CZ233191A3 - vasopressin analogs, and process for preparing thereof - Google Patents

Abstract

Analogues of vasopressin of general formula Y-X-Phe-Gln-Asn-Cys-Pro-D-Har-GlyNH2 are prepared by synthesis into the solid phase, where X is D-O-methyl tyrosine or D-O-ethyl tyrosine and Y is cysteine or β-mercaptopropionic acid. The given analogues have very high inhibitive action on the activity of oxytocin on the uterus and almost zero anti-diuretic activity.

Description

Technical field

The invention relates to novel amino and deamino vasopressins and to a process for their preparation.

BACKGROUND OF THE INVENTION

The basicity of the amino acid side chain at position 8 is very important for vasopressin activities (for a review of structure-activity dependence in neurohypophyseal hormone analogs see, e.g., CRC Handbook of Neurohypophyseal Hormone Analogs (Jost K., Lebl M., and Brtnik F., Eds. (CRC Press,

Boca Raton 1987: Gross V.J., Smith C.W. in The Peptides, Vol. 8 (Udenfriend S. an'd Meienhofer J., Eds) p. 77. Academic Press, New York 1987: Sawyer WH, Manning M. in Oxytocin: Clinical and Laboratory Studies (Amico JA and Robinson AG, Eds), page 423. Elsevier, Amsterdam, 1985). Surprisingly, this basic amino acid also plays an important role in the design of the uterotonic inhibitor structure. Currently synthesized oxytocin inhibitors in vitro, and in vivo oxytocin-derived uterotonic assays have bulky substituents at the 3-carbon at the 1 or n position of the D-substituted aromatic amino acid at the 2-position or both modifications simultaneously. The inhibitory activity of these analogs * is increased by the presence of a basic amino acid at position 8.

The literature review shows that for uterotonic inhibitors derived from the vasopressin molecule (i.e., with Phe at the 3-position), there is no bulky substituent on the 3-carbon in the

8 position 1 not only a sufficient condition (see Cpp, Arg vaso 8 presin or Pen, Lys vasopressin with pA ^^ e.S and 6.60 in vitro) but also a necessary condition (Lebl M. in Handbook of Neurohypophyseal Hormone Analogs) K., Lebl M. and Brtnik F., Eds., Volil, Part 1, p. 17. CRC Press, Baca Raton 1987). However, several vasopressin analogues with uterotonic inhibitory activity and having no bulky substituent on the 3-carbon cysteine at position 1 have been synthesized.

likely a number of recently prepared analogs having inhibitory activity in the presoric and antidiuretic test (see, eg, Manning M., Stoyev S., Kolodziejczyk A., Klis W.A.,

Kruszynski M., Misicka A., Olma A., Wo N.C., Sawyer W.H .:

J. Med. Chem. 33, 3079 (1990) would also be uterotonic inhibitors but have never been tested in this regard. It has been shown that a sufficient condition for inhibitory activity in the absence of a bulky substituent on the 3-carbon amino acid at position 1 is substituted L-tyrosine at position 2. It can be assumed that the tyrosine configuration is not important for inhibitory activity but no analogue to D-tyrosine at position 2 with the only modification was not tested. The presence or absence of the amino group at position 1 and the configuration of the basic amino acid at position 8 is also not important. However, for analogs with the basic amino acid D-configuration at position 8, the presence of D-Tyrosine (even unsubstituted) at position 2 is sufficient for inhibitory uterotonic activity.

Several new amino acids have already been prepared (Žertová M., Procházka Z. _t Bláha I., Barth T., Slaninová J., Maletinská L., Lebl M .: Collect. Czech. Chem. Commun. 55, 3000 1990) and deamino (Zertova M., Prochazka Z., Barth T., Slaninova J.,

- Skopko-v — J — B I hah_I — or 1. M. : Coli ec-t, - C zech. Chem. Commun -.—

56, XXX 1991) analogs wherein the tyrosine at the 2-position has been replaced by L- or O-p-methyl or p-ethylphenylalanine, showing? - inhibitory uterotonic activity. Therefore, ^f were synthesized and the analogs both with and without the amino group in the 1-position were studied

and with an appropriately substituted D-tyrosine at the 2-position and a D-homoarginine at the 8-position.

SUMMARY OF THE INVENTION

The present invention provides analogs of vasopressin of the general formula

Y-X-Phe-Gln-Asn-Cys-Pro-D-Har-GlyNH 4

where X = D-O-methyltyrosine Y = Cys (substance formulas AND) X = D-O-ethyltyrosine Y = Cys (substance formulas II) . - X-, = D-O-methyltyrosine y = Mpr (substance formulas III) X = D-O-ethyltyrosine Y = Mp r (substance formulas IV)

Another object of the present invention is to provide a process for the preparation of these compounds by subjecting a chain of hydroxybenzotriazole esters of BOC amino acids to the solid phase method, after which the peptides are released from the resin by liquid hydrogen fluoride while cleaving the side chain protecting groups. , and then the linear peptides formed are cyclized in the presence of potassium ferricyanide and the isomers are separated with the L and D O-methyltyrosine or the analogue with DO-ethyltyrosine at position 2 is purified by high pressure liquid chromatography.

The synthesis of these compounds was performed on the solid phase using a benzhydrylamine or p-methylbenzhydrylamine resin by sequential coupling of amino acids protected by generally known groups. N-tert-butyloxycarbonyl NG-nitrohomoarginine was used as a suitable derivative of D-homoarginine for the synthesis of all analogs. The O-methyltyrosine unnecessary was introduced in the form of the racemate and the O-ethyltyrosine in the form of the D-enantiometer. The side chain protecting groups were cleaved simultaneously when the peptide chain was cleaved from the carrier with liquid hydrogen fluoride. Oxidation of SH-groups and subsequent cyclization were performed with ferricyanide solution. The substances were purified by high pressure liquid chromatography (HPLC), separating the peptides with the D and L amino acids at position 2.

The obtained substances were characterized by thin layer chromatography (TLC) on silica plates (Silufol,

In the following systems. 2-butane 1-98% formic acid-water (10: 3: 8) or 1-butanol acetic acid-pyridine (15. : 3: 10: 6), by electrophoresis on paper in 1 M acetic acid or in pyridine-acetate buffer (pH 5.7) on Whatman 3 MM paper for 1 hour at a gradient of 20 V / cm. Mass spectra were measured on a ZAB-EQ spectrometer (VG Analytical Ltd., Menchester) with xenon at 8 kV. Amino acid analysis was performed on the T 339 or D-500 amino acid analyzer (Durrum Corp. U.S.A.). Analytical HPLC was performed on a Vydac 218TP54 column, preparative on a Vydac 218TP510 column.

The biological activity of the peptides was determined in rats:

uterotonic in vitro according to Holton (Holton J .: 9r. J.

Pharmacol. 3, 328, 1948) in Munsick's treatment (Endocrinology

66, 451, 1961), in vivo according to Pliska (Pliska V .: Eur. J.

Pharmacol. 5, 253, 1969). Inhibitory activity was expressed as pA (Eggena P. et al .: J. Gen. Physiol. 56, 250,

1970), pressoric activity was determined on despinalized male rats according to Krejci et al. (Br. J. Pharmacol.

Chemother. 30, 497, 1967) and Burni antidiuretic (Burn et al., Biol. Standards, Oxford Univ. Press, London 1950).

/ Deamino, g was used as a standard in the anti-diuretic test

D-arginine / -vasopressin. An overview of biological activities is. listed in Table.

DETAILED DESCRIPTION OF THE INVENTION

Scheme of synthetic procedure

The protected amino acid attachment cycle in the construction of the peptide-chain-to-the-nose-1-of-e-step scheme of EMI1.1. Cleavage of the BOC group 20 ml of 50% trifluoroacetic acid in dichloromethane containing 2% anisole for 2 min. After 30 min.

2. Wash with dichloromethane (3 x 20 mL, 1 wash 30 sec)

3. Wash with isopropanol (3 X 20 mL, 1 wash 30 sec.)

4. Wash with dichloromethane (3 X 20 mL, 1 wash 30 sec)

5. Neutralization of 10% triethylamine in dichloromethane (2 x 20 mL, 2 min. Cycle)

6. Wash with dichloromethane (3 x 20 mL, 30 sec. Cycle)

7. Wash with dimethylformamide (3 x 20 mL, 30 sec cycle)

8. Add BOC-protected amino acid hydroxybenzotriazole ester in dimethylformamide (60-180 min cycle).

9. Wash with dimethylformamide (3 x 20 mL, 30 sec cycle)

10. Wash with isopropanol (3 x 20 ml, 30 sec. Cycle)

11. Wash with dichloromethane (3 x 20 mL, 1 minute cycle).

Preparation of Heptapeptide-Bound Resin p-Methylbenzhydralamine resin (8 g, 0.79 mmol / g) was slurried in dichloromethane and after washing with 5% diidopropylamine in dichloromethane and dimethyl formamide was mixed with a 3 molar excess of BOC-Gly-OBt. The reaction was discontinued after 2 h and the resin was washed sequentially with dimethylformamide (3 x 20 mL) and dichloromethane (3 x 20 mL). The degree of substitution (0.55 mmol / g) was determined by amino acid analysis. The free amino groups on the polymer were acetylated. Thereafter, the procedure of Example 1 was applied. BOC amino acids in the form of active esters (in 3 molar excess) were coupled to the amino-linked amino acids in the following sequence. BOC-D-Har (NO 2) -OH, BOC-Pro-OH, BOC-Cys (4-Me-Bz 1) -OH, BOC-Asn-OH, BOC-Gln-OH, and BOC-Phe-OH.

The following are examples of preparation of compounds 1 to IV.

Example 1

Preparation of .beta.-D-2-O-methyltyrosine, 8-D-homoarginine.

B0C-D, L-Tyr (Me) -0H (1.1 equivalents after 24 h and repeated with 0.5 equivalents for another 24 h) were bound to the heptapeptide-bound resin (2 g, 0.6 mmol) according to the general instructions and

BOC-Cys (4-Me-Bzl) -OH. The nonapeptide-bound resin was treated with liquid HF (20 mL, 60 min., 0 ° C) in the presence of 2.0 mL of anisole. The HF was then evaporated. The released nonapeptide together with the resin was triturated with ether after removal of HF, filtered and washed with ethyl acetate. The free peptide was sequentially extracted with acetic acid, 50% acetic acid, water and finally lyophilized. The lyophilisate was dissolved in water (600 mL) and the pH of the solution was adjusted to 7.0 with 0.1 N NaOH. Potassium ferricyanide (0.01 M) in a persistent yellow color was added to the solution. During the oxidation (20 min) the pH was maintained at 7.2 with NaOH solution (0.1 N), then reduced with acetic acid 4.5. The product was then adsorbed to

-sl-oupee-AmbeT-litu-GG-50-I- (-30-m-1-) -co-l-she-was-la-straight ta-Q-2 5% --- acetic acid, and the product was eluted with 50% acetic acid (90 mL), lyophilized (592 mg) and purified on a Vy.dac 21 column 8TP51 0 eluting with methanol (linear gradient from 30 to 50% MeOH-0.05 TFA over 60 min) . Lyophilization yielded 192 mg

L-2-O-methyl-tyrosine-8-D-homoarginine-vasopressin and 25 mg of D-2-O-methyltyrosine, 8-D-homoarginine-vasopressin. For compound I: HPLC (k 1.13, methanol-0.05% trifluoroacetic acid)

35:65) R _f 0.02 (51), 0.43 (54). E ^Gly 1.05, E ^HlS 0.43.

M, ₀ 2.4 '5.7

D-33.7 (c 0.1: 1M acetic acid). Amino Acid Analysis: Asp 1.00, Glu 1.00, Pro 0.96, Gly 1.00, Cys 1.80, Tyr (Me) 0.21,

Tyr 0.71, Phe 1.02, Har 0.65.

For C ₄₀ HN 0 ₂ & 3.5TFA.3.5H 0 (1574.4) calculated: 41.95% C,

% H, 5.09%;% N, 13.34%. Found:% C, 41.72%;% H, 4.70%;

FAB M5 (m / z): 1113 (M + H < + & gt ^; ).

Example 2

Preparation of β-2-O-ethyl-tyrosine, 8-D-homoarginine -vasopressin (compound of formula II).

Boc-D-Tyr (Et) -OH and BOC-Cys (4-Me-Bz1) -OH were coupled to the heptapeptide-bound resin (0.5 g, 0.15 mmol) according to the general instructions. The nanopeptide-bound resin was undercoated with liquid HEL- (-00-0-mi, -60-min-0-C-) ----- in the presence of 1.0 ml. anisole. The HF was then evaporated. The released nanopepptide together with the resin was spread after removal of HF

with ether, filtered and washed with ethyl acetate. The free peptide was sequentially extracted with acetic acid, 50% acetic acid, water and finally lyophilized. The lyophilisate was dissolved in water (200 mL) and adjusted to pH 7.0 with 0.1 N NaOH. Potassium ferricyanide (0.01 M) was added to the solution until a persistent yellow color. During the oxidation (20 min), the pH was maintained at 7.2 with NaOH (0.1 N), then reduced to 4.5 with acetic acid. The product was then adsorbed onto an Amberlite CG-50 I column, the column was washed with 0.25% acetic acid and the product was eluted with 50% acetic acid, decanted (104 mg) and purified on a Vydac 218TP510 column eluting with methanol (linear gradient from 20 to 20). 40% MeOH-0.05 TFA over 60 min). Lyophilization yielded 34 mg of pure product II. HPLC (to 2.48: methanol-0.05% trifluoroacetic acid: 6). R _f 0.02 (S 1), 0.46 (S 4). E l _D

Gly

1.01, E

His

0.44

Mp -27.9 ° (c 0.1, 1M acetic acid). Amino Acid Analysis: Asp 1.00, Glu 1.00, Pro 1.00, Gly 1.00, Cys 1.70, Tyr 0.85, Phe 1.00, Har 0.87. For C 24 H 25 N 2 O 2 TFA. AcOH (1414.4) calculated: 46.70% C, 5.48% H, 14.85% N, found: 46.34% c, 5.27% H, 15.13% N. FAB MS (m / z): 1126.7 (M + H ⁺ ).

Example 3

Preparation of β-deamino, D-2-O-methyltyrosine, 8-D-homoarginine-vasopressin (compound of formula III).

B0C-D, L-Tyr (Me) -0H (1.1 equivalents after 5 h and repeated with 0.7 equivalents for another 24 h) and B0C-Cys were bound to the heptapeptide-bound resin (2 g, 0.6 mmol) according to the general instructions. 4-Me-Bzl) -OH. The nonapeptide-bound resin was treated with liquid HF (20 mL, 60 min., 0 ° C) in the presence of 2.0 µl of anisole. The HF was then evaporated. The released nonapeptide together with the resin was triturated with ether after removal of HF, filtered and washed with ethyl acetate. The free peptide was sequentially extracted with acetic acid. 50% acetic acid, water and finally lyophilized. The lyophilisate was dissolved in water (600 mL) and the pH of the solution was adjusted to 7.0 with 0.1 N NaOH. Potassium ferricyanide (0.01 M) was added to the solution until the yellow color persisted. During oxidation (20 min), the pH was maintained at 7.2 with NaOH (0.1 N), then reduced to 4.5 with acetic acid. The product was then adsorbed onto an Amberlite CG-50 I column (30 mL), the column was washed with 0.25% acetic acid and the product eluted with 50% acetic acid (90 mL), lyophilized (405 mg) and purified on a Vydac 218TP510 column. elution with methanol (linear gradient from 25 to 45% MeOH.-0.05 TFA over 60 min). Lyophilization gave 108 mg of β-deamino, L-2-O-ethyltyrosine, 8-D-homoarginine vasopressin and 20 mg of β-deamine, D-2-O-methyltyrosine, S-D-homparginine vasopressin. For compound III: HPCL (to 3.95, methanol-0.05% trifluoroacetic acid 4: 6). Rj-0.05 (S1)

0.51 (S4) W _D -6 3 Asp 1.00, Tyr 0.79, (1379.4) 44.92 ° C,. E ^y 0.64, E _ 0.30 (detection by chlorination method), o 2.4 3.7.7 (c 0.1, 1H acetic acid). Amino Acid Analysis Glu 1.01, Pro 0.95, Gly 1.00, Cys 0.40, Lyr (Me) 0.25, Phe 1.03, Har 0.87. For ^C 48 ^H 68 ^N ₂ ^S 2 · ^2ΤΓΑ · ^5Η 2 ° calculated: 45.27 ?; C, 5.55% H, 14.2%; N: found:

5.15 µ H, 14.49% N. Fab MS (M / z): 1097 (M ⁺ ).

Example 4

Preparation of [1-deamino, D-2-O-ethyltyrosine, 8-D-homoarginine] vasopressin (Formula IV).

Heptapeptide-bound resin (0.5 g, 0.15 mmol) was coupled to 80C-D-Tyr (Et.) - OH and BOC-Cys (4-Me-Bz1) -OH according to the general instructions. The nonapeptide-bound resin was treated with liquid HF (10 mL, 60 min. 0 ° C) in the presence of 1.0 mL of anisole. The HF was then evaporated. The released nonapeptide together with the resin was triturated with ether after removal of HF, filtered and washed with ethyl acetate. The free peptide was sequentially extracted with acetic acid, 50% acetic acid, water and finally lyophilized. Lyofilisát was dissolved in water C2 ~ 00 m Γ) á ^_ pH "extensible"bk'ubylo adjusted 0ΤΓ Ν ~ '0 ~ H' to 'no t h TRR 7.0. Potassium ferricyanide (0.01 M) was added to the solution until the yellow color persisted. During oxidation (20 min), the pH was maintained at 7.2 with NaOH (0.1 N), then reduced to 4.5 with acetic acid. The product was then adsorbed onto an Amberlite cG-50 L column, the column was washed with 0.25 µm; acetic acid and the product eluted with 50% acetic acid, lyophilized (95 mg) and purified on a Vydac 218TP510 column eluting with methanol (linear gradient from 25 to 45% MeOH-0.05 TFA over 60 min). Lyophilization yielded 21 mg of pure product IV. HPLC (k 3.22, methanol-0.05%, trifluoroacetic acid 4: 6).

R _f 0.60 (S 1), 0.53 (S 4). E ^Gly 0.64, 0.30 (detection ^Γ Γ / Ί ² · ⁴ o ^{5.7 by} chlorination method). 37.0 (c 0.1: 1M acetic acid).

Amino Acid Analysis: Asp 1.00, Glu 1.02, Pro 0.97, Gly 0.97, Cys 0.26, Tyr (Et) 0.05, Tyr 0.89, Phe 1.03, Har 0.92.

For ^c 49 ^H 70 ⁿ 14 ⁰ 12 ^S 2 ' ^2TFA - ^3H 2 ° ⁽¹³⁹³ * ⁴⁾ calculated: 45.68% C, 5 (63% H, 14.06% N, found: 4.76? C, 5.55% H, 14.15 ?; N.

FAB MS (m / z): 1111.7 (M + H < + & gt ^; ).

Industrial applicability

The amino and deamino vasopressin analogs of the invention have a very high inhibitory activity on oxytocin in the uterus and an almost zero antidiuretic activity and can be expected to be used as medicaments.

Claims (3)

PATENT CLAIMS 1. Vasopressin analogs of the general formula, λΛ3Γ90Ϋ. λΖ3ΙΫι · · λ Ofcd. j avyj? __ i ί l 6 ΠΛ> Z j 'SK.Q *! c with IS C o Y-X-Phe-Gln-Asn-Cys-Pro-D-Har-Gly-NH4 where X = D-O-methyltyrosine Y = Cys (substance formulas AND) X = D-O-ethyltyrosine Y = Cys (substance formulas II) X = D-O-methyltyrosine Y = Mpr (substance formulas III) X = D-O-ethyltyrosine Y = Mpr (substance formulas IV) and Mpr is β-mercaptopropionic acid 2. A process for the preparation of analogues according to claim 1, which comprises exposing a chain z1 of hydrobenzotriazole esters of BOC amino acids in a solid phase method, after which the peptides are released from the resin by liquid hydrogen fluoride while cleavage of the side protecting groups. and then the linear β-e-id-V-s-cyclic acid is formed in the presence of potassium ferricyanide and the isomers are separated with L and D. -methyltyrosine or purification of the DO-ethyltyrosine analogue in position 2 by high pressure liquid chromatography. 3. A process according to claim 2 wherein the solid phase is a p-methylbenzhydrylamine or benzhydrylamine resin.