GB2121052A - Analogues of neurphypophysial hormones with inhibitory properties - Google Patents

Abstract

Analogues of neurophypophysial hormones with inhibition properties of the general formula I <IMAGE> where all chiral amino acids are of the L-series and the aromatic amino acid in the position 2, indicated with an asterisk*, is of the D-series, and where R<1> and R<2> mean hydrogen or methyl group, R<3> means hydrogen, amino group, or a triglycylamine grouping, R<4> means hydrogen, methyl, ethyl or theoxyl, R<5> means a group S-CH2 or S-S, X<1> means a residue of isoleucine or phenylalanine, X<2> means a residue of leucine or lysine, and X<3> means a glycinamide residue or hydroxyl group, are described. These analogues exhibit inhibition effects towards the uterotonic and pressor activity of natural hormones. The common structural feature of all these analogues is the presence of an aromatic hydrophobic D-amino acid in the position 2.

Description

SPECIFICATION Analogues of neurohypophysial hormones with inhibitory properties The invention pertains to analogues of neurohypophysial hormones, oxytocin and vasopressin, which exhibit inhibitory effects towards the uterotonic and pressor activity of natural hormones.

The common structural feature of all these analogues is the presence of an aromatic hydrophobic D-amino acid in the position 2. This structural feature is mainly responsible for the inhibitory effects, whereas further structural modifications performed in the molecule make the inhibitory effect more pronounced or change its quality.

It is known that neurohypophysial hormones possess several biological activities (Handbook of Experimental Pharmacology, Vol. XXIII. Neurohypophysial Hormones and Similar Polypeptides, B. Berde, Ed.; Springer-Verlag, Berlin 1968), which are utilized in medicine. Compounds which negate (inhibit) some of these activities are also useful in certain cases. We have found that such compounds may be obtained by introducing an aromatic hydrophobic D-amino acid into the molecule of neurohypophysial hormones instead of a tyrosine residue in the position 2.

Compared to the earlier published analogues (Rudinger J., Krejci I. in "Handbook of Experimental Pharmacology. Vol. XXIII. Neurohypophysial Hormones and Similar Polypeptides" (B.Berde, Ed.), pp 748-801, Springer-Verlag, Berlin 1968; Sawyer W.H., Grzonka Z., Manning M.: Mol.Cell. Endocrinol. 22, 117-134 (1981)), the compounds according to the invention are more efficient inhibitors with the possibility of qualitative modifications of biological activities (e.g. a prolonged inhibitory effect).

The invention pertains to analogues of neurohypophysial hormones of general formula I, which can be prepared by known methods of organic chemistry (some synthetic routes are shown in the examples of performance), where all chiral

amino acids are of the L-series and the amino acid indicated with an asterisk* is of the D-series and where R1 and R2 mean hydrogen or methyl group, R3 means hydrogen or a tri-glycylamino grouping, R4 means hydrogen, methyl, ethyl or ethoxyl, R5 means a group S-CH2 or S-S, X' means an isoleucine or phenylalanine residue, X2 means a leucine or lysine residue and X3 means a glycinamide residue or hydroxyl group.

The following compounds are particularly concerned: Compound R' R2 R3 R4 R5 X' X2 X3 la H H H H S-CH2 lle Leu GlyNH2 Ib H H H OC2H5 S-CH2 lle Leu GlyNH2 Ic H H H CH3 S-CH2 lle Leu GlyNH2 Id H H H C2H5 S-CH2 lle Leu GlyNH2 le H H H C2H5 S-S lle Leu GlyNH2 If H H NH2 C2H5 S-S lle Leu GlyNH2 lg CH3 CH3 NH2 C2H5 S-S lle Leu GlyNH2 Ih H H NH2 C2H5 S-S Phe Lys GlyNH2 li H H Gly-Gly-Gly-NH C2H5 S-S lle Leu GlyNH2 li H H H CH3 S-CH2 lle Leu OH All above given analogues exhibited strong inhibitory effect.Table I contains numeral values of these activities, while pA2 is the negative decadic logarithm of an inhibitory constant according to the relationship B pA2 = - log 1 A50 where B is the concentration of ihnibitor (in mole per liter), A50 is the concentration of an agonistic compound (oxytocin) leading to 50% of the maximum effect, and A505 is the concentration of an agonistic compound giving 50% of the maximum effect in the presence of an inhibitor of concentration B (Eggena P., Schwartz l.L., Walter R.: J. Gen. Physiol. 52, 465 (1968)).

Table I. Values pA2 in the test with an isolated rat uterus Compound of formula pA2 la 8.26 Ib 7.54 Ic 8.73 Id 8.73 le 8.06 If 8.15 lg 8.09 Ih 7.05a li b.14b Ij 8.8 The negative decadic logarithm of the analgoue concentration which leads to suppression of the biological effect of vasopressin (i.e. of blood pressure in a despinalized rat) to 50% of the original response (Schild H.O.: Brit. J. Pharmacol.Chemother. 2, 189 (1947)); the blood volume of rat 6.7 ml/100 g of rat weight is assumed in the in vivo test (Nestor J.J., Ferger M.F., du Vigneaud V.: J. Med. Chem. 18, 284 (1975)).

b The analogue has an prolonged inhibitory effect towards the uterotonic activity of oxytocin in the in vivo test with uterus in comparison with the equipotential dose of the assumed generated compound ((2-D-p-ethylphenylalanine]oxytocin): The inhibitory effect is retained even if the glycinamide residue is removed from the molecule (compound Ij); it is known, that this type of replacement eliminates endocrine activities (Czechoslovak Patent Applications PV 2803-82, PV 2099-81, and PV 8301-82), which is very favourable for the application of inhibitors. Acylation of the primary amino group with a short peptide chain gave an inhibitor with prolonged effect.This prolonged effect is caused by a sequential enzymatic cleavage of the added part of molecule accompanied with the gradual generation of the inhibitor, i.e. utilization of the principle of hormonegen effect (Beránkova- Ksandrová Z., Bisset G.W., Jost K., Krejct I., Pliska V., Rudinger J., Rychltk I., Sorm F.: Brit. J.

Pharmacol. 26, 615-632 (1966)) in the field of inhibitors.

The method for producing analogies of neurohypophysial hormones is further described in the examples of performance.

Example 1 (2-D-Phenylalaninejdeamino-6-carba-oxytocin o-N itrobenzenesulfenyl-D-phenylalanyl-isoleucyl-glut-aminyl-asparaginyl-S-(ss-carboxyethyl)ho- mocysteinyl-prolyl-leucyl-glycinamide (150 mg) was dissovled in dimethylformamide (4 ml), a solution of hydrogen chloride in ether (3.2M, 0.4 ml) was added, and the mixture was allowed to stand for 6 min. at ambient temperature. Hydrochloride of octapeptide precipitated after addition of ether, was reprecipitated from dimethylformamide with ether, filtered and dried in vacuum. Then it was dissolved in dimethylformamide (4 ml), mixed with hyroxybenzotriazole (181 mg) cooled to 0 C and dicyclohexylcarbodiimide (200 mg) was added to this mixture. The solution was stirred for 1 h at 0 C and another 1.5 h at ambient temperature. The separated crystals of urea were filtered off, the solution was introduced into methanol (150 ml) heated to 45"C and the mixture was adjusted to pH 8.5 by addition of N-ethylpiperidine. The mixture was heated to 45'C for 2 h and then concentrated to a small volume. The product was precipitated by addition of ether, filtered and dried in vacuum (140 mg). A part of the product (30 mg) was dissolved in the mixture methanol-water (1:1, 10 ml), applied on a column of silica gel modified with octadecyl chains (50 x 0.9 cm) and eluted with the mixture methanol-0.05% aqueous trifluoroacetic acid (55:45). The fraction containing a compound of k = 7.3 was concentrated in vacuum and freeze-dried giving 6.5 mg of the compound, which was pure according to HPLC and TLC in four solvent systems.For C44H67N,,O,1S.2H20 (994.2) calculated: 53.16% C, 7.20% H, 15.50% N; found: 52.88% C, 7.39% H, 15.29% N. Analysis of amino acids: Asp 1.04, Glu 1.05, Pro 0.95, Gly 1.04, lle 0.94, Leu 1.05, Phe 0.98, Hcy(C2H4COOH) 0.97.

Example 2 [2-D-o-Ethyltyrosin]deamino-6-carba-oxytocin o-N itrobenzenesulfenyl-D-O-ethyltyrosyl-isoleucyl-glutami-nyl-asparaginyl-S-(ss-carboxyethyl) homocysteinyl-prolyl-leucyl-glycinamide (130 mg) was cyclized in the same way as in Example 1.

The crude product was obtained in the amount of 1 35 mg and its part (30 mg) was dissolved in the mixture methanol-water (4:6), applied on a column of silica gel modified with octadecyl chains (50 X 0.9 cm) and eluted with the mixture methanol-O.05% aqueous trifluoroacetic acid (1:1). The fraction containing a compound of k = 26.3 was concentrated and freeze-dried giving 6.2 mg of pure compound according to HPLC and TLC. For C46H7,N"O,2S.4H20 (1074) calculated: 51.43% C, 7.41% H, 14.34% N; found 51.27% C, 7.65% H, 14.08% N. Analysis of amino acids: Asp 1.02, Glu 1.03, Pro 1.00, Gly 1.04, lle 0.93, Leu 0.98, Tyr(Et) 0.41, Tyr 0.56, Hcy(C2H4COOH) 0.92.

Example 3 [2-D-p-Methylphenylalanine]deamino-6-carba-oxytocin o-N itrobenzenesulfenyl-D-p-methylphenylalanyl-isoleu-cyl-glutaminyl-asparaginyl-S-(ss-carboxye- thyl)homocysteinyl-prolyi-leucyl-glycinamide (220 mg) was cyclized in the same way as in Example 1. A part of the crude product (30 mg) was purified analogously as in Example 1. The fraction containing a compound of k = 1 3.6 was concentrated in vacuum and freeze-dried giving 7.3 mg of pure product according to HPLC and TLC. For C45H69N11O11S.3H2O (1026) calculated: 52.67% C, 7.37% H, 15.01% N; found 52.40% C, 7.61% H, 14.83 N. Analysis of amino acids: Asp 1.00, Glu 1.00, Pro 1.05, Gly 1.01, lle 0.91, Leu 1.04, Phe(Me) 0.99, Hcy(C2H4COOH) 0.99.

Example 4 [2-D-p-Ethylphenylalanine]deamino-6-carba-oxytocin O-Nitrobenzenesulfenyl-D-p-ethyíphenylalanyl-isoleucyl-glutaminyl-asparaginyl-S-(ss-carboxye- thyl) homocysteinyl-prolyl-leucyl-glycinamide (200 mg) was cyclized and a part of the product (30 mg) was purified in the same way as in Example 1 giving 5.3 mg of pure compound according to HPLC and TLC. For C46H7,N"O"S.4H2O (1055) calculated: 52.21% C, 7.52% H, 14.56% N; found: 51.90% C, 7.54% H, 14.41% N. Analysis of amino acids: Asp 1.01, Glu 1.03, Pro 0.94, Gly 0.99, lle 0.92, Leu 1.05, Phe(Et) 1.00, Hcy(C2H4COOH) 0.95.

Example 5 [2-D-p-Ethylphenylalanine]deamino-oxytocin S-Benzyl mercaptopropionyl-D-p-ethylphenylalanyl-isoleucyl-glutaminyl-asparaginyl-S-benzylcys- teinyl-prolyl-leucyl-glycinamide (50 mg) was dissolved in liquid ammonia and reduced with a sodium rod until a blue colouration stable for 1 minute was obtained. The blue colouration was removed with a drop of acetic acid and the mixture was freeze-dried. The lyophilizate was dissolved in 0.1 M HCI (8 ml), diluted to 100 ml with water, and adjusted to pH 7 by addition of 0.1M NaOH. Then a solution of K3Fe(CN)6 (25 mg in 5 ml water) was added within 20 minutes while pH 7 was kept during this time. The mixture was stirred for another 40 min at ambient temperature.Then, pH was adjusted to 4 by addition of acetic acid an the solution was applied on a column with silica gel modified by octadecyl chains (25 x 0.4 cm) by means of a high-pressure pump. The column was washed with water and peptides were eluted with the mixture methanol-0.05% aqueous trifluoroacetic acid (80:20). The aluate was concentrated in vacuum and freeze-dried, again dissolved in the mixture methanol-water (1:1, 8 ml), applied on a column with silica gel modified with octadecyl chains (50 X 0.9 cm) and eluted with the mixture methanol-0.05% aqueous trifluoroacetic acid (65:35). The fraction containing a compound of k = 16.6 was concentrated in vacuum and freeze-dried giving 8.6 mg of the product which was pure according to HPLC and TLC.For C45H69N11O11S2.4H2O (1076) calculated: 50.22% C, 7.21% H, 14.32% N; found: 49.94% C, 7.24% H, 14.18% N.

Analysis of amino acids: Asp 1.02, Glu 0.97, Pro 0.89, Gly 1.00, Cys 0.53, lle 0.91, Leu 1.00, Phe(Et) 1.00.

Example 6 (2-D-p-Ethylphenylalaninejoxytocin Na-Benzyloxycarbonyl-S-benzylcysteinyl-D-p-ethylphenyl-alanyl-isoleucyl-glutaminyl-asparaginyl- S-benzylcystein-yl-prolyl-leuoyl-glycinamide (200 mg) was reduced and oxidized similarly as in Example 5. The solution after cyclization was applied on a column of carboxylic cationexchanger (30 ml), the column was washed with 0.25% acetic acid and peptides were eluted with 50% acetic acid. A part of product obtained by freeze-drying (one third) was dissolved in the mixture methanol-water (1:1, 8 ml), applied on a column of silica gel modified with octadecyl chains (50 X 0.9 cm), and eluted with the mixture methanol-0.1M aqueous ammonium acetate (60:40) of pH 7.The fraction containing a compound of k = 21.1 was concentrated in vacuum and freeze-dried giving 16.3 mg of the product which was pure according to both HPLC and TLC. For C45H70N12O11S2.2H20.C2H4O2 (1115) claculated: 50.61% C, 7.05% H, 15.07% N; found: 50.36% C, 6.81% H, 15.23% N. Analysis of amino acids: Asp 0.96, Glu 0.98, Pro 1.02, Gly 0.96, Cys 1.73, lle 1.06, Leu 1.03, Phe(Et) 1.02.

Example 7 [1-Penicilamine, 1 -Penicilamine, 2-D-p-ethylphenylalanine] oxytocin Na-Benzyloxycarbonyl-S-benzylpenicilaminyl-D-p-ethyl-phenylalanyl-isoleucyl-glutaminyl-aspara- ginyl-S-benzylcysteinyl-prolyl-leucyl-glycinamide (200 mg) was reduced and oxidized in the same way as in Example 5. The mixture after cyclization was worked out and purified analogously as in Example 6 with the exception that the mixture containing 65% methanol was used for elution. The pure product according to both HPLC and TLC was obtained in the amount of 8.2 mg. For C47H74N12011S2.3H2O.C2H402 (1161) calculated: 50.67%, C, 7.29% H, 14.47% N; found: 50.78% C, 7.43% H, 14.31% N. Analysis of amino acids: Asp 0.98, Glu 1.00, Pro 0.89, Gly 1.00, lle 1.03, Leu 1.02, Phe(Et) 0.97, Cys + Pen 1.48.

Example 8 (2-D-p-Ethylphenylalanine, 8-lysine] vasopressin N-Benzyloxycarbonyl-S-benzylcysteinyl-D-p-ethylphenyl-alanyl-phenlalanyl-glutaminyl-asparagi- nyl-S-benzylcysteinyl-prolyl-N'-p-toluenesulfonyllysyl-glycinamide (100 mg) was reduced with sodium in liquid ammonia (40 ml) until a blue colouration stable for 60 seconds was formed.

This colouration was then removed by addition of acetic acid and the solution was freeze-dried.

The residue was dissolved in 0.1 M HCI (10 ml), diluted with water to 200 ml, and adjusted to pH 7. After addition of K3Fe(CN)6 solution to a permanently yellow colouration, this pH was maintained under stirring for 1 h. The solution was then applied on a column of carboxylate cation-exchanger, the column was washed with 0.05% acetic acid and eluted with 50% acetic acid. The product was freeze-dried, dissolved in 10 ml water, applied on a column of silica gel modified with octadecyl chains (column dimension 50 X 0.9 cm), and eluted with the mixture methanol-0. 1 % aqueous trifluoroacetic acid (40:60). The pertinent fractions were freeze-dried.

Purity of the product was checked by thin-layer chromatography (4 solvent systems) and paper electrophoresis (2 buffers of different pH). Analysis of amino acids: Lys 1.07, Asp 1.03, Glu 0.92, Pro 0.94, Gly 1.04, Cys 1.84, Phe 1.06, Phe(Et) 0.92.

Example 9 N -Glycyl-glycyl-glycyl (2-D-p-ethylphenylalanine] oxytocin. The solution of N-hydroxysuccinimide ester of o-nitrobenzenesulfenylglycyl-glycyl-glycine (10 mg) in 0.3 ml dimethlformamide was added to the solution of (2-D-p-ethylphenylalaninei oxytocin (4 mg) in 0.3 ml water. After stirring at ambient temperature for 2 h, further active ester of triglycine (14 mg) was added and the solution was alkalized to pH 8 by addition of 11 ,ul IM NaOH. The reaction was followed by means of the reversed-phase high-pressure liquid chromatography.After the starting compound disappeared (20 h), the solution was evaporated in a vacuum on rotation evaporator at ambient temperature, the residue was dissolved in methanol (1 ml), 2.3M HCI in ether (60 ,ul) was added, the solution was allowed at ambient temperature for 5 min and again evaporated. The residue was dispersed in 1 ml 3M acetic acid and filtered. The filtrate was diluted with 5 ml water, applied on a column of silica gel, modified with octadecyl chains (column dimension 50 x 0.9 cm), and eluted under the following conditions: the mobile phase methanol-0.1% aqueous trifluoroacetic acid (40:60) for 40 min and then a linear gradient to the methanol content 60% for 20 min, flow rate 4 ml/min. The pertinent fractions were concentrated in vacuum and freeze-dried to give 2.3 mg of product, which was chromatographically (TLC, HPLC) and electrnphoretically pure. Analysis of amino acids: Asp 0.99, Glu 1.05, Pro 0.90, Gly 4.08, Cys 1.29, lle 0.96, Leu 1.02, Phe(Et) 0.99.

Example 10 2-D-p-Methylphenylalanine, 9-desglycinamide] deamino-6-carba-oxytocin (2-D-p-Methylphenylalanine] deamino-6-carba-oxytocin (2.5 mg) was dissolved in 50 iLl methanol and 300 iLl 20mM phosphate buffer of pH 7.7, 2.5 mg chymotrypsin was added and the mixture was incubated at 37"C for 12 hours. The reaction was followed by means of highperformance liquid chromatography. After the starting compound disappeared, the reaction mixture was applied on a column (25 X 0.4 cm) of silica gel modified with octadecyl chains and eluted with the mixture methanol-0.05M ammonium acetate of pH 7.0 (40:60) as a mobile phase. The compound I, which proved chromatographically pure in four solvent systems used in thin-layer chromatography and in two systems in high-performance liquid chromatography, was obtained by freeze-drying in the amount of 1.6 mg. Analysis of amino acids; Asp 0.96, Glu 0.98, Pro 1.02, Hcy(C2H4CO2H) 0.93, lle 1.03, Leu 1.03, Phe(Me) 1.05.

Claims (2)

1. Analogues of neurohypophysial hormones with inhibition properties of the general formula I

where all chiral amino acids are of the L-series and the aromatic amino acid in the position 2, indicated with an asterisk*, is of the D-series, and where R1 and R2 mean hydrogen or methyl group, R3 means hydrogen, amino group, or a triglycylamino grouping, R4 means hydrogen, methyl, ethyl or ethoxyl, R5 means a group S-CH2 or S-S, X' means a residue of isoleucine or phenylalanine, X2 means a residue of leucine or lysine, and X3 means a glycinamide residue or hydroxyl group.

2. An anologue of neurohypophysial hormones as claimed in Claim 1 and substantially as described in any one of the examples disclosed herein.