GB1563356A

GB1563356A - Peptides

Info

Publication number: GB1563356A
Application number: GB5203276A
Authority: GB
Original assignee: American Home Products Corp
Current assignee: Wyeth LLC
Priority date: 1975-12-16
Filing date: 1976-12-14
Publication date: 1980-03-26

Description

(54) PEPTIDES (71) We, AMERICAN HOME PRODUCTS CORPORATION, a corporation organised and existing under the laws of the State of Delaware, United States of America, of 685, Third Avenue, New York 10017, New York, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement: The invention relates to cyclic undecapeptides and intermediates therefor including processes for their preparation and pharmaceutical compositions containing the active compounds.

The cyclic somatotropin-release inhibiting factor (SRIF), known as somatostatin, has been shown [Brazeau et al., Science, 179, 77 (1973)] to have the following structure:

where all the amino acid residues have the L-configuration.

Several methods for synthesizing somatostatin have been reported in the literature including the solid phase method of Rivier, J. Am. Chem. Soc., 96, 2986 (1974), and the solution methods of Sarantakis et at., Biochemical Biophysical Research Com- munications, 54, 730 (1974); and there is much peptide research whose goal is to enhance somatostatin's pharmacological activity by synthetically modifying its structure.

The present invention provides novel cyclic analogs of somatostatin wherein its tryptophyl residue may be either of the L or D stereochemical configuration and wherein its Alas, Gly-, Cys and Cysl' residues have been replaced by an amino acid of the general formula H,N--(CH1),~,,-COOH. Replacement of the L-Trp residue in somatostatin by D-Trp is discussed by J. Rivier et at., Biochem, Biophys.

Res. Commun., 65, 746 (1975). Accordingly this invention provides compounds of formula

and the non-toxic salts thereof, wherein (T) represents D-tryptophyl or L-tryptophyl, n is an integer from 3 to 8, R2 and R3 are attached to the nitrogen of the Amino group of lysine and independently represent hydrogen or a protecting group for the side chain amino group, R4, R5 and Rs are the hydrogen atom of the alcoholic hydroxyl group of threonine and serine, or a protecting group therefor, with the provisos (i) that when R4, R5 and R6 are t-butyl then R2 and R3 are other than t-butyloxycarbonyl and (ii) that at least R2 or R3 or the group Rl, R and RG are other than hydrogen.

When R is a protecting group for the e-amino substituent of lysine, examples are benzyloxycarbonyl and substituted benzyloxycarbonyl, said substituent being selected from halo (e.g chloro, bromo, fluoro) and nitro (e.g. 2-chlorobenzyloxy carooiiyl, p-narooe.lzrLuxycarDonyl, ,4-dichiorobenzyloxycarbonyl), tosyl, t-amyloxy- carbonyl, t-butyloxycarbonyl and diisopropylmethoxycarbonyl.

R' is selected from the lysine protecting groups defined by R2.

When R4, R and R6 are protecting groups for the alcoholic hydroxyl group of threonine and serine, examples are lower alkanoyl (e.g. acetyl), benzoyl, tert-butyl, trityl, benzyl and 2,64ichlorobenzyl. The preferred protecting group is benzyl. Preferably n is 6.

The nomenclature used to depict the peptides follows that adopted by Schroder & Lubke, "The Peptides", 1, pp viii-xxix (Academic Press 1965). All chiral amino acid residues identified in formula I and the other formulae hereinafter are of the natural or L-configuration, except for Trp, which is optionally of the L- or configuration.

The invention also provides compounds having the formula R1NH(CH2)11CO-Lys(R2YAsn-Phe-Phe-(T)-Lys(R3)-Thr(R1)-Phe-Thr(R')-Ser(R6)-X (11) and its non-toxic salts, wherein (T), n, R2, R3, R4, R and R6 are as defined above, R1 is hydrogen or an amino protecting group which is removable under conditions that will not remove the protecting groups R2 and R3; X represents OH, NHNH2, lower allryl), --O(benzyl)

Preferably n is 6.

Preferably Rl is hydrogen or t-butyloxycarbonyl, R2 and R3 are 2-chlorobenzyloxycarbonyl and R4, R and R6 are benzyl. The term "lower" as used herein to qualify a group or molecule means the group or molecule contains 1 to 6 carbon atoms.

Among the preferred classes of amino protecting groups covered by Rl are (1) acyl type protecting groups illustrated by the following: formyl, trifluoroacetyl, phthalyl, p-toluenesulfonyl (tosyl), benzenesulfonyl, o-nitrophenylsuifenyl, tritylsulfenyl, o-nitrophenoxyacetyl and tchlorobutyryl; (2) aromatic urethan type protecting groups illustrated by benzyloxycarbonyl and substituted benzyloxycarbonyl such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, l-(pwbiphenylyl)-l-methylethoxycarbonyl, 12,l2 - dimethyl - 3,5 dimethoxybenzyloxycarbonyl and benzhydryloxycarbonyl; (3) aliphatic urethan protecting groups illustrated by tert-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; (4) cycloalkyl urethan type protecting groups illustrated by cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl; (5) thio urethan type protecting groups such as phenylihiocarbonyl; (6) alkyl type protecting groups as illustrated by triphenylmethyl (trityl), benzyl; (7) trialkylsilane groups e.g. tri(lower alkyl) silane groups such as trimethylsilane. The preferred amino protecting group defined by R1 is tert-butyloxycarbonyl.

When X is

the polystyrene resin support may be any suitable resin conventionally employed in the art for solid phase preparation of polypeptides and is preferably a copolymer of styrene and divinyl benzene in which the degree of cross linking is 1 to 2% which causes the polystyrene polymer to be insoluble in certain organic solvents. It has also preferably been chloromethylated to provide sites for ester formation with the initially introduced amino protected amino acid. The polystyrene polymer is composed of long alkyl chains bearing a phenyl ring on every second carbon and the terminal amino acid residue is joined through a covalent carbon to oxygen bond to this resin support.

The selection of the amino protecting groups R2 and R3 iS not critical except that they must be groups which are stable to conditions that will remove the amino protecting group R1. Hence R' cannot be the same as R2 or R3.

The compounds of formula I(a) possess the inherent physical properties of being white to light tan coloured solids, are substantially insoluble in chloroform and benzene but exhibit solubility in water and aqueous acid solutions such as hydrochloric and acetic. These compounds display no clearly discernible melting points and may be purified by, for example, chromatographic means. Hydrolysis of these compounds in, for example, 4N me!hanesulfonic acid allows determination of their amino acid content, which is consistent with the structures as hereinbefore set forth.

The compounds of formula Ia possess pharmaceutical activity, for example they inhibit the release of growth hormone, insulin and glucagon as evidenced by standard pharmacological test procedures.

Particularly preferred compounds of this invention have the formulae

and the non-toxic salts thereof.

Illustrative of the non-toxic acids for preparing these salts are hydrochloric, acetic, sulfuric, maleic, benzoic, fumaric and citric.

This invention also provides processes for preparing compounds of this invention.

Accordingly this invention provides a process for preparing a compound of formula

or a non-toxic salt thereof which comprises removing the protecting group or groups from a corresponding compound of formula:

in which formulae n is an integer from 3 to 8 and (T) represents D-tryptophyl or L-tryptophyl, R2 and R3 independently represent hydrogen or a protecting group for the amino group of lysine, R4, R5 and R6 are all hydrogen or all protecting groups for the alcoholic hydroxyl group of serine and threonine, with the provisos (i) that at least R2 or R3 or the group R4, R5 and R3 is other than hydrogen, and (ii) that when R4, R5 and R6 are t-butyl then R2 and R3 are other than t-butyloxycarbonyl.

Protecting groups and methods for removing them are disclosed in the literature e.g. Schroder and Lubke, The Peptides, Vol. 1. Academic Press, 1965, and Greenstein and Winitz, Chemistry of the Amino Acids, Vol. 2, John Wiley & Sons, Inc., 1961.

The compound of formula I may be prepared by cyclising a linear undecapeptide intermediate having the requisite, suitably protected amino acid sequence and having N-terminal and C-terminal groups which may be intramolecularly coupled to form an amide bond. Examples of such methods are disclosed in the above mentioned text books. For example the formation of the cyclic amide bond may be carried out by intramolecularly condensing a free amino terminal group in the presence of a condensing or functionalising agent, for example, carbonyl diimidazole; a carbodiimide e.g.

dicyclohexylcarbodiimide (DCC); in the presence of N-hydroxysuccinimide. or 1 - hydroxybenzotriazole; and alkoxyacetylene, e.g. ethoxyacetylene. Alternatively a terminal group may be activated to promote facile condensation, in which case activated forms of the amino or carboxyl groups are employed. For example, activated forms of the terminal carboxyl group comprise the acid chloride, anhydride or mixed anhydride, azide and activated ester, e.g. p-nitrophenylester; which activated terminal groups may be condensed with an N-terminal amino group. When the terminal amino group is activated, examples of activated forms are the phosphorazo or isocyanato derivatives.

It will be apparent to those skilled in the art that the method for carrying out the cyclisation reaction should be compatible with the protecting groups on the linear undecapeptide intermediate.

A preferred method of cyclising is to employ the azide derivative of the C-terminal carboxylic acid group. Preferred conditions for the azide method of coupling comprise reacting the hydrazide derivative of the linear undecapeptide with organic nitrite, e.g.

t-butyl or isoamylnitrite, in the presence of a mineral acid in an anhydrous inert organic solvent, for example, dimethyl formamide, ethyl acetate, methylene dichloride or tetrahydrofuran, at 300 to 20"C, preferably --25"C, for 10--60 minutes to obtain the corresponding azide, diluting with reaction inert solvent and rendering the resulting mixture alkaline by the addition of a strong base, preferably an organic base, for example, [(CH3)2CH] 2EtN, triethylamine or N-ethylmorpholine, and thereafter cyclising the azide at temperatures about 0 C for several days, e.g. 3-5 days. See also Kopple, "Peptides and Amino Acids", Benjamin, N.Y. 1%6 and Schroder and Lubke (cited above) for additional descriptions of this method.

Because of the cyclic nature of the undecapeptides of formula I it is immaterial which amino acid in the sequence is chosen to initiate the synthesis of the linear intermediate therefore; provided that this linear sequence is assembled in the order one would encounter by moving counterclockwise around the undecapeptide cycle from the amino acid so chosen.

The linear sequences from which the cyclic undecapeptides of this invention are prepared are themselves preferably prepared by solid phase synthesis, following techniques generally known in the art for building an amino acid sequence from an initial resin supported amino acid. Merrifield, J.A.C.S., 85, 2149 (1963), generally illustrates the technique involved. The linear sequences are then removed from the resin support and cyclized intramolecularly producing the novel cyclic undecapeptides of the invention. For reasons of convenience, the undecapeptides of the present invention have been assembled using L-serine as the starting amino acid, thus L-serine appears (reading right to left) as the first amino acid residue in the sequence of the linear intermediates. It thus follows that the w-amino acid of formula HJN(CH2),8COOH will appear as the last amino acid residue in the sequence of the linear intermediates (again reading right to left). An amide bond produced by reacting the carboxyl group of the L-serine residue and the amino group of the amino acid residue completes the synthesis.

This invention therefore further provides a process for preparing a compound of formula I as defined above which comprises cyclising a compound of formula

or an activated derivative thereof wherein (T) and n are as defined above and R2, R3, R4, R5, R6 are protecting groups as defined above, and if desired selectively removing one or more of the protecting groups from the compound of formula I obtained to give other compounds of formula I. By the term "activated derivative" is meant a derivative in which one or both terminal groups are activated so that they are capable of coupling together to form an amide bond. Preferably the azide-derivative of the compound of formula II(a) is employed. The azide derivative may be prepared by reacting a compound of formula: R'NH(CH2)aCO-Lys(R2)-Asn-Phe-Phe-(T)-Lys(R3)-Thr(R4)-Phe- Thr(R3)-Ser(RB)-NHNH, II(c) wherein R2 to R6, n and (T) are as defined above and Rl represents hydrogen or an a-amino protecting group which is removable under conditions that will not remove the protecting groups R2 and R", with an organic nitrite, e.g. isoamylnitrite or t-butylnitrite, in the presence of a mineral acid at --30"C to 20C; and, if necessary, removing the R' protecting group. It will be obvious to those skilled in the art that if an amino acid other than L-serine is chosen for the starting point in the synthesis, the sequence of the linear intermediates will vary but, once cyclized, the required amino acid sequence will be presented by the product undecapeptide cycle. Thus, for example, the following amino acid sequence is required of the linear intermediate (reading right to left) to produce the cyclic undecapeptides of the invention if D-tryptophan is chosen to initiate the linear sequence: Lys-Thr-Phe-Thr-Ser- [NH(CH2)3sCO] -Lys-Asn-Phe-Phe-D-Trp The formation, intramolecularly, of an amide bond between the carboxyl group of D-tryptophan and the a-amino group of the end chain lysine residues will produce the cyclic undecapeptides of the invention. A particular benefit which may be derived from employing an amino alkanoic acid to initiate the linear sequence, i.e. the first amino acid attached to the resin, is freedom from racemization either on removal from the resin or on activation.

Accordingly this invention also provides a process for preparing a compound of formula II wherein X is

which comprises coupling the requisite suitably protected and/or activated amino acids in the desired order of succession to a chloromethylated polystyrene resin support, and if desired removing the R1 protecting group from the compound of formula II formed.

In a preferred sequence, the eamino-,$-hydroxy protected amino acid t-Boc-Obenzyl-L-serine is coupled at its carboxyl group to the chloromethylated resin according to the procedure of Gisin, Helv. Chim. Acta., 56, 1476 (1973). Following the coupling of eamino-,,B-hydroxy di-protected L-serine to the resin support, the amino protecting group is removed by standard methods preferably employing trifluoroacetic acid in methylene chloride containing 5 out by volume 1,2-ethanedithiol, or by means of trifluoroacetic acid alone or by means of HCl in dioxane. The deprotection is preferably carried out at a temperature between O"C. and room temperature. The remaining amino protected amino acids are coupled, seriatim, in the desired order to obtain the linear sequence which may be cyclized to the desired product. Alternatively, multiple amino acid groups may be coupled by the solution method prior to coupling with the resin supported amino acid sequence, to produce the desired linear intermediate. The selection of an appropriate coupling reagent is within the skill of the art. A particularly suitable coupling reagent is N,N'-diisopropylcarbodiimide (DIC).

Another suitable coupling agent is N,N'-dicyclohexylcarbodiimide (DCC).

Each protected amino acid or amino acid sequence is introduced into the solid phase reactor in a two to six fold excess and the coupling is carried out in a medium of dimethylformamide; methylene chloride or in either dimethylformamide or methylene chloride alone. In cases where incomplete coupling occurs the coupling procedure is repeated before removal of the amino protecting group, prior to the introduction of the next amino acid to the solid phase reactor. The success of the coupling reaction at each stage of the synthesis is monitored by the ninhydrin reaction as described by E. Kaiser et al., Analyt. Biochem., 34, 595 (1970).

The fully protected, resin supported undecapeptide presents the preferred amino acid sequence

RlNHCH-Sincotys(R2)-Asn-phe-phe(T)-L > s(R3)-Thr(R4)-phQ-Thr(Q5)-ser(R O-cHt- < in which the group (lib) Cf 6) -O-CH2 2cr represents the ester moiety of one of the many functional groups present in the polystyrene resin, the R2 to R6 groups, n, and (T) are as defined above.

The fully protected linear amino acid sequence is separated from the polystyrene resin support, the R1 group is removed, and the linear peptide is subjected to intra molecular cyclization, the remaining blocking groups i.e. RZ, R3, R4, R5 and R6 are removed, the cyclic deblocked undecapeptide is purified by, for example, chromatographic means, and if desired, the free cyclic undecapeptide is converted to a pharmacologically acceptable acid addition salt.

Compounds of formula II wherein R1, R2, R3, R4, Rs and R6 are as hereinbefore defined and X represents OH, NHNH2, O(lower alkyl) orO(benzyl) may be preprepared by cleaving a corresponding compound of formula II wherein X is an anchoring bond linked to a polystyrene resin support; i.e. a compound of formula II(b). For example, transesterification using a lower alkanol or benzyl alcohol produces a compound of formula II wherein X is O(lower alkyl) or O(benzyl).

Compounds of formula II wherein X is OH may be prepared by hydrolysis of a compound of formula II wherein X isO(lower alkyl) or -O(benzyl).

Compounds of formula II wherein X is NHNH may be prepared from the corresponding compounds of formula II wherein X isO(lower alkyl) orO(benzyl) by hydrazinolysis. Alternatively compounds of formula II wherein X is NHNH2 may be prepared by hydrazinolysis of compounds of formula II(b) as defined above.

Hydrazinolysis reactions may be carried out by reacting the ester of formula II with hydrazine in a suitable solvent, e.g. dry dimethylformamide, at ambient temperature.

A particularly convenient and efficacious sequence for performing the above steps is described below. This outlined procedure discloses preferred methods, other procedures for performing the requisite reaction steps will be obvious to those skilled in the art, and are considered equivalents to those herein disclosed.

The fully protected linear amino acid sequence may be removed from the polystyrene resin support by treatment with hydrazine (97% grade), this treatment produces a linear undecapeptide of the following structure: RlNH(CH2)"CO-Lys(R2)-Asn-Phe-Phe-(T)-Lys(R3)-Thr(R4)- Phe-Thr-(R5)-Ser(R6)-NHNH2 II(c) wherein R1 to R6, n and (T) are as defined above. The Rl group, when t-Boc, may next be removed by treatment with, for example, anhydrous trifluoroacetic acid after which the intramolecular cyclization is carried out Alternatively, the R1 group, when t-Boc, may be removed from the resin supported linear sequence by treatment with, for example, anhydrous trifluoroacetic acid prior to the hydrazinolysis procedure. The free amino undecapeptide hydrazide is treated with, for example, t-butylnitrite in a mineral acid and inert solvent at about -25 0C. for about 30 minutes. At this time a negative reaction to Tollens Reagent indicates the absence of hydrazide. The mixture is next diluted by a factor of 30 to 40 times the volume with a reaction inert solvent such as dimethylformamide at about -20C., the pH is adjusted to c. 8 with, for example, diisopropylethylamine and is allowed to remain at 0 C. for 3 to 5 days. The high dilution favours intramolecular cyclization. The solvent is next evaporated and the residue may be precipitated by, for example, addition to 1% by volume acetic acid and allowing to stand at 0 C. overnight. The product is next completely deblocked (i.e. the R2 to R6 groups are removed) by treatment with, for example, anhydrous hydrogen fluoride and anisole at 0 C. for about 1 hour. After removal of the hydrogen fluoride, the residue may be purified by procedures similar to those skilled in the art, for example trituration, followed by chromotography.

The in vive pharmacological activity of the cyclic undecapeptides of formula I(a) was established by the following procedures. For purposes of convenience in reporting the test results, the compound cyclic(7 - aminoheptanoyl - L - lysyl - L -asparaginyl L - phenylalanyl - L - phenylalanyl - L - tryptophyl - L lysyl - L - threonyl - L phenylalanyl - L - threonyl - L - seryl) is referred to as compound A; and the com pound cyclic(7 - aminoheptanoyl - L - lysyl- L - asparaginyl - L - phenylalanyl - L phenylalanyl - D - tryptophyl - L - lysyl - L - threonyl - L - phenylalanyl - L - threonyl - L - seryl) is referred to as compound B.

Test Procedure I A subcutaneous (sc) injection of peptide solubilized or suspended in physiological saline5 is given to Charles River CD nonfasted male rats. Matched saline control solution sc injected rats serve as control animals so that every experimental rat is paired with 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 intraperitoneal (i.p.) injection of Nembutal (Nembutal is a Registered Trade Mark) at a dose of 50 mg/kg. Blood samples are obtained by cardiac puncture and the plasma separated for the radio-immunoassay of growth hormone (GH) concentration (ng/ml). Time periods after injection of 1, 2 or 4 hours are generally used to test the duration of the activity of the peptide to supress 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. The Student "t" test is discussed in "Statistical Tables of Biological, Agricultural and Medical Research", Fisher and Yates, 5th Edition, 1957, Oliver & Boyd. Results are tabulated below: Compound (Dose) 1 Hr. (No. Rats) 2 Hr. (No. Rats) 4 Hr. (No. Rats) A (1 mg/kg) 95+47 (7) 80#12 (9) Control 370+l103 (10) 270*90 (10) p=0.05 p=0.05 B (2 mg/kg) 13+1.7 (10) 15#2.8 (10) Control 301+73 (10) 103*31(9) p= < 0.001 p= < 0.01 B (1 mg/kg) 44+12 (8) 46+6 (10) Control - 233+42 (10) 90+l15 (9) p= < 0.001 p=0.02 B (0.25 mg/kg) - 108* 18 (9) 84* 17 (9) Control - 159+36 (9) 156+43 (8) p=' > O.05 p= > 0.05 Test Procedure II Albino male rats are arranged in three groups (nine rats/group) and injected i;p.

with Nembutal (Nembutal is a registered Trade Mark) at 50 mg/kg. Fifteen minutes after the Nembutal injection they are injected s.c. according to group with (a) test compound, typically 500-2000 g/kg; (b) SRIF 200 sDg/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 growth hormone, glucagon and insulin. An active compound is one which significantly changes the plasma level of any of these hormones from that of the saline controls. Com parisons between control and experimental values are statistically evaluated by the analysis of variants method and statistical significance (p) at 0.05 or lower is used as the index of activity. Results are tabulated below: Compound (Dose) GH (ng/ml) Insulin (a units/ml) Glucagon (pg/ml) A (3.35 mg/kg) 31+6 157+8 3.0+0.7 Control 149+23 181+6 6.4+1.3 p= < 0.01 p= < 0.05 p= < 0.05 A (1 mg/kg) 38+11 127+10 57+13 Control 191+38 141+6 96+15 p= < 0.01 p= > 0.05 p= < 0.05 A (0.2 mg/kg) 57+12 152+15 32+5 SRIF (0.2 mg/kg) 65+16 111+10 12+3 Control 287+53 151+15 20+1 p=- < 0.05 p= < 0.05 p= < 0.05 A (0.05 mg/kg) 55 10 - SRIF (0.05 mg/kg) 47 8 Control 170 49 p < 0.05 Compound (Dose) GH (ng/ml.) Insulin (Es units/ml.) Glucagon (pg/ml.) B (3.0 mg/kg.) 92+9 74+13 Control 1009+298 280+37 p=' < 0.01 p= < 0.01 B (30 ,ag/kg.) 180+49 SRIF (20 ag/kg.) 232+87 Control 619+103 p= < 0.01 B (2 ,ug/kg.) 211+18 SRIF (10 sg/kg.) 241+15 Control 543#86 P= < 0.01 B (200 ,ag/kg) 66+i6 21+5 SRIF (200 g/kg.) - 117+12 13+4 Control - 167 + 12 62+8 p=1 < 0.01 p= < 0.01 The above test results demonstrate that the cyclic undecapeptides having formula I(a) are useful in depressing the secretion of somatotropin, insulin, and glucagon in domestic animals and for the control of the immuno-reactive pituitary growth hormone in comparative and experimental pharmacology. From the known relationship between growth hormone control in standard experimental animals and the human, the demon strated pharmacological activity of the disclosed cyclic undecapeptides characterizes the compounds as useful in the treatment of acromegaly and juvenile diabetes in the same manner as somatostatin itself. Administration of the cyclic undecapeptides of formula I(a) may be by convenhonal routes common to s moles) in 1: 1 by volume dimethylformamide-methylene chloride solution and 1 molar dicyclohexylcarbodiimide (DCC) in methylene chloride (470 ml.) overnight. The resin was washed successively with dimethylformamide (twice), methanol (twice) and methylene chloride (twice). To test for completeness of reaction, the peptide resin was subjected to a ninhydrin in color test following the procedure of E. Kaiser, et al., Analytical Chemistry, 34, 595 (1970), and found to be weakly positive. The coupling step was repeated using t-Boc-O-benzyl-L-threonine (43.8 g., 0.14 moles in 1:1 by volume dimethylformamide-methylene chloride, 70 ml. 1 molar DCC). After stirring overnight and the washing sequence described above, the reaction with ninhydrin was still slightly positive, so a further coupling with t-Boc-O-benzyl-L-threonine (43.8 g., 0.14 moles in dimethylformamide containing l-hydroxybenzotriazole 21.9 g., 0.14 moles, 143 ml. 1 molar DCC) was performed. After stirring overnight and the washing sequence, the ninhydrin reaction was negative indicating complete coupling. Removal of the t-Boc,a-amino protecting group was carried out as described in steps (3) through (11) above.

The following amino acid residues were then introduced consecutively. (A coupling time of 18 hr. was used for each amino acid unless specified otherwise. Each coupling between consecutive amino acids were separated by both the washing schedule and the deblocking sequence (3) through (11) described for the t-Boc-Obenzyl-L-serine resin). t-Boc-L-phenylalanine (187 g., 0.7 mmoles in dimethylformamide comaining l-hydroxybenzotriazole 107.9 g., 0.705 moles, 705 ml. 1 molar DCC). A 48.4 g. portion of peptide resin was removed and the synthesis continued with the addition of t-Boc-O-benzyl-L-threonine (145.3 g., 0.47 moles in dimethylformamide containing l-hydroxybenzotriazole 71.9 g., 0.47 moles, 470 ml. 1 molar DCC), t-Boc-N6-(2-chlorobenzyloxycarbonyl)-L-lysine (194.8 g., 0.47 moles in di methylformamide containing 1-hydroxybenzotriazole 35 g., 0.23 moles, 470 ml. 1 molar DCC), t-Boc-L-tryptophan (142.9 g., 0.47 moles in dimethylformamide, 470 ml., 1 molar DCC). Incomplete coupling occurred at this stage, so after the washing sequence coupling with t-Boc-L-tryptophan was repeated using amino acid (72 g., 0.237 moles) in dimethylformamide containing l-hydroxybenzotriazole (36 g., 0.237 moles) 235 ml. 1 molar DCC, 40 g. of peptide resin was removed and the synthesis continued with t-Boc-phenylalanine (124.6 g., 0.46 moles in dimethylformamide containing 1-hydroxybenzotriazole 71.9 g., 0.47 moles, 470 ml. 1 molar DCC). The synthesis was continued by repeating the addition of t-Boc phenylalanine. 43.3 g. of peptide resin was removed and the synthesis continued with the addition of t-Boc-L- asparagine-p-nitrophenyl ester (199.1 g., 0.56 moles in dimethylformamide containing 1% by volume glacial acetic acid). A reaction time of 4 days was allowed for this stage. 52.4 g. of pep tide resin was removed and the synthesis concluded with the addition of t-Boc-N(2-chlorobenzyIoxycarbonyl)-LAysine (165.8 g., 0.4 moles in dimethylformamide containing l-hydroxybenzotriazole 61.2 g., OA moles, 400 ml.

1 molar DCC). The dried and washed resin weighed 568.1 g.

Example 2 7-t-Butyloxycarbonylaminoheptanoic Acid 7-Aminoheptanoic acid (20 g., 1.38X 10-1 moles) was converted to the title compound using the method of Ulf Ragnarrson et al., Org. Syn., 53, 25 (1973), using tetramethylguanidine (17.3 g., 1.49x10-t moles) and t-butylphenylcarbonate (30 g., 1.54 X 10-1 moles) in dimethylsulfoxide (100 ml.) for three days.

The product (24 g.) had m.p. 558 after recrystallization from ethyl acetatehexane-diethyl ether.

Analysis for: Cl2H,3NO4 Calculated: C, 58.75; H, 9.45; N, 5.71 Found: C, 59.14; H, 9.61; N, 5.86 Example 3 7-t-Butyloxycarbonylaminoheptanoyl-N(2ChIornbenzyloxycarbo nyl) -L-Lysyl-L Asparaginyl-L-Phenylalanyl-L-Phenylalanyl-L-Tryptophyl-Ne- -(2-Chlorobenzyloxy- carbonyl)-L-Lysyl-O-Benzyl -L-Threonyl-L-Phenylalanyl-O-Benzyl-L-Threonyl- O-Benzyl-L-Seryloxymethyl polystyrene Resin The peptide resin I (15 g.) from Example 1 was treated according to schedule (3) through (11) of Example I and the deblocked peptide resin stirred with 7-t-butyloxycarbonylaminoheptanoic acid (30 mmoles in 1:1 by volume dimethylfonommide- methylene chloride) and 60 ml. of an 0.5 molar solution of diisopropyl carbodiimide (DIC), in methylene chloride.

The peptide resin was washed with methylene chloride (twice), dimethylformamide (thrice) and methylene chloride (twice) to give the title resin.

Example 4 7-t-Butyloxycarbonylaminoheptanoyl-N-(2-Chlorobenzyloxycarbonyl)-L-LySyl-L- Asparaginyl-L-Phenylalanyl-L-Phenylalanyl-D-Tryptophy1-N(2-Chlorobenzyloxy carbonyl)-L-Lysyl-O-Benzyl-L-Threonyl-L-Phenylylanyl-O-Benzyl-L-Threonyl- O-Benzyl-L-Seryloxymethyl polystyrene Resin The peptide resin II from Example 1, t-Boc-L-phenylalanyl-O-benzyl-L-threonyl O-benzyl-L-serine resin (10 g.), was converted into the title compound by the stepwise coupling of the appropriate amino acids in 1:1 by volume methylene chloridedimethyl-formamide (50 ml.) with an 0.5 molar solution (45 ml.) of dicyclohexylcarbodiimide (30 ml.). A coupling time of 18 hours was used for each amino acid.

Each coupling was preceeded by the deblocking schedule and followed by the washing schedule as described.

Deblocking Schedule 1. Methylene chloride wash (3x).

2. Prewash with 40% by volume trifluoroacetic acid in methylene chloride containing 0.5X w/v of dithioerythreitol.

3. Wash with 40% by volume trifluoroacetic in methylene chloride containing 0.5% w/v of dithioerythreitol (2 X) for 15 minutes each.

4. Wash with methylene chloride (2 x).

5. Dimethylformamide wash (2x).

6. 15% by volume triethylamine in dimethylformamide (2x) for 10 minutes each.

7. Dimethylformamide wash (2x).

8. Methylene chloride wash (3x).

9. Dimethylformamide wash.

10. Methylene chloride.

A contact time of 5 min. was allowed for each wash period.

Washing Schedule (i) Methylene chloride (2 X).

(ii) Dimethylformamide (3 X).

(iii) Methylene chloride (2 X ).

Amino acids in order of addition (20 moles).

t-Boc-O-benzyl-Lthreonine (6.18 g.) t-Boc-N#-(2-chlorobenzyloxycarbonyl)-L-lysine (8.34 g.).

t-Boc-D-tryptophan (6.1 g.).

t-Boc-Lphenylalanine (5.3 g.).

t-Boc-L-phenylalanine (5.3 g.).

t-Boc-L-asparagine c & p-nitrophenyl ester (7.1 g.), no DIC was used with this coupling which was run in dimethylformamide containing 1 l ó by volume acetic acid.

t-Boc-Ne-(2-chlorobenzyloxycarbonyl)-L-lysine (8.34 g.).

7-t-Boc-aminoheptanoic acid (4.9 g.).

Example 5 7-t-Butyloxycarbonylaminoheptanoyl-N-(2-Chlorobenzyloxycarbonyl)-L-Lysyl-L Asparaginyl-L-Phenylalanyl-L-Phenylalanyl-L-Tryptophyl-N#-(2-Chlorobenzyloxy- carbonyl)-L-Lysyl-O-Benzyl-L-Threonyl-L-Phenylalanyl-O-Benzyl-L-Threonyl- O-Benzyl-L-Seryl Hydrazide The peptide resin (15 g.) of Example 3 suspended in dry dimethylformamide (100 ml.) was stirred with hydrazine (10 mi, 97% grade) for 2 days at ambient temperatures under nitrogen. The resin was filtered and washed thoroughly with dimethylformamide. The combined filtrate and washings were concentrated under reduced pressure at temperatures not exceeding 305C. The residue was triturated with methanol (100 ml.) and filtered. The solid precipitate was stirred with methanol during 1 hour and again filtered. The precipitate dried in feecuo over phosphorous pentoxide afforded 5.8 g. hydrazide.

Example 6 7-t-Butyloxycarbonylaminoheptanoyl-Nt'-(2-Chlorobenzyloxycarbonyl)-L-Lysyl-L- Asparaginyl-L-Phenylalanyl-L-Phenylalanyl-D-Tryptophyl-N#-(2-Chlorobenzyloxy- carbonyl)-L-Lysyl-O-Benzyl-L-Threonyl-L-Phenylalanyl-O-Benzyl-L- Threonyl-O-Benzyl-L-Seryl Hydrazide The total peptide resin of Example 4 was washed with dimethylformamide and then suspended in dry dimethylformamide (100 ml.) and stirred with hydrazine (10 ml., 97% grade) for two days at room temperature under nitrogen. The resin was filtered and washed thoroughly with dimethylformamide. The combined filuate and washings were concentrated under reduced pressure at temperatures not exceeding 30 C. The residue was triturated with methanol and filtered. The solid precipitate was stirred with methanol during 1 hour and again filtered. The precipitate dried in vacuo after phosphorous pentoxide afforded 4.9 g. crude hydrazide.

Example 7 Cyclic(7-Aminoheptanoyl-N#-(2-Chlorobenzyloxycarbonyl)-L-Lysyl-L- Asparaginyl-L-Phenylalanyl-L-Phenylalanyl-L-Tryptophyl-Nb-(2-Chlorobenzyloxy- carbonyl)-L-Lysyl-O-Benzyl-L-Threonyl-L-Phenylalanyl-O- Benzyl-L-Threonyl-O-Benzyl-L-Seryl) The crude dried hydrazide of Example 5 suspended in anhydrous trifluoroacetic acid (60 ml.), methylene chloride (40 ml.) anisole (5 ml.) was stirred with 0.5 g.

dithioerythreitol during 15 minutes at 0 C and then for a further 45 mins. at room temperature. The clear solution was evaporated to an oil which, on trituration with large volumes of diethyl ether afforded a flocculent solid (5.65 g.), which was dried in uno over phosphorous pentoxide.

The above solid (5.5 g., 2.i mmoles) dissolved in dimethylformamide (60 ml.) at room temperature was cooled to -300C and treated with 12.2 ml. of 0.76M hydrogen chloride (9.27 moles) in tetrahydrofuran followed by t-butylnitrite (0.36 ml., 3.1 moles). The solution had a negative reaction with ammoniacal silver nitrate (Tollen's reagent) after 30 min. in the -20' to +30"C temperature range indicating absence of hydrazide. The reaction mixture was transferred to a flask containing anhydrous dimethylformamide (21) at -200C and the pH adjusted to 8 with diisopropylethylamine. The reaction mixture was stored at 0 0C, during 3 days and then evaporated to a small volume under reduced pressure. The residue was poured into 500 mL 1% by volume acetic acid stood overnight at 0 C and filtered. The solid was washed with water and dried in vacuo to give the title product (4.7 g.).

Example 8 Cyclic(7-Aminoheptanoyl-N#-(2-Chlorobenzyloxycarbonyl)-L-Lysyl-L- Asparaginyl-L-Phenylalanyl-L-Phenylalanyl-D-Tryptophyl-N6-(2-Chlorobenzyloxy- carbonyl)-L-Lysyl-O-Benzyl-L-Threonyl-L-Phenylalanyl-O-Benzyl-L- Threonyl-O-Benzyl-L-Seryl) The crude hydrazide of Example 6 (4.9 g.) was stirred in a mixture of anhydrous trifluoroacetic acid (100 ml.), and anisole (5 ml.) containing dithioerythreitol (0.5 g.) during 10 mins. at 0 C and then for a further 50 mins. at room temperature. The clear solution was evaporated to an oil which on trituration with large volumes of diethyl ether afforded a solid (4.8 g.) after drying in vacuo over phosphorous pentoxide.

The above solid (4.8 g., 2.1 mmoles) dissolved in dimethylformamide (50 mi.) at room temperature was cooled to -300C and treated with 2 ml. 4M hydrogen chloride (8 mmoles) in dioxane followed by t-butylnitrite (0.31 ml., 2.68 mmoles).

The solution had a negative reaction with ammoniacal silver nitrate (Tollen's reagent) after 30 minutes in the 20 to --30"C temperature range indicating absence of hydrazide. The reaction mixture was transferred to a flask containing anhydrous dimethylformamide (21) at -200C and the pH adjusted to 8 with diisopropylethylamine. The reaction mixture was stored at 0 C during 4 1/2 days and then evaporated in vacua at temperatures < 35 C. The residue was poured into 500 ml. 1% by volume acetic acid and the solid recovered by filtration. After washing with water and drying in vacao over phosphorous pentoxide, 4.2 g. product was obtained.

Example 9 Cyclic(7-Aminoheptanoyl-L-Lysyl-L-Asparaginyl-L-Phenylalanyl-oPhenylalanyl-L- Tryptophvl-L-Lvsyl-L-Threonyl-L-Phenylalanyl-L-Threonyl-L-Seryl) Cyclic(7 - arninopentanoyl - N - (2 - chlorobenzyloxycarbonyl) - L - lysyl - L asparaginyl - L - phenylalanyl - L - phenylalanyl - L - tryptophyl - Ne (2 - chlorobenzyloxycarbonyl) - L - lysyl - O - benzyl - L - threonyl - L - phenylalanyl - 0 - benzyl - L - threonyl - 0 - benzyl - L - seryl) (4.6 g.) was treated in uacuo with anhydrous hydrogen fluoride (100 ml.) and anisole (30 ml.) at 0 C during 1 hour.

Hydrogen fluoride was removed under reduced pressure and the residue triturated thoroughly with ether prior to extraction with degassed 2000 by volume acetic acid (100 mi.). The acid fraction was diluted with water and lyophyllized to leave the crude title compound (3.15 g.).

Purification The crude deprotected peptide (3.1 g.) in 20% by volume acetic acid was applied to a column of Sephadex G 25 (fine), 200x2.5 cm. in 200ó acetic acid (Sephadex is a Registered Trade Mark). 180 fractions (5.6 ml. each) were collected. Column effluent was monitored at 254 ma with an Altex Model 153 Analytical UV Detector-using an Altex preparative Flow Cell and omniscribe recorder. Four major fractions were taken A (82-94) 267 mg.; B (99-107) 883 mg.; C (111-119) 970 mg.; D (120--136) 700 mg. Fraction D (120--136) was selected for further purification on the basis of tlc, amino acid analysis, and positive tryptophan reaction with Ehrlich reagent. Resizing on the same column afforded a single peak in fractions 11-127 (5.6 ml. each) 491 mg. (70% recovery). Further purification was effected on a partition column (150x2.5 cm.) of Sephadex G-25 fine prepared by equilibration with lower phase and then upper phase of the solvent system: n-butanol : acetic acid: water 4: 1: 5 by volume.

Column effluent monitored as above afforded homogeneous material in fractions 4861. The fractions were collected, evaporated and lyophyllized from 10% by volume acetic acid. The solid was dissolved in 1%: acetic acid filtered through an 0.3 im Millipore filter and lyophyllized to give 250 mg. product [a]D26 --27.5 c., 1.18 1U" by volume acetic acrid. (Millipore is a Registered Trade Mark).

Amino acid analysis for a sample hydrolyzed 18 hr. at 110 in an evacuated sealed tube containing 4N methane sulfonic acid and 0.2% 3-(2-aminoethyl) indole.

Asp (1.0), Thr (1.88), Ser (.94), Phe (2.97), NH,(CH2)6C0,H (1.0), Lys (2.03), NH, (.79), Trp (0.95).

No loss of 7-aminoheptanoic acid was observed relative to phenylalanine after exhaustive free amine dansylation and subsequent amino acid hydrolysis under conditions causing complete loss of lysine.

Thin Layer Chromatography Rf values I It III Silica gel 0.3 0.77 0.54 Cellulose 0.6 0.88 0.60 Rr values determined on S X 20 cm. Brinkinican SiO, gel 60 F-254 and on a 5 x20 am Avicel Analtech plates (Avicel is a Registered Trade Mark). The spots were visualized by iodine and Ehrlich reagent.

Solvent Systems I Butanol: acetic acid: water (4: 1:5 by volume).

II Ethyl acetate: butanol: acetic acid: water (1:1:1:1 by volume).

III Isoamyl alcohol : pyridine: water (7:7:6 by volume).

Example 10 Cyclic (7-Aminoheptanoyl-L-Lysyl-L-Asparaginyl-L-Phenylalanyl-L-Phenylalanyl-D- Tryptophyl-L-Lysyl-L-Threonyl-L-Phenylalanyl-L-Threonyl-L-S eryl) Cyclic(7 - aminoheptanoyl) - Ne (2 - chlorobenzyloxycarbonyl) - L - lysyl - L asparaginyl - L - phenylalanyl - L - phenylalanyl - D - tryptophyl - N - (2 - chlorobenzyloxycarbonyl) - L - lysyl - O - benzyl - L - threonyl - L - phenylalanyl - 0 - benzyl - L - threonyl - 0 - benzyl - L - seryl) (4.3 g.) was treated in vacuo with anhydrous hydrogen fluoride (100 ml.) and anisole (30 ml.) at 0 during 1 hour.

Hydrogen fluoride was removed under reduced pressure and the residue triturated thoroughly with diethyl ether prior to extraction with degassed 20% by volume acetic acid (100 ml.). The acid fraction was diluted with water and lyophyllized to leave the title compound (2.55 g.).

Purification The crude deprotected peptide (2.50 g.) in 20% by volume acetic acid was applied to a column of Sephadex G-25 (fine), 200 X2.5 cm. in 20% by volume acetic acid. 180 fractions (5.6 ml. each) were collected. Column effluent was monitored as described for Example 9. Tryptophan positive fractions were collected lyophyllized to give 1.0 g. peptide which was reapplied to the column. Fractions 129-148 (295 mg.) were selected, on the basis of tlc and amino acid analysis, for further purification.

Fractions 129-148 were reapplied to a partition column (150X 2.5 cm.) of Sephadex G-25 fine prepared by equilibration with lower phase and then upper phase of the solvent system: n-butanol: acetic acid: water < : 1:5 by volume. Column effluent monitored as above afforded homogeneous material in fractions 74-94. The fractions were collected, evaporated and the residue lyophyllized from 10% acetic acid. The solid was dissolved in 1% acetic acid filtered through an 0.3 'm Millipore filter and lyophyllized to give 120 mg. product. [ ]D36 --31.5"C., 1.11% acetic acid.

Amino acid analysis for a sample hydrolyzed 18 hours at 110 in an evacuated sealed tube containing 4N methane sulfonic acid and 0.2 , by weight 3-(2-amino- ethyl)indole.

Asp (1.0), Thr (1.83), Ser (1.05), Phe (2.88), NH (CH*),;CO H (1.0), Lys (1.99), NH, (1.03), Trp (0.87).

Thin Layer Chromatography R, values I II III Silica gel 0.32 0.77 0.54 Cellulose 0.60 0.88 0.64 Origin of plates and meaning of solvent systems I, II, and III as in Example 9.

WHAT WE CLAIM IS: 1. A compound having the formula

or a non-toxic salt thereof wherein (T) represents D-tryptophyl or L-tryptophyl and n is an integer from 3 to 8.

2. A compound as claimed in Claim 1 wherein n is 6.

3. Cyclic(7 - aminoheptanoyl - L - lysyl - L - asparaginyl - L - phenylalanyl - L phenylalanyl - L - tryptophyl - L - lysyl - L - threonyl - L - phenylalanyl - L - threonyl - L - seryl).

4. Cyclic(7 - aminoheptanoyl - L - lysyl - L - asparaginyl - L - phenylalanyl - L phenylalanyl - D - tryptophyl - L - lysyl - L - threonyl - L - phenylalanyl - L threonyl - L - seryl).

5. The hydrochloric, acetic, sulphuric, maleic, benzoic, fumaric or citric acid salt of a compound as claimed in any one of Clairns 1 to 4.

6. A compound having the formula

and the non-toxic salts thereof, wherein (T) represents D-tryptophyl or L-tryptophyl, n is an integer from 3 to 8, R2 and R3 are attached to the nitrogen of the side chain e-amino group and independently represent hydrogen or a protecting group for the amino group, R4, R5 and R" are the hydrogen atom of the alcoholic hydroxyl group of threonine and serine, or protecting groups therefor, with the provisos (i) that when R4, R5 and R6 are t-butyl then R2 and R" are other than t-butoxycarbonyl and (ii) that at least R2 or R" or the group R4, R5 and R6 are other than hydrogen.

7. A compound as claimed in Claim 6 wherein R and R3 independently represent benzyloxycarbonyl, halobenzyloxycarbonyl, nitrobenzyloxycarbonyl, tosyl, t-amyloxycarbonyl, t-butyloxycarbonyl or diisopropylmethoxycarbonyl.

8. A compound as claimed in Claim 6 or Claim 7 wherein R4, R5 and R6 all represent acetyl, benzoyl, tert-butyl, trityl, benzyl or 2,6-dichlorobenzyl.

9. A compound as claimed in any one of Claims 6 to 8 wherein n is 6.

10. A compound having the formula

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. solvent system: n-butanol: acetic acid: water < : 1:5 by volume. Column effluent monitored as above afforded homogeneous material in fractions 74-94. The fractions were collected, evaporated and the residue lyophyllized from 10% acetic acid. The solid was dissolved in 1% acetic acid filtered through an 0.3 'm Millipore filter and lyophyllized to give 120 mg. product. [ ]D36 --31.5"C., 1.11% acetic acid. Amino acid analysis for a sample hydrolyzed 18 hours at 110 in an evacuated sealed tube containing 4N methane sulfonic acid and 0.2 , by weight 3-(2-amino- ethyl)indole. Asp (1.0), Thr (1.83), Ser (1.05), Phe (2.88), NH (CH*),;CO H (1.0), Lys (1.99), NH, (1.03), Trp (0.87). No loss of 7-aminoheptanoic acid was observed relative to phenylalanine after exhaustive free amine dansylation and subsequent amino acid hydrolysis under conditions causing complete loss of lysine. Thin Layer Chromatography R, values I II III Silica gel 0.32 0.77 0.54 Cellulose 0.60 0.88 0.64 Origin of plates and meaning of solvent systems I, II, and III as in Example 9. WHAT WE CLAIM IS:

1. A compound having the formula

2. A compound as claimed in Claim 1 wherein n is 6.

6. A compound having the formula

9. A compound as claimed in any one of Claims 6 to 8 wherein n is 6.

10. A compound having the formula

RlNH{CH2)CO-Lys(R2)-Asn-Phe-Phe-(T)-Lys(R3)-Thr(R I)- Phe-Thr(R5)-Ser(R6)-X (II) and non-toxic salts thereof, wherein n, R4, R5, R6 and (T) are as defined in Claim 1, R2 and R3 are protecting groups for the amino group of lysine, R1 represents nitrogen on an amino protecting group which is removable under conditions that will not remove the R2 and R3 protecting groups, and X represents OH, NHNH, O(lower alkyl), -O(benzyl) or

11. A compound as claimed in Claim 10 wherein R1 is selected from formyl, trifluoroacetyl, phthalyl, p-toluenesulfonyl, benzenesulfonyl, 0-nitrophenylsulfenyl, tri tylsulfenyl, nitrophenoxyacetyl, y-chlorobutyryl, benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, pmethoxybenzyl- oxycarbonyl, l-(p-biphenylyl)-l-methylethoxycarbonyl, ey-dimethyl-3,5-dimethoxy- benzyloxycarbonyl and benzhydryloxycarbonyl; tert-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, tri phenylmethylbenzyl and trimethylsilyl.

12. A compound as claimed in Claim 11 wherein R1 is tert-butyloxycarbonyl.

13. A compound as claimed in any one of Claims 10 to 12 wherein R2 and R3 are as defined in Claim 7.

14. A compound as claimed in any one of Claims 10 to 13, wherein R4, R5 and Rss are as defined in Claim 8.

15. A compound as claimed in Claim 10 wherein R1 represents hydrogen or t-butyloxycarbonyl, R2 and R3 are 2-chlorobenzyloxycarbonyl and R4, R5 and R6 represent benzyl.

16. 7 - t - Butyloxycarbonylariinoheptanoyl - Ne - (2 - chlorobenzyloxycarbonyl) L - lysyl - L - asparaginyl - L - phenylalanyl - L - phenylalanyl - L - tryptophyl - Ne - (2 - chlorobenzyloxycarbonyl) - L - Iysyl - O - benzyl - L - threonyl - L - phenyl alanyl - 0 - benzyl - L - threonyl - 0 - benzyl- t; - seryloxyrnethyl polystyrene resin.

17. 7 - t - Butyloxycarbonylaminoheptanoyl - Ne - (2 - chlorobenzyloxycarbonyl) L - lysyl - L - asparaginyl - L - phenylalanyl - L - phenylalanyl - D - tryptophenyl N' - (2 - chlorobenzyoxycarbonyl) - L - lysyl - O - benzyl - L - threonyl - phenylalanyl - 0 - benzyl - L - threonyl - 0 - benzyl - L - seryloxymethyl polystyrene resin.

18. 7 - t - Butyloxycarbonylaminoheptanoyl - Ne - (2 - chlorobenzyloxycarbonyl L - lysyl - L - asparaginyl - L - phenylalanyl - L - phenylalanyl - L - tryptophyl N - (2 - chlorobenzyloxycarbonyl) - L- lysyl- O - benzyl - L - threonyl - L - phenyl alanyl - O - benzyl - L - threonyl - 0 - benzyl - L - seryl hydrazide.

19. 7 - t - Butyloxycarbonylaminoheptanoyl - Ne - (2 - chlorobenzyloxycarbonyl) L - lysyl - L - asparaginyl - L - phenylalanyl - L - phenylalanyl - D - tryptophyl Ne - (2 - chl;ordbenzyloxycarbonyl) - L - lysyl - O - benzyl - L - threonyl - L phenylalanyl - 0 - benzyl - L - threonyl - 0 - benzyl - L - seryl hydrazide.

20. Cyclic(7 - aminoheptanoyl - N - (2 - chlorobenzyloxycarbonyl) - L - lysyl L - asparaginyl - L - phenylalanyl - L - phenylalanyl - L - tryptophyl - Ne - (2 - chlorobenzyloxycarbonyl) - L - lysyl - O - benzyl - L - threonyl - L - phenylalanyl O - benzyl - L - threonyl - O - benzyl - L - seryl).

21. Cyclic(7 - aminoheptanoyl - Ne - (2 - chlorobenzyloxycarbonyl) - L - lysyl L - asparaginyl - L - phenylalanyl - L - phenylalanyl - D - tryptophyl - Ne - (2 chlorobenzyloxycarbonyl) - L - lysyl - O - benzyl - L - threonyl - L - phenylalanyl O - benzyl - L - threonyl - O - benzyl - L - seryl).

22. A process for preparing a compound of formula

in which formulae n is an integer from 3 to 8 and (T) represents D-tryptophyl or L-tryptophyl, R2 and R3 independently represent hydrogen or protecting group for the amino group of lysine, R4, R5 and R6 are all hydrogen or all protecting groups for the alcoholic hydroxyl group of serine and threonine, with the proviso (i) that at least R2 or Rs or the group R4, R5 and R6 is other than hydrogen and (ii) when R4, R5 and R8 are t-butyl then R2 and R3 are other than t-butyloxycarbonyl and, if desired, converting to a non toxic salt thereof.

23. A process for preparing a compound of formula

as defined in Claim 22 which comprises cyclising a linear undecapeptide having the requisite, suitably protected amino acid sequence, and having N-terminal and Cterminal groups which may be intrmolecularly coupled to form an amide bond.

24. A process as claimed in Claim 23 in which the linear undecapeptide is a compound of formula:

or an activated derivative thereof as hereinbefore defined wherein R2 to R6, (T) and n are as defined in Claim 22.

25. A process as claimed in Claim 24 in which the activated derivative of the compound of formula II(a) is the azide.

26. A process as claimed in Claim 25 wherein the azide functional derivative is prepared by reacting a compound of formula: RNH(CH2)nCO-Lys(R)-Asn-phe-Phe-(T)-Lys(R )- Thr(R4The-Thr(R5Ser(R6)-NHNHi II(c) wherein R2 to R6, n and (T) are as defined in Claim 22, and R1 represents hydrogen or an amino protecting group which is removable under conditions that will not remove the protecting groups R2 and R3, with an organic nitrite in the presence of a mineral acid at --30"C to 20"C; and, if necessary, removing the R1 protecting group.

27. A process as claimed in Claim 26 in which the organic nitrite is isoamylnitnte or t-butylnitrate.

28. A process for preparing a compound of formula II(c) as defined in Claim 26 which comprises hydrazinolysing (i) an ester of formula II as defined in Claim 10 wherein X represents --O(lower alkyl) or --D(benzyl), or (ii) a resin supported peptide of formula

in which the group

represents the ester moiety of one of the many functicnal groups present in the polystyrene resin; R2 to R6, n and (T) are as defined in Claim 22 and R1 represents hydrogen or an amino protecting group which is removable under conditions which do not remove the R2 and R3 protecting groups; and if desired removing the R protecting group.

29. A process for preparing a compound of formula II as defined in Claim 10 wherein X representsO(lower alkyl) or --O(benzyl) which comprises transesterifying a compound of formula II(b) as defined in Claim 31, with a lower alkanol or benzyl alcohol.

30. A process for preparing a compound of formula II as defined in Claim 10 wherein X represents OH which comprises hydrolysing a compound of formula II wherein X representsO(lower alkyl) or -O(benzyl).

31. A process for preparing a compound of formula 11(b) as defined in Claim 28 which comprises coupling the requisite suitably protected and/or activated amino acids in the desired order of succession to a chioromethylated polystyrene resin support, and if desired removing the R' protecting group from the compound of formula II(b) formed.

32. A process as claimed in Claim 22 in which the compound of formula I as defined in Claim 22 is prepared by a process as claimed in any one of Claim 23 to 27.

33. A process as claimed in any one of Claims 26 to 31 wherein Rl represents a protecting group as defined in Claim 11.

34. A process as claimed in any one of Claims 22 to 33 wherein R2 and R3 represent a protecting group as defined in Claim 7.

35. A process as claimed in any one of Claims 22 to 34 wherein R4, R5 and R6 are protecting groups as defined in Claim 8.

36. A process as claimed in any one of Claims 22 to 26 and 28 to 31, wherein R1 when present represents hydrogen or t-butyloxycarbonyl, R2 and R3 represent 2-chlorobenzyl and R4, R5 and R6 represent berizyl.

37. A compound of formula II as defined in Claim 10 substantially as hereinbefore described with reference to any one of Examples 3 to 6.

38. A compound of formula I as defined in Claim 6 substantially as hereinbefore described with reference to either Example 7 or Example 8.

39. A compound of formula I(a) as defined in Claim 1 substantially as hereinbefore described with reference to either Example 9 or Example 10.

40. A compound of formula I(a) as defined in Claim 1 whenever prepared by a process as claimed in any one of Claims 22, 32 and 34 to 36 as dependent on Claim 22.

41. A compound of formula I as defined in Claim 6 whenever prepared by a process as claimed in any one of Claims 23 to 27 and 33 to 35 as dependent on Claims 23 to 27.

42. A compound of formula II(c) as defined in Claim 26 whenever prepared by a process as claimed in Claim 28 and 33 to 35 as dependent on Claim 28.

43. A compound of formula II(b) as defined in Claim 28 whenever prepared by a process as claimed in Claim 31 and 32 to 35 as dependent on Claim 31.

44. A compound of formula I(a) or I as defined in Claim 1 or Claim 6 whenever prepared by a process as claimed in Claim 36 as dependent on Claims 22-27.

45. A pharmaceutical composition comprising a compound of formula I(a) as defined in Claim 1 or non toxic salt thereof together with a pharmaceutically acceptable carrier.

46. A pharmaceutical composition as claimed in Claim 45 when in unit dosage form.

47. A pharmaceutical composition as claimed in Claim 45 or Claim 46 whenever n is 6 in the compound of formula I(a).