5 degrees C. After 1 hour of stirring at 0 degrees C., the 
reaction mixture was permitted to stand for 12 hours at room temperature, 
so resulting in the separation of N, N-dicyclohexylurea which was filtered 
off. The filtrate was evaporated; the evaporation residue dissolved in a 
mixture of ethyl acetate and water. The organic phase formed was gradually 
shook with 1M hydrochloric acid, water, 5% sodium hydrogen carbonate, with 
water, dried with anhydrous sodium sulphate and evaporated. The 
evaporation residue was crystallized from a mixture of 30 ml. of ether and 
30 ml. of petroleum ether. 5.4 g of a product having a melting point of 99 
to 101 degrees C. was obtained. 
[.alpha.].sub.D.sup.20 -93.4.degree. (c 0.3; methyl alcohol). 
For C.sub.18 H.sub.25 N.sub.3 O.sub.4 (347.4): calculated: 62.23% C, 7.25% 
H, 12.09% N; found: 62.13% C, 7.44% H, 11.70% N. 
Alanyl Proline Ethylamide 
This compound was prepared by a process analogous to that employed for 
proline ethylamide in quantitative yield from the corresponding 
benzyloxycarbonyl derivative. R.sub.f 0.13/S.sub.1 ; 0.57/S.sub.2. 
3-Carboxypropionylalanyl Proline Ethylamide 
500 mg of succinic anhydride was added to a solution of 500 mg of alanyl 
proline ethylamide in form of dioxane (10 ml) and 0.25 ml of 
dimethylformamide and the reaction solution heated under reflux for 30 
minutes. Then the solution was evaporated and the residue crystallized 
from 10 ml of acetone and 30 ml of ether. 510 mg of a product having a 
melting point of 139 to 141 degrees C. was obtained. 
[.alpha.[.sub.D.sup.20 -129.2 degrees (c 0.3; 50% methyl alcohol). 
For C.sub.15 H.sub.26 N.sub.4 O.sub.6 (359.2): calculated: 53.53% C, 7.38% 
H, 13.38% N; found: 52.90% C, 7.45% H, 12.98% N. 
EXAMPLE 4 
Benzyloxycarbonylalanyl-ananyl Proline Ethylamide 
1.6 g of N,N-dicyclohexylcarbodiimide was added to a solution of alanyl 
proline ethylamide prepared from 2.45 g of a benzyloxycarbonyl derivative 
by the reaction of 36% hydrogen bromide in acetic acid followed by 
liberation at Merolit FF in OH-cycle in 20 ml of dimethylformamide and 
1.55 grams of benzyloxycarbonyl alanine cooled to -5 degrees C. After 1 
hour of stirring at 0 degree C and 12 hours standing at 3 degrees C., 
N,N-dicyclohexylurea separated and was filtered off, the filtrate being 
evaporated. A non-crystalline evaporation residue was then dissolved in 50 
ml of ethyl acetate and after 12 hours standing at 3 degrees C. the 
separated product was filtered off. 1.75 g of a product having a melting 
point of 138 to 139 degrees C. was obtained. [.alpha.].sub.D.sup.20 -47.0 
degrees (c 0.3; dimethylformamide). 
For C.sub.21 H.sub.30 N.sub.4 O.sub.5 (418.5): calculated: 60.27% C, 7.23% 
H, 13.39% N; found: 59.97% C, 7.21% H, 13.12% N. 
Benzyloxycarbonylalanyl-alanyl Proline Ethylamide 
11 grams of N,N-dicyclohexylcarbodiimide was added to a solution of 14.7 
grams of benzyloxycarbonylalanyl alanine in 100 ml of dimethylformamide 
and 7.6 g of proline ethylamine (prepared from the corresponding 
benzyloxycarbonyl derivative) cooled to -5 degrees C. and the reaction 
mixture treated by conventional techniques. 10.6 g of product having a 
melting point of 139 to 140 degrees C. was obtained. 
[.alpha.].sub.D.sup.20 -46.5 degrees (c 0.3; dimethylformamide). 
For C.sub.21 H.sub.30 N.sub.4 O.sub.5 (418.5): calculated: 60.27% C, 7.23% 
H, 13.39% N; found: 60.08% C, 7.55% H, 13.32% N. 
Alanyl-alanyl Proline Ethylamide 
This compound was prepared from the corresponding benzyloxycarbonyl 
derivative by the procedure employed in Example 1 for the preparation of 
alanyl-alanyl alamine ethylamide. The product ws obtained in a 69% yield 
with a melting point of 109 to 111 degrees C. R.sub.f 0.14/S.sub.1 ; 
0.42/S.sub.2. 
[.alpha.].sub.D.sup.20 -126.1 degrees (c 0.3; methyl alcohol). 
For C.sub.13 H.sub.24 N.sub.4 O.sub.3 (284.4) calculated: 54.91% C, 8.51% 
C, 8.51% H, 19.70% N; found: 54.43% C, 8.61% H, 19.44% N. 
4-Carboxybutyrylalanyl-alanyl Proline Ethylamide 
1.7 g of glutaric anhydride was added to a solution of 4.0 grams of 
alanyl-alanyl proline ethylamide in 25 ml of dimethylformamide and the 
reaction solution heated for 1 hour at 60 degrees C. Then, the solution 
was evaporated and the resultant non-crystalline evaporation residue 
dissolved in 30 ml of ethyl acetate. After 48 hours of standing at 3 
degrees C., 3.6 g of separated product was filtered off with a melting 
point of 154 to 156 degrees C. ([.alpha.].sub.D.sup.20 -137.3 degrees (c 
0.3; 50% methyl alcohol). 
For C.sub.18 H.sub.30 N.sub.4 O.sub.4 (398.5): calculated: 54.26% C, 7.59% 
H, 14.06% N; found: 53.96% C, 7.75% H, 13.87% N. 
3-Carboxypropionylalanyl-alanyl Proline Ethylamide 
This compound was prepared in the same manner described for 
3-carboxypropionylalanyl-alanyl alanine ethylamide in a yield of 71% and 
it exhibited a melting point of 185 to 186 degrees C. 
[.alpha.].sub.D.sup.20 -69.3 degrees (c 0.3; dimethylformamide). 
For C.sub.17 H.sub.28 N.sub.4 O.sub.6 (385.2): calculated: 53.01% C, 7.33% 
H, 14.55% N; found: 52.75% C, 7.45% H, 14.44% N. 
EXAMPLE 5 
Benzyloxycarbonyl Alanine Isobutylamide 
14 ml of chloro-sec. butyl formate added at -10 degrees C. to a solution of 
23 grams of benzyloxycarbonyl alanine in 200 ml of methylene chloride and 
14 ml of N-ethyl piperidine, 20.7 g of a product having a melting point of 
109 to 111 degrees C. was obtained by the procedure described in Example 1 
by crystallization from 60 ml of ethyl acetate and 200 ml of petroleum 
ether [.alpha.].sub.D.sup.20 8.7 degrees (c 0.3; dimethylformamide). 
For C.sub.15 H.sub.22 N.sub.2 O.sub.3 (278.4): calculated: 64.73% C, 7.97% 
H, 10.06% N; found: 64.45% C, 8.01% H, 9.83% N. 
Alanine Isobutylamide 
This compound was prepared from the corresponding benzyloxycarbonyl 
derivative by a process analogous to that described in Example 1. 
R.sub.f 0.32 (S.sub.1 ; 0.69)S.sub.2. 
Benzyloxycarbonylalanyl-alanyl Alanine Isobutylamide 
This compound was prepared by a process analogous to that employed for the 
corresponding benzyloxycarbonylalanyl-alanyl alanine ethylamide in a 58% 
yield having a melting point of 260 to 262 degrees C. 
[.alpha.].sub.D.sup.20 -10.9 degrees (c 0.3; dimethylformamide). 
For C.sub.12 H.sub.32 N.sub.4 O.sub.5 (420.5): calculated: 59.98% C, 7.67% 
H, 13.32% N; found: 59.63% C, 7.70% H, 13.20% N. 
Alanyl-alanyl Alanine Isobutylamide 
This compound was prepared by a process analogous to that employed from the 
corresponding alanyl-alanyl alanine ethylamide in a 76% yield with a 
melting point of 249 to 252 degrees C. R.sub.f 0.28(S.sub.1 ; 
0.68)S.sub.2. 
[.alpha.].sub.D.sup.20 -66.4 degrees (c 0.3; methyl alcohol). 
For C.sub.13 H.sub.26 N.sub.4 O.sub.3.H.sub.2 O (304.4) calculated: 51.30% 
C, 9.27% H, 18.41% N; found: 51.10% C, 8.73% H, 18.71% N. 
An analytical sample of alanyl-alanyl alanine isobutylamide hydrobromide 
was crystallized from methyl alcohol and ether, having a melting point of 
214 to 216 degrees C. 
For C.sub.13 H.sub.26 N.sub.4 O.sub.3.HBr (367.3) calculated: 42.51% C, 
7.41% H, 15.25% N; found: 42.54% C, 7.36% H, 15.23% N. 
3-Carboxypropionylalanyl-alanyl Alanine Isobutylamide 
This compound was prepared by a process analogous to that employed for the 
corresponding ethylamide derivative in an 81% yield with a melting point 
of 283 to 286 degrees C. [.alpha.].sub.D.sup.20 -58.2 degrees (c 0.3; 
dimethylformamide). 
For C.sub.17 H.sub.30 N.sub.4 O.sub.6 (387.2): calculated: 52.74% C, 7.81% 
H, 14.47% N; found: 52.34% C, 7.93% H, 14.58% N. 
4-Carboxybutyrylalanyl-alanyl Alanine Isobutylamide 
This compound was prepared by a process analogous to that employed for the 
ethylamide derivative described in Example 1 in a 69% yield with a melting 
point of 292 to 293 degrees C. [.alpha.].sub.D.sup.20 -82.7 degrees (c 
0.3; 50% methyl alcohol). 
For C.sub.18 H.sub.32 N.sub.4 O.sub.5 (400.5): calculated: 53.99% C, 8.05% 
H, 13.99% N; found: 54.33% C, 7.88% H, 14.19% N. 
EXAMPLE 6 
Benzyloxycarbonyl Alanine Propylamide 
This compound was prepared by a process analogous to that employed in the 
preparation of carbonyl alanine ethylamide in Example 1 in an 87% yield 
with a melting point of 118 to 119 degrees C. [.alpha.].sub.D.sup.20 8.4 
degrees (c 0.3; dimethylformamide). 
For C.sub.14 H.sub.20 N.sub.2 O.sub.3 (264.3) calculated: 63.62% C, 7.63% 
H, 10.60% N; found: 63.64% C, 7.74% H, 10.46% N. 
Benzyloxycarbonylalanyl-alanyl Alanine Propylamide 
This compound was prepared by a process analogous to that employed in 
preparing the corresponding ethylamide described in Example 1 in a 56% 
yield with a melting point of 268 to 269 degrees C. [.alpha.].sub.D.sup.20 
-10.6 degrees (c 0.3; dimethylformamide). 
For C.sub.20 H.sub.30 N.sub.4 O.sub.5 (406.5): calculated: 59.10% C, 7.44% 
H, 13.78% N; found: 58.74% C, 7.61% H, 123.78% N. 
Alanyl-alanyl Alanine Propylamide 
This compound was prepared by a process analogous to that employed in 
preparing alanyl-alanyl alanine ethylamide described in Example 1 in a 71% 
yield with a melting point of 265 to 268 degrees C. R.sub.f : 0.18/S.sub.1 
; 0.57/S.sub.2. 
[.alpha.].sub.D.sup.20 -82.1 degrees (c 0.3; 50% methyl alcohol). 
For C.sub.12 H.sub.24 N.sub.4 O.sub.3 (272.4): calculated: 52.92% C, 8.88% 
H, 20.57% N; found: 53.15% C, 8.87% H, 20.54% N. 
3-Carboxypropionylalanyl-alanyl Alanine Propylamide 
This compound was prepared by a process analogous to that employed in 
preparing the corresponding ethylamide derivative described in Example 1 
in an 88% yield and a melting point of 284 to 287 degrees C. 
[.alpha.].sub.D.sup.20 -59.1 degrees (c 0.3; 50% methyl alcohol). 
For C.sub.16 H.sub.28 N.sub.4 O.sub.6 (373.2): calculated: 51.50% C, 7.56% 
H, 15.01% N; found: 51.69% C, 7.52% H, 14.67% N. 
4-Carboxybutyrylalanyl-alanyl Alanine Propylamide 
This compound was prepared by a process analogous to that employed in 
preparing the corresponding ethylamide derivative described in Example 1 
in an 89% yield with a melting point of 283 to 285 degrees C. 
[.alpha.].sub.D.sup.20 -84.7 degrees (c 0.3; 50% methyl alcohol). 
For C.sub.17 H.sub.30 N.sub.4 O.sub.5 (386.5): calculated: 52.84% C, 7.82% 
H, 14.50% N; found: 53.24% C, 7.92% H, 14.55% N. 
EXAMPLE 7 
Benzyloxycarbonyl Proline Propylamide 
This compound was prepared by a process analogous to that employed in 
preparing the corresponding ethylamine derivative of benzyloxycarbonyl 
alanine described in Example 1 in an 83% yield with a melting point of 74 
to 75 degrees C. 
For C.sub.16 H.sub.22 N.sub.2 O.sub.3 (290.4): calculated: 66.19% C, 7.64% 
H, 9.65% N; found: 66.05% C, 7.41% H, 9.52% N. 
Proline Propylamide 
This compound was prepared by a process analogous to that employed in 
preparing proline ethylamide in Example 3. R.sub.f 0.21(S.sub.1 ; 
0.62)S.sub.2. 
Benzyloxycarbonylalanyl Proline Propylamide 
This compound was prepared by a process analogous to that employed in 
preparing the corresponding ethylamide described in Example 3 in a 73% 
yield with a melting point of 117 to 120 degrees C. 
For C.sub.19 H.sub.27 N.sub.3 O.sub.4 (361.4): calculated: 63.14% C, 7.53% 
H, 11.63% N; found: 63.03% C, 7.39% H, 11.28% N. 
Alanyl Proline Propylamide 
This compound was prepared by a process analogous to that employed in 
preparing the corresponding ethylamide described in Examples 1 and 3 and a 
79% yield. R.sub.f 0.14 (S.sub.1 ; 0.49)S.sub.2. 
Benzyloxycarbonylalanyl-alanyl Proline Propylamide 
This compound was prepared by a process analogous to that employed in 
preparing benzyloxycarbonyl alanine ethylamide from benzyloxycarbonyl 
alanine and alanyl proline propylamide in a 71% yield with a melting point 
of 101 to 103 degrees C. [.alpha.].sub.D.sup.20 -47.7 degrees (c 0.3; 
dimethylformamide). 
For C.sub.22 H.sub.32 N.sub.4 O.sub.5 (432.5): calculated: 61.09% C, 7.46% 
H, 12.95% N; found: 61.11% C, 7.69% H, 12.98% N. 
Alanyl-alanyl Proline Propylamide 
This compound was prepared by a process analogous to that employed in 
preparing the corresponding ethylamide described in Example 4 in a 68% 
yield with a melting point of 103 to 105 degrees C. R.sub.f 0.12(S.sub.1 ; 
0.56)S.sub.2. 
[.alpha.].sub.D.sup.20 -135.3 degrees (c 0.3; methyl alcohol). 
For C.sub.14 H.sub.26 N.sub.4 O.sub.3 (298.4): calculated: 56.35% C, 8.78% 
H, 18.78% N; found: 55.87% C, 8.93% H, 18.78% N. 
4-Carboxybutyrylalanyl-alanyl Proline Propylamide 
This compound was prepared by a process analogous to that employed in 
preparing the corresponding ethylamide described in Example 4 in a 73% 
yield with a melting point of 131 to 134 degrees C. [.alpha.].sub.D.sup.20 
=148.7 degrees (c 0.3; 50% methyl alcohol). 
For C.sub.19 H.sub.32 N.sub.4 O.sub.4 (412.5): calculated: 55.32% C, 7.82% 
H, 13.58% N; found: 55.25% C, 7.99% H, 13.74% N. 
EXAMPLE 8 
Benzyloxycarbonylprolyl Alanine Methyl Ester 
11.0 grams of N,N-dicyclohexylbodiimide was added to a solution of 12.45 
grams of benzyloxycarbonyl proline and alanine methyl ester released from 
7.0 grams of the corresponding hydrochloride using 7 ml of N-ethyl 
piperidine in 40 ml of methylene chloride cooled to -5 degrees C. The 
resultant reaction suspension was stirred for 1 hour at 0 degree C. 
N,N-dicyclohexylurea separated from the suspension and was filtered off 
after it was allowed to stand for 12 hours at 3 degrees C. Then, the 
filtrate was successively washed with 1M hydrochloric acid, water, 5% 
sodium hydrogen carbonate, with water, dried with anhydrous sodium sulfate 
and evaporated. A solid evaporation residue was crystallized from 100 ml 
of ethyl acetate and 500 ml of petroleum ether. 12.2 g of a product having 
a melting point of 45 to 49 degrees C. was obtained. 
Benzylcarbonylprolyl Alanine Hydrazide 
7.5 ml of 80% hydrazine-hydrate was added to a solution of 10.0 grams of 
benzyloxycarbonylprolyl alanine methyl ester in 100 ml of methyl alcohol 
and the reaction solution heated under reflux for 2 hours. The solution 
was then evaporated and a solid evaporation residue was crystallized from 
40 ml of water. 6.8 g of a product having a melting point of 142 to 143 
degrees C. was obtained. 
For C.sub.16 H.sub.22 N.sub.4 O.sub.4 (334.4): calculated: 57.47% C, 6.63% 
H, 16.76% N; found: 57.41% C, 6.85% H, 16.90% N. 
Benzyloxycarbonylprolyl-alanyl Alanine Ethylamide 
This product was prepared by a process analogous to that employed in 
preparing the corresponding benzyloxycarbonylalanylalanyl alanine 
ethylamide described in Example 1 from benzyloxycarbonylprolyl alanine 
hydrazide and alanine ethylamide in a 49% yield with a melting point of 
219 to 220 degrees C. [.alpha.].sub.D.sup.20 -36.2 degrees (c 0.3; 
dimethylformamide). 
For C.sub.21 H.sub.30 N.sub.4 O.sub.5 (418.5): calculated: 60.27% C, 7.23% 
H, 13.39% N; found: 60.08% C, 7.41% H, 13.22% N. 
Propyl-alanyl Alanine Ethylamide 
This compound was prepared by a process analogous to that employed in 
preparing alanyl-alanyl alanine ethylamide described in Example 1 in a 53% 
yield with a melting point 216 to 219 degrees C. R.sub.f : 0.07(S.sub.1 ; 
0.54)S.sub.2. 
For C.sub.13 H.sub.24 N.sub.4 O.sub.3 (284.4): calculated: 54.91% C, 8.51% 
H, 19.17% N; found: 54.49% C, 8.42% H, 19.39% N. 
4-Carboxybutyrylprolyl-alanyl Alanine Ethylamide 
This compound was prepared by a process analogous to that employed in 
preparing 4-carboxybutyryl-alanyl-alanyl alanine ethylamide described in 
Example 1 in a 44% yield with a melting point of 172 to 177 degrees C. 
For C.sub.18 H.sub.30 N.sub.4 O.sub.4 (398.5): calculated: 54.26% C, 7.59% 
H, 14.06% N, found: 54.81% C, 7.43% H, 14.38% N. 
EXAMPLE 9 
3-Carboxyacryolylalanyl-alanyl Alanine Ethylamide 
200 mg of maleic anhydride was added to a solution of 260 mg of 
alanyl-alanyl ethylamide in 20 ml of dimethylformamide heated to 60 
degrees C. and the reaction solution heated for 30 minutes at 80 degrees 
C. Then, the reaction mixture was evaporated and the evaporation residue 
crystallized from dimethylformamide and 2-propyl alcohol. 185 mg of a 
product having a melting point of 260 to 266 degrees C. (decomposition) 
was obtained. [.alpha.].sub.D.sup.20 -35.6 degrees (c 0.3; 
dimethylformamide). 
For C.sub.15 H.sub.24 N.sub.4 O.sub.6 (356.4): calculated: 50.55% C, 6.79% 
H, 15.72% N; found: 49.97% C, 7.13% H, 15.83% N.