IE47454B1 - Somatostatin analogs - Google Patents

Description

This invention relates to somatostatin analogs which contain certain 0-amino acid residues. Wore particularly this invention relates to somatostatin analogs uhich g contain, in addition to a D-Trp residue, glycyl or JL 2 D-amino acid residues in positions 4 and 5. The Η-Ala-Gly residues may either be present or replaced uith Η,Η-Gly, H-L-Ala Gly, H-IrAla-IrAla or H-Gly-Gly-Gly.

This invention also relates to processes for the preparation of such compounds, pharmaceutical compositions containing them and intermediates for preparing them.

The cyclic somatotropin-release inhibiting factor (SRIF), known as somatostatin, has been shown [Brazeau et ai., Science, 179, 77 (1973)] to have the following structure: all amino acids being of the natural or L configuration. Several methods for synthesizing somatostatin have bee.n reported in the literature including the solid phase method of Rivier, 3. Am. Chem. Soc., 96., 2986 (1974), and the solution methods of Sarantakis et al., Biochemical 8io'physical Research Communications, .54, 234 (1973), and Immer et al., Helv. Chim. Acta, 57, 730 (1974); and there is* *much on-going peptide research whose goal is to enhance somatostatin's pharmacological activity by synthetically modifying its structure.

The present invention provides novel analogs of 1 2 somatostatin wherein the Η-Ala -Gly residues may either - 3 be present or are replaced uith H, H-Gly, H-ptSla-L-Ala, or 8 8 H-Gly-Gly-Gly; the L-Trp residue is replaced uith D-Trp ; and the L-Lys^-L-flsn5 residues are replaced either uith Gly or a residue derived from a specified D amino acid.

These compounds inhibit the release of pituitary grouth hormone , glucagon and insulin.

Accordingly this invention provides compounds of formula SA X1-L-Cys-X2-X3-L-Phe-L-Phe-D-Trp-L-LysL-Thr-L-Phe-L-Thr-L-Ser-L-Cys-OH Ia (i) uherein A is hydrogen or the tuo A groups from a direct bond betueen the sulfur atoms; X is H, H-Gly, H-L-Ala-Gly, 3 H-L-Ala-L-Ala, or H-Gly-Gly-Gly? X and X may be the same or different and are chosen from Gly, D-Leu, D-Phe, O-Tyr, D-Trp, D-I*let, D-His, D-Arg, D-Lys, D-Ser, D-Asp, or D-Asn, uith the proviso that both may not simultaneously be Gly; and the pharmacologically acceptable addition salts thereof. 3 Preferably X and X are selected from D-Arg, D-His, D-Trp and D-Tyr. Preferably X1 is H.

The tangible embodiments of the invention possess the inherent physical properties of being uhite to light tan coloured solids, are substantially insoluble in chloroform, benzene, but' exhibit bolubility in water and aqueous acid solutions such as hydrochloric and acetic.

The compositions of the invention display no clearly discernible melting points and may be purified by, for example, chromatographic means. Hydrolysis of the compositions of the invention in, for example, 4 N methanesulfonic acid allous determination of their amino acid content,. uhich is consistent uith the structures as hereinbefor^'se.^, forth. - 4 The tangible embodiments of the invention possess the applied use characteristic of inhibiting the release of hormones somatotropin, glucagon, and insulin as evidenced by standard pharmacological test procedures. g The invention sought to be patented in its first subgeneric aspect resides in the concept of a chemical compound of the structure: SA H-L-Cys-X2-X3-L-Phe-L-Phe-D-Trp-L-LysL-Thr-L-Phe-I_-Thr-L-Ser-L-Cys-OH uherein A is hydrogen or the tuo A groups form a direct bond between the sulfur atoms; X2 and XJ may be the same or different and are chosen from Gly, D-Leu, D-Phe, D-Tyr, D-Trp, D-flet, D-His, Ο-Arg, D-Lys, D-Ser, D-Asp, or D-Asn,uith the proviso that both may not simultaneously be Gly; and the pharmacologically acceptable addition salts thereof.

The invention sought to be patented in its second subgeneric aspect resides in the concept of a chemical compound of the structure: SA H-l-Cys-X4-X5-L-Phe-L-Phe-0-Trp-L~LysL-Thr-L-Phe-L-Thr-L-Ser-L-Cys-OH ^A uherein A is hydrogen or the tuo A groups form a direct bond between the sulfur atoms; X4 and X3 may be the same or different and are chosen from D-Tyr, D-Trp, D-His, or D-Arg; and the pharmacologically acceptable addition salts thereof. - 5 The invention sought to be patented in a specific aspect resides in the concept of the chemical compound which is: H-l-Cys-D-Arg-D-Tyr-L-Phe-L-Phe-D-TrpI S-----g L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L-iys-OH The invention sought to be patented in a second specific aspect resides in the concept of the chemical compound which is: H-I_-Cys-D-Tyr-D-Trp-L-Phe-L-Phe-D-TrpI—-S L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L-iys-QH The invention sought to be patented in a third specific aspect resides in the concept of the chemical compound uhioh is: H-L-Cys-O-His-D-Tyr-L-Phe-L-Phe-D-Trpi------g L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L-liys-OH The polypeptide final products and their requisite intermediates may be prepared by the well known solid phase method as described by, for example, Merrified, 3. Am. Chem. Soc., 85, 2149 (1963).

Thus this invention also provides intermediates for the compounds of formula I, which intermediates have the peptide sequence of the compounds of formula I, wherein the C-terminal cysteine residue is bonded to a polystyrene resin support, the terminal amino group is- 6 protected by an α-amino protecting group, and uhere necessary the side chain groups on the amino acid residues are protected.

In performing solid phase synthesis as applied to the compounds of this invention, α-amino and sulfhydryl protected cysteine may be first attached to a chloromethylated polystyrene resin folloued by removal of the α-amino protecting group uith trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or HCl in dioxane. The deprotection is preferably 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, 1_, 72-75 (Academic Press, 1955). After removal of the α-amino protecting group, the next desired protected amino acids are coupled individually to the resin supported sequence, seriatim. Alternatively, small peptide fragments may be prepared by, for example, 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 tuo solvents. The success of each coupling reaction at each stage of the synthesis may be 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 reported before the α-amino protecting group is removed for introduction of the next amino acid or amino acid sequence.

The preferred coupling reagents are 1-hydroxybenzotriazole and diisaprapylcarbodiimide; other such reagents uill be familiar to those skilled in the art.

Accordingly this invention provides a process for preparing the above mentioned intermediates for compounds - 7 47434 of formula I uhich comprises coupling, under solid phase peptide synthesis conditions, ths requisite suitably protected and/or activated amino acids in the desired order to a polystyrene resin support.

After the desired amino acid sequence has been synthesized, the polypeptide is deprotected -and removed fran the resin support by treatment uith, for example, hydrogen fluoride and anisole to obtain the fully deprotected linear polypeptide. The cyclic disulfide may be produced by air oxidation or use of potassium ferricyanide.

Accordingly this invention also provides a process for preparing a compound of formula X1-L-Cys-X2-X3-L-Phe-L-Phe-D-Trp-L-Lys έ-5 L-Thr-L-Phe-L-Thr-L-Ser-L-iys-OH (la) 3 uherein X , X and X are as defined in Claim 1 uhich comprises oxidising a corresponding compound of formula X1-L-Cys-X2-X3-L-Phe-L-Phe-D-Trp-L-Lys SH SH 1 Z V -L-Thr-L-Phe-L-Thr-L-Ser-L-Cys-OH (lb) and if desired isolating the compound of formula la produced as a pharmacologically acceptable addition salt.

Non-toxic addition salts of the linear and cyclic polypeptides are produced by methods uell knoun in the art frcm, for exemple, hydrochloric, hydrobromic, sulfuric, Ehosjhanic, polyphosphoric, maleic,aceric, citric, benzoic, succinic, malonic or ascorbic acid. The acetic acid salt is preferred.

The protecting groups employed throughout the solid phase synthesis are uell knoun to the art. The a-amino - θ protecting groups employed with each amino acid introduced in sequence of the ultimate polypeptide are of the (1) acyl type protecting groups illustrated by the following: formyl, trifluoroacetyl, phthalyl, £-toluenesulfonyl (tosyl), nitrophenylsulfenyl; (2) aromatic urethane type protecting groups illustrated by benzyloxycarbonyl and substituted benzyloxycarbonyl such as £-chlorobenzyloxycarbonyl, £-nitrobenzyloxycarbonyl; (3) aliphatic'urethane protecting groups illustrated by tert-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxyoarbonyl, allyioxycarbonyl, 2,2,2trichloroethoxycarbonyl, amyloxycarbonyl; (4) eycloalkyl urethane type protecting groups illustrated by cyclopentyloxycarbonyl, adamantyloxycarbonyl, 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-butyioxycarbonyl. The side chain nitrogen atoms of arginine, denoted N^n ar8 protected by a group which may be nitro, tosyl, benzyloxycarbonyl, adamantyloxycarbonyl or tertbutyloxycarbonyl, preferably the tosyl group.

Protection for the side chain amino group of lysine, may be by, for exanple, tosyl, t-amyloxycarbonyl, t-butyloxycarbonyl, diisopropyloxycarbonyl, benzyloxycarbonyl, halobenzyloxycarbonyl or nitrobenzyloxycarbonyl; the 2chlorobenzyloxycarbonyl group being preferred. Protection for the hydroxyl group of threonine and serine may be with the acetyl, benzoyl, tert-butyl, benzyl. The benzyl group is preferred for this purpose.

The protecting group for the sulfhydryl group of the cysteinyl amino acid residue is , for example, benzyl; substituted benzyl wherein the substituent is at least one of methyl, methoxy, nitro cr halo (e.g. 3,4-dimethylbenzyl, £-methoxybenzyl, £chlorobenzyl, £-nitrobenzyl ); trityl, benzyloxy47454 - 9 carbonyl, benzhydryl, £-methoxybenzyloxycarbonyl, benzylthiomethyl, ethylcarbamoyl, thioethyl, tetrahydropyranyl, acetamidomethyl, benzoyl and s-sulfonate salt; the £-methoxybenzyl group being preferred. The demonstrated pharmacological activity of the peptides of the invention characterizes the compounds as useful in the treatment of acromegaly and juvenile and adult-onset diabetes in the same manner as somatostatin itself. Administration of the peptides may be by conventional routes common to somatostatin and related polypeptides, under the guidance of a physician in an amount dictated by the extent of the dysfunction as determined by the physician. The compounds may be administered alone or in conjunction uith conventional pharmaceutically acceptable carriers and adjuvants, if desired, in unit dosage form.

Accordingly, this invention also provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable addition salt thereof and a pharmaceutically acceptable carrier. - 10 The folio,‘ring Exawles 1-10, 12 and 15 further illustrate the invention. Examples 11, 13 and 14 are oanparative Examples.

Example 1 N-q-Tert-Butyloxycarbonyl-S-p-Wethoxybenzy1-L-Cysteinyll\l9n-Tosyl-0-Arginyl-0-2,6-Dichlorobenzyl-D-Tyrosyl-LPhenylalanyl-L-Phenylalanyl-D-Tryptaphyl-W&-2-Chlorobenzyloxycarbonyl-L-Lysyl-O-Benzyl-L-Threonyl-L-Phenylalanyl-0-8enzyl-L-Threonyl-0-Benzy1-L-Seryl-S-pWethoxybenzyl-L-Cysteinyl Hydroxymethyl Polystyrene Ohloromethylated polystyrene resin (Lab Systems, Inc.) in vhich. the degree of cross-linking by divinylbenzene is IS, was esterified with Boc-Cys (SPIBzl)-OH according to Gisin, Helu. Chim. Acta, 56, 1976 (1973). The polystyrene resin ester was treated according to Schedule A for the incorporation of Boc-Ser (Bzl)-OH, Boc-Thr(Bzl)-0H, Boc-Phe-OH, Boc-Thr(Bzl)-OH, 8oc-Lye(ClZ)-0H, Boc-D-Trp-OH, Boc-Phe-OH, Boc-Phe-OH, Boc-O-Tyr(Cl28zl)-0H, Boc-D-Arg(Tos)-OH and Bac-Cys(SWBzl)OH to afford the title peptidoresin.

Schedule A 1. Wash with CB^C^ x 3. 2. Treat with TFAS3LC1- (1:1 v/v) containing 5S (v/v) 1,2-ethanedithiol for 5 minutes. 3. Treat as in 2 for 25 minutes. 4. Uash uith CH2C12 x 3.

. Uash uith DWF. 6. Treat with 12% (v/v) triethylamine in DMF x 2 fer 3 minutes. 7. Uash uith DFIF.

S. Uash uith CHgClg x 3. 9. Treat uith 4 equivalents of the corresponding amino acid derivative in CHgCl^DPlF and stir for 5 minutes.

. Add in two portions 5 equivalents of diiscprcpylcarboniimide dissolved in CH2C12 and over a period of 30 minutes. Reaction time 6 hours. 474 54 - 11 11. Uash with DHF x 3. 12. Uash with CH^Cl^ x 3. 13. Test ninhydrin reaction according to Kaiser et al., Annal. Biochem., 34, 595 (1970). In case of incomplete reaction, repeat lines 9 to 13 as above.

Example 2 L-Cysteinyl-D-Arginyl-O-Tyrosy1-L-Phenylalanyl-L-Phenylalanyl- D-Tryptophyl-L-Lysyl-L-Threonyl-L-Phenylalanyl-LThreonyl-L-Seryl-L-Cystein (Cyclic 1-12) Disulfide (A) The peptidoresin of the previous example (10.2 g.) was mixed with anisole (20 ml.) and treated with liquid HF (200 ml.) for 45 minutes in an ice-bath. The excess HF uas removed in vacuo and the residue was extracted with 25% (v/z) aqueous AcOH. lhe aqueous solution was extracted with diethyl ether and then the aqueous phase uas neutralized uith dilute NH^OH to pH 7 and left to stand for 2 days in the open air. The mixture uas acidified uith glacial AcOH to pH 5 and lyophilyzed. Tha crude product uas applied onto a column of Sephadex (Registered Trade Mark) G-15 and eluted with 10% (v/v) aqueous AcCH. Fractions of 5.5 ml. were collected and the material which emerged betueen fractions 96 to 126 uas collected to yield 260 mg. of L-cysteinyl-D-arginyl-Otyrosyl-L-phenylalanyl-L-phenylalanyl-O-tryptophyl-Llysy1-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-Lcysteine (cyclic 1-12) disulfide as the diacetic acid salt.

Rf (BUA, 4:5:1 v/v) 0.52 Rp (BUAP, 30:24:6:20 v/v) 0.77 BUA = n-Butanol:Whter:Acetic Acid BUAP = n-Butanol:Water;Acetic Acid:Pyridiras flmino Acid Analysis: Thr (2) 1.76, Ser (1) 0.88, Tyr (1) 0.95, Phe (3) 3, Lys (l) 1.09, Arg (1) 0.89, Trp and Cys not determined.

Example 3 The in vivo pharmacological activity of the compound prepared in Example 2 (denoted compound A) uas established by the following procedures uith the indicated results: Suppression of Grouth Hormone A subcutaneous (sc) injection of peptide solubilized or suspended in physiological saline, is given to Charles River, (. Trade Hark) CD nonfasted male rats: Matched saline control solution sc injected rats serve as control animals so that every experimental rat is paired uith a control rat.

The rats are kept in separate cages and 20 minutes before the end of the test time period they are given an intraperitanaal (i.p) injection of Nentoutal (registered Trade Mark) at a dose of 50 mg/kg. Blood samples are obtained by cardiac puncture and the plasma separated for the radioimmunoassay of grouth hormone (GH) concentration (ng/ml.). Time periods after injection of 2, 4, 5 and 6 hours are generally used to test the duration of the activity of the peptide to suppress circulating peripheral GH levels. Comparisons between control and experimental GH values at each time are evaluated by the Student t test and statistical significance (p) at the 0.05 level or lower is used as the index of activity.

Compound 2 Hr.

(Dose) _ Control 171^20 A(lmg/kg.) 17^8 p= Hr. 5 Hr. 6 Hr. 73Ϊ21 6θίΐ1 S2—21 23^8 32^7 74±14 p=<0.05 p=<0.05 p=>0.05 474S4 Suppression of Grouth Hormone, Glucagon and Insulin Albino male rats are arranged in three groups (nine rats/group) and injected i.p. uith Nembutal at 50 mg/kg. Fifteen minutes after the Nembutal injection they are injected s.c. according to group uith (a) test compound, typically 10-2000 pg/kg.; (b) SRIF 2OQ μg/kg.; or (c) physiological saline. Ten 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 into Trasylol-EDTA. Appropriate aliquots are then assayed for grouth hormone, glucagon and insulin. An active compound is one uhich significantly changes the plasma level of any o.f these hormones from that of the saline controls. Comparisons betueen control and experimental values are statistically evaluated by the analysis of variants method and statistical significance (p) at 0.05 or louer is used as the index of activity.

Compound (Dose pq/kq) GH(nq/ml.) Insulin (pU/ml.) Glucagon (pq/ml.) Control 164^38 213-12 37Ϊ8 A(2000) 20±2 97^18 8±5 p=<0.001 p=<0.01 p=<0.01 Control ...... 221-9 71±6 A (100) —- 127^20 24Ϊ4 p=<0.01 p=<0.01 Control 163±29 278-32 69-13 A (10) 11-3 312-52 39-7 p=<0.01 p=>0.05 p=<0.05 - 14 Example 4 Tert-Butyloxycarbonyl-S-p-Flethoxybenzyl-L-Cysteinyl-O-?, 6-Dichlorobenzyl-D-Tyrosyl-D-Tryptophyl-LPhenylalanyl-L-Phenylalanyl-D-Tryptophyl-N£-2-Chlorobenzyloxycarbonyl-L-Lysyl-Q-Benzyl-L-Threonyl-L-Phenylalanyl-OBenzyl-L-Seryl-S-p-Methoxybenzyl-L-Cysteinyl Hydroxymet hylpolystyrene Chloromethylated polystyrene resin (Lab Systems Ino.) in which the. degree of cross-linking fcy divinylbenzene is 1% was esterified with BOC-Cys-(S/1Bzl)-0H according to Gisin, ibid. The polystyrene resin ester uas treated according to Schedule A of Example 1 for the incorporation of BOC-Sar(Bzl)OH, B0C-Thr(Bzl)0H, BOC-Phe-OH, B0C-Thr(Bzl)0H, BOC-Lys(Clz)OH, BOC-D-Trp-OH, BOC-Phe-OH, BOC-Phe-OH, BOC-O-Trp-OH, BOC-D-Tyr(Cl2Bzl)OH and BOC-Cys(SNBzl)OH to afford the title peptidoresin.

Example 5 L-Cysteinyl-D-Tyrosyl-D-Tryptophyl-L-Phenylalanyl-LPhenylalanyl-D-Tryptophy1-L-Lysyl-L-Threony1-L-Pheny1alanyl-L-Threonyl-L-Seryl-L-Cysteine Cyclic (1-12)Disulfide (B) The peptidoresin of Example 4 (7.9 g.) uas treated uith liquid HF 050 ml.) in the presence of anisole 05 ml.) in vacuo and an ice bath for 45 minutes. The excess HF uas removed as fast as possible under vacuum (ca. 45 minutes) and the residue uas taken in 25% (v/v) aqueous AcOH, The polymeric support uas filtered off and the filtrate uas uashed with diethyl ether then the aqueous phase was poured into 3.5 1. of deaerated water and the pH uas brought ta 7 uith dilute NH^OH. The disulfhydryl dodecapeptide uas oxidized uith KgFe(CN)g and then the pH uas brought to 5 uith glacial AcOH. The excess oxident uas - 15 removed with ion exchange resin Bio Rad ( Trade Mark) AG 3 and the peptide was absorbed on a Bio Rex ( Trade Mark) 70 resin. Elution uith pyridine buffer pH 7 afforded the crude dodecapeptide (300 mg.).

The crude material (150 mg.) uas chromatographed through Sephadex G 15 (1.5 x 90 cm.) and eluted uith 15% (v/v) aqueous AcCH. The material which emerged in fractions (3.1 ml. each) 49 to 77 uas pooled and lyophilyzsd to yield L-cysteinyl-D-tyrosyl-D-tryptophyl10 L-phenylalanyl-L-phenylalanyl-D-tryptophyl-L-lysyl-Lthreonyl-L-phenylalany1-L-threony1-L-seryl-L-cysteine cyclic (l-12)-disulfide (69 mg.).

Rf (BUA, 4:1:1 (v/v))0.68 Rf (SWAP, 30:24:6:20 (v/v)) 0.86.

Amino Acid Analysis: Thr (2) 2.05, Ser (1) 1.05, Tyr (1) 0.96, Phe (3) 3, Lys (l) 1.04, Cys and Trp not determined.

Example 6 The in vivo pharmacological activity of the compound prepared in Example 5 (denoted compound B) uas established by the procedures set forth in Example 3 uith the indicated results: Suppression of Growth Hormone Compound (Dosb) Control 8 (1 mg/kg.) 2Hr. 199—27 111±14 2 Hr. 303^95 133-34 4 Hr. 161±30 67Ϊ14 5 Hr 15ΒΪ26 151-18 p=<0.01 p=>0.05 p=<0.01 p=>0.05 SuDPression of Growth Hormone, Glucaqon and Insulin Compound (Dose u.q/kq.) Control B (500) GH (nq/ml.) 197*26 38*6 p=<0.01 Insulin (uU/ml.) 166*8 116*8 p=<0.01 Glucagon (pq/rnl.) 50*5 18*4 p=<0.01 Control 171-6 86*6 B (300) 153*9 38*8 p=>0.05 p=<0.01 10 Control 278*26 71*9 B (200) 239-17 44*9 p=>0.05 p=<0.05 Control 171*29 251*50 32*6 B (100) 43*7 170*15 5*1 15 p=<0.01 p=>0.05 p=<0.01 Control 197*26 166*8 50*5 B (10) 125*26 162*13 36*4 p=>0.05 p=>0.05 p=<0.05 Example 7 Tert-Butvloxycarbonyl-S-p-Wethoxybenzy1-L-Cysteinyl-Nln1Tasyl-D-Histidy1-0-2,6-Dichlorabenzyl-D-Tyrosyl-L-Phenyl£ alanyl-L-Phenylalanyl-O-Tryptophyl-W -2-Chlorobenzyloxycarbonyl-L-Lysyl-O-Benzyl-L-Threonyl-L-Phenylalanyl-OBenzy1-L-Threony1-0-Benzy1-L-Seryl-S-p-Msthoxybenzy1-L25 Cysteinyl Hydroxymethyl Polystyrene Ester Chloromethylated polystyrene resin was esterified with B0C-Cys(5flBzl)0H according to Gisin, ibid., and the polymeric ester was treated according to Schedule A of Example 1 for the incorporation of B0C-Ser(Bzl)0H, - 17 BOC—Thr(Bzl)OH, B0C-Phe-0H, B0C-Thr(8zl)0H, BOC-Lys(Clz), BOC-D-Trp-OH, BOC-Phs-OH, BOC-Phe-OH, BOC-D-Tyr(ClBzl)OH, B0C-D-His(Tos)0H, and BOC-Cys(SI*lBzl)OH, to afford the title peptidoresin.

Example 8 L-Cystsinyl-D-Histidyl-D-Tyrosyl-L-Phenylalanyl-L-Phenylalanyl-D-Tryptophyl-L-Lysyl-L-Threonyl-L-Phenylalanyl-LThreonyl-L-Seryl-L-Cysteine Cyclic (1-12)-Disulfide(c) The peptidoresin of Example 7 (9.8 g.) uas mixed uith 18 ml. anisole and the mixture uas treated uith liquid HF in the absence of air and in an ice bath for 60 minutes, after uhich time the excess HF uas removed under vacuo and the residue uas extracted uith 2ΙΊ aqueous AcOH and filtered. The filtrate uas poured into 7 litres of dearated uater and the pH uas adjusted to 7.1 uith NH^OH.

The mixture uas stirred overnight (oxidation uas complete) and then the pH uas adjusted to 5 uith gl. AcOH and the peptide was absorbed onto Anberlite (registered Tracte Mark) CG-50 (II+ form). The peptidic material was eluted with 30% (v/v) aq. AcCH and lyophilized to yield 250 mg. of crude product.

Ths above material uas chromatographed through a column of Sephadex G25 (2.5 x 57 cm.) and eluted uith 10% (v/v) aq. AcOH. The material uhich emerged in fractions (5.4 ml. each) 61-111 uas pooled and lyophilized to yield the title dodecapeptide. TLC, Avioal ( Trade Marie) preooated glass plates, chloroxtolidine spray: Rf (BUA, 4:1:1, v/v) 0.62 Rj, (tertAmOH, P, U, 7:7:6, v/v) 0.80.

Amino Acid Analysis: Thr (2) 1.89, Ser (1) 0.91, Cys (2) 1.52, Tyr (1) 0.95, Phe (3) 3, Lys (l) 1.03, His (1) 0.93 Trp (1) 0.85. - 18 (tert AmOH, P, U = tertiary amyl Alcohol, pyridine water).

The following compounds were prepared in a fashion similar to that of the preceding examples: Example 9 L-Cysteinyl-D-Arginyl-D-Tryptcphyl-L-Phenylalanyl-LPhenylalanyl-D-Tryptophyl-L-Lysyl-L-Threonyl-L-Phenylalanyl-L-Threonyl-L-Seryl-L-Cysteine Cyclic (1-12)Disulfide (D) TLC, Avicel precoated glass plates, chlorox-tolidine 10 spray: Rf (BUA, 4:1:1, v/v) 0.49 Rf (BUAP, 30:24:6:20, v/v) 0.77.

Amino Acid Analysis: Thr (2) 2.04, Ser (l) 1.09, Cys (2) 1.42, Phe (3) 3, Lys (1) 1.04, Trp (1) 0.77, Arg (1) 0.99.

Example 10 L-Cysteinyl-D- Histdyl -D-Histidyl-L-PhenyJ alany1-L-Phenylalany1-D-Tryptophy1-L-Lysy1-L-Threonyl-LPhenylalanyl-L-Threonyl-L-Seryl-L-Cysteine Cyclic (1-12)20 Disulfide (E) TLC, Avicel precoated glass plates, chlorox-tolidine spray: Rf (BUA, 4:1:1, v/v) 0.43 Rj, (BUAP, 30:24:6:20, v/v) 0.67 Amino Acid Analysis: Thr (2) 1.93, Ser (1) 0.91, Cys (2) 1.53, Phe (3) 3, Lys (l) 1.0S, His (2) 1.78.

Example 11 L-Cysteinyl-Q-Tyrosy1-Q-Arginyl-L-Phenylalanyl-L-Phenyl5 alanyl-0-Try pt op hy 1-L-Lysyl-L-Thr eony 1-L-Pheny lalanyl-LThreonyl-L-Seryl-O-Cysteine Cyclic (1-12)-Disulfide (F) TLC, Auicel precoated glass plates, chlorox-tolidine spray: Rf (BUA, 4:1:1, v/v) 0.55 Rp (tert AmOH, U,P, 7:6:7, v/v) 0.81.

Amino Acid Analysis: Thr (2) 1.91, Ser (1) 0.83, Cys (2) 1.45, Phe (3) 3, Tyr (1) 0.89, Lys (1) 1.02, Arg (1) 0.93, Trp (N.D.) Example 12 L-Cysteinyl-D-Leucyl-D-Tyrosyl-L-Phenylalanyl-L-Phenylalanyl-D-Tryptophyl-L-Lysyl-L-Threonyl-L-Phenylalanyl-LThreonyl-L-Seryl-L-CystBire Cyclic (1-12)-Disulfide (G) TLC, Avicel precoated glass plates, chlorox-tolidine spray: Rp (BUA, 4:1:1, v/v) 0.53 Rp (BUAP, 30:24:6:20, v/v) 0.85 Amino Acid Analysis: Thr(2) 1.93, Ser (1) 0.86, Cys (2) 1.28, Leu (1) 0.95, Tyr (1) 0.91, Phe (3), Trp (N.D.) - 20 Example 13 L-Cysteiny1-D-Arginyl-D-Tyrosy1-L-Phenylalanyl-L-Pheny1alany1-D-Tryptophy1-L-Lysy1-L-Threonyl-L-Phenylalanyl-LThreonyl-L-Seryl-O-Cysteine Cyclic (1-12)-Oisulfide (H) TLC, Avicel precoated glass plates, chlorox-tolidine spray: Rf (BUA, 4:1:1, v/v) 0.48 Rf (BUAP, 30:24:6:20, v/v) 0.69 Amino Acid Analysis: Thr (2) 1.79, Ser (1) 0.85, Phe (3) 3, Tyr (1) 0.95, Lys (1) 1.03, Trp (1) 0.69, Arg (1) 0.96, Cys (2) 1.22.

Example 14 L-Cysteinyl-D-Tyrosyl-D-Tryptophyl-L-Phenylalany1-L-Phenylalany1-0-Tryptcphy1-L-Lysy1-L-Threonyl-L15 Phenylalanyl-L-Threonyl-L-Seryl-D-Cysteine Cyclic (1-12)Disulfide (I) TLV, Avicel precoated glass plates, chlorox-tolidine spray: Rp (BUA, 4:1:1, v/v) 0.55 20 Rf (BUAP, 30:24:6:20, υ/ν) 0.87 Amino Acid Analysis: Thr (2) 1.89, Ser (1) 0.86, Cys (2) 1.29, Tyr (l) 1, Phe (3) 3, Lys (1) 1.07, Trp (2) 0.93.

Example 15 L-Cysteiny1-D-Tyrosy1-D-Glutamyl-L-Phenyla.Lanyl-L-Phenylalany1-D-Tryptophyl-L-Lysyl-L-Threonyl-L-Phenylalanyl-LThreonyl-L-Ssryl-L-Cysteine Cyclic (1-12)-Disulfide (0) TLC, Avicel precoated glass plates, chlorox-tolidins spray: Rf (BUA, 4:1:1, v/v) 0.55 Rf (BUAP, 30:24:6:20, v/v) 0.63 Amino Acid Analysis: Thr (2) 1.83, Ser (1) 0.82, Glu (1) 0.9S, Cys (2) 1.33, Tyr (1) 0.95, Phe (3) 3, Lys (1) 1.01, Trp (1) 0.81.

Example 16 Ihe catipounds of Examples 8 through 10, 12, and 15 and conparative Examples, 11, 13 and 14 were assayed far suppression of growth hantcne and far suppression of growth hantcne, glucagon and insulin, according to the procedures set forth in Example 3. The following results uers obtained: Compound Suppression of Grouth Hormone Dose P9/kg. Hours GH ng/ml. Control 2 100±16 C 1000 2 34-1* Control 4 145^35 C 1000 4 33-2* Control 5 161^28 C 1000 5 51-8** - 22 Compound Dose Hours μ9/*<9 GH ng/ml.

Control 6 C 1000 6 Control ----- 7 C 1000 7 Control 8 C 1000 8 Control 2 D 1000 2 Control 4 D 1000 4 Control 2 E 1000 2 Control _____ 4 E 1000 4 Control _____ 2 F 1000 2 Control 4 F 1000 4 Control 2 G 1000 2 Control 4 G 1000 4 202-39 36^3** 105—13 37±5** 175-52 37-6+ 302^67 28^3** 177*50 11*2* 93*17 36*16+ 34*11 45*10 170*23 113*34 133*25 99*11 212*37 84*11* 173*25 94*23+ Compound Dose Hours pg/kg Control 5 G 1000 5 Control 6 G 1000 6 Control 2 H 1000 2 Control 4 H 1000 4 Control 2 I 1000 2 Control 4 I 1000 4 Control 2 J 1000 2 Control 4 J 1000 4 GH ng/ml. 243±44 93±30 109±61 72±68 191±18 75ί4** 169^32 144+21 187ί28 4gi5** 148±23 86±11+ 392-59 311Ϊ68 18S±27 179-29 * p<0.01; **p<0.001; + p<0.05 - 24 Suppression of Growth Hormong> Glucagon.and Insulin Compound Dose ixg/kg. GH μg/ml. INS pU/ml. GLUN pg/ml. Control 258*46 323*28 379*109 5 C 200 76*14* 254*31 84*25+ Control 365*31 379*47 336*51 C 200 109*10* 273*36 184*22+ C 10 149*23* 252*41 165*13* Control ___ 122*26 195*18 99*22 10 D 100 21*2* 35*2* 53*9 Control ___ 190*28 295*38 93*12 E 200 34*3* 182*18+ 29*12* Control 109*21 226*29 40*4 F 100 11*3* 103*24* 24*5+ 15 G 100 29*16+ 218*44 28*16 Control ___ 171*29 224*20 74*4 H 25 23*4* 128*11* 56*6+ Control— ___ 279*53 323*30 42*6 I 200 57*14* 236*27+ 13*2* 20 Control _____ 240*34 211*26 42*7 3 100 128*24+ 263*28 45*4 * p

Claims (32)

1. A compound of formula SA X 1 -L-Cys-X 2 -X 3 -L-Phe-L-Phe-D-Trp-L-LysL-Thr-L-Phe-L-Thr-L-Ser-L-Cys-OH ^A (I) uherein A is hydrogen or the two A groups form a ι direct bond between the sulfur atoms; X is H, H-Gly, H-L-Ala-Gly, H-IrAla-L-Ala, car H-Gly-Gly-Gly; X 2 and X may be the same or different and are chosen from Gly, D-Leu, D-Phe, D-Tyr, D-Trp, D-flet, D-His, D-Arg, D-Lys, D-Ser, Ο-Asp, or D-Asn, uith the proviso that both may not simultaneously be Gly; and the pharmacologically acceptable addition salts thereof. 15

2. A compound as claimed in Claim 1 uherein X is D-Arg, D-His, D-Trp or D-Tyr.

3. A compound as claimed in Claim 1 or Claim 2 uherein X is D-Arg, D-His, D-Trp or D-Tyr,

4. A compound as claimed in any one of Claims 1 to 3 20 uherein X is hydrogen.

5. The compound of formula H-L-Cys-D-Arg-D-Tyr-L-Phe-L-Phe-O-T rp b I L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L-Cys-OH - 26 10

6. The compound of formula H-L-Cys-D-Tyr-D-Trp-L-Phe-L-Phe-D-TrpS-—S L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-l-Cys-OH

7. The compound of formula H-L-Cys-D-His-D-Tyr-L-Phe-L-Phe-D-Trpi--S L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L-iys-OH 3. The compound of formula H-L-Cys-Q-Arg-D-Trp-L-Phe-L-Phe-O-Trp5L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L-Cys-OH

8. 9. The compound of formula H-L-Cys-D-His-O-His-L-Phe-L-Phe-O-Trp-.-s L-Lys-L-Thr-l-Phe-L-Thr-L-Ser-l-liys-OH

9. 10. The compound of formula H-L-Cys-O-Leu-D-Tyr-L-Phe-L-Phe-O-Trp1-------- s 15 L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L-Cys-OH

10. 11. The compound of formula

H-L-Cys-O-Tyr-D-Glu-L-Phe-L-Phe-D-Trp2-----------5

L-Lys-L-Thr-l-Phe-L-Thr-L-Ser-l-Cys-OH 47 454 27 12. A compound as claimed in any one of Claims 1 to 11 which is in the form of the hydrochloric, hydrobromic, sulphuric, phosphoric, polyphasphoric, maleic, acetic, citric, benzoic, succinic, malonic or ascorbic acid addition salt.

13. A modification of a compound of formula I as defined in any one of Claims 1 to 4 in which the C-terminal cysteine residue is bonded to a polystyrene resin support, the terminal amino group is protected by an α-amino protecting group, and where necessary the side chain groups on the amino acid residues are protected.

14. A compound as claimed in Claim 13 in which the side chain amino group of lysine is protected by tosyl, t-amyloxycarbonyl, t-butyloxycarbonyl, diisopropyloxycarbonyl, benzyloxycarbonyl, halobenzyloxycarbonyl or nitrobenxyloxycarbonyl.

15. A compound as claimed in Claim 13 or Claim 14 wherein the hydroxyl group of threorine or serine is protected by an acetyl, benzoyl, tert-butyl or benzyl group.

16. A compound as claimed in any one of Claims 13 to 15 wherein the sulphydryl group of cysteine is protected by benzyl; substituted benzyl wherein the substituent is at least one of methyl, methoxy, nitro or halogen; trityl; benzyloxycarbonyl; benzhydryl; p-methoxybenzyloxycarbonyl; benzylthiomethyl; ethylcarbamoyl; thioethyl; tetrahydropyranyl, acetamidomethyl; benzoyl or s-sulfonate salt.

17. A compound as claimed in any one of Claims 13 to 16 in which the α-amino group is protected by formyl, trifluoroacetyl, phthalyl, p-toluenesulphonyl, 28 4 7454 nitrophenylsulphenyl, benzyloxycarbonyl, p-nitrobenxyloxycarbonyl, tert-butyloxycarbonyl, diisopropyImethoxycarbonyl, isopropyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 5 amyloxycarbonyl, cyclopentyloxycarbonyl, admantyloxy carbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, trityl or trimethylsilyl.

18. A compound as claimed in any one of Claims 13 to 17 uherein the side chain nitrogen atoms of arginine, 10 when present, are protected by nitro, tosyl, benzyloxycarbonyl, adamantyloxycarbonyl or tert-butyloxycarbonyl.

19. N-«-Tert-butyloxycarbonyl-S-p-methoxybenzyl-Lcystainyl-N 9n -tosyl-0-arginy1-0-2,6-dichlorobenzyΙΟ- tyrosy1-L-phenylalanyl-L-phenylalanyl-D-tryptophylN e -2-chlorobenzyloxycarbonyl-L-lysyl-0-benzyl-Lthreony1-L-phenylalany1-0-benzyl-L-threonyl-0-benzy1L-seryl-S-p-mathoxybenzyl-L-cystainyl hydroxymethyl polystyrene ester.

20. Ν-α-Tert-butyloxycarbony1-5-p-methoxybenzyl-Lcysteinyl-0-2,6-dichlarabenzyl-D-tyrosyl-D-tryptophyl L-phenylalanyl-L-phenylalanyl-O-tryptaphyl- Ν ε -2chlorabenzyloxycarbonyl-L-lysyl-O-benzyl-L-threonylL-phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L-serylS-p-methoxybenzyl-L-cysteinyl hydroxymethylpclystyrene ester.

21. N-g-Tert-butyloxycarbonyl-S-p-methoxybenzyl-Lcysteinyl-l\l ln, -tosyl-D-histidyl-0-2,6-dichlorobenzylD-tyrosyl-L-phenylalany1-L-phenylalany1-0-tryptophylβ 30 N -2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-Lthreony1-L-phenylalany1-0-benzyl-L-threonyl-O-benzyl47454 -29L-seryl-S-p-methoxybenzy1-L-cysteinyl hydroxymethyl polystyrene ester.

22. A process far preparing a compound of formula X 1 -L-Cys-X 2 -X 3 -L-Phe-L-Phe-D-Trp-L-Lys5---S I L-Thr-L-Phe-L-Thr-L-Ser-L-Cys-OH (la) 11. 12 3 wherein X , X and X are as defined in Claim 1 which comprises oxidising a corresponding compound of formula X 1 -L-Cys-X 2 -X 3 -L-Phe-L-Phe-D-Trp-L-Lys!oH SH I L-Thr-L-Phe-L-Thr-L-Ser-L-Cys-OH (lb) and if desired isolating the compound of formula Ia produced as a pharmacologically acceptable addition salt thereof.

23. A process as claimed in Claim 22 wherein oxidation is effected by using oxygen or potassium ferricyanide.

24. A process for preparing a compound of formula X 1 -L-Cys-X 2 -X 3 -L-Phe-L-Phe-O-Trp-L-LysSH SH L-Thr-L-Phe-L-Thr-L-Ser-L-iJys-OH (lb) 1 2 3 wherein X , X and X are as defined in Claim 1 which emprises removing the protecting groups ard the polystyrene resin support frcm a carpourd as claimed in any one of Claims 13 to 18.

25. A process for preparing a compound as claimed in any one of Claims 13 to 1S which comprises coupling, 47434 - 30 under solid phase peptide synthesis conditions, the requisite suitably protected and/or activated amino acids in the desired order to a polystyrene resin support.

26. A process as claimed in Claim 22 or Claim 23 in which the compound of formula lb as defined therein is prepared by a process as claimed in Claim 24.

27. a process far preparing a ooirpound of formula I as claimed in claim 1 substantially as hereinbefore described with reference to any one of Examples 2, 5, 8, 9, 10, 12 and 15.

28. A process for preparing a Claim 13 substantially as with reference to any one compound as claimed in hereinbefore described of Examples 1, 4 and 7

29. A ocnpound of formula I as claimed in claim 1 whenever prepared by a process as claimed in any one of Claims 22, 23, 24, 26 and 27.

30. A compound as claimed in Claim 13 whenever prepared by a process as claimed in Claim 25 or Claim 28.

31. A pharmaceutical composition comprising a compound as claimed in any one of Claims 1 to 12 and a pharmaceutically acceptable carrier.

32. A pharmaceutical composition as claimed in Claim 31 in unit dosage form.