CA1083143A - Carba derivatives of somatostatin and process therefor - Google Patents

Abstract

CARBA DERIVATIVES OF SOMATOSTATIN AND PROCESS THEREFOR

Compounds of the formula I

in which X is (CH2)k wherein k is an integer from 1 to 9, or (CH2)n-S-(CH2)m wherein m and n each is an integer from 1 to 4 and Y is Trp or D-Trp, and pharmaceutically acceptable salts thereof are disclosed.
The compounds of formula I are useful for the management of diabetes and the treatment of acromegaly in mammals. Compositions and methods for their use are also disclosed.

Description

A~IP-6635-1-Cl .
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Background of the Invention a. Field of Invention This invention relates to derivatives of the tetradecapeptide soMatostatin. More particularly, this invention concerns carba deri-vatives 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.
b. Prior Art The name "somatostatin" has been proposed for the factor found in hypothalamic extracts which inhibits the secretion of growth hormone ~somatostropin). 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:
H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH
The abbreviations used herein for the various amino acids are Ala, L-alanine; Asn,~L-asparagine; Cys, L-cysteine; Gly, glycine;
Lys, L-lysine; Phe, L-phenylalanine; Ser, L-serine; Thr, L-threonine;
~ Trp, L-Tryptophan; and D-Trp, D-Tryptophan.
The cons~itution of the cyclic disulfide peptide somatostatin has been confirmed by synthesis; for example, see D. Sarantakis and W. A. McKinley, Biochem. Biophys. Res. Comm., 5~ 234 ~1973), J. Rivier e~ al., Compt. ~end. Ser. D, 276, 2737 (1973) and H. U. Immer et al , Helv. Chim. Acta~ 57, 730 (1974).
25~ - The important physiological activity of this cyclic disulfide peptide established it as a compound of significance for clinical pharmacology relating to the treatmen~ of acromegaly and the Management of diabetes; for example, see K. Lundbaek et al., Lancet, 2, 131 ~1970) and ;

A~-IP-66~5-1-CI
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R. Guillemin in "Chemistry and ~iology of Peptides", J. Meienhofer, Ed., 3rd hmerican 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 somatostatTn 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 structurb than somatostatin and are prepared readlly by a convenient process, which includes the following advantages: the process starts from read71y available materials, . ..
avoids noxious reagents, is executed facilely, utllizes easily removable protecting groups and provides a pure product having a high degree of physiological potency.
A "carba analog" of a cyclic disulfide peptlde refers to a ... ~,- :
compound in whlch one or two of the sulfur atoms In the cyclic backbone have been substituted by one or two methylene radlcals, respectlvely. For example, carba analogs of oxytocin have bean prepared, see a revlew by K. Jost, Colli Czech. Chem. Commun.j 36, 218(1971) and also carba analogs ~:
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of vasopress1n, see a review by K. Jost, et al., Coll- Czech. Chem.

Commun.j 39, 2835 (1974).
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3~4;3 The foregoing advantages and attributes render the carba derivatives of this invention useful for management of diabetes and for the treatment of acromegaly.
Summarv of the Invention The compounds of this invention are represented by formula 1 ÇO-Lys-Asn-Phe-Phe-Y-Lys-Thr-Phè-Thr-Ser-NH,", X
in which X is (~i2)k wherein k is an integer from I to 9; or - ' ' (CH2)n-S-tCH2)m wherein m and n each is an i,nteger from I to 4. ~
and Y is L-Trp;or D-Trp. - ~ ' , A: i3re~ferred~group of~compound~of this inventio~ is ~epresented , -' by~f!or~u!a 1 1n which X is (CH2)n-S-tCH23m wharein m and n each is an integer from I 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 thts inventlon.
: :
The compounds of formula L in which X and Y are as defined ~herein are prepared by a process which comprlses: subJecting the cyclic protected pept7de of formula X
~O-Lys(Boc?Asn-Phe-Phe-Y-Lys(Boc)-Thr(i3u~)-Phe Thr(~ut)-Ser~But)-NH '' -- - X - ~- ) (X) ::
- in whlch X and Y are as deflned hereln to moderately acldlc conditlons to remove the protectT,ve groups, and isolatlng the correspondtng compound of formula 1.
:
The compounds of formula X in which X-and Y are as defined '' herein are prepared by a pracess which compr7ses: ~ -reacting according to the~azlde ,coupllng method a tetrapeptidé hydrazide of for~ula~ll ' - Rl-Lys~Boc)-AsD-Phe-Phe-NHNH2 in which Rl Is i~dz or Z wlth a hexapoptld~ of formula ~
~ ' H-Y-Lys(~oc)-Thr~B~t)-i~he-Thr~Bu ~-Ser~But)'OMe - 7n whtch Y is as deftneci he~eln to obtaln the correspondlng decapeptlde ~ ;, of formula IV- - '-. ~

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Ai-lP-6635 -I-CI
~083~43 Rl-Lys(Boc)-Asn-Phe~Phe-Y-Lys(Boc)-Thr(But`)-Phe- `
Thr(Bu )-Ser(But)-OMe (IV) in which RI and Y are as defined herein,subjecting the latter compound -i-o hydrazinolysis to obtain the decapeptide hydrazide of formula V
R -Lys(Boc)-Asn-Phe-Phe-Y-Lys(Boc)-Thr(Bu j-Phe-Thr(But)-Ser(Bu )-NHNH2 (V), in which Rl and Y are as defined herein, reac-iing the last-named . .
compound according to the azide coupllng method with a compound of formula Vi H2N-X-COOR (Vl~
in which X is as def-lned herein and R2 jS lower alkyl to obtain the corresponding compound of formula Vll Rl-Lys(Boc)-Asn-Phe-Phe-Y-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH-X-CCOR2 (Vll) in which Rl, R , X and Y are as defined herein, subjecting the latter compound to hydrazinolysis or alkaline hydrolysis to obtain the .: . :
corresponding compound of formula V!ll -~
Rl-Lys(Boc)-Asn-Phe-Phe-Y-Lys(Boc)-Thr(Bu )- .~.-Phe-Thr(But)-Ser(But)-NH-X-C0-R3 (Vlll .
in which Rl, X and Y are as dafined herein and R3 is OH or ~ :
NHNH2 respectlvely, removlng the protectlng group Rl ~rom the :~:
compound of formula Vlll when Rl is Ddz under ~lldly acidic : condttions or when R is Z and X is (Cii2)k with hydrogen in the . .
presence of a noble metal catalyst to obtain the corresponding compound of formula IX
25~ H-Lys(eoc)-Asn-Phe-Phe-Y-Lys(Boc)-Thr(But)-i'he-ThrtBut)-Sar(But)-NH X-C0-R3 (IX) Ai-lP-6635-1-CI
- ~0~31~L3 in which R , X and Y are as defined herein, and cyclizing the compound of formula 1~ with 3 carboxyl group àctivating : :
agent to obtain the corresponding cyclic protectbd peptide of formula X
in which X and Y are as defined herein. : :
Another embodiment of the process to prepare the compound of formula X in which X is tCH2)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 ~.
hydrazTde, Ddz-Y-LystBoc)-Thr(But)-Phe-Thr(But)-Ser(But~-NHNH2 in which Y is as defined herein with a compound of formula VIJH2N-x-cooR
in which X is (CH2) -S-(CH2)nwherein m and n are as defined herein and R is lower alkyl to ob-i-ain the corresponding compound of formula Xl Ddz-Y-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH-(CH2)m-S-(CH2)n-COOR2 (Xl) in which m, n, R and Y are as defined herein, subjec-ing the latter compound to hydrazinolysis to obtain the corresponding hydrazide of formula Xll :
Ddz-y-Lys(Boc)-Thr(But)-phe-Thr(But)-ser(But)-NH-(cH2)m-s-(cH2)ncoNHNH2 (Xll) ~ :
in which m, n and Y are as defined herein, reacting the last-namedcompound according to the azide coupling method with the tetrape;~ttde H-Lys(Boc)-Asn-Phe-Phe-OMe to obtaln the corresponding compound of formula Xlll Ddz-Y-Lys(Boc~-Thr(i3ut)-Phe~Thr(But)-Ser(But~-NH~(CH2)m-5-(CiJ2)n-: CO-Lys(Boc)-Asn-Ph0-Phe-O~b (Xlll) Tn whTch m n and Y are as de~Tned hereTn, subJectTng the latter compound .
: to mTIdly acldtc condltTons to obtaTn th0 correspondlng compound of~ formula XIV
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~0~3~43 AIIP-6635 I-Cl -Y-Lys(Boc)-TI-r~Bu )-Phe-TIlr(Bu )-Ser~Bu )-Nll-(C~l2)m-S-(Cll2)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 ~he corresponding hydrazide of forMula XV
I-l-Y-I.ys(Boc)-Thr(Bu )-Phe-Thr(But)-Ser(But)-NH-(C~l2)m-S-(CH2)n-C0-Lys(Boc)-Asn-Phe-Phe-N~lNIi2 (~
in which m, n and Y are as defined herein, and cyclizing the latter compowld according to the azide coupling method to obtain the corresponding cyclic protected peptide of formuLa X in which X is (CH2)n-S-(CH2)m wherein m and n are as defined herèin,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 managing diabetes in a mammal which comprises administering to said mammal an effective amount of a compound of formula I.
Details of the Invention 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, Il, 1726 - 1732 (1972). For instance, L,ys, Asn, Asp, Phe, Trp, D-lrp, Thr and Ser represent the "residues" of L-lysine, L-asparagine, L-aspartic acid, L-phenylalanine, L-tryptophan, D-trypto~phan, L-threonine and L-serine, respectively. The term "resldue" 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.

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.A number of procedures or techniques for the preparation of peptides has hitherto been well established. For instance, the functional groups which are not involved in the péptide 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 trepresented by Z), t-butoxycarbonyl ~represented by Boc), ~,~-dimethyl-3,5-10 dimethoxy benzyloxycarbonyl (represented by Ddz), 2-(D-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 toluenesuIfonyl; the aIkyl 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 ~,~-dimethyl-3,5-dimethoxy-benzyloxycarbonyl. The protecting groups for the hydroxyl of serine and threontne are represented .
by acetyl, tosyl, benzoyl, teri-butyl (represented by Bu ) and benzyl; the preferred protecting group is tert-butyl. The ~carboxyl1c acld function of a peptide or am1no ac1d can be cons1dered .: . .
protectad by a lower alkyl or lower aralkyl ester which.include methyl (represented by OMe), ethyl (represented by O~t), or benzyl trepresented by OBzl) esters; and also by suhstltuted hydraz1des whlch 1nclude t-butoxycarbonyl hydraz1de (represented by NilNH-Boc), .. ~ :
benzyloxycarbonyl hydraz1de ~represented by NHNH-Z), or ~ ~

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dimsthyl 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.~enjamin, Inc., New York, 1966, pp. 45-51 and E. Schr~der and K.LUbke, "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.gO~ dicyclohexyIcarbodiimide; N-hydroxysuccinimide, 2,4,5-trichloro-phenol, pentachlorophenol, p-nitrophenol or l-hydrobenzotriazole in the presence ofa dialkylcarbodiimide; and in the case of a hydrazids 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 d7alkylcarbodi7mide.
The following activated esters have proved to be particularly suitable in the process of this invention: 2,4,5-trTchlorophenyl (represented by OTcp)j pentachlorophenyl (represented by OPcp), p-nitrophenyl (represented by O~p), or l-benzotrTazolyl; the succlnimtdo group is also useful for such actlvat70n.
The term "azide coupllng method" as us0d herein refers to the method of activating the term1nal carboxyl of a peptlde fragment wlth an azide and condenslng the latter peptide azlde with another pepttde havlng a free amlno group. The peptlde azlde ts conveniently prepared by reactlng a peptide hydrazld0 wlth a r~agent which ~ ,., :.

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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 nitr7te in the presence of a strong acid, preferably hydrogen chloride, (pH
ranging usually ,~rom 0.1 to 2~, in an anhydrous inert organic solvent, for example, dimethylformamide, dimethyl sulfoxide, ethyl acetate, methylene dichloride, tetrahydrofuran, dioxane, and the like at -30C to 20C, preferably at about -15C for 10 to 30 ~ -minutes to obtain the corresponding azide. The peptide azide can be isolated and crystallized but is preferably allowed to rema7n ~
in the reaction m7xture. Thereafter the a7ide 7n the above mixture is ~ ;
~ . .
reacted w7th the peptide unit or compound having the free amino ~ ;group at temperatures ranging from -30C to 20C for about one to ~20 ;two hours and then at 0 to 30C for 10 to 30 hours. An acid acceptor, preferably an organ7c base, for example N-ethyld77so-j propylam7ne, N-ethyimorphol7ne or tr7ethylam7ne~ Is present in the ` ~ react70n m7xture in order to make the react70n medium sl7ghtly alkal7ne, preferably pH 7.0 to 9Ø See also the above c7ted textbooks ~25~

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33~L~3 of Kopple or Schr~der and LUbke for additional descriptions of ~.
-Ihis 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 represen-i-s ~-an ethyl group, Pr represents a propyl group and NHNH2 represents a hydrazide group.
The term "lower alkyl" as used harein contemplates hydro- :
carbon radicals having one to three carbon atoms and includes methyl, ethyl, and propyl.
15~ The term "mineral acid" as used herein contemplates the ;
: :
strong inorganic acids and includes hydrochloric, hydrobromic, sulfuric, or phosphoric acid. When tha term is used in conJunction `.
:: . : .
with an anhydrous system, anhydrous hydrogen chlorlde is the : preferred minera1 acid. ~ .
The term "mildly acidic conditions" as used herein contemplates conciitions in which a dilute aqueous solution of an organic acid, for example 30 - 80% aqueous formic, acettc or propionlc acld, preferably 70 - 80~, or mixtures thereof, Is a .
; ~ principal component of the reactlon medtum.

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The term "moderately acidic conditions" as used herein contemplates conditions in which concentrated organic acids or solutions of the mineral acids are used as a principal component of the reaction medium at temperatures ranging from about -30 -to 30C. Examples ef preferred conditions in this case include the use of 50 to 100% trifluoroacetic acid at 0 to 30C 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 10C.
: .
The term "organic nitrite" includes the commercially available alkyl nitrites, for instance, t-butyl nitrite, isoamyl nitrite, and the ! ike.
The term "organic base" as used herein includes tri=thylamine, N-ethylmorpholine, N-ethyldiisopropylamine and the like.
; 15 The peptides of formula I are obtaTned in the form of the fr=e base or as~an acid addition~salt either directly from the process r 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, sa!icylic, methanesulfonic or -toluenesulfonic acid; as well as polymeric acids such as tannic acid or carboxymethyl cellulose, and salts with inorganic acids such as hydrohalic acids, e.g. hydrochloric acid, or suifuric acid, ~ I -or phosphoric acid. It should be noted that the peptides have two basic nitrogens giving rise to addition salts with 4ne to possibly ~ two equivalents of acid. If desired, a particular acid addi-ilon sali 1'' , ' ~ ,, ,,.' ,1/ , , , , '.''';' ' ' "
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~3~3 AIIP-6635-1-Cl is converted lnto 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 tlle Sephadex* C type, and strongly baslc 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.
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"
lssued November 4, l975 and U.S. Patent No. 3,917,581, entitled "Derivatives of Somatostatin and Process Therefor" issued November 4, 1975.
The petptides produced by the process of this invention, as well as their corresponding pharmaceutically acceptable salts, are useful because tlley possess the pharmacological activity of the natural tetradecapeptide somatostatin. Their activity is demonstrated readily 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)-~Sephadex is a Trade Mark A~IP-6635-1-CI

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The activlty of the peptides of formula i of this invention is demonstrated also In vivo in a modification of the pentobarbital-induced increase in plasma growth hormone level in the rat as described by Brazeau et al., cited above. In this test the peptides of this invention show a level o-f activity which is greater than or o-f 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 management of diabetes in mammals; see for example, iO P. Brazeau et al., cited above. When the peptides or salts thereof are employed for such treatment or management, they are 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 psptide or a salt thereof is used in a sterile aqueous solution, such solution may also contain other solutes such as buffers or preservatives, j as well as sufficient amountsof pharmaceutically acceptable salts or glucose : .
to make the solut70n 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 I mcg to 300 mcg per kilogram body we~ight. However, a dosage le~el in the range of from about I mcg to about 50 mcg per kllogram body weight is most desirably employed ;
in order to achieve effective results.

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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 intrarnuscular injection or by implantation.
Such dosage forms are designed to release from about 0.1 mcg to about 50 mcg per kilogram body weight per da~.
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 saIts described below, or they 10 may contain the peptide in the form of a water-soluble salt together --with a protective carrier which prevents rapid release. In the Iatter case, for exampie, the peptide may be formulated with a non-antigenic partially hydrolyzed gelatin in the form of a viscous liquid;
or the peptide may be absorbed on a pharmaceutically acceptable solid carrierj for example, zinc hydroxide, and may be administered in suspension in a pharmaceutically acceptable liquid vehicle; or the i peptide may be formulated in gels or suspensions wi~h a protective ; non-antlgenic hydrocolloid, for example sodium carboxymethylcellulose, polyvinylpyrrolidone, sodium alglnate, gelatine, polygalacturonic ~ 1 ZO ~acids, for example, pectin,; or certain mucopolysaccharides, together - wTth aqueous or non-aqueous pharmaceutically acceptable liquTd vehicles, preservatTves, or surfactants. Examples of such formulations are found Tn standard pharmaceutical texts, e.g. in RemTngton's /: ~ ' , ,.

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Pharmaceutical Sciences, 14th Ed., Mack Publishing Co., Easton, Pennsylvania, 1970. Long-acting, slow-release preparations of the peptide produced according to the process o-f 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 about 1.0 mcg to about 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 disperslons in a neutral hydrogel of a polymer ; ;
;~ of ethylene glycol methacrylate or similar monomers cross-linked as described in U.S. Patent 3J551,556,may also be formulated in the form of pellets releasing about the same amounts as shown above , .
and may be implanted subcutaneously or intramuscularly.

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~83~4;3 Process The process of this invention will be illustrated by the following embodiments in which specific peptides of formula I are prepared.
In one embodimeni 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 11, Rl-Lys~Boc)-Asn-Phe-Phe-NHNH2 in which Rl is Z or Ddz is readily obtained by hydrazinolysis of the corresponding tetrapeptide ester Rl-Lys(Boc)-Asn-Phe-Phe-OMe in which Rl is as defined herein. The latter tetrapeptide in which R is Z is described by H.U.lmmer et al.j cited above, and the tetrapeptide of formula Ddz-Lys(Boc)-Asn-Phe-Phe-OMe (i.e. R is Ddz) is readily prepared by following the procedure described in the latter reference but replacing Z-LystBoc)-OH with Ddz-Lys(Boc)-OH. The hydrazinolysis is conveniently carried out by dissolvlng the tetrapeptide ester in an inert organic solvent, for example, methanol, ethanol, dimethylformamide and the like, 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 about 0 to 10C for about 12 to 30 hours.
The correspond7ng tetrapeptide hydrazide of formula 11 is obtained by conventional means, for example, evaporation or preclpitation with water.
The requisite second starting material, the hexapeptide of formula 111, H-Y-Lys(Boc)-Thr(8a~)-Phe-Thr(But)-Ser(But)-OMe in whlch Y is ~ ~ Trp is obtained by a-amTno deprotection of the hexapeptlda Ddz-Trp-Lys(Boc)-; ~ t t t Thr~Bu )-Phe-Thr(Bu )-Ser(Bu )-OMe (described by H.U. Immer et al., cTted above) under miIdly acldic condTtions, preferably by allowlng ; ~ the latter hexapeptide to stand in a solutlon of acetic acld-formTc acTd-water (7:1:2) at about 20C to 30C for 12 to 30 hours.

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By following the procedure described by H~U. Immer et al., cited above, but replacing L-tryptophan with D-tryptophan, +he hexapeptide ~ -Ddz-D-Trp-Lys(Boc)-Thr(But)-Phe-Thr(Bu~)-SertBut)-OMe is obtained.
Deprotectlon of the latter compound under mildly acidic conditions gives the hexapeptide oF formula 111 in which Y is D-Trp.
In the next step of the present process, the tetrapeptide hydrazide of formula 11 and the hexapeptide of formula 111 are coupled according to the azide coupling method to obtain the corresponding decapeptide of formula IV,RI-Lys(Boc~-Asn-Phe-Phe-iO Y-Lys(Boc)-Thr(Bu )-Phe-Thr(Bu )-Ser(But~OMe in which Rl and Y are as defined herein.
A convenient and efficacious procedure for this step comprises dissolving the tetrapeptide hydrazide of formula 11 In an inert organic solvent, preferably dimethylformamide, and cooling ; 15 the mixture to about -20 to -10Co A solution of about two to five molar equivalents of a mineralacid 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 nitrite, for example, 1.2 molar equivalents of t-butyl nitrite. In this manner the corresponding tetrapeptide - ~ azide, Rl-Lys(Boc)-Asn-Phe-Phe-N3 7n which Rl is as defined herein, is obtalned. After about 10 to 20 minutes at about -20 to -10C
an organic base, preferably N-ethyldiisopropylamine, is added until pH 7.1 to 9 is attained. The mixture is cooled to about -30 to -20C and a solution of substantially one molar equivalent of - the hexapeptide of formula 111 in an inert organic solvent, preferably ~ -dimethylformamide, is added to +he above solution containing saTd azide. The reaction mixture Is then stirred at about -20 to -10C -~ for one to two hours and then at about 20 to 30C for Z0 to 30 hours.

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AHP-6635~
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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 Rl and Y are as defined herein.
The decapeptide of formula IV is readily converted to the corresponding decapeptide hydrazide of formula V, Rl-Lys(Boc)-Asn-Phe-Phe-Y-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NHNH~ in which Rl 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 about 20 to 30C 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 Vl, H2N-X-COOR2 in which R2 and X are as defined herein, are coupled according to tha azide coupling method, in the same manner as described hereinabove, to obtain the corresponding compound of formula Vll, Rl-Lys(Boc)-Asn-Phe-Phe-Y Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH-X-COOR2 in which Rl, R2, X and Y are as defined herein.

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1~3~L~3 Al-IP-6~35-1-Cl ; The compounds of formula VI in which X is (C~l2)k wherein k is as defined herein are either known or are readily prepared by known procedures, for example, 4-aminobutanoic 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-aminononanoic 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)n wherein m and n are as defined herelh,are readily prepared by alkylation of an w-aminoalkanethiol. For example, a convenient preparation comprises reacting liquid ammonia wi~h lithium and hydrated ferric nitrate untilthe blue color disappears, adding an ~-aminoalkanethiol, H2N-(CH2)~-, SH in which m is as defined herein, adding an w-bromoalkanoic acid ester, `I ,, " .
Br-(C~12)n-COOR~ in which n and R are as defined herein, and isolating the correspanding compound of fDrmula 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 matter as described above to obtain the corresponding hydrazide `¦ of formula VIII, R -Lys(Boc)-Asn-Phe-Phe-Y-Lys(Boc)-Thr(Bu )-Phe-Thr(Bu )-i ~ Ser(But)-NH-X Co-R3 in which Rl, X and Y are as defined herein and R3 is NHNH .
l . 2 The o!-amino protecting group (Rl) is removed from said last-named compound of formula VIII to obtain the corresponding ~-amino .
~¦ ~ 25 deprotected compound of formula IX, H Lys(Boc)-Asn-Phe-Phe-Y-Lys(Boc)-¦ ~ ~ Thr(But)-Phe-Thr~But)-Ser(But)-NH-X-C0-R3 in which X and Y are as defined ~5 herein and R3 is NHNH .

~' .

~(~8;~
/\IIP-()635-1-Cl When Rl is the ~-amino protecting group Z and X is (Cll2)k, the protecting group ~Z) is conveniently removed by hydrogenolysis, preferably by agitating ~mder an atmosphere of hydrogen a mixture of the compound of formula VIII in which R3 is N~12 and a noble metal catalyst, for example, platinum or palladium on carbon in an inert organic solvent, for example, methanol, ethanol or acetic acid.
The ~-amino protecting group (Ddz) is readily removed from the compound of formula VIII in which Rl is Ddz and Y and X are as defined herein and R3 is NHNH2 by treatment under mildly acidic conditions, preferably by allowing the compound of formula VIII to stand in a solution of acetic acid-formic acid-water ~7:1.2) at about 20 to 3~ C for 12 to 24 hours to obtain the corresponding compound of formula IX in which X and Y are as defined herein and R3 is NHNH2.
Alternatively, the compound of formula VII is hydrolyzed under alhaline conditions, preferably with two to six molar equivalents of sodium or potassium hydroxide in an inert solvent, for example, wa~er, methanol, ethanol, isopropanol, acetone and the like or mixtures thereof, :
at about 0to 30C for about 10 to 60 hours. The latter alkaline solution : is acidified with citric acid and the corresponding compound of formula . 20 VIII, Rl-Lys~Boc)-Asn-Phe-Phe-Y--Lys~Boc)-Thr~But)-Phe-Thr~But)-Ser~But)-NH-X-Co-R3, in which Rl, X and Y are as defined herein and R3 is OH is isolated by conventional means, for instance) filtration, extraction and the like.
The amino protecting group Rl is removed from the latter compound ~ 25 of formula VIII in which R3 is OH, in the same manner as described above for the compound of formula VIII in which R3 is NI~INH2, to give the corresponding compound of formula IX, H-Lys~Boc)-Asn-Phe-Phe-Y-Lys~Boc)-Thr~But)-Phe-Thr-I ~But)-Ser~But)-NH-X-CO-R3, in which X and Y are as defined herein and R3 is OH.
:

~83~9~3 Al-IP-6635-l-Cl The compound of formula IX is cycli~ed in the presence of a carboxyl group activating agent to obtain the corresponding compound of formula X, C~-Lys(Boc)-Asn-Phe-Phe-Y-Lys(Boc)-Thr(Bu )-Phe-Thr(Bu )-Ser(Bu )-rll, I 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 termina~ 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 R 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 M~INH2 in an inert organic solvent, preferably dimethylformamide, cooling the mixture to about -20~to -10C, adding a solution of about two to five molar equivalents ;

of a mineral acid in an inert organic solvent, preferably three molar 20 equivalents of hydrogen chloride in ethyl acetate, adding 1.0 to 1.5 molar equivalenLs of an organic nitrite, for example, 1.2 molar equivalents of t-butyl nitrite, and stirring the mixture for 10 to 20 minutes at about -2~

,: ~
to -10C. In this manner the corresponding a~ide, ~-I-Lys(Boc)-Asn-Phe-Phe-Y-Lys(Boc)-Thr~But)-Phe-Thr(Bu )-Ser(Bu )-NH-X-CON3 in which X and Y are 25 as defined herein, as the hydrochloride salt i5 present in the solution. The l~ mixture containing the latter azide is diluted with ten times the amount of ¦ its volume of an inert organic solvent, pref~rably dimethylformamide, precooled to about -ZO to -10C. An organic base, preferably N-ethyldiisopropylamine, is addecl until pll 7,1 to ~ is attained.

3~ 3 AIIP-~635-1-Cl The mixture is stirred at about -20 to -10C for 30 minutes to two hours, then at about OC for two days and finally at about 20 to 30C
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.
Similarily, the terminal carboxyl in the compound of formula IX
in which R3 is OII 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 cycli7ing 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 R3 is OH with two eo six molar equivalents of N-hydroxysuccinimide, 2,4,5-trichlorophenol, pentachlorophenol, p-nitrophenol or l-hydroxybenzotriazale and one to five molar equivalents of dicyclohexylcarbodiimide in an inert organic soIvent at 0~ to 5C ~or 10 to 30 hours and at 20 to 30C for two to ten days. An organic proton acceptor, preferably triethylamine, N-ethylmorpholine, N-ethyldiisopropylamine and the like, is added to the reaction solution in order to maintain the solution at a pH of about 7 to 9. Suitable inert organic solvents can be selected from methylene :
chloride, chloroform, ethyl acetate, dimethylformamide and the like. The corresponding compound of formula X is isola-ted from the reaction mixture by conventional methods.
Finally, the aforementioned cyclic protected compound of formula X is transformed to the correspondin~ compound of formula I
in whlch~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. Generallythis step is carried out by dissolYing the cyclic compound in an aqueous reaction medium containing a mineral acid at 0 to 20C for lO to about 60 minutes. P~xamples of such media are trifluoroacetic .

~3143 ~-IP-6635-1-Cl acid, lO to 20~ aqueous sulfuric acid, 10% phosphoric acid, 10 to 30% hydrobromic acid and 10 to 30% 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 nlinimum of concentrated hydrochloric acid cooled to 0C and allowing the mixture to stand at 0C for five to ten minutes under a nitrogen atmosphere. Thereafter, about 5 to 15 volumes of glacial acetic acid is added and the solution is cooled to about -70C 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) 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)n-S-(CH2)m is described. In this embodiment the requisite first star~ing material of formula XI, Ddz-Y-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH-(CH2) -S-(CH2)n-COOR

-2'~-~0 ` ~)83143 A~IP-6635-1-CI

in which m, n and Y are as defined herein and R is lower alkyl, is readily obtain by coupling the hexapeptide hydrazide, Ddz-Y-L.ys(Boc)-Thr(Bu )-Phe-Tllr(But~-Ser(Bu )-NI~H2 in which Y is as defined herein with a compound of formula VI in which X is (CH2)m-S-(Cl-l2)n whereill m and n are as defined herein and ~2 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)-NI-~H2 in which Y is Trp is described by ~I.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-O~I.
In the next step of the present process the compound of formula XI is reacted with a molar excess of hydrazine hydrate, preferably at about 20 to 30C for two to five days, to obtain the correspollding hydrazide of formula XII, Ddz-Y-Lys(Boc)-Thr(Bu )-Phe-Th~(But)-Ser(Bu )-NH-(CH2)m-S-(CH2)n-CONIINH2 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)-N~I-(C~l2)1n-S-(CI-I2)rl-CON3 in which m, n and Y are as defined herein and said azide is coupled with the tetrapeptide of formula ~I-Lys(Boc)-Asn-Phe-Phe-OMe, described by Il.U. Im~ler 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)n-CO-Lys(Boc)-Asn-Phe-Phe-OMe in which m, n and Y are as defined herein.
, .

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~0~33L43 .

The a-amino protecting group (Ddz) is readily removed from the compound of formula Xlll by treatment under miIdly acidic conditions, preferably by allowing -~he compound of formula Xlll to stand in a solution of acetic acid-formic acid-water (7:1:2) at about 20 to 30C for 12 to 2~ hours to obtain the corresponding compound of formula XIV, H-Y-Lys(Boc~-Thr(But)-Ph~-Thr(But~-Ser-(But)-NH-(CH2)m-S-(CH2)n-C0-Lys(Boc)-Asn-Phe-Phe-OMe in which m, n and Y are as defined herein. Said compound of formula XIV
is reacted with a molar excess of hydrazine hydrate at about 20 10 -to 30C for 20 to 30 hours to obtain the correspondinq hydrazide of formula XV, H-Y-Lys(~oc~-Thr(Cut~-Phe-Thr(But)-Ser(But)-NH-(~H2~m-; ~-(cH2)n-co-Lys(Boc)-Asn-phe-phe-NHNH2 in which m, n and Y are as defined herein.
The hydrazide of formula XV Ts cyclized accordi~g to the ; ~ 15 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 ,<
in which X is (CH2)n-S-(CH2)m whereln m and n are as defined herein.

.. . ..
and Y is as defined herein. The latter compound of formula X is .. : .
identical in all respects to the corresponding compound of formula X
obtained from the previously described process. Finally, the latter compound of formula X is sub3ected to moderately acid7c conditions ~ and purified as described above to obtain the corresponding compound ; ~ of formula I In which X is (CH2)n-S~(CH2)m wherein m and n are as defined herein and Y is as deflned herein which is identical to the ::,:, .
~` corrsspondtng compound of formula I obtatned from the previously descrtbed process.

The foilowtng examples illustrate further thts inventton.

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~ 3 ~IP-6635-1-CI
., EXAMPLE i ~-Dimethy1-3.5-dimethoxy-benzy_loxvcarbonyl-(N -t-butoxycarbonyl)-lysYI-asparaqi-nyl-phenylalanyl-phenvlalanine Msthyl Ester tDdz-Lys(Boc)-Asn-Phe-Phe-OMe) A mixture of N -tert-butyloxycarbonyllysine (10 9 ~0 7 mmole) in methanol (500 ml) and benzyltrimethylammonium hydroxide (35% in methanol 19.4 9 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 200 ml) and dried under reduced pressure. The residue is dissolved in pyridlne-dimethylformamide (2:1) (600 ml) and stirred at 47C under a nitrogen atmosphere. ~ a-DTmethyl-3 5-dimethoxy-benzyloxycarbonyl azide (10.78 9 40.7 mmole) tn pyridine (~OO ml) is added dropwise over 0.5 hour to the mixture. The temperature is main-taTned at 47C and stirring is contTnued for 3 days. The reaction mTxture ls concsntrated to a vTscous oil. The oil is disolved in O.IN sodium hydroxide (300 ml) and extracted with ether (3 X 150 ml).
The aqueous layer Ts acTdTfied wTth 50% aqueous citric acid to approximately pH 4.0 and extracted wTth ether (3 X 200 ml). The ~` 20 ~ combTned ether layers are dried wTth sodium sulfate and concentrated to give Ddz-Lys(Boc)-OH as a colourless foam.
~ .
Ddz-Lys(Boc)-OH (9045 9. 0.02 mole described above and ~ ~ I-hydroxybenzotrTazole (2.99 9 0.02 mole) are dtssolved in ; dTmethyl~ormamide (40 ml) and the solutTon is coole~ tn Tce.
DTcyclohexylcarbodTTmide (4.36 9 0.021 mole) is added and the mtxture is stTrred at 0C for one hour and at room temperature for ~ -2 hours. The mTxture is cooled to 0C and a solution of H-Asn-Phe-;'~ ~ ":.`''' .-: ,:

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, EXAMPLE 1_- continued Phe-OMe CF3COOH (10.65 9, 0.0192 mole, described by H.U. Immer et ai., cited above) and N-ethylmorpholine t2.46 ml, 0.0192 mole) in dimethylformamide (40 ml) is added and the mixture i5 stirred.
Another portion of N-ethylmorpholine (2.46 ml) is added to bring the pH to 7 and the mixture is stirred at room temperature for 2 days.
The mixture is filtered and the filtrate is evaporated. The residue is dissolved in methanol and diethyl ether is added. The precipitate is collected and dried to give the title compound, [~]D5~24.81 (c = 1, DMF) and nmr (DMS0-d6) 6 1.36 (s, 15 H), 3.58 (s, H),

3.58(s, 311), 3.77~s, 6H1 and 7.22 - .27 (m, 13 H).

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~ 43 ~1l) 6635-1-Cl EX~MPLE 2 Benzyloxycarbonyl-~N~t-butoxycarbonyl)lysyl-asparaginyl- -phenylalanyl phenylalanine Hyclrazide (Z Lys~Boc) Asn-Phe~Phe-N~-IN~12) II; Rl ~ Z
1-1ydrazine hydrate (2 ml) is added to a suspension of Z-Lys(Boc)-Asn-Phe-Phe-OMe (1.2 g, described by 1-1.U. Immer et al., cited above) in dimethylformamide (l0 Ml) at 0C. The mixture is stirred at 0~C overnight during which time all the starting material dissolved. Water is added-to the solution and the i precipitate is collected. The precipitate is washed with water and dried under reduced pressure over phosphorus pentoxide to give the title compound.
In the same manner but replacing Z-Lys(Boc)-Asn-Phe~Phe-OMe with an equivalent amount of Ddz-Lys~Boc)-Asn-Phe-Phe-OMe (described in Example 1), Ddz-Lys~Boc)-Asn-Phe-Phe-NHN112~ nmr ~DMSO-d6) ~ 1.37 (s, 15H), 3.72 ~s, 6H~ and 7.5 - 7.3 ~mJ 13H) is obtained.

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~8314~ AIIP-6635-1-Cl ~,~-Dimethyl-3~5-dimethoxy-benzyloxycarbonyl-D-~ryptophyl-(N~-t-butoxycarbonyl)lysyl-(O-t-butyl)threonyl-phenylalanyl-. .
(O-t-butyl)threonyl-(O-t-butyl) serine Methyl Ester (Ddz-D-Trp-Lys(Boc)-Thr(Bu )-Phe-Thr~Bu )-Ser(Bu )-OMe) A solution of 0C of dicyclohexylcarbodiimide (1.5 g~
7.3 mmole) in tetrahydrofuran (15 ml) is added dropwise to a solution at 0C of Ddz-D-Trp-OH (3.11 g, 7.3 mmole) and l~hydroxy-benzotriazole (1.97 g, 14.6 mmole) ln tetrahydrofuran (55 ml). The mixture is stirred for one hour at 0C and one hour at 20 to 25C.
The mixture is added to a solution of H-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser~Bu )-OMe (6.32 g, 7.3 mmole, described by H.U. Immer et al., cited above) and N-ethylmorpholine (0.935 ml, 7.3 mmole) in tetrahydrofuran (20 ml). The resulting mixture is stirred for one l$ hour at 20 to 25C and cooled to 0C. The mixture is filtered and the filtrate is evaporated. The residue is dissolved in ethyl acetate.
The solution is washed with saturated sodium bicarbonate solution, - saturated sodium chloride solution, dried over magnesium sulfate and evaporated. The residue is subjected to chromatography on ; 20 silica gel using ethyl acetate-benzene (1:1) and the eluates are ovaporated. The residue is crystallized from acetone-benzene to give the title compound, mp 162~166C.

' '' ~ -30-~3~3 AIIP-663s l Cl Tryptophyl-(N~ t-butoxycarbony])lysyl-(O-t-butyl)threonyl phenylalanyl-(O-t-butyl)threonyl-(O-t-butyl)serine ..... . .
Methyl Ester (H-Trp-Lys(Boc)-Thr(But)~Phe-TIIr(But)-Ser(Bu )-OMe) III; Y = Trp A solution of Ddz-Trp-Lys(Boc)-Thr(But)-Phe'Thr(Bu )-Ser(Bu )-OMe (2.037 g, described by ~I.U. Immer et al., cited above) in 28 ml of a solvent consisting of acetic acid-formic acid-water (7:1:2) is stirred at room,temperature ovérnight. 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(Bu )-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-D-Trp Lys(Boc)-Thr(Bu )-Phe-Thr(Bu )-Ser(Bu )-OMe is obtained.

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3lL~3 Benzyloxycarbonyl-(N~-t-butoxycarbonyl)lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyl-(N~-t-butoxycarbonyl)lysyl-_ _ (O-t-butyl)threonyl-phenylalanyl-(O-t-butyl)threonyl-.
(O-t-butyl)serine Methyl Ester (Z-Lys(Boc)-Asn-Phe-Phe~Trp-. ~
Lys(Boc)-Thr(Bu )-Phe-Thr(But)-Ser(Bu )-OMe) IV; ~1 _ z and _ _ _ _ . . . . . _ _ . _ _ Y - Trp A solution of Z-Lys(Boc)-Asn-Phe-Phe-NHN~12 ~1.023 g~
described in Example 2 in dimethyl~formamide (20 ml) is cooled to -20C. Hydrogen chloride (2.7M) in ethyi acetate (1.2 ml) is added followed by t-butyl nitrite (0.18). The solution is stirred at -~5C for 15 min. and N-ethyldiisopropylamine (0.79 ml) is added. The mixture is cooled to -30C 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~ethyldiisopropyl- -- amine (0.1 ml) in order to bring the pll to 8. The mixture is stirred at -15C for one hour, at 0C 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 pre-cipitate is subjected to chromatogr~phy on silica gel using 4%
methanol and 0.1% triethylamine in chloroform. The eluates are evaporated to give the title compound, amino acid analysis: Lys 8), Asp (0.99), Thr (1.89), Ser (0.93), Phe ~3.00~ and Trp (presont).

: "

~ 3~ AHP-6635-1-CI

EXAMPLE 5 - con-tinued ; In the same manner Z-LystBoc)-Asn-Phe-Phe-NHNH2 or Ddz-Lys(80c)-Asn-Phe-Phe-NHNH2 tdescribed 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(Bu )-OMe (described in Example ~) to obtain the following compounds of ~ormula IV:
Z Lys(Boc)-Asn-phe-phe-D-Trp-Lys(Boc)-Thr(But)-phe-Thr(But)-ser(Bu~)-oMe~
Ddz-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(Bu )-Phe-Thr(But)-Ser(Bu )-OMe, mp 210-212C and C~]D -2.5 (c = 1, DMF), and Ddz-Lys(Boc)- : -10 10 Asn-Phe-Phe-D-Trp-Lys(Boc)-Thr(Bu )-Phe-Thr(But)-Ser(But)-` ~ :
: OMe, mp 179-183C.
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, . . , . . : , ~3~3 AHP-6635-1-CI

Benzyloxycarbonyl-(N -t-butoxycarbonyl)lysyl-asparaginyl- -phenylalanyl-phenylalanyl-tryptophyl-(N -t-butoxycarbonyl)-.
Iysyl-(O-t-butyl)threonyl-phenylalanyl-(O-t-butyl)threonyl-. .
S (O-t-butyl)serine Hydrazide (Z-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(Bu~)-Phe-Thl-(But)-Ser(But)-NHNH ) V; Rl - Z and Y = Trp Hydrazine hydrate (I ml) is added to a solution of the compound of formula IV, Z-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(Bu~)-; 10 Ser(But)-OMe (1.15 9, described in Example 5), in dimethylformamide (13 ml) at 0C and the solution is stirred at room temperature for 5 days. The solution is cooled to 0C and water is added. The ;I precipitate is collected and dried under reduced pressure over .
phosphorus pentoxide to give the title compound: ~]25= -2.12 (c = 1, DMF); amino acid analysis: Lys (1.98), Trp (trace), Asp (1.00), Thr (1.83), Ser (0.97) and Phe (3.19).

In the same manner but replacing the above starting ~-~` material of formula IV with an equivalent amount of the other compounds of formula IV described in Example S, 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 Bu and CH3), 3.73 (slnglet for OCH3) and 7.13 to 7.23 ~tmultfplet for aromatlc~, Ddz-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NHNH2, .

.1 .

: ' ,. . .
-:, , :
: ~ . - , :
,: , ' , . .. , ~' ,. ' .. , :
$

3~3 EXAMPLE 6 - continued nmr (DMSO-d6) 6 1.07, 1.1 and 1.17 (sinylets 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(Bu )--NHNH2, nmr (DMSO-d6) 6 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).
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~IP-6635-1-CI
~33~43

4-[2-Amino-ethylthlo]-butanoic Acid Ethyl Ester tH2N-(CH2)2-S-(CH2)3-COoEt) Vl; X = (CH2)2-S-(CH2~3 and R2 = Et .
Lithium (1.5 g, 0.2 mole) is stirred with 150 ml

5 of liquid ammonia containing S0 mg of ferric nitrate (hydrated) till the blue color of lithium 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 9, 0.1 mole) is added and the ammonia is removed.
The ether solution is washed with water, dried over sodium sulfate and the solvent is removed. The oily residue is subjected to chromatography on silica gel using 10~ methanol and 0.1% triethylamine in chloroform. The eluates are evaporated to give the title compound, nmr (CDC13) ~ 1.28 (t, J=7Hz, 3H), 1.53 (s, 2H), 2.0 (m, 2H), 2.3 tm, 8H) and ~.8 (q, J-7Hz, 2H~.

~: :

.

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.. . .. .

83~

EXAMPLE 7 - continued In the same manner by following the above procedure other compounds of -formula Vl 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-amino-propylthio)-propanoic acid ethyl ester ~H2N-(CH2)3-S-(CH2)2-COOEt]is obtained-Also, in the same manner the following compounds of formula Vl in which X is (CH2)m-S-(CH2)n are obtained: H2N-(CH2)3-S-OOM H N (CH ) -S-(CH2)4-COOMe and H2N (CH2)4 2 4 . ,' "' ,.
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33~.43 ~ P-6635-1-CI

Ben7yloxycarbonyl-(N -t-butoxycarbonyl)lysyl-asparaginyl-.. _ -phenylalanyl-phenylalanyl-tryptophyl-(N~-t-butoxycarbonyl)-.
Iysy l-(0-t-butyl)threonyl-phenylalanyl-(0-t-butyl)threonyl-(0-t-butyl)5eryl-7-aminoheptano~ acid methyl ester . . . _ (Z-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But~-Phe-ThrtBut)-Ser(But)-NH (CH2)6-COOMe) Vll; Rl = Z, R2 = Me, X = (CH2)6 and Y = Trp ~`

A solution of the compound of formula V, Z-Lys(Boc)-Asn-t t t Phe-Phe-Trp-Lys(Boc)-Thr(Bu )-Phe-Thr(Bu )-Ser(Bu )-NHNH2 (10.4 g, described in Example 6)J in dimethylformamide (91 ml) is cooled to -20C. 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 -15C for 15 minutes and N-ethyldiisopropylamine (3.3 ml) is added.

The mixture is cooled to -30C and the compound of formula Vl, 7-aminoheptanoic acid methyl ester hydrochloride [1.08 9, 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 -15C for one hour, at 0C 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).
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~` 9.U~33~

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 Vl, other compounds o~ formula Vll in which Rl, R2 and Y are as defined herein are . I
obtained. Examples of the latter compounds of formula Vll are listed as products in Table li together with the appropriate starting materials of formula V and Vl used for the preparation of the compound of formula Vll.
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~83~43 AHP-6635-1-CI

TA ELE
, , ~.
StartingStarting Material Ex. Material of Formula Vl Product of Formula Vll Formula V _ _ ~
Rl Y R R2 X Y
~ . _ _ _ . . ._._ 9 Z Trp UNH-(CH2)3-COOEt Z Et (CH2)3 Trp Z Trp HNH-(cH2)5-cooEt Z Et (CH2)5 ~ Trp 11 Z Trp HNH-(CH2)8-COOMe Z Me (CH2)8 Trp 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-CMe Z Me (CH2)8 D-Trp 15 Ddz Trp HNH-(CH2)4-COOEt d ¦Et tCH2)4 Trp 16 Ddz Trp HNH-(CH2)5-COOEt dz Et (CH2)5 Trp 17 Ddz Trp HNH-(CH2)6-COOMe dz Me (CH2)6 Trp nmr(DMSO-d6) 6 1.05, 1.16, 3.47 and 3.2 (singlets) and 6.82 to 6.95 l (muItiplet) ; 18 Ddz Trp HNH-(CH2)8-COOMe dz Me (CH2)8 Trp ¦ Ddz Trp HNH-(CH2)25(CH2)3-COOEt dz Et (CH2)2S(CH2)3 Trp ` 20 Ddz Trp HNH-(CH2)3S(CH2)2-COOEt dz Et (CH2)35(CH2)2 Trp 21 IDdz Trp HNH-(CH2)2S(CH2)4-COOMe dz Me (CH2)25(CH2)4 Trp22 Ddz D-Trp HNH-(CH2)3-COOEt dz Et (C~l2)3 D-Trp 23 Ddz D-Trp HNH-(CH2)6-COOMe dz Me (CH2)6 D-Trp I am7no acid analysis: Lys(2.0), Thr-(1.85), Phe(3.06), Asn(1.03) and ~ Ser(O.99) 25~ 24 Ddz D-Trp HNH-(CH2)8-COOMe ~
,, _... _ . . .' '.

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.

, , ~ 83~43 AHP-6635-1-CI

TABLE I - continued _ . . _ .. --- - - - ------!
Starting Starting Material 5 Ex. Material o~ Formula Vl Product of Formula Vll of Fc rmula V _ _ ~
x r 25 Ddz D-Trp HNH-(cH2)2s(cH2)3-cooEt Ddz Et (CH2)2S(CH2)3 D-Trp ~ ~ -; 10 nmr(CDC13) 6 1.18(m), 1.45(s), :
; 2.45(m~, 3.78(s), 4.1(m) and ~ ;;
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3 A~IP-6635 1 -C I

Benzyloxycarbonyl-(N -t-butoxycarbonyl)lysyl-asparaginyl-.
phenylalanyl-phenylalanyl-tryptophyl-(N -t-butoxycarbonyi)lysyl-~ _ .
(0-t-butyl)threonyl-phenylalanyl-(0-t-butyl)threonyl-.
(0-~-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)6CONHNH2) Vlll; Rl = Z, R = NHNH2, X = (CH2)6 and Y = Trp ~ :
A solution of the compound of formula Vll, Z-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(8ut)-Ser(Bu )-NH-(CH2)6-COOMe (10.3 9, described in Example 8), and hydrazine hydrate (3.4 ml) in dimethyl~ormamide (60 ml) is stirred at room temperature for 60 hours.
Water is added and the precipitate is collected and dr-ied over phosphorus pentoxide to give the title compound; nmr (DMS0-d6) ~ 1.15 (m), ~ -1.45 (s), 5.05 (s) and 7.25 (m).
. ~, , By following the procedure of Example 28 using the appropriate ~1 15 starting material of formula Vll, other compounds of formula Vlll in which R is - NHNH2 are obtained. Examples of the latter compounds of formula Vlll are listed as products in Table 2 together with the appropriate starting ~, mater7al of formula-VII. In each case the starting material is noted by the number of the example in which it is prepared.
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-~ ~o~ 3 ~ TABLE 2 _ . . . ..
No. o~ the Example in Example which starting material which R3 is NHNH ~ .
.~ prepared __ . .~ _ :
, . . -. .__ . . ,.
; 5 29 9 Z (CH2)3 Trp Z (CH2)5 Trp :-: 31 11 . Z (CH2)~ Trp ~ ;
: 32 . 12 Z (CH2)~ D-Trp 33 13 Z ( CH2 ) 5 D-Trp : - :
34 14 Z ( CH2 ) 8 ~ D-Trp ;~
. 35 15 Ddz (CH2) 4 Trp 36 ~ 21 Ddz (CH2)2S(CH2)4 Trp -37 24 Ddz (CH2)~ D-Trp ~ :
'~ 15 38 Ddz (CH2)25(CH2)3 D-Trp :~ 39 26 Ddz (CH2)3S(CH2)2 D-Trp ~ (CH2)4S(CH2~4 D-Trp ' '~; :

~, ': :: : : . :
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: ' ., ': ''~' , , . ','' ' ' , ' '', , ' ~ , . ' ' ~3~L~3 : EXAMPLE 41 : (N -t-Butoxycarbonyl)lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyl-(N -~-butoxycarbonyl)lysyl-(0-t-butyl)threonyl-phenylalanyl-(0-t-butyl)threonyl-(0-t-butyl)seryl-7-amino-~ ~:
Thr(Bu )-Phe-Thr(But)Ser(But)-NH(CH2)6CONHNH2) IX; R3 = NHNH2, .. _ , A mixture of the compound of formula Vlll, Z-Lys(Boc)-Asn-. ;'he-Phe-Trp-Lys(Boc3-Thr(But)-Phe-Thr(Bu )-Ser(But)-NH-(CH2)6-CONHNH2 (3.0 9, described in Example 28), and 10~ palladium on carbon (1.5 9) in acetic acid (45 ml) is rapidly stirred under an atmosphere of hydrogen for 21 hrs. The mixture is fiItered 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 Vlll in which R is NHNH2 with other compounds of , formula Vlll described in Examples 29,30, 31, 32, 33 and 34, the following : compounds of formula IX are obtained respectively: :
'~ H-Lys(Boc~)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-,~ NH(CH2)3-CONHNH2, H-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(Butj-NHtCH2)5-CONHNH2, H-Lys(Boc)-Asn Phe-Phe-Trp-LystBoc)-Thr(But)-Phe-ThrtBut)-SertBut)-NHtCH2)8-CONHNH2, . H-LystBoc)-Asn-Phe-Phe-D-Trp-LystBoc)-ThrtBu~l-Phe-ThrtBu~`)-SertBut)-.
NHtCH2)3-CONHNH2, H-LystBoc)-Asn-Phe-Phe-D-Trp-LystBoc)-ThrtBut)-Phe-ThrtBut)-SertBut)-NH(CH2)5-CONHNH2 and H-Lys(Boc)-Asn-Phe-Phe-: D-Trp-Lys(Boc)-ThrtBu )-Phe-ThrtBu )-SertBu )-NHtCH2)~-CONHNH2.

.

.. . . .

' .~ . .

.

(N -t-Butoxycarbonyl)lysyl-asparaginyl-phenylalanyl-phenylalanyl-.... . . . .
tryptophyl-(N -t-butoxycarbonyl)lysyl-tO-t-butyl)threonyl-._ _ . phenylalanyl-(O-t-butyl)threonyl-(O-t-butyl)sery1-5-amino-, pentanoic Acid Hydrazide (H-Lys~Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-_ _ _ _ Thr(Uu ~-Phe-Thr(8u)-Ser(But)-NH(cH ) CONHNH ) IX; R3 = NHNH~, . _ ~_4 ~ _ ` X = (CH2)4 and Y = Trp :
A solution of the compound of formula Vll, Ddz-Lys(Boc)- ~ -Asn-Pha-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH(CH2) CONHNH2 t2.0 9, described in Example 35), in 17 ml of a solvent : consisting of acetic acid-formic acid-water (7:1:2) is stirred at room temperature for 16 hours. The solvent is removed under reduced pressure and the residue is dissolved in methanol.
, ;,. Dlethyl 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 Vlll with other compounds of formula Vlll ~-described in Examples 36, 37, 38, 39 and 40, the following compounds of formula IX are obtained respectively:
H-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH(CH2)2S(CH2)4-CONHNH2, H-Lys(Boc)-Asn-Phe Phe~D-Trp-Lys(Boc)-Thr(But)-Phe-Thr(Bu )-Ser(Bu )-NH(CH2~8-CONHNH2, H-LystBoc)-Asn-Phe-Phe-D-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH(CH2)2-: ~25 S(CH2)3-CONHNH2, H-Lys(Boc)-Asn-Phe-Phe-D-Trp-Lys(Boc)-Thr(Bu~)-Phe-Thr-(But)-Ser(But)-NHtCH2)3S(CH2)2-OONHNH2 and H-Lys(Boc)-Asn-Phe-Phe-D-Trp-LystBoc)-Thr(Bat)-Phe-Thr(But)-Ser(But)-NHtCH2)4S(CH2)4-CONHNH2.

"' ' ' ' ~ ,, -45- :

' .~ ,: ' : ' ' ,. , , . . . . , :
- : : . - ,..... ..
,: ,., . , . . ., .. , , : . . . .
: :, ' ' ''. . , , ' ' , .

~!8314;~
AHP-6635-1 Cl Cyclic Amide of (N -t-Butoxycarbonyl)lysyl-asparaginyl-phenyialanyl-phenylalanyl-tryptophyl-tN -t-butoxycarbonyl~lysyl-(0-t-butyl)threonyl-phenylalanyl-(0-t-butyl)threonyl-(0-t-butyl)-seryl-7-amlno-heptanoic acid .
(CO-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(~oc?-Thr(But)-Phe-Thr(But~Ser(~ut~NH) ' H2'6 - --~ --- - --X; X = (CH2)6 and Y = Trp The compound of formula IX , H-Lys(~oc)-Asn-Phe-Phe-Trp-Lys-(Boc)-Thr(Bu )-Phe-Thr(But)-Ser(But)-NH(CH2)6CONHNH2 (2.94 9, described in Example 41) is dissolved in dimethylformamide (20 ml), `~ cooled to -20C and 2.39 M hydrogen chloride in ethyl acetate (1.67 ml) is added, tollowed by t-butyl nitrite (0.19 ml).
The solution is stirred at -15C for 15 minutes and cold (-15C) dimethyiformamide (231 ml) is added, followed by N-ethyldiisopropyl-amine (0.957 ml). The mixture is stirred at -15C for I hour, at . ~ . ., 0C for 2 days and at room temperature for I day. The solvent is removed under reduced pressure, the residue is dissolved in methanoi and the latter solution is added to water. The preclpitate is collected and subJected to chromotography on sllica gel uslng 5%
methanol and 0.1% triethylamine in chloroform. The eluates are evaporated to glve the title compound; amino acid analysis:
Trp (present), Asp (0.89), Ser ~0.85), Lys (1.77), 1hr (2.04) and Phe (3~o)~

In the same manner by following the above procedure, other compounds of formula X are obtalned. For example, replaclng `- the starting materlal with an equivalent amount of other ~ ~
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~ILU~3~L~3 .. .
:
- EXAMPLE 43 - continued starting materials of formuia IX, as described in Examples 41 and 42, the following compounds of formula X are obtained, respectively:
'j fO-Lys~Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH, (CH.~) C0-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(Bu )-Ser(Bu )-NH, ,; ( CH2 ) 5 . ._, , ,,, ., ,__ , ,. ) , CO-Lys(Boc)-Asn-Phs-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH, ~:
~10 (CH2)8 ------ ---- - , ....................... :': .' CO-Lys(Boc)-Asn-Phe-Phe-D-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH, ' ( CH2 )3 ' - - -- - - - ----- - . ........... . _ ) . .. ' 70-Lys(Boc)-Asn-Phe-Phe-D-Trp-Lys(80c)-Thr(Bu )-Phe-Thr(Bu )-Ssr(Bu )-NH, ( CH2 ) 5 ) .
~15 ~ -Lys(Boc)-Asn-Phe-Phe-D-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH, `~ ( H2)8 ' -.............. _ - . . ) . '' ', C(,-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH, (CH ) ;~ 2 4 C0-Lys(Boc)-Asn-Phe-Phs-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH, ~0 . (CH ) -S-(CH ) ; : 2 4 2 2 ~ -~-- -- -- ~ - - -~
' CO-Lys(Boc):-Asn-Phe-Phe-D-Trp-Lys~Boc)-Thr(But)-Phe-Thr(Bu )-Ser(But)-NH, '.
( CH2 ~ 8 - -- --- - - --- - - -- - ---- ) .
.

: . . .

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r .

- Al-IP-6635- 1 ~C I
33~43 EXAMPLE ,43 - cont i nued CO-Lys(Boc)-Asn-Phe-Phe-D-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But~-Ser(But)-NH, 3_5_(cH2)3 CO-Lys(Boc)-Asn-Phe-Phe-D-Trp-Lys(Boc?-Thr(Bu )-Phe-Thr(But)-Ser(Bu )-NH and (CH2) -S-(CH ) 2 2 ~
C10-Lys(Boc)-Asn-Phe Phe-D-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH.
( CH2 ) 4 S ( CH2 ) 4 ,'~ . .
; -' :^` -, :`. ~ ' . ! :
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~ ~3143 A~IP-6635-1-CI
EXAMPLE~44 Cyclic Amide of Lysyl-asparaginyl-phenylalanyl-phenylalanyl-trptophyl- :
. Iysyl-threonyl-phenylalanyl-threonyl-seryl-7-amino-heptanoic acid :
(C0-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH) (C~12)6 _ , l; X = (CH2)6 and Y = Trp .
A solution of the compound of formula X, ~C0-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(Bu )-Ser(Bu )-NH
( H2)6 - -- ) (0.92 9, described in .~xample 42), in concentrated hydrochloric acid (4.16 ml) is rapidly stirred at O~C under an atmosphere of - nitrogen for 10 minutes. Acetic acid (300 ml) is added and the solut7on is Iyophilized. The residue is dissolved in water (100 ml) and again Iyophilized. The residue jc dissolved in the .-~15 upper phase of the solvent system butanol-acetic acid-water (4:1:5),.~ applied to a column of a chemicaliy modified cross-linked dextran(Sephadex LH-20) and the column is eluted with the upper phase solvent. The eluates are evaporated, the resldue is dissQlved in water and lyophtlized to give the tltle compound; ~MmaxH
~20 290 (~ 4,695), 2al (~ 5,315), 274 (6 4,955), 269 ~ ~,675), . ..
:. 265 t~ 4,260) and 215 nm (~ 47,335).
.. . .
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.

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~(~83~.143 EXAMPLE 44::55~Li~

In the same manner by following the above procedure other compounds of formula I are obtained. For example, replacing the starting ma+erial 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:
fO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH, ( 2)~ . . J
ICO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH, tCH2)~ .. . J
ICO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH, CO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-N~H, : 15 (CH ) ~
ICO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-l'~H, (CH2 ) 5 - '- -- . -~
fO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-NH, (CH2)0 --- -~ -- ~-- :._ _ ) :
. .' ', .
CO-Lys-Asn-Phe-Phe-Trp-Lys-Thr~Phe-Thr-Ser-N~H, :~ ;
(CH2)4 ----- --- - ---J :
fO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH, ; (C~I2 ) 4- S- (CH2 ) J
~O-Lys-Asr-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-~25 ( H2)~

'' ', , ' '.' .: ' . .
':

.

~L()t'33.~43 EXAMP_E 44 - continued CO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-NH, 2)3 tC~2)2 ~ ---- J
CO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-NH,and (CH2)2-S-(CH2)_ _ J
CO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-NH.
2 4 ( H2)4 ----- --~

. ~~ ... .
. , ' . .. " ':
.''~ ' .

,, . :
:, :
;, ' ,:
: ' '': . .'' '' ~ :, i: , :' ;. :
'; '; ' ' :

. ~ .

~"` , ~ ' .
.
. ~ .

~51- .

3~43 Dimethvl-3.5-dimethoxY-benzvlox~carbonyl-~N~=t-butoxYcarbonY131ysyl asparaqinyl-phenYlalanvl-Dhenylalanyl-trypt-ophyl-(Ne-t-batoxycarbonyl) lysY~ -buty!)threonyl-rhenvlalany!-(t)-t-buty!)threonyl-(o-t-buty~
seryl-i-aminohentanoTc Acid (Ddz-LYs(Boc~-hsn-i3he-i'he-Trp-Lys(Boc)-Thr(But)-Ser(Bu )-NH(CH2~6COOH) ~ ; Rl - DdZJ R3 = OH, Y = Trp and X = (CH ) A solution of the compound o~ formula Vll, Ddz-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc~-Thr(Bu )-Phe-Thr(Bu )-Ser(But)-NH(CH2)6COOMe (3.39, described in Example 17), in acetone (32 ml) and N sodium hydroxide (6.4 ml) is stirred at room temperature for 42 hrs~ A solution of 5%
citric acid 7s added until the solution is neutral. The precipttate is i collected, washed with water and dried to give the title compound, nmr (DMSO-d6) ~ 1.02, 107 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 ~11, other compounds of formula Vl !l in which R3 is OH are obtained. Examples of the latter compounds of formula Vlll are listed as products in Table 3 together with the appropriate startlng material of formula Vl ! . In each case the start7ng material Is noted by the number of the example 7n which 7t 7s prepared.

- ~.
. : .
. ' ' ' . .
I ' ' . , . ' . .

~:

, -52 :

33~43 TAB~ 3 ' . . ~
No. of the Example in ~ Product of Formula Vlll in . which starting material of __ ~hich R3 is OH
Ex~mple . formula V!l-ls-prepared RI ~ Y~
47 14 Z (CH2)8 D-Trp 16 Ddz tCH2)5 Trp . 48 18 Ddz Trp .~: 49 19 Ddz . Trp . 50 20 Ddz 2 3 (c~l2j2 Trp 51 23 Ddz 2)6 D-Trp . .:
.: . amino acid analysis: Lys( .23), : Thrt2.03), Phet3024), Asntl.OO), SertO.94) and H NtCH2)6Ct~DH
.: . (present) 2 '. : : .'",' .
52~ 25 Ddz ¦ (CH2)2S(CH2)3 ¦D-Trp nmr(DMSO-d ) d 1.07(s), I.l(s), . 1.131(s~, ~.38(s), 2 5(m,), . 3.72(s) and 6.9 to 7 3(m~
.'' ~ . .,~':
. , ~'.'''' ;', '~ .

~ 2~ ¦ Dd~ ¦tCH2~SICH2)~

~.

:~, ' ' , ' .
. . .

. . ~ , . . ~ . .
':,' ~ ' ; ..
, ~ ~33~43 A~IP-6635- 1 -c I

(N~-t-butoxycarbonyl)lysyl-as~araqinyl-pheny!a--a-nyl-phenylalanyl-tryptoph (N~-t-butoxvcarbonyl)lysyl-(~-t-buty!~threonyl-phenylalanyl-(o-t-butyl)-serY1-7-aminoheptanoic Acid (H-Lys(Boc)-A_n Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr¢But~-ser(qut~-~H(cH2)6cooHj IX, R3 - OH, X = (CH ) and Y = Trp ~ - - ? 6 A solution of the compound of formula Vlll, Ddz-Lys(Boc)-Asn- .
Phe-Phe-Trp-Lys(Boc)-Thr(Bu )-Phe-Thr(Bu~)-Ser(But)-NH(CHz)6COOH (2.9 g, .
described in Example 45) in acetic acid-formic acid-water (7:1:2, 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 compoundsO
In the same manner by following the above procedure other compounds ~-~ of ~ormula IX in which R is O~-are obtained. For example, replacing the -~15 starting material with an equivalent amount of other starting materials of formula Vlll described in Examples n, ~8, 49, 50, 51, 52 and 53, the following compounds of formula IX are obtained respectively: :
H-Lys(Boc)-Asn-phe-phe-Trp-Lys(Boc)-Thr(But)-phe-Thr(But)-ser(But)-NH(cH2)5cooH~
: ~ H-Lys(Bocj-Asn-phe-phe-Trp-Lys(Bocj-Thr(Butj-phé-Thr(But)-ser(But)-NH(cH2)8cooH~

~20 H-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr~But)-Phe-Thr(But)-Ser(But)-(CH2)2StCH2)3COOH, H-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Bocj-Thr(But)-Phe-Thr(But)-Ser(But)-NH(CH2)3S- , ' (CH2 )2COOH, .,~ H-Lys(Boc)-Asn-Phe-Phe-C-Trp-Lys(Boc)-Thr(Bu )-Phe-Thr(But)-Ser(Bu~)-NH(CH2)6- -~5 COOH, H-Lys(Boc)-Asn-Phe-Phe-D-Trp-Lys(Boc)-Thr(But)-Phe-Tllr(But)-Ser(But?-NH(CH2)2-S(CH2)3COOH and : H-Lys(Boc)-Asn-Phe-Phe-D-Trp-Lys(Boc)-Thr(fl~ )-Phe-Thr(Bu )-Ser(But)-NH(CH2)3-S (CH2 )2COOH .
;O .:~ ', ''1 ' ' ' '', -5~

.. , . : : . . . .
. .
. .. . ~ . . . ~ ~

~ 3~43 Al-lp-6635-1-CI

- tN~-,t-ButoxycarbonYl)lysyl-aspara~inyl-phen,ylalanyl-phenylalanyl-D-tryptophyl-,' (N~-t-butoxycarbonyl~lvsyl-(O.-t-butyl~threonyl-phenylalanvl-(C-t-butyl)- ~, '. threonyl-(O-t-buty-l)seryl-9-amino-nonano!c Acid (H-LYs(Boc)-Asn-phe-phe-D : 5 Trp-LYs¦Boc~-Thr(Bu ~-Phe-Thr(Bu )-Ser(Bu )-NUI~ 15 _~2~_ X = (CH2)~ a~d Y = D-Trp A mixture of tne compound of formula Vll! , 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 46j, and 10% palladium on carbon (1.5 9) in acetic acid (45 ml) is rapidly stirred under an atmosphere of hydrogen for 24 hrs. The mixture is fiItered and the fiItrate is evaporated under reduced pressure ; to givç the title compound.

i15 :
''; '. ' ~', ~'~ ' ,'' ' ~i .
. . ..
; ~'~ ' . i : , ~ , .

.
: -~:., . , .

-55- ~
~, . .

~, . , ,.: `

. :' . .

- ~)133~L43 Cyclic Amide of (N~-t-Butoxy-carbony-l-?lvsv!~3~ raqinyl-phenylalanvl-DhenYlalanYI-trYptoehyl-~N6-t-~Ihnx~3rbonyl)1vsyl-(0=t-butYl)threonvl-phenyla!any!-(O-t-butyl)threony~(o-t-butyl)seryl-7-amino-heD~anoic Acid (~O-LYs(Boc)-Asn-phe-phe-Tr~ cc, rhr(3u~)-Ph Thr(But~-Ser~Bu )-~H) ' ~12'~
X; X = (CH2)6 and Y = Trp A solution of the compound of formula Vlll, H-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH(CH2)6COOH (0.45 9, described in Example 54), N-hydroxysuccinimide (00115 g) and triethyl-10 amine (0.07 ml) in methylene ch!oride (118 ml) is cooled to 0UC. A

solution of dicyclohexylcarbodiimide (0.154 9) in methylene chloride (3 ml) Is added and the resulting mixture is stirred at 0UC for 2 hrs, at 5C
; for 18 hrs and at 25-C for 7 days~ The mixture is fiItered and the filtrate is evaporated. The residue is washed with lN citric acid, water and dried over phosphorous pentoxide to give the title compound.
In the same manner but replacing N-hydroxysuccinimide with an equivalent amount of l-hydroxybenzotriazole, 1,3,5-trichlorophenol, pentachlorophenol or ~-nitrophenol, the title compound is obtaintedO
In the same manner by following the above procadure other 2 compounds of formula X are obtained. For example, replaclng the starting mater7al with an equivalent amount of other starttng mater7als - of formula IX described In Exaples 54 and 55, the following compounds ~ of formula X are obtained respect7vely:
~.
:. -, '" ' ' . .~ ~ :
, ~

, . .. .. ~
-, , . . : .
, ' : . ':, ' ' ' ~ 3~43 AHP-6635-1-CI

. ~

CO-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(Butl-Ser(Bu )-NH, (CH2)~
fO-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-ThrlBut~Ser(Bu )-NH, (C~12 ) CO-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc~-Thr(But~-Phe-Thr(But)-Ser(But)-NH, .' (CH2)3-S-(CH2)2 .
CO-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH, (CH2)2 1 2 3r-~
0-Lys(Boc)-Asn-Phe-Phe-D-Trp-Lys(Boc~-Thr(But)-Phe-Thr(But)-Ser(But)-NH, -~ .
~;~ ( H2) ., .
CO-Lys-(Boc-)-Asn-Phe-phe-D-Trp-LystBoc)-Th~r(But)-Phe-Thr(But)-Ser(Bu )-NH, -~15 2 3 (CH2)~ -------- ----- ~ - ------ _ ~
CO-Lys(Boc)-Asn-Phe-Phe-D-Trp-Lys(Boc)-Thr(But)-Phe--Thr(But)-Ser(But)-NH and (CH2)2-s(cH2) - - ~ --~ J
CO-Lys-(Boc)-Asn-Phe-Phe-D-Trp-Lys(Boc~-Thr(But~-Phe-Thr(But)-Ser(Bu ~-hH.
( H2)~
~ O
: . .

. ~ :', ' ,., . .

:
. i: :

, , ~, . .

.
: ' . ,., ~

:-' :
~57~

.

3~43 AHP-6635-1-CI

EXAMPLE ~7 _y~lic Amide of Lysyl-asp~raqi nv ! -Dhen~lala~YI-phenvlalanY ! _tr~ phyl-lysyl-threon~l-ph-enylalany!--tllreonyl-seryl-7-aminohe~ oic acTd (ICO-Lys-Asn Phe-Phe-Tr~-Lys-Thr-Phe-Thr-Ser-NH) (C~12~ ---- - - -- J
l; X ~ (CH2~6 and Y ~ Trp .:
A solutlon of the compound of formula X, Lys(Boc)-Asn-phe-phe-Trp-Lys(Boc)-Thr(But)-phe-Thr(But)-ser(Bu )-NH - -( H2 ) ~
~10.20 g, described in Example 56), in concéntrated hydrochloric acid (9 ml) is rapidly stirred at 0UC under an atmosphere of nitrogen for 10 minutes. Cold acetic acid (0C, 90 ml) is added and the solution is Iyophilized. The residue is dissôlved in water (30 ml) and again Iyophilized. The residue is dissolved in the upper phase of the solvent system butanol-acetic acid-water (4:1:5), applied to a column of a chemically mod7fied cross-iinked 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 Iyophilized to give the title compound; ~maexoH ;
289 (~ 4,558), 280 (~ 5,148), 275 (~ 4,334), 270 (~ 4,671), 265 (~ 4,292), 259 (~ 3,717) and 254 nm (~ 3,0i2). ;
~, . . , ~. .
The title compound of this Example Is identical in all ..
; respects to the title compound of Example 44.

In the same manner by followlng the above procedure other compounds of formula I are obtained. For example, replaclng the startlng material with an equivalent amount of other starting materlals of formula X described In Example 56, the followlng compounds of formula I are obtalned respectively:

::

--5~8-' " ' ,', ' ; ~ :' :' .
.. . .
.

.t~)133~L4;~
, . .

AHP-6635~1-CI
EXAMPL~ 57 - con~i nu~

CO-Lys~Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH, (CH2~5 ~ . ` J
0-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-~lH, 2 D~ J
CO-Lys-Asn-Phe-phe-Trp-Lys-Thr-Phe-Thr-Ser-N~H, CH2)3-S-(CH2)2 ,J
fO-Lys~Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-~iH, (CH j -S-(CH ) : `
2 2 2 :
O-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-NH; amino acid analysis:
(CH )G- - : ) Lys(1089), Thr(1.93), Phe(3.00), Asn(1000)~ Ser(O.99) and Trp(present), ~mexo~l 290(6 5,i62), 282 (6 5,737), 2i3 (~ 5,498j, 269 (~ 5,302), i5 265 (~ 5,077), 260 (~ 4,685) and 253 nm (e 4,189), CO-Lys-Asn-Phe-Phe~D-Trp-Lys-Thr-Phe~Thr-Ser-NH, -~ ~CH ) -S- (ch ) fO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe~Thr-Ser~NH and (CH2)2-S-(CH2)~
fO~Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe~Thr~Ser~NH, -( ' . :
:, ; '':

:, ' .' .
: , , , ~ : .:

: :

,- .: : :. : : . : .
: :
:, , - ~ ::: :

~ Al-IP-6635-1-CI

.
~,~-Dimethy1-3,5-dimethoxy-benzyloxycarbonyl-tryptophyl-.. ...... . . _ . _ .
(N~-t-butoxycarbony!11ysyl-(0-t-bu~yl~tbrconyl-phellylalanyl-.
(0-t-butyl)threonyl-(0-t-butyl)seryl-4-(2-amino-ethyl-thio)-butanoic Acld Ethyl Ester (Ddz-Trp-Ly$~Boc?-ThrtBu ?-, Phe-Thr(Bu~)-Ser(But)-NH-~CH213-C00Et) XI, m = 2, n - 3, R2 = Et and Y = Trp - ... . .
A solution of Ddz-Trp-Lys(Boc)-Thr(But)-Phe-Thr(Bu )-t - - -Ser(Bu )-NHNH2 (3.64 9, described by H. U. Immer et al., cited above) in dimethylformamide (40 ml~ is cooled to -20UC.
Hydrogen chloride (2.7 M) in ethyl acetate (2.64 ml) is added followed by t-butyi nitrite (0.40 ml). The solution is stirred :: .
- 15 at - 15UC for 15 minutes and N-ethyldiisopropylamine (1.73 ml) ` is addedO The mixture is cooled to -30UC and the compound of ormula Vl, H2N-(CH2)2-S-(CH2)3-C00Et (0.545 9, described in , Example 7) is added. The mixture is stirred at -15~C for one hr, at 0UC for one hr and at room temperature overnight. The solvent is removed under reduced pressure and the residue is suspended in methanolO Water is added, the preclpltate is collected and ;washed with methansl~ The precipitate 7s subjected to chromatography on silica gel using 3~ methanol and 0.1% trtethylamine 7n chloroform.
The eluates are evaporated to glve the tltle compound: ~]25 =
(c = 1, DMF); ~MmaxH 289 (6 5,490), 281 ~6 7,915), 275 (~ 7,S30) and ~-~
217 mn (~ i7,988).

,' ~ ' ;~'' ': , ' ~, ,~ , ;~
; . .:
' ~ ' ' , ' ,, ',.' ' : .' .
: ., :

~L0~3~43 EXAMPLE 58- con~L~
In the same manner by following the above procedure, other compounds of formula Xl are obtained~ For example, rep~acing H2N-(CH2)2-5-(CH2)3-COOEt with an equiva!ent amount of other compounds of formula Vl as described in Example 7, the following compounds of formula Xl are obtained, respectively:
Ddz-Trp-Lys(Boc)-Thr(Bu~)-Phe-Thr(But)-Ser(But)-NH-(CH2)3-S-(CH2)2-COOEt,. ' ' Ddz-Trp-Lys(Bocj-Thr(Bu j-Phe-Thr(Butj-Ser(Butj-NH-(CH2j3-S-CH2-COOMe, Ddz-Trp-Lys(Bocj-Thr(Butj-phe-Thr(Butj-ser(Butj-NH-(cH2j2-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(æut)-NHNH2 with an equivalent amount of Ddz-D-Trp-Lyst3Oc)-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 M~
H. U. Immer et al " cited above] and using one of the following compounds ,, , 1 ~ H2N (~H2)2 S~~ ,CH2~3~CEt~ H2~ (cH2)3-s-(cH2)2 ` . COOEt and H2N-(CH2)4-S-(CH2)4-COOMe, the following compounds of formula Xl ~i are obtained, respectively:
,, Ddz-D-Trp-Lys(Boc)-Thr(But)-Phe-Thr~But)-Ser(But)-NH-(CH2)2-S-(CH2)3-COOEt, 3 20 Ddz-D-Trp-Lys(Bocj-Thr(Butj-phe-Thr~Butj-ser(Butj-NH-~cH2j3-s-(cH2j2-cooEt and . Ddz-D-Trp-Lys(Bocj-Thr(But)-phe-Thr(Butj-ser(Butj-NH-(cH2j4-s-(cH2j4-cooMe.
, ~ , :, , .
~ ' ' .' `:' ''` ~. , ~ : .
: :
, : .
~' . ~ . .
.' ' - .
. ' , :

.. . _ .. _ .. ... ....
:' ~ , ': ,' . . ' . :

1~83~43 AHP-6635-1-Cl a,(x-Dimethy!-3.5-dimethoxY-benzylQxycarbonvl-tryp ~ phy1-IN~ -buto~y~g~scy~ysyl-(o-t-botyl~threon~ henylalany !-tO-t-butyl)threonYI-(O-t-but~l_ser~1-4-~2-amino-ethvlthio)-butanoic Acld H~ razide (Ddz-Trp-Lys~Boc)-Thr~But-)-phe-Thr(But) Ser(But)-NH-(CH2)~~S-(CH2).-CONHNH~) Xll: m = 2, n = 3 and Y ~ _r~
A solution of Ddz-Trp-Lys(Bocj-Thr(But)-phe-Thr(But)-Ser(Bu )-NH-(CH2)2-S-(CH2)3-COOEt (4.6 9, described in Example 58) .. . .
and hydrazine hydrate (406 ml) in dimethylformamide (20 ml) . .
is stirred at room temperature for four dqys. Water is added and the ; precipitate is collected and dried over phosphorus pentoxide to give the title compound: [a]D5 = ~13.8~ (c = 1, DMF);
289 (~ 5,530), 281 (~ 7,950), 275 (~ 7,550) and 217 nm ~, .
(~ 50,160).
In the same manner by followlng t~e above procedure, ather compounds of formula Xll are obtained. For example, replacing the starting matertal with an equivalent amount of other staring materials -of formula Xl, as described tn Example 58, the following compounds of formula Xll are obtained, respect7vely:
Ddz-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-$er(But)-NH-(CH2)3 - S-(CH2)2-CONHNH2, Ddz-Trp-Lys(Bocj-lhr(Butj-Phe-Thr~Butj-Ser(But;-NH-(Cl-12j3 5-CH2-CONHNH2~
.. . . . . . .
',. Ddz-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH-(CH2)2-5-(CH2)~,-CONHNH2, ,' Ddz-Trp-Lyst~ocj-Thr(Butj-phe-ThrtButj-sertBut)-NH-(cH2)4-s-(ch2j4-coNHNH2~

Ddz-D-Trp-LystBoc)-Thr(But)-Phe-Thr(But)-Ser(But}NH-(CH2)2-S-(CH2)3-CONHNH2, Ddz-D-Trp-Lys(Boc)-Thr(But) Phe-Thr(But)-Ser(But)-N~(CH2)~-S (CHz)2-CONHNH2 and Ddz-D-Trp-Lys(Boc)-Thr(Bu~)-Phe-Thr(But~-Ser(Bu~)~NH-(CHz)4-S-(CH2)~-... .. . . . ..
NHNH2 ,.... .

: ' ' .. . ...

.

1()~3~43 AHP-6635-1-CI

EXAMPLE~O
a~a-Dimethyl-3.5-dimethoxy-t)en7yl~x~ -t (N -t-butoxycarbonyl)lxsyl-~o-t-butyl~threony~!-phenylalan ~O-~-butyl)~thre~ -(O-t-butvl)serY
I-oxobu~YI-~N6-t but~x~carbonyi)'lysv!-asp~raqin~- ' '-Dhenylalanyi-phenylalanine_Methy~_Ester ~Ddz _ rD-L~s(Boc)-Thr~Bu~-Phe-Thr~But)-S~ u )-NH-( ~ ~ } C~-Lvs(Boc)-As~-,Ph, e-Phe-OMe) Xlllj_ m = 2~n = 3 and Y ~ Tre A solution of the hydrazide of formula Xll, Ddz-Trp-Lys(Boc)- ' ~' o 10 Thr(But)-Phe-Thr(But)-Ser(But)-NH-(CH2)2-S-(CH2)3-CONHNH2, (3D83 9, described in Example 59) in dimethylformamide (41 ml) is cooled to -20VC. Hydrogen chlor7de (2.7 M) tn ethyl acetate (2.5 ml) is added followed lvy _-butyl nitrite (0.38 ml). The solution is stirred at -15VC for 15 minutes and N-ethyidiisopropylamine (1.64 ml) 5 15 is added. The mixture is cooled to -30VC and ~-Lys(Boc)-Asn-Phe-Phe-OMe (1.94 9, prepared as described by H. U. Immer et 3L., cited above) is addedO The mixture is sttrred at -15VC for one hr, at OVC
for one hr and at room temperature overnight. The solvent is ,~
'~ removed under reduced pressure and the re~idue 7s trlturated with methanol. The solid is collected and dried under reduced pressure over phosphorus pentoxide to g7ve the title compound; ~maxoH 289 (6 5,655), 281 (6 8,070) and 275 'nm (~ 7,700).~
.
~ ' ' , ' ., , .

' , ~0~33'1L9L3 ~K~M~
In the same manner by following the above procedure, other compounds of formula Xlll are obtained. For example replacing the starttng material of formula Xll with an equivalent amount of other starting materials of formula Xll, as described in Example 59, the following compounds of formuia Xl ! ! are obtairled, respecti~ely;
Ddz-Trp-Lys(Boc)-Thr~But)-Phe-Thr(But)-Ser(But)-NH-(CH2)3-S- , (CH2)2CO-Lys(Boc)-Asn-Phe-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(Bocj-Thr(But)-phe-Thr(But)-ser(But)-NH-tcH2) ': (CH2j4-CO-Lys(Bocj-Asn-Phe-Phe-OM'e, Ddz-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH-(CH2)4-S- :
(CH2j4-CO-Lys(Bocj-Asn-Phe-Phe-OMé, .. . . . . ..
Ddz-D-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-N~(CH ) -S-, (CH2)3-CO-Lys(Boc)-Asn-Phe-Phe-OMe, : ':' '~ Ddz-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 1, ~ , Ddz-D-Trp-Lys~Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH-(CH2)4-S- , ~- (CH2)4-CO-Lys(Bocj-Asn-phe-phe-oMe~ .
: . .
~ ' ;
. ' .
' ~ ..... .

",~
' "' . .

;
'' : : 64 , , :,.: . ... .. .... . . ... . . .... . .. .
. , ".. - .. " ,. .. . ...... ... ...... . . . ... . . . .
:" , , " ". : . . ", ~: . " . .. . . , .. ., , . .. . :

1~83.143 AHP-6635-1-CI

Tryptophv~l_(N -t-butox~ca~bonYI)IYSYI~~-t ~5~ __ eonyl-Dheny I a ! anvi-{O-t-but~l~th ~ O-t~t~ 4- (2-amino-ethylthlo~-l-oxobutvl-(N~-t-butoxYcarbonyl)lYsVI-asparaqiny~-phenylalanYI-phenYlalanlne Methy ! Ester (H-Tr~-LYs(Boc)~Thr(But~--phe--Thr(But)-ser(But~-NH-(cH
tys5Boc)-Asn ~he-Phe-OMe) XlV, m = 2~n = ~i and Y = 7'rD
A solution of Ddz-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-H2)2 S (CH2)3-CO-Lys(Boc)-Asn-ihe-ihe OM (2 05^
o 10 descrtbed in Example 60) tn 17.5 ml of a solvent conststtng of acetic actd-formtc acid water ~7:1:2) ts sttrred at room t0mperature for 16 hours. The solvent ts removed under reduced pressure and the restdue is dissolved tn methanol. Water ts added and the prectpttate ts collected and drted over phosphorus pentoxtde. 5 15 The drted prectpttate ts washed wtth 40 ml of petroleum ether-ether (1:1) and dried to gtve the title compound,amtno acid analysts Lys( î ~93), Asp (0.91), Thr (1.87), Ser(0.9S) and Phe (3.00), - ' ' ' ' .

!

, ' ` ~ ' . ' ' ; ' ,~ ' ' .

' ., , ' '' '. ','.', ' ,'. .

~ 3~43 ,, AHP-b635- 1 -C I

EXAMPLE 61-continued : In the same manner by foilowing the above procedure, other compounds of formula XIV are obtained. For example, replacing the i :
starting material with an equivalent amount of other starting materials of formula Xlll, as described in Example 60, the following compounds of formula XIV are obtained, respectively:
H-Trp-LystBoc)-Thr(Bu ~-Phe-Thr(Bu )-Ser(But)-NH-(CH2)3-S-tCH2)2- . ~
CO-l.ystBoc)-Asn-Phq-Phe-OMe. ~ .
H-Trp-LystBoc)-Thr(But)-Phe-ThrtBut)-Ser(Bu~)-NH-(CH2)3-S-CH2-- 10 CO-Lys(Boc)-Asn-Phe-Phe-OMe, :.
H-Trp-Lys(Boc)-Thr~But)-Phe-Thr(8ut)-Ser(But)-NH-(CH2)2-S-(CH2)4- ~ '~
CO-LystBoc)-Asn-Phe-Phe-OMe, , ~ :' H-Trp-LystBoc)-Thr~But)-Phe-Thr(But)-Ser(But)-NH-(CH,~)4-~-(CH2)4-. CO-Lys(Boc)-~sn-Phe-Phe-OMe, : :
, 15 H-D-Trp-Lys(Boc)-ThrtBut)~Phe-Thr(But)-Ser(But)-NH-(CH2)2-S-(CH2)3-- , CO-LystBoc);Asn~Phe-Phe-OMe, . . .
H-D-Trp-Lys(Boc)-Thr(But)-phe-Thr(But)-ser~But)-NH-(cH2)3-s- (cH2)2-.
--. CO-Lys(Boc)-Asn-Phe-phe-OM6 and ~ .
: H-D-Trp Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH-(CH2)~-S-(CH2)~- :
, 20 CO-Lys(Boc)-Asn-Phe-Phe-OMe.

. . .
, '` , . .

~' ' ~.; .

' i~ ,. .

.
, . . .
.~ ' ' .
. . .
. .
., : .
~ .

''':'. ~, .

'.:.: ' ' ' ;'. '' ' ' . ' ' ' ', ' ',.. :': ;, :

~LV~143 Al-IP-6635-1-CI

Trvptophyl-(N~-t-butoxvcarbonyl~y~yl-(0-t-butvl)thraonYI-phenYla,anyl-~O-t-buty,l~threonyl-~ t-botyl)serYI-4-(2-amino-ethYlthio)-l-oxobutYI-~N~-t~butoxycarbonyl)lYsyl-as~araqinYl-~heny!alanyl-phe~lalanine HYdrazide ~H~Trp-LYs(Boc)-Thr~But)-Phe-Thr~Bu )~er~But)-NH-~C11~ CO-LYs(Boc)-Asn-Phe Phe-NHN~2) XV. m - 2in = 3 and Y - TrP
HydrazTne hydra~e (1.7 ml) is added to a solution at 0UC of H-Trp-Lys(Boc?-Thr(But)-Phe-Thr(But)-Ser(But)-NH-(CH2)2-S-(CH2)3-C0-Lys(Boc)-Asn-ihe-Phe-OMe (1.7 9, described in Example 61) in dimethylformamide (2 mi) and the solution is stirred at room temperature overnight. Water is added and the precipitate is colie~ted and dried over phosphorus pentoxTde to give the fitle compound, ~ aOH 290 (e 5,515), 281 (e 6,435), 275 (~ 6,065), 269 (~ 5,680) and 265 nm (~ 5,195);
.
In the same manner by following the above procedure, other compounds of formula XV are obtained. For example, replacing the starting material wTth an equTvalent amount of other startTng ~ . :
materTals of formula XIV, as described Tn Example 61, the foltowing compounds of formula XV are obtatned, respectively: H-Trp-Lys(Boc)-Thr (But)-Phe-Thr(But)-Ser(But)-N ~ (CH2)3-S-(CH7)2-CO-Ly6(Boc)-Asn-Phe-Phe-NHNtl2, '".:; "' ',:

~ ' :

" . ., .... .

~(~1 il3~3 ~3~ .
H-Trp-Lys(Boc)-Thr(But)-Phe-Thr(Bu )-Ser(But)-NH-(CH2)3-5-CH2- :
CO-Lys(Boc)-Asr.-Phe-Phe-NHNH2, H-rrp-Lys(Boc)-Thr(Bu )-Phe-Thr(But)-Ser(Bu )-NH-(CH2)2-S-(CH2)4-CO-Lys(Boc)-Asn-Phe-Phê-NHNH2, H-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But~NH-(CH2)4-5-(CH2)4-- . . , . . , . . . ~.
CO-Lys(Boc)-Asn-Phs-Phe-NHNH2, . H-D-Trp~Lys(Boc)-T~r(But)-Phe-Thr(But)-Ser(But)-NH-(CH2)2-S-(CH2)3 ..
.. . . . . . .
CO-Lys(Boc)-Asn-Phe-Phe-NHNH2, 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 H-D-Trp-Lys~Boc;) Thr(Bu~)-Phe-Thr(But)-Ser~But)-NH-(CH2~4-S-(CH2)4-C()-Lys(Boc)-Asn~Phe-Phe-NHNH2~ , ' . . . .
'' ' ~ :' :, ~

' .

'',: ~ , ~'. '' ' , ,1 ~ . ' . ' ':

'' ' ',",, ,', ' . - " ~' '' ' ' , ' ' ' "
68~
.. . .

.- ~ , . , :
~ .

3' ~ 143 AHP-6635-1-CI

CYCIjC Amide of (N~-t-ButoxYcarbonYI)lysyl-as~araqinyl-ehenyla-an ~_ .
(O-t-butY!-?threo~-y-l-phenylalanyl-(--o--t-but~t)thr onY
(0-t-butyl;seryl-4-~2-amino-ethYlthio?-butanoic Acid (CO-Lys(Boc~-Asn-Phe-Phe-Trp Lys~Boc -Thr(But)-Phe-Thr(But)~er(But?_N~) (CH ) ~ - - - - - ~- - ~- ~- -(CH ) .
X; X = tCH2)3-$-(CH~?2 and Y = Trp A solution of H-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(Bu )-NH-(cH2)2-s-tcH2)3-co-Lys~Boc)-Asn-phe-phe-NHNH2 (1.57 g, described in Example 62) in dimethylformamide (10 ml) is cooled to -20~C ar,d 2.64 M hydrogen chloride in ethyl acetate (0.8 ml) is added, followed by t-butyi nitrite (0.117 ml). The solution is stTrred at -15~C for 15 minutes and cold (-15~C) dimethylformamide .
(123 ml) is added, followed by N-ethyldiisopropylamine tO.363 ml).
The mixture Is st7rred at -15~C for I hr, at 0UC for 2 days and at room temperature for I day. The solvent is removed under reduced pressure, the res7due is dissolv d in methanol and the latter solution is added to water. The precipitate is collected and ~` subjected to chromatography on sllica gel using 5% methanol and 0.1% triethylamine Tn çhloroform. The eluates are evaporated to give the tltle compour,d; ~MexoH 290 (c 4,915), 281 (6 5,670), 273 (~ 5~350) and 269 nm (~ 5,020).
' . ' ' .
:. '. .: ' -69- ~

.. .

1~83143 AHP-663S-I-CI

In the same manner by follow7ng the above procedure, other compounds of formula X in which X is (CH2)n-S-(CH2) are obtainedO
For example, replacing the starting material with an equivalent amount o~ other starting materials o~ formula XV, as described in Example 62, the followTng compounds of formula X are obtained, respectively;
ICO-Lys(Boc~-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser~But)~llH, ( C ~l2 ) ~ _ _ (CH2 ) 3 8O-LYS(BOC)-ASn-Phe-Phe-TrP-LYS(BOC)-Thr(BUt)-Phe-Thr~BUt)-Ser~BUt)- H, H_ S _ ~ ~ ~ -- - - (I~H2)3 CO-Lys(Eoc)-A~n-Rhe-Ph~-Trp1-Lys(Boc)-Thr(But)-Phe-Thr~But)-Ser(But)-NH, (1H2 ) ~ ~ ~ (IH2 ~2 .~ CO-Lys(Boc~-Asn-Phe;Phe-Trp-Lys(Boc)-Thr(But)-Phe-Thr(aut)-Ser(But)-~H, (CH2)4 ~ CH2)4 15 CO-LYS(~OC)-ASn-Phe-Phe D-TrP-LYS(BOC)-Thr(BUt)-Phe-1hr(BUt)-Ser(BUt)- H, (I H2) ~ ~ H2)2 ` ICO-Lys(Boc)~,~sn-Phe-Phe-G-Trp.Lys(Boc)-Thr(But)phe-Thr(But)-Ser(But)-~H and (CH2) ~ (CH ) CO-LYS(~OCj-ASn-Phe-Phe-D-TrP-LYS(BOC)-Thr(BUt)-Phe-Thr(BUt)-Ser(BUt)- H.
1 - - S ~ H2)~
i .

.. . . . .. . . .
'. . .

. ..
.
. , -, .
, , .; -70-, . ,~ .
.. . .

~ 3143 ~IP-6635~1-CI

Cyclic Amide of Lysy~-asparaqinYI-phenYlalanyl-phen~lalany!-fry~lto~h lYsyl-threonvl~phenvlalanyl-threonyl-seryl-4r(2~amino-ethvlth~lo)-- butanoic Acid (~Q~s~s~Phe-Phe-l~p-Lys-Thr-phe-Thr-~er-yt~
2T~ (CH2)2 1 X - ~C~!2~ ~ ~2 and Y = Trp A solu~ion of ~0-Lys(Boc)-Asn-Phe-Phe-Trp-Lys(Boc)-Thr(Bu~) I - t f ~
(CH2)3-S-(CH2)2-NH ~Ser(Bu ) ~Thr(Bu )~Pha~ --(0.729 9, described in Examplq 63) in concentrated hydrochloric acid (4.0 ml) is r~pidly stirred at 0VC under an atmasphere of nTtrogen for 10 mTnute$. Acstic acTd (300 ml) Is added and fhe solution is Iyophilized.
The residue ts taken ~In w3ter tlO0 ml ) and again Iyophilized. The residue is dissolved in the upper phase of the solvent system butanol-acetic acid-water t4:!:5), applie~ to~a column of chemically modifled 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 res7due is dissolved tn , 20 water and iyophilized to glve the tttle compound; ~:maxoH 290 ~E 4,825), . 281 (c 5,550), 275 ~6 S~195)! 269 ~E 4 ,aso:), 265 ~ 6 4,485) and 216 nm (E 49,750)~
' ,' ' ~ .
~ ', ' ' ' -7;-., .

: ' .,:

3.~43 AHP-6635-1-CI

,,.
In the same manner by following the above procedure, other compounds of formula I in which X is (CH2)n-5-~CH2)m are obtainsdO
For example, replacing the starting material with an equivalent amount of ather starting materials of formula X in whlch X is (CH2)n-S-(CH2)m, as described in Example 63, the followTng compounds of formula I are obtained, respectively:
fO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-lllH
(CH2)~ ' '' '---' S - , ., (CH2)3 : ~O-Lys-Asn-Phe-Phe-rrp~Lys-Thr-Phe-Thr-Ser-~H
H~ S ' ~ ~-- ( H2)3 J ~ :

. fO-LysoAsn-Phe~Phe-Trp-Lys-Thr-Phe-Thr-Ser-~H -.
(CH2)4 5 ~ . ~CH2)2 CO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH
)~ 5 , (CH2)4 1O-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-~H
~CH2)~ - ( H2~2 ~' ' .
: fO-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr~Phe-Thr-Ser-~H ~ :
. ._ ~ ~.(CH2)3 and : ~O-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-~H
H2 ~ 4 - - (~H2 ) 4 , `; ' ' ' ' . , , ' .
, :' .

. . . .

..

Claims (26)

1. A process for preparing a compound of formula I

(I) in which X is (CH2)k wherein k is an integer from 1 to 9, or (CH2)n-S-(CH2)m wherein m and n each is an integer from 1 to 4 and Y is Trp or D-Trp which comprises:
subjecting a cyclic protected peptide of formula 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.

2. A process as claimed in Claim 1 in which said cyclic protected peptide of formula X is reacted with hydrochloric acid and the corresponding compound of formula I is isolated.

3. A process as claimed in Claim 1 in which X is (CH2)6 and Y is Trp.

4. A process as claimed in Claim 1 in which X is (CH2)6 and Y is D-Trp.

5. A process as claimed in Claim 1 in which X is (CH2)3-S-(CH2)2 and Y is Trp.

6. A process as claimed in Claim 1 in which X is (CH2)3-S-(CH2)2 and Y is D-Trp.

7. A process as claimed in Claim 1 in which X is (CH2)2-S-(CH2)3 and Y is Trp.

8. A process as claimed in Claim 1 in which X is (CH2)2-S-(CH2)3 and Y is D-Trp.

9. A process as claimed in Claim 1 in which said cyclic protected peptide of formula X in which X and Y are as defined therein is prepared by:
reacting the tetrapeptide of formula R1-Lys(Boc)-Asn-Phe-Phe-OMe in which R1 is Ddz or Z with hydrazine hydrate to obtain the tetrapeptide hydrazide of formula II
R1-Lys(Boc)-Asn-Phe-Phe-NHNH2 (II), in which R1 is as defined herein, coupling said last-named compound by means of the azide method 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
R1-Lys(Boc)-Asn-Phe-Phe-Y-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-OMe (IV), in which R1 and Y are as defined herein, subjecting said last-named compound to hydrazinolysis to obtain the corresponding decapeptide hydrazide of formula V
R1-Lys(Boc)-Asn-Phe-Phe-Y-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NHNH2 (V), in which R1 and Y are as defined herein, reacting said last-named compound by means of the azide coupling method with a compound of formula VI
H2N-X-COOR2 (VI), in which X is as defined herein and R2 is lower alkyl to obtain the corresponding compound of formula VII
R1-Lys(Boc)-Asn-Phe-Phe-Y-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH-X-COOR2 (VII), in which R1, R2, X and Y are as defined herein, subjecting said last-named 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)-NH1-X-CO-R3 (VIII) in which R1, X and Y are as defined herein and R3 is OH or NHNH2, respectively, removing the protecting group R1 from said compound of formula VIII when R1 is Ddz under mildly acidic conditions or when R1 is Z and X is (CH2)k wherein k is as defined herein with hydrogen in the presence of a noble metal 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)-in which R3, X and Y are as defined herein, and cyclizing said compound of formula IX with a carboxyl group activating agent to obtain said cyclic protected peptide of formula X in which X and Y
are as defined herein.

10. A process as claimed in Claim 1 in which said cyclic protected peptide of formula X in which X is (CH2)n-S-(CH2)m wherein m and n are as defined therein and Y is as defined therein is prepared by:

coupling the hexapeptide hydrazide of formula Ddz-Y-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NHNH2 in which Y is as defined herein by means of the azide coupling method with a compound of formula VI H2N-X-COOR2 (VI) in which X is (CH2)m-S-(CH2)n wherein m and n are as defined herein and R2 is lower alkyl to obtain the corresponding compound of formula XI
Ddz-Y-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH-(CH2)m-S-(CH2)n-COOR2 (XI) in which m, n, R2 and Y are as defined herein, subjecting said last-named compound to hydrazinolysis to obtain the corresponding hydrazide of formula XII
Ddz-Y-Lys(Boc)-Thr(But)-Phe-Thr(But)-Ser(But)-NH-(CH2)m-S-(CH2)n-CONHNH2 (XII) in which m, n and Y are as defined herein, reacting said last-named compound by means of the azide coupling method with the tetrapeptide of formula 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, reacting said last-named compound under mildly acidic conditions 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 (XIV) in which m, n and Y are as defined herein, subjecting said last-named 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 said last-named compound by means of the azide coupling method to obtain said cyclic protected peptide of formula X
in which X and Y are as defined herein.

11. A process as claimed in Claim 9 in which X is (CH2)6 and Y is Trp.

12. A process as claimed in Claim 9 in which X is (CH2)6 and Y is D-Trp.

13. A process as claimed in Claim 9 in which X is (CH2)3-S-(CH2)2 and Y is Trp.

14. A process as claimed in Claim 9 in which X is (CH2)3-S-(CH2)2 and Y is D-Trp.

15. A process as claimed in Claim 9 in which X is (CH2)2-S-(CH2)3 and Y is Trp.

16. A process as claimed in Claim 9 in which X is (CH2)2-S-(CH2)3 and Y is D-Trp.

17. A process as claimed in Claim 10 in which X is (CH2)3-S-(CH2)2 and Y is Trp.

18. A process as claimed in Claim 10 in which X is (CH2)3-S-(CH2)2 and Y is D-Trp.

19. A process as claimed in Claim 10 in which X is (CH2)2-S-(CH2)3 and Y is Trp.

20. A process as claimed in Claim 10 in which X is (CH2)2-S-(CH2)3 and Y is D-Trp.

21. A process as claimed in Claim 1 in which X is (CH2)n-S-(CH2)m wherein m and n each is an integer from 1 to 4 and Y is Trp or D-Trp.

22. A compound of formula I

(I) in which X is (CH2)n-S-(CH2)m wherein m and n is an integer from 1 to 4 and Y is Trp or D-Trp, or a pharmaceutically acceptable salt thereof, when prepared by the process of Claim 21, or an obvious chemical equivalent thereof.

23. The compound of Claim 22 in which X is (CH2)3-S-(CH2)2 and Y is Trp, when prepared by the process of Claim 5, or an obvious chemical equivalent thereof.

24. The compound of Claim 22 in which X is (CH2)3-S-(CH2)2 and Y is D-Trp, when prepared by the process of Claim 6, or an obvious chemical equivalent thereof.

25. The compound of Claim 22 in which X is (CH2)2-S-(CH2)3 and Y is Trp, when prepared by the process of Claim 7, or an obvious chemical equivalent thereof.

26. The compound of Claim 22 in which X is (CH2)2-S-(CH2)3 and Y is D-Trp, when prepared by the process of Claim 8, or an obvious chemical equivalent thereof.