CS242062B1 - Vasopressin and vasotocin analogues and method for their preparation - Google Patents

Abstract

Vasopressin and vasotocin analogs of the general formula I I--------■-------- 1 X-Y-Z-R-Asn-Cys-Pro-W-Gly-NH2 (1). in which X is Cys or Mpr, Y is D-Tyr or D-Phe, Z is Ile or Phe, R is Gln or Abu and W is Arg or D-Arg, which are prepared by cleavage of a protected peptide precursor of the general formula 11 S.Bzl S.Bzl CH -X-Y -Z-R-Asn-Cys-Pro-W-Gly-NH2 (II), where CH denotes a tosyl or benzyloxycarbonyl protecting group, P a polymeric carrier, Bzl is benzyl, Toa, is tosyl and X, Y, Z, R and W have the meanings given above. from a polymer carrier, by removing the protecting groups from the peptide, closing the disulfide ring by oxidation and purifying the obtained crude product of formula I. Analogs of formula I have a high and specific antidiuretic effect.

Description

The invention relates to vasopressin and vasotocin analogues with a high and specific antidiuretic effect and a method for their preparation. The compounds are characterized by the general formula I

I 1

X-Y-Z-R-Asn-Cys-Pro-W-Gly-NHg (I),

3 4 567 89 in which

X is Cys or Mpr,

Y is D-Tyr or D-Phe^

Z is Ile or Phe R is Gln or Abu and W is Arg or D-Arg.

Vasopressin analogues with a high and specific antidiuretic effect are used mainly for the substitution therapy of thirsty dysentery (diabetes insipidus), but also for the therapy of nocturnal enuresis in children (enuresis nocturna) and for the treatment of milder forms of hemophilia, including von Willebrand syndrome. (Marek □., Loutocký A., Pacovský V., Zaoral M.j Endokrinologie 72, 188 (1978); Sov.Med.

10, 106 (1976); Proc. lst Int. Synp. on DDAVP in bleeding disorders (Á.H. Sutor, Ed.). Schattauer Verlag, Stuttgart - New York (1980)). In clinical diagnostics they are mainly used to determine the concentration function of the kidneys and in excretory urography of the kidneys. (Aronson A.S. et al. Arch. Dis. Child. 49, 654 (1974)· Némethová V. et al. Brat. lék. listy 61, 229 (1974)). The most widely used preparation in this area is still £1-3-mercaptopropionic acid, 8-D-apginin]vasopressin (DDAVP). (Zaoral M., Kole □ ., Sorm F.: Collect. Czech. Chem. Gommun. 32, 1250 (1967); Czechoslovak patent 132 685 ). The disadvantage of DOAVP is the residual effect on blood pressure and especially the residual uterotonic effect, which represents a certain risk when applying the preparation to very sensitive pregnant women.

242,062

The key to the preparation of DDAVP-type substances is a change in the configuration at position 8 of the vasopressin molecule (Zaoral M., §orm F,: Collect, Czech. Chem. Commun. 31, 310 (1966)). The magnitude and purity of the antidiuretic effect depends on other structural changes. Substances containing 3-mercaptopropionic acid at position 1 and D-arginine at position 8 have a particularly high and highly specific antidiuretic effect, such as the above-mentioned DDAVP. Later, other vasopressin analogues with a high and specific antidiuretic effect were prepared. Manning and co-workers (Manning et al. □ . Med. Chem. 19, 842 (1976)) increased the specificity of the antidiuretic effect of DDAVP by introducing valine at position 4. Smith and Waltar prepared a vasopressin analog containing 3,4-dehydroproline at position 7, phenylalanine at position 2, and mercaptopropionic acid at position 1. However, none of these analogs has found practical use. The properties of the substances discussed are listed in Table 1, in which the properties of lysine- and arginine-vasopressin are listed for comparison.

Table 1

Compound AD BP UT G AD/BP Ly si n-vasopress si n 250® 243 - 3 4.7 0.5 60 - 10 1.0 Arginine-vasopressin 430 í 45® 380 í 40 16-4 64 í 9 1.1 DDAVP 50 450 ^points 0.96 5.1 - 52,550 ^4-valinejDDAVP 1 230 ^C 0.01 - - 123,000 £2- phenylalanine. 13,250 - 1 250 ^days 0 - ooo

7-(3,4-dehydroproline)}

DDAVP

Handbook of Exp. Pharmacol. XXIII, Neurohypophysial Hormones and

Peptides (B. Berde, Ed.), p 802. Springer 1968.

b'

Zaoral M. et al. Collect. Czech. Chem. Commun. 43, 511 (1978).

C

Manning M. et al. Febs. Latter 44, 229 (1974).

^b Smith CW and Waltar R.: Science 199, 297 (1978).

The values of biological effects are given in international units per mg of active substance (IU/mg). AD « antidiuretic effect, BP » pressor effect, UT · uterotonic effect, 0 = galactogogue effect, AD/BP specificity of the antidiuretic effect.

242,062

The inventors have now found that a change in the configuration at position 2 of the vasopressin molecule has a similar effect on both typical effects of vasopressins as a change in the configuration at position 8, i.e. it leads to analogues with increased specificity of the antidiuretic effect. This discovery was completely unexpected. Hruby and co-workers described the preparation and properties of £D-Tyr arginine-vasopressin. According to Hruby and co-workers, the biological effects of the analogue are very close to lysine-vasopressin. According to the results obtained by the inventors, this analogue has a significantly high and specific antidiuretic effect. The situation is illustrated by Table 2jv, which lists the biological effects found by Hruby and co-workers and the inventors.

Table 2

Compound AD BP AD/BP £2-D-tyrosine arginine vasopressin Rude and cooperative. 207 1 10® 194 í 11 1.1 according to the invention found values 5,000 39.5 126 ^and Hruby V. and co-workers. 3. American Chem. i. Soc. 101, 2717 (1979). Abbreviations and figures they have the same meaning as in table 1

Furthermore, the inventors have found that a change in configuration at position 2 increases the specificity of the antidiuretic effect not only in the vasopressin but also in the vasotocin series and also in analogues containing phenylalanine at position 2. The properties of the respective substances are listed in Table 3. Finally, it has been found that the antidiuretic effect of 2-D substituted vasopressin and vasotocin analogues can be advantageously modified by further structural changes, such as deamination at position 1, substitution of position 4 with a neutral amino acid and a change in configuration at position 8. The effect of the change in configuration at position 2 alone and in combination with other changes is demonstrated by the properties of the substances prepared according to the invention and which are listed in Table 3.

The vasopressin and vasotocin analogs of the above formula I according to the invention contain cysteine or 3-mercaptopropionic acid at position 1, D-tyrosine or D-phenylalanine at position 2, qf-aminobutyric acid at position 4 and arginine or D-arginine at position 8.

242 062 nin. The essence of the method for preparing these analogues consists in preparing a linear precursor of the analogue of general formula II on a polymer carrier, e.g. on a styrene-divinylbenzene copolymer, by the method of solid-phase peptide synthesis.

S.Bzl S.Bzl Tos

CH- X - Y - Z - R -Asn-Cys-Pro- W -Gly-(p) (H), where CH denotes a tosyl or benzyloxycarbonyl protecting group (p) denotes a polymeric carrier and Bzl denotes benzyl,

That's right, and

X,Y,Z,R and W have the meanings given above.

it is cleaved from the support by the action of ammonia in methanol or in methanol - dimethylformamide. The linear protected precursor thus obtained is purified by crystallization or reprecipitation from methanol - water or dimethylformamide - water. The protecting groups are removed from it by the action of sodium in liquid ammonia, the disulfide ring ee is closed by oxidation with potassium ferricyanide, the product is desalted on a carboxylate-type ion exchange resin (Amberlite IRC-50 or Ostion AT) and purified by continuous carrier-free electrophoresis and high-pressure liquid chromatography (HPLC).

The substances listed in Table 3 sub la -ld (Table 3 see page 5) have a lower and less specific antidiuretic effect compared to DDAVP. They are therefore not more advantageous for medical use than DDAVP.

□however, they are very valuable as parent substances for the preparation of vasopressin analogues and have very interesting properties, as shown by the properties of substances le) alf), whose parent substance can be considered compound lb). From a practical point of view, for medical use, substances le) and lf are very interesting. The first of them has a clearly higher antidiuretic and almost 10x lower pressor effect in comparison with DDAVP, i.e. it has about 12x higher specificity of antidiuretic effect than DDAVP. It acts as a significant inhibitor of the uterotonic effect of oxytocin, both in vitro and in vivo. This is very important from the point of view of therapeutic application. Compared to DDAVP, the risk of administering the preparation to pregnant women is completely excluded. Substance lf) has a lower antidiuretic effect than DDAVP, but it acts as a significant inhibitor of the pressor effect of vasopressin and the uterotonic effect of oxytocin. From this point of view, it is still advantageous compared to DDAVP. 5 Table 3

Compound AD BP AD/BP la) [D-Tyr ² ]AVT X /0 60 8 1b) [Mpr ¹ , D-Tyr ² ]AVP 100% 483 103 lc) [Mpr ¹ , D-Tyr ² ]AVT O ť C /0 34.6 29 ld) [Mpr ¹ , D-Phe ² , D-Arg ⁸ ] 10% inh. ooo VP le) [Mpr ¹ , D-Tyr ² , D-Arg ⁸ ] 120 to 150% 0.1 706 30C lf) [Mpr ¹ , D-Tyr ² , Abu ⁴ 40 to 60% inh. <GO D-Arg ⁸ ]VP Abbreviations and numerical values have the same meaning as in table 1,

AVP and arginine vasopressin, AVT arginine vasotocin, VP = vasopressin. The values given in % indicate the magnitude of the activity given in % of the effect of DDAVP. Substance le) inhibits the uterotonic effect of oxytocin in situ and in vitro (pA ₂ 6.3, 7.9). Substance 14) and lf) inhibit the pressor effect of vasopressin (pA ₂ 7.1) and the uterotonic effect of oxytocin in situ and in vitro (pA ₂ 6.7, 7.6).

than substance le). Both substances, substance le) and substance lf) represent a new type of vasopressin analogues, which combine the properties of strong antidiuretic agonists with the properties of significant inhibitors of uterotonic and pressor effects. Both substances are more advantageous than DDAVP from the point of view of medical use. Another advantage of substance le) and substance lf) is that they contain amino acids of configuration D at both the N-terminus and the C-terminus of the molecule. This makes them much better protected against attack by inactivating enzymes than DDAVP, which is very advantageous especially for oral administration.

The essence of the invention is illustrated and substantiated by the following examples:

Melting points were measured on a Kofler block and were not corrected. Optical activity was measured on a polarimeter. Solid-phase synthesis was performed on an automatic peptide synthesizer. Free peptides were purified by carrier-free continuous electrophoresis. The purity of the products was checked by thin-layer chromatography on silica gel in the system tert-butyl alcohol: n-butyl alcohol: acetic acid: water (2:2:1:1), detection by chlorination

B

- 6 —

242 062 procedure, control paper electrophoresis on paper (Whatman No 3) at 700 V (about 50 V/cm) in aqueous acetic acid (pH 2.5), detection with ninhydrin and HPLC on reverse phase, column 250 mm x 4 mm, mobile phase 4 M CH3COOH : MeOH, 30 : 70 (v/v), detection UV 280 nm. Samples for amino acid analysis were hydrolyzed for 20 h in 6M HCl at 110 °C. Buffered hydrolysates (pH 2.2) were analyzed on an amino acid analyzer.

HPLC of the protected precursors of vasopressin and vasotocin analogues was carried out in aqueous methanol (65 to 75%) containing 0.1% CF3CO2H, isocratic. As adsorbent, 10 µm phthalodecylsilyl silica gel (Separon SI C 18) and 10 µm phthalosilicate gel (Lichrosorb RP 8) were used. Detection was carried out by measuring the absorbance of the effluents at 225 and 254 nm. HPLC of vasopressin and vasotocin analogues was carried out on the same supports. Gradient elution (40 to 80% aqueous methanol, containing 0.1% _CF3CO2H ) was used. Detection was carried _out by measuring the absorbance at 220 nm.

Example 1* la) £2-0-Tyrosine^arginine-vasotocin

Amide N-tosyl-S-benzylcysteinyl-D-tyros^1-isoleucyl-glutaminyl-asparagyl-S-benzylcysteinyl-prolyl-N-tosylarginyl-glycine

The synthesis was performed on a chloromethylated polystyrene resin crosslinked with 2% divinylbenzene (DVB) (chlorine content 5.9%), esterified with Boc-Gly (content 0.75 mmol Gly/g resin). The synthesis was performed according to the procedure described previously (Collect. Czech.

Chem. Commun. 44, 1173 (1979). 2 1.33 g of esterified resin was obtained after cleavage of the peptide from the resin by ammonolysis 1.12 g (73%) of product with mp 142-145 °C. The sample for analysis was recrystallized twice from methanol-water, mp 147-149, °Cj. The product was chromatographically uniform. Amino acid analysis: Cys(S-Bzl) 1.98, Tyr 0.90, Ile 0.90, Glu 1.04, Asp 1.07, Pro 1.04, Arg 1.01, Gly 1.06. Pro ^C 7i ^H 93 ^N i ₅ °i6 ^S 4 . 3 HgO (1595) calculated: 53.47% C, 6.26% H, 13.17% N; found: 53.30% C,

5.93% H, 13.44% N.

- 7 £2-D-Tyrosine3erginine-vasotocin 242,062

The protected peptide was reduced with sodium in liquid ammonia, desalted on an Amberlite IRC-50 column and purified by carrier-free continuous electrophoresis. 2,340 mg of the protected peptide yielded 183 mg of crude product and, after purification, 79 mg of the compound 1a)^ [α ^2O = -66.9° (c ≤ 0.2, 1M acetic acid). The product was homogeneous by paper electrophoresis and HPLC purity control. Amino acid composition analysis: Tyr 0.91, Ile 0.90, Glu 1.04, Asp 1.07,

Pro 1.09, Arg 0.94, Gly 1.06, Pro ^c 43 ^H 67 ^N 15 ⁰ 12 ^S 2 * ^{2.5 CH} 3 ^C00H 2.5 H ₂ 0 (1245) calculated: 46.29% C, 6.64% H, 16.87% N; found 46.54% C, 6.44% H, 16.60% N.

Example 2 lb) [1-3-Mercaptopropionic acid,

2-D-tyrosineJarginine-vasopressin

β-Benzylthiopropionyl-D-tyrosyl-phenylalanyl-glutaminyl-asparaginyl-S-benzylcysteinyl-prolyl-N -tosylarginyl-glycine amide The synthesis was carried out using chloromethylated polystyrene resin crosslinked with 2% divinylbenzene (chlorine content 5.9%), esterified with Boc-Gly (content 0.75 mmol Gly/g resin). From 1.33 g (1 mmol Gly) of resin, after synthesis and subsequent ammonolysis, 1.05 g (75%) of the product with a melting point of 130 to 145 °C was obtained. The analytical sample was recrystallized twice from methanol-water, melting point 142 to 145 °C and was chromatographically uniform. Amino acid analysis: Tyr 0.91, Phe 0.98, Glu 1.06, Asp 1.05,

Cys (S-Bzl) 0.95, Pro 1.03, Arg 0.94, Gly 1.08. For

C ₆₇ H ₈₄ N ₁₄ 0 ₁₄ S ₃ . 2.5 H ₂ 0 (1451) calculated: 55.47% C, 6.18% H, 13.52% N·, found: 55.63% C, 5.97% H, 13.86% N.

£1-3-Mercaptopropionic acid,

2-D-tyrosineJarginine-vasopressin

The protected peptide was reduced with sodium in liquid ammonia, desalted on an Amberlite IRC-50 column and purified by carrier-free continuous electrophoresis. From 300 mg of the substance, 161 mg of crude product was obtained and after purification 77 mg of the analogue. The lyophilisate contained 81% of the substance (average value from polarographic determination of the content and from

ΛΛ Λ nitrogen content in the lyophilisate), £ofJ _D · -60.7 (c = 0.2, 1M acetic acid), HPLC and electrophoretically homogeneous. Amino acid analysis: Tyr 1.01, Phe 1.04, Glu 1.05, Asp 1.05, Pro 0.93, Arg 0.92, Gly 1.00. For ^C 46 ^H 64 ^N 14°12 ^S 2 · ^{1.5 CH} 3 ^C00H · ^H 2° (H ⁷⁷ ) calculated:

242 062 — 8 —

49.99% C, 6.16% H, 16.66% N; found: 49.80% C, 6.01% H, 16.69% N.

Example 3 lc) [1-1<3-mercaptopropionic acid,

S-D-tyrosine^arginine-vasotocin

Amide 3-benzylthiopropionyl-D-tyrosyl-isoleucyl-glutaminylG

-asparaginyl-S-benzylcysteinyl-prolyl-N-tosylarginyl-glycine The substance was prepared analogously to the protected product sub la) (1 mmol Gly bound to the resin). Yield 1.0 g (73, m.p.

139 to 143 °C. The analytical sample was recrystallized twice from methanol-water, mp 149 to 151 °C. _f chromatographically uniform. Amino acid analysis: Tyr 0.90, Ile 0.91, Glu 1.07,

Asp 1.09, Cys(S-Szl), 0.94, Pro 1.04, Arg 0.99, Gly 1.06. For ^C 64 ^H 86 ^N 14°14 ^S 3 · ^{3 H} 2° ( ¹⁴²⁶ ) calculated: 53.91% C, 6.50% H. 13.75% Nj found: 53.75% C, 6.68% H. 14.05% N.

[1-1<3-mercaptopropionic acid,

2-D-tyrosine^Jargini n-vasotocin

The substance was prepared analogously to product la, 2320 mg of protected peptide yielded 195 mg of crude product and after purification 81 mg of analogue. The lyophilisate contained 85% of the substance, -57.5° (c β 0.2, 1M acetic acid), HPLC and electrophoretically homogeneous. Amino acid analysis: Tyr 0.99, Ile 0.90, Glu 1.05, Asp 1.07, Pro 1.02, Arg 0.99, Gly 1.07. Pro ^C 43 ^H 66 ^N 14 ⁰ 12 ^S 2 . 2 CHgCOOH .

2.5 H ₂ 0 (1200) calculated: 47.03%C, 6.63%H, 16.34%N,· found: 47.27%C, 6.36%H, 16.20%N.

Example 4 ld) [1-3-Mercaptopropionic acid, 2-D-phenylalanine,

8-D-arginine]vasopressin

Amide-3-benzylmercaptopropionyl-D-phenylalanyl-phenylalanyl-glutaminyl-asparaginyl-S-benzylcysteinyl-prolyl-N-tosyl-D-arginyl-glycine t

The synthesis used a chloromethylated resin of Czech origin (1% DVB, 1.02 mmol Cl/g), which after esterification contained 0.74 mmol Gly per 1 g of resin. The synthesis was carried out with 2 g of esterified resin (1.48 mmol Gly) in an autom.

242 062 synthesizer of our own design. In the condensations, 3 eq. of symmetrical anhydrides and the program mentioned earlier were used. After drying, 3.75 g (97%) of the substance was obtained. Cleavage of the peptide from the support was carried out ammonolytically, yield of crude nonapeptide 1.77 g (86%), m.p. 160 to 165 °C. After double crystallization from a methanol-water mixture, 1.15 g (56%) of the chromium product was obtained, uniform in the system 1-butanol - acetic acid - water (4:1:1) and on HPLC. m.p. 164 to 166 °C, -8.85° (c = 0.508, dimethylformamide (DMF)). Amino acid composition: Phe 1.96,

Glu 1.06, Asp 1.01, Cys(Bzl) 0.91, Pro 0.99, Arg 1.05, Gly 1.02. Pro C ₆ 7 ^H 84N ₁₄ 0 ₁₃ S3 . 2 « ₂ 0 (1426) calculated: 56.43% C, 6.22% H, 13.75% N· found: 56.52% C, 5.99% H, 13.61% N.

£l-3-Mercaptopropionic acid, 2-D-phenylalanine, e-D-arginine^vasopressin

Reduction with sodium in liquid ammonia, oxidation with potassium ferricyanide, desalting on Amberlite IRC-50 and lyophilization gave 454 mg (61%) of crude product from 1 g (0.72 mmol) of the protected peptide. After purification by continuous carrier-free electrophoresis (2,500 V) the yield was 201 mg (27%). The lyophilizate contained 82% of the substance (average value determined from nitrogen content and polarographic determination of the content), -22.5° (c - 0.142, 1M CH3COOH).

The HPLC record contained a single peak. Amino acid analysis:

Phe 1.90, Glu 1.01, Asp 0.97, Pro 0.97, Arg 1.08, Gly 1.07. For ^C 46 ^W 64 ^N 14°11 ^S 2 * ^{2 CH} 3 ^C00H * ^H 2 ⁰ ( ¹¹⁹¹ ) calculated: 50.41% C,

6.26% H, 16.46% Nj found: 50.30% C, 6.11% H, 16.38% N.

Example 5 le) £1-3-Mercaptopropionic acid, 2-O-tyrosine,

8-D-arginine^vasopressin

3-Benzylmercaptopropionyl-D-tyrosyl-phenylalanyl-glutamyl-\asparaginyl-S-benzylcysteinyl-prolyl-N-tosyl-D-arginyl-glycine amide

In the synthesis, 2 g of resin esterified with glycine (1.48 mmol Gly) were used. In the condensations, 3 equiv. of symmetrical anhydrides were used, only in the condensation of Boc-D-TyrOH was N,N ^z -dicyclohexylcarbodiimide (OCC) + N-hydroxybenzotriazole (HOBt) (3 equiv.) used. After drying, 3.67 g (92%) of the substance were obtained. After ammonolytic cleavage, 1.09 g (91%) of the crude nonapeptide was obtained, mp 158 to 161 °C. After double crystallization from the mixture

242 062 methanol - water - ether yielded 1.05 g (50%) of product, chromatographically uniform in the system 1-butanol - acetic acid - water (4:1:1) and on HPLC, mp 172 to 174 °C. -8.06° (c » 0.422, DMF). Amino acid composition: Tyr 0.97, Phe 1.01,

Glu 1.02, Asp 1.03, Cys(Bzl) 0.89, Pro 1.05, Arg 0.98, Gly 1.04. ^{For C} 67 ^H 84 ^N 14°14 ^S 3 ( ^14θθ ) calculated: 57.25% C, 6.02% H, 13.95% N; found: 57.29% C, 6.03% H, 14.17% N.

£l-3-Mercaptopropionic acid, 2-D-tyrosine, S-D-arginine^vasopressin

Reduction with sodium in liquid ammonia, oxidation with potassium ferricyanide, desalting on Amberlite IRC-50 and lyophilization gave 624 mg (82%) of crude product from 1 g (0.71 mmol) of the protected peptide. After purification by continuous carrier-free electrophoresis (2,500 V) the yield was 250 mg (33%). The lyophilizate contained 80% of the substance, Μθ° ^β -65.7° (c = 0.548, 1M CHgCOOH). The HPLC record contained a single peak. Amino acid analysis: Tyr 0.94, Phe 1.00,

Glu 1.01, Asp 1.03, Pro 1.02, Arg 0.92, Gly 1.07. For ^C 46 ^H 63 ^N 14°12 ^S 2 * ^CH 3 ^C00H · ^H 2° ( ¹¹⁴⁶ ) calculated: 50.30% C,

6.07% H. 17.11% N; found: 50.06% C, 6.01% H, 16.84% N.

Example 6

If) £1-3-Mercaptopropionic acid, 2-O-tyrosine, 4-of-aminobutyric acid, 8-D-arginine^vasopressin

3-Benzylthxopropionyl-D-tyrosyl-phenylalan^1-q-aminobutyryl-asparaginyl-S-benzylcysteinyl-prolyl-N-tosyl-O-arginyl-glycine amide

Solid phase synthesis was performed on a chloromethylated polystyrene resin screened with 1% DVB (chlorine content 2.9%) esterified Boc-Gly (0.3 mmol/g carrier). The synthesis was carried out with 4 g of the esterified carrier (2.2 mmol Gly) on an automatic peptide synthesizer. Removal of the tert-butyloxycarbonyl protecting group was carried out by a 3-minute treatment with 3% HBr and CHCl3-CH3COOH (5:1), condensation with symmetrical anhydrides (3 equiv., 30 to 60 min). After ammonolysis, 1.04 g (64%) of the product was obtained, mp 144 to 152 °C._ An analytical sample was recrystallized twice from methanol- ^water , mp ¹⁵⁴ °C ( ^{10 ° C} , ^{dimethylformamide} ). It was chromatographically uniform. Amino acid analysis: Tyr 0.92, Phe 0.98, Abu 0.99,

Asp 0.95, Cys(S-Bzl) 1.02, Pro 1.07, Arg 0.99, Gly 1.06. For

- 11 242 062 ^C 66 ^H 83 ^N 13°e ^S r ^{1.5 H} 2° ( ¹³⁹ °) calculated: 57.04% C, 6.24 _H , 13.10% N; found: 56.88% C, 5.97% H. 12.84% N.

£1-3-Mercaptopropionic acid 2-D-tyrosine, 4-acid

3-aminobutyric, 8-D-arginine]vasopressin Reduction with sodium in liquid ammonia, oxidation with potassium ferricyanide, desalting on an IRC-50 ion exchange resin and lyophilization yielded 410 mg (91%) of crude product from 600 mg (0.44 mmol) of the protected peptide. After purification by HPLC, the yield was 150 mg (33%) of electrophoretically homogeneous substance. The lyophilizate contained 77% of the substance, “θ5.5 ^ο (c = 0.5, 1M CHgCOOH), amino acid composition:

Phe 1.00, Tyr 0.91, Abu 1.00, Asp 1.02, Pro 1.00, Arg 0.97,

Gly 1.09. For ^C 4 ₅ ^H 63 ^N l3 ⁰ il ^S 2 · ^{3 CH} 3 ^C00H · ^{2 H} 2° U ²⁴² ) calculated: 49.30 % C, 6.41 % H, 14.66 % N; found: 49.00 % C,

6.12% H, 14.63% N.

The substances that are the subject of the invention can be used for therapeutic purposes in the areas indicated in the introductory part, in a form suitable for intranasal, sublingual, subcutaneous, intravenous and oral use, i.e. in aqueous solutions, in emulsions with vegetable oils, in liposomes, adsorbed, encapsulated and covalently bound to polymeric carriers, etc. The presence of D-Tyr in position 2 increases their resistance to proteolytic enzymes and makes them particularly advantageous for oral application.

Claims (4)

SUBJECT OF THE INVENTION CLAIMS 1. Vasopressin and vasotocin analogues of the general formula I j-j X-Y-Z-R-Asn-Cys-Pro-W-Gly-NH ₂ (I) 1 2 3 4 5 6 7 8 9 wherein X is Cys or Mpr, Y is D-Tyr or D-Phe. Z is Ile or Phe _of R is G 1 H or Abu and W is Arg or D-Arg. 2. ! ---- 1 Mpr-D-Tyr-Phe-Gln-Asn-Cys-Pro-D-Arg-Gly-NHg 3., -, Mpr - D - Tyr - Phe - Abu - Asn - Cys - Pro - D - Arg - Gly - NH ₂ , 4. A process for the preparation of the vasopressin and vasotocin analogs of formula (1) according to claim 1, wherein said polymeric carrier bound peptide precursor of formula (II) is selected. S.Bzl CH - X - Y S.Bzl Tos ^{1 2} . zx - Z - R - Asn - Cys - Pro - W. - Gly - (p) (II), where CH represents a tosyl or benzyloxycarbonyl protecting group Θ polymerni carrier / Bzl indicates benzyl / It with is tosyl and X, Y, Z, R and W are as defined above, cleave the protected peptide precursor, then remove the protecting groups from the peptide, dissolve the disulfide ring by oxidation and purify the product of formula (I).