CS229893B1 - Oxytocin and vasopressin analogues with inhibitory effects and method of their production - Google Patents

Abstract

The invention relates to oxytocin and vasopressin analogues of the chemical formula I where Pen represents a penicillamine residue, X represents a hydrogen atom or an acetyl group, Y represents a hydrogen atom or a methyl group, A represents a leucine or phenylalanine residue and B represents a leucine or lysine residue, and a method for their production. These analogues are characterized by having inhibitory (antagonistic) effects on some biological activities of neurohypophyseal hormones. The essence of the method for the production of oxytocin and vasopressin analogues lies in the fact that linear peptides of the formula II, where Pen, X, Y, A β B have the same meaning as in formula I, containing two sulfhydryl groups, are oxidized to form a disulfide bond by a known procedure.

Description

The subject of the invention are analogs of oxytocin and vesopressin and modified amino acids in position 1 and optionally 2 and a method for their production.

These are analogues that have an inhibitory effect on some biological activities of neurohypophyseal hormones.

Oxytocin analogues, having an inhibitory effect on the biological activities of neurohypophyseal hormones, are of considerable importance both in proving the presence of these hormones in various body fluids and in terms of potential medical use, i.e. in pathological overproduction of these hormones.

So far, two main structural features have been known, the presence of which in the oxytocin molecule causes inhibitory effects: replacement of the hydroxyl group of tyrosine in position 2 with a methoxyl group (Beránková Z., Rychlík I., Joět K., Rudinger J., Šorm F.: Collection Czechoalov. Chám. Commun. 26. 2 673 /196)/), while the inhibitory effect can be enhanced by simultaneous acetylation of the alpha-amino group of cysteine in position 1 (Joět K., Šorm F.: Collection Czechoalov. Chám. Commun. 36. 297 /1971/). The introduction of 2 methyl groups (Schulz H., du Vigneaud V.: J. Med. Chem. 2, 647 /1966/) or two ethyls (Vevrek RJ, Ferger MF, Allen 0. A., Hloh DH, Blomquiat AT, du Vigneaud V.: J. Med. Chám. 12, ¹²⁴ /1972/) or a polymethylene chain (Nestor JJ, Ferger MF, du Vigneaud V.: J. Med. Chem. J8, 284 /1975/), into the beta position - beta-mercaptopropionic acid into position 1 in combination with alkyl tyrosine (Sawyer WH, Grzonka Z., Manning M.: Mol. Cell. Endocrinol. 22, 117 /1981/) creates a different type of effective inhibitors from these substances.

We have now discovered that even the mutual combination of structural modifications, such as alkylation of tyrosine and introduction of two methyl groups into the beta position of cysteine in position 1 of the peptide chain and possible acetylation of the primary cysteine group, gives rise to substances with significant inhibitory effects on the action of oxytocin or vesopressin.

The invention is based on new analogues of oxytocin and vesopressin of the general formula I. X-Pen-Tyr(Y)AA-Gln-Asn-Žy s-Pro-B-Gly-NHg (I) where all amino acids are L-series. Pan denotes a pentylamine (beta,beta-dimethylcysteine) residue, X denotes a hydrogen atom or an acetyl group, Y denotes a hydrogen atom or a methyl group, A denotes an isoleucine or phenylalanine residue, B denotes a leucine or lysine residue, Tyr denotes tyrosine, Gln denotes glycine, Asn Aaparagin, Cya denotes cysteine, Pro proline,

Gly glycine.

The essence of the process for producing new oxytocin and vesopressin analogues of formula I according to the invention is that the linear peptide of formula II

H H

I I

X-Pen-Tyr(YJ-A-Gln-Aan-Cys-Pro-B-Gly-NHg (II), where Pan, X, Y, A and B have the same meaning as in formula I, is oxidized to form a disulfide bond in a known manner.

Some biological effects of oxytocin and vasopressin analogues are listed in Table I. The inhibitory effects of substances in the in vivo test are expressed in terms of pAg values, which is the negative logarithm of the molar concentration of the inhibitor that reduces the response of 2X units of agonist to be equal to the response of 1X units in the absence of the inhibitor (Schild H. 0.: Brit. J. Pharmacol. 2, 189 /1974/; Dyckea DF, Nestor JJ, Ferger MF, du Vigneaud V.: J. Med. Chem. 17, 260 /1974/). In the in vitro test (uterus in vitro), the pAg value corresponds to the negative logarithm of the inhibition constant according to the relationship pA ₂ = -log where B is the concentration of the Inhibitor (Μ), A^θ is the concentration of the agonist leading to 50% maximal effect and A ₅₀ B is the concentration of the agonist leading to 50% maximal effect in the presence of inhibitor B (Eggena P., Schwertz IL, Valter R.: J. Gen. Physiol. 52. 465 /1968/).

T a b l e 1 k a 1

X Analog I Y A B Inhibitory activity (pA ₂ ) in determining the effect of uterotonic presorl galactogogue in vitro in vivo CH-jCO H Ile Leu 4.4 - ineffective 0.04® H ch ₃ Ile Leu 8.0 6.9 7.2 6.8 _CH3CO ch ₃ Ue Leo 5.4 6.4 0.45® H H Phe Lys 6.6 6.3 6.9 - CH-jCO H Phe Lys 5.5 6.3 ineffective ineffective H ch ₃ Phe Lys 8.1 6.9 7.4 6.8 ch ₃ what ch ₃ Phe Lys 6.7 ineffective

⁸ intrinsic activity, IU/mg

The method of producing oxytocin analogs is explained in more detail in the examples.

Methods used in the examples:

Amino acid analyses were performed on an automatic instrument (Development Workshops of the Czechoslovak Academy of Sciences, type 6020). Peptide samples were hydrolyzed with 6M-HCl in a vacuum of 150 Pa at 105 °C for 20 hours. Thin-layer chromatography was performed on silica gel plates (Silufol, Kevalier) in the systems: 2-butanol - 98% formic acid - water (75:13.5:11.5; Sl), 2-butanol - 25% aqueous ammonia - water (85:7.5:7.5, S2), 1-butanol - acetic acid - water (4:1:1, S3) and 1-butanol - pyridine - acetic acid - water (15:10:3:6, S4) and methanol-chloroform-acetic acid - water (10:15:3:2, S6). Analytical electrophoresis was performed on Vhatman 3MM paper in a humid chamber at a potential gradient of 20V/cm for 1 hour in 1M acetic acid (pH 2.4) and in pyridine-acetate buffer (pH 5.7). The substances were detected by ninhydrin or chlorination method. For countercurrent shaking, an electrophoresis apparatus with the possibility of shifting the upper and lower phases was used.

Preparative carrier-free electrophoresis was performed on a previously described apparatus (Prusík Z., Sedlákové E., Barth T.: Hoppe-Seyler s Z. Physiol. Chem. 353. 1 .837 /1972/). For high-performance liquid chromatography, an apparatus assembled from commercial parts and a column packed with Separon SI-C-18 (Laboratory Instruments, Prague) was used.

Example 1

o-Nltrobenzenesulfenyl-O-tert.butyltyrosyl-isoleucyl-glutaminyl-asparaginyl-S-benzylcysteinyl-propyl-leucyl-glyein amide

To a solution of isoleucyl-glutaminyl-eapareglnyl-S-benzyl-leuyl-propyl-leuyl-glycine amide (1.27 g; Jošt K., Rudinger J., Šorm F.: Collection Czechoslov. Chem· Commun. 26. 2 496 /196,/) in dimethylformamide (25 ml) was added N-hydroxysuccinimide ether o-nitrobenzenesulfenyl-O-tert.butyltyrosine (0.56 g).

After two days of stirring, another portion of the active ester (0.05 g) was added and stirring was continued for another 24 hours. The dimethylformamide was evaporated, the residue was triturated with petroleum ether and ether, filtered off and washed with water, 0.1 M-H^SO., water, 0.25 M-NaHCOj and water. 1.2 g (65 *) of product were obtained, m.p. 225-229 °C, 0° (c 0.2, dimethylformamide). Rj, 0.51 (S1), 0.36 (S2), 0.65 (S3), 0.74 (S4). Amino acid analysis: Tyr 0.94,

Ile 0.94, Olu 1.03, Aap 1.06, Cys (Bzl) 0.95, Pro 0.95, Leu 1.08 Oly 1.04.

For C ₅₇ H ₈₀ N, ₂ 0, ₃ S ₂ . H ₂ O (1223):

calculated: 55.96 * C, 6.76% H, 13.74 * N;

found: 55.97% C, 6.57% H, 13.70% N.

Benzyloxycarbonyl-S-benzylpenicyllaminyl-tyrosyl-isoleucyl-glutaminyl-aaparaginyl-S-benzylcysteinyl-prolyl-leucyl-glycine amide

To a solution of the above prepared octepeptide (1.1 g) in dimethylformamide (15 ml) was added 2.05 M-HCl in ether (1 ml). After standing for 7 minutes, the hydrochloride was precipitated with ether. E® ¹ ^

Ej ¹ ® 0.33; Ry 0.25 (S1), 0.17 (S2), 0.24 (S3), 0.64 (S4). The hydrochloride was dissolved in dimethylformamide (10 ml), the pH of the solution was adjusted with N-ethylpiperidine to a value of 7.5 to 8.0 (moistened pH paper) and to the solution was added p-nitrophenyl ester of benzyloxycarbonyl-S-benzylpenicillamine (0.4 g) in dimethylformamide (3 ml). After 2 days of stirring, another portion of the active ester (0.04 g) was added and stirring was continued for another 3 days.

The dimethylformamide was evaporated and the residue was triturated with petroleum ether and ether, filtered off and washed with water, 1H-HCl, water, 0.25M-NaHCO^ and water. The obtained substance (0.77 g) was dissolved in trifluoroacetic acid (77 ml). After standing for 1 hour at room temperature, toluene (77 ml) was added to the reaction mixture and the solution was evaporated to dryness. The residue was triturated with ether, filtered off and dried in a desiccator over PgO^ and KOH. 0.7 g (57 *) of the product was obtained. 0.5 g of this substance was further purified by gel filtration in two portions of 0.25 g each. The product was isolated by evaporation of the dimethylformamide and trituration of the residue with ether. 0.27 g (54 *) of product was obtained, mp 228-231 °C, [d] _D -42.1° (c 0.2, dimethylformamide). Rf 0.54 (S1), 0.33 (S2), 0.66 (S3), 0.77 (S4). Amino acid analysis: Pen(Bzl) 0.96, Tyr 0.93, Xle 0.95, Olu 0.99, Aap 1.02, Cys(Bzl) 0.89, Pro 1.04, Leu 1.02, Oly 0.97.

For Cg ₇ H ₉₀ N, ₂ O, ₄ S ₂ . Η _£ Ο (1370):

calculated: 58.75% C, 6.77% H, 12.27% N;

found: 58.70*0, 6.53% H, 12.33*N.

Acetyl-S-benzylpenicileminyl-tyrosyl-lsoleucyl-gluteminyl-asparaginyl-S-benzylcysteinyl-prolyl-leucyl-glycine amide

To a solution of the above-prepared nonepeptide (245 mg) in acetic acid (2 ml) was added 35% HBr in acetic acid (2 ml). After standing for 15 minutes at room temperature, the hydrobromide was precipitated with ether. E® ¹ ^ 0.51; Rj, 0.42 (S1), 0.09 (S2), 0.43 (S3), 0.62 (S4). The hydrobromide was dissolved in dimethylformamide (2.5 ml), the pH of the solution was adjusted to 7.5-8.0 (moistened pH paper) with N-ethylpiperidine and 5-chloro-8-hydroxyquinolinyl acetate (106 mg) was added. After 2 days of stirring, another portion of the active ether (20 mg) was added and stirring was continued for 24 hours. The dimethylformamide was evaporated, the residue was triturated with petroleum ether and ether, filtered off and washed with water, 1 M-HCl, water, 0.25 M-NaHCO^ and water. .85 mg (70%) of the product was obtained, which was further purified by gel filtration. 151 mg (82%, total yield 57%) of the product was obtained, mp 226-231 °C, [α] _D -39.7° (c 0.1, dimethylformamide). R _p 0.49 (SI), 0.24 (S2), 0.58 (S3), 0.73 (S4).

For C ₆₁ Hg ₆ N, ₂ O ₁₃ S ₂ . 0.5H ₂ O (1269):

calculated: 57.76%C, 6.91%H, 13.25%N; not found: 57.73%C, 6.89%H, 13.57%H.

N°-Acetyl [l-penieylamine] oxytoein

The protected nonepeptide described above (138 mg) was dissolved in boiling liquid ammonia (150 ml) and reduced with sodium to a blue coloration of the reaction mixture lasting for 10 seconds. The solution was decolorized with ammonium chloride and the ammonia was lyophilized. The residue was dissolved in 0.01 M-HCl (100 ml), diluted with water to a total volume of 300 ml and the pH of the solution was adjusted to 7.0 with 0.01 M-NeOH. Oxidation was carried out with 3.3 . 10 ^-2 MK ₃ [Fe(CN)g], which was added until the yellowish coloration of the reaction mixture lasting for 1 hour. During the oxidation, the pH of the reaction mixture was maintained in the range of 6.9 to 7.0.

The reaction mixture was acidified with 1 M HCl to pH 4.1 and applied to a column packed with a weakly basic anion exchange in a chloride cycle (50 ml). The column was washed with water (50 ml) and the combined eluates were desalted on a column packed with Seperon SI-C-18. After washing the column with water, the product was eluted with 90% methanol. The eluate was acidified with acetic acid to pH 4.5 and the methanol was evaporated. After lyophilization, 72 mg of product was obtained. 35 mg of this product was purified by high-pressure liquid chromatography (methanol-water /4:6/). 11 mg of product was obtained. [α]jj -47.0 °C (c 0.18, 3 M acetic acid). R _p 0.64 (S4), 0.76 (S6). Amino acid analysis: PeníOýDt +Cys(O ₃ H) (2.00), Tyr 0.97 ( 0.66), Ile 1.00 0.00), Glu 0.97 (1.00), Asp 0.97 (1.00),

Pro 1.03 (0.77), Leu 1.06 (1.00), Oly 1.00 (1.00). (Values for the sample oxidized with performic acid are given in brackets).

For Ο ₄₇ Ηγ ₂ Ν, ₂ Ο ₎₃ 3 ₂ . 2CH ₂ C00H. 6H ₂ O (1305):

calculated: 46.92% C, 7.10% H, 12.87% N;

not found: 46.87% C, 6.72% H, 12.81% H.

Example 2

Amide target butyloxycarbonyl-O-methyltyrosyl-isoleucyl-glutaminyl-eapereginyl-S-benzylcysteinyl-prolyl-leucyl-glycine

The above-described heptapeptide (2.1 g) was dissolved in dimethylformamide (50 ml) and 2,4,5-trichlorophenyl tert-butyloxycarbonyl-O-methyltyroein ester (1.1 g) was added to the solution. After two days of stirring, a longer portion of the active ester (0.2 g) was added and stirring was continued for the second day. The dimethyldiformamide was evaporated, the residue was triturated with petroleum ether and ether, filtered off and washed with water, 5% citric acid, water, 0.25 M-NaHCO ₃ and water. 1.9 g (67 St) of product with mp 225-228 °C, -35.2° (e 0.2 dimethylformamide) was obtained.

R _p 0.50 (SIb 0.32 (32), 0.59 (S3), 0.72 (S4). Amino acid analysis: Tyr(0Me + Tyr 0.93, Ile 0.96, Glu 0.99, Asp 1.01, Cys(Bzl) 1.03, Pro 1.02, Leu 1.04, Gly 1.00.

in

For C ₅₃ H ₇₉ N,,O, ₃ S . 0.5H ₂ O (1119):

calculated: 56.87% C, 7.20% H, 13.76% N;

not found: 56.62% C, 7.10% H, 13.73%»·

Benzyloxycarbon1-S-benzylpenicillaminyl-O-methyltyrosyl-isoleucyl-glutaminyl-asperaginyl-S-benzylcysteinyl-prolyl-leucyl-glyein amide

To a solution of the above-prepared octepeptide (1.5 g) in acetic acid (15 ml) was added 35% HBr in acetic acid (15 ml). After 5 minutes at room temperature, the hydrobromide was precipitated with ether. 0.57, E^ 0.27; R _p 0.23 (S1), 0.20 (S2), 0.34 (S3), 0.59 (S4). The hydrobromide was dissolved in dimethylformamide (15 ml), the pH of the solution was adjusted with N-ethylpiperidine to a value of 7.5 to 8.0 (moistened pH paper) and to the solution was added p-nitrophenyl benzyl oxycarbonyl-S-benzylpenicillamine ester (0.7 g) in dimethylformamide (4 ml). After 4 days of stirring, the dimethylformamide was evaporated, the residue was triturated with petroleum ether and ether, filtered off and washed with water, 1 M-HCl, water, 0.25 ll-NaHCO^ and water. 1.0 g (54%) of the product was obtained, which was further purified by Sten Bél filtration in four portions of 0.25 g each.

0.59 g (59%, total yield 32%) of product was obtained, mp 233-239 °C, [α] _D -40.2 (c 0.2, dimethylformamide). R _p 0.54 (S1), 0.33 (S2), 0.66 (S3), 0.76 (S4). Amino acid analysis: Pen(Bzl) 0.87, Tyr(OMe) + Tyr 0.87, Ile 0.94, Glu 0.99, Aap 1.04,

Cys(Bzl) 0.88, Pro 1<p04, Leu 1.04, Gly 0.99. ^C 68 ^H 92 ^N 12° 14 ^S 2 ' ^H 2° (1384): calculated: 59.03% C, 6.85% h, , 12.15% N; found: 59.05% C, 6.79% H, , 12.26% of the.

[l-Penicilamine, 2-O-methyltyrosine] oxytocin

The protected nonapeptide described above (238 mg) was dissolved in boiling liquid ammonia (250 ml) and reduced with sodium to a blue coloration of the reaction mixture lasting for 30 seconds. The solution was decolorized with ammonium chloride, the ammonia was lyophilized, and the residue was dissolved in 0.01 M-HCl (100 ml). The solution was diluted with water to a total volume of 500 ml, the pH of the solution was adjusted to 7.0 with 0.1 M-NaOH. Oxidation was carried out with 3.3 . 10~ ² lMC ₃ [Pe(CH)g], which was added to the yellow coloration of the reaction mixture lasting for 1 hour. During the oxidation, the pH of the reaction mixture was maintained in the range of 6.9 to 7.0. The solution was acidified with 1 N-HCl to pH 4.5 and applied to a column packed with a weakly basic anion exchange in a chloride cycle. The column was washed with water (50 ml) and the eluates were lyophilized. The material was purified by continuous carrier-free electrophoresis. 23.1 mg of product was obtained. [α]·ρ 0° (o 0.14, 3-M acetic acid). E^4 EÍ? ¹ ® o.23. Ry 0.62 (S4), 0.56 (S6). Amino acid analysis: Pen(0^í) + CysťO-jH) 2.09, Tyr(OMe) + Tyr 0.95 (0.82), Xle 0.96 (0.93), Glu 1.02 (1.02), Asp 1.00 (1.02), Pro 0.98 (1.02), Leu 1.02 (1.00), Gly 1.00 (1.00). (Values for the sample oxidized with performic acid are given in parentheses.)

For ^c 45H ₇₂ N _{) 2} O ₁₂ S ₂ . 0.5 CH ₃ COOH . 2.5H ₂ 0 (1124):

calculated: 50.21% C, 7.08% H, 14.95 * Kj found: 50.30% C, 6.72% H, 14.94% N.

Example 3

Acatyl-S-benzylpenicillaminyl-O-methyltyrosyl-lsoleucyl-gluntaminyl-asparaginyl-S-benzyleystelnyl-prolyl-leueyl-glycine amide

To a solution of the nonapeptide (190 mg) in acetic acid (2 ml) was added 35% HBr in acetic acid (2 ml). After standing for 10 minutes at room temperature, the hydrobromide was precipitated with ether. Εθ ¹ * 0.50; Rj, 0.47 (S1), 0.28 (S2), 0.44 (S3), 0.63 (S4). The hydrobromide was dissolved in dimethylformamide (5 ml), the pH of the solution was adjusted with N-ethylpiperidine to a value of 7.5 to 8.0 (moistened pH paper) and 5-chloro-8-hydroxychlorolinyl acetate (39 mg) was added to the solution. After two days of stirring, another portion of the active ester was added and stirring was continued for two more days. The dimethylformamide was evaporated, the residue was triturated with petroleum ether and ether, filtered off and washed with water, 1 M-HCl, water, 0.25 N-NaHCO^ and water. 140 mg (79%) of the product was obtained, which was further purified by gel filtration. 97 mg of the product was obtained, mp 234-238 °C, [ _a ] _D -47.2° (o 0.1, dimethylformamide). Rj. 0.49 (S1), 0.27 (S2), 0.55 (S3), 0.73.(S4).

For ^c 62 ^H 88 ^H 12 ^S 2 ' ^2H 2° ( ¹ ^ ¹⁰ ) calculated: 56.86% C, 7.08% H, 12.83% N;

not found: 56.61% C, 6.84% H, 12.76% N.

N “-Acetyl[l-penicillamine, 2-O-methyltyrosine] oxytocin

The protected nonapeptide described above (87 mg) was dissolved in boiling liquid ammonia (100 ml) and reduced with sodium to a blue coloration of the reaction mixture lasting for 10 seconds. The solution was decolorized with ammonium chloride and the ammonia was lyophilized. The residue was dissolved in 0.01 U HCl (100 ml). The solution was diluted to a total volume of 400 ml. Oxidation was carried out

3.3.10 ^-2 l-Kj[Fe(CN)g], which was added until the yellow color of the reaction mixture remained constant for 1 hour.

The solution was acidified with 1 Li-HCl to pH 4.5 and applied to a column packed with a weakly basic enex in a chloride cycle (50 ml). The column was rinsed with water (50 ml) and the eluates were lyophilized. The material was further purified by countercurrent shaking in the system e.g. butanol-0.05% aqueous acetic acid (1:1). After 100 shifts of the upper and 100 shifts of the apophase, the material from tubes 44 to 56 was isolated and further purified by gel filtration. 7.1 mg of the product was obtained at -67.0° (c 0.10, 3 M acetic acid); Rp 0.64 (S4), 0.83 (S6). Amino acid analysis: Pen(OjH) + + Cys(O^J) (2.05), Tyr(ííe) + Tyr 0.98 ( 0.35), Ile 0.98 (1.00), Clu 1.05 (1.03), Asp 1.00 (1.10), Pro 0.96 (0.95), Leu 1.00 (1.00), Gly 1.00 (1.00). (Values for the sample oxidized with performic acid are given in parentheses.)

For C ₄₈ H ₇₄ N, ₂ O, ₃ S ₂ . 6H ₂ 0 (1199):

calculated: 48.07% C, 7.23% H, 14.01% N;

not found: 48.09%C, 7.34%H, 1.23%N.

Example 4

o-Nitrobenzenesulfenylasperaginyl-S-benzylesteinyl-prolyl-N^-p-toluenesulfonylylglycine amide

S-Benzylcysteinyl-prolyl-H ^£ -p-toluenesulfonyl-lysyl-glycine amide (14.0 g) was dissolved in dimethylformamide (70 ml) and the pH of the solution was adjusted to 7.5-8.0 (moistened pH paper) with N-ethylpiperidine. To the solution was added o-nitrobenzenesulfenylasparagine 2,4,5-trichlorophenyl ester (11.0 g) and the mixture was stirred at room temperature.

After 24 hours, another portion of the active ester (1.0 g) was added and stirring was continued for another 24 hours. The diethylformamide was evaporated and the residue was triturated with petroleum ether and ether, filtered and washed with water, 0.1 M HgSO4, water, 0.25 M NaHCO4 and water. 15.1 g (76%) of product were obtained, mp 175-179 °C, [(<1>]]) -62.1° (c 0.2, diethylformamide).

Rp 0.55 (S1), 0.39 (S2), 0.60 (S3), 0.69 (S4. Amino acid analysis: Asp 0.97,

Cys(Bzl) 0.98, Pro 1.03, Oly 1.01.

*r° θ ₄ 0 ^Η 51 ^Ν 9 ^Ο 1Ο ^δ 3 calculated: 52.65%C, 5.62%H, 13.79%N;

not found: 52.23%C, 5.54%H, 13.70%N.

o-Nitrobenzenesulfenylgluteminyl-aspareginyl-S-benzyl-cysteinyl-prolyl-N ^E -p-toluenesulfonylysyl-glycine amide

To a solution of the above-prepared product (14.0 g) in dimethylformamide (50 ml) was added 2.26 M-HCl in ether (14.2 ml). After standing for 7 minutes, the hydrochloride was precipitated with ether and extracted with ether from the dimethylformamide solution. 12.3 g of hydrochloride was obtained.

0.76, E^ ¹ ® 0.42. Rp 0.23 (S1), 0.21 (S2), 0.21 (S3), 0.64 (S4). The hydrochloride was dissolved in dimethylformamide (50 ml), the pH of the solution was adjusted with N-ethylpiperidine to a value of 7.5 to 8.0 (moistened pH paper) and 2,4,5-trichlorophenyl ester of o-nitrobenzenesulfenylglutamine (7.9 g) was added to the solution. After 24 hours of stirring, another portion of the active ester (0.8 g) was added. After another 24 hours, the dimethylformamide was evaporated, the residue was triturated with petroleum ether and ether, filtered off and washed with water, 0.1 M-HgSO^, water, 0.25 M-NaHCO^ and water.

10.5 g (65%) of product were obtained, mp 187-192 °C, [ _a ] _D -43.0° (c 0.2, dimethylformamide). Rp 0.38 (S1), 0.24 (S2), 0.49 (S3), 0.69 (S4). Amino acid analysis: Glu 1.08, Asp 1.07, Cys(Bzl) 0.72, Pro 1.01, Gly 1.06.

For C^H^NpO^S-j . HgO (1060):

calculated: 50.98% C, 5.80% H, 14.53% N;

not found: 50.79% C, 5.74 » H, 14.61% N.

Amide o-nitrobenzenesulfenylphenylalsnyl-glutaminyl-aspereginyl-S-benzylcystelnyl-prolyl-N ^f: -p-toluenesulfonylysyl-glycine

To a solution of the above-prepared product (10 g) in dimethylformamide (50 ml) was added 2.26 M-HCl in ether (8.9 ml). After standing for 7 minutes, the hydrochloride was precipitated with ether and triturated several times with ether. 7.9 g of hydrochloride was obtained. Eg^ 0.70, E^·® 0.40. Rp 0.09 (S1), 0.10 (S2), 0.09 (S3), 0.55 (S4). The hydrochloride was dissolved in dimethylformamide (40 ml), the pH of the solution was adjusted with N-ethylpiperidine to 7.5 to 8.0 (moistened pH paper) and 2,4,5-trichlorophenyl o-nitrobenzenesulfenylphenylalanine ester (4.6 g) was added to the solution. After 24 hours of stirring, another portion of active ester (1.0 g) was added and stirring was continued for another 24 hours.

The dimethylformamide was evaporated and the residue was triturated with ether and petroleum ether, filtered and washed with water, 0.1 M HgSO^ with water, 0.25 M NaOH^,β with water. 8.5 g (74%) of product were obtained, mp 206-209 °C, [α] _D -24.0° (c 0.2, dimethylformamide).

Rp 0.53 (S1), 0.29 (S2), 0.58 (S3), 0.72 (S4). Amino acid analysis: Phe 1.05, Glu 1.05, Asp 1.03, Cys(Bzl) 0.83, Pro 1.03, Gly 1.01.

For ^C 54 ^H 68 ^N 12°1 3 ^S 3

_5H2O (1198):

calculated: 54.12% C, found: 54.02% C,

5.80% H, 14.02% N; 5.64% H, 14.32% N.

Amide o-nitrobenzenesulfenyl-O-tert.butyltyrosyl-phenylalanyl-glutaminyl-asparaginyl-S-benzylcysteinyl-prolyl-N ¹ -p-toluenesulfonylysyl-glycine

To a solution of the above-described hept8peptide (2.4 g) in dimethylformamide (100 ml) was added 2.05 M-HCl in ether (5.1 ml). After standing for 7 minutes, the hydrochloride was precipitated with ether. 0.63,

0.37. Rp 0.18 (S1), 0.18 (S2), 0.17 (S3), 0.63 (S4). The hydrochloride was dissolved in dimethylformamide (50 ml), the pH of the solution was adjusted with N-ethylpiperidine to a value of 7.5 to 8.0 (moistened pH paper) and the N-hydroxysuccinimide ester of o-nitrobenzenesulfenyl-O-tert.butyltyrosine (1.1 g) was added to the solution. After 24 hours of stirring, another portion of the active ester (0.2 g) was added and stirring was continued until the next day.

The dimethylformamide was evaporated, the residue was triturated with petroleum ether and ether, filtered off and washed with water, 0.1 M HgSO., water, 0.25 M-NaHCO^ and water. 2.5 g (88%) of product were obtained, mp 193-197 °C [<J _D -21.1° (c 0.2, dimethylformamide). Hp 0.58 (S1), 0.34 (S2),

0.63 (S3), 0.77 (S4). Amino acid analysis: Tyr 0.97, Phe 1.02, Glu 1.05, Asp 0.97,

Cys(Bzl) 0.85, Pro 1.06, Gly 1.01.

For ^c 67^85^13®15®3 · ^H 2° <' ⁴² 7> ^: calculated: 56.41% C, 6.18% H, 12.76% N;

found: 56.24% C, 6.03% H, 12.94% N.

Benzyloxycarbonyl-S-benzylpenicillinaminyl-tyrosyl-phenylalenyl-glutaminyl-esparaginyl-S-benzylcysteinyl-prolyl-N ^ε -p-toluenesulfonylylsyl-glycine amide

The hydrochloride was prepared from the octepeptide (1.5 g) by the procedure described above (using 1.1 ml of 2.05 M-HCl in ether). The hydrochloride was dissolved in dimethylformamide (10 ml), the pH of the solution was adjusted with N-ethylpiperidine to a value of 7.5-8.0 (moistened pH paper) and to the solution was added p-nitrophenyl ester benzyloxycarbonyl-S-benzylpenicillamine (0.5 g) in dimethylformamide (4 ml). After two days of stirring, another portion of the active ester (0.05 g) in dimethylformamide (1 ml) was added and stirring was continued for two more days. The dimethylformamide was evaporated and the residue was triturated with petroleum ether and ether, filtered off and washed with water.

M-HCl, water, 0.25 M-NaHCO 3 and water. The product obtained (1.2 g) was dissolved in trifluoroacetic acid (120 ml) and left to stand for 1 hour at room temperature.

Toluene (120 ml) was added to the solution and the mixture was evaporated in vacuo. After trituration of the residue with ether, 1.1 g (66%) of the product was obtained: 1.0 g of this product was further purified by gel filtration in four 0.25 g portions. 438 mg (58%) of the product were obtained, mp 136-141 °C, [alpha] _D -31.2° (c 0.1 dimethylformamide). Rp 0.56 (S1), 0.30 (S2), 0.68 (S3), 0.71 (S4). Amino acid analysis: Pen(Bzl) 0.88, Tyr 0.87, Phe 1.05, Glu 1.01, Asp 0.99, Cys (Bzl) 0.88 Pro 0.99, Gly 1.01.

For C ₇₇ H ₉₅ N ₁₃ O, ₆ Sj . H ₂ O (1573):

calculated: 58.80*0, 6.22% H, 11.58% N;

not found: 58.83 * C, 6.10% H, 11.76% N.

[i -Pěni cilamln, 8-ly sin] in s opre sin

The protected peptide (144 mg) was dissolved in boiling liquid ammonia (150 ml) and reduced with sodium until the reaction mixture turned blue and remained for 20 seconds. The reaction mixture was decolorized with ammonium chloride and the ammonia was lyophilized. The lyophilized solution was dissolved in 0.01 M HCl (50 ml), diluted with water to 400 ml and extracted with ether. The residue was removed from the solution in vacuo. Oxidation was carried out with 3.3· 10“ ² MK ₃ DFe(CN)g], which was added until the yellow color remained for 1 hour; the pH of the reaction mixture was maintained between 6.9 and 7.0 with 0.1 M NaOH.

After oxidation, the reaction mixture was acidified with acetic acid to pH 3.5 and the solution was chromatographed on a column packed with carboxylate cation exchange resin (30 ml, 100 to 200 mesh). After washing the column with 0.25% acetic acid, the product was displaced with 50% acetic acid and lyophilized. The obtained lyophilized product (71 mg) was further resolved by support-free electrophoresis. 13.5 mg of the product was obtained at -12.1° (c 0.14i 3 M acetic acid). E^ ¹ ® 0.63, E^ ⁸ 0.61. Rj, 0.32 (S4),

0.03 (S6). Amino acid analysis: Pan(O^H) + Cys(OjH) (1.94), Tyr 0.97 (0.58), Phe _s 1.01 (0.96), Olu 1.05 (1.03), Asp 1.04 (1.03), Pro 0.99 (0.93), Oly 1.03 (1.00). Values for the sample oxidized with performic acid are given in parentheses.

For ^C 4e ^H 69 ^N ,3®12®2 '3>5CH ₃ COOH . 6.5 H ₂ O (1412):

calculated: 46.80% C, 6.85% H, 12.90% N.

not found: 46.61% C, 6.06% H, 12.82% N.

Example 5

Acetyl-S-benzylpenicllaminyl-tyroyl-phenylalanyl-gluteminyl-aapereginyl-S-benzyleateinyl-prolyl-N ^E -p-toluenesulfonylysyl-glycine amide

The above-described protected nonapeptide (200 g) was dissolved in acetic acid (2 ml) and 35% HBr in acetic acid (2 ml) was added to the solution. After standing for 10 minutes at room temperature, the hydrobromide was precipitated with ether. E® ¹ ^ 0.53, Rp 0.55 (SI), 0.16 (S2), 0.54 (S3), 0.62 (S4). The hydrobromide was dissolved in dimethylformamide (5 ml). The pH of the solution was adjusted to 7.5-8.0 (moistened pH paper) with N-ethylpiperidine and 5-chloro-8-hydroxychlorolyl acetate (56 mg) was added to the solution. After 2 days of stirring, another portion of the active ester was added and stirring was continued for another two days.

The dimethylformamide was evaporated, the residue was triturated with ether and petroleum ether, filtered off and washed with water, 1H-HCl, water, 0.25M-NaHCO^, water; 126 mg (76%) of the product were obtained, mp 136-140 °C. Mq -7.9° (c 0.1, dimethylformamide). R _p 0.54 (SI), 0.24 (S2), 0.60 (S3), 0.72 (S4).

For C ₇₁ H ₉ , N, ₃ O, ₅ S ₃ . 4H ₂ 0 (1S35): calculated: 55>56% C, 6.50% H, 11.86% N; found: 55.38% C, 6.02% H, 11.83% N.

Nfl-Acetyltl-penicillamine, 8-lysine]vasoprasin

The protected nonapeptide (105 mg) was dissolved in boiling liquid ammonia (100 ml) and reduced with sodium to a blue coloration of the reaction mixture lasting for 10 seconds. The solution was decolorized with ammonium chloride and the ammonia was lyophilized. The lyophilized solution was dissolved in 0.01 M-HCl (100 ml), diluted with water to a volume of 400 ml and shaken with ether. The ether residues were removed from the solution in vacuo. Oxidation was carried out with 3.3.10 ^-2 MK ₃ [Pe(CN)gl, which was added to a yellow coloration lasting for 1 hour; the pH was maintained between 6.9 and 7.0 with 0.1 M-NaOH. After oxidation, the reaction mixture was acidified with acetic acid to pH 3.5 and chromatographed on a column packed with carboxylate cation exchange resin (20 ml, 100 to 200 mesh).

The column was washed with 0.25% acetic acid and the product was displaced with 50% acetic acid. After lyophilization, 59 mg of product was obtained, which was further purified by support-free electrophoresis. 14.8 mg of product was obtained with [α]j) -51.0° (c 0.14, 3 M acetic acid). Eg ¹ ^ ®»51,

0.38. Rj. 0.40 (S4), 0.26 (S6). Amino acid analysis: PenCO^H) + CysCO^H), (1.88),

Tyr 0.94 (0.61), Phe 0.96 (1.02), Glu 1.05 (1.00), Asp 1.12 (1.00), iTro 1.21 (1.00), Lys 0.96 (0.98), Gly 1.02 (0.95). (Values for the sample oxidized with performic acid are given in parentheses.)

For . 2CH ₃ COOH . 4.5 HgO (1328):

calculated: 48.86% C, 6.68% H, 13.72% N;

not found: 48.80% C, 5.94% H, 13.76% N.

tert-Butyloxycarbonyl-O-methyltyrosyl-fsnylelenyl-glutaminyl-sepereginyl-S-benzylcysteinyl-prolyl-N ^E -p-toluenesulfonylysyl-glycine amide

The hydrochloride was prepared from the heptapeptide (2.4 g) by the procedure used previously (5.1 ml of 2.05 M-HCl in ether was used). The hydrochloride was dissolved in dimethylformamide (50 ml), the pH of the solution was adjusted with N-ethylpiperidine to a value of 7.5 to 8.0 (moistened pH paper) and 2,4,5-trichlorophenyl tert-butyloxycarbonyl-O-methyltyrosine ester (1.0 g) was added to the solution.

After 24 hours of stirring, a larger portion of the active ester (0.2 g) was added and stirring was continued for another 24 hours. The dimethylformamide was evaporated and the residue was triturated with petroleum ether and ether, filtered off and washed with water, 10% citric acid, water,

0.25 M-NaHCO ₃ and water. 2.2 g (84%) of product were obtained, mp 196-200 °C, -21.6° (c 0.2, dimethylformamide). Rf, 0.55 (SI), 0.25 (S2), 0.46 (S3), 0.66 (S4). Amino acid analysis: Tyr+Tyr(OMe) 0.92, Phe 0.98, Glu 1.00, Asp 1.00, Cys(Bzl) 0.82, Pro 1.05, Gly 1.00.

^C 63 ^H e4 ^N 12°15 ^S 2 · ² > ⁵ «2° <1359):

calculated: 55.70% C, 6.60% H, 12.37% N;

not found: §5.54% C, 6.22% H, 12.43% N.

Benzyloxycarbonyl-S-benzylpenicylamihyl-O-methyltyrosyl-phenylalenyl-gluteminyl-asperaginyl-S-benzylcysteinyl-prolyl-N ^c -p-toluenesulfonylylglycine amide

The hydrobromide was prepared from the above-described octapeptide (1.35 g) dissolved in acetic acid (10 ml) in a conventional manner (5 ml of 35% HBr in acetic acid were used). The hydrobromide was dissolved in dimethylformamide (10 ml), the pH of the solution was adjusted with N-ethylpiperidine to a value of 7.5-8.0 (moistened pH paper) and to the solution was added benzyloxycarbonyl-S-benzylpenicillamine p-nitrophenyl ester (0.51 g) in dimethylformamide (2 ml).

After two days of stirring, a longer portion of the active ester (0.25 g) in dimethylformamide (2 ml) was added and stirring was continued for another 2 days. The dimethylformamide was evaporated, the residue was triturated with petroleum ether and ether, filtered off and washed with water, 1 M-HCl, water, 0.25 U-NaHCO ₃ and water. 1.2 g of product (74%) was obtained. 0.75 g of this substance was further purified by gel filtration in three 0.25 g portions. 361 mg (49%) of product were obtained, mp 202 to 205 °C (Wd “ ³⁴ > ⁶ ° < ^{c 0} >', dimethylformamide). R _p 0.60 (SI), 0.31 (S2), 0.68 (S3), 0.76 (S4). Amino acid analysis: Pen(Bzl) 1.04, Tyr+Tyr(Me) 0.94, Phe 1.03, Glu 1.00, Asp 0.99, Cys(Bzl)

0.92; For 1 ,00, Gly 1 1 .00. PrO ΟγθΗ^γΝ 13°16 ^S 3 * 1.5 _H2O (1596): calculated: 58.70% c, 6.31% H, 11.41 » N; not found: 58.65% c, 6.25% H, 11.57% N.

[i-Penicilamine, 2-O-methyltyrosine, 8-lysine] vasopreein

The above-described nonapeptide (145 mg) was dissolved in boiling liquid ammonia (150 ml) and reduced with sodium to a blue color that persisted for 10 seconds. The solution was decolorized with ammonium chloride and the ammonia was lyophilized. The lyophilizate was dissolved in 0.01 M-HCl (100 ml), diluted with water to a total volume of 450 ml, and the solution was extracted with ether. The residual ether was removed by evaporation in vacuo.

The oxidation was carried out with 3.3 . 10”2M-K ₃ [Fe(CN)g], which was added to the reaction mixture. The yellow color of the reaction mixture remained constant for 1 hour. During the oxidation, the pH of the reaction mixture was maintained at 6.9 to 7.0 with 0.1 M-NaOH. After oxidation, the solution was acidified with acetic acid to pH 4.0 and chromatographed on a column packed with carboxylate ketone (20 ml, 100 to 200 mesh).

The column was washed with 0.25% acetic acid, the product was displaced with 50% acetic acid and lyophilized. 86 mg of lyophilized product was collected, which was further purified by carrier-free electrophoresis. 10.3 mg of product β [«Jp 0° (c 0.15, 3 M acetic acid) was obtained.

eÍJ ¹ ® 0.63, Egl* 0.60. R _p 0.34 (S4), 0.05 (S6). Amino acid analysis: PeníOýi) + + Cye(O^H) (1.94), Íyr+Tyr(OMe) 0.94(0.82), Phe 1.06 (1.04), Glu 0.96 (1.00), Asp 1.06 (1.00), Pro 1.00 (0.99)> Lys 0.97 (1.04), Oly 1.00 (0.99). (The values for the sample oxidized with performic acid are given in parentheses).

For C^H^N^O,^ . 3CH ₃ COOH . 5HgO (1369):

calculated: 48.27% C, 6.85% H, 13.30% N;

found: 48.30% C, 6.27% H, 13.42% N.

Example 7.*

Acetyl-S-benzylpenicilerainyl-O-methyltyrosyl-phenylelanyl-gluteminyl-esperaginyl-S-benzylcysteinyl-prolyl-N ^c -p-toluenesulfonylylglycine amide

The above-described protected nonapeptide (200 mg) was dissolved in acetic acid (2 ml) and 35% HBr in acetic acid (2 ml) was added to the solution. After standing for 10 minutes at room temperature, the hydrobromide was extracted with ether. 0.49, Rp 0.07 (S2), 0.63 (S4). The hydrobromide was dissolved in dimethylformamide (2 ml), the pH of the solution was adjusted with N-ethylpiperidine to 7.5-8.0 (moistened pH paper) and 5-chloro-8-hydroxyquinolinyl acetate (43 mg) was added to the solution.

After 2 days of stirring, another portion of the active ester (15 mg) was added and stirring was continued for another two days. The dimethylformamide was evaporated and the residue was triturated with petroleum ether and ether, filtered off and washed with water, 1 M HCl, water, 0.25 M NaHCO3 and water. 142 mg (74%) of the product was obtained, which was further purified by gel filtration. 109 mg of the product was obtained, mp 190-195 °C, -31.2° (c 0.1, dimethylformamide). R _f 0.56 (S1), 0.26 (S2),

0.59 (S3), 0.74 (S4).

For C ₇₂ H ₉₃ N ₁₃ O, ₅ S ₃ . 2.5 HgO (1522):

calculated: 56.83% C, 6.49% H, 11.96% N;

found: 56.50% C, 6.30% H, 11.99% N.

N ^a -Aoethyl[l-penicillamine, 2,0-methyltyrosine, 8-lysine]vasopressin

The protected nonapeptide described above (92 mg) was dissolved in boiling liquid ammonia (150 ml) and reduced with sodium to a blue coloration of the reaction mixture lasting for 10 seconds. The solution was decolorized with ammonium chloride and the ammonia was lyophilized. The lyophilizate was dissolved in 0.01 M-HCl (100 ml), diluted with water to a volume of 450 ml and extracted with ether.

The ether residues were removed from the solution by evaporation in vacuo. Oxidation was carried out with 3.3.10~ ^of MK ₃ tFe(CN) ₆ ], which was added until the yellow color of the reaction mixture was stable for 1 hour. During the oxidation, the pH of the reaction mixture was maintained with 0.1 M.NaOH in the range of 6.9 to 7.0. After oxidation, the solution was acidified with acetic acid to pH 4.0 and chromatographed on a column packed with carboxylate cation exchange resin (25 ml, .00 to 200 mesh). The column was washed with 0.25% acetic acid; the product was displaced with 50% acetic acid and the solution was lyophilized. 60 mg of lyophilizate was obtained.

After purification by carrier-free electrophoresis, 2.4 mg of the product was obtained with [α] -32.8° (c 0.13, 3 M acetic acid). Εθ ¹ ^ 0.51, 0.35. E _p 0.43 (S4), 0.39 (S6). Amino acid analysis; Pen(O ₃ H) + Cys 3 O-H) (1.96), Lyr+Tyr(OMe) 0.96 (0.69), Phe 1.04 (0.99),

Glu 1.01 (1.00), Asp 1.03 (1.00), Pro 1.03 (0.97), Lys 0.95 (0.98), Gly 1.03 (1.01).

For . 6CH ₃ COOH . 7H ₂ O (1628):

calculated: 46.51% C, 6.88% H, 11.19% N; not found: 46.40% C, 6.43% H, 11.08% N.

Claims (2)

1. Oxytocin and vasopressin analogs of formula I X-en-Tyr (Y) -N-Gln-Asn-Lys-Pro-B-Gly-NH ₂ (I) wherein věechny amino acids are the L-series, penicillamine Pen represents the radical, X represents hydrogen atom or acetyl group, Y is hydrogen or methyl, A is isoleucine or phenylalanine, and B is leucine or lysine. 2. A process for the preparation of novel oxytocin and vasopressin analogs of formula (X), characterized in that the linear peptide of formula (II): Η H X-Pen-Tyr (Y) -A-Gln-Asn-Eys-Pro-B-Gly-NH ₂ (II) wherein Pen, X, Y, A, and B have the same meaning as in Formula I, oxidizing to form disulfide bonds in a known manner.