IE49831B1 - Truncated somatostatin analogs - Google Patents

Description

This invention relates to polypeptides, more particularly to truncated somatostatin analogs and to pharmaceutically acceptable salts thereof possessing pharmaceutical activity, to processes for preparing them to pharmaceutical compositions containing them and to precursor intermediates thereof.

In accordance uith this invention there is provided a group of polypeptides of the formula I: S-A A-S I | X1-Cys-Phe-X2-X3-Lys-Thr-Phe-D-Cys-0H (I) in uhich X1 is hydrogen, des-amino, H-flla-Gly or H-Ala-D-Ala; X2 is Trp, Lsu, flat or ja-Cl-Phe; X^ is Ρ,-Trp or 5- or 6-fluoro-Di-Trp; and the A groups are hydrogen or a direct bond betueen the tuo sulfur atoms; and pharmaceutically acceptable salts thereof. These compounds, uhile possessing the common ability to suppress grouth hormone, differ frpm somatostatin in their selective activity toward grouth hormone and glucagon uithout suppression of insulin, in their long term biological activity and structurally in 4 5 that they omit the amino acid residues of Lys , Asn , 13 7 Thr and Ser of somatostatin and replace Phe uith 14 Trp, Trp uith D-Trp or 5- or 6-fluorp-D-Trp and Cys uith D-Cys. In addition, tha amino acid moieties appearing in 1- and 2-positions of somatostatin are either present as Ala-Gly- or they may be substituted uith Ala-D-Ala, entirely omitted uith or uithout the 3 presence of the alpha amino substituent of Cys . - 48831 - 3 Basically, the compounds of this invention may be vieued as cyclic or linear octapeptides optionally modified N-terminally.

The preferred compounds of this invention, from the 5 standpoint of differentiation between suppression of growth hormone, glucagon and insulin are those depicted above which contain Trp as Xg· Preferably Xg is D-Trp.

The pharmaceutically acceptable salts of the compounds of this invention include non-toxic addition salts produced by known methods from acids conventionally employed with pharmaceuticals such as hydrochloric, hydrobromic, sulfuric, phosphoric, polyphosphoric, maleic, acetic, citric, benzoic, succinic, malonic or ascorbic acid. Acetic acid is the preferred acid.

The octapeptides of formula I selectively inhibit release of growth hormone and glucagon without materially altering blood levels of insulin. As such, they are useful in treatment of hyperglycemia in general and specifically in diabetes mellitus uhich is characterised by excessive glucagon secretion and deficient insulin release. Thus, fcr example, the postprandial hyperglycemic state in insulin-dependent diabetes may be improved through suppression of excessive glucagon by administration of the compounds of this invention uith or uithout concomitant administration of suboptimal amounts of exogenous insulin.

Likewise, the compounds of this invention are useful in the treatment of glucagon secretion by benign and malignant v - 4 islet-cell tumors to obtain the normoglycemic state.

In addition, the increased blood levels of growth hormone in diabetics and acromegalics can be controlled with the compounds of this invention.

Precursor intermediates of the cyclic octapeptides are provided by this invention. These include linear or cyclic octapeptides having the amino acid sequence as defined above and carrying at least one protecting group for a side chain group and/or terminal α-amino group when present and/or terminal carboxy group. In particular, included are intermediates of formula: S (R1) (R2)S ' 3 4 I 5 (R)-Cys-Phe-X2-X3-Lys(R)-Thr(R )-Phe-D-Cys-OR (II) in which X2 and X^ are as defined above, R is hydrogen, an alpha amino protecting group, des-amino or a-amino protected Ala-Gly or α-amino protected Ala-D-Ala; R1 and 1 R are hydrogen, a sulfhydryl protecting group or R and 3 R are a direct bond between sulphur atoms, R is hydrogen ε 4 or a N protecting group of Lys; R is hydrogen or a hydroxyl protecting group of Thr; and R$ is hydrogen, a carboxy protecting group or -CH0(polystyrene resin), with z 1 the proviso that when R is hydrogen or des-amino and R 2 and R are a direct bond or both hydrogen then at least one 3 4 5 of R ,R and R is other than hydrogen. These intermediates comprise the fully protected and partially protected octa25 peptides bound to a hydroxy methylated polystyrene resin support employed in solid phase synthesis of the polypeptide as well as the fully deprotected linear polypeptide removed from the resin support.

The protecting groups employed during preparation of the linear intermediates are conventional in classical or solid phase polypeptide synthesis. Thus, in the above 48831 - 5 formula, α-amino protecting groups may be (1) acyl type protecting groups illustrated by the follouing: formyl, trifluoroacetyl, phthalyl, £-toluenesulfonyl (tosyl) and o-nitrophenylsulfenyl; (2) aromatic urethane type protecting groups illustrated by benzyloxycarbonyl and substituted benzyloxycarbonyl such as £-chlorobenzyloxycarbonyl, £-nitrobenzyloxycarbonyl; (3) aliphatic urethane protecting groups illustrated by butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, amyloxycarbonyl; (4) cycloalkyl urethane type protecting groups illustrated by cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxy carbonyl; (5) thiourethane type protecting groups such as phenylthiocarbonyl; (6) alkyl type protecting groups as illustrated by triphenylmethyl (trityl); (7) trialkylsilane groups such as trimethylsilane.

The preferred α-amino protecting group is butyloxycarbonyl.

Examples of the hydroxyl protecting group R of threonyl are benzyl, £-methoxybenzyl, £-chlorobenzyl, β,-nitrobenzyl, trityl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, acetyl, tert-butyl or benzoyl. The benzyl group is preferred for the threonyl moiety. 9831 - 6 1 2 Examples of sulfhydryl protecting groups R and R ar8 benzyl, p-methoxybenzyl, p-chlorobenzyl, p-nitrobenzyl, trityl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, thiaethyl, ethylcarbamoyl, tetrahydropyranyl, acetamido5 methyl, benzoyl, benzhydryl, 3,4-dimethylbenzyl, benzyl1 2 thiomethyl or S-sulfonio salt Preferably R and R represent p-methoxybenzyl.

Protecting groups for the nitrogen (c) atom of lysine include tosyl, benzyloxycarbonyl, 2-chloro1Q benzyloxycarbonyl, and tert-butyloxycarbonyl, preferably the 2-chlorobenzyloxycarbonyl group.

The support employed in the solid phase synthesis of these compounds is a chloromethylated or hydroxymethylated polystyrene resin preferably cross-linked uith divinylbenzene. These resins are prepared by known methods and are commercially available in the art.

Examples of R5 protecting groups for carboxyl are lower alkyl having 1 to 6 carbon atoms (e.g. methyl, t-butyl) and benzyl.

The invention also provides processes for preparing the compounds of the invention.

The compounds of formula I may be prepared by removing all the protecting groups and the polystyrene resin support when present, from a protected precursor peptide, e.g. a compound of formula II as defined above; if desired oxidising or 48831 - 7 reducing the product to give the cyclic or open chain form, and further if desired isolating as the free bass or as a pharmaceutically acceptable salt.

Removal of the protecting groups may be effected by methods known in the art for the respective protecting groups. Preferably the protecting groups are removed by using hydrogen fluoride in the presence of anisole.

The polystyrene resin support may bs cleaved at the same time as removal of the protecting groups, e.g. using hydrogen fluoride in the presence of anisole, or it may be cleaved by transesterification to give a carboxyl protected intermediate of formula II wherein is an ester function. For example, methanolysis gives a methyl ester intermediate of formula II wherein R^ is methyl.

Hydrolysis of such esters provides the required C-terminal carboxyl function.

Sy appropriate choice of protecting groups the 1 2 protecting groups R and R may be selectively removed from the compounds of formula II to give the free disulphydryl derivatives of formula II uherein R and 12 R represent hydrogen. For example, uhen R and R are trityl or acetamido, they may be selectively removed by the action of a mercuric or silver salt to obtain the corresponding disulphydryl derivative in the form of its corresponding mercuric or disilver salt. The latter salt may be subjected to the action of hydrogen sulfide to - 8 obtain tha corresponding free disulphydryl derivative of 1 2 formula II uherein R and R are hydrogen.

The free disulphydryl compounds of formulae I and II may bs cyclised by oxidizing using an oxidizing agent, for example using iodine, oxygen (e.g. air), 1,2diiodoethane, or sodium or potassium ferricyanide, to obtain the corresponding compounds of formulae I and II 1 2 uherein R and R form a direct bond.

The polypeptide final products and their requisite intermediates may be prepared by the uell knoun solid phase method following techniques generally knoun in the art for building an amino acid sequence from an initial resin supported C-terminal amino acid. Such techniques are described by J.Pl. Steuart et al., Solid Phase Peptide Synthesis, Freeman and Co., San Francisco, 1969. As applied to the synthesis of the polypeptides of formula II, the initial C-terminal amino acid is D-cysteine, and the resin support preferably a chloromethylated polystyrene resin. The chloromethyl groups provide sites for attach20 ment of D-cysteine to the resin support by means of ester formation.

For example, in carrying out the synthesis, the chloromethylated polystyrene resin is esterified uith α-amino and sulfhydryl protected D-cystBina (e.g.

Boc-D-Cys(SPlBzl)-OH) according to tha procedure of Gisin, Helv. Chim. Acta., 56, 1976 (1973). The protected amino acid is linked by ester formation between the carboxyl group of D-cysteine and a chloromethyl group 48831 - 9 of the resin. The α-amino protecting group is then ’ removed uith trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or hydrogen chloride in dioxane. The deprotection is conducted at a temperature betueen about 0°C and room temperature. Other standard cleaving reagents and conditions for removal of specific α-amino protecting groups may be used as described in Schroder 4 Lubke, The Peptides, 1_, 72-75 (Academic Press 1965). After removal of the α-amino protecting group, the subsequent protected amine acids are coupled individually to the resin supported sequence, seriatim. Alternatively, - small peptide fragments may be prepared by 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 about a 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 at each stage of the synthesis is determined by the ninhydrin reaction as described by E.Kaiser et al., Analyt. Biochem., 34. 595 (1970).

Uhere incomplete coupling has occurred, the reaction is repeated before the α-amino protecting group is removed for introduction of the next amino acid sequence.

Diisopropylcarbodiimide is a preferred coupling reagent, although other agenta uill be apparent to those skilled in the art. - 10 Hanca this invention also provides a process for 5 preparing compounds of formula II wherein R represents a polystyrene resin support uhich comprises sequentially coupling the requisite amino acids, protected and/or activated as necessary, under solid phase synthesis conditions to a chloromethylated or hydroxymethyl polystyrene resin support, if dssired removing one or more protecting groups.

After the desired amino acid sequence has been 10 synthesized, the polypeptide is preferably removed from the resin support by treatment uith hydrogen fluoride and anisole to obtain the fully deprotactad linear polypeptide. The cyclic disulfide is produced by oxidation of the linear polypeptide, such as by treatment uith K3Fe(CN)5 or by contact uith air.

Non-toxic acid addition salts of the linear and cyclic polypeptides are produced by methods uell-knoun in the art from organic or inorganic acids uhich are nontoxic and acceptable for pharmaceutical purposes, such as hydrochloric, hydrobromic, sulfuric, phosphoric, polyphosphoric, maleic, acetic, citric, benzoic, succinic, malonic, or ascorbic acid and the like.

In the classical method as applied to the compounds of this invention the desired peptide is built up by25 condensing amino acids or groups of amino acids uhich are protected as necessary. The condensation reactions may ba carried out using methods generally knoun to form - 11 amide bonds in peptide and penicillin chemistry. To promote facile condensation of the amino acids it is preferred to employ a condensing agent. Examples of condensing agents are carbodiimides; e.g. N,N’dicyclohexylcarbodiimide (DCC), N,N’-diisopropylcarbodiimide. Alternatively the condensation may be effected by activating one or both of the terminal groups. Examples of the activated form of the terminal carboxyl are the acid chloride, anhydride, azide and activated ester. It uill be apparent to those skilled in the art that the proposed method of carrying out the condensation reactions should be compatible uith the protecting groups on the amino acids. 2 The compounds of formula II uherein R and R are protecting groups and R5 is hydrogen or a carboxyl protecting group may be prepared by coupling the requisite amino acids protected and/or activated, as necessary, or groups of amino acids by methods knoun for the formation of peptide bonds to give the desired 1 2 sequence of amino acids. The R and R protecting groups can then be selectively removed and the product oxidized to give the corresponding cyclic disulfide of 1 2 formula II uherein R and R form a direct bond. Alternatively ail the protecting groups can be removed to give the linear peptide uhich may be converted to the cyclic peptides of formula I by oxidation.

Methods of activating amino acids prior to coupling and coupling methods themselves are uell knoun in the 48831 - 12 art - sea for example the textbook of Schroder and Lubke mentioned above.

In selecting a particular side chain protecting group to be used in the solid phase or classical synthesis of the peptides of this invention, the following Rules should bs followed: (a) the side chain protecting group must be stable to the reagent and under the reaction conditions selected for removing the α-amino protecting group at each step of the synthesis, (b) ths protecting group must retain its protecting properties (i.e. not be split off under coupling conditions), and (c) the side chain protecting group must be removable upon the completion of the synthesis containing tha desired amino acid sequence under reaction conditions that uill not alter the peptide chain.

Preferred protecting groups for the amino acids employed in the solid state synthesis arB as follows: (a) for an α-amino group: ^-butyloxycarbonyl (BOC); (b) for a sids-chain hydroxyl group; benzyl (Bzl); (c) for a side chain £ -amino group: 2-chlorobenzylaxycarbonyl (C1Z); and (d) for a side chain mercapto group: £-methoxybenzyl (MBzl) In ths compounds of formula II the polystyrene resin may be any suitable resin support conventionally employed in the art for the solid-phase synthesis of polypeptides, preferably polystyrene uhich has been cross linked uith from 0.5 to about 3% divinyl benzene, uhich has been chloromethylated or hydroxymethylated to provide sites - 13 for estar formation betueen the initially introduced α-amino protected O-cysteine. A chloromethylated polystyrene resin is commercially available from Bio Rad Laboratories, Richmond, California and the prepara5 tion of such a resin is described by Steuart et al., Solid Phase Peptide Synthesis, Freeman and Co., San Francisco, 1969, Chapter 1, pages 1-6.

This invention also provides pharmaceutical compositions comprising an active compound of formula I or pharmaceutically acceptable salts thereof and a pharmaceutical acceptable carrier. Suitable pharmaceutical carriers uill be apparent to those skilled in the art. Preferably the pharmaceutical composition is in unit dosage form. - 14 .

The following examples illustrate the preparation of Cys-Phe-Trp-D-Trp-Lys-Thr-Phe-D-Cys cyclic (1-8) disulfide uhich is representative, in its solid phase preparation and biological activity, of the other compounds of the invention, supra.

Example 1 tert-Butyloxycarbony1-S-p-methoxybenzy1-L-cysteinyl-LΛ phenylalanyl-L-tryptophyl-D-tryptophyl-N -2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-threoriyl-L-phenyl alanyl-S-p-methoxybenzyl-O-cysteine hydroxymethyl polystyrene ester.

Chloromethylated polystyrene resin (Lab. Systems Inc. ) uas esterified uith Boc-D-Cys(SNBzl)OH according to Gisin, Helv. Chim. Acta.56, 1976 (1973). The polymeric ester (8 g.) uas placed in a reaction vessel of a peptide synthesizer Beckman 990 A and subjected to subsequent cycles of amino group deprotection and amino acid couplings as described in Program No 1 and Program NO. 2. (Beckman is a Trade-Mark). Die last program was performed in order to insure ccnplete coupling of each amino acid. The following amino acids were incorporated into the resin as described above: Boc-Phe-OH, BocThr(8zl)-0H, 8oc-Lys(ClCBz)-0H, Boc-D-Trp-OH, BocTrp-OH, Boc-Phe-OH, and Boc-Cys(SNBzl)-OH, to afford the title peptidoresin. - 15 Program No 1 Peptide Synthesizer-Beckman 990 1. Uash uith CHgClg x 3. 2. Treat uith TFA-CHgClg-EDT, 1:1:5% for 5 minutes. 3. Repeat (2) for 25 minutes. 4. Uaah uith CHgOlg * 4.

. Treat uith TEA 12% in DMF for 1 minute. 6. Repeat (5) for 5 minutes. 7. Uash uith CHgClg x 3. 8. Add 4 equivalents of Boc-protected amino acid and stir for 5 minutes. 9. Add 2 equivalents of 1M-DIC solution in DMF and stir for 25 minutes.

. Add 2 equivalents of 1M-DIC solution in DMF and stir far ISO minutes. 11. Uash uith CHgClg x 3. 12. Uash uith methanol x 3. 13. Uash uith CHgClg x 3. Proqram No 2. Peptide Synthesizer, Beckman 990 1. Uash uith CHgClg x 3. 2. Add 2 equivalents of Boc-protected amino acid and stir for 5 minutes. 3. Add 2 equivalents of 1M-DIC solution in DMF and stir for 180 minutes. 4. Uash uith DMF x 3. 5. Uash uith CHgClg x 3. 5. Uash uith methanol x 3. 7. Uash uith CHgClg x 3 - 16 Example 2 L-Cysteinyl-L-phenylalanyl-L-tryptophyl-D-tryptophyl L-lysyl-L-threonyl-L-phenylalanyl-O-cysteine Cyclic (1-8) Disulfide, The peptide resin of the previous example (15 g.) uas mixed uith anisole (30 ml.) and treated uith anhydrous HF (100 ml.) for 60 minutes in an tpe-bath and under exclusion of air. The excess HF uas removed as fast as possible (ca. 45 minutes) and the residue uas uashsd uith ether then taken in 20% aqueous acetic acid and added into 6 liters of degassed water. The pH of the solution uas brought to 7 uith dilute NH^OH and then oxidized uith a solution of K3Fe(CN)g (3 g. par 1000 ml). The pH was adjusted to 5 uith glacial acetic acid and the solution uas treated uith Bio-Rad flG 3-X4A (100 g.) for 30 minutes and filtered. The filtrate uas passed through Amberlite CG-50 (H + form) and the absorbed peptide was eluted with 50% aqueous acetic acid. (Amberlite is a Trade Mark). Ihe fractions containing the peptidic material were pooled and lyophilized to yield 1.1 g. of crude material.

This crude product uas chromatographed through a column of Sephadex G-25 (2.5 cm. x 150 cm) and eluted with 10% aqueous acetic acid. (Sephadex is a Trade Mark).

Fractions 138 to 160 (180 drops each) were pooled and lyophilized to yield the title compound, 167 mg.

TLC, silica gsl G precoated glass plates (n-Bu0H-Ac0H~H20-Et0Ac, 1:1:1:1, v/v) 6.75.

Amino acid analysis: Thr(l) 0.98, Cys(2) 1.60, Pha(2) 2, Lys(l) 0.98, Trp(2) 1.78.

The product of the preceding examples illustrates the selective activity of the compounds of this invention for grouth hormone and glucagon suppression in the follouing standard procedure: Albino male rats are administered Nembutal intra10 peritcneally at a dose of 50 milligrams per kilogram. (Namtntal is a Trade liark). Fifteen minutes later a subcutaneous injection of the test ccnpound ot physiological saline (control, is administered. Ten minutes later 0.5 milliliters of arginina (300 milligrams per milliliter, pH 7.2) is injected into the heart. Five minutes after rsceipt of the arginine the rats are decapitated and blood is collected into trasylol-EDTA. An appropriate aliquot is assayed for grouth hormone (GH), insulin, and glucagon by radioimmunoassay. Tha results of the 2Q assay are as follows : Dose GH INS GLUN Compound uo/ko no/ml_uU/ml oo/ml Control Example 2 100 *p<0.05 488+82 191+60* 74+4 133+13 69+4 60+12* duration of activity of the product □ uas as follows : Dose GH Compound pq/kq Hours no/ml Control 2 369+60 Example 2 1,000 2 60+6* Control - 4 354+68 Example 2 1,000 4 75+10* *p<0.01 As uith administration of any therapeutic agent used in the treatment of diabetes mellitus, the compounds of this invention must be individualized for the patient under guidance and close control of the attending physician to reach optimum blood levels of growth hormone, insulin and glucagon.

Doses for achieving the desired stats vary uith the condition of the patient, such as ags, amount of endogenous insulin produced, the presence of glucagon secreting tumors, ths route of administration, the duration of treatment, severity of the condition being treated etc.

Thus, the compounds of this invention may be administered alone or in combination uith insulin, uith or uithout carriers or excipients conventional to the route of administration selected, uhich may - 19 bs oral, intravenous, subcutaneous, intramuscular, intranasal,intrarectally, etc. Suitable pharmaceutical compositions for application are apparent to those skilled in the art.

Claims (6)

claims 1. 2 II uherein R and R are hydrogen to give a compound 1 2 of formula II uherein R and R are a direct bond. 20. A process for preparing a compound of formula II as defined in Claim 7 substantially as hereinbefore 25 described uith reference to Example 1. 21. A process for preparing a compound of formula I as defined in Claim 1 substantially as hereinbefore described uith reference to Example 2. 22. A compound of formula I as defined in Claim 1 20 uhensvsr prepared by a process as claimed in any one of Claims 14 to 17 and 21. 23. A compound of formula II as defined in Claim 7 uhenever prepared by a process as claimed in any one of Claims 18, 19 and 2D. 25 24. A pharmaceutical composition comprising a compound of formula I as defined in any one of Claims 1 to 5 or a pharmaceutically acceptable salt 48831 - 25 thereof, in conjunction with a pharmaceutically acceptable carrier. 25. A pharmaceutical composition as claimed in Claim 24 when in unit dosage form. 1 2 uherein R and R are benzyl, 3,4-dlmethylbenzyl,pmethoxybenzyl, p-chlorobenzyl, p-nitrobenzyl, trityl, benzyloxycarbonyl, benzhydryl, p-methoxybenzylcarbonyl, benzylthiomethyl, ethylcarbamoyl, thiosthyl,tetrahydropyranyl, acatamidomethyl, benzoyl or S-eulfonic salt. 9. A compound of formula II as claimed in claim 7 or claim 8 uherein R represents formyl, tri fluoro-acetyl, phthalyl, tosyl, o-nitrophenylsulfenyl, benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, ]>butyloxycarbonyl,diisopropylmethoxy, 49831 - 22 carbonyl, isopropyloxycarbonyl, allyloxycarbonyl,

1. A compound of formula : S-A A-S 1 I X 1 -Cys-Pha-X 2 -X 3 -Lys-Thr-Pha-D-Cys-0H (I) in uhich X 1 is hydrogen, das-amino, H-Ala-Gly or 5 H-Ala-D-Ala; X 2 is Trp, Leu, flat or g.-Cl-Phe; Xj is D-Trp or 5- or6-fluoro-D-Trp; and the A groups are hydrogen or a direct bond betueen the tuo sulfur atoms; and pharmaceutically acceptable salts thereof. 10 2. A compound as claimed in claim 1 uherein X 2 is Trp.

2. 5 R are protecting groups and R represents a carboxy protecting group, uhich comprises coupling the 5 requisite amino acids, protected and/or activated as necessary, or groups of amino acids by methods knoun for the formation of amide bonds to give the desired sequence, and if desired selectively removing one or some of the protecting groups and 10 further, if desired, oxidising a compound of formula 2,2,2-trichloroethoxycarbonyl, amyloxycarbonyl, cyclopsntyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiooarbonyl, trityl, 5 or trimethylsilyl, or Ala-Gly or Ala-D-Ala protected by one of the aforementioned groups. 10. A compound of formula II as claimed in any one of claims 7 to 9 uherein R is tosyl,benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl or .t-butyloxycarbonyl. Iq 11. A compound of formula II as claimed in any one of claims 7 to 10 uherein R^ ia benzyl, p-methoxybenzyl, p-chlorobenzyl, p-nitrobenzyl, trityl, benzyloxycarbonyl , p-methoxybenzyloxycarbonyl,acetyl, tert-butyl and benzoyl. 15 12. A compound of formula II as claimed in any one of claims 7 to 10 uherein R^ is t-butyl or benzyl. 13. t,-Buty loxy carbo nyl-S-p-methoxybenzyl-L-cysteinyl L-phenylalanyl-L-tryptophyl— £ -D-tryptophyl-N -2-chlorobenzyloxycarbonyl-L-lysyl2o -O-benzyl-L-threonyl-L-phenylalanyl-S-p-methoxybenzyl-D-cysteinyi-hydroxymethyl polystyrene ester. 14. A process for preparing a compound of formula I as defined in claim 1 uhich comprises removing all the protecting groups and the polystyrene resin support 25 uhen present from a compound as claimed in any one of claims 6 to 13, if desired oxidising a compound of formula I or reducing a compound of formula I - 23 produced tc give the cyclic or open chain Form, and further if desired isolating as the free base or a pharmaceutically acceptable salt. 15. A process as claimed in Claim 14 uherein the polystyrene resin support and/or protecting groups are removed by treatment uith hydrogen fluoride in the presence of anisole. 16. A process for preparing a cyclic compound of formula I as defined in Claim 1 uherein the A· groups represent a direct bond uhich comprises oxidising a corresponding compound of formula I as defined in Claim 1 uherein the A groups represent hydrogen. 17. A process as claimed in any one of Claims 14 to 16 in uhich the oxidation is effected using oxygen or potassium ferricyanide. 18. A process for preparing a compound of formula II, as claimed in any one of Claims 7 to 11 and 13 uherein r5 represents a polystyrene resin support linked via a methylene group uhich comprises sequentially coupling the requisite amino acids, protected and/or activated as necessary, cr groups of amino acids, under solid phase synthesis conditions to a chlaromethylated or hydroxymethyi polystyrene resin support, end if desired removing one or some of the protecting groups, and further if desired oxidising a compound 1 2 of formula II uherein R and R are hydrogen to give 1 2 a compound of formula II uherein R and R are a direct bond. - 24 19. A process for preparing a compound of formula ΙΓ as claimed in any one of Claims 7 to 12 uherein R and 3. 4 5 of R , R and R is other than hydrogen. 8. A compound of formula II as claimed in claim 7

3. A compound as claimed in claim 1 uherein is D-Trp.

4. L-Cysteinyl-L-phenylalanyl-L-tryptophyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-D-cysteins cyclic (1-8) disulfide. 15 5. A compound as claimed in any one of Claims 1 to 4 uhich is in the form of a salt from one of the follouing acids : hydrochloric, hydrobromic,sulphuric, phosphoric, polyphosphoric, maleic, citric, benzoic, succinic, malonic and ascorbic. 20 6. A compound having the amino acid sequence as defined in any one of claims 1 to 4 carrying at least one protecting group far a sida chain group and/or terminal α-amino group when present, and/or terminal carboxy group. 48831 21 7. A compound of formula : S(R 1 ) (R 2 )S (R)-Cya-Phe-X 2 -X 3 -Ly8(R 3 )-Thr(R 4 )-Phe-D-Cy8-0R 5 (II) in uhich X 2 and Xj are ae defined in Claim 1, R ia . hydrogen, an α-amino protecting group, des-amino or α-amino protected Ala-Gly or α-amino protected 1 2 Ala-D-Ala; R and R are hydrogen, a sulphydryl 1 2 protecting group or R and R are a direct bond 3 betueen sulphur atoms, R is hydrogen or a PP protecting group of Lye; R 4 is hydrogen or a hydroxyl protecting group of Thr; and R 3 is hydrogen, a carboxy protecting group or -CH 2 (polystyrene resin), uith the ή proviso that uhen R is hydrogen or des-amino and R and 2 R are a direct bond or both hydrogen then at least one

5. 2

6. A compound of formula I as defined in any one of Claims 1 to 5 for use as a pharmaceutical.