IE48500B1 - Peptides related to somatostatin - Google Patents

Description

This invention relates to synthetic peptides structurally related to somatostatin possessing pharmacological activity, to processes for preparing them, to intermediates employed in such processes and to pharma5 ceutical compositions containing them. In the compounds of thB invention, the somatostatin peptide chain is modified at the 4, 5 and 13 position, uith the 1, 2, 8 and 14 poations being preferably modified also.

Somatostatin is the cyclic disulfide tetradecapeptide of the formula: H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp s·^ I s\ HO-Cys-Ser-Thr-Phe-Thr-Lys (A) This peptide (fi) has been identified as the somatotropinrelease inhibiting factor (SRIF) uhich is secreted by the hypothalamus and regulates the secretion of pituitary grouth hormone (GH) (somatotropin). [See Brazeau et al., Science, 179, 77 (1973), Burgus et al., Proc.Nat.Acad.Sci. (USA), 70., 684 (1973), and Ling et al., Biochemical and Biophysical Res. Communication, 50, 127 (1973)]. The reduced form of somatostatin (RS) is the linear tetrade20 capeptide of the formula: SH SH I I H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH (B) 48300 - 3 The reduced Form (B) has been prepared by total synthesis, [see Rivier et al., C.R.Aoad. Sci. Ser. p.

Sci. Natur. (Paris), 276, 2737 (1973) and Sarantakis and McKinley, Biochem. and Biophys. Res. Communications, 54, 234 (1973)] and it (B) can be converted to· somatostatin (A) by oxidation whereby a bridging bond is Formed between the two eulphydryls oF the two cysteinyl amino acid residues in the tetradecapeptids.

Various polypeptides uhich may be regarded as structural modiFications oF somatostatin have been prepared synthetically and are reported in the chemical literature. Such polypeptides have 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 missing or are replaced by other amino acid residues or functional groups. The present invention relates to novel synthetic biologically active polypeptides uhich may be regarded as a structural modification of somatostatin. The polypeptides of the invention differ From somatostatin in the Following respects: 2 (a) the H-Ala -Gly segment is either present, missing, or replaced by H-Gly-Gly-Gly, H-Ala-D-Ala, acetyl or benzoyl; «7 (b) the Cys residue is either present or replaced by a g-mercaptopropionic acid residue. (c) the Lys4 residue is replaced by Arg or His; » 48500 - 4 (d) The Asn5 residue is replaced by His, Glu, Tyr, Trp, cr Phe; and θ (e) the Trp residue is either present or replaced by D-Trp dt 6-F-D-Trp; (f) the Ser residue is replaced by a D-ct-amino acid residue; and Ί 4 (g) the Cys residue is either present or replaced by D-Cys.

Accordingly this invention provides a peptide of 10 formula I; -S I CHr I X-CHCO -X1-X2-Phe~Phe-X3-Lys-Thr-Phe-Thr-X4-NH-CH-COOH (I) wherein X is H, NHj-, H-Ala-Gly-ΝΗ-, H-Ala-D-Ala-NH-H-GLy-Gly-Gly-HH-, acetyl-iffi- or benzoyl-NH-; X^ is His or Arg; X2 is His, Glu, Tyr, Trp or Phe; is Trp, D-Trp, or 6-F-D-Trp; X^ is D-a-amino acid; ΞΙ ϊ2 the residue X-CHCO has the L-configuration; and 25 _ς Ϊ CH_ I 2 the residue NH-CH-COOH has either the L- or Dconfiguration 5 - 5 or a non-toxic, pharmaceutically acceptable acid addition salt thereof.

In addition, this invention provides the linear form of the compounds of formula I (i.e. the non-cyclic reduced compounds of formula II which differ in that they contain two free sulphhydzyl groups), or a non-toxic, pharmaceutically acceptable acid addition salt thereof! SH SH I I CH„ CH, I 2 I 2 X-CHC0-X1-X2-Phe-PhB-X3-Lys-Thr-Phe-Thr-X4-NH-CH-C00H (ID The linear peptides of formula II are also precursors for the cyclic peptide of formula I.

All optically active amino acids and amino acid residues in the polypeptides depicted and described herein are in thB natural or L-cdnfiguration, unless otherwise noted. The symbols identifying the amino acids and the amino acid residues in the polypeptides described herein are those adopted by the IUPAC-IUB Committee on Biochemical Nomenclature Recommendation (1971), and are described in the Archives of Biochemistry and Biophysics, 150, 1-0 (1972). The symbol 6-F-D-Trp means D-tryptophan in which the 6-position is substituted by fluorine. The term D-ct-amino acid means an optically active α-amino acid in which the α-carbon is in the D-configuration. Preferred examples of such 0-amino acids are: D-proline, D-alanine, D-valine, D-leucine, - 6 D-isoleucine, D-serine, D-threonine, D-methionine, D-aspartic acid, D-glutanic acid, D-lysine, D-arginine, D-asparagine, D-histidine, D-tryptophan, D-phanylalanine, and D-tyrosine.

It will be apparent to those skilled in the art that the α-carbon of the cysteine residues (corresponding to ν 14 Cys^ and Cys of somatostatin) contain an asymmetric carbon atom and optical isomers of such amino acid residues are possible. In the peptides depicted by the formulae set forth herein (formula I, II, III or IV), the Cys8 residue must be in the natural (L-) configuration, 14 uhile the Cys residue can be in either the natural (L-) or unnatural (D-) configuration.

The compounds of formula I and II possess pharma15 cological activity, in particular they inhibit the secretions of growth hormone, insulin, and/or glucagon as demonstrated in standard pharmacological test procedures, and are useful in controlling serum glucose in the treatment of diabetes or in the treatment of hyperinsulinemia. The compounds have also shoun a prolonged period of action.

Preferred values for Xj are His and Glu.

Preferably X is -NH^· Preferably X^ is D-Trp.

Preferred compounds of formula I and ΊΙ are those 25 uherein X is NH2; X^ is Arg; is His, X^ is D-Trp; and is D-Tyr. Thsir preparation is described in Examples 1, 2(a) and 2(b). Also preferred are compounds -4-8 50 0 of formula I and II wherein X is NH^j X^ is Arg; X2 is Glu; Xg is D-Trp; and X^ is D-Tyr, which inhibit the release of growth hormone and insulin without materially inhibiting the release of glucagon. Their preparation is described in Examples 3, 4(a) and 4(b). Further preferred compounds of formula I and II are those uherein X is NH2; X1 is His; Xg is His; Xg is D-Trp and Χή is D-Ser, which inhibit the release of growth hormone and glucagon without materially inhibiting the release of insulin. Their preparation is described in Examples 5, 6(a) and 6(b). Other embodiments can be prepared using similar methods or obvious modifications thereof. A desired embodiment can be prepared using the exemplified technique by substituting a desired protected amino acid for a particular moiety illustrated.

This invention also provides protected intermediates for the compounds of formula I and II, which intermediates have the same amino acid sequence as compounds of formula I and II and carry at least one protecting group for a side chain group and/or terminal α-amino group when present and/or terminal carboxy group.

More particularly this invention also provides intermediates having the formula III SZ CH, Y-tHCO-Y.-Y_-Phe-Phe-X_-Y„-Y/-PhB-Y/-Y,.-NH ο O 4 4 0 8 500 - 8 wherein Rg represents a carboxy protecting group or a polystyrene resin support linked uia a methylene group; the residue Y-CH (CHjSZ^) CD has the (.-configuration; the residue -NHCH(CH2SZ2)C0 has either the L- or D5 configuration; X3 represents Trp, D-Trp or 6-F-D-Trp; Y is K, R-NH-, R-Ala-Gly-ΝΗ-, R-Gly-Gly-Gly-NH-R-flia-D-Ala-NH-, acety1-NH- or benzoyl-NH-; Y1 is His^) or Arg(R.,); Y2 is His^), G1u(R2), Tyr(R3), Trp or Phe; Y3 is Lys(R4); Y4 is Thr(R5); Y5 is an D-a-amino acid residue in uhich any protectable side chain group is protected Z^ and Zj are each sulphhydryl protecting groups or hydrogen or Z^ and Z2 together represents a direct bond betueen sulphur atoms; and R is an α-amino protecting group and R^, Rj, Rg, R^ and Rg are each independently a side chain protecting group.

Examples of Z^ and Z2 are benzyl, 3,4-diroethylbenzyl, p-methoxybenzyl, p-chlorobenzyl, p-nitrobenzyl, trityl, benzyloxycarbonyl, benzhydryl, £-methoxybenzylcarbonyl, benzylthiomethyl, ethylcarbamoyl, thioethyl, tetrahydro25 pyranyl, acetamidomethyl, benzoyl or s-sulfonic salt. Preferably and Z. represent p-methoxybenzyl.

Preferred intermediates are those of formula IU; 48300 - 9 S-Z I CH„ I 2 Y-CHC0-Y1-Y2-Phe-Phe-X3-Y3-Y4-Phe-Y4Yg-NH-CHio-CH-[polystyrene | z resin] CH1 S-Z (IV) wherein X3 is Trp, D-Trp, or 6-F-D-Trp; Y is H, R-NH-, R-Ala-Gly-NH-, R-Gly-Gly-Gly-ΝΗ-, R-Ala-D-Ala-NH-, 5 acetyl-ΝΗ-, or benzqyl-NH-; Y1 is His(R,)) or Arg^); Y2 is His(R^), G1u(R2), Tyr(R3), Trp, or Phe; Y3 is Lys(R4); Υή is Thr(Rg); Yg is D-Pro, D-Ala, D-Val, D-Leu, D-isoLeu, D-Ser(Rg), D-Thr(Rg), D-Asp(R2), D-Glu(R2), D-Lys(R4), D-Arg^), D-Asn, D-HisiRp, D-Trp, D-Phe, or D-Tyr(Rg); and Z is the £-methoxybenzyl protecting group; wherein R is an α-amino protecting group and Rp R2, Rg, R^ •^g and Rg are each, independently, a side-chain protecting group, the residue YCH(CH2SZ)C0- is in the L-configuration, and the residue -NHCH(CH2SZ)C0 is in either the L- or 20 D-configuration. examples of α-amino protecting groups fcr R in formulae (III) and (IV) are (1) acyl type protecting groups illustrated by the following: formyl, trifluoroacetyl, phthalyl, - 10 ^-toluenesulfonyl (tosyl) and o-nitrophenylsulfenyl; (2) aromatic urethane type protecting groups illustrated by benzyloxycarbonyl and substituted benzyloxycarbonyl such as jo-chlorobenzyloxycarbonyl, £-nitrobenzyloxycarbonyl; (3) aliphatic urethane protecting groups illustrated by tert-butvloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, amyloxycarbonyl; (4) cyeloalkyl urethane type protecting groups illustrated by cyclopentyloxy10 carbonyl, adamantylcarbonyl, cyclohexyloxycarbonyl; (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 tert-butyloxycarbonyl.

Examples af are tosyl, benzyloxycarbonyl, adamantyloxycarbonyl or tert-butyloxycarbonyl; preferably R1 is tosyl.

Protection via the tosyl group is on either the * or Nnitrogen atoms of arginine, while the oxycarbonyl type protecting groups protect the and either one of the M or nitrogen atoms.

Preferably Rg is benzyl. Preferably Rg is 2,6dichlorobenzyl.

The side chain amino group of lysine is protected by Εή which is exemplified by tosyl, t^-amyloxycarbonyl, jtbutyloxycarbonyl, diisopropyloxycarbonyl, benzyloxycarbonyl, halobenzyloxycarbonyl and p-nitrobenzyloxycarbonyl, the - 11 2-chlorobenzyloxycarbonyl group being preferred.

Examples of Rg protecting groups for the hydroxyl group of threoninB and serine are acetyl, benzoyl, tert-butyl, and benzyl. The benzyl group is preferred for this purpose.

This 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 uhen present, from a protected precursor peptide, e.g. a compound of formula III or IV as defined above; if desired oxidising or reducing the product to give tha cyclic -or open chain form, and further if desired isolating as the free base 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 presense of anisole.

The polystyrene resin support may be 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 Rg is an ester function. For example, methanolysis gives a methyl ester intermediate of formula IH uhBrein Rg is methyl. Hydrolysis of such esters provides the required C-terminal carboxyl function. - 12 By appropriate choice of protecting groups the protecting groups Z^ and Z2 may be selectively removed from the compounds of formula III to give the free disulphhydryl derivatives of formula III wherein and Z2 represent hydrogen. For example, when Z^ and Z2 are trityl or acetamido, they may be selectively removed by the action of a mercuric or silver salt to obtain the corresponding disulphhydryl derivative in the form of its corresponding mercuric or disilver salt. The latter salt may be subjected to the action of hydrogen sulfide to obtain the corresponding free disulphhydryl derivative of formula III.

The free disulphhydryl derivatives of formula II and III may each be 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 formula I and III wherein Z^ and Z2 form a direct bond.

The polypeptide final products and their requisite intermediates may be prepared by the well known solid phase method following techniques generally known in the art for building an amino acid sequence from an initial resin supported C-terminal amino acid. Such techniques are described by 0.ΙΊ. Stewart et al., Solid Phase Peptide Synthesis, Freeman and Co., San Francisco, 1969. As applied to the synthesis of the polypeptides of formula III and IV, the initial C-terminal amino acid is L-or Dcysteine, and the resin support preferably a chloromethylated polystyrene resin. The chloromethyl groups provide - 13 sites for attachment of L- or 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 sulphhydryl protected D- and L-cysteine (e.g. Boc-Cys(SMBzl)-OH) according to the procedure of Gisin, Helv. Chim. Acta., 56, 1476 (1973). The protected amino acid is linked by ester formation between the carboxyl group of L- or D-cysteine and a chloromethyl group of thB 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 from O°C. to room temperature.

Other standard cleaving reagents and conditions for removal of specific α-amino protecting groups may be used as described in Schroder E. Lubke, The Peptides, 72-75 (Academic Press, 1965). After removal of the α-amino protecting group, the subsequent protected amino 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 dBsired 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 tuo solvents. The success of each coupling reaction at each stage of the synthesis OO - 14 is determined by the ninhydrin reaction as described by E.Kaiser et al., Analyt. Siochem., .34, 595 (1970). Where 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 agents will be apparent to those skilled in the art.

Hence this invention also provides a process for preparing compounds of formula III wherein and Z2 are protecting groups and Rg represents a polystyrene resin support and compounds of formula IU which 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.

After the desired amino acid sequence has been synthesized, the polypeptide is removed from the resin support by treatment with hydrogen fluoride and anisole to obtain the fully deprotected linear polypeptide. The cyclic disulfide is produced by oxidation of the linear polypeptide, such as by treatment with K^Fe(CN)g or by contact with air.

Non-toxic acid addition salts of the linear and cyclic polypeptides are produced by methods well-known in the art from organic or inorganic acids which are nontoxic and acceptable for pharmaceutical purposes, such as hydrochloric, hydrobromic, sulfuric, phosphoric, polyphosphoric, maleic, acetic, citric, benzoic, succinic, malonic, or ascorbic acid. 48300 - 15 In the classical method as applied to the compounds of this invention the desired peptide is built up by condensing amino acids or groups of amino acids uhieh are protected if necessary. The condensation reactions may be carried out using methods generally known to form amide bonds in peptide and penicillin chemistry. To promote facile condensation of thB 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 οπβ 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 will 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.

The compounds of formula III uherein and are protecting groups and Rg is a carboxyl 'protecting group may be prepared by coupling the requisite amino acids protected and/or activated, as necessary, or groups of amino acids to give the desired sequence of amino acids. The Z^ and Z2 protecting groups can then be selectively removed and the product oxidized to give the cyclic disulfide of formula III wherein and Z2 form a direct bond. - 16 Methods of activating amino acids prior to coupling and coupling methods themselves are well known in the art - see 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 be 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) the 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 1E completion of the synthesis containing the desired amino acid sequence under reaction conditions that will not alter the peptide chain.

Preferred protecting groups for the amino acids employed in the solid state synthesis are as follows: (a) for an a-amino group: jt-butyloxycarbonyl (BDC); (b) for a side-chain hydroxyl group: benzyl (Bzl); (c) for a side chain aromatic hydroxyl group: 2,6oichlorobenzyl (ClgEfcl); (d) for a side chain carboxy group: benzyl (Bzl); (e) for a side chain 6-amino group: 2-chlorobenzyloxycarbonyl (C1Z); (f) for the side chain guanidine nitrogens: tosyl (Tos); (g) for the secondary imidazol nitrogen: tosyl (Tos); and (h) for a side chain mercapto group: £-methoxybenzyl (MBzl). 4-8500 - 17 In the compounds of formulae ITI and IU, the polystyrene resin may be any suitable resin support conventionally employed in the art for the solid-phase synthesis of polypeptides, preferably polystyrene in which the degree of crosslinking by divinyl benzene is from 0.5 to 3%, which has been chloromethylated or hydroxymethylated to provide sites for ester formation between the initially introduced α-amino protected L- or Dcysteine. ft chloromethylated polystyrene resin is commercially available from Bio Rad Laboratories, Richmond, California and the preparation of such a resin is described by Stewart et al., Solid Phase Peptide Synthesis, freeman and Co., San francisco, 1969, Chapter 1, pages 1-6. In formulaIU, -C-O-CHj- t represents the ester linkage betueen the C-terminal carboxy group of the polypeptide and one of the many methylene groups originally introduced into the phenyl moieties of the polystyrene chain by chloromethylations.

The compounds described herein may be administered to uarm-blooded mammals, either intravenously, subcutaneously, intramuscularly, or orally to control serum glucose in the treatment of diabetes or hyperinsulinemia. The required dosage will vary uith the particular conditions being treated, the severity of the condition and the duration of treatment.

The active ingredient may be administered alone or 8 500 -Ιθin combination uith pharmaceutically acceptable carriers or excipients. Suitable pharmaceutical compositions will be apparent to those skilled in the art.

Preferably the pharmaceutical composition is in unit dosage form. 4850Q - 19 The methods of making and using the compounds of the invention are illustrated in the follouing examples: Example 1 „ 9 N -tert-Butyloxycarbonyl-S-p-Methoxybenzyl-L-Cysteinyl-N 5 Tosyl-L-Arginyl-Ninl-Tosyl-L-Histidyl-L-Phenylalanyl-L£ Phenylalanyl-D-Tryptophyl-N -2-Chlorobenzyloxycarbonyl-LLysyl-D-Benzyl-L-Threonyl-L-Phenylalanyl-O-Benzyl-LThreonyl-0-2,6-Dichlorobanzyl-D-Tyrosyl-S-p-MethoxybenzylL-Cysteinyl-HydroxyniBthyl-Polystyrene Ester 10 Chloromethylated polystyrene resin [Lab Systems, Inc.] (0.75 meq. per g.) is esterified uith Boc-Cys(SMB2l)-DH cesium salt, according to the procedure of Gisin, Helv. Chim. Acta., 56, 1976 (1973). The amino acid resin was then treated according to Schedule A (set forth below), Boc-D-Tyr(CljBzlJ-OH being employed as the protected amino acid in Step 9 thereof. Schedule A was then repeated as necessary in order to incorporate consecutively the follouing amino acids into the peptido resin: 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)-0H Boc-Arg(Tos)-OH Boc-Cys(SMBzl)-OH - 20 Schedule A: LPr°toc°l For the deprotection of the a-amino group and coupling of thB protected amino acids on the Boc-Cys-(SMBzl)-0-CH2-(protected resin)] 1. Uash uith methylene chloride (CH2C12), three times. 2. Treat uith trifluoroacetic acid-methylene chloride 9:1, v/v containing 5% v/v 1,2-ethane dithiol for 5 minutes. 3. Repeat Step 2 for 25 minutes. 4. Uash uith CH2C12> three times.

. Uash uith dimethylformamide (DMF).

. Treat uith 12% v/v triethylamine in DMF for 3 minutes. 7. Uash uith DMF. 8. Uash uith CK2C12, three times. 9. Treat uith 4 equivalents of the appropriate protected amino acid in CH2C12-DMF and stir for 5 minutes.

. Add in tuo portions over a 30 minute period 5 equivalents of diisopropylcarbodiimide dissolved in 0Η2012· Allou reaction to proceed for S hours. 11. Uash uith DMF, three times. 12. Uash uith CH2C12> three times. 13. Test by ninhydrin reaction according to the procedure of Kaiser et al., Annal. Biochem., 34, 595 (1970). In case of incomplete reaction, repeat Steps' 9 to 13, as above. - 21 Example 2 L-Cysteinyl-L-Arginyl-L-Histidyl-L-Phenylalanyl-lPhenylalanyl-D-Tryptophyl-L-Lysyl-L-Threonyl-L-Phenylalanyl-L-Threcnyl-D-Tyrosyl-L-Cysteine Cyclic (1—> 2) Disulfide (a) A mixture of the peptidoresin of Example 1 (12 g.), anisole (20 ml.) and liquid hydrogen fluoride (200 ml.) mas allowed to stand in an ice-bath (air excluded) for 50 minutes, after which excess hydrogen fluoride was removed under vacuo. The residue was extracted with 20% (v/v) aq. acetic acid, and the extract containing L-cysteinyl-L-arginyl-L-histidyl-L-phenylalanyl-Lphenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-D-tyrosyl-L-cysteine was poured into litres of deaerated water. (b) The pH of the water from Example 2(a) was brought to 7.4 by adding dilute ammonium hydroxide, and the mixture was oxidized by adding a solution of KgFe(CN)g (1.5 g. in 500 ml. water). The oxidation mixture was acidified uith glacial acetic acid to pH 5, and excess oxidant was removed by adding Bio Rad AG 3. The peptide material was absorbed onto Amberlite CG-50 (H+ form) and « then eluted uith a mixture of water-acetic acid-pyridine (56:4:30 v/v). The fractions containing peptidic material ware pooled and lyophilized tc yield crude product, which was applied to a column of Sephadex G-25 (2.5 x 160 cm.) and eluted with 10% v/v aq. acetic acid.

(BioRad, Amberlite and Sephadex are Registered Trade Marks). - 22 Fractions 113 to 133 uere collected and lyophilized to yield the title peptide, 855 mg.

TLC: Avicel coated glass plates, chlorcsx spray Rf (n-butanol-waterglacial acetic acid, 4:1:1, v/v) 0.41. (Avicsl is a Registered Trade Mark) Amino Acid Analysis: Thr (2) 1.87; Phe (3) 3; Cys (2) 1.49; Tyr (1) D.87; Lys (1) 1.02; His (l) 0.89; Arg (1) 1.01; Trp (1) 0.67.

Example 3 Na-tert-BtrtylaxycarbGr^l - S-p-^thoxsi3en2yl-J>-Ciysteinyl-N^10 Tosyl-L-Arginyl-·}-Benzyl-L-Glutamyl-L-Phenylalanyl-LPhenylalanyl-D-Tryptophyl-l\l€-2-ChlorobenzyloxycarbonylL-Lysyl-0-Benzy1-L-Threonyl-L-Phenylalanyl-O-Benzyl-LThreonyl-0-2,6-Dichlorobenzyl-D-Tyrosyl-S-p-HethoxybenzylL-Cysteinyl Hydroxymethyl Polystyrene Ester The above peptidoresin uas prepared in a fashion similar to Example 1, substituting the requisite amino acid at the appropriate point in the synthesis.

Example 4 L-Cysteinyl-L-Arginyl-L-a-Glutamyl-L-Phenylalanyl-L20 Phenylalanyl-D-Tryptophyl-L-Lysyl-L-Threonyl-L-Phenylalanyl-L-Threonyl-Q-Tyrosyl-L-Cysteine Cyclic (1—>12) Disulfide (a) In a manner similar to Example 2(a) the peptidoresin of Example 3 uas cleaved and deprotected to give L-cysteinyl-L-arginyl-L-a-glutamyl-L-pbenylalanyl-L4850ο -23phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-D- tyrosyl-L-cysteine. (b) The title dodecapeptide was prepared From the linear peptide of Example 4(a) in a fashion similar to Example 2(b) as the triacetate salt TLC: Avicel preccated glass plates.

Rf (n-Butanol-uater-gl. acetic acid, 4:1:1, v/v) 0.57 Rf (n-Butanol-ethyl acetate-uater-gl. acetic acid, 1:1:1:1, v/v) 0.B5 Rf (tert-Amylalcohol-pyridine-uater, 7:7:6, v/v) 0.92.

Amino Acid Analysis: Thr (2) 1.96; Glu (1) 1.04; Cys (2) 1.79; Tyr (l) 1.02; Phe (3) 3; Lys (1) 1.04; Trp (1) 1.09; Arg (1) 1.01.

Example 5 Na-tert-Butyloxycadxxiyl-S-p-MethQxyt)enzyl-L-Cysteinyl-NlmTosyl-l-Histidyl-N^m-Tosyl-L-Histidyl-L-Phenylalanyl-Lc, Phenylalanyl-D-Tryptophyl-N -2-Chlorobenzyioxycarbonyl-lLysyl-O-Benzyl-L-Threonyl-L-Phenylalanyl-O-Benzyl-LThrBony 1-0-Benzyl-0-Seryl-S-p-l*lethoxy benzyl-L-Cy s teinyl Hydroxymethyl Polystyrene Ester The above peptidoresin was prepared in a fashion similar to Example 1, substituting the requisite amino acid at the appropriate point in the synthesis. - 48500 - 24 Example 6 L-Cysteinyl-L-Histidyl-L-Histidyl-L-Phenylalanyl-LPhenylalanyl-D-Tryptophyl-L-Lysyl-L-Threonyl-LPhenylalanyl-L-Threonyl-D-Seryl-L-Cysteine Cyclic (1—^12) Disulfide (a) In a manner similar to Example 2(a) the peptido resin of Example 5 uas cleaved and deprotected to give L-cysteinyl-L-histidy1-L-histidy1-L-phenylalanyl-Lphenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenyΙΙΟ alanyl-L-threonyl-D-seryl-L-cysteine. (b) The linear peptide of the Example 6(a) was treated in similar fashion as in Example 2(a) to provide the title compound, as the triacetate salt.

TLC: Silica gel precoated glass plates.

Rf (n-Butanol-ethyl acetate-water-gl. acetic acid, 1:1:1:1:, v/v) 0.52.

Avicel precoated glass plates.

Rf (π-Butanol-water-gl. acetic acid, 4:5:1, v/v) 0.60 Rf (tert- Amyl alcohol-pyridine-water, 7:7:6, v/vj 0.76.

Amino Acid Analysis: Thr (2) 1.98; Ser (1) 0.98; Cys (2) 1.64; Phe (3) 3; Lys (l) 1.1; His (2) 2.1; Trp (1) 0.84.

Example 7 The ability of the compounds of this invention to inhibit the secretions of growth hormone, insulin and/or glucagon can be demonstrated in the following test procedure: 4-8500 - 25 Male albino rats are divided into two groups (e.g. nine rats per group). Each animal is administered sodium pentobarbital I.P., at a dose of mg/kg. Fifteen minutes later, one group received a S.C. injection of the tBst compound in saline (typically 5 to 200 pg/kg.), and tha other group (control) receives saline alone. Ten minutes latBT, each animal is given a 0.5 ml. injection of arginine (300 mg/ml., pH 7.2) into the heart. Five minutes after receipt of the arginine, the animals are decapitated and blood is collected into Trasylol-EDTA. An appropriate aliquot of each sample is assayed for GH, glucagon, and insulin by radioimmunoassay. GH is determined by the method of Sinha et al., Endocrinol., 91, 704 (1972); glucagon is determined by the method of Faloona and Unger, Methods of Hormone Radioimmunoassay, Caffe et al., Ed., Academic Press, New York, 1974, pages 317-330; and insulin is determined by the method of Hales and Randle, Biochem. 0·, 68, 137 (1963). The blood concentrations of growth hormone, glucagon, and insulin for the animals given the test compound are compared by standard statistical methods to the blood concentrations for the control group. Inhibition is demonstrated by a significant decrease in hormone concentration.

The results of the testing of the peptides of Examples 2(b), 4(b) and 6(b) are set forth below: 8 5 0 0 Compound Dose (u.q/kq) GH . Ka/ka). Insulin pll/ml Glucagon (pq/ml) A Control 508 i 13, 298 γ 33 61 i S Example 2(b) 100 181 i 47T 154 - 39T 22 - 7 * B Control ___ 234 ί 36 187 t 21 41 ί 5 Example 2(b) 400 67 - 13* 70 - 23* 9-1* E Control 354 ί 57 336 ί 31 62 j 10 Example 4(b) 200 81 ί 6* 128 1 33* 41 i 14 F Control 363 ί 74 75 t S 162 ΐ 29 Example 5(b) 100 83 ί 16* 60 Ϊ 10 55 ί 12* t = p<0.05 * =: p<0.01 The ability of cqmpounds of this invention to provide prolonged inhibition of growth hormone can be demonstrated in the following test procedure; Albino male rats are arranged in tuo groups (nine rats per group). One group received a subcutaneous (S.C.) injection of the test compound (typically 1 mg/kg.) in saline, and the other group received a S.C. injection of saline alone (control). Tuenty minutes before the end of the test period (tuo to four hours after injection) each animal is given an I.P injection of sodium pentobarbital, mg/kg. At the end of the experiment, blood samples are taken from each animal by cardiac puncture, and each sample is mixed uith Trasylol-EDTA. An aliquot of each sample is assayed for GH by radioimmunoassay. The blood grouth hormone concentrations for the animals receiving the test compound are compared by standard statistical methods to the grouth hormone concentrations for the - 27 control animals. Compounds considered long-acting produce a significant decrease in blood growth hormone concentration at a dose of 1 mg/kg. at least two hours after injection.

Uhen tested in the above-described test procedure, the compound of Example 2(b) gave the following test results: Exp. Compound Dose (ug/kq.) Time (hour) Plasma GH ,(,09/.^,.-).. C Control 2 213 ί 14 10 Example 2(b) 1000 2 89 ί 5** Control 4 182 χ 12 Example 2(b) 1000 4 101 ί 13** D Control 5 337 ί 36, Example 2(b) 1DD0 5 230 ί 31* 15 Control 6 234 ί 24 Example 2(b) 1000 6 172 ί 34 ** = p<0.001 f = p<0.05 The results demonstrate that the compound of Example 20 2(b) shows prolonged inhibition of growth hormone.

Claims (11)

1. A compound of ths formula: 5 .....— —------- — — — — — 1 · S in, CHI 2 j 2 X-CHC0-X 1 -X 2 -PhB-Phe-X 3 -Lys-Thr-Phs-Thr-X 4 -NH-CH-C00H (I) or SH SH I I CH- CHI 2 | 2 X-CHCO-X 1 -X 2 -Pbe-Phe-X 3 -Lys-Thr-Phe-Thr-X 4 -NH-CH-COOH (II) wherein: X is H, ¢^-, H-Ala-Gly-NH-, Η-Ala-D-Ala-NH-, H-Gly-Gly-Gly-NH-, acetyl-ΝΗ- or b^nzoyl-NH-; fe is His or Arg; x 2 is His, Glu, Tyr, Trp or Phe X 3 is Trp, D-Trp or 6-F- D-Trp; X 4 is a D-a-amino S acid; | CH, I 2 the residue X-CHCD- has the L-configuration; and the S f H 2 residue-NH-CH-CDOH has either the L- or D-configuration or a non-toxic, pharmaceutically acceptable acid addition salt thereof.

2. A compound as claimed in claim 1 wherein is D-Pro, D-Ala, D-Val, D-Leu, D-isoLau, D-Ser, D-Thr, D-Asp, D-Glu, D-Lys, D-Arg, D-Asn, D-His, D-Trp, D-Phe or D-Tyr.

3. A compound as claimed in claim 1 or claim 2 wherein - 29 - X is NHg.

4. A compound as claimed in any one of claims 1 to 3 wherein Χ^ is D-Trp.

5. A compound as claimed in any one of claims 1 to 4 wherein Xg is His or Glu.

6. L-Cys teiny 1-L-arginy 1-L-histi dyl-L-pheny lalany 1-Lpheny lalany 1-D-tryp top hy 1-L-ly syl-L-threony 1-L-pheny lalany 1L-threonyl-D-tyrosyl-L-cysteine cyclic (1—>12) disulfide.

7. L-Cyetein y1-L-arginy1-L-histidy1-L-phenylalany1-Lpheny lalanyl-D-tryp top hyl-L-lysyl-L-threonyl-L-phenylalany1-L-threony1-D-tyrosy1-L-cysteine.

8. L-Cysteinyl-L-arginyl-L-a-glutamyl-L-phenylalanyl-Lphenylalany1-D-tryptophy1-L-lysyl-L-threony1-L-pheny1alanyl-L-threonyl-D-tyrosyl-L-cysteine cyclic (1—>12) disulfide.

9. L-Cysteinyl-L-arginyl-L-a-glutamyl-L-phenylalanyl-Lphenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-D-tyrosy1-L-cysteine.

10. L-Cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl-Lphenylaianyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-D-seryl-L-cysteine cyclic (1—»12) di20 - 30 sulfide.

11. L-Cysteinyl-L-histidyl-L-histidyl-L-phenylalanyl-Lphenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-D-seryl-L-cysteine. 5 12. A compound as claimed in any one of claims 1 to 11 uhich is in the form of a pharmaceutically acceptable acid addition salt from an acid selected from hydrochloric, hydrobromic, sulphuric, phosphoric, polyphosphoric, maleic, citric, benzoic, succinic, malonic and ascorbic. 10 13. A compound having the amino acid sequence as defined in any one of claims 1 to 11 carrying at least one protecting group for a side chain group and/or terminal aamino group uhen present and/or terminal carboxy group. 14. A compound having the formula 15. ΐ' 1 Γ 2 CH„ CH„ I 2 | 2 Y-fcHCD-Y 1 -Y 2 -Phe-Phe-X 3 -Y 3 -Y 4 -Phe-Y 4 -Y 5 -NHCHC00R g (III) uherein Rg represents a carboxy protecting group or a polystyrene resin support linked via a methylene group; the residue Y-CHiCH^SZ^)CQ has the L-configuration; the 20 residue -NHCH (CH 2 SZ 2 ) COORg has either the L- or D-configuration; X 3 represents Trp, D-Trp or 6-F-D-Trp; Y is H, R-NH-, R-Ala-Gly-ΝΗ-, R-Gly-Gly-Gly-NE- r R-Ala-D-Ala-NH-, acety1-NH- or benzoyl-NH-; Y 1 is HisCRp or Arg(R^); Y 2 is HisiR^, G1u(R 2 ), Tyr(R 3 ), Trp, or Phe; Y 3 is Lys(R 4 ); Y 4 is Thr(Rg); Yg is an D-a-amino acid residue in which any protectable side chain group is protected; Z^ and Z 2 are each sulphydryl protecting groups or hydrogen or Ty and Z 2 together represent a direct bond between sulphur atoms; and R is an α-amino protecting group and R 1 , R 2 , Rj, R^ and Rg are each independently a side chain protecting group. 15. A compound of formula III as claimed in claim 14 wherein Zy and Z 2 are benzyl, 3,4-dimethylbenzyl, pmethoxybenzyl, p-chlorobenzyl, p-nitrobenzyl, trityl, benzyloxycarbonyl, benzhydryl, E.-methoxybenzylcarbonyl, benzylthiomethyl, ethylcarbamoyl, thioethyl, tetrahydropyranyl, acetamidomethyl, benzoyl or s-sulfonic salt. 16. A compound of formula III as claimed in claim 14 wherein Ty and Z 2 represent p-methoxybenzyl. 17. A compound of formula IV: S-Z CH, , 48500 uherein - oz XgisTrp, D-Trp, or 6-F-D-Trp; Y is K, R-NH-, R-Ala-GLy-NH-, R-Gly-Gly-Gly-ΚΗ-, R-Ala-D-Ala NH-, Acetyl-NH- or benzoyl-t®-; 5 Y. ] is HisiRp or Arg^); Y 2 is His(Rp, G1u(R 2 ), Tyr(R 3 ), Trp, ar Phe; Y 3 is Lys(R 4 ); Y 4 is Thr(Rg); Yg is D-Pro, D-Ala, D-Ual, D-Leu, D-isoLeu, D-Ser(Rg) 10 D-Thr(Rg), D-Asp(R 2 ), D-Glu(R 2 ), D-Lys(R 4 ), D-Arg(R.j) D-Asn, D-His(Rp, D-Trp, D-Phe, or D-Tyr(R 3 ); and Z is the £-methoxybenzyl protecting group; uherein R is an α-amino protecting group and R^, R 2 , R 3 , R^ and Rg are each, independently, a side-chain protecting 15 group; the residue YCH(CH 2 SZ)CO- is in the L-configuration and the residue -NHCH(CH 2 SZ)C00H is in either the L- or D configuration. 18. 20 IB. A compound of formula III as claimed in any one of claims 14 to 16 uherein Yg is D-Pro, D-Ala, D-Ual, DLeu, D-isoLeu, D-Ser(Rg), D-Thr(Rg), D-Asp(R 2 ), D-Glu(R 2 ), D-Lys(Rp, D-Arg(Rp, D-Asn, D-His(Rp, D-Trp, D-Phe or D-Tyr(Rg). 23 19. fl compound of formula III or IU as claimed'in any one of claims 14 to 18 uherein R represents formyl, trifluoroacetyl, phthalyl, tosyl, o-nitrophenylsulfenyl, benzyloxy48500 - 33 carbonyl, p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, _t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, amyloxycarbonyl, cyclopentyloxycarbonyl, 5 adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, trityl or trimethylsilyl. 20. A compound of formula III or IV as claimed in any one of claims 14 to 19 wherein is tosyl, benzyloxycarbonyl, adamantyloxycarbonyl, or £-butyloxycarbonyl. 10 19. 21. A compound of formula III or IV as claimed in any one of claims 14 to 20 wherein Rj and/or Rg is benzyl. 20. 22. A compound of formula III or IV as claimed in any one of claims 14 to 21 wherein Rg is 2,6-dichlorobenzyl. 21. 23. A compound of formula III or IV as claimed in any one 15 of claims 14 to 22 wherein R^ is tosyl, t_-amyloxycarbonyl, t_-butyloxycarbonyl, diisopropyloxycarbonyl, benzyloxycarbonyl, halobenzyloxycarbonyl or nitrobenzyloxycarbonyl. 22. 24. N 01 -_t-Butyloxycarbonyl-S-p-methcixybenzyl-L-cysteinyl-I?tosyl-L-arginy 1-N 1 ' 11 - to syl-L-histidy 1-L-pheny lalany 1-Lc 20 phanylalanyl-D-tryptophyl-N -2-chlorobsnzyloxycarbonyl-Llysyl-O-benzyl-L-threonyl-L-phenylalanyl-O-benzyl-L-threonyl-0-2,6-dichlorobenzyl-D-tyrosyl-S-p-methoxybenzyl-Lcysteinyl-hydroxymethyl-polystyrene ester. - 34 2 5. N a -_t-Butyloxycarbonyl-S-p-irethoxyhsnzyl-Ij-CYSteinyl-N^tosyl-L-arginyl-y-benzyl-L-glutamyl-L-phenylalanyl-L¢. pheny lal any 1-0- tryptophyl-Ρ) - 2-chloro benzyloxy carbonyl-Llysyl-O-bsnzyl-L-threonyl-L-phenylalanyl-O-benzyl-L-threo5 nyl-Q-2,6-dichlorobenzyl-D-ftyrosyl-S-p-methoxybenzyl-Lcysteinyl-hy droxymethy 1 polystyrene ester. 26. N a -±-Butyl02^carbonyl-S-p-methax^3enzyl-L-cysteinyl-N lm tosyl-L-his tidy l-N ln, -tosyl-L-histidyl-L-phenylalanyl-L£ phenylalanyl-D-tryptophyl-N -2-chlorobenzyloxycarbonyl10 L-lysyl-O-benzyl-L-threonyl-L-phenylalanyl-O-benzyl-Lthreonyl-O-benzyl-Q-seryl-S-p-methoxybenzyl-L-cysteinyl -hydroxymethyl polystyrene ester. 27. A process for preparing a compound of.formula I or H as defined in Claim 1 which comprises deprotecting a canpound.as claimed in claim 15 13 or any one of claims 14 to 23 wherein Rg is a carboxy protecting group, if desired isolating as the free base or pharmaceutically acceptable acid addition salt. 28. A process for preparing a compound of formula I or II as defined m claim 1 uhich comprises removing all the pro20 tecting groups and the po.ystyrene resin support uhen present from a compound of formula III or IV as defined in any one of claims 14 to 23, if desired oxidising a compound of formula II or reducing a compound of formula I produced to give the cyclic or open chain form, and further if 23. 25 desired isolating as the free base or a pharmaceutically acceptable acid addition salt. - 35 29. A process as claimed in claim 28 wherein the polystyrene resin support and/or protecting groups are removed by treatment uith hydrogen fluoride in the presence of anisole. 5 30. A process for preparing a compound of formula I as defined in claim 1 uhich comprises oxidising a corresponding compound of formula II as defined in claim 1. 31. A process as claimed in any one of claims 28 to 30 in uhich the oxidation is effected using oxygen or potass10 ium ferricyanide. 32. A process for preparing a compound of formula III or IV as claimed in any one of claims 14 to 23 uherein and Z 2 are protecting groups and Rg represents a polystyrene resin support linked via a methylene group uhich comprises 15 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. 33. A process for preparing a compound of formula III as 20 claimed in any one of claims 14 to 17 uherein Z^ and Z 2 are protecting groups and Rg represents a carboxy protecting group, uhich comprises coupling the requisite amino acids, protected and/or activated as necessary, or groups of amino acids to give the desired sequence. - 36 34. A process for preparing a compound of formula IV as defined in claim 17 substantially as hereinbefore described uith reference to any one of Examples 1, 3 and 5 35. A process for preparing a compound of formula II as 5 defined in claim 1 substantially as hereinbefore described uith reference to any one of Examples 2a, 4a and 6a. 36. A process for preparing a compound of formula 1 as defined in claim 1 substantially as hereinbefore described uith reference to Example 2b, 4b and 6b. 10 37. A compound of formula I or II as defined in claim 1 whenever prepared by a process as claimed in any one of claims 27 to 31, 35 and 36. 38. A compound of formula III as defined in claim 14 uhenever prepared by a process as claimed in claim 32 or 15 claim 33. 39. A compound of formula IV as defined in claim 17 uhenever prepared by a process as claimed in claims 32 or claim 34. 40. A pharmaceutical composition comprising a compound of 20 formula I or II as defined in any one of Claims 1 to 11 and 37, or a pharmaceutically acceptable acid addition salt thereof, in conjunction uith a pharmaceutically acceptable carrier. 41. A pharmaceutical composition as claimed in claim 40 uhen in unit dosage form. 42. A compound of formula I or XI as defined in any one of Claims 1 to 11 for use in the treatment of diabetes or hyperinsulinemia.