SE447482B - PROCEDURE FOR PREPARING SOMATOSTATIN ANALOGS - Google Patents

Description

447 482 r Puon 2, 0fl ÅUTY The reduced form (open chain form) of somatostatin (RS) is the linear tetradecapeptide of the formula: sH S "H-Ala ~ Gly-Lys ~ Lys-Asn-Dhe-Dhe-Trp-Lys -Thr-Dhc-Thr-Ser-àyß-UH (B) The reduced form (B) has been prepared according to a total synthesis [see Rivier et al., CR Acad. Sci. Ser. P. Sci. Natur.

(Paris), gig, 2737 (1973) and Sarantakis and McKinley, Biochem. and Biophys. Res. Communications, âg, 234 (1973l] and the (B) can be transferred to somatostatin (A) by oxidation, whereby a bridging bond is formed between the two sulfhydryl groups on the two cysteinyl amino acid residues in the tetradecapeptide.

Various polypeptides, which can be considered as structural modifications of somatostatin, have been synthetically prepared and described in the chemical literature. Such polypeptides exhibit certain structural features in common with somatostatin and differ from somatostatin in that specific amino acid residues or functional groups originally present in the somatostatin molecule are either absent or replaced by other amino acid residues or functional groups. The present invention relates to novel synthetic biologically active polypeptides which can be considered as structural modifications of somatostatin. The polypeptides of the invention differ from somatostatin in the following respects: (a in the Alal-Glyl segment is missing; (b) the Lys4 residue is replaced by Arg or His; (dl Asns residue is replaced by His or Glu; and 8 (d} Trp - the remainder is either present or is replaced by D-Trp.

All optically active amino acid residues and amino acids are present in the L-configuration, unless otherwise stated. 447 482 The invention thus relates to chemical compounds of the formula: H-cys-X1-X2-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-OH (I) X.- | 4 SS wherein X1 is His or Arg, X 2 is His or Glu and X 4 is Cys or D-Cys, and the pharmacologically acceptable salts thereof.

The symbols identifying the amino acids and amino acid residues in the polypeptides described herein are those adopted by the IUPAC-IVB Committee on Biochemical Nomenclature Recommendation (1971) and described in Archives of Biochemistry and Biophysics, low, 1-8 (1972). The symbol "6-F-D-Trp" refers to D-tryptophan, wherein the 6-position is substituted with fluorine.

The present embodiments of the invention exhibit the following physical properties: they are white to light yellow-brown solids, substantially insoluble in chloroform, benzene and the like, but exhibit solubility in water and acidic aqueous solutions, such as hydrochloric acid and acetic acid. The compositions according to the invention do not show a sharp melting point and can be purified by, for example, chromatographic means. Hydrolysis of the compositions according to the invention in, for example, 4N methanesulfonic acid enables the determination of their amino acid content, which corresponds to the structures given above.

The present embodiments of the invention show pharmaceutical activity, in particular with respect to inhibition of the release of one or more of the hormones somatotropin, glucagon and insulin, which is determined by means of standard pharmacological test methods. Through dose control, efficacy selectivity enables inhibition of somatotropin and glucagon release without affecting endogenous insulin secretion levels.

Furthermore, the present embodiments of the invention may be used in admixture with insulin for the treatment of warm-blooded animals suffering from diabetes mellitus. According to a subgeneric aspect, the invention comprises the following: a) a compound of formula I, wherein X 1 is His; X2 is His and is Cys. b) a compound of formula I, wherein X 1 is Arg; X 2 is His and is Cys or D-Cys. c) a compound of formula I, wherein X 1 is His; X 2 is His and is Cys or D-Cys. D) a compound of formula I, wherein X 1 is Arg or His; X 2 is Glu and X 4 is Cys or D-Cys; X 1 and X 2 are preferably both His or X is Arg and X 2 is Glu. Particularly suitable compounds of the invention are: H-Cys-Arg-Glu-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys SS H-Cys-His-Glu-Phe-Phe-D- The present invention relates to processes for the preparation of the above-described compounds. Trp-Lys-Thr-Phe-Thr-Ser-Cys I in SS.

The compounds of formula I can be prepared by removing all protecting groups and the polystyrene resin carrier, when present, from a compound of formula II R1-Cys-X -X -Phe-Phe-D-Trp-Lys-Thr-Phe-Thr -Ser-X -OR (II) Ilz Is'7 189 ”Sa. RR RRs / s wherein 1R represents a 4K amino protecting group; R represents a carboxy-protecting group or a polystyrene resin support bonded via a methylene group, i.e. -CH2-poly- 447 482. 5 styrene resin carriers; X 3 is his or? Rg, wherein R 3 is a protecting group for the R 3 side chain nitrogen atoms in arginine and R 2 is hydrogen or a protecting group for the imidazole nitrogen atom in histidine; X is His as above or? Lu, wherein R 4 is a protective | R 2 R 4 group for the side chain carboxy group in glutamic acid; R6 is a protecting group for the side chain amino group in lysine; R 7, R 8 and R 9 are each protecting groups for the hydroxyl groups of threonine and serine; X4 has the meaning given above; and 04 and ß represent a sulfhydryl protecting group; and oxidizing the resulting compound, and optionally isolating the product in the form of the free base or a pharmaceutically acceptable salt.

Examples of R 2 are tosyl, benzyloxycarbonyl, adamantyloxycarbonyl and tert-butyloxycarbonyl; R 2 is preferably tosyl.

Examples of R 3 are nitro, tosyl, benzyloxycarbonyl, adamantyloxycarbonyl and tert-butyloxycarbonyl; R 3 is preferably tosyl.

Examples of R 4 are benzyl and t-butyl.

The side chain amino group of lysine is protected by R6, which is exemplified by tosyl, t-amyloxycarbonyl, t-butyloxycarbonyl, diisopropyloxycarbonyl, benzyloxycarbonyl, halobenzyloxycarbonyl, nitrobenzyloxycarbonyl and the like, wherein the 2-chlorobenbenzyl group is

Examples of R7, R8 and R9 protecting groups for the hydroxyl group of threonine and serine are acetyl, benzoyl, tert-butyl and benzyl.

The benzyl group is the most suitable for this purpose. Elimination of the protecting groups can be performed according to methods known in connection with the respective protecting groups. The protecting groups are preferably removed in a single step, such as using hydrogen fluoride in the presence of anisole.

The polystyrene resin support can be cleaved while the protecting groups are removed, such as with the use of hydrogen fluoride in the presence of anisole, or it can be cleaved by transesterification to form a carboxy-protected intermediate of formula II, wherein R is an ester function. Thus, for example, methanolysis gives a methyl ester intermediate of formula II, wherein R is methyl. Hydrolysis of such esters gives the desired C-terminus carboxy function.

When it is also hydrogen, the free disulfhydryl derivatives of the formula II can be cyclized by oxidation using an oxidizing agent such as iodine, oxygen (for example air), 1,2-diiodoethane or sodium or potassium iron (III) cyanide. to form the corresponding compounds of formula II, wherein N and II form a direct bond.

The polypeptide end products and their intermediates can be prepared according to the well-known solid phase method, described for example by Ixerrifieia, J. Am. Chem. sec. _83, 2149 (1963), or according to classical synthesis. The invention thus also encompasses a process for the preparation of a compound of formula II, wherein e and ß are protecting groups and R is a polystyrene resin support, the successive, suitably protected and / or activated amino acids being successively coupled under solid phase synthesis conditions to a chloromethylated or hydroxymethyl resin carrier. Alternatively, the compounds of formula II, wherein a and ß are protecting groups and R is a carboxy protecting group, can be prepared by coupling the required, suitably protected and / or activated amino acids or groups of amino acids according to methods known in connection with the formation of peptide bonds in formation. of the desired amino acid sequence. The n and fi protecting groups can then be selectively removed and the product oxidized to form the cyclic disulfide of formula II, wherein Q and ß form a direct bond. 447 482 Methods for activating amino acids before coupling and the coupling methods themselves are well known, see for example the textbook mentioned below by Schröder and Lübke.

When selecting a particular side chain protecting group for use in the synthesis of the peptides of the invention, the following rules should be followed: (a) the side chain protecting group must be resistant to the reactant and under the reaction conditions selected to eliminate the n-amino protecting group at each stage of synthesis; b) the protecting group must retain its protecting properties (ie not cleave under coupling conditions) and (c) the side chain protecting group must be able to be removed after completion of the synthesis of the desired amino acid sequence under the reaction conditions so that the peptide chain does not change.

According to the classical method applied to the compounds of the invention, the desired peptide is degraded by condensation of amino acids or groups of amino acids, which are protected if necessary. The condensation reactions can be carried out using methods generally known for the formation of amide bonds in peptide and penicillin chemistry. To promote an elegant condensation of the amino acids, it is advisable to use a condensing agent. Examples of condensing agents are larbodiimides, such as N, N'-dicyclohexylcarbodiimide (DCC) and N, N'-diisopropylcarbodiimide. Alternatively, the condensation can be carried out by activating one or both end groups.

Examples of the activated form of the carboxy end group are the acid chloride, the acid anhydride, the acid azide and the activated ester.

It will be apparent to those skilled in the art that the proposed method of carrying out the condensation reactions should not interfere with the protecting groups on the amino acids. When constructing the molecule via the classical method, it is particularly convenient to condense a protected peptide of the formula 447 432 R1-Ers-B1-82-Phe-UH sa (vra) with a protected peptide of the formula H-Phc ~ X3-Lys Thr-Dhe-Thr-Ser-X4-UR II R6 R7 'RB ng sß wherein R is a carboxy protecting group (preferably benzyl); R 1, R 6, R 7, R 8, R 9, B 1, B2, X 3 and X 4 have the indicated meaning and d and are β sulfhydryl protecting groups, preferably p-methoxybenzyl, to form a compound of formula II, which is deprotected and cyclized .

In the solid phase method applied to the present compounds, uramino- and sulfhydryl-protected cysteine is first bound to a chloromethylated polystyrene resin, after which the α-amino-protecting group is eliminated with trifluoroacetic acid in methylene chloride, trifluoroacetic acid separately or HCl in dioxane. The deprotection is carried out at a temperature between approximately 0 ° C and room temperature.

Other standard cleavage agents and conditions for the elimination of specific amino-protecting groups may be used, as described by Schröder and Lübke, "The Peptides", 1, 72-75 (Academic Press, 1965). After elimination of the d-amino-protected group, the following desired protected amino acids are coupled individually one by one to the resin-bound sequence Alternatively, small peptide fragments can be prepared, for example, according to the solution method and introduced into the solid phase reactor in the desired order.

Each protected amino acid or amino acid sequence is introduced into the solid phase reactor in an approximately four-fold excess. The coupling is carried out in dimethylformamide, methylene chloride or a mixture of the two solvents. The success of each coupling reaction in each step of the synthesis is determined according to the ninhydrin reaction, as described by E. Kaiser et al., Analyt. Biochem., Vol., 595 447 (482 (1970)) When the coupling is incomplete, the reaction is repeated before the α-amino protected group is removed to introduce the following amino acid or amino acid sequence.

The most suitable coupling agents are 1-hydroxybenzotriazole and diisopropylcarbodiimide; other such agents are known to those skilled in the art.

Once the desired amino acid sequence has been synthesized, the polypeptide is removed from the resin support by treatment with, for example, hydrogen fluoride and anisole to form the completely deprotected linear polypeptide. The cyclic disulfide can be obtained by air oxidation or for example by oxidation with K3Fe (CN) 6.

The non-toxic addition salts of the linear and cyclic polypeptides are prepared according to methods well known in the art from hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, polyphosphoric acid, maleic acid, acetic acid, citric acid, benzoic acid, succinic acid, malonic acid or similar ascorbic acid. The acetic acid salt is most suitable.

The protected groups used during the solid phase synthesis are generally known. The namino-protected groups used together with each amino acid introduced into the sequence of the final polypeptide consist of (1) acyl-type protecting groups illustrated by the following: formyl, trifluoroacetyl, phthalyl, p-toluenesulfonyl (tosyl), nitrophenylsulfenyl, etc .; (2) aromatic urethane type protecting groups illustrated with benzyloxycarbonyl and substituted benzyloxycarbonyl, such as p-chlorobenzyloxycarbonyl and p-nitrobenzyloxycarbonyl; (3) aliphatic urethane protecting groups illustrated with tert-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, amyloxycarbonyl; (4) cycloalkylurethane type protecting groups illustrated with cyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl; (5) thiourethane type protecting groups such as phenylthiocarbonyl; (6) alkyl-type protecting groups illustrated with triphenylmethyl (trityl); (7) trialkylsilane groups, such as trimethylsilane. The most suitable α-amino protected group is tert-butyloxycarbonyl.

The imidazole nitrogen atom of histidine, designated Nlm, is protected by a group which may be tosyl, benzyloxycarbonyl, adamantyloxycarbonyl or tert-butyloxycarbonyl, preferably the tosyl group.

Protection for the side chain amino group in lysine can be provided by tosyl, t-amyloxycarbonyl, t-butyloxycarbonyl, diisopropyloxycarbonyl, benzyloxycarbonyl, halobenzyloxycarbonyl, nitrobenzyloxycarbonyl and the like, wherein the 2-chloroxarbonyl group is suitable.

The side chain nitrogen atoms in arginine, designated Ng, are protected by a group which may be nitro, tosyl, benzyloxycarbonyl, adamantyloxycarbonyl or tert-butyloxycarbonyl, preferably the tosyl group. Protection via the nitro or tosyl group is performed on either of the Nw or Nwl nitrogen atoms, while the oxycarbonyl type protecting groups protect Ng and any of the N '; and N ° * 1 nitrogen atoms.

Protection of the hydroxy group of threonine and serine can be performed with acetyl, benzoyl, tert-butyl or benzyl. The benzyl group is most suitable for this purpose.

The protecting group for the sulfhydryl group in the cysteinylamino acid residue is selected from the class consisting of benzyl; substituted benzyl, the substituent being at least one of the groups methyl, methoxy, nitro or halogen (such as 3,4-dimethylbenzyl, p-methoxybenzyl, p-chlorobenzyl, p-nitrobenzyl, etc.); triethyl, benzyloxycarbonyl, benzhydryl, p-methoxybenzyloxycarbonyl, benzylthiomethyl, ethylcarbamoyl, thioethyl, tetrahydropyranyl, acetamidomethyl, benzoyl, s-sulfonic acid salt, etc .; with the p-methoxybenzyl group being most suitable.

.... - .. H- 447 482 ll q- The pharmacological activity of the peptides according to the invention characterizes the compounds as useful in the treatment of acromegaly and diabetes in the same way as somatostatin itself. Administration of the peptides can be performed in a conventional manner in the same manner as for somatostatin and related polypeptides under the guidance of a physician in an amount fl._ determined by the extent of the discomfort, as these are judged ¿¿by the physician. The compounds may be administered separately or in admixture with conventional pharmaceutically acceptable carriers "and adjuvants in unit dosage form.

As stated above, the compositions of the invention are also useful in admixture with insulin for the treatment of diabetes mellitus. See, for example, U.S. Pat. No. 3,912,807, which discloses the use of an effective amount of a compound containing somatostatin in admixture with insulin for the treatment of a warm-blooded animal suffering from diabetes mellitus. treatment of acromegaly, juvenile diabetes and diabetes mellitus, the treatment is initiated with small doses, which are less than the optimal dose for the compound. Thereafter, the dosage is increased with small additives, until the optimal effect under the prevailing conditions is reached. In general, the compounds of the invention are administered at a dosage level which usually gives effective results without causing any harmful side effects. However, the dosages may be varied according to the requirements of the patient and the compound used. The total daily dosage may conveniently be divided and administered in portions during the day, if desired.

According to a further aspect of the invention there is provided a pharmaceutical composition containing a compound of formula I or a pharmacologically acceptable salt thereof in admixture with a pharmacologically acceptable carrier. Preferably, the pharmacological composition is in unit dosage form. The invention is further illustrated by the following examples, in which the temperatures indicated refer to degrees Celsius.

Example 1

Tert-butyloxycarbonyl-Sp-methoxybenzyl-L-cysteinyl-Nim-tosyl--L-histidyl-Nim-tosyl-L-histidyl-L-phenylalanyl-L-phenylalanyl- -D-Tryptophyl-Ng -2-chlorobenzyloxycarbonyl-L- lysyl-O-benzyl-L- -trecnyl-L-phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L-Seryl-S- -p-methoxybenzyl-L-cysteinyl-hydroxymethyl polystyrene Chloromethylated polystyrene resin (Lab Systems, Inc. .), 1% crosslinked with divinylbenzene, was esterified with BOC-Cys- (SMBzl) OH according to Gisin, Helv. Chim. Acta., §§, 1976 (1973). The polystyrene resin ester was treated according to Scheme A to incorporate BOC-Ser (Bzl) OH, BOC-Thr (Bzl) OH, BOC-Phe-OH, BOC-Thr (Bzl) OH, BOC-Lys (ClZ) OH, BOC- D-Trp-OH, BOC-Phe-OH, BOC-Phe-OH, BOC-His (Tos) OH, BOC-His (Tos) OH and BOC-Cys (SMBzl) OH in addition to form the title peptido resin. Scheme A: 1. Wash with CH 2 Cl 2 x 3 2. Treat with TFA-c fl zclz-EDT (l = 1 = 5% f VOIYm / VOlYm) for 5 minutes 3. Treat according to 2 for 25 minutes 4. Wash with CH 2 Cl 2 x 3 5. Wash with DMF. Treat with 12% TEA in DMF twice for 3 minutes 7. Wash with DMF 8. Wash with CH2Cl2 x 3 7 9. Treat with 4 equivalents of the corresponding amino acid derivative in CH2Cl2-DMF and stir for 5 minutes 10. Add in two portions 5 equivalents of DIC dissolved in CH 2 Cl 2 hours. ll. Wash with DMF x 3 l2. Wash with CH 2 Cl 2 x 3 13. Test the ninhydrin reaction according to Kaiser et al., Annal.

Biochem., Yyyy, 595 (1970). In case of incomplete reaction, repeat steps 9-13 above. for a period of 30 minutes. Reaction time 6 447 482 13 Example gel 2.

L-cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-cysteine cyclic (I ~ 12) disulfide 9 g of the peptido resin from Example 1 was mixed with 18 mg of anisole and treated with 180 ml of liquid HF for 45 minutes in an ice bath. The excess HF was removed in vacuo as soon as possible (about one hour) and the residue was extracted with deaerated 2M glacial acetic acid, after which it was filtered off. The filtrate was washed with ether and the aqueous layer was oxidized with K 3 Fe (CN) 6 at pH 7. The excess oxidant was removed with ion exchange resin, Bio Rad AG-3, after which the peptide was adsorbed on an ion exchange resin, Bio Rex 70, and eluted with a pH 7 pyridine buffer. to give 1.5 g of material after lyophilization. This was applied to a column of Sephadex R G-15 (2.5 x 160 cm) and eluted with 15% ACOH in water. The fractions (5.2 ml each) in tubes 88-90 were pooled and lyophilized to give the title compound, 174 mg.

Rf (BWA, 4: 1: 1) 0.45 Rf (BWAP, 30: 24: 6: 20) 0.64 Amino acid analysis: Thr (2) 1.87, Ser (1) 0.89, Cys (2) l, l6, Phe (3) 3, Lys (l) l, 05, His (2) l, 76, Trp ND.

Exemgel 3.

The in vivo pharmacological activity of the dodecapeptide prepared according to Example 2 was determined according to the following procedures with the following results: Suppression of growth hormone A subcutaneous (sc) injection of peptide solubilized or suspended in physiological sodium chloride solution is administered to male Charles River CD rats. not put on the fast diet. Injectable sodium chloride control solution sc-injected rats were used as control animals, so that each experimental rat could be compared to a control rat. The rats are maintained in separate cages and 20 minutes before the end of the test period they are given an intraperitoneal (i.p.) injection of nembutal at a dose of 50 mg / kg.

Blood samples are taken by cardiac puncture and the plasma is separated for radioimmunoassay of the concentration (ng / ml) of growth hormone (GH). Time periods after injection of 2 and 4 hours were used to test the duration of peptide activity regarding suppression of circulating peripheral GH levels. Comparison between control and experimental GH values at each time point is determined according to the Student's t test and statistical significance (p) at the 0.05 level or below as an index of activity.

Compound (dose) 2 hours 4 hours Control 118 1 20 ll5 1 23 Example 2 (1 mg / kg) 32 1 6 62 i 13 P = <0.0l p = <0.0l Suppression of growth hormone, glucagon and insulin Albinoan rats are arranged in three groups (9 rats / group) and injected ip with Nembutal R at 50 mg / kg. 15 minutes after the Nembutal line injection, it is injected subcutaneously according to the group with (a) the test compound, typically 10-2000 Ag / kg; (b) SRIF (200, ug / k9); or (c) physiological sodium chloride solution. 10 minutes later, 0.5 ml of arginine (300 mg / ml, pH 7.2) is injected into the heart.

The rats are decapitated five minutes after receiving the arginine and the blood is collected in Trasylol-EDTA. Appropriate proportions are then analyzed for growth hormone, glucagon and insulin. An active compound is one that significantly alters the plasma levels of any of these hormones compared to the sodium chloride solution controls.

Comparison between the control and experimental values is performed statistically by analysis according to the variance method and statistical significance (p) at 0.05 or lower as an index of the activity.

Compound GH (ng / ml) Insulin Glucagon (dose q / kq) (U / ml) (pg / ml) Control 163 1 29 278 1 32 68 i 13 Example 2 (100) 20 1 8 202 i 49 20 1 8 p = <0.0l p =) 0.05 p = <0.0l 447 482 Example Tert-butyloxycarbonyl-Sp-methoxybenzyl-L-cysteinyl-Ngn-tosyl- -L-arginyl-N1m-tosyl-L- histidyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-Nc-2-chlorobenzyloxycarbonyl-L-1syl-O-benzyl-L-threonyl-L-phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L -seryl-S- p-methoxybenzyl-L-cysteinyl-hydroxymethyl-polystyrene ester Chloromethylated polystyrene resin (Lab Systems, Inc.), 1% crosslinked with divinylbenzene, was esterified with Boc-Cys- (SMBzl) -OH according to Gisin, Helv. Chim. Acta, äâ, 1976 (1973). The polystyrene resin ester was treated according to Scheme A to introduce Boc-Ser (Bzl) -OH, Boc-Thr (Bzl) -OH, Boc-Phe-OH, Boc-Thr (Bzl) -OH, Boc-Lys (ClZ) - OH, Boc-D-Trn-OH, Boc-Phe-OH, Boc-Phe-OH, Boc-His (Tos) -OH, Boc-Arg (Tos) -OH and Boc-Cys (SMBzl) -OH in and to form the title peptido resin.

Scheme A: 1. Wash with CH2Cl2 x 3 2. Wash with TFA-CH2Cl2-EDT (1: 1: 5%, v / v) for 5 minutes 3. Treat according to 2 for 25 minutes 4. Wash with CH2Cl2 x 3 5 Wash with DMF 6. Wash with 12% TEA in DMF 2 times for 3 minutes 7. Wash with DMF 8. Wash with CH2Cl2 x 3 9. Treat with 4 equivalents of the corresponding amino acid derivative in CH2Cl2-DMF and stir 5 minutes 10. Add in two portions 5 equivalents of DIC dissolved in CH 2 Cl 2 over a period of 30 minutes. Reaction time 6 hours ll. Wash with DMF x 3 12. Wash with CH2Cl2 x 3 ll. Test the ninhydrin reaction according to Kaiser et al., Anal. Biochem., Åí, 595 (1970). In case of incomplete reaction, repeat steps 9-13. 447 482 16 gšempel 5.

L-cysteine-L-arginyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-cysteine cyclic (1-12) disulfide 8.5 g of the peptido resin from the previous example were mixed with 16 ml of anisole and treated with 100 ml of liquid HF for 45 minutes. The excess HF was removed in vacuo as soon as possible and the residue was taken up in 25% AcOH in water. The polymeric support was filtered off and the filtrate was washed with ether.

The aqueous layer was poured into 3.5 liters of water, the pH was adjusted with NH 4 OH to 7 and then the sulfhydryl compound was oxidized with K 3 Fe (CN) 6. The pH was adjusted to reach 5 with glacial acetic acid and the inorganic oxidizing agent was removed with Bio Rad AG 3. The peptide material was adsorbed on Bio Rex 70 and eluted with pyridine buffer pH 7 to give the crude compound, 2 g.

G-15 (2.5 x 160 cm) and eluted with 15% AcOH in water. The material This material was passed through a column of Sephadex in fractions 47-93 (5.2 ml in each fraction), 886 mg, applied to a column of CM-Sephadex; G ~ 25 and eluted with a stepwise NH 4 OAc gradient (0.1 to 0.3 molar NH 4 OAc) to give the title compound. This material was applied to a column of Sephadex RILH 20 (2.5 X 92 cm 3) and eluted with 10% -q AcOH in water. The pure title compound was obtained in fractions 55-63 (4.1 ml each). Yield 159 mg.

Rf (BWA, 4: 1: 1) 0.46, Rf (BWAP, 30: 24: 6: 20) 0.65.

Amino acid analysis Thr (2) 1.94, Ser (1) 0.91, Cys (2) 1.79, Phe (3) 3, His (1) 1.02, Lys (1) 0.85, Arg (1 ) 0.95, Trp. N.D.

Example 6.

The in vivo pharmacological activity of the title compound prepared in Example 5 was determined according to the following procedure with the following results: 447 482 17 Suppression of growth hormone, glucagon and insulin Albinohan rats are arranged in two groups (nine rats per group) and injected iip. with Nembutal Rti 50 mg / kg. 15 minutes after the Nembutal 5¿ injection, the test compound or physiological sodium hydride solution is injected subcutaneously. 10 minutes later, 0.5 ml of arginine (300 mg / ml, pH 7.2) is injected into the heart. The rats are decapitated five minutes after receiving the arginine and the blood is collected in Trasylol-EDTA. Appropriate portions are then assayed for growth hormone (GH), glucaton (GLUN) and insulin (INS). An active compound is one that significantly changes the plasma levels of any of these hormones from those in the sodium hydride saline control. Comparison between control and experimental values is performed statistically by analysis according to the variance method and statistical significance (p) at 0.05 or below as an index of the activity.

Results Try nos GH ms GLUN war / kg nq / ml / ß-N / ml pq / ml 1 ---- 257 i 49 177 i 19 61 i 9 1oo 61 + sx 52 i lax oi ox 2 ---- 131 i 43 262 i 26 42 i lo 71 i 21x 238 i 72 4 i XX p 0.01 gšemwel 7.

Tert-butyloxy-carbonyl-β-methoxybenzyl-L-cysteinyl-im-tosyl--1-histidyl-im-tosyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-E-2-chlorobenzyloxycarbonyl- L-lysyl-D-benzyl-L-threonyl-L-phenylalanyl-O-benzyl-L-threonyl-O-benzyl-1-seryl-S-igimethoxybenzyl-D-cysteinyl-hydroxymethyl-Dolystyrene ester Chloromethylated polystyrene resin (Lab Systems, Inc. .), 1% crosslinked with divinylbenzene, was esterified with Box-D-Cys- (SMBzl) OH.

The polystyrene resin was treated according to Scheme A to incorporate Boo-Ser (Bzl) OH, Boc-Thr (Bzl) OH, Boo-Phe-OH, Boc-Thr (Bzl) OH, Boc-Lys (Cl7) OH, Boc-D- Trp-OH, Boc-Phe-OH, Boc-Phe-OH, Boc-His 447 482 18 (Tos) OH, Boc-His (Tos) OH and Boc-Cys (SMBzl) OH to form the title peptide resin .

Example 8.

L-cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-: D-tryptophil-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-D-cysteine Cyclic (1-12) -disulfide diacetate salt 10 g of the peptido resin from the previous example were mixed with .20 ml of anisole and treated with 150 ml of liquid HF in an ice-K bath for 45 minutes with exclusion of air. The excess liquid HF was removed in vacuo and the residue was taken up in 50% ACOH in water. The mixture was filtered and the filtrate was diluted with water to 3.5 liters. The pH was adjusted to 7 with dilute NH 4 OH and the disulfhydryl peptide was oxidized with K 3 F (CN) 6 to cyclic disulfide. The pH of the mixture was adjusted to 5 with glacial acetic acid and treated with Bio-Rad AG 3. The peptide material was adsorbed on Bio Rex 70 (H + form) and eluted with pyridine buffer (Py-H 2 O-AcOH, 30: 66: 4, v / v) . The fractions containing the peptide material were combined and lyophilized to give 1.6 g of crude material. This crude product was applied to a column of Sephadex \ LH-20 (2.5 x 90 cm) and eluted with 10% ACOH in water (each fraction 5.8 ml). The material obtained in fractions 97-101 was combined and lyophilized to give the title dodecapeptide, 171 mg.

TLC, Avicel precoated glass plates, Rf (BWA, 4: 1: 1, v / v) 0.32, Rf (BWAP, 30: 24: 6: 20, v / v) 0.56.

Amino acid analysis: Thr (2) 1.85, Ser (1) 0.84, Cys (2) 1.29, Phe (3) 3, Lys (1) 1.05, His (2) 1.41, Trp ( 1) 0.75.

The in vivo pharmacological activity of the dodecapeptide prepared in Example 8 was determined according to the procedures described in detail in Example 3. The following data were obtained: "íïíöíløw uUAUlY i 447 482 19 Suppression of growth hormone Compound (doseL 2 hours 4 hours Control 127 in 22 59 i 20 Example s (1 mg / kg) zo i axx 1o7 i 34 XX p <0.001 Suppression of growth hormone, glucagon and insulin Compound GH (ng / ml) Insulin (AU / ml) Glucagon [dose Ueg / kq)] ( P9 / ml) xentre11 279 _ ss 323 i se 42 i 6 Example s (100) 64 i 20 * 165 1 21 ”si 2 * xentreil 201 3 35 397 i se 117 1 11 Example s (100) 110 3 22+ 318 i 54 7o ¿7 * x) p <0.01 +> p It is clear from this that at low doses the peptide from Example 8 is specific for the suppression of glucagon and growth hormone without affecting insulin secretion.

Example 9.

Tert-butyloxycarbonyl-Sp-methoxybenzyl-L-cysteinyl-Ngn-tosyl-L-arginyl-1'-benzyl-L-glutamyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-NF-2-chloro-benzyloxycarbonyl -L-lysyl-O-benzyl-L- -treonyl-L-phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-S- -p-methoxybenzyl-L-cysteine hydroxymethyl polystyrene ester Chloromethylated polystyrene resin esterified with Boc- Cys (SMBzl) -OH, according to Gisin, Helv. Chim. Acta., §§, 1976 (1973). The amino acid resin ester is then treated according to Scheme A (as follows) using Boc-Ser (Bzl) -OH as the protected amino acid in step 9. Scheme A is then repeated for consecutive incorporation of the following amino acids into the peptido resin: Boc-Thr (Bzl) -OH Boc-Phe-OH Boc-Thr (Bzl) -OH Boo-Lys (ClZ) ~ OH Boc-D-Trp-OH 447 482 20 Boc-Phe-OH Boc-Phe-OH Boc-Glu (OBzl) -OH Boc -Arg (Tos) -OH Boc-Cys (SMBzl) -OH âghema A: (for treatment of the resin ester) 1. Wash three times with methylene chloride (CH2Cl2) 2. Treat with trifluoroacetic acid / methylene chloride (1: 1, v / v) containing 5% 1,2-ethanedithiol for 5 minutes 3. Repeat step 2 for 25 minutes 4. Wash 3 times with CH 2 Cl 2 5. Wash with dimethylformamide (DMF) 6. Treat with 12% triethylamine in DMF for 3 minutes 7. Wash with DMF 8. Wash three times with CH2Cl2 9. Treat with 4 equivalents of the appropriate protected amino acid in CH2Cl2-DMF and stir for 5 minutes 10. Add in two portions over 5 minutes 5 equivalents of diisopropylcarbodiimide dissolved in CH2Cl2. Allow the reaction to proceed for 6 hours. ll. Wash three times with DMF - = l2. Wash three to 9% with CH 2 Cl 2 13. Test the ninhydrin reaction according to Kaiser et al., Annal. Biochem., Qi, 595 (1970). In case of incomplete reaction, repeat steps 9-13 above.

Example 10.

L-cysteinyl-L-arginyl-L-glutamyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-cysteine cyclic (1-2) disulfide 8.5 g of the peptido resin from Example 9 were suspended in 16 ml of anisole and treated with anhydrous liquid hydrogen fluoride (HF) for 45 minutes in an ice bath, after which the excess HF was removed in vacuo and the residue was extracted with 10% - in acetic acid in water. The filtrate was washed with ether and the aqueous layer was poured into 5 liters of water, after which the pH was adjusted to 7 with dilute ammonium hydroxide. The mixture was stirred in the open air during jïrïlíñ fi in ÛUALÜÜY _. -___, ..._. ma-, 447 482 21 48 hours, after which the pH was adjusted to 5 with glacial acetic acid and the peptide time was adsorbed on Amber1ite <š) CG-50 (H + form). The peptide material was eluted with 50% acetic acid in water and lyophilized to give 850 mg of solid.

The crude product was applied to a column of Sephadex 6: LH 20 (2.5 x 150 cm 3) and eluted with 10% acetic acid. Fractions 94-130 were pooled and lyophilized to give 483 mg of material. This was applied to a column of Sephadex R'G25 (1.5 x 115 cm) and eluted with 10% acetic acid in water. Fractions 43-53 were pooled and lyophilized to give the title peptide, 286 mg.

TLC, Avicel <š \ "precoated" glass plates, Rf (BWA, 4: 1: 1, v / v) 0.40, Rf (BWAP, 30: 24: 6: 20, v / v) 0.65.

Amino acid analysis: Thr (2) 1.92, Ser (1) 0.89, Glu (1) 1.03, Cys (2) 1.49, Phe (3) 3, Lys (1) 1.05, Trp ( 1) 0.81, Arg (1) 1.02.

Example 11. _ _. in Tert-butyloxycarbonyl-Sp-methoxybenzyl-L-cysteinyl-N-tosyl-L- -histidyl-β-benzyl-L-glutamyl-L-phenylalanyl-L-phenylalanyl-D--tryptophyl-N-2-chlorobenzyloxycarbonyl- L-1syl-O-benzyl-L-threonyl-L-phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-Sp- -methoxybenzyl-L-cysteine-hydroxymethyl-polystyrene ester Chloromethylated polystyrene resin esterified with Boc-Cys (SMBzl) -OH, according to Gisin, Helv. Chim. Acta., §§, 1976 (1973). The amino acid resin is then treated according to Scheme A (see Example 9), using Boc-Ser (Bzl) -OH as the protected amino acid in step 9. Scheme A is then repeated for consecutive incorporation of the following amino acids into the peptido resin: Boc-Thr (Bzl) -OH Boc-Phe-OH Boc-Thr (Bzl) -OH Boc-Lys (C1Z) ~ OH Boc-D-Trp-OH Boc-Phe-OH 447 482 22 Boc-Phe-OH Boc-Glu (0Bzl) - OH Boc-His (Tos) -OH Boc-Cys (SMBzl) -OH Example 12.

L-cysteinyl-L-histidyl-L-glutamyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-cysteine cyclic (1-12) disulfide The peptido resin from Example 11 (8 g) is treated with liquid anhydrous HF according to Example 10 and the linear disulfhydryl dode capeptide is cyclized by oxidation with K 3 Fe (CN) 6 at pH 7.3 in high dilution. The pH is adjusted to 5 with glacial acetic acid and the mixture is treated with Bio Rad AG3-X4 (C1 form) ion exchange resin and then adsorbed on Amberlite R CG 50 (H + form).

The peptide material is eluted with 30% acetic acid in water and lyophilized to give 500 mg of crude material. This is chromatographed on a column of Sephadex R LH 20 to give the title dodecepteptide.

TLC, Avicel <š "precoated" glass plates, Rf (BWA, 4: 1: 1, v / v), 0.43, Rf (tert-AmOH-Py-W, 7: 7: 6, v / v) 0 , 74.

Amino acid analysis: Th (2) 1.92, Ser (1) 0.93, Glu (1) 0.94, Cys (2) 1.59, Phe (3) 3, Lys (1) 1.02, His ( l) 0.91, Trp (1) 0.81.

Example 13

The biological activity of the peptides from Examples 10 and 12 was determined according to the following procedure: For albino male rats, Nembutaldà is administered intraperitoneally at a dose of 50 mg / kg. 15 minutes later, a subcutaneous injection of the test compound or physiological sodium hydride solution is administered. 10 minutes later, 0.5 ml of arginine (300 mg / ml, pH 7.2) is injected into the heart. 5 minutes after the introduction of the arginine, the rats are decapitated and the blood is collected in 447,482 trasylol-EDTA. An appropriate portion is assayed for growth hormone (GH), insulin and glucagon by radioimmunoassay. The results of the analysis are as follows: Dos GH "Ins lin Förening (uw / xq) nfs / lr-g lin / ru. Âflfßäïæn Ad njflarl Kontmn -_ av? I cs sol i 27 46 is 10 EXem9el 10 ZOO 42 | i 6 * ll5_i 1G * l.5¿¿ 1 * lQ Control ~~ 2l5_i 35 285 i 28 55_: 5 10 Example 10 40 '28_¿ 5 * 158 i 5? 12 i 2 * 10 Control _ ~ - 454 i_lOG 269_i 37 60_i 10 _ 10 Example 12 200 lO7_i 29 * 205: _56 4 ih 2 * 10 Control - 233 _ ~ _x ~ _ 35 342 _-x _-_ 45 44 i IL 10 Example 12 50 $ 4_¿ 17 * 322: _40 lO | These data show that the peptides of Examples 10 and 12, representative of the other peptides of the invention, are effective agents for reducing growth hormone levels and glucagon levels without significantly interfering with insulin levels at a single dose. of 40 mg / kg and 50 mg / kg, respectively, The peptides from Examples 10 and 12 were also tested for the duration of their effect on GH secretion in rats treated with Nembutal The peptide from Example 10 showed significant inhibition of GH for at least 4 hours at a dose of 100 mg / kg (subcutaneous). The pentide from Example 12 did not show any significant inhibition of GH at 2 and 4 hours. The compounds described herein may be administered to warm-blooded animals, either intravenously, subcutaneously, intramuscularly or orally, for the control of serum glucose in the treatment of diabetes. The required dosage varies with the particular condition to be treated, the severity of the condition and the duration of treatment.

The active ingredient may be administered separately or in combination with pharmaceutically acceptable carriers or excipients.

Suitable pharmaceutical compositions will be apparent to those skilled in the art. gšemnel 14.

Classical synthesis of L-cysteinyl-L-histidyl-L-histidyl-L-phenyl-alanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl--L-seryl-L-cysteine Cyclic (1-§12) disulfide (a) N-benzyloxycarbonyl-im-benzyloxycarbonyl-L-histidyl-L-phenylalanyl-methyl ester (I) A suspension of 42.3 g (0.1 mol) of Z-His (Z) -OH and 21.8 g (0.1 mol) of Phc-owe-Hcl (98%) 1 000 ml of cH 2 Cl 2 were cooled to 0 °, after which 22 g (0.2 mol) of DCC and 10.1 g (0 mol 10 mol) triethylamine was added. The mixture was allowed to warm to room temperature overnight.

The separated dicyclohexylurea was filtered off and the filtrate was concentrated to a sticky material. The material was dissolved in ethyl acetate and insoluble material was filtered off. The ETOAC layer was washed with 5% citric acid, water, ice-cooled 5% sodium bicarbonate solution in water and water. The organic layer was dried over sodium sulfate, concentrated to 1/3 of its original volume, added with 500 ml of ether, until the solution became cloudy, and stored in the cold overnight.

The white solid formed was dried, 32 mg. Melting point 131-13s °.

TLC: silica gel F-254, CH 2 Cl 2: MeOH (9: 1) Rf = 0.8. (b) Im-benzyloxycarbonyl-L-histidyl-L-phenylalanylmethyl ester dihydrobromide salt (II) 30 g (0.5 mol) Z-His (Z ) -Phe-OMe (I) was dissolved in 300 ml of HBr-HOAc (32% -iq) (the solution became cloudy within 2 minutes) and the mixture was stored for 30 minutes at room temperature and added with ether to form a precipitate. The soft solid was filtered off and dried overnight (32 g). Melting point TLC: the stain did not move. (c) N-Benzyloxycarbonyl-im-benzyloxycarbomyl-L-histidyl-im-benzyloxycarbonyl-L-histidyl-L-phenylalanyl-methyl ester (III) A suspension of 30.5 g (0.05 mol) of His (Z) -Phe -OMe-2HBr (II), 21.7 g (0.05 ml) of Z-His (Z) -OH in 500 ml of CH 2 Cl 2 were cooled to 0 DEG.

Then, 11 g of DCC and 1.0 g (1.0 mol) of triethylamine were added.

This mixture was allowed to warm to room temperature overnight.

The dicyclohexylurea formed was filtered off and the filtrate was concentrated to a glassy material. The residue was dissolved in ethyl acetate and the EtOAc layer was washed with 10% citric acid, water, ice-cooled 5% sodium bicarbonate solution and water.

The EtOAc layer was dried over sodium sulfate, concentrated to about 1/3 of its original volume, added with ether to give a crumbly solution and stored in a cold room.

White solid precipitated, 29.5 g. (D) L-histidyl-L-histidyl-L-phenylalanyl-methyl ester trihydrochloride salt (IV) ZZ The mixture of 29 g of Z-ice-ice-Phe-OMe (IV ), 10.8 ml of concentrated HCl, 180 ml of MeOH and two spatulas of 10% Pd-C were hydrogenated for 5 hours using a Parr hydrator.

After filtration, the methanol layer was concentrated to an oil and ether was added to give a solid. 18 g of crude material were used for the next reaction without further stirring. 447 482 26 (e) tert-butyloxycarbonyl-Sp-methoxybenzyl-L-cysteine-L-histidyl- -L-histidyl-L-phenylalanyl-methyl ester (V) 13 g Boc-Cys (MBzl) -OH (0.038 mol), 5.4 g (0.04 mol) of hydroxybenstriazole and 8.3 g (0.040 mol) of dicyclohexylcarbodiimide were stirred for 30 minutes in liters of ice-cold methylene chloride. 19.5 g (0.0345 mol) of His-His-Phe-OMe '3HCl and 100 ml (0.096 mol) of N-methylmorpholine were added and the mixture was stirred overnight at room temperature. The suspension was filtered to eliminate dicyclohexylurea and the filtrate was washed successively with water, 10% H 2 CO 3 and water and dried over Na 2 SO 4. After filtration, the solvent was removed in vacuo to give 21.0 g of crude product. This material was stirred in 100 ml of acetonitrile for 30 minutes at room temperature and filtered. The precipitate was washed with acetonitrile and then with ether and dried. Yield 11 g TLC: Rf 0.12 silica gel 60, CHCl 3: CH 3: OH-HAC, 9: 1: 0.5 v / v.

The filtrate from the acetonitrile extraction contains product and impurities and can be used for the extraction of other crude batches. (f) tert-butyloxycarbonyl-Sp-methoxybenzyl-L-cysteinyl-L-histidyl-L-histidyl-L-phenylalanine (VI) 11.0 g (0.04242 mol) Boc-Cys (MBZI) -His-His- Phe-Me was dissolved in 200 ml of methanol and 30 ml of 1N potassium hydroxide was added.

The solution was stirred for 2 hours at room temperature and the methanol was removed in vacuo. The residue was diluted with 200 ml of water and filtered. The filtrate was adjusted to pH 5 with 10% citric acid and the sticky precipitate was stored until it crystallized. The product was filtered off, washed with water and dried in vacuo over P 2 O 5. Yield 7.3 g, 67%.

TLC: Rf 0.52 silica gel 60 n-butanol: ethyl acetate acetic acid H 2 R 0.43 silica gel 60 tert-amyl alcohol: pyridine: H 2 O, f 7: 6: 7 v / v).

0.1: 1: 1: 1 v / v) 447 482 27 (g) N-tert-butyloxycarbonyl-O-benzyl-L-threonyl-L-phenylalanine-methyl ester (VII) A solution of 61.8 g ( 0.2 mol) of Boc-L-Thr (Bzl) -OH and 22.4 ml (0.2 mol) of N-methylmorpholine in THF were cooled to -150. 26.2 ml (0.2 mol) of isobutyl chloroformate were added in portions, maintaining the temperature between -15 and -100. After stirring at -150 for 15 minutes, a cold mixture of 43.1 g (0.2 mol) of L-Phe-OMeHCl and 22.4 ml (0.2 mol) of N-methylmorpholine in DMF was added in portions, while the temperature maintained between -10 and -50. The mixture was stirred for two hours at 0 ° C and then overnight at room temperature. The filtered reaction mixture was concentrated in vacuo and the residue was taken up in ethyl acetate. The ethyl acetate solution was washed consecutively with 5% KHSU4, 5% KHCO3 and sodium chloride solution and dried over Na2SO4. After concentration in vacuo, an oil was obtained which crystallized on storage. The solid was recrystallized from isopropyl ether / hexane, 74.9 g (80%). melting point 7s-s1 °, [α] +4 + 10.75 (c 1.023, meon), Rf (cHc13) 0.35.

C. HN 06 (470.55) δ 6 34 2 Calculated: C 66.36 H 7.28 N 5.95 Found: C 66.72 H 7.32 N 5.85 '(h) N-tert-butyloxycarbonyl -O-benzyl-L-threonine-L-phenyl-alanine (VIII) 23.5 g (0.05 mol) of Boc-Thr (Bzl) -Phe-OMe were dissolved in a mixture of 100 ml of MeOH / dioxane (1: l) and treated with 55 ml of 1N-NaOH for 3 hours (until starting material can no longer be detected with TCI). The majority of the solvent was evaporated in vacuo and the residue was diluted with water, after which it was acidified with a 5% citric acid. The separated sticky substance was taken up in EtOAc and washed with water (concentrated sodium chloride solution) and evaporated to dryness. The residue was crystallized from Et 2 O / hexane, 19.8 g (87%), mp 118-119.

R; (chloroform / methanol / glacial acetic acid, 85: 10: 5) 0.80. 447 482 (i) N-tert-butyloxycarbonyl-O-benzyl-L-threonyl-O-benzyl-L- -serine methyl ester (IX) 30.9 g (0.1 mol) of N-tert-butyloxycarbonyl-O- benzyl-L-threonine was dissolved in 200 ml of dry tetrahydrofuran cooled to -200 and treated with 11 ml of N-methylmorpholine and then with 13.4 ml of isobutyl chloroformate. The cold reaction mixture was stirred for 5 minutes at -200 and then treated with a solution of 25 g (about 0.1 mol) of O-benzyl-L-serine methyl ester hydrochloride, containing 11 ml of N-methylmorpholine, in DME and the mixture had to assume room temperature overnight.

The solvent was removed in vacuo and the residue partitioned between water and ethyl acetate. The organic phase was washed with 5% citric acid, water, KHCO 3 in water and water and dried over MgSO 4, then evaporated to dryness to give an oily residue which was crystallized from diethyl ether / hexane to a gel-like solid (29 g) , Rf (CHCl 3 -MeOH, 25: 1) 0.85, Rf (EtOAc-hexane, 1: 1) 0.65. (j) N-tert-butyloxycarbonyl-O-benzyl-L-threonyl-O-benzyl-L-serine (X) 28.2 g (0.0563 mol) Boc-Thr (Bzl) -Ser (Bzl) -OMe was dissolved in about 50 ml of methanol and treated for 1.5 hours at room temperature with 75 ml of 1N sodium hydroxide. The alkaline solution was neutralized to pH 7 with 10% citric acid and most of the methanol was removed in vacuo. The residue was diluted with a small amount of water and acidified with 5% KHSO 4 in water, after which it was extracted with EtOAc. The organic phase was washed with water, dried over Na 2 SO 4 and evaporated to dryness in the form of an oil. Exchange quantitatively. Rf (EtOAc-hexane, 1: 1) 0.15 (long spot). (k) N-tert-butyloxycarbonyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-Sp-methoxybenzyl-L-cysteine-benzyl ester (XI) 24.3 g (SO mmol) Boc-Thr (Bzl ) -Ser (Bz1) -OH was dissolved in acetonitrile and 250 ml of dichloromethane (2: 3) and mixed with 25 g (50 mmol) of HCl (SMBzl) -Bzl-TosOH and then with 6.8 ml of triethylamine and 6.8 g N-hydroxybenzotriazole and the mixture was cooled in an ice bath. A solution of 11 g (53 mmol) of DCC in 50 ml of acetonitrile was added and the reaction mixture was stirred for two hours in the cold and then for 2 days at room temperature. The separated DCU was filtered off and the filtrate was evaporated to dryness. The oily residue was partitioned between ethyl acetate and water and the organic phase was washed with 10% citric acid, water, 5% KHCO 3 Na 2 SO 4. The solvent was evaporated and the oily residue and concentrated sodium chloride solution and dried over it were crystallized from Et 2 O / hexane to give a white solid, 24.5 g, mp 87-900. Rf (chloroform / methanol, 25: 1) 0.54, traces at 0.4 and 0.3 (heptane-EtOAc, 1: 1), 0.64, traces at 0.25, I2-positive [a ] š5 -14.7 (c 0.98, DMF).

CMHSZNBSQQ (798.9) Calculated: C 66.15 H 6.56 N 5.26 Found: C 66.22 H 6.95 N 5.29 (1) O-benzyl-L-threonyl-O-benzyl-L -seryl-Sp-methoxybenzyl-L- -cycine-benzyl ester trifluoroacetate (XII) 8 g (10 mmol) of Boc-Thr (Bzl) -Ser (Bzl) -Cys (SMBzl) -OBzl were mixed with 100 mmol of anisole and treated with 100 ml TFA for 45 minutes. The solvent was evaporated in vacuo and the residue was dissolved in dry Et 2 O, after which it was evaporated to dryness in high vacuum to give an oily compound, 7.2 g (90%).

Rf n-hutanol / water / glacial acetic acid 4: 1: 0.9, Rf (heptane / EtOAc) 0.0-0.1 long spot, I2-positive and ninhydrin-positive. (m) N-tert-butyloxycarbonyl-O-benzyl-L-threonyl-L-phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-Sp-methoxybenzyl-L-cysteine benzyl ester (XIII) 9.2 g (20 mmol) of Boc-Thr (Bz1) -Phe-OH (prepared according to Example 3) were dissolved in 100 ml of DMF and mixed with 3.4 g of N-hydroxysuccinimide and a solution of 20 mmol of Thr (Bzl). ) -Ser (Bzl) -Cys (SMBzl) - OBzl'TFA salt in 20 ml DMF neutralized with triethylamine to pH 7. The mixture was cooled in an ice bath and treated for two hours with 4.5 g DCC in cold and then 2 days at room temperature.

The DCU was filtered off and the filtrate was evaporated to dryness. The residue was triturated with water to give a precipitate, which was taken up in EtOAc, washed with 5% citric acid, water, 5% Na 2 CO 3 and water, dried over Na 2 SO 4 and evaporated to dryness. The residue was solidified in Et 2 O-hexane; it can be crystallized from MeOH, very soluble in CHCl3. 17.3 g (70%), mp 105-1070, Rf (CHCl 3 -MeOH, 10: 1) 0.90; lalšs -3.6 (Q 1, DMF).

C64H75N5SO12H2O (1156.2) Calculated: C 66.46 H 6.71 N 6.05 Found: C 66.68 H 6.59 N 6.20 (n) tert-butyloxycarbonyl-D-tryptophyl-N9 -benzyloxycarbonyl- -L-lysyl methyl ester (XIV) 30.4 g (0.1 mol) of Boc-D-Trp-OH, 33 g (0.1 mol) of Lys (Z) -OMe.HCl and 13.6 ml of triethylamine were dissolved in 400 ml of DM and cooled in an ice bath, then 22 g of dicyclohexylcarbodiimide were added and the mixture was allowed to warm to room temperature overnight.

The separated dicyclohexylurea was filtered off and the filtrate was concentrated to a small volume, after which an excess of water was added to form a gummy material. This material was taken up in ethyl acetate and washed with 10% citric acid in water, water, saturated NaHCO 3 solution and water, dried over Na 2 SO 4 and evaporated to dryness. The residue was triturated with ether and then with hexane to give a crystalline solid, 28 g.

TLC: silica gel G, hexane / EtOAc, 1: 1, v / v, Rf 0.3. (0) tert-Butyloxycarbonyl-L-phenylalanyl-D-tryptophyl-N57-benzyloxycarbonyl-L-lysine (XV) A. 22 q (44 mmol) Boc-D-Trp-Lys (Z) -OMe was added to a pre-cooled mixture of 220 ml TFA and 25 ml anisole and stirred for 30 minutes in an ice bath. The TFA was evaporated as rapidly as possible at 270 on a rotary evaporator and the residual oil was triturated several times with a hexane / ether solution, 2: 1, and decanted to finally form a sticky product. Yield 21.3 g, 36 mmol. ... __.-- _.- _ _ _ _ _. _-......_._.._ .., .._._............_...._, _..._.__ .. 447 482 31 B. 9.6 g (36 mmol) of Boc-Phe-PH, 5.3 g (39 mmol) of HOBT and 8 g (39 mmol) of DCC were stirred in 500 ml of cold dichloromethane for 20 h. minutes. 21.3 g (36 mmol) of D-Trp-Lys (Z) -OMe.TFA salt in 200 ml of dichloromethane were added, after which 4.25 ml (39 mmol) of N-methylmorpholine were added. The reaction mixture was stirred overnight while warming to room temperature. The mixture was filtered to eliminate DCC and the filtrate was washed twice with water, twice with 10% citric acid, twice with saturated NaHCO 3 solution and twice with water and dried over Na 2 SO 4. After filtration and evaporation, the residue weighed 21.4 g (82%) and was used without further purification. TLC: silica gel F ~ 254 / CHCl 3 9: CH OH 1: HAC 0.5. Rf 0.76 plus track 3 of minor contaminants.

C. 2.9 g (31.4 mmol) of Boc-Phe-D-Trp-Lys (Z) -OMe were dissolved in 450 ml of p-dioxane and 100 ml of methanol and 39 ml of 1N KOH solution were added, after which the mixture was stirred overnight at room temperature. The solution was evaporated on a rotary evaporator. The residue was dissolved in 500 ml of hot water, acidified to pH 3 with fo '-iq citric acid, stirred for 30 minutes and filtered.

After drying in vacuo overnight, 21.6 g (96%) were obtained.

Smårtnunkt 85-900. Pdlase = +7.09 (c 0.987 MeOH). RF 0.63 and 13.9 = cn3on, 1 = HAc, 0.5. siiikaqei F-254. * (on tert-butyloxycarbonyl-L-phenylalanyl-D-tryptophyl-NP -benzyloxycarbonyl-L-lysyl-O-benzyl-L-threonyl-L-phenylalanyl-O-benzyl-L-threonyl-O- benzyl-L-seryl-Sp-methoxybenzyl-L-cysteinyl: tensyl ester (XVI) A. 40.5 q (35 mmol) Boc-Thr (Bzl) -Phe-Thr (Bzl) -Ser (Bzl) -Cys- (SMBzl) -OBzl (XIII) was added to a cold solution of 400 ml of TFA and 40 ml of anisole and stirred in the cold for 30 minutes, the TFA was evaporated on a rotary evaporator and the residue was triturated with 1 liter of ether / hexane (1: 1). After filtration, washing with ether / hexane and drying in vacuo over P 2 O 5 and NaOH pellets, 34.6 g (82%), m.p. 136-139 °, softens at 120 DEG. 9.93 (C 1.04 MeuH; Ry. 0.63 silica gel CHCl 3, 9: CH 3 OH, 1: HAc, 0.05 minor, more polar impurity, Rf 0.53. 447 482 32 B. 20.0 g (27, 9 mmol) of Boc-Phe-D-Trp-Lys (Z), 4.15 g (30.8 mmol) of HOBT and 6.3 g (30.8 mmol) of DCC were stirred in 2.0 liters of cold dichloromethane in an ice bath for 30 minutes 32.6 g (27.9 mmol) H-Thr (Bzl) -Phe-Thr (B zl) -Ser (Bzl) -Cys (SMB21) -OBzl-TFA salt was added and then 3.35 ml (30.8 mmol) of N-methylmorpholine, after which stirring was continued overnight while the mixture was warmed to room temperature. TLC confirmed a substantially complete reaction after two hours.

The mixture was filtered and the filtrate was washed with water, 10% citric acid, water, saturated NaHCO 3 solution and water and dried. After filtration and evaporation, the TLC showed the product and a more polar impurity, which was removed by dissolving the crude product in 200 ml of hot DMF and adding 1 liter of 2% NaHCO 3 solution in water. The product was filtered off, washed with dilute NaHCO 3 solution and water and dried in vacuo over PZOS. Yield 36.5 g, 75%. Melting point lea-isf (shrinks at 150 °>. Mšö = 0 °, c 0.988, cHc13. Rf (a single spot) 0.76 silica gel CHCl 3, 9: CH 3 OH, 1: HAc, 0.05. (Q) Preparation of H-Phe-D-Trp-Lys (Z) -Thr (Bzl) -Phe-Thr (Bzl) - -Ser (Bzl) -Cys (SMBzl) -OBzl-trifluoroacetate salt (XVII) 32 g Boc-Phe-D- Trp-Lys (Z) -Thr (Bzl) -Phe-Thr (Bz1) -Ser (Bzl) -Cys (SMBzl) -OBzl and 33 ml of anisole were dissolved in 50 ml of dichloromethane and 100 ml of trifluoroacetic acid were added and stirred for 30 minutes at room temperature The solution was evaporated on a rotary evaporator at 300. The residue was triturated with anhydrous ether to a powder which was filtered off, washed with ether and dried in vacuo over P 2 O 5 and sodium hydroxide pellets.

TLC on silica gel 60 in CHCl 3: CH 3 OH: HOAC 9: 1: 0.5 showed a single spot, Rf 0.65 HNMR showed no peak for t-butyl.

Poon .SIQALITY 447 482 33 (r) tert-Butyloxycarbonyl-Sp-methoxybenzyl-L-cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl-L-phenylalanyl-D-tryptophy1- ~ N <- benzyloxycarbonyl-L- lysyl-O-benzyl-L-threonyl-L-phenylalanyl-O-benzyl-L-threonyl-D-benzyl-L-seryl-Sp-methoxybenzyl-L-cysteine benzyl ester (XVIII) 5.5 g (7 , 2 mmol) Boc-Cys (SMBz1) -His-His-PHEOH, 1.07 g (7.9 mmol) of 1-hydroxybenstriazole monohydrate and 1.65 g (7.9 mmol) of dicyclohexylcarbodiimide were stirred in 100 ml dry dimethylformamide in an ice bath for 30 minutes. 12.5 g (7.2 mmol) H-Phe-D-Trp-Lys (Z) - -Thr (B21) -Phe-Thr (Bzl) -Ser (Bz1) -Cys (SMBzl) -OBZ1-trifluoroacetate- salt and 0.855 g (7.65 mmol) of N-methylmorpholine were added and the mixture was allowed to warm to room temperature overnight with stirring. The suspension was filtered to eliminate dicyclohexylenamide and the filtrate was evaporated to 50 ml and precipitated with 500 ml of saturated aqueous sodium bicarbonate solution. The resulting solid was filtered off, washed with water and dissolved in 200 ml of DMF. The solution was diluted with 1 liter of 5% -fw citric acid in water and filtered. The solid was washed with water, dissolved in 200 ml of DMF and precipitated with 1 liter of saturated sodium bicarbonate solution, filtered, washed with water and dried in vacuo over P 2 O 5 at room temperature.

Yield 15 q.

Amino acid analysis His (2.0) 1.66, Lys (1.0) 1.05, Phe (3.0), Ser (1.0) 1.02, Thr (2.0) 2.2. (s) L-cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl-L-phenyl-alanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl- L ~ cysteine ~ cyclic (1-12) disulfide (XIX) 5 q Boc ~ Cys (SMBzl) -Hin-His-Phe-Phe-D ~ Trp-Lyz (Z) -Thr (Bzl) ~ Phe- -Thr ( Bzl) -Ser (Bzl) -Cys (SMBz1) OBZ1 was mixed with 10 ml of anisole and treated with 100 ml of liquid HF under exclusion of air and in an ice bath for 60 minutes. the excess HF was removed in vacuo and the residue was taken up in 200 ml of 10% AcOH in water and poured into 7 liters of deaerated water. The pH was adjusted to 7 with dilute NH 4 OH and stirred in the open air for 24 hours, where Ffef was adjusted to 5 with glacial acetic acid and the solutions were passed through a column of Amberlite CG 50 (H4 form). Peptide-Boiler The material adsorbed on the ion exchange resin was eluted with 30% acetic acid in water and the fractions containing the peptide material were combined and lyophilized to give 2 g of the crude material. This crude dodecane peptide was purified by R'G-25 and eluted with 10% -iq {§COH in water and partition chromatography by column chromatography through Sephadex through Sephadextš G-25 with the two-phase system N-BuOH-water-glacial acetic acid, 4: 5: volume / volume.

TLC, silica gel, Merck A.G., chloropeptide spray, D.E. Nitecki et al., Biochem. g ess' (1966).

Rf (BWA, 4: 1: 1) 0.45, Rf (BWAP, 30: 24: 6: 20) 0.64.

Amino acid analysis: Thr (2) 1.85, Ser (1) 0.79, Phe (3) 3, Lys (1) 1.02, His <2) 1.75, Cys, Trp N.D. WEGALITY F: 447 482 Comparative Pharmacological Data U.S. Patent 4,061,608 (American Home Products Corporation), issued December 6, 1977, relates to Arg4-somatostatin and analogs thereof. These polypeptides inhibit the secretion of growth hormone and in low doses they are especially distinguished by lowering the glucagon level without drastically changing the insulin concentration, thereby providing a significant advantage in the treatment of diabetes, as better regulation of glucose concentration in the blood can be obtained by avoiding insulin. In comparison with the Arg4, Glu5-SS-derivatives of the present invention, the compounds of U.S. Patent 4,061,608 are not as long-acting or as specific in lowering the glucagon content. Column 5, lines 35-45 of US 4,061,608 states that the Arg4-SS derivative, compared to a blank, reduced glucagon from 18 to 11 pg / ml at 400 pg / kg and from 16 to 4 pg / ml at 20 pg / kg. The product of Example 10, representing Arg4, the Glu5-SS analog of the above compound of U.S. Patent 4,061,608, reduced glucagon from 46 to 1.5 pg / ml at 200 pg / kg and from 55 to 12 pg / ml. at 40 pg / kg (see Example 13). Furthermore, the Arg4, Glu5-SS analog was active in reducing growth hormone for at least 4 hours at a dose of 1000 mg / kg (subcutaneously), while the Arg4 ~ SS analog is comparable to somatostatin in the 0.25 hour activity time. .

The compounds listed in "Biochemical and Biophysical Research Communications" (hereinafter referred to as BBRC) §2¿ 1202 (1974) and Bßnc _6_5, 752 (1975), namely des- / Ala fi clyzß SS and various N ~ acylated derivatives, are not long-acting when administered in a conventional manner. They cannot be compared with the compounds of the present invention because they do not show any specificity for growth hormone and glucagon lowering. This is well known in the art.

The compounds listed in BBRC 14, 630 (1977) are selective for the inhibition of growth hormone and glucagon, but they are 447,482 36 in + e long acting.

Arqi, the Glu5-SS derivatives of the present invention combine in one and the same compound the desirable properties of being selective in their action in regulating growth hormone and glucagon with long-lasting action (up to 4 hours).

hrs

Claims (8)

447 482 37 EÉJ ____ 'ENTÉ? AY 1. A process for the preparation of a compound of formula I H-Cys-X1-x2 ~ Phe ~ Phe-D ~ Trp-Lys-Thr-Phe-Thr-Ser- ¥ 4-OH (I) ISS wherein X1 is His or Arg, X2 is His or Glu and X4 is Cys or D-Cys, and pharmaceutically acceptable salts thereof, may be derived therefrom, by removing all protecting groups and the polystyrene resin carrier, when present, from a compound of formula II R1 -Cye-X -X.-Phe-Phe-D-Trp-Lys-Thr-Phe ~ Thr ~ Ser-X -OR (II) I 1 ¿I5 17 13 lg 14 su RR RRs / s wherein R1 denotes a u -amino protecting group; R represents a carboxy-protecting group or a polystyrene resin support bonded via a methylene group, i.e. -CH2 polystyrene resin carrier; is His or Arg, wherein R 3 is a protecting group for I? the side chain nitrogen atoms of arginine and R 2 are hydrogen or an X1 protecting group for the imidazole nitrogen atom of histidine; X is His as above or Glu, wherein R 4 is a protecting 2 | 2 | 4 R R group for the side chain carboxy group of glutamic acid; R6 is a protecting group for the side chain amino group in lysine; R 7, R 8 and R 9 are each protecting groups for the hydroxyl groups of threonine and serine; X4 has the meaning given above; and cc and ß represent a sulfhydryl protecting group; and oxidizing the resulting compound, and optionally isolating the product in the form of the free base or a pharmaceutically acceptable salt. 447 482 38 2. A process according to claim 1, characterized in that the polystyrene resin carrier and / or protecting groups are removed in a single step by treatment with hydrogen fluoride in the presence of anisole. 3. A process according to claim 1 or 2, characterized in that the oxidation is carried out using acid or potassium iron (III) cyanide. A process according to any one of the preceding claims, characterized in that R 2 is tosyl, benzyloxycarbonyl, adamantyloxycarbonyl or tert-butyloxycarbonyl. Process according to any one of the preceding claims, characterized in that R 3 is nitro, tosyl, benzyloxycarbonyl, adamantyloxycarbonyl or tert-butyloxycarbonyl. Process according to any one of the preceding claims, characterized in that R 4 is benzyl. Process according to any one of the preceding claims, characterized in that R 6 is tosyl, t-amyloxycarbonyl, t-butyloxycarbonyl, diisopropyloxycarbonyl, benzyloxycarbonyl, halobenzyloxycarbonyl or nitrobenzyloxycarbonyl. Process according to any one of the preceding claims, characterized in that at least one of R 7, R 8 and R 9 is acetyl, benzoyl, tert-butyl or benzyl.