GB2156822A - Biologically active tridecapeptides - Google Patents

Abstract

Peptides of the formula: <IMAGE> wherein X represents a neutral or acidic L- alpha -amino acid residue; A represents hydrogen or an acyl, alkoxycarbonyl, aralkoxycarbonyl, alkyl or aralkyl amino-protecting group; B represents hydrogen or an acyl, alkyl, aralkyl, alkoxycarbonyl or aralkoxycarbonyl phenolic hydroxy-protecting group; Y and W independently represent a neutral L- alpha -amino acid residue C represents hydroxy, amino or a group of the formula OR, NHR, NR2 or NH-NH-R' wherein R represents a straight chain, branched chain or cyclic alklyl group having up to 11 carbon atoms, phenyl or C7-C14 aralkyl and R' represents hydrogen, any of the groups which R may represent, a straight chain, branched chain or cyclic aliphatic acyl group having from 1 to 11 carbon atoms, a straight chain, branched or cyclic alkoxycarbonyl group having from 3 to 11 carbon atoms, or an aralkoxycarbonyl group.

Description

SPECIFICATION Biologically active tridecapeptides The Invention relates to biologically active peptides, their preparation and pharmaceutical compositions containing them.

In this specification symbols and abbreviations are those commonly used in peptide chemistry (see Eur. J. Biochem. (1984) 138, 9-37). Consequently, the three-letter amino acid symbols denote the L-configuration of chiral amino acids. Other symbols and abbreviations used are: AcOEt, ethyl acetate; AcOH, acetic acid; DCC, dicyclohexylcarbodiimide; DCEU, dicyclohexylurea; DMF, dimethylformamide; ECC, ethylchloroformate; Et2O, diethyl ether; FC, flash chromatography on silica gel (Merck) 0.040-0.063 mm; HCI/THF, dry HCI in anhydrous tetrahydrofuran; HOBT, 1-hydroxybenzotriazole; HOTcp, 2,4, 5-trichlorophenol; iBCC, isobutylchloroformate; iPr2O, diispropyl ether; iPrOH, isopropyl alcohol; MeOH, methyl alcohol; Msc, methylsulphonylethoxycarbonyl; NMM, N-methylmorpholine; PE, petroleum ether;THF, tetrahydrofuran. The invention provides peptides of formula

wherein X represents a neutral or acidic L-a-amino acid residue; A represents a hydrogen atom or an acyl, alkoxycarbonyl, aralkoxycarbonyl, alkyl or aralkyl amino-protecting group; B represents a hydrogen atom or an acyl, alkyl, aralkyl, alkoxycarbonyl or aralkoxycarbonyl phenolic hydroxy-protecting group Y and W independently represent a neutral L-a-amino acid residue;; C represents a hydroxy group, an amino group or a group of the formula 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, a phenyl group or an aralkyl group having from 7 to 1 4 carbon atoms and R' represents a hydrogen atom, any of the groups which R may represent, a straight chain, branched chain or cyclic aliphatic acyl group having from 1 to 11 carbon atoms, a straight chain, branched chain or cyclic alkoxycarbonyl group having from 3 to 11 carbon atoms, or an aralkoxycarbonyl group; and pharmaceutically acceptable salts thereof.

Preferred L-a-amino acid residues which X may represent include Glu and Gln.

Preferred terminal amino-protecting groups which A may represent include (of the acyl type) formyl, acetyl, trifluoroacetyl, propionyl and benzoyl groups; (of the aralkoxycarbonyl type) benzyloxycarbonyl, 4-nitrobenzyloxycarbonly, 4-methoxybenzyloxyczrbonyl, 2,4-dichlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl and 3, 5-dimethoxy-a,a'- dimethylbenzyloxycarbonyl groups; (of the alkoxycarbonyl type) t-butyloxycarbonyl, 1-methylcyclobutyloxy-carbonyl, adamantyloxycarbonyl, isobornyloxycarbonyl and methylsulphonylethoxycarbonyl groups and (of the alkyl and aralkyl type) trityl, benzyl, methyl and isopropyl groups.

Preferred phenolic hydroxy-protecting groups which B may represent include: (of the acyl type) acetyl and benzoyl groups; (of the alkyl type) a butyl group; (of the aralkyl type) benzyl, 2,6-dichlorobenzyl and 2-nitrobenzyl groups; (of the alkoxycarbonyl type) t-butyloxycarbonyl group; and (of the aralkoxycarbonyl type) benzyloxycarbonyl and 2-bromobenzyloxycarbonyl groups.

Preferred L-a-amino acid residues which Y may represent include lle, Leu, Ahx and Phe.

Preferred L-a-amino acid residudes which W may represent include Met, MetO and Ahx.

Preferred meanings which R and R' may represent include methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, 2,2,2-trifluoroethyl, cyclohexyl, adamantyl, phenyl, benzyl, phenethyl and fluorenylmethyl groups.

Examples of acyl groups which R' may represent are formyl, acetyI, trifluoroacetyl, propionyl, butyryl, adamantylcarbonyl, benzoyl, phenylacetyl and cinnamyl. The preferred alkoxycarbonyl and aralkoxycarbonyl groups which R' may represent are those groups mentioned above as preferred amino- protecting groups A of the alkoxycarbonyl and aralkoxycarbonyl type.

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

The present invention also provides a process for the preparation of a peptide of the invention or a pharmaceutically acceptable salt thereof, which process comprises condensing suitably protected amino acids or peptides in the order of the amino acids of the desired peptide, as required removing the protecting groups and optionally converting the resulting peptide into a pharmaceutically acceptable salt thereof.

The synthesis consists essentially of appropriate successive condensations of protected amino acids or peptides. The condensation is carried out so that the resulting peptides have the desired sequence of thirteen amino acid residues. The amino acids and peptides, which can be condensed according to methods known in themselves in polypeptide chemistry, have their amino and carboxyl groups, which are not involved in the formation of the peptide linkage, blocked by a suitable protecting group.

The protecting groups are capable of being removed by acid or alkali treatment and by hydrogenolysis. For the protection of an amino group the following protecting groups may, for example, be employed: benzyloxycarbonyl, t-butyloxycarbonyl, trityl, formyl, trifluoroacetyl, onitrophenylsulphenyl, 4-methoxybenzyloxycarbonyl, 94luorenylmethoxycarbonyl, 3, 5-dimethoxy a-a'-dimethylbenzyloxycarbonyl or methylsulphonylethoxycarbonyl. For the protection of a carboxyl group the following protective groups may for example, be employed: methyl, ethyl, tbutyl, benzyl, p-nitrobenzyl or fluorenylmethyl.

De-protection reactions can be carried out according to methods known per se on polypeptide chemistry.

The condensation between an amino group of one molecule and a carboxyl group of another molecule to form a peptide linkage may be carried out through an activated acyl-derivative such as a mixed anhydride, an azide or an 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 Nhydroxysuccinimide or 1-hydroxybenzotriazole. The condensation may be carried out in a solvent such as dimethylformamide, dimethylacetamide, pyridine, acetonitrile, tetrahydrofuran or Nmethyl-2-pyrrolidone.

The reaction temperature may be from - 30"C to ambient temperature. The reaction time is generally from 1 to 1 20 hours. The scheme of synthesis, the protecting groups and the condensing agents are selected so as to avoid the risk of racemization.

Peptides wherein C represents OR are prepared, for example, starting from the C-terminal amino acid esterified by the appropriate alcohol. Peptides wherein C represents OH can be prepared, for example, by hydrolysis of peptides wherein C 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.

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-butyldicarbonate or adamantylfluoroformate.

The peptides of the invention and their pharmaceutically acceptable salts are useful in methods of treatment of the human or animal body by therapy. By intracerebroventricular administration, the compounds according to the invention have been found to stimulate the secretion of pituitary prolactin, to exert sedative and deconditioning effects in behavioural tests for emotionality in laboratory animals, to inhibit the intestinal transit of a meal, in particular a carbon meal test, to lower body temperature and to possess an antipyretic effect.

The increase of prolactin release has been assessed by the method proposed by G.D.

Niswender, C.L. Chen., A.R. Midgley Jr., J. Meites and S. Ellis in Proc. Soc. Exp. Biol. Med.

130, 793-797 (1969).

The behavioural effects have been evaluated by tests carried out on rats according to the procedure described by J. Archer in Anim. Behav. 21, 205-235 (1973).

The inhibition of intestinal motility has been assessed on rats employing the "transit charcoal meal" test according to B.B. Brown and H.W. Werner, J. Pharmacol. Exp. Ther. 97, 1 57 (1949). The invention further provides a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable diluent or carrier; in addition, these preparations can have directed or delayed liberation of the active ingredient.

Preferred peptides according to the invention are reported below Glp-Glu-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-lle-Tyr-Pro-Met-OH Glp-Glu-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-lle-Tyr-Pro-Met-OH --------------------------------------- -----------OMe ----------------NH2 ----Gln- " ------------------------------------------OH ----Gin OMe ----Gin NH2 ------------------------------------Leu- Leu OMe ------------------------------------Leu-----------NH2 --------c---------------------------Ahx- Ahx OMe --------c---------------------------Ahx- ------------------------------------Phe- Phe OMe - Phe NH2 Me tO-OH Me tO-NH 2 Ahx-OH Ahx-OMe The following Example intends to illustrate the invention without limiting it.The R, are 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/benzine (60-80)/ethyl acetate = 70/10/40 by volume.

System B: benzene/ethyl acetate/acetic acid/water = 100/100/20/10 by volume (upper phase).

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

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

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

"Merck" is a trade-mark.

TLC analysis are carried out at a temperature ranging from 18 to 25 C: the R, values can therefore change + 5%. Melting points are determined in open capillaries with a 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 Schleicher and Schüll 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 are characterized by their mobilities relative to Glu at pH 1.2 (E12), and at pH 5.8 (E58).

Optical rotations are measured photoelectrically with a Perkin-Elmer 141 polarimeter at a concentration c = 1 in methanol unless otherwise stated.

Example Preparation of Glp-Glu-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-lle-Tyr-Pro-Met-OH (XXIV) Step 1. Boc-Tyr-Pro-OH (I) 14.06 g (30 mmol) of

(R. de Castiglione et al., Int. J. Peptide Protein Res. (1981), 17, 263-272) dissolved in 60 ml of methanol were hydrogenated at room temperature and atmospheric pressure in the presence of 3.51 g of 10% palladium-on-charcoal. The catalyst was removed by filtration and the solution was concentrated in vacuo. The residue was dissolved in ethyl acetate and concentrated in vacuo up to the precipitation of the product. Precipitation was completed by diluting with a mixture of diethyl ether and petroleum ether. 11.35 g (quantitative yield) of compound I were obtained: m.p. 124-132"C (foam); [a]%B -28.0'; RfB 0.45; E58 0.55.

Step 2. Boc-Tyr-Pro-Met-OMe (II) To a solution of 11.35 g (30 mmol) of Boc-Tyr-ProOH (I) in 60 ml of anhydrous THF, 3.37 ml (30 mmol) of NMM and 2.97 ml (30 mmol) of ECC were successively added at a temperature of - 12'C.

After stirring at this temperature for 2 minutes, a cold solution of 5.99 g (30 mmol) of HCl.H-Met-OMe (H. Yajima, Chem. Pharm. Bull (1968), 16, 1342) and 3.37 ml of NMM (30 mmol) in 40 ml of DMF was added. The reaction mixture was stirred for 45 minutes at - 1 2'C and for 90 minutes at 0-15"C, then 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 sulfate and the solvent removed in vacuo. 14.92 g (95% yield) of compound II were obtained as a solid foam from AcOEt: [a] 28 44.9 ; RfA 0.37; Rfa 0.67.

Step 3. HCl.H-Tyr-Pro-Met-Ome (Ill) 14.70 g (28.07 mmol) of Boc-Tyr-Pro-Met-OMe (II) were dissolved at room temperature in 1 50 ml of a 3.5 M solution of HCI/THF. After 30 minutes Boc-removal was complete and the solution was diluted with a great excess of Et2O obtaining the precipitation of the product. After recrystallization from iPrOH/Et,O, 11.75 g of compound 111(91% yield) were obtained: m.p.

78 C (foam); [a]2D - 32.7; RfD 0.52; E1 2 0.78.

Step 4. Boc-lle-Tyr-Pro-Met-OMe (IV) To a precooled (0 C) solution of 17.42 g (75.33 mmol) of Boc-lle-OH in 50 ml of anhydrous THF, 7.77 g (37.66 mmol) of DCC were added. After 30 minutes, the formed DCEU was removed by filtration and a solution of 11.55 g (25.11 mmol) of HCl.H-Tyr-Pro-Met-OMe (III) and 2.82 ml (25.11 mmol) of NMM in 50 ml of DMF was added and allowed to react for 90 minutes at room temperature. Solvents were removed in vacuo and the product partially purified by washings as described in Step 2.The compound IV was obtained in pure form after purification by FC eluting with AcOEt: n-hexane = 9:1. 10.39 g (65% yield) of compound IV were isolated from Et2O/iPr2O: m.p. 55"C (foam); [a] 2D6 - 62.40; Rf, 0.30; RfB 0.69.

Step 5. HCl.H-lle-Tyr-Pro-Met-OMe (V) 10.20 g (16.018 mmol) of Boc-lle-Tyr-Pro-Met-OMe (IV) were dissolved in 100 ml of a saturated solution of hydrogen chloride in acetic acid at room temperature.

After 30 minutes Boc-removal was complete and the solvent removed in vacuo. The residue was dissolved in methanol and the solution concentrated to dryness. 7.34 g 80% yield) of compound V were obtained from iPrOH/iPr,O: m.p. 115-129 C; [a] 2D2 - 34.7 ; RfD 0.68; E1 2 0.68.

Step 6. Boc-Trp-Pro-OH (VI) 3.45 g (30 mmol) of H-Pro-OH were dissolved at room temperature in 1 5 ml of 2 M sodium hydroxide. The solution was cooled to 0 C, diluted with 60 ml of DMF and the solvents were removed in vacuo at 35"C. The residue was suspended in 120 ml of DMF and 12.09 g (25 mmol) of Boc-Trp-OTcp (W. Broadbent et al., J. Chem. Soc. (C) (1967) 2632-2636) were added. The reaction mixture was stirred for 2 hours at room temperature, then evaporated in vacuo.

The residue was dissolved in water and washed several times with ethyl acetate. The aqueous layer was cooled to 0 C, acidified with a 5 N aqueous solution of hydrogen chloride to pH 2, then extracted with ethyl acetate. The organic layer was washed to neutrality with saturated sodium chloride (aqueous) solution and dried over anhydrous sodium sulphate. The solvent was removed in vacuo and compound VI was obtained from Et2O/PE (9.53 g, 95% yield): m.p.

100"C; [a]2D - 24.2"; RfB 0.55; E5.8 0.39.

Step 7. Boc-Trp-Pro-Pro-Pro-OBzl (VII) Starting from 9.35 g (23.29 mmol) of Boc-Trp-Pro-OH (Vl) and 7.89 g (23.29 mmol) of HCI.H-Pro-Pro-OBzl (C.M. Deber et al., J. Am. Chem. Soc. (1970) 92, 6191-6198) and operating as described in Step 2, but using iBCC as condensing agent, 1 2.14 g (76% yield) of compound VII were obtained from iPrOH/iPr,O: m.p. 137"C; [a]2D - 154.9"; RfB 0.50.

Step 8. Boc-Trp-Pro-Pro-Pro-OH (VIII) Starting from 11.95 g (17.42 mmol) of Boc-Trp-Pro-Pro-Pro-OBzl (VII) and operating as described in Step 1, 7.78 g (75% yield), of compound VIII were obtained from iPrOH/iPr,O; m.p. 139-147"C (d); [a]22 - 147.9'; Rfc 0.43; E5.8 0.32.

Step 9. Boc-Trp-Pro-Pro-Pro-lle-Tyr-Pro-Met-OMe (IX) To a precooled (0 C) solution of 7.39 g (12.40 mmol) of Boc-Trp-Pro-Pro-Pro-OH (VIII) and 1.78 g (12.40 mmol) of HOBT in 100 ml of anhydrous THF, 2.56 g (12.40 mmol) of DCC were added. After five minutes the solution of the activated ester was added to a cold solution of HCl.H-lle-Tyr-Pro-Met-OMe (V) (7.11 g, 12.40 mmol) and 1.39 ml (12.40 mmol) of NMM in 100 ml of DMF. The mixture was allowed to react at 0 C for 30 minutes, then at room temperature until completion of the reaction. DCEU was removed by filtration and the solvents by evaporation in vacuo. The product was then purified by washings as described in step 2.

The compound IX was finally isolated from iPrOH/iPr2O: (9.67 g, 70% yield): m.p.

125-135"C; [aI2D2 - 155.7'; Roc 0.52 Step 10. Boc-Trp-Pro-Pro-Pro-lle-Tyr-Pro-Met-OH (X) 9.30 g (8.35 mmol) of Boc-Trp-Pro-Pro-Pro-lle-Tyr-Pro-Met-OMe (IX) were saponified with 80 ml of 1 N sodium hydroxide for 1 hour at room temperature. The solution was diluted with water, cooled to 0 C, acidified to pH 2 with 5 M aqueous solution of hydrogen chloride, then extracted several times with chloroform.

The collected organic layers were washed to neutrality with saturated sodium chloride (aqueous) solution and dried over anhydrous sodium sulphate and the solvent removed in vacuo.

7.16 g (78% yield) of compound X were obtained from iPrOH/iPr,O; m.p. 147-163"C: [a] 2D - 148.9'; Rfc 0.26; E5.8 0.17.

Step 11. HCOOH.H-Trp-Pro-Pro-Pro-lle-Tyr-Pro-Met-OH (XI) 6.95 g (6.32 mmol) of Boc-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met-OH (X) were dissolved in 70 ml of HCOOH at room temperature. After complete Boc-removal the product was precipitated by diluting the solution with a great excess of Et2O.

5.95 (90% yield) of compound XI were obtained after recrystallization from MeOH/Et2O: m.p. 163-171 C (d); [a]2D - 127.3'; RfD 0.44; E12 0.45 Glu.

Step 12. HCI.H-Tyr-NH-NH-Z (XII) 12.88 g (30 mmol) of Boc-Tyr-NH-NH-Z (Yajima et al. (1972), J. Am. Chem. Soc. 94, 6170-6178) were dissolved at room temperature in 130 ml of 3.5 M solution of HCI/THF.

After 30 minutes Boc-removal was complete and the solvent was removed in vacuo. 9.88 g (90% yield) of compound XII were obtained from PE: m.p. 87-92 (foam); [a] - 26.6"; RfD 0.74; E 2 0.82.

Step 13. Boc-Pro-Tyr-NH-NH-Z (XIII) Starting from 5.65 g (26.24 mmol) of Boc-Pro-OH and 9.60 (26.24 mmol) of HCl.H-Tyr-NH-NH-Z (XII) and operating as described in Step 2, 11.05 g of crude product XIII were obtained. Compound XII was further purified by FC eluting with AcOEt: n-hexane = 8:2, obtaining 8.43 g (61 % yield) from MeOH/iPr2O: m.p. 105-117"C; [a] 2D2 -55.9'; RfA 0.34; RfB 0.68.

Step 14. HCI.H-Pro-Tyr-NH-NH-Z (XIV) Starting from 8.30 g (15.76 mmol) of Boc-Pro-Tyr-NH-NH-Z (XIII) and operating as described in Step 3, 7.30 g (90% yield) of compound XIV were obtained from iPrOH/iPr2O: m.p. 128-1 33'C (foam); [a]2D2 - 28.1'; RfD 0.49; E12 0.76 Glu.

Step 1 5. Boc-Lys(Msc)-OTcp (XV) To a cold (O"C) solution of 7.30 g (20 mmol) of Boc-Lys(Msc)-OH (A. Eberle et al. (1975) Helv. Chim. Acta 58, 2106-2129) and 3.95 g (20 mmol) of HOTcp in 200 ml of AcOEt, 4.127 g (20 mmol) of DCC were added. After 30 minutes at 0 C the mixture was allowed to react overnight at room temperature. DCEU was removed by filtration and the solvent removed in vacuo. 7.83 g (68% yield) of compound XV were obtained from AcOEt/iPr2O: [a] 2D2 - 24.7"; RfB 0.67; m.p. 112-117"C.

Step 16. Boc-Lys(Msc)-Pro-Tyr-NH-NH-Z (XVI) To a solution of 6.15 g (13.28 mmol) of HCI.H-Pro-Tyr-NH-NH-Z (XIV) and 1.49 ml (13.28 mmol) of NMM in 70 ml of DMF, 7.65 g (13.28 mmol) of Boc-Lys(Msc)OTcp (XV) were added and the mixture was allowed to react at room temperature overnight.

The solvent was removed in vacuo and the residue purified by FC eluting with AcOEt containing increasing amount of MeOH (from 2 to 5%). 7.48 g (70% yield) of compound XVI were obtained from iPrOH/iPr,O: m.p. 74-82"C (foam); [a]2D - 51.0'; Rfc 0.42.

Step 17. HCl.H-Lys(Msc)-Pro-Tyr-NH-NH-Z (XVII) 7.35 g (9.13 mmol) of Boc-Lys(Msc)-Pro-Tyr-NH-NH-Z (XVI) were dissolved in 75 ml of a 3.5 M solution of HCI/THF. After 40 minutes Boc-removal was complete and the solvent removed in vacuo. The residue was dissolved in methanol and treated with activated carbon.

5.75 g (85% yield) of compound XVII were obtained from MeOH/AcOEt/PE: m.p. 105-117"C (foam); [a] 2D2 -26.7'; RfD 0.37; E,2 0.62.

Step 18. HCl.H-Glu(OBzl)-ONb (XVIII) Starting from 7.09 g (15 mmol) of Boc-Glu(OBzl)ONb (R. Schwyzer and P. Sieber (1959) Helv. Chim. Acta 42, 972-977) and operating as described in Step 12, 5.83 g (95% yield) of compound XVIII were obtained from MeOH/iPrOH/iPr2O: m.p. 112-119"C; [a]2D + 6.7 ; RfD 0.70; E,2 0.96.

Step 1 9. Z-Glp-Glu(OBzl)-ONb (XIX) Starting from 3.64 g (13.82 mmol) of Z-Glp-OH and 5.65 g (13.82 mmol) of HCl.H-Glu(- OBzl)ONb (XVIII) and operating as described in Step 2, 7.25 g (85% yield) of compound XIX were obtained from MeOH/AcOEt/iPr2O: m.p. 147-151"C; [a]2D - 12.60 (c= 1, DMF); RfA 0.43; RfB 0.64.

Step 20. Z-Glp-Glu(OBzl)-OH (XX) To a solution of 7.109 (11.49 mmol) of Z-Glp-Glu(OBzl)-ONb (XIX) in 300 ml of 90% acetic acid cooled to 0 C, 3.6 g of zinc dust were added dropwise.

After 2 hours excess of zinc dust was removed by filtration and the solvent removed in vacuo.

The oily residue was taken up with AcOEt saturated with water and the precipitate was filtered off. The organic layer was extracted several times with 1 M sodium bicarbonate solution. The aqueous solution was cooled to 0 C, acidified with 5 N aqueous solution of hydrogen chloride to pH 2, then extracted several times with AcOEt. The collected organic layers were washed to neutrality with saturated sodium chloride (aqueous) solution and dried over anhydrous sodium sulphate.

4.71 g (85% yield) of compound XX were obtained from MeOH/H2O: Rfc 0.30; RfD 0.65; E58 0.54.

Step 21. Z-Glp-Glu(OBzl)-Lys(Msc)-Pro-Tyr-NH-NH-Z (XXI) Starting from 3.64 g (7.55 mmol) of Z-Glp-Glu(OBzi)-OH (XX) and 5.60 g (7.55 mmol) of HCl.H-Lys(Msc)-Pro-Tyr-NH-NH-Z (XVII) and operating as described in step 2, but using methylene chloride as solvent during washings, 7.07 g of partially purified product were obtained. Further purification was accomplished by FC eluting with CH2CI2: MeOH = 95:5. 5.74 g (65% yield) of compound XXI were obtained from iPrOH/iPr2O: Rfc 0.17; RfD 0.67.

Step 22. Glp-Glu-Lys(Msc)-Pro-Tyr-NH-NH2 (XXII) 5.60 g (4.79 mmol) of Z-Glp-Glu(OBzl)-Lys(Msc)-Pro-Tyr-NH-NH-Z (XXI) in 70 ml of DMF were hydrogenated at room temperature in the presence of 2.8 g of 10% palladium-oncarbon. The catalyst was removed by filtration and the solvent removed in vacuo. 3.30 g (85% yield) of compound XXII were obtained from iPrOH/iPr,O: RfD 0.24; E, 2 0.52.

Step 23. Glp-Glu-Lys(Msc)-Pro-Tyr-Trp-Pro-Pro-Pro-lle-Tyr-Pro-Met-OH (XXIII) To a solution of 4.04 g 3.87 mmol) of HCOBH.H-Trp-Pro-Pre -Pro-lle-Tyr-Pro-Met-OH (Xl) in 40 ml of DMF cooled to 0 C, 1.1 6 ml of a 4 M solution of hydrogen chloride in anhydrous THF were added. The solution was concentrated in vacuo, the residue taken up with 40 ml of DMF and concentrated again in vacuo.

To a solution of 3.10 g (3.87 mmol) of Glp-Glu-Lys(Msc)-Pro-Tyr-NH-NH2 (XXII) in 50 ml of anhydrous DMF, 2.42 ml (9.67 mmol) of a 4 M solution of hydrogen chloride in anhydrous THF and 0.50 ml (4.25 mmol) of n-butyl nitrite were successively added at a temperature of - 30"C. After stirring at - 1 5'C for 30 minutes, 1.52 ml (13.53 mmol) of NMM were added, followed by a cold (- 30'C) solution of 4.01 g (3.87 mmol) of HCl.H-Trp-Pro-Pro-Pro-Ile- Tyr-Pro-Met-OH (prepared as described above in this step) and 0.87 ml (7.73 mmol) of NMM in 40 ml of DMF.The reaction mixture was allowed to react at - 9"C for three days and then the salts were filtered off; the solvents were removed in vacuo and the product was partially purified by gel chromatography on Sephadex LH-20 eluting with DMF. 3.99 g (58% yield) of compound XXIII were obtained from iPrOH/AcOEt/iPr2O: RfD 0.43; RfE 0.58; E58 0.36.

Step 24. Glp-Glu-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-lle-Tyr-Pro-Met-OH.HCI (XXIV) 3.7 g (2.08 mmol) of Glp-Glu-Lys(Msc)-Pro-Tyr-Trp-Pro-Pro-Pro-lle-Tyr-Pro-Met-OH (XXIII) were dissolved at room temperature in 37 ml of MeOH. 184 ml of a solution constituted by dioxane/methanol/8M sodium hydroxide = 7.5/2.25/0.25 were added. The mixture was allowed to react at room temperature for five minutes then, after cooling to 0tC, 46 ml of HCI 1 N were added. Solvents were removed in vacuo and the crude product was isolated from MeOH/Et20 The compound XXIV was purified by counter current distribution using the solvent system nbutanol/acetic acid/ethyl alcohol/water = 5:1:1:8, obtaining 1.25 g (60% yield) from MeOH/ iPr0H/iPr20: RfD 0.23; RfE 0.40; E,20.38; E58 0.13.

Claims (22)

1. A peptide of formula:

wherein X represents a neutral acidic L-ex-amino acid residue; A represents a hydrogen atom or an acyl, alkoxycarbonyl, aralkoxycarbonyl, alkyl or aralkyl amino-protecting group; B represents a hydrogen atom or an acyl, alkyl, aralkyl, alkoxycarbonyl or aralkoxycarbonyl phenolic hydroxy-protecting group; Y and W independently represent a neutral L-a-amino acid residue;; C represents a hydroxy group, an amino group or a group of the formula OR, NHR, NR2 or NH-NH-R' wherein R represents a straight chain, branched chain or cyclic alkyl group having up to 11 carbon atoms, a phenyl group or an aralkyl group having 7 to 14 carbon atoms and R' represents a hydrogen atom, any of the groups which R may represent, a straight chain, branched chain or cyclic aliphatic acyl group having from 1 to 11 carbon atoms, a straight chain, branched or cyclic alkoxycarbonyl group having from 3 to 11 carbon atoms, or an aralkoxycarbonyl group; and pharmaceutically acceptable salts thereof.

2. A compound according to claim 1 in which X represents Glu or Gln.

3. A compound according to claim 1 or 2 in which A represents a formyl, acetyl, trifluoroacetyl, propionyl, benzoyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethoxy- carbonyl, 3,5-dimethoxy-a, a'-dimethylbenzyloxycarbonyl, t-butyloxy-carbonyl, 1-methyl-cyclobu- tyloxycarbonyl, adamantyloxycarbonyl, isobornyloxycarbonyl, methylsulphonylethoxycarbonyl, trityl, benzyl, methyl or isopropyl group.

4. A compound according to any one of the preceding claims in which B represents an acetyl, benzoyl, t-butyl, benzyl, 2,6-dichlorobenzyl, 2-nitrobenzyl, t-butyloxycarbonyl, benzyloxycarbonyl or 2-bromobenzyloxy-carbonyl group.

5. A compound according to any one of the preceding claims in which Y represents lle, Leu, Ahx or Phe.

6. A compound according to any one of the preceding claims in which W represents Met, MetO or Ahx.

7. A compound according to any one of the preceding claims in which R or R' represents methyl, ethyl, n-propyl isopropyl, n-butyl, sec.butyl, isobutyl, tert.butyl, 2,2,2-trifluoroethyl, cyclohexyl, adamantyl, phenyl, benzyl, phenethyl or fluoroenylmethyl.

8. A compound according to any one of the preceding claims in which R' represents formyl, acetyl, trifluoroacetyl, propionyl, butyryl, adamantylcarbonyl, benzoyl, phenylacetyl, cinnamyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, 3,5-dimethoxy-a,a'-dime- thylbenzyloxycarbonyl, t-butyloxycarbonyl, 1 -methyl-cyclobutyloxycarbonyl, adamantyloxycarbonyl, isobornyloxycarbonyl or methylsulphonylethoxycarbonyl.

9. A compound according to claim 1 which is Glp-Glu-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-lle -Tyr-Pro-Met-OH .

1 0. A peptide as claimed in claim 1 or a pharmaceutically acceptable salt thereof for use in a method of treatment of the human or animal body by therapy.

11. A peptide or salt thereof according to claim 10 for use in stimulating the secretion of pituitary prolactin.

1 2. A peptide or salt thereof according to claim 10 for use as a sedative or deconditioning agent.

1 3. A peptide or salt thereof according to claim 10 for use in slowing the intestinal transit of a meal.

14. A peptide or salt thereof according to claim 10 for use in lowering body temperature or as an antipyretic agent.

1 5. A process for preparation of a peptide as claimed in claim 1 or a pharmaceutically acceptable salt thereof which process comprises condensing suitably protected amino acids or peptides in the order of the amino acids of the desired peptide, as required removing the protecting groups and optionally converting the resulting peptide into a pharmaceutically acceptable salt thereof.

1 6. A process according to claim 1 5 in which the starting C-terminal amino acid W is esterified so as to obtain a peptide in which C represents OR.

1 7. A process according to claim 1 6 in which the peptide thus obtained is hydrolysed to obtain a peptide in which C represents a hydroxy group.

1 8. A process according to claim 1 6 in which the peptide thus obtained is subjected to ammono- or aminolysis so as to obtain a peptide in which C represents NH2, NHR or NR2.

1 9. A process according to claim 1 5 in which the starting C-terminal amino acid W is amidated so as to obtain a peptide in which C represents NH2, NHR or NR2.

20. A process according to claim 1 5 in which the starting C-terminal acid W or the Nprotected resulting peptide is condensed with a substituted hydrazine so as to obtain a peptide in which C represents NH-NH-R' or with hydrazine and subsequently an alkylating agent so as to obtain a peptide in which C represents NH-NH-R' wherein R' is an alkyl group as defined in claim 1.

21. A process for the preparation of a peptide as claimed in claim 1, said process being substantially as hereinbefore described in the Example.

22. A pharmaceutical composition comprising as active ingredient a peptide as claimed in claim 1 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable diluent or carrier.