GB2130590A

GB2130590A - Peptides

Info

Publication number: GB2130590A
Application number: GB08329425A
Authority: GB
Inventors: Castiglione Roberto De; Luigia Gozzini; Pier Carlo Montecucchi; Giuseppe Perseo
Original assignee: Farmitalia Carlo Erba SRL
Current assignee: Pfizer Italia SRL
Priority date: 1982-11-10
Filing date: 1983-11-03
Publication date: 1984-06-06
Also published as: GB8329425D0; GB2130590B

Abstract

There are provided peptides of the formula X - A - B - C - Trp - D - Y wherein X is H, a terminal N atom protecting group of acyl, aromatic urethane, alkyl or aralkyl or aliphatic urethane type. A and D are independently a valence bond or a L- alpha -amino acid residue; B is a L- alpha -imino acid residue or a L- alpha -amino acid residue. C is a L- alpha -imino acid residue or a neutral L- alpha -amino acid residue, Y is OH, NH2, OR, NHR, NR2 or NH-NH-R'; R is a straight chain, branched chain or cyclic (including fused or bridged ring), substituted or unsubstituted C1- C11 alkyl group, phenyl, or C7-C9 aralkyl; R' is H, any value which R may have, C3-C8 alkenyl, a straight chain, branched chain or cyclic aliphatic C1-C11 acyl group) optionally substituted by OH, NH2, or a halogen atom), an aromatic acyl group similarly optionally substituted, a straight chain, branched chain or cyclic aliphatic C3-C11 urethane type group or an aromatic urethane type group. Pharmaceutically acceptable salts of these peptides are also provided, as are pharmaceutical compositions containing peptides or their salts. The peptides can be prepared by classical solution synthesis, display useful endocrinological activity and are active as growth promoters.

Description

SPECIFICATION Peptides The invention relates to biologically active peptides, their pharmaceutically acceptable salts, processes for their preparation and application as therapeutic agents.

In this specification symbols and abbreviations are those commonly used in peptide chemistry (see J. Biol.

Chem. 1972, 247, 977-983). Boc, t-butyloxycarbonyl; Bzl, benzyl; C, concentration; d, decomposition; HOTcp, trichlorophenol; MeOH, methanol; Met(O), methionine sulphoxide; (4-CI)Phe, 4-chloro-L-phenylalanine; (4-NH2)Phe, 4-amino-L-phenylalanine; (4-NO2)Phe, 4-nitro-L-phenylalanine; Pip, L-pipecolic acid; Thz, 4-L-thiazolidine carboxylic acid; TLC, thin layer chromatography.

The invention provides peptides of the general formula: X-A-B-C-Trp-D-Y wherein X represents a hydrogen atom, a terminal nitrogen protecting group of acyl, aromatic urethane, alkyl aralkyl or aliphatic urethane type; A represents a valence bond or a L-a-amino acid residue; B represents a L-a-imino acid residue or a L-a-amino acid residue; C represents a L-a-imino acid residue or a neutral L-a-amino acid residue; D represents a valence bond or a L-o-amino acid residue; Y represents a hydroxy group, an amino group or a group oftheformula OR, NHR, NR2 or NH-NH-R' wherein R represents a straight chain, branched chain or cyclic (including fused or bridged ring) alkyl group having up to 11 carbon atoms, and being substituted or unsubstituted, a phenyl group or an aralkyl group having from 7 to 9 carbon atoms; and R' represents a hydrogen atom, any of the groups which R may represent, an alkenyl group having from 3 to 8 carbon atoms, a straight chain, branched chain or cyclic aliphatic acyl group having from 1 to 11 carbon atoms, unsubstituted or substituted by a hydroxy or an amino group or a halogen atom, an aromatic acyl group, unsubstituted or substituted by a hydroxy or amino group or a halogen atom, a straight chain, branched chain or cyclic aliphatic urethane type group having from 3 to 11 carbon atoms, or an aromatic urethane type group.

Preferred terminal nitrogen atom protecting groups which X may represent include (of acyl type) formyl, acetyl, trifluoroacetyl, propionyl and benzoyl groups; (of aromatic urethane type) benzyloxycarbonyl) (Z), 4-nitrobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 2 bromobenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc) and 3,5-dimethoxy--of'- dimethylbenzylocarbonyl (Ddz) groups; (of aliphatic urethane type) t-butoxycarbonyl, 1methylcyclobutoxycarbonyl, adamtanyloxycarbonyl and isobornyloxycarbonyl groups; and (of alkyl and aralkyl type) trityl, benzyl, methyl and isopropyl groups.

Preferred L-a-amino acid residues which A may represent may include Phe, (4-NO2)Phe, (4-NH2)Phe, (4-Cl)Phe, and Tyr. Preferred L-a-imino acid residues which B may represent include Pro, Thz and Pip; when A represents a valence bond preferred L-(x-amino acid residues which B may represent include Pyr, Phe and Tyr. Preferred L-a-imino acid residues which C may represent include Pro, Thz and Pip; preferred neutral L-a-amino acid residues which C may represent include Ala, Val and Leu. Preferred L-a-amino acid residues which D may represent include Val, Leu, Met, Met(O), lle and Phe. Preferred groups which R may represent include methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, 2,2,2-trifluoroethyl, cyclohexyl, adamantyl, phenyl, benzyl and phenethyl groups.

Examples of acyl groups which R' may represent are formyl acetyl, trifluoroacetyl, propionyl, butyryl, adamantylcarbonyl, benzoyl, phenylacetyl and cinnamyl. The aliphatic and aromatic urethane type groups which R' may represent are preferably those groups mentioned as preferred terminal nitrogen protecting group X of aliphatic and aromatic urethane type.

Salts of peptides according to the invention with pharmaceutically or veterinarily acceptable acids or bases are included. Such acid addition salts can be derived from a variety of inorganic and organic acids as sulphuric, phosphoric, hydrochloric, hydrobromic, hydroiodic, nitric, sulphamic, citric, lactic, pyruvic, oxalic, maleic, succinic, tartaric, cinnamic, acetic, trifluoroacetic, benzoic, salicylic, gluconic, ascorbic and related acids. Such base addition salts can be derived from a variety of inorganic and organic bases as sodium hydroxide, potassium hydroxide, diethylamine, triethylamine and dicyclohexylamine.

The synthesis of the peptides of the invention is accomplished by classical solution methods. The synthesis consists essentially in appropriate successive condensations of protected amino acids or peptides.

The condensation is carried out so that the resulting peptides have the desired sequence of 4 or 5 amino acid residues. The amino acids and peptides, which are condensed according to methods known in themselves in polypetide chemistry, have such of their amino and carboxyl groups as are not involved in the formation of peptide linkage, blocked by a suitable protecting group. The protecting groups are capable of being removed by acidolysis, saponification or hydrogenolysis.

For the protection of the amino groups the following protective groups may for example be employed: benzyloxycarbonyl, t-butoxycarbonyl, trityl, formyl, trifluoroacetyl, o-nitrophenylsulphenyl, 4 methoxybenzyloxycarbonyl 94luorenylmethoxycarbonyl or 3,5-dimethoxy-cl-a'-di methyl benzyloxyca rbonyl.

For the protection of the carboxy groups the following protective groups may for example be employed: methyl, ethyl, t-butyl, benzyl orp-nitrobenzyl.

The condensation between an amino group of one molecule and a carboxyl group of another molecule to form the peptide linkage may be carried out through an activated acyl-derivative such as a mixed anhydride, an azide oran activated ester, or by direct condensation between a free amino group and a free carboxyl group, in the presence of a condensing agent such as dicyclohexylcarbodiimide, alone or together with a racemization preventing agent, such as N-hydroxysuccinimide or 1-hydroxybenzotriazole.

Hydrazido or substituted hydrazido derivatives according to the invention are prepared by condensation of the N-protected peptide or amino acid with a suitably substituted hydrazine, such as benzylcarbazate, t-butylcarbazate, adamantylcarbazate, phenylhydrazine or adamantylhydrazine, or reacting the N-protected peptide or amino acid hydrazide with a suitable alkylating agent, such as an alkyl chloride, or with a suitable acylating agent such as benzylchloroformate, t-butylchloroformate, di-t-butyl-dicarbonate or adamantylfluoroformate. The condensation may be carried out in a solvent such as dimethylformamide, pyridine, acetonitrile, tetrahydrofuran or N-methyl-2-pyrrolidone. The reaction temperature may be from -30 C to ambient temperature. The reaction time is generally from 1 to 120 hours.The scheme of synthesis, the protecting groups and the condensation agents are selected so as to avoid the risk of racemization.

De-protecting reactions are carried out according to methods known per se in polypeptide chemistry.

Peptides wherein W represents OR are prepared, for example starting from the C-terminal amino acid esterified by the appropriate alcohol. Peptides wherein W represents OH can be prepared, for example, by hydrolysis of peptides wherein W represents OR. Peptides wherein W represents NH2, NHR or NR2 can be prepared by ammonolysis of the corresponding esters or starting from a C-terminal amino acid amidated by an appropriate amine.

The final condensation in the preparation of a peptide according to the invention is preferably between a compound X-A-B-W-OH or a mixed anhydride, activated ester or azide thereof and a compound H-J-Trp-D-Y wherein one of W and J represents a residue C and the other represents a valence bond, and A,B,C,D,X and Y are as herein defined save that X does not represent a hydrogen atom and Y does not represent a hydroxy or hydrazine group.

The compounds according to the invention possess interesting growth promoting activity in animals as indicated by in vivo - in vitro test system on the protein synthesis of liver tissue as described by K. Kammerer and A. Dey-Hazra in Veterinar-Medinische Nachrichten, 99 112(1980). They also show interesting endocrinological activity such as prolactin and luteinizing hormone release.

The peptides of the invention, for example H-Phe-Pro-Pro-Trp-Met-NH2, were tested in rats in daily doses ranging from 1 to 100 ng/Kg administered subcutaneously for a period of one to four weeks. They showed an increase of liver protein synthesis as determined by the method of Kämmerer and Dey-Hazra and an increase of body weight at the end of the four weeks experiments. In addition the feed conversion ratio was improved.

For veterinary use, the administration of the compounds of the invention to food producing animals can be effected in a dose range from 1 to 100 ng/kg according to the usual veterinary techniques for treatment with anabolic or growth promoting agents, namely by subcutaneously implant or in a suitable stabilized form mixed through the feed.

Accordingly, the invention further provides a pharmaceutical or veterinary composition comprising a compound of the invention or a pharmaceutically or veterinarily acceptable salt thereof in admixture with a pharmaceutically or veterinarily acceptable diluent or carrier; in addition, these preparations can have directed or delayed liberation of the active ingredient.

The preferred peptides according to the invention are: Pyr-Pro-Trp-Met-OH Pyr-Pro-Trp-Met-OMe Pyr-Pro-Trp-Met-NH2 Pyr-Pro-Trp-Met(O)-OH Pyr-Pro-Trp-Met(O)-OMe Pyr-Pro-Trp-Met(O)-NH2 Pyr-Ala-Trp-Met-OH Pyr-Ala-Trp-Met-OMe Pyr-Ala-Trp-Met-NH2 Pyr-Ala-Trp-Leu-OH Pyr-Ala-Trp-Leu-OMe Pyr-Ala-Trp-Leu-NH2 Pyr-Pro-Trp-Val-OH Pyr-Pro-Trp-Val-OMe Pyr-Pro-Trp-Val-NH2 H-Phe-Pro-Pro-Trp-OH H-Phe-Pro-Pro-Trp-OMe H-Phe-Pro-Pro-Trp-NH2 H-Phe-Pro-Trp-Met-OMe H-Phe-Pro-Trp-Met-N H2 H-Tyr-Pro-Trp-Met-OH H-Tyr-Pro-Trp-Met-OMe H-Tyr-Pro-Trp-Met-N H2 H-Tyr-Pro-Trp-Leu-OH H-Tyr-Pro-Trp-Leu-OMe H-Tyr-Pro-Trp-Leu-NH2 H-Phe-Pro-Pro-Trp-Leu-OH H-Phe-Pro-Pro-Trp-Leu-OMe H-Phe-Pro-Pro-Trp-Leu-NH2 H-Phe-Pro-Pro-Trp-Met-OH H-Phe-Pro-Pro-Trp-Met-OMe H-Phe-Pro-Pro-Trp-Met-NH2 H-Phe-Pro-Pro-Trp-Val-OH H-Phe-Pro-Pro-Trp-Val-OMe H-Phe-Pro-Pro-Trp-Val-NH2 H-Tyr-Pro-Pro-Trp-Met-OH H-Tyr-Pro-Pro-Trp-Met-OMe H-Tyr-Pro-Pro-Trp-Met-N H2 H-(4-Cl)Phe-Pro-Pro-Trp-Met-OH H-(4-Cl ) Phe-Pro-Pro-Trp-Met-O Me H-(4-CI)Phe-Pro-Pro-Trp-Met-NH2 H-(4-NH2)Phe-Pro-Pro-Trp-Met-OH H-(4-NH2)Phe-Pro-Pro-Trp-Met-OMe H-(4-NH2)Phe-Pro-Pro-Trp-Met-NH2 The following Examples illustrate the invention. The Rf values were determined on pre-coated plates of silica gel 60 F254 (Merck) layer thickness 0.25 mm, length 20 cm. using the following development systems: System A: benzene/ethyl acetate/acetic acid/water 100/100/20/10 by volume (upper phase).

System B: benzene/ethyl acetate/acetic acid/water = 100/100/40/15 by volume (upper phase).

System C: n-butanol/acetic acid/water = 4/1/1 by volume.

System D: chloroform/methanol/32% ammonium hydroxide = 55/45/20 by volume.

"E. Merck" is a Trade Mark.

TLC analyses were not carried out under standard conditions. The Rf values can therefore change, particularly at different temperatures. Melting points were determined in open capillaries with Tottoli apparatus and are uncorrected.

Most of the derivatives soften and decompose before melting. Solvents for crystallization, precipitation or grinding are reported in brackets. High voltage paper electrophoresis is carried out with a Pherograph Original-Frankfurt Type 64 apparatus on Schleicherand Scull paper No.2317 at pH 1.2 (formic acid: acetic acid:water = 123:100:777) at 1600 V (40 V/cm), and at pH 5.8 (pyridine:acetic acid: water = 450:50:4500) at 1400 V (32.5 V/cm). The products were characterized by their mobilities at pH 1.2 relative to Glu (E1.2)r and at pH 5.8 relative to His or Glu (E58), according to the migration direction.

EXAMPLE 1 Preparation of Pyr-Pro- Trp-Met-NH2 rIV) Step 1. Boc-Trp-Met-NH2 61) To a solution of 3.043 g (10 mmol) of Boc-Trp-OH in 30 ml of anhydrous tetrahydrofuran, 1.12 ml (10 mmol) of N-methyl-morpholine and 0.99 ml (10 mmol) of ethylchloroformate were successively added at a temperature of -12 C. After stirring for 2 minutes, a cold solution of 1.482 g (10 mmol) of H-Met-NH2 (P.

Chillemi, Gazz. Chim. Ital., 1963, 93, 1079) in 30 ml of dimethylformamide was added. The reaction mixture was stirred for 1 h at -12C, and for 2 h at 0-15 C, filtered from salts and evaporated in vacuo. The residue was dissolved in ethyl acetate and washed several times successively with sodium chloride saturated solutions of 1 M citric acid, 1 M sodium bicarbonate and water. The organic layer was dried over anhydrous sodium sulphate, and the solvent removed in vacuo.

4.041 g (93% yield) of compound I were obtained from ethyl acetate: m.p. 143"C, [a]20 = -12.3" (c= 1 meOH); RfA 0.61; Rf5 0.84.

Step 2. HCI.H-Trp-Met-NH2 {11) 3.911 g (9 mmol) of Boc-Trp-Met-NH2 (I) were dissolved in 40 ml of formic acid at room temperature. After complete Boc-removal (TLC monitoring) the solvent was evaporated in vacuo at 300C. The residue was dissolved in methanol cooled to 0 C, and 3.6 ml (10.8 mmol) of 3 M solution of hydrogen chloride in anhydrous tetrahydrofuran were added. Solvents were removed in vacuo, and 3 g (90% yield) of compound II were obtained from MeOH/AcOEt: m.p. 114C(d); [ai20= +20.4'(c= 1 MeOH); Rfc 0.62; E1.2 0.82.

Step 3. Pyr-Pro-Oll (III) 3.604 g (10 mmol) of Z-Pyr-Pro-OH (R. de Castiglione et al., Gazz. Chim. Ital., 1964,94,875) dissolved in 30 ml of a mixture methanol:dimethylformamide (1 :1 by volume) were hydrogenated at room temperature and atmospheric pressure in the presence of 1.2 g of 10% by weight palladium on charcoal. The catalyst was removed by filtration and the solution concentrated in vacuo. 2.149 g (95% yield) of compound III were obtained from diethyl ether: m.p.168 C; [a]20 = -105.1 (c= 1 MeOH); Rfc 0.21; RfD 0.62; E58 0.98.

Step 4. Pyr-Pro-Trp-Met-NH2 (IV) To a solution of 1.584 g (7 mmol) of Pyr-Pro-OH (III) dissolved in 20 ml of anhydrous tetrahydrofuran, 0.79 ml (7 mmol) of N-methyl-morpholine and 0.69 ml (7 mmol) of ethylchloroformate were successively added at - 12"C. After stirring for 2 minutes, a cold solution of 2.596 g (7 mmol) of HCI. H-Trp-Met-NH2 (II) and 0.79 ml (7 mmol) of N-methyl-morpholine in 20 ml of dimethylformamide was added. The reaction mixture was stirred for 1 hour at -12"C and for 2 hours at 0-15"C, then filtered from salts and evaporated in vacuo.The crude product was purified by column chromatography on silica gel (Merck) 0.040-0.063 mm eluting with chloroform:methanol:water 87:13:1 by volume. 2.545 g (67% yield) of IV were obtained from diisopropyl ether: m.p.207-209 C, [a]C2J3 = -95.3"(c=1 MeOH); Rfc 0.43. Amino acid ratio: Glu 1.00; Pro 0.98; Met 1.00.

EXAMPLE 2 Preparation of HCI. H-Phe-Pro-Pro-Trp-Met-NH2 fIX) Step 1. Boc-Phe-Pro-OH(V) 1.151 g (10 mmol) of proline was suspended in 10 ml of water and 10 ml 1 N NaOH were added. The obtained solution was diluted with dimethylformamide, and the solvents were evaporated in vacuo; dimethylformamide was added and again evaporated in vacuo. A solution of Boc-Phe-OTcp (4.447 g, 10 mmol) (E. Sandrin and R. A. Boissonnas, Helv. Chim. Acta, 1963,46, 1637) in 50 ml of dimethylformamide was added and the reaction mixture stirred at room temperature overnight.After solvent removal by evaporation in vacuo, the crude product was transformed into the corresponding free acid in the usual manner and purified by column chromatography on silica gel (Merck) 0.040-0.63 mm eluting with chloroform: methanol = 9.1 by volume. 3.262 g of compound V (90% yield) was obtained as a foam by evaporation from petroleum either: RfA 0.67.

Step 2. Boc-Pro- Trp-Met-NH2 (Vl) Starting from 1.722 g (8 mmol) of Boc-Pro-OH and 2.967 g (8 mmol) of HCl.H-Trp-Met-NH2 (II) and operating as in Step 1 of the Example 1,3.828 g (90% yield) of VI were obtained from isopropyl alcohol: [oL]DO = -70.2 (c=1 MeOH); RfA 0.38; RfB 0.73.

Step 3. HCl.H-Pro-Trp-Met-NH2 (VII) Starting from 3.722 g (7 mmol) of Boc-Pro-Trp-Met-NH2 (VI) and operating as in Step 2 of Example 1, 2.621 g (80% yield) of VII were obtained from absolute ethyl alcohol; [D20 = -23.0" (c= 1 MeOH); Rfc 0.41; E.2 0.76.

Step 4. Boc-Phe-Pro-Pro-Trp-Met-NH2 (VIII) Asolution of 2.340 g (5 mmol) of HCI.H-Pro-Trp-Met-NH2 (Vil) in 20 ml of dimethylformamide was cooled to OOC and 0.56 ml of N-methyl-morpholine were added, followed by 1.812 g of Boc-Phe-Pro-OH (V), 0.676 g (5 mmol) of 1 -hydroxy-benzotriazole and 1.135 g (5.5 mmol) of dicyclohexylcarbodiimide. The reaction mixture was stirred at room temperature for 3 hours then filtered and evaporated in vacuo. The crude product was purified by column chromatography on silica gel (Merck) 0.040-0.063 mm eluting with ethyl acetate: methanol:water = 70:30:2 by volume. 1,940 g (50% yield) of compound (VIII) were obtained from ethyl acetate/diethyl ether: RfA 0.15; RfB 0.60.

Step 5. HCl.H-Phe-Pro-Pro-Trp-Met-NH2 (IX) Starting from 1.552 g (2 mmol) of Boc-Phe-Pro-Pro-Trp-Met-NH2 (VIII) and operating as in Step 2 of Example 1, 1.282 g of crude product were obtained. The crude product was purified by column chromatography on silica gel (Merck) 0.040-0.063 mm eluting with chloroform:methanol = 82:18 by volume.

0.705 g of compound IX (50% overall yield) were obtained from methanol/diethyl ether: m.p. 205-215"C (d), [of]DO = -79.8 (c=1 MeOH); Rfe 0.39; E1.2 0.61. Amino acid ratio: Pro 1.99; Met 1.00; Phe 1.00.

EXAMPLE 3 Preparation of Pyr-Pro-Trp-Val-OMe (XII) Step 1. Boc-Trp-Val-OMe (X) To a solution of 12.17 g (40 mmol) of Boc-Trp-OH in 100 ml ofanhydroustetrahydrofuran, 4.5 ml (40 mmol) of N-methyl-morpholine and 3.96 ml of (40 mmol) of ethylchloroformate were successively added at a temperature of - 1 2'C. After stirring for 2 minutes, a cold solution of 6.70 g (40 mmol) of HCl.H-Val-OMe (E.L.

Smith et al., J. Biol. Chem. 199,801, 1952) and 4.5 ml of N-methyl-morpholine (40 mmol) in 50 ml of dimethylformamide was added. The reaction mixture was stirred for 1 hour at - 12 C and 2 hours at 0 -15 C, filtered from salts and evaporated in vacuo. The residue was dissolved in ethyl acetate and washed several times successively with sodium chloride saturated solutions of 1 M citric acid, 1 M sodium bicarbonate and water. The organic layer was dried over anhydrous sodium sulphate and the solvent removed in vacuo.

14.1 g (84.4% yield) of compound X were obtained from isopropyl alcohol/diisopropyl ether. RfA 0.81; Rfb 0.87.

Step 2. HCI.H-Trp-Val-OMe (XIJ 12.52 g (30 mmol) of Boc-Trp-Val-OMe (X) were dissolved in 15 ml of formic acid at room temperature.

After complete Boc-removal (TLC monitoring) the solvent was evaporated in vacuo at 30"C. The residue was dissolved in methanol cooled to OOC, and 11 ml (33 mmol) of 3 M solution of hydrogen chloride in anhydrous tetrahydrofuran were added. Solvents were removed in vacuo and 9.55 g (90% yield) of compound II were obtained from isopropyl alcohol/diisopropyl ether.

Rfc 0.69, E12 0.89 Glu.

Step 3 Pyr-Pro- Trp- Val-OMe (XII) To a solution of 5.66 g (25 mmol) of Pyr-Pro-OH (III) in 60 ml of anhydrous tetrahydrofuran, 2.81 ml (25 mmol) of N-methyl-morphone and 2.48 ml (25 mmol) of ethylchloroformate were successively added at -12"C. After stirring for 2 minutes, a cold solution of 8.85 g (25 mmol) of HCI.H-Trp-Val-OMe (Xl) and 2.81 ml (25 mmol) of N-methyl-morpholine in 60 ml of dimethylformamide was added. The reaction mixture was stirred for 1 hour at -12 C and for 2 hours at 0"-15"C, then filtered from salts and evaporated in vacuo.The crude product was purified by column chromatography on silica gel (Merck) 0.040-0.063 mm eluting with methylene dichloride: methanol:water (92:8:1) by volume. 8.37 g (63.7% yield) of XII were obtained from isopropyl alcohol/diisopropyl ether. m.p. 120"C [1X]D4 = -76.3 (c= 1 MeOH); RfB 0.21; Rfc 0.56; Amino acid ratio:Glu 1.00; Pro 0.98; Val 1.00.

EXAMPLE 4 Preparation of Pyr-Pro-Trp-Val-OH {XIIIJ 2.63 g (5 mmol) of Pyr-Pro-Trp-Val-OMe (XII), prepared in Example 3, Step 3, were dissolved in 15 ml of methanol and saponified with 7.5 ml 1 N sodium hydroxide at room temperature. The reaction was completed within 4 hrs. The solution was diluted with 40 ml of water and concentrated in vacuo to half the volume, diluted again with 40 ml of water, cooled to 0 C, acidified with a 5 N hydrochloric acid to pH 2 and finally extracted with ethyl acetate. The organic layer was washed to neutrality with sodium chloride saturated solutions, dried over anhydrous sodium sulphate and the solvent removed in vacuo. 2.1 g (82% yield) of compound (XIII) were obtained from isopropyl alcohol/diisopropyl ether.

[a]D5 = - 47.6 (c=1 MeOH); RFB 0.11; Rfc 0.48; E5..8 0.43 Glu.

Amino acid ratio Glu 1.00; Pro 0.99; Val 1.00.

EXAMPLE 5 Preparation ofPyr-Pro-Trp- Va I-NH2 (XIV) 2.63 g (5 mmol) of Pyr-Pro-Trp-Val-OMe (XII) prepared in Example 3 Step 3, were dissolved in 20 ml of methanol and 0.4 ml (2% v/v) of ethylene glycol. The solution was saturated at 5"C with gaseous ammonia and kept in the refrigerator until completion of the reaction (TLC monitoring). Excess ammonia was removed under vacuum and the solution concentrated in vacuo.After purification by column chromatography (silica gel 0.040-0.063 mm; eluent system CH2C12: MeOH = 87:13) the desired compound XIV was obtained (1.99 g, 78% yield) from isopropyl alcohol/diisopropyl ether. m.p. 128"C; [a]o = -69.3" (c=1 MeOH); Rf5 0.10; Rfe 0.43. Amino acid ratio: Glu 0.99; Pro 0.97; Val 1.00.

Operating as in previous Examples, the following other peptides have been synthesized: XV) H-Phe-Pro-Pro-Trp-Leu-NH2.HCI Rfe 0.43; E120.61.

XVI) Pyr-Ala-Trp-Met-OH Rfc0.57.

XVII) Pyr-Pro-Trp-Met-OH RfcO.42.

XVIII) H-Tyr-Pro-Trp-Leu-NH2.HCI m.p. 160-1 70"C (d) (methanol/diethylether); E1.2 0.63.

XIX) H-Phe-Pro-Trp-lle-NH2HCI m.p. 125"-130"C (d) (diethyl ether); E12 0.60.

Claims

1. A peptide having the general formula X - A - B -C - Trp - D - Y as herein defined, or a pharmaceutically or veterinarily acceptable salt thereof.

2. A peptide according to claim 1 wherein X represents a hydrogen atom, or a formyl, acetyl, trifluoroacetyl, propionyl, benzoyl, benzyloxy-carbonyl, 4-nitrobenzyloxycarbonyl, 4-methoxybenzyl oxycarbonyl,2,4-dichlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethylcarbonyl and 3,5-di methoxy-a ' di methyl benzyloxy-carbonyl, t-butoxycarbonyl 1 -methyl-cyclobutoxycarbonyl, adamantyloxycarbonyl, isobornyloxycarbonyl, trityl, benzyl, methyl or isopropyl group, A represents a valence bond or a Phe, (4-NO2)Phe, (4-NH2)Phe, (4-Cl)Phe or Tyr residue, B represents a Pro, Thz or Pip residue or when A represents a valence bond, a Pyr, Phe or Tyr residue, C represents a Pro, Thz, Pip, Ala, Val or Leu residue, D represents a valence bond or a Val, Leu, Met, Met(O), lle or Phe residue, and Y represents a hydroxy group, an amino group or a group of the formula OR, NHR, NR2 or NH-NH-R' wherein R represents a methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, 2,2,2-trifluroethyl, cyclohexyl, adamantyl, phenyl, benzyl or phenethyl group, R' represents a hydrogen atom, any of the groups specifically named in this claim for R, or a formyl, acetyl, trifluoroacetyl, propionyl, butyryl, adamantylcarbonyl, benzoyl, phenylacetyl, cinnamyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4- methoxybenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 9- fI uorenyl methoxycarbonyl, 3,5-di methoxy-a a'-di methylbenzyloxycarbony, t-butoxycarbonyl, methyl cyclobutoxycarbonyl, adamantyloxycarbonyl or isobornyloxycarbonyl group; or an acid or base addition salt thereof, said acid addition salt being derived from sulphuric, phosphoric, hydrochloric, hydrobromic, hydroiodic, nitric, sulphamic, citric, lactic, pyruvic, oxalic, maleic, succinic, tartaric, cinnamic, acetic, trifluoroacetic, benzoic, salicilic, gluconic or ascorbic acid and said base addition salt being derived from sodium hydroxide, potassium hydroxide, diethylamine, triethylamine or dicyclohexylamine.

3. A compound according to claim 1 or claim 2, wherein X represents a hydrogen atom.

4. Any one of the peptides Pyr-Pro-Trp-Met-OH Pyr-Pro-Trp-Met-OMe Pyr-Pro-Trp-Met-NH2 Pyr-Pro-Trp-Met(O)-OH Pyr-Pro-Trp-Met(O)-OMe Pyr-Pro-Trp-Met(O)-N H2 Pyr-Ala-Trp-Met-OH Pyr-Ala-Trp-Met-OMe Pyr-Ala-Trp-Met-N H2 Pyr-Ala-Trp-Leu-OH Pyr-Ala-Trp-Leu-OMe Pyr-Ala-Trp-Leu-NH2 Pyr-Pro-Trp-Val-OH Pyr-Pro-Trp-Val-OMe Pyr-Pro-Trp-Va I-NH2 H-Phe-Pro-Pro-Trp-OH H-Phe-Pro-Pro-Trp-OMe H-Phe-Pro-Pro-Trp-NH2 H-Phe-Pro-Pro-Met-OMe H-Phe-Pro-Trp-Met-NH2 H-Tyr-Pro-Trp-Met-OH H-Tyr-Pro-Trp-Met-OMe H-Tyr-Pro-Trp-Met-NH2 H-Tyr-Pro-Trp-Leu-OH H-Tyr-Pro-Trp-Leu-OMe H-Tyr-Pro-Trp-Leu-NH2 H-Phe-Pro-Pro-Trp-Leu-OH H-Phe-Pro-Pro-Trp-Leu-OMe H-Phe-Pro-Pro-Trp-Leu-NH2 H-Phe-Pro-Pro-Trp-Met-OH H-Phe-Pro-Pro-Trp-Met-OMe H-Phe-Pro-Pro-Trp-Met-N H2 H-Phe-Pro-Pro-Trp-Val-OH H-Phe-Pro-Pro-Trp-Val-OMe H-Phe-Pro-Pro-Trp-VaI-NH2 H-Tyr-Pro-Pro-Trp-Met-OH H-Tyr-Pro-Pro-Trp-Met-OMe H-Tyr-Pro-Pro-Trp-Met-NH2 H-(4-Cl)Phe-Pro-Pro-Trp-Met-OH H-(4-Cl)Phe-Pro-Pro-Trp-Met-OMe H-(4-CI)Phe-Pro-Pro-Trp-Met-NH2 H-(4-N H2)Phe-Pro-Pro-Trp-Met-OH H-(4-NH2)Phe-Pro-Pro-Trp-Met-OMe H-(4-NH2)Phe-Prn-Prnjrp-Met-NH2 or a pharmaceutically or veterinarily acceptable salt thereof.

5. A pharmaceutical or veterinary composition comprising a peptide according to any preceding claim or a pharmaceutically or veterinarily acceptable salt thereof in admixture with a pharmaceutically or veterinarily acceptable diluent or carrier.

6. An animal feed in which there is admixed a peptide according to any of claims 1 to 4 or a veterinarily acceptable salt thereof.

7. A method of stimulating animal growth, the method comprising administering to an animal an effective amount of a peptide according to any of claims 1 to 4 or a veterinarily acceptable salt thereof.

8. A process for the preparation of a compound according to claim 1, the process comprising condensing, optionally in the presence of dicyclohexylcarbodiimide, a compound having the general formula X-A-B-W-OH or a mixed anhydride, activated ester or azide thereof with a compound having the general formula H-J-Trp-D-Y wherein one of W and J represents a residue C and the other represents a valence bond, and A,B,C,D,X and Y are as herein defined save that X does not represent a hydrogen atom and Y does not represent a hydroxy or hydrazino group, and, if desired, performing on the resultant peptide according to claim 1 one or more of the additional steps of (a) converting the group Y to another group Y by hydrazinolysis, esterification, hydrolysis or ammonolysis, (b) deprotecting the peptide, (c) salifying the free peptide and (d) obtaining a free peptide from a salt thereof.

9. A process according to claim 8 in which W represents a residue C and J represents a valence bond.

10. A process for the preparation of a peptide according to claim 1, the process being substantially as described herein with reference to any of the Examples.