GB2078755A

GB2078755A - Oxytocin analogs and method for preparation thereof

Info

Publication number: GB2078755A
Application number: GB8118815A
Authority: GB
Original assignee: Czech Academy of Sciences CAS
Current assignee: Czech Academy of Sciences CAS
Priority date: 1980-06-24
Filing date: 1981-06-18
Publication date: 1982-01-13
Also published as: FR2485527A1; JPS5753448A; CA1179328A; SE448303B; CS216722B1; JPS6330318B2; GB2078755B; FR2485527B1; IT1136670B; CH649090A5; DE3124818A1; IT8122390A0; SE8103778L

Abstract

Analogs of oxytocin of general formula I <IMAGE> where all amino acids are L-isomers and X is phenylalanine or phenylalanine substituted in the p-position by alkyl, ethoxy, amino, substituted amino, or nitro group, suitable for medical applications, and a method for their preparation consisting in the cyclization of linear octapeptides of general formula II <IMAGE> where X has the aforesaid meaning and Act is a group activating the carboxylic group of S-carboxyethylhomocysteine residue, e.g. an active ester. Two methods may be used for the preparation of [2-p-aminophenylalanine] deamino-6-carba-oxytocin, either the reduction of [2-p-nitrophenylalanine]- deamino-6-carba-oxytocin or deprotection of [2-p- benzyloxycarbonylaminophenylalanine] deamino-6-carba-oxytocin.

Description

SPECIFICATION Oxytocin analogs and method for preparation thereof The invention relates to a method for the preparation of oxytocin analogs. More specifically, the present invention relates to a method for the preparation of deamino-6-carba-oxytocin having a modified amino acid in position 2, and to the oxytocin analogs so produced.

It has been recognized that some of the numerous activities of the neurohypophysial hormone oxytocin may be enhanced or suppressed by chemical modification of the molecule, and the attainment of biological effects is a condition precedent for its successful clinical application. Thus, for example, studies have revealed that replacement of a tyrosine group in the 2 position of a deamino-6carba-oxytocin molecule by a phenylalanine or a phenylalanine substituted in the para position yield compounds evidencing a high and specific natriuretic effect which may be suitable for use in treating renal and heart in-sufficiency or in some cases hypertension.

Workers in the art (Fraser, A.M. J. Pharmacol. Ex. Therapy 60, 89 (1937)) have long recognized that oxytocin evidences a natriuretic effect. Unfortunately, that effect is very weak and generally has superimposed thereon a strong antidiuretic activity. The deamino-6-carba-oxytocin evidences a substantially greater natriuretic effect and, even so, is combined with high values of other biological activities, such as oxytoxic, galactogogic and pressor activities, which preclude use of the natriuretic effect of the compound in clinical applications. These analogs are not interesting only with respect to the absolute magnitude of natriuretic effect but also for specificity of the effect of the ratio of different activities.

Moreover we cannot exclude apriori, that by different animals (including man), the value as well as specificy of the natriuretic effect can be different.

In accordance with the present invention, these prior art limitations are effectively obviated by means of novel analogs of oxytocin of the general formula:

wherein the amino acids are of the L-series and X represents the amino acid phenylalanine or a phenylalanine substituted in the para position with a group selected from among alkyl, ethoxy, amino or substituted amino, or nitro groups.

The described novel oxytocin analogs (I) may be conveniently prepared by cyclization between an amino group of an amino acid X and the carboxylic group of the S-carboxyethylhomocysteine residue of a linear peptide of the general formula, (ill):

wherein X represents the amino acid phenylalanine or phenylalanine substituted in the para position by a group selected from among alkyl ethoxy, amino or substituted amine, or nitro groups and Act represents an activator for a carboxylic group such as an active ester group.

The analog of oxytocin of the following general formula, (III):

may conveniently be prepared either by (a) reduction of [2-p-nitrophenylalanine] deamino-6-carbaoxytocin with sodium in liquid ammonia or (b) from [2-p-benzyloxycarbonylaminophenylalanine deamino-6-carba-oxytocin by cleavage of the protecting group with hydrogen bromide in acetic acid.

Some biological activities of the analogs of oxytocin of general formula I are given in Table I in comparison to oxytocin.

TABLE I Analogy I Biological Activity (in Rats) Utero- Galacto- Natri tonic gogic Pressor uretic X (a) (a) (a) (b) oxtytocin 450 450 3.0 100 NH-CH(CH2-QH)-C0 70 170 0.9 143 NH-CH(CH2-C6H4-CH3)CO 45 35 1 326 NH-CH(CH2-C8H4-C2H5)-CO 27 1.4 < 0.2 254 NH-CH(CH2-C6H4-0C2H)-C0 < 0.001 5.6 < 0.2 31 NH-CH(CH2-C6H4-NH2)-CO 15 7 < 0.2 87 NH-CH(0H2-C6H4-N(CH3)2)-CO < 0.14 4.5 < 0.2 75 NH-CH(CH2-C6H4-NH-CO2CH2.

.C6Hs)CO 0.07 < 0.2 10 NH-CH(CH2-C6H4 NO2) CO < 0.001 1.4 < 0.2 66 (a) International units per mg; (b) percent of oxytocin activity.

The method for producing the analogs of oxytocin is further illustrated in examples of performance: EXAMPLE 1 This example described the preparation of [2-p-Nitro-phenylalanine] deam ino-6-carba-oxytocin.

Prior to the initiating of the preparation thereof, an octapeptide of formula II was prepared, as follows: 0.29 grams of 2,4,5-trichlorophenol and 0.33 grams of dicyclohexylcarbodiimide were added to a solution of ortho-nitrobenzenesulfenyl-para-nitrophenylalanine (0.53 grams) in a mixture of 1 5 cm3 of dichlorornethane and 1 5 cm3 of dimethylformamide cooled to -1 00C. The resultant mixture was stirred for one hour at-10 C and then for 12 hours at room temperature. Following, the mixture was concentrated in vacuum, resulting in the formation of crystals which were filtered by suction. The filter cake so produced was then washed with dichloromethane and the filtrate evaporated to dryness (at the 200C bath temperature). The yellow oil resulting was next triturated several times with petroleum ether and dissolved in 12 cm3 dimethylformamide.Following, the amide of isoleucyl-glutaminylasparaginyl-S-(2-carboxyethyl)homocysteinyl-prolyl-leucyl-glycine (0.8 g) was suspended in this solution and after stirring the resultant solution for 135 hours at ambient temperature it was evaporated to dryness (bath temperature 350C). The resultant oily product crystallized on trituration with petroleum ether and was then successively washed on fritted glass with water, 0.05 M sulfuric acid, water and ether, yielding 720 mg of an octapeptide melting within 215-219 C having the characteristics set forth in Table II, below:: TABLE II Some characteristics of octapeptides of general formula

His RF E 5.7 S1 S2 Gly Calculated Gly E Molecular formula Found Asp Glu Pro Hcy (C2H4) X 2.4 S3 S4 (Mol. weight) %C %H %N Ile Leu Xa COOH) Phe(NO2) 0.14 0.37 0.07 C50H71B13O6S2. 61.15 6.09 15.51 1.05 1.04 1.05 1.07 0.57 0.49 0.62 (1174) 51.10 6.29 15.21 0.91 1.07 0.95 0.92 Phe(OEt) 0.14 0.36 0.07 C52H76N12O5S2. 52.03 6.63 14.00 1.07 1.02 1.07 1.03 0.56 0.48 0.62 1.5 H2O (1200) 51.97 6.55 12.98 0.92 1.07 0.96 0.85 Phe 0.10 0.40 0.17 C50H7N12O14S2. 50.72 6.64 14.19 1.05 1.01 1.01 1.04 0.66 0.49 0.65 3 H20 (1183) 50.69 6.38 14.31 0.93 1.02 0.95 0.99 Phe(NHZ) 0.11 0.47 0.19 C58H79N3O16S2. 53.00 6.36 13.85 1.07 1.06 0.98 1.08 0.65 0.57 0.69 2 H2O (1315) 53.03 6.45 14.09 0.93 1.10 0.86 0.89 Phe(Me) 0.11 0.35 0.07 C51H74N12O14S2. 52.74 6.60 14.47 1.04 1.10 0.87 1.06 0.68 0.49 0.66 H2O (1161) 52.75 6.67 14.66 0.91 1.05 1.03 0.95 Phe(Et) 0.09 0.48 0.10 C52H76N12O14S2. 52.34 6.76 14.08 1.00 1.02 0.99 1.01 0.61 0.41 0.69 2 H2O (1193) 52.48 6.50 13.81 0.90 1.01 1.00 0.84 Phe(NMe2) 0.21 0.11 0.06 C52H77N13O14S2. 51.69 6.75 15.07 1.08 1.01 1.10 1.06 0.96 0.10 0.66 2 H2O (1208) 51.81 6.56 14.98 1.00 1.07 0.74 0.84 aThe sum Tyr + Phe(OEt) is given for compounds containing Phe (OEt) ; Phe(NH2) was determined for the compound containing Phe (NHZ).

Z=C6H5CH2OCO- 0.7 gram of bis(p-nitrophenyl) sulfite was then added to a solution of 200 milligrams of the octapeptide so produced in 7 cm3 of dimethylformamide and 7 cm3 of pyridine through which nitrogen had been bubbled. After stirring for 9 hours at room temperature, an additional 0.7 gram of sulfite was added and after stirring for another 12 hours an additional 0.35 gram of sulfite was added. After 4 hours, the reaction mixture was concentrated in vacuum and the product precipitated by ether, filtered by suction and thoroughly washed with ether. The product was then dried, dissolved in 7 cm3 of dimethylformamide and 2.26 M hydrogen chloride in 0.52 cm3 of ether added thereto. The mixture so obtained was permitted to stand for 7 minutes and diluted with 100 cm3 of ether.The hydrochloride precipitate of the compound of formula II was then filtered by suction, washed with ether and dried in vacuum.

Cyclization to form a peptide bond was effected as follows: The hydrochloride prepared in the foregoing manner was dissolved in 7 cm3 of dimethylformamide and added at the rate of 2 cm3 Rer hour with a vigorously stirred mixture of 200 cm3 of pyridine and 50 microliters of N-ethylpiperidine which had nitrogen bubbled therethrough and which had been warmed to 500C. Following completion of the addition, the mixture was heated to 500C for 4 hours and permitted to stand at ambient temperature for 2 hours.Then, the solution was concentrated to a small volume (bath temperature -300C) and the product precipitated by ether. 1 70 mg of microcrystalline material were obtained. 100 mg of the product were then dissolved in 4 cm30 3 M acetic acid and applied on a column packed with polyacrylamide gel [Bio-gel-P-4(100 x 1 cm)]. Freeze-drying of the corresponding fractions yielded 70 mg of a compound which was again dissolved in 3 cm3 of 3 M acetic acid and applied on a column packed as above described. Freeze-drying of the corresponding fraction yielded 42 my of a compound.

1 5 mg of this product was dissolved in 2 cm3 of a methanol-water (2:3) mixture, and the solution applied on a column packed with modified silica gel (Separon SI-C18, 1 5 x 0.6 cm). Elution was then performed with a methanol-water mixture (44:56) ata pressure of 20 MPa. The fraction of k = 8.2 was concentrated in vacuum and freeze-dried, yielding 6.3 mg of the compound whose characteristics are set forth in Table Ill, on the following two pages.

EXAMPLE 2 This example describes the preparation of [2-para-ethoxyphenylalanine] deamino-6-carba-oxytocin.

The starting compound of formula II was prepared in the manner described in example 1. 0.23 gram of a suspension of the dicyclohexylammonium salt of ortho-nitrobenzenesul phenyl-p-ethoxyphenylalanin in 50 cm3 of ethyl acetate was then shaken with 0.05 M sulfuric acid and the resulting solution dried over sodium sulfate and evaporated to dryness. The oil so formed was then dissolved in dichloromethane and the active ester prepared in the manner described in example 1. Condensation with 0.25 gram of heptapeptide was performed in the same manner, yielding 200 mg of a compound having a melting point of 215-21 80C and evidencing characteristics of the type shown in Table II.

Next, the cyclization described in example 1 was conducted. The yield resulting from 200 mg of protected peptide was 196 mg of the product of cyclization. A part of the reaction mixture was purified by repeated gel filtration, yielding 8.3 mg of a pure compound evidencing the characteristics set forth in Table Ill,ff: TABLE III Some characteristics of cyclopeptides of general formula

RF Calculated S1 S2 Molecular Found Gly formula Asp Glu Pro Hcy X S3 S4 (Mol. weight) %C %H %H Ile Leu Xa (C2H4COOH) k' sytemb Phe(NO2) 0.19 0.13 C44H66N12O13S. 47.56 7.07 15.12 1.06 1.04 0.99 1.05 4.23 MeCH-H2O 1::1 0.24 0.65 6 H2O (1111) 47.58 6.63 14.80 0.95 1.04 0.89 0.95 Phe(OEt) 0.18 0.12 C45H71H11O12S. 53.51 7.10 13.73 1.07 1.04 0.94 1.00 2.27 MeOH-buffer pH 0.22 0.63 2 C2H4O2 (1122) 53.25 7.22 13.75 0.97 1.07 0.89 1.00 4.2 3:2 Phe 0.15 0.07 C44H67N11O11S. 53.15 7.20 15.50 0.96 1.01 1.02 0.97 7.00 MeOH-H2O 3:2 0.15 0.62 2 H2O (994.2) 52.90 6.95 15.26 0.97 1.06 1.01 1.01 Phe(NHZ) 0.33 0.12 C52H74H12O13S. 53.79 6.43 14.48 1.08 1.00 0.95 1.04 3.58 MeOH-buffer pH 0.21 0.62 3 H2O (1161) 53.95 6.32 14.30 0.92 1.00 0.82 1.00 4.2 3:2 Phe(Me) 0.28 0.14 C45H69H11O11S. 51.76 7.43 14.75 1.03 1.01 0.94 1.03 4.15 MeOH-buffer pH 0.23 0.69 4 H2O (1044) 51.89 7.15 14.73 0.96 1.05 1.08 0.90 4.4 3:2 Phe(Et) 0.27 0.14 C46H71H11O11S. 55.52 7.29 15.48 1.00 1.00 1.04 0.99 4.50 MeOH buffer pH 0.22 0.70 0.5 H2O (995.2) 56.03 7.33 15.08 0.97 1.00 1.09 0.96 4.4 13::7 TABLE III (Continued) RF Calculated S1 S2 Molecular Found Gly formula Asp Glu Pro Hcy X S3 S4 (Mol. weight) %C %H %H Ile Leu Xa (C2H4COOH) k' sytemb Phe(NMe) 0.10 0.32 C46H72N12O11S. 53.28 7.00 16.21 1.09 1.04 1.09 1.04 2.62 MeOH-buffer pH 0.5 0.61 2 H2O (1037) 52.89 6.80 15.81 0.97 1.08 0.83 0.81 4.4 3:2 Phe(NH2) 0.08 0.05 C14H68N12O11S. 50.57 6.56 16.09 0.96 1.10 0.96 1.02 0.71 MeOH-buffer pH 0.05 0.57 4 H2O (1045) 50.90 6.39 15.70 1.03 1.12 0.79 0.95 4.4 3:2 a - The same meaning as in Table II.

b - The buffer of pH 4.2 is the 1% solution of trifluoroacetic acid with addition of triethylamine ; the buffer of pH 4.4 is 0.015 M sodium phosphate.

EXAMPLE 3 This example describes the preparation of [2-phenylalaninet deamino-6-carba-oxytocin. The starting compound of formula II was prepared in the manner set forth in example 1. Then, 2,4,5trichlorophenyl ester of o-nitro-benzensulfenylphenylalanine was added to 0.8 gram of a suspension of free heptapeptide in 1 5 cm3 of dimethylformamide. After stirring for 96 hours at ambient temperature, the reaction mixture was treated as described in example 1. 1 gram (85%) of a compound having a melting point of 223-2250C was obtained, the characteristics thereof being set forth in Table II.

Cyclization was then performed as described in example 1. The product, which contained ninhydrin-positive material was dissolved in a 1:1 methanol water mixture and filtered through a column of sulfonate cation exchanger (Dowex 50 in the H+ cycle, 5 cm3). Concentration and freezedrying yielded 1 25 mg of a compound which was then purified by gel filtration. 1 5 mg of the product so obtained was then chromatographed on a column with modified silica gel (Separon SI C'8, 15 x 0.6 cm) in a 3:2 methanol water mixture. Concentration of the k = 7.0 fraction and freeze-drying yielded 4.2 mg of a compound, the characteristics of which are set forth in Table Ill.

EXAMPLE4 This example describes the preparation of [2-para-benzyloxycarbonylaminophenylalanine] deamino-6-carba-oxytocin. The starting compound was prepared (formula II) in the manner set forth in example 1. Ortho--nitrnbenzensulfenyl-para-benzyl-oxycarbonylaminophenylalanine was liberated from 1.05 grams of its dicyclohexylammonium salt in the manner described in example 2 and it was transferred into the active ester in the manner set forth in example 1.On trituration with petroleum ether, 1.0 gram of a crystalline compound was obtained having a melting point from 54--570C. The active ester (0.8 g) was added to a suspension of 0.6 gram of heptapeptide in 1 5 cm3 of dimethylformamide and processed as described in example 1.0.53 gram (55%) of a compound melting at 220-2220C was obtained having the characteristics set forth in Table II.

Cyclization was conducted as described in example 1, starting with 200 mug. 155 mg of a product was obtained and 30 mg thereof was purified by repeated gel fitration. Further refining was effected by chromatography on a column with reverse phase (modified silica gel Separon SI C18: 1 5 x 0.6 cm) in a mixture of methanol with trifluoroacetate buffer (3:2) of pH 4.4. The fraction containing the compound k x 3.56 was freeze-dried and 4.2 mg of a product was obtained having the characteristics set forth in Table ill.

EXAMPLE 5 This example describes the preparation of [para-aminophenylalanine] deamino-6-carba-oxytocin.

Sodium was added to 3.6 mg of a solution of [2-p-nitro-phenylalenine] deamino-6-carba-oxytocin in liquid ammonia (5 cm3) until a blue coloration which was stable for 30 seconds arose. Then, the solution was decolorized by addition of acetic acid and the residue, after evaporation of ammonia, was purified by gel filtration. The corresponding collected fractions gave by freeze-drying 2.1 mg of a compound which is characterized in Table ill.

EXAMPLE 6 A solution of hydrogen bromide in acetic acid (35% 1 cm3) was added to a suspension of [2-p benzyloxycarbonylaminophenylalaninej deamino-6-carba-oxytocin (30 mg) in acetone (1 cm3) and the solution formed was allowed to stand for 1 hour at ambient temperature. After repeated evaporation from acetone and reprecipitation from methanol with ether, the product was dissolved in 3 M acetic acid (3 cm3) and refined by gel filtration. Freeze-drying gave 8.7 mg of the compound which corresponded by its properties to the product according to Example 5.

EXAMPLE 7 This example describes the preparation of t2-p-methylphenylalanine] deamino-6-carba-oxytocin.

The active ester of o-nitrobenzenesulphenyl-p-methylphenylalanine was prepared from the corresponding dicyclohexylamonium salt (0.7 g) in the same manner as in Example 4. 0.58 gram of a compound melting at 127-1 33CC was obtained. This active ester was added to a suspension of heptapeptide (0.65 g) in dimethylformamide (13 cm3) and 0.83 g of a compound of m.p. 220-2260C was obtained in the same way as in example 1; the characteristics can be found in Table II.

Cyclization of 200 mg of peptide was carried out in the same way as in example 1. The product was obtained in the amount of 180 mg and a part of it (50 mg) was purified by gel filtration and a column chromatography (Separon SI C'8) in a mixture of methanol-water (3:2). The fraction of k = 5.03 yielded by freeze-drying 13.6 mg of the compound which is characterized in Table III.

EXAMPLE 8 This example described the preparation of [2-p-ethylphenylalanine] deamino-6-carba-oxytocin.

The protected octapeptide was prepared as described in example 1 from o-nitrobenzenesulphenyl-pethylphenylalanine (0.4 g) and the free heptapeptide (0.3 g). A compound melting 21 8-2240C was obtained in the amount of 0.23 g and its properties are given in Table II.

Cyclization and refining of the octapeptide were effected in the same manner as in example 7.

There was obtained 8.3 mg of the compound (k = 7.4, methanol-water (3:2) which exhibited properties shown in Table Ill.

EXAMPLE 9 This example describes the preparation of [2-p-dimethylaminophenylalaninej deamino-6-carbaoxytocin. The active ester of o-nitrobenzenesuíphenyl-p-dimethylaminophenylalanine was prepared from the dicyclohexylammonium salt (0.5 g) in the same manner as in example 4. A compound of m.p.

113-11 70C was obtained in the amount of 405 mg. This active ester was added to a suspension of heptatpeptide (0.3 g) in dimethylformamide and 0.43 g of a compound melting within the range of 201-2040C was obtained by the same procedure as in example 1 (omitting washing with diluted sulphuric acid). Its properties are shown in Table II.

Cyclization was carried out in the same way as in example 1 and yielded 190 mg of a product which was refined by gel filtration. The product obtained by freeze-drying (100 mg) was dissolved in 20% acetic acid and subjected to purification by free-flow electrophoresis. The compound obtained in the amount of 54 mg was further refined by gel filtration. Properties of the product (28 mg) are given in Table ill.

Claims

1. Oxytocin analogs of the general formula

wherein the designatedamino acids are of the L-series and X represents phenylalanine of phenylalanine substituted in the para position with a member selected from the group consisting of (a) alkyl groups, (b) ethoxy group, (c) amino group of substituted amino groups and (d) a nitro group.

2. Method for preparing oxytocin analogs in accordance with claim 1 which comprises cyclizing a linear peptide of the formula

wherein X represents an amino acid phenylalanine or phenylalanine substituted in the para position with a member selected from the group consisting of (a) alkyl group, (b) an ethoxy group, (c) an amino or substituted amino group, and (d) a nitro group; and Act represents a carboxyl group activator, the cyclization being effected between the amino group of amino acid X and the carboxyl group of Scarboxyethylhomocysteine residue.

3. A method for the preparation of a compound of the formula

which comprises reducing [2-p-nitrophenylalanine] deamino-6-carba-oxytocin with sodium in liquid ammonia.

4. Method in accordance with claim 2 wherein said activator is an ester.

5. Method in accordance with claim 3 wherein the protecting group is cleaved from [2-pbenzyloxycarbonylaminophenylalanine] deamino-6-carba-oxytocin by the action of hydrogen bromide in acetic acid.

6. An oxytocin analog as claimed in claim 1 substantially as described in any of the examples disclosed herein.

7. A method of preparing oxytocin analogs as claimed in claim 2 substantially as described herein.