FR2485527A1 - OCYTOCIN ANALOGUES AND PROCESS FOR THEIR PREPARATION - Google Patents

Abstract

THE INVENTION RELATES TO OLYTOCIN ANALOGUES AND A PROCESS FOR THEIR PREPARATION; TO PREPARE OLYTOCIN ANALOGUES RESPONDING TO GENERAL FORMULA (I): (CF DRAWING IN BOPI) OR ALL AMINO ACIDS ARE L AND X ISOMERS REPRESENT PHENYLALANINE OR PHENYLALANINE SUBSTITUTED IN THE PARA POSITION BY A RADICAL ETHOXY, AMINO, AMINO SUBSTITUE OR NITRO, WE CYCLIZE LINEAR OCTAPEPTIDES OF GENERAL FORMULA (II): (CF DRAWING IN BOPI) OR X WITH THE SAME MEANING AS PREVIOUSLY AND ACT REPRESENTS AN ACTIVATOR OF THE RADICAL CARBOXY OF THE EXEMPLE PAREMPLE PAREMYSTEHYL-CARBOXY RESTEOCYSTEHYL-CARBOXY AN ACTIVE ESTER; TO PREPARE THE P-AMINOPHENYL-2 ALANINE DESAMINO CARBA-6 OCYTOCIN, THE P-NITROPHENYL-2 ALANINE DESAMINO CARBA-6 OCEYTOCIN OR DEPROTECT THE P-BENZYLOXYCARBONYLAMINYTROPHENYL-2 ALANINE DESAMINO CARBA-6 OCHYTOCIN OR DEPROTECT P-BENZYLOXYCARBONYLAMINYTOPHENY-6 ALANINE DESAMINO CARBA-6.

Description

The present invention relates to analogues of

  and a process for their preparation.

  More particularly, the invention relates to a method

  to prepare desamino carba-6 oxytocins having an amino

  2-position acid and oxytocin analogues

thus produced.

  We know that some of the many activities of hor-

  Neurohypophyseal neuropathy may be increased

  reduced by chemical modification of the molecule and

  that the achievement of biological effects is a necessary condition

  to a successful clinical application. So for example studies have revealed that the replacement of a tyrosine radical in position 2 of a molecule of desamino

  oxytocin carba-6 by a radical phenylalanine or phenylalanine

  substituted para in the form of compounds having a

  high and specific natriuretic effect that can be used

  be appropriate for the treatment of insufficiency

  kidney and heart or in some cases hypertension.

  Those skilled in the art (Fraser, A.M. J. Pharmacol.Ex., Therapy 60, 89 (1937)) have long established that oxytocin has a natriuretic effect. Unfortunately this effect is very weak and usually associated with significant antidiuretic activity. Desamino carba-6 oxytocin has

  a significantly superior natriuretic effect but

  to other important biological activities such as

  oxytocic, galactagogue and vasomotor activity, which

  the use of the natriuretic effect of the compound in

  clinical applications. The absolute importance of the natri

  uretic and the specificity of the effect of the report of activities

  different types do not make these compounds interesting.

  Moreover, it can not be excluded a priori that in animals

  different ailments (including man) the value as well as the

  specificity of the natriuretic effect may be different.

  The invention effectively removes these limitations

  of the prior art by means of new analogues of the octo-

  wherein the amino acids belong to the L and X series and have the general formula CH 3 CH 2 CH 2 -CO-X-Ile-Gln-AsnNH-CH-CO-Pro-Leu-Gly-NH 2 (I) represents the amino acid phenylalanine or a phenylalanine substituted in the para position by an alkyl, ethoxy, amino or amino radical

substituted or nitro.

  The novel oxytocin analogs (I) described can be conveniently prepared by cyclization between an amino radical of the amino acid X-and the carboxy radical of the S-carboxyethylhomocysteine residue of a linear peptide of the general formula (II ):

CH2-S-CH2 (II)

  CH2CO-Act X-Ile-Gln-Asn-NH-CH-CO-Pro-Leu-Gly-NH2

  o X represents the amino acid phenylalanine or a phenylalanine

  nine substituted in the para position by an alkyl, ethoxy,

  amino or substituted amino or nitro and Act represents an activa-

  of the carboxylic group such as an active ester group.

  One can conveniently prepare the analogue of the oc-

  tocine having the following general formula (III):

CH2 S CH2 CH2 (III)

  CH2-CO-NH-CH-CO-Ile-Gln-Asn-NH-CH-CO-Pro-Leu-Gly-NH2

CH2

  NH 2 is (a) by reduction of 2-p-nitrophenyl-2-alanine] desamino carbamate oxytocin by sodium in liquid ammonia or (b) from Zp-benzyloxycarbonylaminophenyl-2-alanine-7-desamino carba-oxytocin by cleavage of protective group

  by hydrobromic acid in acetic acid.

  Some biological activities of oxytocin analogues

  of general formula (I) appear in Table I relating to

with oxytocin.

TABLE I next page

TABLE I

  Compound I Biological Activities (in rats)

X Urethonic Galacta- vasonatri

  uretic motor (a) (a) (a) (b) oxytocin 450 450 3.0 100

NH-CH (CH2-C6H5) -CO 70 170 0.9 143

NH-CH (CH2-C6H4-CH3) CO 45 35 1 326

  NH-CH (CH2-C6H4-C2H5) -CO 27 1.4 <0.22

  NH-CH (CH2-C6H4-OC2H5) -CO <0.001 5.6 <0.2 31

NH-CH (CH2-C6H4-NH2) -CO 15 <0.28

  NH-CH (CH2-C6H4-N (CH3) 2) -CO 40,14 4, -5 0.2 75

NH-CH (CH2-C6H4-NH-CO2CH2.

C6H5) -CO 0.07 0.2 10

  NH-CH (CH2-C6H4-NO2) -CO 40.001 1.4 40.2 66

  (a) International Units per mg; (b) Percentage of activity relative to oxytocin The process for preparing the oxytocin analogs is further illustrated by the following nonlimiting examples;

EXAMPLE 1

  This example describes the preparation of [p-nitrophenyl-2-alanine] desamino carba-6 oxytocin. Before starting the preparation, an octapeptide of formula (II) is prepared in the following manner: 0.29 g of 2,4,5-trichlorophenol and 0.33 g of

  dicyclohexylcarbodiimide has a solution of o-nitrobenzene-

  -sulfenyl p-nitrophenylalanine (0.53 g) in a mixture of 15 ml of dichloromethane and 15 ml of dimethylformamide cooled to -10 ° C. The mixture obtained is stirred for one hour at -10 ° C. and then for 12 hours at room temperature. . Then the mixture is concentrated in vacuo to obtain crystals which are filtered with the aid of vacuum. The filter cake thus produced is then washed with dichloromethane and evaporated.

  the filtrate to dry (with a bath at 20 C). Then triturate

  the yellow oil obtained with petroleum ether and dissolved in 12 ml of dimethylformamide. The isoleucyl-glutaminyl-asparaginyl-S- (carboxy-2) amide is then added.

  ethyl) homocysteinyl-prolyl-leucyl-glycine (0.8 g) suspended

  in this solution and after stirring the solution for 135 hours at room temperature, evaporated to dryness (with a 35 C bath). The oily product obtained crystallized when triturated with petroleum ether and then washed on a sintered glass plate successively with water, 0.05 M sulfuric acid, water and

  ether to give 720 mg of an octapeptide melting at 215-

  219 C and having the characteristics which appear in the table

  Table II, page 2, is then added. 0.7 g of bis (p-nitrophenyl) sulfite is then added to a solution of 200 mg of the octapeptide thus obtained in 7 ml of dimethylformamide and 7 ml of pyridine. made

  splash with nitrogen. After 9 hours of stirring at room temperature

  0.7 g of sulphite are added, after stirring for a further 12 hours. After 4 hours, the reaction mixture is concentrated under vacuum and the product is precipitated with ether. filtering with help

  vacuum and washed thoroughly with ether. We then dry the

  duit, dissolve in 7 ml of dimethylformamide and add

  2.26 M hydrochloric acid in 0.52 ml of ether. We let

  The mixture thus obtained is allowed to stand for 7 minutes and is diluted with 100 ml of ether. Using the vacuum, the hydrochloride of the compound of formula (II) which precipitates is filtered off

  washed with ether and dried under vacuum.

  The cyclization is carried out to form a pep-

  in the following way: the hydrochloride pre-

  prepared as described above in 7 ml of dimethylformamide and a mixture of 2 ml / h

  stirred vigorously with 200 ml of pyridine and 50 ml of N-ethyl

  -piperidine where nitrogen was bubbled and heated

  When the addition is complete, the heat is heated to 50 ° C.

  at 50 ° C for 4 hours and let stand at room temperature.

  room temperature for 12 hours. The solution is then concentrated under a small volume (with a bath at 30 ° C.) and

  precipitates the product with ether. We obtain 170 mg of a

  microcrystalline content. Then 100 mg of the product is dissolved

TABLE II

  Some of the octapeptide characteristics of general formula:

CH2CH25CH2CH2COOH

  Nps-X-Ile-Gln-Asn-NH-CH-Co-Pro-Leu-Gl1-N H2-EHis RF Molecular formula Theoretical analysis * 5.7 (weight found Asp Gl u Pro G1 / S1lzS molecular S2)% CHN Io LuCOO )

2.4 63 S4

  Phc (NO2) 0.10> 37.0107 C50H71N13016S2. 51y15 6109 15.51 ilt05 1t04 1l05 1107 0.57 0y49 0162 (1174) 51] .0 6y29 15121 0t91 1107 0195 0102 Phe (OEt) 0.14 0 to 36 0107 C52l 76N12015S2. ## EQU2 ## where: ## STR2 ## 50.72 6164 14119 1.05 1.01 1.01 1 04

  0.66 019 0965 3 H20 (1183) 50 169 6.38 14131 0.93.1 02 0.95 0199

  Phe (NHZ) 0.107 07 19 C58H79N13016S2. 53.00 6> 36 13 85 11 07 1r06 0198 1.08

  0157 0.69 2 FH20 (1315) 53103 6145 14 09 0193 1] 10 0186 0189

  Phe (Me) 0.111 0.35 0.07 C51H74N12014S2. 52.74 6.60 14.47 1/04 1.10 0.87 1o06 0168 0> 49 066 lH20 (11.61) 52175 6167 14.66 0 91 1105 1103 0195 CP6807>? - 1 1 y yI f Phe (and) 009 0? 48 Ot10 C52H76N12014S2. 52134 6.76 14108 100 1.02 099 101 0161 0.41 0.69 2 H20 (1193) 52148 6150 13t81 0190 1101 1tOO 0.84 Phe (lNMe2) 0.21 0.11 0i06 C52H77N1301462 51.69 6.75 15.07 1.08 1101 1110] 106

  01 96 01 10 01 66 2 14H20 (1208) 5181 6 156 14198 1.00 1 07 0 174 0 184

  The sum Tyr + Phe (OEt) is indicated for the compounds containing Phe (OEt) Phe (NH2) for the compound containing Phe (NHZ) was determined

Z = C6H5CH20C00-

  Ln co Ln Ln 1r - in 4 ml of 3M acetic acid and applied to a column

  filled with polyferrylamide gel fBio-gel-P-4 (100 x 1 cm)].

  The corresponding fractions are lyophilized to give 70 mg of a compound which is dissolved in 3 ml of 3M acetic acid and applied to a packed column as above.

  described. The corresponding fractions are lyophilized to

  hold 42 mg of a compound. 15 mg of this product are dissolved in 2 ml of a 2/3 mixture of methanol and water and the solution is applied to a column packed with a modified silica gel (Separon SI-C 18, 15 × 0, 6 cm). Eluted with a 44/56 mixture of methanol and water under a pressure of 20 MPa. We concentrate

  under vacuum and freeze-dried the fraction of k '= 8.2 to obtain

  6.3 mg of the compound, the characteristics of which are shown in

Table III below.

EXAMPLE 2

  This example describes the preparation of [p-ethoxyphenyl]

  2 alaninel desamino carba-6 oxytocin. The starting compound of formula (II) is prepared as described in Example 1.

  0.23 g of a suspension of the dicyclohexyl salt are then stirred

  -ammonium of o-nitrobenzenesulfenyl p-ethoxyphenylalanine in 50 ml of ethyl acetate with 0.05 M sulfuric acid and the resulting solution is dried over sodium sulfate and then evaporated to dryness. The oil thus formed is then dissolved

  in dichloromethane and the active ester is prepared as a

  described in Example 1. The same is done in the same way

  0.25 g of the heptapeptide to obtain 200 mg of a compound having a melting point of 215-2180C and

  the characteristics shown in Table II.

  The cyclization described in the example is then carried out.

  1 mg. 196 mg of cyclization product is obtained from 200 mg of protected peptide. Part of the reaction mixture is purified by several gel filtrations to obtain 8.3 mg of a pure compound having the indicated characteristics.

in the following Table III.

TABLE III pages 7 and 8

TABLE III

  Some of the characteristics of cyclopeptides of general formula:

0H 2 S ..... CH2 ...

CH2 -S ---- CH2 CH2

  CH2-CO-X-dl-Gln-Asn-NH-CH-CO-Pro-Leu-Gly-NH2 R Formula b Molecular molecular analysis Asp Glu Pro Gly system XS 2 (Molecular weight of S3 S4)% C% ll Leu X (CHCO)

(2H4COH

  PhlO (N02) 0, 19 0 24 Phe (OEt) 0118 01 22 Pho 0/15 0.15 Phle (NJI-IZ) 0133 0 21

Phe (ile) 0) 28.

  0 23 Pho (and) 0127 0 12 0,] 2 0t 14 0 169

C44H66 N12013S.

6 H20 (1111)

C45671Nll 12S.

2 C2H402 (1122)

C44H67NllOllS. 2 Il20 (994.2)

C521474N12013S.

3 (1161) C45H69N1Oll.

4 hours (1044)

C46 71 Nil ilS.

0122 0170 0.5 H20 (995.2)

47f56 7'07 151 12 47158 6t63 14.80

53751 7110 13173

53 25 7122 13175

  53f15 7 20 15r50 52190 6195 15f26 53y79 6143 14148 53195 6y32 14.30 51176 7143 14y75

51189 7715 14173

55152 7729 15148

56.03 7133 15108

1S06 0 97 1l 08 1t03 0.96 1 007

1.04 0199

1104 0y89 1.04 0y94

1., 07 0.89

1t01 1102 1.06 1i01

1.00 01 95

1100 0182

1.01 0.94

1705 1.08

1.00 1.04

1.00 1, 09

  1 05 0 95 1.00 if 00 01 97 1.01 1 04 1.100 1.03 0 90 0y99 0.96 4f23 leCHH20 1: 1 2.27 MeOH-buffer pH 4f2 3: 2 7.00 MeCH-H 2 O 3: 2. 3.56 MeOH-H-buffer. pH

4.2 3: 2

  4115 MeOH-tamon fold 4t4 3: 2 4 50 MeOI pH-buffer

414 13: 7

  ## EQU2 ## TABLE III (cont.) Phe (NNe2) 0, 1.0 0.5 Phe (NHI2) (), 0.80 Oo05 0o 32 0.61 f0, (50%)

C46 1721, 1201, 1S.

2:20 (1037)

44 {68N12011 S.

4 hours (1045 hours)

53, 28

  52f 89, 57 90 7, 00 61 80 6,56 6, 39

16, 21

  , 81 16 09 70 1 09 0f97 01 96 1 03 1104 1109 1.04 2.62 MeOH-pH1 buffer

4T4 3: 2

1.08 0 83 0.81 44 32

  1.10 0.96. 1; 02 0X71 MeOH-buffer pHi

1., 1.2 0.79 0.95 4! 4 3: 2

  a - Same meaning b - Buffer pH 4.2 pH 4.4 buffer as in Table II is a 1% trifluoroacetic acid solution supplemented with triethylamine; is sodium phosphate 0.015 M. co 4- o $ V1 ro

EXAMPLE 3

  This example describes the preparation of Zhenyl-2 alanine.

  not] desamino carba-6 oxytocin. The compound of

  of the formula (II) as indicated in Example 1. Then the 2,4,5-trichlorophenyl ester of o-nitrobenzenesulfenylphenylalanine is added to 0.8 g of a suspension of the free heptapeptide. in 15 ml of dimethylformamide. After stirring for 96 hours at room temperature, the reaction mixture is treated as described in Example 1. 1 g (85%) of a compound having a melting point of 223-225 ° C. are obtained.

  whose characteristics are shown in Table II.

  The cyclization is then carried out as described in Example 1. The product containing a material is dissolved.

  giving a positive reaction with nynhydrin in a

  1/1 mixture of methanol and water and filtered through a sulfonate cation exchanger column (Dowex 50 in H + cycle, 5 ml). Concentrate and lyophilize to obtain 125 mg

  of a compound which is then purified by gel filtration.

  15 mg of the product thus obtained is chromatographed on a

  Modified silica gel (Separon SI C18, 15 x 0.6 cm) in a 3/2 mixture of methanol and water. The fraction of k '= 7.0 is concentrated and lyophilized to obtain 4.2 mg

  of a compound whose characteristics appear in the table

III.

EXAMPLE 4

  This example describes the preparation of [p-benzyloxy-

  -carbonylaminophenyl-2-alanine] desamino carba-6 oxytocin.

  The starting compound of formula (II) is prepared as indicated

  in Example 1. The o-nitrobenzenesulfenyl p-benzyl-

  -oxycarbonylaminophenylalanine from 1.05 g of its diocyclohexylammonium salt, as described in Example 2 and

  the active ester is formed as described in Example 1.

  purified with petroleum ether, 1.0 g of a crystalline compound having a melting point of 54-57 ° C. is added. 0.8 g of the active ester is added to a suspension of 0.6 g. of the heptapeptide in 15 ml of dimethylformamide and treated as described in Example 1. 0.53 g (55%) of a

  compound melting at 220-222 ° C. and having the characteristics

listed in Table 2.

  The cyclization is carried out as described in Example 1 from 200 mg. 155 mg of a product are obtained which is purified 30 mg by repeated gel filtrations. Additional purification is carried out by chromatography on a reverse phase column (modified silica gel Separon SI

  C18; 15 x 0.6 cm) in a 3/2 mixture of methanol and

  trifluoroacetate at pH 4.4. The freeze-dried fraction is

  taking the compound of k '= 3.56 and 4.2 mg of a

  with the characteristics shown in Table III.

  EXAMPLE 5 This example describes the preparation of Zp-aminophenyl-2

  alaninej desamino carba-6 oxytocin.

  Sodium is added to 3.6 mg of a Zp-nitro-

  2-phenylalanine? desamino carba-6 oxytocin in 5 ml of amines

  liquid moniac until a blue color

  standing for 30 seconds. The solution is then discolored by ad-

  addition of acetic acid and, after evaporation of the ammonia,

  the residue is purified by gel filtration. The fraction

  respondent collected gives by lyophilization 2.1 mg of a

  compound whose characteristics are shown in Table III.

EXAMPLE 6

  A suspension of hydrobromic acid is added to the acid.

  of acetic acid (35%, 1 ml) to a suspension of 30 mg of

  benzyloxycarbonylaminophenyl-2alanine] desamino carba-6 oxytocin in 1 ml of acetone and the resulting solution is allowed to stand for 1 hour at room temperature. After having evaporated several times in acetone and reprecipitated in methanol with ether, the product is dissolved in 3 ml of 3M acetic acid and purified by gel filtration. By

  lyophilization, 8.7 mg of a compound whose

  The properties correspond to those of the product of Example 5.

EXAMPLE 7

  This example describes the preparation of 2-p-methylphenylalanine.

  ne7 desamino carba-6 oxytocin. The active ester of onitrobenzenesulfenyl p-methylphenylalanine is prepared from

  0.7 g of the corresponding dicyclohexylammonium salt, as de-

  Crystalline in Example 4. 0.58 g of a compound melting at 127 ° -133 ° C. is added. This active ester is added to a suspension of 0.65 g of the heptapeptide in 13 ml of dimethylformamide and the same way as in Example 1, 0.83 g of a

  compound having a melting point of 220-226 C; the characteristics

  This compound is listed in Table II.

  200 mg of the peptide are cyclized in the same way as in Example 1. 180 mg of product are obtained, 50 mg of which are purified by gel filtration and column chromatography (Separon SI C18) in a 3/2 mixture of methanol and of water. The fraction of k '= 5.03 was lyophilized to give 13.6 mg of a compound whose characteristics are shown in Table III.

EXAMPLE 8

  This example describes the preparation of [p-ethylphenyl-2-alanine] desamino carba-6 oxytocin. The octapeptide is prepared

  protected as described in Example 1 from 0.4 g of o-

  nitrobenzenesulfenyl p-ethylphenylalanine and 0.3 g of the free heptapeptide. 0.23 g of a compound melting at

  218-224 C whose properties are shown in Table II.

  The octapeptide is cyclized and purified as in Example 7. 8.3 mg of a compound (k '= 7.4, 3/2 mixture of methanol and water) having the characteristics indicated in

Table III.

EXAMPLE 9

  This example describes the preparation of [p-dimethylamino]

  -phenyl-2-alanine, desamino carba-6 oxytocin. We are preparing 1 '

  active ester of o-nitrobenzenesulfenyl p-dimethylaminophenyl

  -alanine from 0.5 g of the dicyclohexylammonium salt in the same manner as in Example 4. This gives 405 mg of a compound having a melting point of 113-117 C. This active ester is added to a suspension of 0.3 g of the heptapeptide in dimethylformamide and 0.43 g of a compound melting between 201 and 204 ° C. are obtained according to the same procedure as in Example 1 (but without washing with sulfuric acid) diluted). The

  The characteristics of the compound are shown in Table II.

  The cyclization is carried out in the same manner as in Example 1 to obtain 190 mg of a product which is purified by

  gel filtration. The product obtained is dissolved by freeze-drying

  (100 mg) in 20% acetic acid and purified by free electrophoresis. 54 mg of compound are obtained

  purifies by gel filtration. The characteristics of the

  duit (28 mg) are shown in Table III.

Claims (5)

  1. Analogues of oxytocin characterized in that they correspond to the general formula: CH2 S CH2 CH2   The amino acids belong to the L series and X represents phenylalanine or phenylalanine substituted in the para position by a radical. selected from (a) alkyl radicals, (b) ethoxy radical, (c) amino radical or amino radical substituted and (d) the nitro radical.   2. Process for preparing oxytocin analogs according to claim 1, characterized in that it consists of cyclizing a linear peptide of formula: CH2 S-CH CH2 1 21   CH 2 -CO-Act X-Ile-Gln-Asn-NH-CH-CO-Pro-Leu-Gly-NH 2 X represents an amino acid consisting of phenylalanine or phenylalanine substituted in the para position by a radical chosen from a) an alkyl radical, (b) the ethoxy radical, (c) the amino radical or a substituted amino radical and (d) the nitro radical, and Act represents an activator of the carboxy radical, the cyclization occurring between the amino radical   amino acid X and the carboxy radical of the S-carboxy-   -éthylhomocystéine. 3. Process for preparing a compound of formula CH2 S CH2- - CH2 I I   CH 2 -CO-NH-CH-CO-Ile-Gln-Asn-NH-CH-CO-Pro-Leu-Gly-NH 2 C L NH 2   characterized by reducing Zp-nitrophenyl-   2 alaninej desamino carba-6 oxytocin by sodium in 1 ' liquid ammonia.   4. Method according to claim 2, characterized in that that the activator is an ester.   5. Method according to claim 3, characterized in that   cleave the protecting group of Zp-benzyloxycarbonyl-   -aminophenyl-2-alanine] desamino carba-6 oxytocin by means of   hydrobromic acid in acetic acid.