NO134296B - - Google Patents

Description

the present invention relates to the production of

new, pharmacologically active hepta- and octa<p>eptides with the formula:

where 'R means hydrogen, succinyl, L-aspartyl, sarkosyl, L-seryl, succinamyl, L-prolyl, glycyl or D- or L-asparaginy1, R. means an L-

alanine, L- or D-leucine, glycine, L-isoleucine or P-alanine residue and Rg means L-valyl, L-isoleucyl or L-alanyl.

The compounds produced according to the invention have a pharmacological effect. They can inhibit the pressor effect of angiotensinamide on blood pressure. Thus, when administered by intravenous infusion to live rats in as little as 20 micrograms/kg/minute, the pressor effect of angiotensinamide administered in a similar manner is inhibited. Because of this inhibitory property on a blood pressure increase caused by angiotensinamide, the above peptides are valuable agents for counteracting hypertension resulting from angiotensinamide. They can also inhibit hypertension in rats with acute unilateral renal hypertension after intravenous infusion.

The procedure for foremost in II. ing of the new, pharmacologically active polypeptides with the above-mentioned formula is characterized by the stepwise linking of the acids HR-^, proline, histidine, Rg-OH, using common methods within the peptide core. tyrosine, valine, arginine and, in the event that R is not hydrogen, also the acid ROH, where R-^ and Rg and R have the above meanings s for the formation of amide bonds between the acids, whereby amino and carboxyl groups that one does not wish to participate in the reaction, on

in a manner known per se is temporarily masked with a protecting group or prevented from participating in the reaction in another known manner.

The hepta- and octapeptides are thus produced in a simple manner using known methods for the production of peptides. Such methods involve the building up of a linear chain of amino acids by repeated amide bonds, whereby in the resulting chain formation the necessary protective groups are used which are amenable to rapid removal by means of normal cleavage methods. The application of such methods to the above-mentioned peptides will be described in the following with reference to the following exemplary embodiment. L- asparaginyl- L- arginyl- L- valyl- L- tyrosyl- L- valyl- L- histidyl- L- prolyl- L- alanine: BOC-Asn-ArgfN02)-Val-Tyrf0-Bzl)-Val-HisfM< lm->Bzl)-Pro-Ala polymer fA)

30 C-Ala resin ester (5 g, 0.5 mmol/g) was placed in a 20 C ml Merrifield rocking type reaction vessel. The resin was swollen in chloroform (analytical grade) by rocking for 20 minutes and then washed with three 50 ml portions of glacial acetic acid. The time for each washing operation was 3-5 minutes, the t-butoxycarbonyl protecting group (BOC) was removed with 1 N HCl in anhydrous acetic acid by rocking

for 40 minutes. The resin was washed three times and in the same order with acetic acid, absolute ethanol and N,N-dimethylformamide. The resulting hydrochloride of alanyl resin ester was neutralized with a 10% solution of triethylamine in dimethylformamide by rocking for 10 minutes. Then the resin was washed with three portions each of dimethylformamide, absolute ethanol, chloroform and methylene chloride and a solution of 8.5 mmol (threefold excess) of BOC-proline in 40 ml of methylene chloride was added. The reaction container was then rocked for 20 minutes to give the amino acid derivative sufficient time to penetrate the resin. Then 8.5 mmol of N,N'-dicyclohexylcarbodiimide (DDC) in 10 ml of methylene chloride were added and the coupling was allowed to take place for 12 hours under rocking. The resin was then washed with three portions each of methylene chloride, absolute ethanol and acetic acid and was thus ready for the next removal operation of protecting groups with HC1 in acetic acid as described above. The washing, neutralization and coupling steps were carried out according to the -described method using BQG-His(N<lm->Bzl)--OH, BOC-Vai-OH, BOC-Tyr(0-Bzl)-OH and BOC -ArgfNO 2 )-OH. A change was made in the coupling steps for B0C-His(N<im->B-zl)-OH and B0C-Arg(N02)-OH, and in these cases a dimethylformamide/methylene chloride mixture was used as solvent (2 :1). The coupling of Asn to the deprotected resin heptapeptide was carried out using BOG-Asn-ONP (BOC-Asparagine-p-nitrophenyl ester) in dimethylformamide by rocking the mixture for 7 hours.

After the final coupling step, the resin peptide was washed with dimethylformamide, ethanol, acetic acid and ethanol and dried in vacuo over P 2 O 3 . The weight of the resin peptide was 7-3 g.

H- Asn- Arg( NO2 )- Val- Tyr( O- Bzl)- Val- His( Nim- Bzl)- Pro- Ala- OH. 2HBr (B)

The resin peptide (A) (7-3g) prepared as described above was suspended in 25 ml of dry trifluoroacetic acid and a stream of dry HBr was quickly passed through the solution at low speed. After 20 minutes, the resin was filtered off and treated a further time with HBr/CFoCOOH for 40 minutes. The filtrate was evaporated to dryness in vacuo at 20 oC, the oily residues were precipitated with absolute ether and the product dried in a desiccator over potassium hydroxide. The total yield was 1980 mg (59-8%) of the protected octapeptide dihydrobromide.

H- Asn- Arg- Val- Tyr- Val- His- Pro- Ala- OH (O

The protected peptide (B) (1.0 g). was dissolved in 20 ml of

an acetic acid/dioxane/water mixture (4:4;1>v/v) and was hydrogenolyzed

over Pd/BaSO^(10% catalyst (0.5 g)) for 48 hours at atmospheric pressure. A new portion (0.2 g) of catalyst was then added and the hydrogenation was continued for 24 hours. The filtered and diluted solution was lyophilized. The yield was 750 mg (m.p. 150<0>-165°C).

(Calculated for dihydrobromide: 15-32% Br. Found: 13.36% Br).

The crude product was purified by gel filtration chromatography on Sephadex G-25n in 0.2 M acetic acid. The fractions containing the pure peptide were combined and lyophilized. The purified compound contained only traces of halogen.

TLC: Merck cellulose F plates, n-butanol/acetic acid/water solvent system (6:2:3 v/v), R- 0.35- Merck

silica gel F-254 plates with the same solvent system, R^0.13.

l~ ajl°= -59-4° (c = 0.2, 1 N AcOH) /"a_/|°8=-6l.l° (c = 0.2, 1 N AcOH)

Amino acid analysis:

Ala: 1.00; Arg: 1.00; Donkey: 1.09; Elevator: 0.90; .Pro: 1.04;

Taurus: 0.75; Val: 1.94-

In a similar way, other peptides were prepared according to the invention by introducing the necessary unit on . a suitable stage. The peptides produced in this way are listed in the tables below:

In the above tables, in accordance with the UIFAC nomenclature to distinguish the L- and D-forms of amino acids in <p>olype<p>times, L-amino acids are marked with a large initial while D-amino acids are marked with a small initial.

Claims (1)

Analogous method for the production of new, therapeutically effective hepta- and octapeptides with the formula: where R means hydrogen, succinyl, L-aspartyl, sarkosyl, L-seryl, succinamyl, L-prolyl, glycyl or D- or L-asparaginy1, R^ means an L-alanine-, L- or D-leucine-, glycine -, L-isoleucine or 3-alanine residue and R2 means L-valyl, L-isoleucyl or L-alanyl, characterized in that, using common methods in peptide chemistry, the acids HR-, , proline, histidine, R2-OH , tyrosine. valine, arginine and, in cases where R is not hydrogen, also the acid ROH. where R-^, R2 and R have the above-mentioned meanings, to form amide bonds between the acids, as amino and carboxyl groups that you do not want to participate in the reaction are masked•temporarily with a protecting group in a manner known per se another known way is prevented from participating in the reaction.