GB1570210A - Peptides - Google Patents

Description

(54) PEPTIDES (71) Ye, AYERST McKENNA & HARRISON LIMITED, a corporation organised and existing under the laws of Canada, of 1025 Laurentien Boulevard, St.

Laurent, Province of Quebec, Canada, 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: This invention relates to derivatives of the tetradecapeptide somatostatin. More particularly, this invention concerns carba derivatives and salts thereof, a process for preparing said derivatives and salts, intermediates used in the process and methods for using the carba derivatives and their salts.

The name " somatostatin" has been proposed for the factor found in hypothalamic extracts which inhibits the secretion of growth hormone (somatotropin). The structure of this factor has been elucidated by P. Brazeau et al., Science, 179, 77 (1973). It is a cyclic disulfide peptide having the following structure:

S!-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Sar-Cys-OH I 1 The abbreviations used herein for the various amino acids are Ala, L-alanine; Asn, L-asparagine; Cys, L-cysteine; Gly, glycine; Lys, L-lysine; Pher, L-phenylalanine; Ser, L-serine; Thr, L-threonine: Trp, L-Tryptophan; and D-Trp, D-Tryptophan.

The constitution of the cyclic disulfide peptide somatostatin has been confirmed by synthesis; for example, see D. Sarantakis and W. A. McKinley, Biochem. Biophys.

Res. Comm., 54, 234 (1973), J. Rivier et al., Compt. Rend. Ser. D, 276, 2737 (1973) and H. U. Immer et al., Helv. Chim. Acta, 57, 730 (1974).

The important physiological activity of this cyclic disulfide peptide established it as a compound of significance for clinical pharmacology relating to the treatment of acromegaly and the management of diabetes; for example, see K. Lundbaek et al., Lancet, 2, 131 (1970) and G. Guillemin in " Chemistry and Biology of Peptides ", J.

Meienhofer, Ed., 3rd American Peptide Symposium Boston 1972, Ann Arbor Science Publications, Ann Arbor, Mich., 1972. On the other hand, somatostatin is metabolized rapidly in the body. This property limits its clinical usefulness. Furthermore, supplies of this hormone depend on synthetic preparations which are complicated by the fact that somatostatin is made up of 14 amino acids.

The present invention discloses carba derivatives of somatostatin which show a level of activity greater than or of the same order as the natural hormone as well as a duration of activity which is greater than that of somatostatin. These derivatives are simpler in structure than somatostatin and are prepared readily by a convenient process, which includes the following advantages: the process starts from readily available materials, avoids noxious reagents, is executed facilely, utilizes easily removable protecting groups and provides a pure product having a high degree of physiological potency.

A "carba analog " of a cyclic disulfide peptide refers to a compound in which one or two of the sulfur atoms in the cyclic backbone have been substituted by one or two methylene radicals, respectively. For example, carba analogs of oxytocin have been prepared, see a review by K. Jost, Coll. Czech. Chem. Commun., 36, 218 (1971) and also carba analogs of vasopressin, see a review by K. Jost, et al., Coll. Czech. Chem.

Commun., 39, 2835 (1974).

The foregoing advantages and attributes render the carba derivatives of this invention useful for management of diabetes and for the treatment of acromegaly.

The compounds of this invention are represented by formula I

CO-Lys-AsnPhe-Phe-Y-Lys-Thr-Phe-Thr-Ser < J (I) X , in which X is (CH,)k wherein k is an integer selected from 1, 2 and 9; or (CH2),^S~(CH2)m wherein m and n each is an integer from 1 to 4 and Y is Trp or D-Trp.

A preferred group of compounds of this invention is represented by formula I in which X is (CH2)nS-(CH2)rn wherein m and n each is an integer from 1 to 4 and Y is Trp or D-Trp.

The pharmaceutically acceptable salts of the compounds of formula I are also included within the scope of this invention.

This invention also provides compounds having the formula:

R4 R5 R6 R7 R8 I I I I CO-ys-Asn-Phe-Phe-Y-Lys-Thr-pha-Thr-Ser-NH (Xa) X in which X is (CH2)" wherein k is an integer selected from 1, 2 and 9; or (CH2)n-S-(CH2)m wherein m and n each is an integer from 1 to 4, Y is Trp or D-Trp, R4 and R5 are protecting groups for the side chain #-amino substituent of lysine and R6, R7 and R8 are protecting groups for the hydroxyl groups of threonine and serine.

Examples of R4 and R are benzyloxycarbonyl, t-butoxycarbonyl, 2-(p-biphenyl)isopropoxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, formyl, trifluoroacetyl, phthalyl, acetyl, tosyl, trityl and benzyl.

Examples of R6, R7 and Rh are selected from acetyl, tosyl, benzoyl, tert-butyl and benzyl.

Preferably R4 and Rj both represent t-butoxycarbonyl and R6, R7 and R8 all represent t-butyl.

Examples of n or m are the integers 2 and 3.

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.

It wil be obvious to those skilled in the art that the protecting groups chosen should be stable under the reaction conditions employed in the processes for preparing the cyclic intermediates.

The invention also provides processes for preparing compounds of formula I as defined above as well as compounds having formula I wherein k is an integer from 3 to 8, or pharmaceutically acceptable salts thereof.

One such process for preparing a compound of formula I wherein X is (CH2)k wherein k is an integer selected from 1, 2 and 9; or (CH2)n-S-(CH2)m wherein n and m are as defined above comprises deprotecting a compound of formula

R4 R5 R8 R? R8 I I I I CO-Lys-Asn-Phe-Phe-Y-Lys-Thr-Phe-rhr-Ser-NH (Xa) x in which formula X is (CH2)k wherein k is an integer selected from 1, 2 and 9; or (CH2)n-S-(CH2)m wherein m and n each is an integer from 1 to 4, Y is Trp or D-Trp, R4 and R5 are protecting groups for the side chain #-amino substituent of lysine and R6, R7 and R8 are independently protecting groups for the hydroxyl groups of threonine and serine, and if desired converting to a pharmaceutically acceptable salt thereof.

The compound of formula (Xa) may be prepared by cyclising a linear undecapeptide intermediate having the requisite, protected amino acid sequence and having Nterminal and C-terminal groups which can be intramolecularly coupled by methods known in peptide and penicillin chemistry to form amide bonds. 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 activating the terminal carboxyl group using, for example, carbonyl diimidazole; a carbodiimide e.g. dicyclohexylcarbodiimide (DCC); in the presence of N-hydroxysuccinimide or 1-hydroxybenzotriazole; and alkoxyacetylene, e.g. ethoxyacetylene. Examples of activated derivatives of the carboxyl group are the acid chloride, anhydride or mixed anhydride, azide and activated ester, e.g. p-nitrophenylester; which terminal groups may be condensed with a N-terminal amino group.

By the term "activated derivative" is meant a derivative in which the terminal carboxyl group is activated so that it is capable of coupling with an amino group to form an amide bond.

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.

Because of the cyclic nature of the undecapeptides of formula (Xa) it is immaterial which amino acid in the sequence is chosen to initiate the synthesis of the linear intermediate therefor; provided that this linear sequence is assembled in the order one would encounter by moving counter-clockwise around the undecapeptide cycle from the amino acid so chosen.

A preferred process for preparing a cyclic compound of formula (Xa) comprises cyclising an activated derivative of a compound of formula

R4 W R6 RT R8 I I! II H-Lys-Asn-Phe-Phe-Y-Lys-Tlr-Phe-Tlr-Ser-NH-X-CÓR8 (VIlla) wherein X, Y, R4, Rl, R6, R1 and R8 are as defined above and R5 is OH.

A preferred activated derivative is the azide which is prepared from the hydrazide (i.e. compound of formula VIIIa wherein RS = NHNH2).

Alternatively a linear undecapeptide of formula

R Rt R7 RS R4 II I H-y-Lys-Tlr-phe-Tlr-ser-NH-x-co-Lys-Asn-phe-phe-NHNH2 (XVa) wherein X, Y, R4, R5, R6, R7 and R8 are as defined above which can be cyclised via the azide coupling method.

It will be obvious to those skilled in the art that if an amino acid other than Llysine or L- or D-tryptophyl is chosen for the first amino acid, the sequence of the linear intermediates will vary but, once cyclized, the required amino acid sequence will be presented by the product undecapeptide cycle.

The compound of formula VIIIa defined herein wherein R3 is OH or NHNH2 may be prepared from a compound of formula:

R4 R Rb RT R8 II II R1-Lys-Asn-Phe-Phe-Y-Lys-Tlr-Phe-Thr-Ser-NH-X-COOR2 (VIIa) wherein X, Y, R4, R5, R6, R7 and R are as defined above, R2 represents lower alkyl and Rl represents an a-amino protecting group removable under conditions which do not remove the protecting groups R4, Rs, R6, R7 and R8 by (i) hydrazinolysis to give a compound of formula

R4 R3 R" R7 Rh II II R'0-Lys-Asn-Phe-Phe-Y-Lys-Tlr-Phe-Tlr-Ser-NH-X-CORll (IXb) wherein X, Y, R4, RS, R6, R7 and R8 are as defined above and R10 represents an a-amino protecting group selectively removable under conditions which do not remove the R4, R5, R6, R7 and R8 protecting groups and R11 represents -NHNH2, followed by selective removal of the R10 protecting group to give a compound of formula (VIIIa) wherein R3 is NHNH2, or (ii) alkaline hydrolysis to give a compound of formula IXb as defined above wherein R11 is OH followed by selective removal of the R10 protecting group to give a compound of formula (VIIIa) wherein R is OH.

The compound of formula VIIa may be prepared by coupling according to the azide coupling method a compound of formula

R4 RS R6 R7 Rb I - II II R1-Lys-Asn-Phe-Phe-Y-Lys-Thr-Phe-Thr-Ser-NHNH2 (Va) with a compound of formula H2N-X-COOR (VI) in which formula X, Y, R4, R5, R6, R7 and R8 are as defined above, R represents lower alkyl, and R is as defined above.

The compound of formula Va as defined above may be prepared by hydrazinolysis of a compound of formula

R4 R R" R7 R8 II R1-Lys-Asn-Phe-Phe-Y-Lys-Thr-Phe-Thr-Ser-O- (lower alkyl) (IVa) wherein X, Y, R, R4, R5, R6, R7 and R8 are as defined above. The compound of formula (IVa) may be prepared by coupling according to the azide coupling method a compound of formula

R4 R1-Lys-Asn-Phe-Phe-NHNH2 (IIa) with a compound of formula

W R6 RT R8 II II H-Y-Lys-Thr-Phe-Thr-Ser-O- (lower alkyl) (IIIa) in which formulae X, Y, R1, R4, R , Rb, R7 and Rb are as defined above.

The compound of formula (IIa) may be prepared by hydrazinolyising of a compound of formula

R4 R1-Lys-Asn-Phe-Phe-O (lower alkyl) wherein R1 and R4 are as defined above.

The compound of formula XVa as defined above may be prepared by hydrazinolysing a compound of formula:

R5 R6 Rb Rb R4 II II I H-Y-Lys-Thr-Phe-Thr-Ser-NHX-CO-Lys-Asn-PhePhe-O (lower alkyl) (XIVa) wherein X, Y, R4, Rs, R6 and R8 are as defined above.

ComDounds of formula (XVb)

R Rb R7 R" R4 II II H-Y-Lys-Tlr-Phe-Thr-Ser-NH-X-CO-Lys-Asn-Phe-Phe-R'2 (XVb) wherein X, Y, R4, R5, R6, R7 and Rb are as defined above and R12 represents -NHNH2 or O(lower alkyl), which includes compounds of formula (XIVa) may be prepared by coupling according to the azide coupling method a compound of formula:

R4 H-Lys-Asn-Phe-Phe-O (lower alkyl) with a compound of formula:

R B R7 R" II II R'-Y-Lys-Thr-Phe-Thr-Ser-NH-X-CO-NHNH2 (XIIa) in which formulae X, Y, R4, R5, R6, R7 and R8 are as defined above and R represents an &alpha;-amino protecting group removable under conditions which do not remove the protecting groups R4, R5, R6, R7 and R8 and selectively removing the R protecting groups, to give a compound of formula (XVb) wherein R12 is -O(lower alkyl), and further, if desired hydrazinolysing to give a compound of formula (XVb) wherein R'2 is -NHNH2.

The compound of formula (XIIa) as defined above may be prepared by hydrazinolysing a compound of formula

W R" R7 R8 II II R'-Y-Lys-Thr-Phe-Thr-Ser-NH-X-COOR2 (XIa) wherein R, X, Y, R5, R6, R7 and R8 are as defined above and R represents lower alkyl.

The compound of formula (XIa) as defined above may be prepared by coupling according to the azide coupling method a compound of formula: H,NX-COOB2- (VI) with a compound of formula

R5 Re RT R8 II II R1-Y-Lys-Thr-Phe-Thr-Ser-NHNH2 in which formulae R, R, X, Y, R5, R6, R7 and R8 are as defined above.

Preferably R represents Ddz, -O(lower alkyl) represents -O(methyl), R represents methyl.

The invention also provides intermediates for preparing the compounds of formula I and Xa, the intermediates having formula (IXb) as hereinbefore defined and wherein Rib may also represent hydrogen, and formula (XVb) as hereinbefore defined.

A particularly preferred porcess for preparing compounds of formula I wherein X is (CH2)k wherein K is an integer from 1 to 9; or (CH2)nS(CH2)m wherein n and m are as defined above comprises; subjecting the cyclic protected peptide of formula X

CO-Lys(Boc)-Asn-Phe-Phe-Y-Lys(Boc)-Thrl8ut)-Phe-Thr(aut)-Ser(Sut)-NH, x .) (X) in which X and Y are as defined herein to moderately acidic conditions to remove the protective groups, and isolating the corresponding compound of formula I.

The compounds of formula (X) in which X and Y are as defined herein are prepared by a process which comprises: reacting according to the azide coupling method a tetrapeptide hydrazide of formula II R'-Lys (Boc) -Asn-Phe-Phe-NHNH2 (11) in which R' is Ddz or Z with a hexapeptide of formula III H-Y-Lys( Boc) Thr(But)-Phe-Thr(But)- -Ser( But) -OMe (III) in which Y is as defined herein to obtain the corresponding decapeptide of formula IV.

R-Lys(Boc)-Asn-Phe-Phe-Y-Lys(Boc)-Thr(But)Phe-Thr(But)-Ser(But)-OMe (IV) in which R' and Y are as defined herein, subjecting the latter compound to hydrazinolysis to obtain the decapeptide hydrazide of formula V R-Lys (Boc) -Asn-Phe-Phe-Y-Lys(Boc) -Thr( But) -Phe-Thr(But) -Ser(But) -NHNH2 (V) in which R and Y are as defined herein, reacting the last-named compound according to the azide coupling method with a compound of formula VI H2N-X-COOB2 (VI) in which X is as defined herein and R2 is lower alkyl to obtain the corresponding com pound of formula VII R-Lys(Boc)-Asn-Phe-Phe-Y-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But) NH-X-COOr (VII) in which R1, R2, X and Y are as defined herein, subjecting the latter compound to hydrazinolysis or alkaline hydrolysis to obtain the corresponding compound of formula VIII R1-Lys(Boc) -Asn-Phe-Phe-Y-Lys( Boc)-Thr( But) -Phe-Thr(But) -Ser(But) - NH-X-CO-Rz (all) in which R, X and Y are as defined herein and R is OH or NHNH2, respectively, removing the protecting group R from the compound of formula VIII when R' is Ddz under mildly acidic conditions or when R is Z and X is (CH2)k with hydrogen in the presence of a noble metal hydrogenation catalyst to obtain the corresponding compound of formula IX H-Lys(Boc)-Asn-Phe-Phe-Y-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But) NH-X-CO-R (IX) in which R , X and Y are as defined herein, and cyclizing the compound of formula IX with a carboxyl group activating agent to obtain the corresponding cyclic protected peptide of formula X in which X and Y are as defined herein. Compounds of formula IX and X as defined herein are within the scope of this invention.

Another embodiment of the process to prepare the compound of formula X in which X is (CH2)n-S-(CH2)m wherein m and n are as defined herein and Y is as defined herein, comprises: reacting according to the azide coupling method the hexapeptide hydrazide, Ddz-Y-Lys (Boc) -Thr(But) -Phe-Thr( But) -Ser(But) -NHNH2 in which Y is as defined herein with a compound of formula VI, H2N-X-COOB2, in which X is (CH2)m-S-(CH2)n wherein m and n are as defined herein and R is lower alkyl to obtain the corresponding compound of formula XI Ddz-Y-Lys(Boc) -Thr( But) -Phe-Thr( But)-Ser(But) -NH-(CH,),-S- (CH, ),-COORZ (Xl) in which m, n, R2 and Y are as defined herein, subjecting the latter compound to hydra zinolysis to obtain the corresponding hydrazide of formula XII Ddz-Y-Lys(Boc) -Thr( Bu' ) -Phe-Thr(Bu' ) -Ser(But ) -NH- ( CH2) S-(CH2)"CONHNH2 (Xll ) in which m, n and Y are as defined herein, reacting the last-named compound according to the azide coupling method with the tetrapeptide H-Lys(Boc)-Asn-Phe-Phe-OMe to obtain the corresponding compound of formula XIII Ddz-Y-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But) NH-(CH2)m-S-(CH2)n-CO-Lys(Boc)-Asn-Phe-Phe-OMe (XIII) in which m, n and Y are as defined herein, subjecting the latter compound to mildly acidic conditions to obtain the corresponding compound of formula XIV H-Y-Lys ( Boc)-Thr(Bu' ) -Phe-Thr(But) -Ser(But) - NH-(CH2)m-S-(CH2)n-CO-Lys(Boc)-Asn-Phe-Phe-OMe (XIV) in which m, n and Y are as defined herein, subjecting the latter compound to hydrazinolysis to obtain the corresponding hydrazide of formula XV h-Y-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH-(CH2)m-s-(CH2)n-CO Lys (Boc) -Asn-Phe-Phe-NHNH2 (XV) in which m, n and Y are as defined herein, and cyclizing the latter compound according to the azide coupling method to obtain the corresponding cyclic protected peptide of formula X in which X is (CH2),1S(CH2)nl wherein m and n are as defined herein and Y is as defined herein.

Another aspect of this invention involves a pharmaceutical composition comprising a compound of formula I and a pharmaceutically acceptable carrier therefor.

Still another aspect involves a method of treating acromegaly or of controlling diabetes in a mammal other than a human which comprises administering to said mammal an effective amount of a compound of formula I.

In general the abbreviations used herein for designating the amino acids and the protective groups are based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature, see Biochemistry, II, 1726-1732 (1972). For instance, Lys, Asn, Asp, Phe, Trp, D-Trp, Thr and Ser represent the "residue" of L-lysine, L-asparagine, L-aspartic acid, L-phenylalanine, L-tryptophan, D-tryptophan, L-threo- nine and L-serine, respectively. The term "residue" refers to a radical derived from the corresponding amino acid by eliminating the hydroxyl of the carboxyl group and one hydrogen of the amino group.

A number of procedures of techniques for the preparation of peptides has hitherto been well established. For instance, the functional groups which are not involved in the peptide bond formation reaction are optionally protected by a protecting group or groups prior to the condensation reaction. For example, protecting groups which may be chosen for an amino function of a peptide or amino acid not involved in the peptide bond formation are: the alkoxycarbonyls which include benzyloxycarbonyl (represented by Z), t-butoxycarbonyl (represented by Boc), &alpha;,&alpha;-dimethyl-3,5-dimethoxybenzyloxycarbonyl (represented by Ddz), 2-(p-biphenyl)-isopropyloxycarbonyl (represented by Bpoc), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, isopropyloxycarbonyl, or ethoxycarbonyl; the acyl type protecting groups which include formyl, trifluoroacetyl, phthalyl, acetyl (Ac), or toluenesulfonyl; the alkyl type protecting groups which include triphenylmethyl or trityl (represented by Trt) or benzyl; the preferred protecting groups in the process of this invention are benzyloxycarbonyl, t-butoxycarbonyl, triphenylmethyl and &alpha;,&alpha;-dimethyl-3,5-dimethoxybenzyloxycarbonyl. The protecting groups for the hydroxyl of serine and threonine are represented by acetyl, tosyl, benz oyl, tert-butyl (represented by But) and benzyl; the preferred protecting group is tertbutyl. The carboxylic acid function of a peptide or amino acid can be considered pro- tected by a lower alkyl or lower aralkyl ester which include methyl (represented by OMe), ethyl (represented by OEt), or benzyl (represented by OBzI) esters; and also by substituted hydrazides which include t-butoxycarbonyl hydrazide (represented by NHNH-Boc), benzyloxycarbonyl hydrazide (represented by NHNHAZ), or a dimethyl-3,5-dimethoxy-benzyloxycarbonyl hydrazide (represented by NHNH-Ddz).

To promote facile condensation of the peptide carboxyl group with a free amino group of another peptide to form a new peptide bond the terminal carboxyl group must be activated. Descriptions of carboxyl activating agents and methods and activated forms are found in general textbooks of peptide chemistry; for example K. D. Kopple, Peptides and Amino Acids", W. A. Benjamin, Inc., New York, 1966, pp. 45-51 and E. Schroder and K. Liibke, "The Peptides"; Vol. 1, Academic Press, New York, 1965 pp. 77-128. Examples of carboxyl group activating agents for a carboxylic acid are thionyl chloride, thionyl bromide, methyl chloroformate, a dialkylcarbodiimide, e.g., dicyclohexylcarbodiimide; N-hydroxysuccinimide, 2,4,5-trichlorophenol, pentachlorophenol, p-nitrophenol or 1-hydrobenzotriazole in the presence of a dialkylcarbodiimide; and in the case of a hydrazide the carboxylic group activating is nitrous acid. Examples of the activated form of the terminal carboxyl are acid chloride, anhydride, azide, activated ester, or O-acyl urea of a dialkylcarbodiimide. The following activated esters have proved to be particularly suitable in the process of this invention: 2,4,5-trichlorophenyl (represented by OTcp), pentachlorophenyl (represented by OPcp), p-nitrophenyl (represented by ONp), or l-benzotriazolyl; the succinimido group is also useful for such activation.

The term "azide coupling method " as used herein refers to the method of activating the terminal carboxyl of a peptide fragment with an azide and condensing the latter peptide azide with another peptide having a free amino group. The peptide azide is conveniently prepared by reacting a peptide hydrazide with a reagent which furnishes nitrous acid in situ. Suitable reagents for this purpose include organic nitrites (e.g. t-butyl nitrite, isoamyl nitrite) or alkali metal nitrite salts (e.g. sodium nitrite, potassium nitrite) in the presence of a mineral acid such as hydrogen chloride or sulfuric or phosphoric acid. The corresponding peptide azide thus obtained is then reacted with a peptide or compound having a free amino group to obtain the desired peptide. Preferred conditions for the azide method of coupling comprise reacting the peptide hydrazide with nitrous acid, generated in situ from an organic nitrite in the presence of a strong acid, preferably hydrogen chloride, (pH ranging usually from 0.1 to 2), in an anhydrous inert organic solvent, for example, dimethylformamide, dimethyl sulfoxide, ethyl acetate, methylene dichloride, tetrahydrofuran and dioxane at - 300C to 20"C, preferably at about - 15 C for 10 to 30 minutes to obtain the corresponding azide. The peptide azide can be isolated and crystallized but is preferably allowed to remain in the reaction mixture. Thereatter the azide in the above mixture is reacted with the peptide unit or compound having the free amino group at temperatures ranging from -300C to 200C for one to two hours and then at 0 to 30"C for 10 to 30 hours. An acid acceptor, preferably an organic base, for example N-ethyldiisopropylamine, N-ethylmorpholine or triethylamine, is present in the reaction mixture in order to make the reaction medium slightly alkaline, preferably pH 7.0 to 9.0. See also the above cited textbooks of Kopple or Schroder and Liibke for additional descriptions of this method.

The terms peptide, polypeptide, tripeptide, hexapeptide, and the like as used herein are not limited to refer to the respective parent peptides but are also used with reference to modified peptides with or without functionalized or protecting groups. The term "peptide" as used herein is used with reference to a peptide with two to ten amino acid residues.

The abbreviation Me represents a methyl group, Et represents an ethyl group, Pr represents a propyl group and NHNH2 represents a hydrazide group.

The term " lower alkyl " as used herein means hydrocarbon radicals having one to three carbon atoms and includes methyl, ethyl, and n-propyl.

The term "mineral acid" as used herein means the strong inorganic acids and includes hydrochloric, hydrobromic, sulfuric, or phosphoric acid. When the term is used in conjunction with an anhydrous system, anhydrous hydrogen chloride is the preferred mineral acid.

The term "mildly acidic conditions" as used herein means conditions in which an aqueous solution of a lower alkanoic acid having 1 to 3 carbon atoms, for example 3080% aqueous formic, acetic or propionic acid, preferably 7080%, or mixtures thereof, is a principal component of the reaction medium.

The term "moderately acidic conditions" as used herein means conditions in which concentrated trifluoroacetic acid or solutions of the mineral acids are used as a principal component of the reaction medium at temperatures ranging from - -300 to 30"C. Examples of preferred conditions in this case include the use of 50 to 100% tri fluoroacetic acid at 0 to 30"C or 0.1-12N hydrochloric acid in aqueous solution or in solution in an organic solvent, or hydrogen chloride in solution in anhydrous organic solvents at - 20 to 10"C.

The term "organic nitrite ' includes the commercially available alkyl nitrites, for instance, t-butyl nitrite and isoamyl nitrite.

The term "organic base" as used herein includes triethylamine, N-ethylmorpholine and N-ethyldiisopropylamine.

The peptides of formula I are obtained in the form of the free base or as an acid addition salt either directly from the process of this invention or by reacting the peptide with one or more equivalents of the appropriate acid. Examples of preferred salts are those with pharmaceutically acceptable organic acids, e.g. acetic, lactic, succinic, benzoic, salicylic, methanesulfonic or toluenesulfonic acid; as well as polymeric acids such as tannic acid or carboxymethyl cellulose, and salts with inorganic acids such as hydrohalic acid, e.g. hydrochloric acid, or sulfuric acid, or phosphoric acid. It should be noted that the peptides have two basic nitrogens giving rise to addition salts with one to possibly two equivalents of acid. If desired, a particular acid addition salt is converted into another acid addition salt, e.g., a salt with a non-toxic, pharmaceutically acceptable acid, by treatment with the appropriate ion exchange resin in the manner described by R. A. Boissonas et al., Helv. Chim. Acta, 43, 1349 (1960). Suitable ion exchange resins are cellulose based cation exchangers, for example carboxymethylcellulose or chemically modified, cross-linked dextran cation exchangers, for example, those of the Sephadex C type, and strongly basic anion exchange resins, for example those listed in J. P. Greenstein and M. Winitz " Chemistry of the Amino Acids ", John Wiley and Sons, Inc., New York and London, 1961, Vol. 2, p. 1456. (SEPHADEX is a Registered Trade Mark.) The peptides of this invention give complex salts with heavy metal ions. An example of a pharmaceutically acceptable heavy metal complex is a complex formed with zinc or with zinc protamine.

Further reference to the terminology, reaction methods and conditions used herein is found in issued U.S. Patent No. 3,917,578, entitled "Process for Producing Somatostatin and Intermediates Therefor" issued November 4, 1975 and U.S. Patent No. 3,917,581, entitled "Derivatives of Somatostatin and Process Therefor" issued November 4, 1975, the disclosures of which are incorporated herein by reference.

The peptides produced by the process of this invention, as well as their corresponding pharmaceutically acceptable salts, are useful because they possess the pharmacological activity of the natural tetradecapeptide somatostatin. Their activity is demonstrated rapidly in pharmacological tests such as a modification [A. V. Schally et al., Biochem. Biophys. Res. Commun., 52, 1314 (1973) and J. Rivier et al., C. R. Acad.

Sci. Paris, Ser. D, 276, 2737 (1973)] of the in vitro method of M. Saffran and A. V.

Schally, Can. J. Biochem. Physiol., 33, 405 (1955).

The activity of the peptides of formula I of this invention is demonstrated also n vivo in a modification of the pentobarbital-induced increase in plasma growth hor saone level in the rat as described by Brazeau et al., cited above. In this test the peptides of this invention show a level of activity which is greater than or of the same order as that of somatostatin.

The peptides of formula I or the salts thereof are useful for the treatment of acromegaly and related hypersecretory endocrine states and in the managament of diabetes in mammals; see for example, P. Brazeau et al., cited above. When the peptides or salts thereof are employed for such treatment or management, they may be administered systemically, preferably parenterally, in combination with a pharmaceutically acceptable liquid carrier. The peptides of formula I have a low order of toxicity. The proportion of the peptide or salt thereof is determined by its solubility in the given carrier, by the given carrier, or by the chosen route of administration. When the peptide or a salt thereof is used in a sterile aqueous solution, such solution may also contain other solutes such as buffers or preservatives, as well as sufficient amounts of pharmaceutically acceptable salts or glucose to make the solution isotonic. The dosage will vary with the form of administration and with the particular species to be treated and is preferably kept at a level of from 1 mcg to 300 mcg per kilogram body weight. However, a dosage level in the range of from 1 mcg to 50 mcg per kilogram body weight is most desirably employed in order to achieve effective results.

The peptides or salts thereof may also be administered in one of the long-acting, slow-release or depot dosage forms described below, preferably by intramuscular injection or by implantation. Such dosage forms are designed to release from 0.1 mcg to 50 mcg per kilogram body weight per day.

It is often desirable to administer the agent continuously over prolonged periods of time in long-acting, slow-release or depot dosage forms. Such dosage forms may either contain a pharmaceutically acceptable salt of the peptide having a low degree of solubility in body fluids, for example one of those salts described below, or they may contain the peptide in the form of a water-soluble salt together with a protective carrier which prevents rapid release. In the latter case, for exarnple, the peptide may be formulated with a non-antigenic partially hydrolyzed gelatin in the form of a viscous liquid; or the peptide may be adsorbed on a pharmaceutically acceptable solid carrier, for example, zinc hydroxide, and may be administered in suspension in a pharmaceutically acceptable liquid vehicle; or the peptide may be formulated in gels or suspensions with a protective non-antigenic hydrocolloid, for example sodium carboxymethylcellulose, polyvinylpyrrolidone, sodium alginate, gelatine, polygalacturonic acids, for example, pectin, or certain mucopolysaccharides, together with aqueous or non-aqueous pharmaceutically acceptable liquid vehicles, preservatives, or surfactants. Examples of such formulations are found in standard pharmaceutical texts, e.g. in Remington's Pharmaceutical Sciences, 14th Ed., Mack Publishing Co., Easton, Pennsylvania, 1970. Longacting, slow-release preparations of the peptide produced according to the process of this invention may also be obtained by microencapsulation in a pharmaceutically acceptable coating, for example, gelatine, polyvinyl alcohol or ethyl cellulose. Further examples of coating materials and of the processes used for microencapsulation are described by J. A. Herbig in "Encyclopedia of Chemical Technology", Vol. 13, 2nd Ed., Wiley, New York 1967, pp. 436 456. Such formulations, as well as suspensions of salts of the peptide which are only sparingly soluble in body fluids, for example salts with pamoic acid or tannic acid, are designed to release from 1.0 mcg to 100 mcg of the active compound per kilogram body weight per day, and are preferably administered by intramuscular injection. Alternatively, some of the solid dosage forms listed above, for example certain sparingly water-soluble salts or dispersions in or adsorbates on solid carriers of salts of the peptide, for example dispersions in a neutral hydrogel of a polymer of ethylene glycol methacrylate or similar monomers crosslinked as described in U.S. Patent 3,551,556, may also be formulated in the form of pellets releasing about the same amounts as shown above and may be implanted subcutaneously or intramuscularly.

The process of this invention will be illustrated by the following embodiments in which specific peptides of formula I are prepared.

In one embodiment for preparing a compound of formula I in which X and Y are as defined herein, the requisite first starting material, the tetrapeptide hydrazide of formula II, R1-Lys(Boc) -Asn-Phe-Phe-NHNH2 in which R1 is Z or Ddz is readily obtained by hydrazinolysis of the corresponding tetrapeptide ester R1-Lys(Boc)-Asn Phe-Phe-OMe in which R1 is as defined herein. The latter tetrapeptide in which R1 is Z is described by H. U. Immer et al., cited above, and the tetrapeptide of formula Ddz-Lys(Boc)-Asn-Phe-Phe-OMe (i.e. R1 is Ddz) is readily prepared by following the procedure described in the latter reference but replacing Z-Lys(Boc)-OH with Ddz-Lys(Boc)-OH. The hydrazinolysis is conveniently carried out by dissolving the tetrapeptide ester in an inert organic solvent, for example, methanol, ethanol and dimethylformamide, preferably dimethylformamide. The solution is treated with an excess of hydrazine hydrate, for example, 15. to 30 molar equivalents. The reaction mixture is kept at 0 to 100C for 12 to 30 hours. The corresponding tetrapeptide hydrazide of formula II is obtained by conventional means, for example, evaporation or precipitation with water.

The requisite second starting material, the hexapeptide of formula III, H-Y Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-OMe in which Y is Trp is obtained by amino deprotection of the hexapeptide Ddz-Trp-Lys ( Boc) -Thr (But) -Phe- Thr(But)-Ser(But)-OMe (described by H. U. Immer et al., cited above) under mildly acidic conditions, preferably by allowing the latter hexapeptide to stand in a solution of acetic acid-formic acid-water (7:1:2 by volume) at 20"C to 300C for 12 to 30 hours. By following the procedure described by H. U. Immer et al., cited above, but replacing L-tryptophan with D-tryptophan, the hexapeptide Ddz-D-Trp Ifys(Boc)-Thr(But)-Phe-Thr(Bit) -Ser(Bu')-OMe is obtained. Deprotection of the latter compound under mildly acidic conditions gives the hexapeptide of formula Ill in which Y is D-Trp.

In the next step of the present process, the tetrapeptide hydrazide of formula II and the hexapeptide of formula III are coupled according to the azide coupling method to obtain the corresponding decapeptide of formula IV, R1-Lys(Boc)-Asn-Phe-Phe-Y Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-OMe in which R1 and Y are as defined herein.

A convenient and efficacious procedure for this step comprises dissolving the tetrapep tide hydrazide of formula II in an inert organic solvent, preferably dimethylform amide, and cooling the mixture to 200 to - 10"C. A solution of two to give molar equivalents of a mineral acid in an inert organic solvent, preferably three molar equivalents of hydrogen chloride in ethyl acetate, is added to the above solution, followed by 1.0 to 1.5 molar equivalents of an organic nitrtite, for example, 1.2 molar equivalents of t-butyl nitrite. In this manner the corresponding tetrapeptide azide, R'- Lys(Boc)-Asn-Phe-Phe-N1 in which Rl is as defined herein, is obtained. After 10 to 20 minutes at - 200 to - 100C an organic base, preferably N-ethyldiisopropylamine, is added until pH 7.1 to 9 is attained. The mixture is cooled to - 300 to - 200C and a solution of substantially one molar equivalent of the hexapeptide of formula III in an inert organic solvent, preferably dimethylformamide, is added to the above solution containing said azide. The reaction mixture is then stirred at - 200 to - 100C for one to two hours and then at 200 to 30"C for 20 to 30 hours. The solvent is evaporated under reduced pressure. The residue is dissolved in methanol and water is added. The precipitate is subjected to chromatography on silica gel to obtain the corresponding decapeptide of formula IV in which R1 and Y are as defined herein.

The decapeptide of formula IV is readily converted to the corresponding decapeptide hydrazide of formula V,R'-Lys(Boc) -Asn-Phe-Phe-Y-Lys( Boc)-Thr(But)-Phe- Thr(But)-Ser(But)-NHNH2 in which R1 and Y are as defined herein, by hydrazinolysis. This hydrazinolysis is carried out by dissolving the decapeptide of formula IV in an inert organic solvent, preferably dimethylformamide, and adding an excess of hydrazine hydrate, for example 10 to 30 molar equivalents. After stirring at 200 to 30"C for four to six days the corresponding decapeptide hydrazide of formula V is isolated by conventional means.

The aforementioned decapeptide hydrazide of formula V and the compound of formula VI, H2N-X-COOB2 in which R2 and X are as defined herein, are coupled according to the azide coupling method, in the same manner as described hereinabove, to obtain the corresponding compound of formula VII, R1-Lys(Boc)-Asn-Phe-Phe-Y Lys(Boc) -Thr(Bu )-Phe-Thr( But)-Ser(But) -NH-X-COOR2 in which R1, R2, X and Y are as defined herein.

The compounds of formula VI in which X is (CH2)k wherein k is as defined herein are either known or are readily prepared by known procedures, for example, 4aminobutanoic acid methyl ester, 5-aminopentanoic acid methyl ester, 6-aminohexanoic acid ethyl ester and 8-aminooctanoic acid ethyl ester are described by E. Neuzil and D. Reiss, J. Chromatog., 21, 355 (1966) (Chem. Abstr., 64, 14262 C) and 9-aminonanoic acid methyl ester is described in French Patent No. 1,087,798 (Chem. Abstr., 53, 6089 h).

The compounds of formula VI in which X is (CH2)",S(CH2)" wherein m and n are as defined herein are readily prepared by alkylation of an w-aminoalkanethiol.

For example, a convenient preparation comprises reacting liquid ammonia with lithium and hydrated ferric nitrate until the blue color disappears, adding an to-aminoalkane- thiol, H,N--(CH,),,,,-SH in which m is as defined herein, adding an w-bromoalkanoic acid ester, Br-(CH2)n-COOR in which n and R are as defined herein, and isolating the corresponding compound of formula VI in which X is (CH2)m-S-(CH2)n by conventional methods.

The compound of formula VII is reacted with hydrazine hydrate in the manner as described above to obtain the corresponding hydrazide of formula VIII, R1 Lys(Boc) - Asn - Phe - Phe - Y - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) NH - X - CO - R3 in which R', X and Y are as defined herein and Ra is NHNH2.

The a-amino protecting group (R1) is removed from said last-named compound of formula VIII to obtain the corresponding Amino deprotected compound of formula IX, H - Lys(Boc) - Asn - Phe - Phe - Y - Lys(Boc) - Thr(But) - Phe - Thr(Bu') Ser(But) - NH - X - CO - B2 in which X and Y are as defined herein and R3 is NHNH2.

When R' is the a-amino protecting group Z and X is (CH2)x, the protecting group (Z) is conveniently removed by hydrogenolysis, preferably by agitaung under an atmosphere of hydrogen a mixture of the compound of formula VIII in which RS is NHNH2 and a noble metal hydrogenation catalyst, for example, platinum or palladium on carbon in an inert organic solvent, for example, methanol, ethanol or acetic acid.

The a-amino protecting group (Ddz) is readily removed from the compound of formula VIII in which R1 is Ddz and Y and X are as defined herein and B2 is NHNH2 by treatment under mildly acidic conditions, preferably by allowing the compound of formula VIII to stand in a solution of acetic-formic acid-water (7:1:2 by volume) at 20 to 30 C for 12 to 24 hours to obtain the corresponding compound of formula IX in which X and Y are as defined herein and R is NHNH2.

Alternatively, the compound of formula VII is hydrolyzed under alkaline condi tions, preferably with two to six molar equivalents of sodium or potassium hydroxide in an inert solvent, for example, water, methanol, ethanol, isopropanol and acetone or mixtures thereof, at 0 to 300C for 10 to 60 hours. The latter alkaline solution is acidified with citric acid and the corresponding compound of formula VIII, B1 - Lys(Boc) Asn - Phe - Phe - Y - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH CO - RS, in which R1, X and Y are as defined herein and R2 is OH is isolated by conventional means, for instance, filtration and extraction.

The amino protecting group R' is removed from the latter compound of formula VIII in which RG is OH, in the same manner as described above for the compound of formula VIII in which B' is NHNH2, to give the corresponding compound of formula IX, H - Lys(Boc) - Asn - Phe - Phe - Y - Lys(Boc) - Thr(But) - Phe - Thr(But) Ser(But) - NH - X - CO - R3, in which X and Y are as defined herein and B1 is OH.

The compounds of formula IX is cyclized in the presence of a carboxyl group activating agent to obtain the corresponding compound of formula X,

90-Lys(Boc')-Asn-Phe-Phe-Y-Lys(Bac)-Thr( )-SerC8u X --- in which X and Y are as defined herein. To achieve this cyclization the terminal carboxyl in the compound of formula IX is activated with a carboxyl group activating agent.

Examples of the carboxyl group activating agents and activated forms of the terminal carboxyl are described above. The preferred activated forms for this cyclization are selected from activated esters, O-acyl urea of a dialkylcarbodiimide and azide.

The compound of formula IX in which B1 is NHNH2 is cyclized according to the conditions of the azide coupling method to obtain the corresponding cyclic protected compound of formula X, in which X and Y are as defined herein. The latter azide coupling is preferably carried out by dissolving the compound of formula IX in which R3 is NHNH2 in an inert organic solvent, preferably dimethylformamide, cooling the mixture to - 200 to - 100C, adding a solution of two to five molar equivalents of a mineral acid in an inert organic solvent, preferably three molar equivalents of hydrogen chloride in ethyl acetate, adding 1.0 to 1.5 molar equivalents of an organic nitrite, for example, 1.2 molar equivalents of t-butyl nitrite, and stirring the mixture for 10 to 20 minutes at -20" to - 100C. In this manner the corresponding azide, H - Lys(Boc) Asn - Phe - Phe - Y - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH CON3 in which X and Y are as defined herein, as the hydrochloride salt is present in the solution. The mixture containing the latter azide is diluted with ten times the amount of its volume of an inert organic solvent, preferably dimethylformamide, precooled to -20 to -10 C. An organic base, preferably N-ethyldiisopropylamine, is added until pH 7.1 to 9 is attained. The mixture is stirred at - 20 to - 10 C for 30 minutes to two hours, then at about 0 C for two days and finally at 200 to 300C for about one day. Said cyclic protected compound of formula X is isolated from the reaction mixture by conventional methods, for example, evaporation, precipitation and chromatography.

Similarly, the terminal carboxyl in the compound of formula IX in which R3 is OH is activated with a carboxyl group activating agent and cyclization occurs to give the corresponding compound of formula X. The preferred method of activating the carboxyl in the compound of formula IX in which R3 is OH and cyclizing is to use the activated ester method. A convenient process for carrying out this latter cyclization is to stir a solution of the compound of formula IX in which B3 is OH with two to six molar equivalents of N-hydroxysuccinimide, 2,4,5-trichlorophenol, pentachlorophenol, p-nitrophenol or 1-hydroxybenzotriazole and one to five molar equivalents of dicyclohexylcarbodiimide in an inert organic solvent at 0 to 50C for 10 to 30 hours and at 20 to 300C for two to ten days. An organic proton acceptor, preferably triethylamine, N-ethylmorpholine or N-ethyldiisopropylamine, is added to the reaction solution in order to maintain the solution at a pH of 7 to 9. Suitable inert organic solvents can be selected from methylene chloride, chloroform, ethyl acetate and dimethylformamide.

The corresponding compound of formula X is isolated from the reaction mixture by conventional methods.

Finally, the aforementioned cyclic protected compound of formula X is transformed to the corresponding compound of formula I in which X and Y are as defined herein by subjecting the former to moderately acidic conditions whereby the remaining protecting groups of the cyclic compound of formula X are removed. Generally this step is carried out by dissolving the cyclic compound in an aqueous reaction medium containing a mineral acid at 0 to 20 C for 10 to 60 minutes. Examples of such media are trifluoroacetic acid, 10 to 20% v/v aqueous sulfuric acid, 10% v/v phosphoric acid, 10 to 30% w/w hydrobromic acid and 10 to 30% w/w hydrochloric acid. An extremely useful medium is concentrated hydrochloric acid. Preferred conditions for the present step include dissolving the cyclic compound of formula X in a minimum of concentrated hydrochloric acid cooled to 0 C and allowing the mixture to stand at 0 C for five to ten minutes under a nitrogen atmosphere. Thereafter, 5 to 15 volumes of glacial acetic acid is added and the solution is cooled to about - 700C and lyophilized to give the cyclic compound of formula I. The latter product is purified further by ion exchange chromatography, preferably using a carboxymethyl cellulose cation exchanger and aqueous ammonium acetate as the eluant. In this case the product is obtained in the form of its acid addition salt with acetic acid. Alternatively, the product is purified by partition chromatography on a chemically modified cross-linked dextran; for example, Sephadex LH-20 using methanol as the eluant, and obtaining the product as the free base, or alternatively using Sephadex G-25 and eluting with the upper phase of butanol-acetic acid-water (4:1:5 by volume) and obtaining the product in the form of its acid addition salt with acetic acid. Evaporation of the eluates, taking up the residue in water and lyophilization yields a substantially pure compound of formula I in which X and Y are as defined herein.

In another embodiment of the process of this invention another process for preparing a compound of formula I in which X is (CH2)nS(CH2)m is described. In this embodiment the requisite first starting material of formula XI, Ddz-Y-Lys(Boc) Thr(Bu")-Phe-Thr(But) -Ser(Bu)-NH-( CH,),-S-(CH2),-COOR2 in which m, n and Y are as defined herein and R2 is lower alkyl, is readily obtained by coupling the hexapeptide hydrazide, Ddz-Y-Lys( Boc) -Thr(But) -Phe-Thr(But) -Ser (But) -NHNH2 in which Y is as defined herein with a compound of formula VI in which X is (CH2)mS(CH2).ji wherein m and n are as defined herein and R2 is as defined herein according to the conditions of the azide coupling method, as described above.

The compound of formula Ddz-Y-Lys(Boc) -Thr (But) -Phe-Thr( But) -Ser( But) - NHNH2 in which Y is Trp is described by H. U. Immer et al., cited above, and the corresponding compound in which Y is D-Trp is- similarly prepared but replacing Ddz-Trp-OH with Ddz-D-Trp-OH.

In the next step of the present process the compound of formula XI is reacted with a molar excess of hydrazine hydrate, preferably at 20 to 300C for two to five days, to obtain the corresponding hydrazide of formula XII, Ddz-Y-Lys(Boc)-Thr (But)-Phe-Thr(But) -Ser(But) -NH-(CH?r),-S-( CHI),-CONHNH, in which m, n and Y are as defined herein. Said last-named compound of formula Xii is converted to the corresponding azide of formula Ddz-Y-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser (But)-NH-(CH2)m-S-(CH2)n-CON3 in which m, n and Y are as defined herein and said azide is coupled with the tetrapeptide of formula H-Lys(Boc)-Asn-Phe-Phe-OMe, described by H. U. Immer et al., cited above, according to the conditions of the azide coupling method, as described above, to obtain the corresponding compound of formula XIII, Ddz - Y - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)m- S - (CH2) - Co - Lys(Boc) - Asn - Phe - Phe - OMe in which m, n and Y are as defined herein.

The amino protecting group (Ddz) is readily removed from the compound of formula XIII by treatment under mildly acidic conditions, preferably by allowing the compound of formula XIII to stand in a solution of acetic acid-formic acid-water (7:1:2 by volume) at 200 to 300C for 12 to 24 hours to obtain the corresponding compound of formula XIV, H - Y - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) NH - (CH2)m - S - (CH2)n - CO - Lys(Boc) - Asn - Phe - Phe - OMe in which m, n and Y are defined herein. Said compound formula XIV is reacted with a molar excess of hydrazine hydrate at 20 to 30 C for 20 to 30 hours to obtain the corresponding hydrazide of formula XV, H - Y - Lys(Boc)- Thr(But) - Phe - Thr(But) - Ser (But) - NH (CH2)" - S - (CH2)n - CO - Lys(Boc) - Asn - Phe - Phe - NHNH, in which m, n and Y areas defined herein.

The hydrazide of formula XV is cyclized according to the conditions of the azide coupling method, in the same manner as described above for the cyclization of the compound of formula IXa, to obtain the corresponding cyclic protected compound of formula X in which X is (CH2)n-S-(CH2)m wherein m and n are defined herein, and Y is as defined herein. The latter compound of formula X is identical in all respect to the corresponding compound of formula X obtained from the previously described process. Finally, the latter compound of formula X is subjected to moderately acidic conditions and purified as described above to obtain the corresponding compound of formula I in which X is (CH2)-S-(CH2) wherein m and n are as defined herein and Y is as defined herein which is identical to the corresponding compound of formula I obtained from the previously described process.

The following examples illustrate further this invention.

EXAMPLE 1.

, - Dimethyl - 3,5 - dimethoxybenzyloxycarbonyl - (N' - t - butoxycarbonyl)lysyl- asparaginyl - phenylalanyl - phenylalanine Methyl Ester (Ddz - Lys(Boc) - Asn Phe- Phe - OMe) A mixture of Ne-tert-butyloxycarbonyllysine (10 g, 40.7 mole) in methanol (500 ml) and benzyltrimethylammonium hydroxide (35 /O w/w in methanol, 19.4 g, 40.7 mmole) is heated on a steam bath until the amino acid completely is dissolved.

The solution is concentrated and the gelatinous product is azeotroped with benzene (3 x 20 ml) and dried under reduced pressure. The residue is dissolved in pyridinedimethylformamide (2:1 by volume) (600 ml) and stirred at 47 C under a nitrogen atmosphere. &alpha;&alpha;-Dimethyl-3,5-dimethoxybenzyloxycarkyl azide (10.78 g, 40.7 mmole) in pyridine (100 ml) is added dropwise over 0.5 hour to the mixture. The temperature is maintained at 47 C and stirring is continued for 3 days. The reaction mixture is concentrated to a viscous oil. The oil is disolved in 0.1N sodium hydroxide (300 ml) and extracted with diethyl ether (3 x 150 ml). The aqueous layer is acidified with 50% w/v aqueous citric acid to washed with saturated sodium bicarbonate solution, saturated sodium chloride solution, dried over magnesium sulfate and evaporated. The residue is subjected to chromatography on silica gel using ethyl acetate-benzene (1:1 by volume) and the eluates are evaporated. The residue is crystallized from acetone-benzene to give the title compound, mp 162--166"C.

EXAMPLE 4.

Tryptophyl - (N - t - butoxycarbonyl)lysyl - (O - t - butyl)threonyl - phenylalanyl (0 - t - butyl)threonyl - (0 - t - butyl)serine Methyl Ester (H - Trp - Lys (Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - OMe) III; Y=Trp A solution of Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) OMe (2.037 g, described by H. U. Immer et al., cited above) in 28 ml of a solvent consisting of acetic acid-formic acid-water (7:1:2 by volume) is stirred at room temperature overnight. The solvent is removed under reduced pressure. The residue is dissolved in methanol, neutralized with ammonium hydroxide and water is added. The solvents are decanted, water is added to the residue and the mixture is stirred until a solid white powder forms. The powder is collected and dried under reduced pressure over phosphorus pentoxide to give the title compound.

In the same manner but replacing Ddz - Trp - Lys(Boc) - Thr(But) - Phe Thr(But) - Ser(But) - OMe with an equivalent amount of Ddz - D - Trp - Lys(Boc) Thr(But) - Phe - Thr(But) - Ser(But) - OMe (described in Example 3), H Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - OMe is obtained.

EXAMPLE 5.

Benzyloxycarbonyl - (N - t - butoxycarbonyl)lysyl - asparaginyl - phenylalanyl phenylalanyl - tryptophyl - (Nb - t - butoxycarbonyl ) lysyl - (O - t - butyl)thre- onyl - phenylalanyl - (0 - t - butyl)threonyl - (0 - t - butyl serine Methyl Ester (Z - Lys(Boc) - Asn - Phe - Phe - Trp - Lys(Boc) - Thr(But) - phe - Thr (But) - Ser(But) - OMe) IV; R1 =Z and Y = Trp A solution of Z-Lys(Boc)-Asn-Phe-Phe-NHNH, (1.023 g, described in Example 2 in dimethylformamide (20 ml) is cooled to -20 C. Hydrogen chloride (2.7M) in ethyl acetate (1.2 ml) is added followed by t-butyl nitrtite (0.18). The solution is stirred at - 15 C for 15 min. and N-ethyldiisopropylamine (0.79 ml) is added. The mixture is cooled to - 30 C and H-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser- (But)-OMe (1.366 g, described in Example 4) is added, followed by N-ethyldiisopropylamine (0.1 ml) in order to bring the pH to 8. The mixture is stirred at - 15"C for one hour, at 0 C for one hour and at room temperature overnight. The solvent is removed under reduced pressure and the residue is suspended in methanol. Water is added, the precipitate is collected and washed with methanol. The precipitate is subjected to chromotography on silica gel using 4% v/v methanol and 0.1% v/v triethylamine in chloroform. The eluates are evaporated to give the title compound, amino acid analysis: Lys (1.98), Asp (0.99), Thr (1.89), Ser (0.93), Phe (3.00) and Trp (present).

In the same manner Z - Lys(Boc) - Asn - Phe - Phe - NHNH2 or Ddz - Lys (Boc) - Asn - Phe - Phe - NHNH2 (described in Example 2) is coupled with H Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - OMe or H - D - Trp Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - OMe (described in Example 4) to obtain the following compounds of formula IV: Z - Lys(Boc) - Asn - Phe - Phe D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - OMe, Ddz - Lys (Boc) - Asn - Phe - Phe - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) OMe, mp 210-2120C and [a]D2D - 2.5 (c= 1, DMF), and Ddz - Lys(Boc) - Asn Phe - Phe - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - OMe, mp 179-1830C.

EXAMPLE 6.

Benzyloxycarbonyl (N - t - butoxycarbonyl)lysyl - asparaginyl - phenylalanyl phenylalanyl - tryptophyl - (Nb - t butoxycarbonyl)lysyl - (O - t - butyl)thre- onyl - phenylalanyl - (0 - t - butyl)threonyl - (0 - t - butyl)serine Hydrazide (Z - Lys(Boc) - Asn - Phe - Phe - Trp - Lys(Boc) - Thr(But) - Phe - Thr- (But) - Ser(But) - NHNH2) V; R;=Z and Y=Trp Hydrazine hydrate (1 ml) is added to a solution of the compound of formula IV, Z - Lys(Boc) - Asn - Phe - Phe - Trp - Lys(Boc) - Thr(But) - Ser (But) - OMe (1.15 g, described in Example 5), in dimethylformamide (13 ml) at 0 C and the solution is stirred at room temperature for 5 days. The solution is cooled to 0 C and water is added. The precipitate is collected and dried under reduced pressure over phosphorus pentoxide to give the title compound: [&alpha;]D25 = -2.12 (c=1, DMF); amino acid analysis: Lys (1.98), Trp (trace), Asp (100), Thr (1.83), Ser (0.97) and Phe (3.19).

In the same manner but replacing theabove starting material of formula IV with an equivalent amount of the other compounds of formula IV described in Example 5, the following compounds of formula V are obtained: Z - Lys(Boc) - Asn - Phe - Phe D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NHNH2, nmr (DMSO-d6) # 1.07, 1.1 and 1.17 (singlets for But and CH3), 3.73 (singlet for OCH3) and 7.13 to 7.23 (multiplet for aromatic), Ddz - Lys(Boc) - Asn - Phe - Phe - Trp Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NHNH2, nmr (DMSO-d6) # 1.07, 1.1 and 1.17 (singlets for But and CH3), 3.73 (singlet for OCH3) and 7.13 to 7.23 (multiplet for aromatic) and Ddz - Lys(Boc) - Asn - Phe - Phe - D - Trp - Lys (Boc) - Thr(But) - Phe -Thr(But) - Ser(But) - NHNH2, nmr (DMSO-d6 # 1.06, 1.11 and 1.18 (singlets for But and CH3 ), 1.38 (singlet for Boc), 3.62 (singlet for OCH3) and 6.85 to 7.1 (multiplet for aromatic).

EXAMPLE 7.

4 - [2 - Aminoethylthio] - butanoic Acid Ethyl Ester (H2N-(CH,)-S-(CH2), COOEt) VI; X=(CH2)2-S-(CH2)3 and R = Et Lithium (1.5 g, 0.2 mole) is stirred with 150 ml of liquid ammonia containing 50 mg of ferric nitrate (hydrated) till the blue color disappears and a grey suspension is obtained. 2-Aminoethanethiol hydrochloride (11.4 g, 0.1 mole) is added with stirring and the mixture is stirred for 30 minutes. 4-Bromobutanoic acid ethyl ester (18.1 g, 0.1 mole) is added and the ammonia is removed. Diethyl ether (200 ml) is added and the solution is washed with water and dried over sodium sulfate. The solvent is removed. The oily residue is subjected to chromatography on silica gel using 10% v/v methanol and 0.1% v/v triethylamine in chloroform. The eluates are evapored to give the title compound, nmr (CDCl3) # 1.28 (t,J = 7Hz, 3H), 1.53 (s,2H), 2.0 (m, 2H), 2.3 (m,8H) and 4.8 (q,J = 7Ht, 2H).

In the same manner by following the above procedure other compounds of formula VI in which X is (CH2)m-s-(CH2)n are obtained. For example, replacing 2-aminoethanethiol with an equivalent amount of 3-aminopropanethiol and replacing 4-bromobutanoic acid ethyl ester with an equivalent amount of 3-bromopropanoic acid ethyl ester, 3-(3-aminopropylthio)-propanoic acid ethyl ester [H2N-( CH2) '-S - (CH2)2-COOEtj is obtained.

Also, in the same manner the following compounds of formula VI in which X is (CH2)m-S-(CH2)n are obtained: H2N-(CH2)3-S-CH2-COOMe, H2N-(CH2)2-S-(CH2)4-COOMe and H2N-(CH2)4-S-(CH2)4-COOEt.

EXAMPLE 8.

Benzyloxycarbonyl - (N - t - butoxycarbonyl)lysyl - asparaginyl - phenylalanyl phenylalanyl - tryptophyl - (N - t - butoxycarbonyl)lysyl - (O - t - butyl)thre onyl - phenylalanyl - (O - t - butyl)threonyl - (O - t - butyl)seryl - 7 - amino heptanoic acid methyl ester (Z - Lys(Boc) - Asn - Phe - Phe - Trp - Lys(Boc) Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)6 - COOMe) VII; R4 = Z R = Me, X = (CH2)6 and Y = Trp A solution of the compound of formula V, Z - Lys(Boc) - Asn - Phe - Phe - Trp Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NHNH2 (10.4 g, described iv Example 6), in dimethylformamide (91 ml )is cooled to -20 C. Hydrogen chloride (2.25 M) in ethyl acetate (6.1 ml) is added followed by t-butyl nitrite (0.763 ml).

The solution is stirred at -15 C for 15 minutes and N-ethyldiisopropylamine (3.3 ml) is added. The mixture is cooled to -30 C and the compound of formula VI, 7-aminoheptanoic acid methyl ester hydrochloride [1.08 g, prepared as described by E.M.

Schultz, J. Am. Chem. Soc., 69, 1056 (1947)], in dimethylformamide (10 ml) is added. The mixture is stirred at -15 C for one hour, at 0 C for one hour and at room temperature overnight. The solvent is removed under reduced pressure and the residue is triturated with methanol to give the title compound, amino acid analysis: Lys (2.00), Asp (0.90), Thr (1.86), Ser (0.94 and Phe (3.00).

By following the procedure of Example 8 using the appropriate starting material of formula V (described in Example 6) and the appropriate starting material of formula VI, other compounds of formula VII in which R1, R2 and Y are as defined herein are obtained. Examples of the latter compounds of formula VII are listed as products in Table 1 together with the appropriate starting materials of formula V and VI used for the preparation of the compound of formula VII.

TABLE 1

Starting Material of Formula V Product of Formula VII Starting Material of Ex. R' Y Formula VI R' R2 X Y 9 Z Trp HNH-(CH2)3-COOEt Z Et (CH2)3 Trp 10 Z Trp HNH-(CH2)5-COOEt Z Et (CH2)5 Trp 11 Z Trp HNH-(CH2)8-COOMe Z Me (CH2)8 Trp 12 Z D-Trp HNH-(CH2)3-COOEt Z Et (CH2)3 D-Trp 13 Z D-Trp HNH-(CH2)5-COOEt Z Et (CH2)5 D-Trp 14 Z D-Trp HNH-(CH2)8-COOMe Z Me (CH2)8 D-Trp 15 Ddz Trp HNH-(CH2)4-COOEt Ddz Et (CH2)4 Trp 16 Ddz Trp HNH-(CH2)5-COOEt Ddz Et (CH2)5 Trp 17 Ddz Trp HNH-(CH2)6-COOMe Ddz Me | (CH2)6 Trp nmr(DMSO-d6) # 1.05, 1.16, 3.47 and 3.2 (singlets) and 6.82 to 6.95 (multiplet) 18 Ddz Trp HNH-(CH2)8-COOMe Ddz Me (CH2)8 Trp 19 Ddz Trp HNH-(CH2)2S(CH2)3-COOEt Ddz Et (CH2)2S(CH2)3 Trp 20 Ddz Trp HNH-(CH2)3S(CH2)2-COOEt Ddz Et (CH2)3S(CH2)2 Trp 21 Ddz Trp HNH-(CH2)2S(CH2)4-COOMe Ddz Me (CH2)2S(CH2)4 Trp 22 Ddz D-Trp HNH-(CH2)3-COOEt Ddz Et (CH2)3 D-Trp 23 | Ddz | D-Trp | HNH-(CH2)6-COOMe | Ddz | Me | (CH2)6 | D-Trp amino acid analysis: Lys(2.0), Thr(1.85), Phe(3.06), Asn(1.03) and Ser(0.99) 24 Ddz D-Trp HNH-(CH2)8-COOMe Ddz Me (CH2)8 D-Trp 25 Ddz D-Trp HNH-(CH2)2S(CH2)3-COOEt Ddz Et (CH2)2S(CH2)3 D-Trp nmr(CDCl3) # 1.18(m), 1.45(s), 2.45(m), 3.78(s), 4.1(m) and 7.2(m) 26 Ddz D-Trp HNH-(CH2)3S(CH2)2-COOEt Ddz Et (CH2)3S(CH2)2 D-Trp 27 DDz D-Trp HNH(CH2)4S(CH2)4-COOEt Ddz Et (CH2)4S(CH2)4 D-Trp EXAMPLE 28.

Benzyloxycarbonyl - (NE - t - butoxycarbonyl)lysyl - asparaginyl - phenylalanyl phenylalanyl - tryptophyl - (Ne - t - butoxycarbonyl)lysyl - (O - t - butyl)thre onyl - phenylalanyl (0 - t - butyl)threonyl - (0 - t - butyl)seryl - 7 - amino heptanoic Acid Hydrazide (Z - Lys(Boc) - Asn - Phe - Phe - Trp - Lys(Boc) Thr(But) - Phe - Thr(But) - Ser(But) - NH(CH2)3CONHNH2) VIII; R1=Z, R3 = NHNH2, X= (CH2), and Y = Trp A solution of the compound of formula VII, 7 - Lys(Boc) - Asn - Phe - Phe Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)6 - COOMe (10.3 g, described in Example 8), and hydrazine hydrate (3.4 ml) in dimethylformamide (60 ml) is stirred at room temperature for 60 hours. Water is added and the precipitate is collected and dried over phosphorus pentoxide to give the title compound; nmr (DMSO-d6) 8 1.15 (m), 1.45 (s), 5.05 (s) and 7.25(m).

By following the procedure of Example 28 using the appropriate starting material of formula VII, other compounds of formula VIII in which R3 is NHNH2 are obtained.

Examples of the latter compounds of formula VIII are listed as products in Table 2 together with the appropriate starting material of formula VII. In each case the starting material is noted by the number of the example in which it is prepared.

TABLE 2

No. of the Example in Product of Formula VIII in which starting material which R is NHNH2 of formula 'VII is Example prepared R X Y 29 9 Z (CM2), Trp 30 10 Z (CH2)s Trp 31 11 Z (CH2)8 TIP 32 12 Z (CM2), D-Trp 33 13 Z (CH2)s D-Trp 34 14 Z (CH2)S D-Trp 35 15 Ddz (CH2)4 Trp 36 21 Ddz (CH2)2S(CH2)4 TIP 37 24 Ddz (CH2)8 D-Trp 38 25 | Ddz (CH2)2S(CH2)3 D-Trp 39 26 Ddz (CH2)3S(CH2)2 D-Trp 40 27 Ddz (CH2)4S(CH2)4 D-Trp EXAMPLE 41.

Nc - t - Butoxycarbonyl)lysyl - asparaginyl - phenylalanyl - phenylalanyl - trypto phyl - (N' - t - butoxycarbonyl)lysyl - (O - t - butyl)threonyl - phenylalanyl (O - t - butyl)threonyl - (0 - t - butyl)seryl - 7 - amino - heptanoic Acid Hydrazide (H - Lys(Boc) - Asn - Phe - Phe - Trp - Lys(Boc) - Thr(But) Phe - Thr(But) - Ser(But) - NH(CH2);CONHNH2) IX; R3=NHNH2, X= (CH2)6 and Y = Trp A mixture of the compound of formula VIII, Z - Lys(Boc) - Asn - Phe - Phe Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)6- CONHNH2 (3.0 g, described in Example 28), and 10% by weight palladium on carbon (1.5 g) in acetic acid (45 ml) is rapidly stirred under an atmosphere of hydro gen for 21 hrs. The mixture is filtered and the filtrate is evaporated under reduced pressure to give the title compound.

In the same manner but replacing the above starting material of formula VIII in which R9 is NHNH2 with other compounds of formula VIII described in Examples 29, 30, 31, 32, 33 and 34, the following compounds of formula IX are obtained respectively: (a) H Lys(Boc) - Asn - Phe - Phe - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) Ser(But) - NH(CH,), - CONHNH2, (b) H - Lys(Boc) - Asn - Phe - Phe - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) Ser(But) - NH(CH2)5 - CONHNH2, (c) H - Lys(Boc) - Asn - Phe - Phe - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But)- Ser(But) - NH(CH2)8 - CONHNH2.

(d) H- Lys(Boc) - Asn - Phe - Phe - D- Trp - Lys(Boc) - Trp(But9 - Phe - Thr (But) - Ser(But) - NH(CH2), - CONHNH2, (e) H - Lys(Boc) - Asn - Phe - Phe - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr (But) - Ser(But) - NH(CH2)6 - CONHNH2 and (f) H - Lys(Boc) - Asn - Phe - Phe - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr (But) - Ser(But) - NH(CH2) - CONHNH2.

EXAMPLE 42.

(N@ - t- Butoxycarbonyl)lysyl - asparaginyl - phenylalanyl - phenylalanyl - trypto phyl - (Ne - t - butoxycarbonyl)lysyl - (O - t - butyl)threonyl - phenylalanyl (O - t - butyl)threonyl - (0 - t - butyl)seryl - 5 - amino - pentanoic Acid Hydrazide (H - Lys(Boc) - Asm- Phe - Phe - Trp - Lys(Boc) - Thr(But)- Phe - Thr(But) - Ser(But) - NH(CH2)4CONHNH2) IX; R3=NHNH2), X= (CH2)4 and Y=Trp A solution of the compound of formula VII, Ddz - Lys(Boc) - Asn - Phe - Phe Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH(CH2)4 - CONHNH2 (2.0 g, described in Example 35), in 17 ml of a solvent consisting of acetic acid-formic acid-water (7:1:2 by volume) is stirred at room temperature for 16 hours. The solvent is removed under reduced pressure and the residue is dissolved in methanol. Diethyl ether is added and the precipitate is collected and dried over phosphorus pentoxide to give the title compound.

In the same manner but replacing the above starting material of formula VIII with other compounds of formula VIII described in Examples 36, 37, 38, 39 and 40, the following compounds of formula IX are obtained respectively: (a) H - Lys(Boc) - Asn - Phe - Phe - Trp - Lys(Boc) - Thr(But) - Phe- Thr(But) Ser(But) - NH(CH2)2S(CH2)4 - CONHNH2, (b) H - Lys(Boc) - Asn - Phe - Phe - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr (But) - Ser(But) - NH(CH2)8 - CONHNH2, (c) H - Lys(Boc) - Asn - Phe - Phe - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr (But) - Ser(But) - NH(CH2)2S(CH2)3-CONHNH2, (d) H- Lys(Boc) - Asn - Phe - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr (But) - Ser(But) - NH(CH2)3S(CH2)2 - CONHNH2 and (e) H - Lys(Boc) - Asn - Phe - Phe - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr (But) - Ser(But) - NH(CH2)4S(CH2)4 - CONHNH2.

EXAMPLE 43.

Cyclic Amide of (N#-#-Butoxycarbonyl)lysyl-asparaginyl-phenylalanyl- phenylalanyl-tryptophyl-(N#-#-butoxycarbonyl)lysyl (0-#-butyl)threonyl-phenylalanyl-(0-#-butyl)threonyl-(0-#-butyl)- seryl-7-amino-heptanoic acid

(CO-Lys(Boc)-Asn-Phe-Phe-Trp-Lys (Soc)-Thr [But)-Phe-Thr( ButySer(ButNH) (CH2)6 X; X = (CH2)6 and Y = Trp The compound of formula IX, H - Lys(Boc) - Asn - Phe - Phe - Trp - Lys (Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH(CH2)6CONHNH2 (2.94 g, described in Example 41) is dissolved in dimethylformamide (20 ml), cooled to - 20"C and 2.39 M hydrogen chloride in ethyl acetate (1.67 ml) is added, followed by t-butyl nitrite (0.19 ml). The solution is stirred at - 150C for 15 minutes and cold (-15 C) dimethylformamide (231 ml) is added, followed by N-ethyldiisopropylamine (0.957 ml). The mixture is stirred at - 15"C for 1 hour, at 0 C for 2 days and at room temperature for 1 day. The solvent is removed under reduced pressure, the residue is dissolved in methanol and the latter solution is added to water. The precipitate is collected and subjected to chromotography on silica gel using 5% v/v methanol and 0.1% v/v triethylamine in chloroform. The eluates are evaporated to give the title compound; amino acid analysis: Trp (present), Asp (0.89), Ser (0.85), Lys (1.77), Thr (2.04) and Phe (3.00).

In the same manner by following the above procedure, other compounds of formula X are obtained. For example, replacing the starting material with an equivalent amount of other starting materials of formula IX, as described in Examples 41 and 42, the fol lowing compounds of formula X are obtained, respectively:

(a) FO-Lys(Boc)-Asn-PheShe-irp-lys (Boc)-Thr( Buf-Phe-Thr( But)-Ser(But)-NH, (cH2)3 (b) (X)-Lys( Boc) C0-Lys(Boc)-Asn-Phe-Phe-Trp-LystBoc)-ThrC8u )-Phe-Thr(Bu )-SerEDu )-NH, (CH2 (c) O-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr 8ut )-Phe-Thr( 8u) -Ser(8u )-NH, CCH2)8 (d) co-Lysseoc)-Asn-phe-phe-D-Trp-Lys(Boc)-Thr(But)-phe-Thr(But)-ser(But)-NH, (CH2)3 - ) (e) C0-Lys(Boc)-Asn-Phe-Phe-D-Trp-Lys(Boc)-Thr(Bu )-Phe-Thr(But)-Ser(But)-NH, (CH2)5 ) (f) P -Lysteoc)-Asn-Phe-Phe-D-Trp-Lyst80c)-Thr(But)-Phe-Thr(But)-Ser(8u )-NH, (H2)8 (g) CO-Lys(Boc)-Asn-Phe-Phe-Trp-Lys (Boc)-Thr(But) -Phe-Thr(But)-Ser(Bu))-NH, (CH2)4 (h) co-LystBoc)-Asn-phe-phe-Trp-Lysseoc)-ThrtBut)-phe-Thr(5ut)-ser(But)-NH, (CH ) -S-(CH ) ) 2 4 2 2 - - Z (i) r -Lys(Boc)-Asn-phe-phe-D-Trp-Lysteoc)-Thresut)-phe-Thr(But)-sersBut)-NH )-Ser(Bu*)-NH.

(CH2)3-S-(CH2)1 (j) C0-Lys(Boc)-Asn-Phe-Phe-D-Trp-Lys(Boc?-Thr(Bu )-Phe-Thr(But)-SersBut)-NH and (CH2)2-S-(CH2) (k) CO-Lys(Boc)-Asn-Phe-Pha-0-Trp-Lys(Boc)-T Bu)-Phe-Thr(aut) -Ser( 8u -NH.

(&commat;)4-S-(CH2) EXAMPLE 44.

CyclIc Amide of Lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyl- Lysyl-threonyl-phenylalanyl-threonyl-seryl-7-aminoheptanoic acid

(ICO-LYs-Asn-Phe-Phe-Trn-Lys-Thr-Phe-Thr~Ser-NH) (CH2 ) e J L; X = (CH2)6 and Y = Trp A solution of the compound of formula X,

t t t Co-Lys(eoc)-Asn-phe-phe-Trp-Lys(goc)-Thr(But)-phe-Thr(eut)-ser(9ut)-NH (CH2)e ) (0.92 g, described in Example 43), in concentrated hydrochloric acid (4.16 ml) is rapidly stirred at 0 C under an atmosphere of nitrogen for 10 minutes. Acetic acid (300 ml) is added and the solution is lyophilized. The residue is dissolved in water (100 ml) and again lyophilized. The residue is dissolved in the upper phase of the solvent system butanol-acetic acid-water (4:1:5 by volume), applied to a column of a chemically modified cross-linked dextran (Sephadex LH-20) and the column is eluted with the upper phase solvent. The eluates are evaporated, the residue is dissolved in water and lyophilized to give the title compound; A MeOH 290 ( 4,695), 281 max ( 5,315), 274 (e 4,955), 269 (e 4,675), 265 (e 4,260) and 215 nm ( 47,335).

In the same manner by following the above procedure other compounds of formula I are obtained. For example, replacing the starting material with an equivalent amount of other starting materials of formula X, as described in Example 42, the following compounds of formula I are obtained, respectively:

(a) fO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH, I (b) fO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH, I (c) CO-Lys-Asn-Pho-Phe-Trp- Lys-ThrPhe-ThrSer-FfH, 2 a (d) ICO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-SHE I 2 (e) CO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-SH, ' H2', J (f) fO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-NH, (C ) o .% (g) O-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-S r I (h) ,CO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH, H2)4 S-(CHZ):

(i) CO-Lys-Asn-Phe-Pha-D-Trp-Lys-Thr-Phe-Thr-Ser-NH, (CH2)3'S-(CH2)2~ (j) CO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe' r-Ser-NH and (CH2)2-S-(CF2) (k) CO-Lys-Asn-Phe-Ph e-D-Trp - Ly s-Th r-Ph e-Th r-Ser-NH .

(CH2)4-S'(CH2)q, EXAMPLE 45.

&alpha;&alpha; - Dimethyl - 3,5 - dimethoxy - benzyloxycarbonyl - (N# - t- butoxycarbonyl)- lysyl - asparaginyl - phenylalanyl - phenylalanyl - tryptophyl - (N# - t - butoxy carbonyl)lysyl - (O - t - butyl)threonyl - phenylalanyl - (O - t- butyl)threonyl (O - t - butyl)seryl - 7 - aminoheptanoic Acid (Ddz - Lys(Boc) - Asn - Phe Phe - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH(CH2)6 COOH) VIII; R= Ddz, R =OH, Y=Trp and X=(CH2)6 A solution of the compound of formula VII, Ddz - Lys(Boc) - Asn - Phe - Phe Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH(CH2)6COOMe (3.3 g, described in Example 17), in acetone (32 ml) and N sodium hydroxyde (6.4 ml) is stirred at room temperature for 42 hrs. A solution of 5% by weight citric acid is added until the solution is neutral. The precipitate is collected, washed with water and dried to give the title compound, nmr (DMSO-d6) 3 1.02, 1.07 and 1.16 (singlets), 3.62 (singlet) and 6.88 to 7.08 (multiplet).

By following the procedure of Example 45 using the appropriate starting material of formula VII, other compounds of formula VIII in which R3 is OH are obtained.

Examples of the latter compounds of formula VIII are listed as products in Table 3 together with the appropriate starting material of formula VII. In each case the starting material is noted by the number of the example in which it is prepared.

TABLE 3

Product of Formula VIII in No. of the Example in which R3 is OH which starting material of Example formula VII is prepared R X Y 46 14 Z (CH2)8 D-Trp 47 16 Ddz (CH2)5 Trp 48 18 Ddz (CH2)8 Trp 49 EXAMPLE 54.

(N - t - butoxycarbonyl)lysyl - asparaginyl - phenylalanyl - phenylalanyl - trypto phyl - (N - t - butoxycarbonyl)lysyl) - (O - t - butyl)threonyl - phenylalanyl (0 - t- butyl)seryl - 7 - aminoheptanoic Acid (H - Lys(Boc) - Asn - Phe - Phe Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH(CH2)tCOOH) IX; B'=OH, X = (CH2),; and Y = Trp A solution of the compound of formula VIII, Ddz - Lys(Boc) - Asn - Phe -Phe Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH(CH2)aCOOH (2.9 g, described in Example 45) in acetic acid-formic acid-water (7:1:2 by volume, 28 ml) is stirred at room temperature for 40 hrs. The solution is evaporated and the residue is dissolved in methanol. Water is added and the precipitate is collected, washed with water and dried to give the title compounds.

In the same manner by following the above procedure other compounds of formula IX in which R3 is OH are obtained. For example, replacing the starting material with an equivalent amount of other starting materials of formula VIII described in Examples 47, 48, 49, 50, 51, 52 and 53, the following compounds of formula IX are obtained respectively: (a) H - Lys(Boc) - Asn - Phe - Phe - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) Ser(But) - NH(CH2)2COOH, (b) H - Lys(Boc) - Asn - Phe - Phe - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But)- Ser(But) - NH(CH2)8COOH, (c) H - Lys(Boc) - Asn - Phe - Phe - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But)- Ser(But) - NH(CH2)2S(CH2)8COOH, (d) H - Lys(Boc) - Asn - Phe - Phe - Trp - Lys(Boc) - Thr(Mut) - Phe - Thr (But) - Ser(But) - NH(CH2)3S(CH2)2COOH, (e) H - Lys(Boc) - Asn - Phe - Phe - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr (But) - Ser(But) - NH(CH2)6COOH, (f) H - Lys(Boc) - Asn - Phe - Phe - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr (But) - Ser(But) - NH(CH2)2S(CH2)aCOOH and (g) H - Lys(Boc) - Asn - Phe - Phe - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr (But) - Ser(But) - NH(CH2)3S(CH2)2COOH.

EXAMPLE 55.

(N - t - Butoxycarbonyl)lysyl - asparaginyl - phenylalanyl - phenylalanyl tryptophyl - (N - t - butoxycarbonyl)lysyl - (O - t - butyl)threonyl - phenyl alanyl - (0 - t - butyl)threonyl - (0 - t - butyl)seryl - 9 - amino - nonanoic Acid (H - Lys(Boc) - Asn - Phe - Phe - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr (But) - Ser(But) - NH(CH2)8COOH) IX; Ra=-OH, X=(CH2)b and Y = D-Trp A mixture of the compound of formula VIII, Z - Lys(Boc) - Asn - Phe - Phe D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH(CH2)8COOH (3.1 g described in Example 46), and 10% palladium on carbon (1.5 g) in acetic acid (45 ml) is rapidly stirred under an atmosphere of hydrogen for 24 hrs. The mixture is filtered and the filtrate is evaporated under reduced pressure to give the title compound.

EXAMPLE 56.

Cyclic Amide of (N#-t-Butoxycarbonyl)lysyl-asparaginyl-phenylalanyl- phenylalanyl-tryptophyl-(N#-t-butoxycarbonyl)lysyl-(0-t-butyl)threonyl- phenylalanyl-(0-t-butyl)threonyl-(0-t-butyl)seryl-7-amino-heptanoic Acid

)-Phe-Thr u )-Ser(Bu )-NH) 1FO-Lys(P,oc)-Asn-Phe-Phe-Tro-Lve t ( H2)e ~ J X; X = (CH2)6 and Y = Trp (i) A solution of the compound of formula VIII, H - Lys(Boc) - Asn - Phe Phe - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH(CH2).6COOH (0.45 g, described in Example 54), N-hydroxysuccinimide (0.115 g) and triethylamine (0.07 ml) in methylene chloride (118 ml) is cooled to OOC. A solution of dicyclo- hexylcarbodiimide (0.154 g) in methylene chloride (3 ml) is added and the resulting mixture is stirred at 0 C for 2 hrs, at 5 C for 18 hrs and at 25 C for 7 days. The mixture is filtered and the filtrate is evaporated. The residue is washed with 1N citric acid, water and dried over phosphorous pentoxide to give the title compound.

(ii) In the same manner but replacing N-hydroxysuccinimide with an equivalent amount of 1-hydroxybenzotriazole, 1,3 ,5-trichlorophenol, pentachlorophenol or p-nitrophenol, the title compound is obtained.

In the same manner by following the above procedure other compounds of formula X are obtained. For example, replacing the starting material with an equivalent amount of other starting materials of formula IX described in Examples 54 and 55 the following compounds of formula X are obtained respectively:

(a) F0-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-PheThr(But)-Ser(But)-N\H, (CH2) - (b) FO-Ly )-Asn-Phe-Phe-Trp-Lys (Boc )-Thr(Sut)-Phe-Thr )-NH, (CH2)0 . ) (c) CO-Lys(Boc)-Asn-Phe-Ph,e-Trp-Lys(Boc)-Thr(But)-Phe-ThrtBut)-Ser(Sut)-NH, (CH2)3-S (CH2)2 (d) CO-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH, (CH2 ) - S- (CH2 ) J (e)co-Lys(Boc)-Asn-phe-phe-D-Trp-Lys(Boc)-Thr(But)-phe-Thr(But)-ser(But)-NH d,,U I (f)CO-Lys-(Boc)-Asn-Phe-Phe-D-Trp-Lys(Boc)-Thr(Bu )-Phe-Thr(aut)-Ser(aut)-NH, (IH2 )3-S- (CH2 (g) CO-Lys(Boc)-Asn-Phe-Phe-O-Trp-Lyr (Boc)-Thr(But)-Phe-Thr(But)-Ser(B~ul)-NH and I (nJco-Lys-(Boc)-Asn-phe-phe-D-Trp-Lys(Boc)-Thr(But)-phe-Thr(But)-ser(But)-NH (H2) EXAMPLE 57.

Cyclic Amido of Lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyllysyl-threonyl-phenylalanyl-threonyl-seryl-7-aminoheptanoic acid

f ! tY s-Asn-Phe-Phe-Trn-Lvs-Thr-Phe-Thr-Ser-NH) ( ) #; x = (CH2)6 and Y = Trp A solution of the compound of formula X,

t t Sut)NH CLys(Soc)-Asn-Phe-Ph0-Trp-Lys(Soc)-Thr(Su Pho-Thr( Bu ) -Ser( I ) (10.20 g, described in Example 56), in concentrated hydrochloric acid (9 ml) is rapidly stirred at 0 C under an atmosphere of nitrogen for 10 minutes. Cold acetic acid (0 C, 90 ml) is added and the solution is lyophilized. The residue is dissolved in water (30 ml) and again lyophilized. The residue is dissolved in the upper phase of the solvent system butanol-acetic acid-water (4:1:5 by volume), applied to a column of a chemically modified cross-linked dextran (Sephadex G-25) and the column is eluted with the upper phase solvent. The eluates are evaporated, the residue is dissolved in water and lyophilized to give the title compound; AMeOH 289 (e 4,558), 280 ( max 5,148), 275 (e 4,334), 270 (e 4,671), 265 (e 4,292), 259 (e 3,717) and 254 nm (e 3,072).

The title compound of this Example is identical in all respects to the title compound of Example 44.

In the same manner by following the above procedure other compounds of formula I are obtained. For example, replacing the starting material with an equivalent amount of other starting materials of formula X described in Example 56, the following compounds of formula I are obtained respectively:

(a) fO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH, (CY ), (b) AC0-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-N\H, ( H2)0 - J (c) CO-Lys-Asn-Phe-Phe-Trp-Lys-Th r-Phe-Thr-S.r-N, (CH2)3-S-(CH2) J (d) Co-Lys-Asn-phe-phe-Trp-Lys-Thr-phe-Thr-ser-tH (t ) ' (C ) J (e) 70-Lys-Asn-Phe-Phe-O-Trp-Lys-Thr-Phe-Thr-Ser-NH; amino acid analysis: (CH2)6 ) Lys (1.89), Thr (1.93), Phe (3.00), Asn (1.00), Ser (0.99) and Trp (present), A MeOH 290 (,e 5,162), 282 (e 5,737), 273 ( 5,498), 269 (e 5,302), 265 (e 5,077), max 260 (e 4,685) and 253 nm (e4,189),

(f) CO Lys-Asn-Phe-Phe-D-Trp-Ly s-Thr-Phe-Thr- Se rNH, (CH2)3-S-(CH2)2 ) O-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-NH snd (CH2)2-5-(CH2)3 ) (h) fO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-NH, (CH2)" EXAMPLE 58.

- Dimethyl - 3,5 - dimethoxy - benzyloxycarbonyl - tryptophyl - (N - t - butoxy carbonyl)lysyl - (O - t - butyl)threonyl - phenylalanyl - (O - t - butyl)threonyl (0 - t - butyl)seryl - 4 - (2 - amino - ethylthio) - butanoic Acid Ethyl Ester (Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH (CH2), - COOEt) XI; m = 2, n = 3, R2 = Et and Y = Trp A solution of Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) NHNH2 (3.64 g, described by H. U. Immer et al., cited above) in dimethylformamide (40 ml) is cooled to -20 C. Hydrogen chloride (2.7 M) in ethyl acetate (2.64 ml) is added followed by t-butyl nitrite (0.40 ml). The solution is stirred at -15 C for 15 minutes and N-ethyldiisopropylamine (1.73 ml) is added. The mixture is cooled to -30 C and the compound of formula VI, H2N-(CH2)2-S-(CH2)3-COOEt (0.545 g, described in Example 7) is added. The mixture is stirred at -15 C for one hr, at 0 C for one hr and at room temperature overnight. The solvent is removed under reduced pressure and the residue is suspended in methanol. Water is added, the precipitate is collected and washed with methanol. The precipitate is subjected to chromatography on silica gel using 3% v/v methanol and 0.1% v/v triethylamine in chloroform. The eluates are evaporated to give the title compound; [&alpha;]D25 = +13.89 (c = 1, DMF) ; #maxMeOH 289 (# 5,490), 281 (# 7,915), 275 (# 7,530) and 217 mn (# 47,988).

In the same manner by following the above procedure, other compounds of formula XI are obtained. For example, replacing H2N-(CH2)2-S-(CH2)2-COOEt with an equivalent amount of other compounds of formula VI as described in Example 7. the following compounds of formula XI are obtained, respectively: Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser (But) - NH - (CH2),- S - (CH2)2 - COOEt, Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser (But) - NH - (CH2)3- S - CH, - COOMe, Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)2 S - (CH2)4 - COOMe and Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)4 S- (CH2)4 - COOEt.

Similarly replacing Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser (But) - NHNH2 with an equivalent amount of Ddz - D - Trp - Lys(Boc) - Thr (But) - Phe - Thr(But) - Ser(But) - NHNH2 [prepared from Ddz - D - Trp - Lys (Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - OMe (described in Example 3) in the same manner as described by H. U. Immer et al., cited above] and using one of the following compounds of formula VI; H2N-(CH2)2-S-(CH2)3-COOEt, H2N-(CH2)3-S-(CH2)2-COOEt and H2N-(CH2)4-S-(CH2)4-COOMe, the following compounds of formula XI are obtained, respectively: Ddz - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)2 S - (CH2)3 - COOEt, Ddz - D - Trp -Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But)- NH - (CH2)3 S - (CH2)2 - COOEt and Ddz - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)4 S - (CH2)4 - COOMe.

EXAMPLE 59.

* &alpha;&alpha; - Dimethyl - 3,5 - dimethoxy - benzyloxycarbonyl - tryptophyl - (N# - t - butoxy- carbonyl)lysyl - (O - t - butyl)threonyl - phenylalanyl - (O - t - butyl)threonyl (O - t - butyl)seryl - 4 - (2 - amino - ethylthio)butanoic Acid Hydrazide (Ddz Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)2 - S (CH2)3 - CONHNH2) XII; m = 2, n = 3 and Y = Trp A solution of Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) NH - (CH2)2 - S - (CH2)2 - COOEt (4.6 g, described in Example 58) and hydrazine hydrate (4.6 ml) in dimethylformamide (20 ml) is stirred at room temperature for four days. Water is added and the precipitate is collected and dried over phosphorus pentoxide to give the title compound: [&alpha;]D25 = + 13.88 (c = 1, DMF) ; #maxMeOH 289 (e 5,530), 281 (e 7,950), 275 (.e 7,550) and 217 nm ( 50,160).

In the same manner by following the above procedure, other compounds of formula XII are obtained. For example, replacing the starting material with an equivalent amount of other starting materials of formula XI, as described in Example 58, the following compounds of formula XII are obtained, respectively: Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH3)3- S - (CH2)2 - CONHNH2, Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)2- S - CH2 - CONHNH2, Ddz - Trp - Lys(Boc) - Thr(Bu') - Phe - Thr(Bu') - Ser(But) - NH - (CH,)3- S - (CH2)4 - CONHNH2, Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)4 S - (CH2)4 - CONHNH2, Ddz - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)2 S - (CH2), - CONHNHi, Ddz - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH,3- S - (CH2)2 - CONHNH, and Ddz - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH,)4- S - (CH2)4 - CONHNH2.

EXAMPLE 60.

&alpha;&alpha; - Dimethyl - 3,5 - dimethoxy - benzyloxycarbonyl - tryptophyl - (N# - t- butoxy- carbonyl)lysyl - (O - t- butyl)threonyl - phenylalanyl - (O - t - butyl)threonyl (O - t - butyl)seryl - 4 - (2 - amino ethylthio) - 1 - oxobutyl - (N# - t - but oxycarbonyl)lysyl - asparaginyl - phenylalanyl - phenylalanine Methyl Ester (Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH (CH2)2 - S - (CH2)3 - CO - Lys(Boc) - Asn - Phe - Phe - OMe) XIII; m=2, n = 3 and Y = Trp A solution of the hydrazide of formula XII, Ddz - Trp - Lys(Boc)- Thr(But) Phe - Thr(But) - Ser(But) - NH - (CH2), - S - (CH2), - CONHNH2, (3.83 g, described in Example 59) in dimethylformamide (41 ml) is cooled to - 200 C. Hydrogen chloride (2.7 M) in ethyl acetate (2.5 ml) is added followed by t-butyl nitrtite (0.38 ml). The solution is stirred at -15 C for 15 minutes and N-ethyldiisopropylamine (1.64 ml) is added. The mixture is cooled to -30 C and H - Lys(Boc) - Asn Phe - Phe - OMe (1.94 g, prepared as described by H. U. Immer et al., cited above) is added. The mixture is stirred at - 150C for one hr, at 0 C for one hr and at room temperature overnight. The solvent is removed under reduced pressure and the residue is triturated with methanol. The solid is collected and dried under reduced pressure over phosphorus pentoxide to give the title compound; #maxMeOH 289 (# 5,655), 281 (e 8,070) and 275 nm ( 7,700).

In the same manner by following the above procedure, other compounds of formula XIII are obtained. For example replacing the starting material of formula XII with an equivalent amount of other starting materials of formula XII, as described in Example 59, the following compounds of formula XIII are obtained, respectively: Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH,)3- S - (CH2)2CO - Lys(Boc) - Asn - Phe - OMe, Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)3 S - CH2 - CO - Lys(Boc) - Asn - Phe - Phe - OMe, Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)2 S - (CH2)4 - CO - Lys(Boc) - Asn - Phe - Phe - OMe, Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)4 S - (CH2)4 - CO - Lys(Boc) - Asn - Phe - Phe - OMe, Ddz - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH3)2- S - (CH,)3-CO-Lys(Boc) -Asn-Phe-Phe-OMe, Ddz - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)2- S - (CH2)2 - CO - Lys(Boc) - Asn - Phe - Phe - OMe and Ddz - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)4 S - (CH,) - CO - Lys(Boc) - Asn - Phe - Phe - OMe.

EXAMPLE 61.

Tryptophyl - (N - t - butoxycarbonyl)lysyl - (O - t - butyl)threonyl - phenylalanyl (O - t - butyl)threonyl - (0 - t - butyl)seryl - 4 - (2 - aminoethylthio) - 1 - oxo butyl - (N# - t - butoxycarbonyl)lysyl - asparaginyl - phenylalanyl - phenylalanine Methyl Ester (H - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) NH - (CH2), - S - (CH2), - CO - Lys(Boc) - Asn - Phe - Phe - OMe) XIV; m = 2, n = 3 and Y = Trp A solution of Ddz - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) NH - (CH3)2 - S - (CH,), - CO - Lys(Boc) - Asn - Phe - Phe - OMe (2.05 g, described in Example 60) in 17.5 ml of a solvent consisting of acetic acid-formic acid water (7:1:2 by volume) is stirred at room temperature for 16 hours. The solvent is removed under reduced pressure and the residue is dissolved in methanol. Water is added and the precipitate is collected and dried over phosphorus pentoxide. The dried precipitate is washed with 40 ml of petroleum ether-ether (1:1) and dried to give the title compound, amino acid analysis: Lys (1.93), Asp (0.91), Thr (1.87), Ser (0.95 and Phe (3.00).

In the same manner by following the above procedure, other compounds of formula XIV are obtained. For example, replacing the starting material with an equivalent amount of other starting materials of formula XIII, as described in Example 60, the following compounds of formula XIV are obtained, respectively: (a) H - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)3 S - (CH2)2-CO-Lys(Boc) -Asn-Phe-Phe-OMe, (b) H - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)3 S - CH2 - CO - Lys(Boc) - Asn - Phe - Phe - OMe, (c) H - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)2 S - (CH2)4-CO-Lys(Boc) -Asn-Phe-Phe-OMe, (d) H - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)4 S - (CH2)4 - CO - Lys(Boc) -Asn-Phe-Phe-OMe, (e) H - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH (CH2)2 - S - (CH2)3 - CO - Lys(Boc) - Asn - Phe - Phe - OMe, (f) H - D - Trp - Lys(Boc) - Thr(But) - Phe- Thr(But) - Ser(But) - NH (CH2)3 - S - (CH2)2 - CO - Lys(Boc) - Asn - Phe - Phe - OMe and (g) H - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH (CH2)A - S - (CH,)4 - CO - Lys(Boc) - Asn - Phe - Phe - OMe.

EXAMPLE 62.

Tryptophyl - (N - t - butoxycarbonyl)lysyl - (O - t - butyl)theronyl - phenylalanyl (O - t - butyl)threonyl - (O - t - butyl)seryl - 4 - (2 - aminoethylthio) - 1 - oxo butyl - N# - t - butoxycarbonyl)lysyl - asparaginyl - phenylalanyl - phenylalanine Hydrazide (H - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH (CH2), - S - (CH2)3 - CO - Lys(Boc) - Asn - Phe - Phe - NHNH2) XV; m=2, n=3 and Y = Trp Hydrazine hydrate (1.7 ml) is added to a solution at 0 C of H - Trp - Lys(Boc) Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2), - S - (CH2)3 - CO - Lys (Boc) - Asn - Phe - Phe - OMe (1.7 g, described in Example 61) in dimethylformamide (2 ml) and the solution is stirred at room temperature overnight. Water is added and the precipitate is collected and dried over phosphorus pentoxide to give the title compound, AMeOH 290 (e 5,515), 21 (e 6,435), 275 (E 6,065), 269 (e 5,680) and 265 nm (z 5,195).

In the same manner by folowing the above procedure, other compounds of formula XV are obtained .For example, replacing the starting material with an equivalent amount of other starting materials of formula XIV, as described in Example 61, the following compounds of formula XV are obtained, respectively: (a) H - Trp - Lys(Boc) - Thr - (But) - Phe Thr (But) - Ser(But) - NH - (CH2)2- S - (CH2)2 - CO - Lys(Boc) - Asn - Phe - Phe - NHNH2, (b) H Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH3)2- S - CH2 - CO - Lys(Boc) - Asn - Phe - Phe - NHNH2, (c) H - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)2 S - (CH2)4 - CO - Lys(Boc) - Asn - Phe - Phe - NHNH2, (d) H - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)4 S - (CH2)4 - CO - Lys(Boc) - Asn - Phe - Phe - NHNH2, (e) H - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH (CH2)2 - S - (CH,)i - CO - Lys(Boc) - Asn - Phe - Phe - NHNH2, (f) H - D - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) - NH - (CH2)3 - S - (CH2)2 - CO - Lys(Boc) - Asn - Phe - Phe - NHNH2 and (g) H - D - Trp - Lys(Boc) - Thr(Bu') - Phe - Thr(But) - Ser(But) - NH (CH,)4 - S - (CH2), - CO - Lys(Boc) - Asn - Phe - Phe - NHNH2.

EXAMPLE 63.

Cyclic Amide of (N#-t-Butoxycarbonyl)lysyl-asparaginyl-phenylalanyl- phenylalanyl-tryptophyl-(N#-t-butoxycarbonyl)lysyl- (O-t-butyl)threonyl-phenylalanyl-(0-t-butyl)threonyl (O-t-butyl)seryl-4-(2-amino-ethylthio)-butanoic Acid

t t t JCO-LYs(Boc)-Asn-Phe-Phe-TrD-LYs(Boc)-Thr(But)-Phe-Thr(Bu )-Ser(Bu )-hTi) (CH2), 5 ---------------- (to2)2 x; xm= (CH2)3-S-(CH2)2 and Y = Trp A solution of H - Trp - Lys(Boc) - Thr(But) - Phe - Thr(But) - Ser(But) NH - (CH2)2 - S - (CH2), - CO - Lys(Boc) - Asn - Phe - Phe - NHNH2 (1.57 g, described in Example 62) in dimethylformamide (10 ml) is cooled to - 200C and 2.64 M hydrogen chloride in ethyl acetate (0.8 ml) is added, followed by t-butyl nitrite (0.117 ml). The solution is stirred at - 150C for 15 minutes and cold ( - 15 C) dimethylformamide (123 ml) is added, followed by N-ethyldiisopropylamine (0.363 ml. The mixture is stirred at - 150C for 1 hr, at 0 C for 2 days and at room temperature for 1 day. The solvent is removed under reduced pressure, the residue is dissolved in methanol and the latter solution is added to water. The precipitate is collected and subjected to chromatography on silica gel using 5% v/v methanol and 0.1% v/v triethylamine in chloroform. The eluates are evaporated to give the title compound; AMeOH 290 (e 4,915), 281 (e 5,670), 273 (e 5,350) and 269 nm (e 5,020). max In the same manner by folowing the above procedure, other compounds of formula X in which X is (CH,),--SS-(CH,),,, are obtained. For example, replacing the starting material with an equivalent amount of other starting materials of formula XV, as described in Example 62, the following compounds of formula X are obtained, respectively:

(a) O-Lys(Boc )-Asn-Phe-Phe-Trp-Lys (Boc )-Thr(But)-Phe-Thr(But)-Ser(8ut)- H, ( H2)Z (CH2)3 )PheThr(But).Ser(But);H, H O-L5(BOC)A5PhPhTPLY5(80)Th(BUt ~ s (CH2)3(CH2)3 (c) FO-Lye (Eoc!-.4e-Pfrft-pse-Trp-Lys ,CO-Lys(eoc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(8ut)-Phe-Thr(Bu )-Ser(But)-NH, (CH2 ({H2)2 (d) CO-Lys(Boc )-Ar;n-Phe-Phe-Trp-Lys (Boc )-Thr(But)-Phe-Thr(But)-Ser(But) (CH ) (CH2 ) 4 (e) C0-Lys(Boc)-Asn-Phe-Phe-D-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-PIH, (LH2)3 S (f) FO-Lys(Boc) Asn-Phe-Phe-G-Trp-Lys(Boc)-Thr(Bu )Phe-Thr(But)-Ser(But)-H and (C)2 S (CH2)3 (g) Co-Lys(Boc)-Asn-Phe-Phe-D-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-tH.

(gH2)4 H (-H2)4 EXAMPLE 64.

Cyclic Amide of Lysyl-asparaginyl-phenylalanyl-phenlalanyl- tryptophyl-lysyl-threonyl-phenylalanyl-threonyl-seryl-4- (2-amino-ethylthio ) butanoic Acid

(CO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr- Ser-NH) -I ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ I (CH2)3 2 (CH2)2 1; X = (CH2)3-S-(CH2)2 and Y = Trp A solution of

CO-Lys(Boc)-Asn-Phe~Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)~Ser(at3;)NH (cm2) S- (cm2)2 (0.729 g, described in Example 63) in concentrated hydrochloric acid (4.0 ml) is rapidly stirred at 0 C under an atmosphere of nitrogen for 10 minutes. Acetic acid (300 ml) is added and the solution is lyophilized. The residue is taken in water (100 ml) and again lyophilized. The residue is dissolved in the upper phase of the solvent system butanol-acetic acid-water (4:1:5 by volume), applied to a column of chemically modified cross-linked dextran (Sephadex G-25, prepared in the lower phase of the above solvent system and then equilibrated in the upper phase of the above solvent system) and the column is eluted with the upper phase solvent. The eluates are evaporated, the residue is dissolved in water and lyophilized to give the title compound; AMeOH 290 (e 4,825), 281 (e 5,550), 275 (e 5,195), 269 ( 4,890), 265 (e 4,485) max and 216 nm ( 49,750).

In the same manner by following the above procedure, other compounds of formula I in which X is (CH2),S(CH2)m are obtained. For example, replacing the starting material with an equivalent amount of other starting materials of formula X in which X is (CH2)n-S-(CH2)m, as described in Example 63, the following compounds of formula I are obtained, respectively:

(a) fO-Lys-Asn-Phe-Phe-Trp-Lys-T

(e) ICO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-l H (cm2) > ( (CH?'2 (f) CO-Ly"2)j CO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-lìlH (CH2)- 3-----------------------5----- S (CH2)3 and (g) ICO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-tH (CH2)4 s ( 2 4

Claims (95)

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

   CO-Lys-Asn-pha-Pha-Y-Lys-Thr-Phe-Thr-Sar-NH -- ~~~ff (I) x in which X is (CH2)k wherein k is an integer selected from 1, 2 and 9; or (CH2)n-S-(CH2)m wherein m and n each is an integer from 1 to 4 and Y is Trp, or a pharmaceutically acceptable salt thereof.
2. 2. A compound of formula I as shown in Claim 1 wherein Y is D-Trp and X is as defined in Claim 1.
3. 3. A compound having the formula

   R4 R5 R6 R7 R8 I I I 1 I CO-LYS-ASn-Phe-Phe-Y-LYS-Thr-Phe-Thr-Ser-NH (Xa) k - X in which X is (CH2)k wherein k is an integer selected from 1, 2 and 9; or (CH2)n-S-(CH2)m wherein m and n each is an integer from 1 to 4 and Y is Trp, R4 and R5 are protecting groups for the side chain e-amino substituent of lysine and R6, R7 and Rb are protecting groups for the hydroxyl groups of threonine and serine.
4. 4. A compound of formula (Xa) as shown in Claim 3 wherein Y is D-Trp and X, R4, RS, R6, R7 and Rb are as defined in Claim 3.
5. 5. A compound of formula (Xa) as claimed in Claim 3 wherein R4 and R5 are selected from benzyloxycarbonyl, t-butoxycarbonyl, 2-(p-biphenyl)isopropoxycarbonyl, p-chloroenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, formyl, trifluoroacetyl, phthalyl, acetyl, tosyl, trityl and benzyl.
6. 6. A compound of formula (Xa) as claimed in Claim 3 or Claim 5 wherein R6, R' and R8 are selected from acetyl, tosyl, benzoyl, tert-butyl and benzyl.
7. 7. A compound of formula (Xa) as claimed in Claim 3 wherein R4 and B both represent t-butoxycarbonyl.
8. 8. A compound of formula (Xa) as claimed in Claim 3 or Claim 7 wherein R6, R7 and Rb all represent t-butyl.
9. 9. A compound of formula (I) or (Xa) as claimed in any one of Claims 1, 3 and 5 to 8 wherein n represents 2 or 3.
10. 10. A compound of formula (I) or (Xa) as claimed in any one of Claims 1, 3 and 5 to 9 wherein m represents 2 or 3.
11. 11. A compound of formula (I) or (Xa) as claimed in Claim 2 or Claim 4 wherein R4, R, R6, R', Rb and n are as defined in any one of Claims 5 to 10.
12. 12. The compound of formula I as defined in Claim 1 wherein X is (CH2),-S-(CH2)2.
13. 13. The compound of formula I as defined in Claim 2 wherein X is (CH2)2-S-(CH2)2.
14. 14. The compound of formula I as defined in Claim 1 wherein X is (CH2)2-S-(CH2)3.
15. 15. The compound of formula I as defined in Claim 2 wherein X is (CH,)3-S-(CH3)3.
16. 16. The compound of formula (Xa) as defined in Claim 3 wherein R4 and R5 are t-butoxycarbonyl; R6, R7 and R8 are t-butyl X is (CH2)3-S-(CHG2)2.
17. 17. The compound of formula (Xa) as defined in Claim 3 wherein R4 and R5 are t-butoxycarbonyl; R6, R7 and Rb are t-butyl X is (CHi)2-S-(CH2)2.
18. 18. The compound of formula (Xa) as defined in Claim 4 wherein R4 and B5 are t-butoxycarbonyl; R6, R7 and R8 are t-butyl X is (CH2)3-S-(CH2)2.
19. 19. The compound of formula (Xa) as defined in Claim 4 wherein R4 and R5 are t-butoxycarbonyl; R6, R7 and R8 are t-butyl X is (CH2)2-S-(CH2)3.
20. 20. A process for preparing a compound of formula I as defined in Claim 1 or a pharmaceutically acceptable salt thereof which comprises deprotecting a compound of formula (Xa) as defined in Claim 3, and if desired converting to a pharmaceutically acceptable salt thereof.
21. 21. A process for preparing a compound of formula I as defined in Claim 2 or a pharmaceutically acceptable salt thereof which comprises deprotecting a compound of formula (Xa) as defined in Claim 4, and if desired converting to a pharmaceutically acceptable salt thereof.
22. 22. A process for preparing a compound of formula (Xa) as defined in Claim 3 which comprises cyclising a linear undecapeptide having the requisite, suitably protected amino acid sequence and having N-terminal and C-terminal groups which can be intramolecularly coupled by methods known in peptide and penicillin chemistry to form amide bonds.
23. 23. A process for preparing a compound of formula (Xa) as defined in Claim 4 which comprises cyclising a linear undecapeptide having the requisite, protected amino acid sequence and having N-terminal and C-terminal groups which can be intramolecularly coupled by methods known in peptide and penicillin chemistry to form amide bonds.
24. 24. A process as claimed in Claim 22 or 23 in which the linear undecapeptide is an activated derivative as hereinbefore defined of a compound of formula:

    B4 R5 R6 Rb R8 II II H-Lys-Asn-Phe-Phe-Y-Lys-T;-Phe-Thr-Ser-NH-X-CORs (VIIIa) wherein X, Y, R4, RS, Rb, B7 and Rb are as defined in Claim 3 or Claim 4 and R2 is OH.
25. 25. A process as claimed in Claim 24 in which the activated derivative of the compound of formula (VIIIa) as defined therein is the azide which is prepared from the corresponding hydrazide having formula VIIIa wherein R is NHNH2.
26. 26. A process as claimed in Claim 22 or Claim 23 in which the linear undecapeptide is a compound of formula

    R" R6 Bb R8 R4 II H-Y-Lys-Thr-Phe-Thr-Ser-NH-XCO-Lys-Asn-Phe-PheNHNH2 (XVa) wherein X, Y, R4, R5, R6 and R8 are as defined in Claim 3 or Claim 4 which is cyclised via the azide coupling method as hereinbefore defined.
27. 27. A process as claimed in Claim 24 or Claim 25 in which the compound of formula (VIIIa) wherein R8 is OH or NHNH2 is prepared from a compound of formula

    R4 Rj Rb Rb R8 I I I I B1-Lys-Asn-Phe-Phe-Y-Lys-Thr-Phe-Thr-Ser-NH-X-COOR2 (VIla) wherein X, Y, R4, R5, Rb, B7 and Rb are as defined in Claim 3 or Claim 4, R2 represents lower alkyl and R represents an &alpha;-amino protecting group removable under conditions which do not remove the protecting groups R4, R5, R6, R7 and R8, by (i) hydrazinolysis followed by selective removal of the R protecting group to give a compound of formula (VIIIa) wherein R is NHNH2 or (ii) alkaline hydrolysis followed by selective removal of the R protecting group to give a compound of formula (VIIIa) wherein R is OH.
28. 28. A process as claimed in Claim 27 wherein the compound of formula VIIa is prepared by a process which comprises coupling according to the azide coupling method a compound of formula

    R4 R R6 Ri RK II R'-Lys-Asn-Phe-Phe-Y-Lys-Thr-Phe-Thr-Ser-NHNH. (via) with a compound of formula: H2N-X-COOB2 (VI) in which formula X, Y, R4, R5, R6, R7 and R8 are as defined in claim 3 or Claim 4, R represents lower alkyl, and R is as defined in Claim 27.
29. 29. A process as claimed in Claim 28 wherein the compound of formula Va as defined therein is prepared by a process comprising hydrazinolysis of a compound of formula

    R4 W Rb Rb R8 I ( I Rl-Lys-Asn-Phe-Phe-Y-Lys-Thr-Phe-Thr-Ser-O (lower alkyl) (IVa) wherein X, Y, R, R4, R5, R6, R7 and R8 are as defined in Claim 27.
30. 30. A process as claimed in Claim 29 in which the compound of formula IVa as defined in therein is prepared by a process which comprises coupling according to the azide coupling method a compound of formula

    R4 R'-Lys-Asn-Phe-Phe-NHNH, (Ila) with a compound of formula

    R5 R" R' R8 II H-Y-Lys-Thr-Phe-Thr-Ser-O (-lower alkyl) (IIIa) in which formulae X, Y, R, R6, R5, R6, R7 and R8 are as defined in Claim 27.
31. 31. A process as claimed in Claim 26 in which the compound of formula XVa as defined therein is prepared by a process which comprises hydrazinolysing a compound of formula:

    R5 Rb R Rb B4 I I I I H-Y-Lys-Thr-Phe-Thr-Ser-NH-X-CO-Lys-Asn-Phe-Phe-O (lower alkyl) (XIVa) wherein X, Y, R4, R5, R6, R7 and R8 are as defined in Claim 3 or Claim 4.
32. 32. A process as claimed in Claim 31 wherein the compound of formula (XIVa) as defined therein is prepared by a process which comprises coupling according to the azide coupling method a compound of formula:

    R4 H-Lys-Asn-Phe-Phe-O ( lower alkyl) with a compound of formula:

    R5 Ru R7 R8 II II B1 -Y-Lys-Thr-Phe-Thr-Ser-NH-X-CO-NHNH2 (XIIa) in which formulae X, Y, R5, R6, R7 and R8 are as defined in claim 3 or claim 4 and R represents an &alpha;-amino protecting group removable under conditions which do not remove the protecting group R, R5, R6, R7 and R8; and selectively removing the R protecting group.
33. 33. A process as claimed in Claim 32 in which the compound of formula (XIIa) as defined therein is prepared by a process which comprises hydrazinolysing a compound of formula

    B5 R6 Ri R9 II II R1-Y-Lys-Thr-Phe-Thr- Ser-NH-X-COOR2 (XIa) wherein X, Y, R5, R6, R7 and R8 are as defined in Claim 3 or Claim 4, R is as defined in Claim 32 and R represents lower alkyl.
34. 34. A process as claimed in Claim 33 in which the compound of formula (XIa) as defined therein is prepared by a process which comprises coupling according to the azide coupling method a compound of formula: H2N-X-COOR (VI) with a compound of formula

    R5 B6 R7 Rb II II Rl-Y-Lys-Thr-Phe-Tlr-Ser-NHNH2 in which formulae X, Y, R5, R6, R7 and R8 are as defined in claim 3 or claim 4, R is as defined in Claim 32 and R represents lower alkyl.
35. 35. A process as claimed in any one of Claim 27 to 30 and 32 to 34 wherein B1 represents Ddz.
36. 36. A process as claimed in any one of Claims 30 to 34 wherein -O(lower alkyl) represents -O (methyl).
37. 37. A process as claimed in any one of Claims 27 to 30, 33 and 34 wherein R2 represents methyl.
38. 38. A process as claimed in any one of Claims 24 to 37 wherein R4 and R5 are selected from benzyloxycarbonyl, t-butoxycarbonyl, 2-(p-biphenyl) isopropoxycarbonyl, ethoxycarbonyl, formyl, trifluoroacetyl, phthalyl, acetyl, tosyl, trityl, benzyl, p-methoxybenzyloxycarbonyl, isopropyloxycarbonyl, and p-chlorobenzyloxycarbonyl.
39. 39. A process as claimed in any one of Claims 24 to 38 wherein R6, R' and Rb are selected from acetyl, tosyl, benzoyl, tert-butyl and benzyl.
40. 40. A process as claimed in any one of Claims 24 to 34 wherein R4 or R5 represent t-butoxycarbonyl, R6, R' and R8 represent t-butyl, R1 represents Ddz and O(lower alkyl) represents -O(methyl).
41. 41. A process as claimed in Claim 20 or Claim 21 in which the compound of formula Xa as defined therein is prepared by a process as claimed in any one of Claims 24 to 40.
42. 42. A process as claimed in Claim 20 or Claim 21 wherein R4 and R5 are selected from benzyloxycarbonyl, t-butoxycarbonyl, 2-(p-biphenyl)isopropoxycarbonyl, ethoxycarbonyl, formyl, trifluoroacetyl, phthalyl, acetyl, tosyl, trityl, benzyl, p-methoxybenzyloxycarbonyl, isopropyloxycarbonyl and p-chlorobenzyloxycarbonyl.
43. 43. A process as claimed in Claim 20 or 42 wherein R6, R' and R8 were selected from acetyl, tosyl, benzoyl, tert-butyl and benzyl.
44. 44. A process as claimed in Claim 20 wherein R4 or R5 represent t-butoxycarbonyl and R6, R7 and R8 represent t-butyl.
45. 45. A compound of formula:

    R4 R5 R6 B7 Rb II II Rl -Lys-Asn-Phe-Phe-Y-Lys-Thr-Phe-Thr-S!r-NH-X-CORll ( IXb) wherein X,R4, R5, R6, R7 and R8 ae as defined in Claim 3, R10 represents hydrogen or an &alpha; amino protecting group selectively removable under conditions which do not remove the R4, R5, R6, R7 and R8 protecting groups and R11 represents -OH or -NHNH2 and Y is D-Trp or Trp.
46. 46. A compound of formula:

    R5 B6 B7 R8 R4 II H-Y-Lys-Thr-Phe-Thr-Ser-NH-X-CO-Lys-Asn-Phe-Phe-B12 (XVb) wherein X, Y, R4, R5, R6, R7 and R8 are as defined in Claim 3 or Claim 4 and R12 represents -NHNH2 or O(lower alkyl).
47. 47. A compound of formula (IXb) or (XVb) as claimed in Claim 45 or Claim 46, wherein R4 and R5 are selected from benzyloxycarbonyl t-butoxycarbonyl, 2-(p biphenyl) isopropoxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, formyl, trifluoroacetyl, phthalyl, acetyl, tosyl, trityl and benzyl.
48. 48. A compound of formula (IXb) or (XVb) as claimed in any one of Claims 45 to 47 wherein R6, R' and R8 are selected from acetyl, tosyl, benzoyl, tert-butyl and benzyl.
49. 49. The compound of Claim 45 in which R10 is H, R4 and ~R5 are Boc, R6, R7 and R8 are all But, R11 is -COOH, X is (CH2)2-S-(CH2)3 and Y is Trp.
50. 540. The compound of Claim 45 in which R10 is H, R4 and R5 are Boc, R6, R7 and R8 are all But, R11 is -NHNH2, X is (CH2)2-S-(CH2), and Y is D-Trp.
51. 51. The compound of Claim 45 in which R10 is H, R4 and R5 are Boc, R6, R7 and R8 are all But, R11 is -COOG, X is (CH2)3-S-(CH2)2 and Y is Trp.
52. 52. The compound of Claim 45 in which R10 is H, R4 and R5 are Boc, R6, R7 and R8 are all But, R11 is -NHNH3, X is (CH2)3-S-(CH2)2 and Y is D-Trp.
53. 53. The compound of Claim 46 in which X is (CH2)3-S-(CH2)2, Y is Trp, R12 is -NHNH2, R4 and R5 are Boc and RO, R' and R8 are But.
54. 54. The compound of Claim 46 in which X is (CH2)3-S-(CH2)2, Y is D-Trp, R12 is NHNH2, R4 and R5 are Boc, and R6, R7 and R8 are But.
55. 55. The compound of Claim 46 in which X is (CH2)2-S-(CH2)3, Y is Trp, R12 is NHNH2, R4 and R5 are Boc and R6, R7 and R8 are But.
56. 56. The compound of Claim 46 in which X is (CH2)2-S-(CH2)3, Y is D-Trp, R12 is NHNH21 R4 and R5 are Boc and R6, R1 and R8 are But.
57. 57. A process for preparing a compound of formula (IXb) as defined in Claim 45 which comprises (i) hydrazinolysis of a compound of formula:

    R4 R5 R6 Rb R8 II (I B1-Lys-Asn-Phe-Phe-Y-Lys-Thr-Phe-Thr-Ser-NH-X-COOB2 (VIIa) wherein X, Y, R4, R5, R6, R7 and R8 are as defined in Claim 3 or Claim 4, R2 represents lower alkyl and Rl represents an a-amino protecting group selectively removable under conditions which do not remove the protecting groups R4, R5, R6, R7 and R8, to give a compound of formula (IXb) wherein R10 is an amino protecting group and R11 is -NHNH2, or (ii) alkaline hydrolysis of a compound of formula (VIIa) as defined above to give a compound of formula (IXb) wherein R11 is -OH, and if desired after the aforementioned processes selectively removing the R10 protecting group to give a compound of formula (IXb) wherein R10 is hydrogen.
58. 58. A process as claimed in Claim 57 wherein the compound of formula (VIIa) is prepared by a process which comprises coupling according to the azide coupling method a compound of formula:

    R4 Rj R6 R7 R3 II II B1-Lys-Asn-Phe-Phe-Y-Lys-Thr-Phe-Thr-Ser-NHNH2 (Va) with a compound of formula: H2N-X-COOR2 (VI) in which formula X, Y, R, R4, R5, R6, R' and R8 are as defined in Claim 57 and R2 represents lower alkyl.
59. 59. A process as claimed in Claim 58 wherein the compound of formula (Va) as defined therein is prepared by a process comprising hydrazinolysis of a compound of formula:

    R4 B R" B1 B0 II II R1-Lys-Asn-Phe-Phe-Y-Lys-Thr-Phe-Thr- Ser-O(lower alkyl) (IVa) wherein X, Y, R, R5, R6, R7 and R8 are as defined in Claim 57.
60. 60. A process as claimed in Claim 59 in which the compound of formula IVa as defined therein is prepared by a process which comprises coupling according to the azide coupling method a compound of formula:

    R4 Rl-Lys-Asn-Phe-Phe-NHNH2 (IIa) with a compound of formula:

    Ra R6 R7 Rg I [ II H-Y-Lys-Thr-Phe-Thr-S er-O (lower alkyl) (IIIa) in which formulae X, Y, R, R4, R5, R6, R7 and R8 are as defined in Claim 51.
61. 61. A process as claimed in Claim 60 in which the compound of formula (IIa) is prepared by a process which comprises hydrazinolysis of a compound of formula:

    R4 R1-Lys-Asn-Phe-Phe-O (lower alkyl) wherein Rl and R4 are as defined in Claim 57.
62. 62. A process for preparing a compound of formula (XVb) as defined in Claim 46 which comprises coupling according to the azide coupling method as hereinbefore defined, a compound of formula:

    R4 H-Lys-Asn-Phe-Phe-O (lower alkyl) with a compound of formula:

    Ro B0 R7 R8 I I II R1 -Y-Lys-Thr-Phe-Thr-S er-NH-X-CO-NHNH2 (XIIa) in which formulae X, Y, R4, R5, R6, R7 and R8 are as defined in Claim 46 and R represents an &alpha;-amino protecting group removable under conditions which do not remove the protecting groups R4, R5, R6, R7, and selectively removing the R protecting group, to give a compound of formula (XVb) wherein R12 is -O(lower alkyl), and further, if desired, hydrazinolysing to give a compound of formula (XVb) wherein Rl2 is -NHNH2.
63. 63. A process as claimed in Claim 62 in which the compound of formula (XIIa) as defined therein is prepared by a process which comprises hydrazinolysing a compound of formula:

    R3 B6 B7 B0 II II Rl-Y-Lys-Thr-Phe-Thr-Ser-NH-X-COOR2 (XIa) wherein X, Y, R5, R6, R7 and R8 are as defined in Claim 46, R represents lower alkyl, and R is as defined in Claim 62.
64. 64. A process as claimed in Claim 63 in which the compound of formula (XIa) as defined therein is prepared by a process which comprises coupling according to the azide coupling method a compound of formula: H2N-X-COOR (VI) with a compound of formula

    R" R6 R1 R8 II II R1-Y-Lys-Thr-Phe-Thr-Ser-NHNH2 in which formulae X, Y, R5, %6, R7 and R8 are as defined in Claim 46, R is as defined in Claim 62 and R represents lower alkyl.
65. 65. A compound of formula (I) as defined in Claim 1 or Claim 2 whenever pre pared by a process as claimed in any one of Claims 20, 21 and 41 to 44.
66. 66. A compound of formula (Xa) as defined in Claim 3 or Claim 4 whenever prepared by a process as claimed in any one of Claims 22 to 40.
67. 67. A compound of formula (IXb) as defined in Claim 45 whenever prepared by a process as claimed in any one of Claims 57 to 61.
68. 68. A compound of formula (XVb) as defined in Claim 46 whenever prepared by a process as claimed in any one of Claims 62 to 64.
69. 69. A compound of formula (I) as defined in Claim 1 substantially as hereinbefore described with reference to any one of Examples 44(h), 57(c), 64 and 64(a) to 64(d).
70. 70. A compound of formula (I) as defined in Claim 2 substantially as hereinbefore described with reference to any one of Examples 44(i), 44(j), 44(k), 57(f), 57(g) and 64(e) to 64(g).
71. 71. A compound of formula (Xa) as defined in Claim 3 or Claim 4, substantially as hereinbefore described with reference to any one of Examples 43(h), 43(i), 43(j), 43(k), 56(c), 56(f), 56(g), 63 and 63(a) to 63(g).
72. 72. A compound of formula (XVb) as defined in Claim 46 substantially as hereinbefore described with reference to any one of Examples 61, 61(a) to 61(g), 62 and 62(a) to 62(g).
73. 73. A compound of formula (IXb) as defined in Claim 45 substantially as hereinbefore described with reference to any one of Examples 36, 38, 39, 40, 42(a), 42(c), 42(d), 42(e), 49, 50, 52, 53, 54(c), 54(d), 54(f) and 54(g).
74. 74. A pharmaceutical composition comprising a compound as claimed in Claim 1 and a pharmaceutically acceptable carrier.
75. 75. A pharmaceutical composition comprising a compound as claimed in Claim 2 and a pharmaceutically acceptable carrier.
76. 76. A method of treating acromegaly in a mammal other than a human which comprises administering to said mammal a compound as claimed in Claim 1 or Claim 2.
77. 77. A method of controlling diabetes in a mammal other than a human which comprises administering to said mammal a compound as claimed in Claim 1 or Claim 2.
78. 78. A compound of formula (X)

    O-Lys(Bod-Asn-Phe-Phe-Y-Lys(Boc)-Thr( But) -Ph e-Th r ( aut)-Se r ( BUt,-NH , (X) x in which X is (CH2)k wherein k is an integer from 1 to 9 and Y is Trp.
79. 79. A compound of formula (X) as shown in Claim 78 wherein Y is D-Trp and X is as defined in Claim 78.
80. 80. The compound of Claim 78 in which X is (CH2)6.
81. 81. The compound of Claim 79 in which X is (CH2)6.
82. 82. A compound of formula (IX) H-Lys(Boc) -Asn-Phe-Phe-Y-Lys(Boc) Thr(But) Phe-Thr( But) Ser(But)-NH-X-CORs (Ix) in which X is (CH2)k wherein k is an integer from 1 to 9, Y is Trp and R3 is OH or NHNH,.
83. 83. A compound of formula (IX) as shown in Claim 82 wherein Y is D-Trp and X and R3 are as defined in Claim 82.
84. 84. The compound of Claim 82 in which X is (CH,)6 and R3 is -NHNH,.
85. 85. The compound of Claim 82 in which X is (CH,)4 and Rs is -NHNH,.
86. 86. A process for preparing a compound of formula (I)

    FO-Lys-Asn-Phe-Phe-Y-Lys-Thr-Phe-Thr-Ser (I) X --- J in which X is (CH2)k wherein k is an integer from 1 to 9, and Y is Trp which comprises deprotecting under moderately acidic conditions as hereinbefore defined, a cyclic protected peptide of formula (X) as defined in Claim 78 and if desired converting to a salt thereof.
87. 87. A process for preparing a compound of formula (I)

    ys-Asn-Phe-Phe-Y-Lys-Thr-Phe-Thr-Ser-NH in which X is (CH2)R wherein k is an integer from 1 to 9, and Y is D-Trp which comprises deprotecting under moderately acidic conditions as hereinbefore defined a cyclic protected peptide of formula (X) as defined in Claim 79 and if desired converting to a salt thereof.
88. 88. A process for preparing a peptide of formula (X) as defined in Claim 78 which comprises cyclizing an activated derivative as hereinbefore defined of a compound of formula (IX) as defined in Claim 82 wherein R3 is OH.
89. 89. A process for preparing a peptide of formula (X) as defined in Claim 79 which comprises cyclizing an activated derivative as hereinbefore defined of a compound of formula (IX) as defined in Claim 83 wherein Rs is OH.
90. 90. A process as claimed in Claim 86 for preparing a compound of formula (I) substantially as hereinbefore described with reference to any one of Examples 44, 44(a) to 44(c).
91. 91. A process as claimed in Claim 88 for preparing a compound of formula (X) substantially as hereinbefore described with reference to any one of Examples 43, 43(a) to 43(c).
92. 92. A process as claimed in Claim 87 for preparing a compound of formula (I) substantially as hereinbefore described with reference to any one of Examples 44(d) to 44(f).
93. 93. A process as claimed in Claim 89 for preparing a compound of formula (X) substantially as hereinbefore described with reference to any one of Examples 54(d) to 43(f), 56(e) and 56(h).
94. 94. A compound of formula (X) as defined in Claim 78 whenever prepared by a process as claimed in Claim 88 or Claim 91.
95. 95. A compound of formula (X) as defined in Claim 79 whenever prepared by a process as claimed in Claim 89 or 93.