CA1074785A - Process for preparing a nonapeptide having lh- and fsh-releasing activity and intermediates therefor - Google Patents

Abstract

PROCESS FOR PREPARING A NONAPEPTIDE HAVING LH- AND
FSH-RELEASING ACTIVITY AND INTERMEDIATES THEREFOR
Abstract of the Disclosure A process for preparing the nonapeptide of the formula I
H-Pyr-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt having LH- and FSH-releasing activity which comprises the following steps: condensing 5-oxoprolyl-histidyl-tryptophane hydrazide by means of the azide method with a protected hydrazide of seryi-tyrosyl-D-alanine, pre-ferably seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester (II), followed by treatment of the reaction product with an acid to obtain 5-oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-allanine hydrazide (IV), condensing the latter compound by means of the azide method with leucyl-arginyl-proline ethylamide (V)to obtain the nonapeptide of formula I isolated as an acid addition salt which is converted to the free base or other pharmaceutically acceptable acid additions salts.

Description

~7~35 Back~round o~ th~ ~nv~ti~
1. Field of Invention This invention relates to a novel process for preparing a nonapeptide having luteinizing hormone (LH)- and follicle stimulating hormone tFSH)-releasing activity. In particular, the present invention relates to a novel process for preparing the nonapeptide of formula (1), H-Pyr-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt tl~ to salts thereof with pharmaceutically acceptable acids, to pharmaceutical compositions containing said LH- and FSH-releasing nonapeptide, and to intermediates obtained in said process.
LH and FSHare both gonadotrophic hormones elaborated by the pituitary gland of humans and of animals~ LH together with FSH stimulates the release of estrogens from the maturing follicles 1~ in the ovary and induces the process of ovulation in the female.
In the male, LH stimulates the interstitial cells and is for that reason also called interstitial cell stimulating hormone (ICSH).
The follicle-stimulating hormone tFSH) induces maturation of the fol!icles in the ovary and together with LH, plays an important role in the cyclic phenomena in the female. FSH promotes the development of germinal cells in the testes of the male. Both LH
and FSH are released rrom the pituitary gland by the action of LH-and FSH-releasing hormone, and there is good evidence that said re-leasing hormone is elaborated in the hypothalamus and reaches the ~5 pituitary gland by a neurohumoral pathway, see e.g. A.V. Schally et al ., Recent Progress in Hormone Research, 24, 497 t1968).
The natural LH- and FSH-releasing hormone has bsen isolated from pig hypothalami and its constitution elucidated by A.V. Schally et al., Biochem. Biophys. Res. Commun., 43, 393 and 1334 (1971), who ' \

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proposed the decapeptide structure (pyro)-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2.
The LH- and FSH-releasing hormone may also be represented by the formula ~~
H-Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2.
This constitution has been confirmed by synthesis for example, see H. Matsuo et al., Biochem. Biophys. Res. Comm., 45, ô22 (1~71) and R. Geiger et al., ibid, 45, 767 (1971).

2. Description of the Prior Art ;

The nonapeptide of formula (I) having LH- and FSH-releasing activity has been synthesized by D.H. Coy, et al., Biochem. Biophys.
Res. Commun., 57, 335 ~1974) using solid-phase peptide synthesis methods; the same hormone has also been synthesized by M.Fujino, et al.~ Arch. Biochem. Biophys., 154, 488 (1973) and M. Fujino, et al., Biochem. Biophys. Res. Commun., 57, 1248 (1974). In con-tradistinction to the processes of the references cited above the process of this invention is simpler and more efficient in giving considerably better over-all yields than any of the known procedures.
It is a particular advantage of the process of this invention that it reqùires only a minimum of protective groups for the intermediates, especially where secondary functions are concerned. Thus, the hydroxyl group in serine does not have to be protected; the NH-groups in tryptophan and in histidine do not require protection; and no pro-tection for the guanidino function in arginine and for the hydroxyl group of tyrosine is necessary in the later s-tages of the process.
The process of this invenTion is thus also more convenient and less cumbersome than the processes of Prior Art. An added advantage of the process of this invention is the fact that the final step thereof consists in the condensation of two unprotected fragments, each of which is well defined~ easy to purify, and each obtainable in a high ,! ~ .. , .. . . ' . `

AHP- 6~97 7~

STate of purity. Also, the final step of the process of -rhis invention utilizes the az;de coupling method for the condensation of the two peptide fragments. A noteworthy advantage of the azide coupling method is that this method gives the least amount o~ racemization compared to the other methods of peptide condensations, or no detectable racemization at all. Thus, the important amino acid, D-alanine, is subjected to the minimum of racemization during the coupling of the peptide fragments. T~e final product is obtained in a high degree of purity and in good yields. The product of the process of this invention thus obtained is thc free, unprotected nonapeptide which does not require any further deprotective steps, in contradistinction to the processes of the references cited above where rather severe deprotection conditions are used to give the final product.
In keeping with the need for a practical synthesis of the IS nonapeptide of formula (1), the present invention discloses a new practical process for the large scale preparation of the nonapeptide.
Furthermore the present process has additional advantages in that it starts from readily available materials, avoids noxious reagents~ is executedf~cilely,utilizes easily removable protecting groups and provides a pure product having a high degree of physiological potency.
Summary of the_lnvention ~
The method for preparing tbe biologically active nonapeptide of formula 1 H-Pyr-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt (1), comprises condensing the hexapeptide hydrazide 5-oxoprolyi-histidyl-tryptophyl-seryl-tyrosyl-D-alanine hydrazide (IV) by means of the azide coupling method with the tripeptide,leucyl-arginyl-proline ethylamide (V) and isolating said nonapeptide as its acid addition salt which is converted to the base or a pharmaceutically acceptable acid , .

~7D~7~5 addition salts. The above mentioned hexapeptide hydrazide, 5-oxo-prolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine hydrazide, is prepared by condensing 5-oxoprolyl-histidyl-tryptophane hydrazide by means of the azide coupling method with tne protected hydrazide of seryl-tyrosyl-D-alanine, preferably seryl-tyrosyl-D-alanine carboxyhydra~ide t-butyl ester (Il), and isolating the protected hydrazide of the hexapeptide 5-oxoprolyl-h,stidyl-tryptophyl-seryl-tyrosyl-D-alanine, preferably S-oxoprolyl-histidyl-tryptophyl~seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester (111), which is treated under moderately acidic conditions to obtain the hexapeptide hydrazide named above.
The tripeptide fragments 11 and IV and the above sequence of reactions, omitting the successive introduction and removal of protecting groups as well as the methods of condensation and the condensing agents used, is shown below in Fig. 1, FIGURE I

5-Oxoproline~
~ Histidine ~ _ ..
Tryptophane IV.
Serine - I _ Tyrosine~
l Nonapeptide of D-Alanine ~ formula (I) ~5 Leucjne -Arginine ~ . `
Proline . 5 AHP-64s7 ~7~5 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 J. Biol.
Chem., 241, 2~91 (1966). For instance, Pyr, His, Trp, Ser, Tyr, D-Ala, Leu, Arg and Pro represent "residues" of L-pyroglutamic acid or 5-oxo-L-proline, L-histidine, L-tryptophane, L-serine, L-tyrosine, D-alanine, L-leucine, L-arginine and L-proline, respectively.
"Residue" means a radical derived from the corresponding amino acid by eliminating the OH portion of the carboxyl group and the H portion of the amino group. Except for alanine, which has the unnatural D-configuration, all the other amino acids have the natural L-configuraticn.
A number of procedures or techniques for the preparation of peptides have hitherto been well established. For instance, the functional group or groups which are not involved in the peptide bond formation reaction areoptjonally protected by a protecting group or groups prior to the condensation reaction. For example, protecting groups for an amino function of a peptide or amino acid not involved in lhe peptide bond formation are the alkoxy-carbonyls which include benzyloxycarbonyl (represented by Z), t-butyloxycarbonyl (represented by Boc),or ~,~-dimethyl-3,5-dimethoxy~
benzyloxycarbonyl trepresented by Ddz). Protecting groups for the hydroxyl of tyrosine include such ether-forming groups as benzyl (represented by 8zl) or t-butyl (represented by Bu~
Protecting groups for the guanidino group of arginine include nitro (represented by N02)or benzyloxycarbonyl 7~7~5 (represented by Z), or salt ~ormation with a strong acid.
The carboxylic acid function of a peptide or amino acid can be con-sidered protected by a lower alkyl or lower aralkyl este, which includes methyl ~represented by OMe), ethyl (represented by OEt) or benzyl ~represented by OBzl), and also by substituted hydrazides which include t-butyloxycarbonyl hydrazide (represented by NHNH-Boc), benzyloxycarDonyl hydrazide (represented by NHNH-Z) or ~,~-dimethyl-3J5-dimethoxybenzyloxycarbonyi hydrazide (represented by NHN~I-Ddz).
To promote facile condensation of the peptide carboxyl group iO with a free amino group of another peptide to form a new peptide bond, the terminal carboxyl group must be activated. Examples of tlle activated form of the terminal carboxyl are acid chlorideJanhydride, azTde, activated ester, or an o-acyl urea of a dialkylcarbodiimide. Des-crtptions of these and other protecting and carboxyl-activating groups are found in general textbooks of peptide chemistry; for example K.D. KoppleJ "Peptides and Amino Acids", W. A. BenjaminJ Inc., New York, 1966, pp. 45 - 51 and E. Schr~der and K. LUbke, "The Peptides";
Vol. 1, Academic PressJ New York, 1965, pp. 77 - 128. The following activated esters have proved to be particularly suitable in the process of this invention: 2,4,5-trichlorophenyl (represented by OTcp), pentachlorophenyl (represented by OPcp), p-nitrophenyl (represented by ONp), succinimido and l-benzotr~iazolylo The abbreviation Me represents a methyl group, Et represents an ethyl gro!~p, NHNH2 represents a hydrazide group, and NH2 or NHEt represents the amide group or the ethylamide group, respectively.
Th~ terms "peptide, polypeptide, tripeptide, hexapeptide, and the like" às used herein are not limited to refer to the respective parent peptides but are also used in reference to .. . .

~7~ 35 modified peptides having functionalized or protecting groups. In addition the term "peptide" as used herein is used in reference to a peptide with one to nine amino acid residues.
The term "lower alkyl" as used herein contemplates hydro-carbon radicals having one to three carbon atoms and includes methyl, ethyl and propyl.
The term "mineral acid" as used herein contemplates the strong inorganic acids and includes hydrochloric, hydrobromicJ
sulfuric, phosphoric and the like. When the term 7s used in con-junction with an anhydrous system, hydrogen chloride is the preferred mineral acid.
The term "miIdly acid conditions" as used herein contemplates conditions in which a dilute aqueous solution of an organic acid, for example 30 - 90~ aqueous formic, acetic or propionic acid, pre-ferably 70 - 80%, or I to 10% aqueous trifluoroacetic acid, is a principal component of the reaction medium.
The term "moderately acidic conditions" as used herein contemplates conditions in which concentrated organic acids or aqueous solutions of the mineral acids are used as a principal component of the reaction medium at temperatures ranging from about -30 to 30C. Examples of preferred conditions in this case include the use of 50 to 100~ trifluoroacetic acid at 0 to 30C or of 0.1 - 12N hydrochloric acid at -20 to 10C.
The term "organic nitrite" includes the commercially available alkyl nitrites, for example, t~butyl nitrite, isoamyl nitrite~ and the like.
The term "organic base" as used herein includes triethyl-amine, N-ethylmorpholine, N-methylpiperidine, pyridine, N-ethyl-diisopropylamine and 1-he like.

~7~

The term "strong base" as used herein contemplates both organic bases, as described above, and strong inorganic bases including the hydroxides and carbonates of sodium and potassium.
The term "azide method" as used herein refers to the method of coupling two pep-tide fragments which comprises the reaction of a peptîde hydrazide with a reagent which furnishes nitrous acid in sjtu. Suitable reagents for this purpose include a lower alkyl nitrite (e.g. t-butyl nitrite,isoamyl nitrite) or an alkali metal nitrite salt (e.g. sodium nitrite, or potassium nitrite) in the presence of a strong acid such as hydrochloric, sulfuric or phosphoric, acid. The corresponding peptide azide thus obtained is then reacted wlth a peptide having a free amino group to obtain the desired pcptide.
Preferred conditions for the azide method of coupling comprise reacting the peptide hydrazide with the organic nitrite in the presence of a mineral acid in an anhydrous inert organic solvent, for example, dimethylformamide, dimethyl sulfoxide, ethyl acetate, methylene dichloride, tetrahydrofuran, dioxane, and the like, at -30 to 20C, preferably at about -15C, for 10 - 60 minutes to obtain the coresponding azide, rendering the resulting mixture alkaline by the addition of a strong base, preferably an organic base, for example N-ethyldiisopropylamjne, N-ethylmorpholine or triethylamine, and thereafter reacting the azide in ~he said mixture with the peptide unit having the free amino group at tempera-tures ranging from -30C to 20C for about one to two hours and then at 0 to 30C for 10 to 24 hours. See also the a~ove cited textbooks of Kopple and Schr~ber and LUbke for additional descriptions of this method.
.

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71~5 The nonapeptide of formula I obTained by the process of th;s invention in the form of an acid addition salt possesses LH- and FSH-releasing properties and is more potent than the natural hormone when tested in the radioimmunoassay described by Niswender et al., Proc. Soc. Exp. Biol. Med., 128, 807 (1968). It is also more potent in the assay determining induction of ovulation in the ha~ster described by Arimura et al., Science 174, 511 (1971), and in a modification of the similar assay in the rat dèscribed by Arimura et ai., in Endrocinology 80, 515 (1967).
The LH- and FS~-releasing propèrties of the present nonapeptide and its corresponding pharmaceutically accepta~le salts, which in turn induce ovulation in animals, render the nonapeptide an useful agent in veterinary practice and in animal husbandry. It is often desirable ~o synchronize estrus in livestock, for example, cattle, sheep, or swine either in order to be able to mate all the females in a given group with a male of the desired genetic quality, or so as to be able to perform artificial insemination on a maximum number of females, both within a comparatively short period of time. In the past, this has been done by administering to the animals an ovutation-inhibiting agent, withdrawing administration of said agent shortly before the date chosen for mating or artificial insemination, and relying either u~pon the natural production of ~H and FSH to induce ovulation and to produce estrus or by administering gonadotrophins. However, this procedure was nol entirely satisfactory because ovulation at a predetermined time occured never in all the animals together but only in a certain proportion thereof when gonadotrophins were not used~ On the other hand, the high cost of gonadotrophins and side effects \, -- i û~

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AHP-6~97 4~S

encountered in -~heir administration made this method impractical.
It is now possible to obtain substantially complete synchronization of ovulation and of estrus, by treating the animals in a given group first with an ovulation inhibitor which is subsequently withdrawn, and then administering the LH- and FSH-releasing agent produced by the process of this invention shortly before the predetermined period of time for mating or artifical insemination, so as to obtain ovulation and estrus within that time interval. The delay in the onset of ovulation and estrus following administration of the nonapeptide produced by the process of this invention varies with the species of animal, and the optimal time interval has to be chosen for each species.
For example, in rodents such as rats or hamsters ovulation takes place within 18 hours following administration of the LH- and FSH-releasing agent of this invention.
The method described above for obtaining ovulation and estrus within a precisely predetermined time interval so as to be certain of a successful mating is particularly important for breeders of race horses and of show animals, where the fees paid for the services of an exceptional male animal often amount to very considerable sums ~0 of money. For example, following adminis~ration of the nonapeptide produced by the process of th~s Invention to cycllng mares the duration of estrus is shortened by about two days and ovulation takes place within ~ - 5 days after the onset of estrus. In this manner the time of ovulation becomes more accurately predictable and the chances of achieving a successful mating are much better than has been possible in the past.
The LH- and FSH-releasing agent produced by the process of this invention is also useful to increase the number of live births per pregnancy in livestock, for example, cattle, sheep or swine~
For this purpose the nonapeptide of this invention is given in

3 series of parenteral doses, preferably by intravenous or sub-cutaneous injections, in the range of 10 ~9 to 2000 ~9 per kilogram A~IP-6~97 ~3'7~ 35 of body weight per day, 9~ to 12 hours prior -~o expecte~ estrus and subsequent mating. A priming injection of 1000 to 5000 IU o~
pregnant mares serum gonadotrophin may also be given one to four days prior to the above injection of LH- and FSH-releasing hormone.
A similar treatment, with or without prior priming, is also useful for inducing puberty in farm animals.
When the nonapeptide produced by ~he process of this invention is employed for the purpose of inducing ovulation and estrus or for inducing puberty in warm-blooded animals~ especially in rodents such as rats or hamsters or in livestock, it is administered systemically, preferably parenterally, in combination with a pharmaceutically acceptable liquid or solid carrier. The proportion of the nonapeptide is determined by its solubility in the given carrier, by the chosen route of administration, and by standard biological practice. For parenteral administration to animals the nonapeptide may be used in a sterile aqueous solution which may also contain other solutes such as buffers or preservatives, as well as sufficient pharmaceutically acceptaDle salts or glucose to malce the solution isotonic. The dosage will vary with the form of administration and with the particular spec7es of animal to be treated and is preferably kept at a level of from 50 ~9 to 2000 ~9 per kilogram body weight-. However, a dosage level in the range of from about IC0 ug to about 1000 ~9 per kilo-gram body weight is most desirably employed in order to achieve effective resuIts.
2~ The nonapeptide may also be administered in one of the long acting, slow-release or depot dosage forms described below, preferably by intramuscular injection or by implantation. Such dosage forms are designed to release from about 5 ~9 to about lOO ug per kilogram body weight ~er day.

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AHP-6~97 ~'7~713~
The nonapeptide produced by the process of this invention is also useful in human medicine. For example, human chorionic gonadotrophin (~G) which contains mainly LH and some FSH has been used for over 30 years to treat certain endocrinological disorders such as disturbances of the cycle, amenorrhea, lack of development of secondary sex characteristics, and infertility in the female, or certain cases of hypogonadism, delayed puberty, cryptorchidism) and non-psychogenic impotence in the male. Lately, infertility in the human female has also been treated with human menopausal gonadotrophin (HMG~ which contains mainly FSH, followed by 1-reatment with HCG. One of the disadvantages of the treatment of infertility in the human female with HCG or with HMG followed by HCG has become apparent in that such treatment often results in superovulation and unwanted multiple births, probably because of the irnpossibility of giving only the exact amounts of FSH and LH which are necessary for ovulation. The administration of the nonapeptide produced by the process of this invention overcomes the above disadvantage, because the nonapeptide induces release of LH and FSH by the pituitary only in the exact quantitites which are required for normal ovulation. For that reason the nonapeptide produced by the process of this invention is not only useful for the above purpose, but it is equally useful in the human female in the treatment of disturbances of the cycle, of amenorrhea, of hypogonadism, and of lack of development of secondary sex characteristicsO
Furthermore, the LH- and FSH-releasing agent produced by ~5 the process of this invention is also useful in contraception. For example, when the hormone is administered to a human female early in the menstrual cycle LH is released at that time and causes premature ovulation. The immature ovum is either not capable of being fertilized, or, if fertilization should nevertneless have taken place, it is highly unlikely that the fertilized ovum will become implanted because the estrogen-progestin balance required to prepare the endometrium is ~7~L7~35 AHP-6497 not present and the endometrium is not in Ihe condition necessary for implantation. On the other hand, when the agent is administered towards the end of the cycle the endometrium is disrupted and menstruation takes place.
In addition, the LH- and FSH-releasing agent produced by the process of this invention is also useful in contraception by the "rhythm" method, which has always been relatively unreliable because of the impossibility of predetermining ovulation in the human female with the required degree of accuracy. Administration of the agent at mid-cycle, i.e. at about the normally expected time for ovulation, induces ovulation shortly thereafter and makes the "rhythm" method both safe and effective.
The LH- and FSH-releasing agent produced by the process of this invention is also useful as a diagnostic tooi for dis-tinguishing between hypothalamic and pituitary malfunctions or lesions in humans. When administering the agent to a patient suspected of such malfunctions or lesions and if a rise in the level of LH is subsequently observed, it constitutes good presumptive evidence to conclude that the hypothalamus is the cause of the malfunction and that the pituitary is intact. On the other hand, when no rise ir circulating LH is seen following the administration of the agent a diagnosis of pituitary malfunction or lesion can be made with a hiqh degree of confidence. ¦~
In the human male, administration of the LH- and FSH- ¦
releasing agent obtained by the process of this invention provides the amounts of LH and of FSH necessary for promoting sexual development in cases of hypogonadism or delayed puberty, and is also useful In the treatment of cryptorchidism. Furthermore, the FSH
released by the administration of the agent stimulates the deve-.. ~ .
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lopment of germinal cells in the testes, and the agent is useful in the treatment of non-psychogenic impotence.
When the LH- and FSH-releasing agent obtained by the process of this invention in the form of an acid addition salt is employed in human medicine, it is administered systemically, either by intravenous, subcutaneous, or intramuscular injection, or by sublingual J nasal, or vaginal administration, in compositions in conjunction with a pharmaceutically acceptable vehicle or carrier.
For administration by injection or by the nasal route as drops or spray it is preferred to use the agent in solution in a sterile aqueous vehicle which may also contain other solutes such as buffers or preservatives, as well as sufficient quantities of pllarn~aceutically acceptable salts or of glucose to make the solution isotonic.
IS The LH- and FSH-releasing agent produced by the process of this invention may also be administered as nasal or vaginal powders or insufflations. For such purposes the hormone is administered in finely divided solid form together with a pharmaceutically accpetable solid carrier, for example, a finely divided polyethylene glycol 2~ ("Carbowax 1540"), finely divided lactose, or, preferably oniy for vaginal administration, very finely divided silica ("Cab-O-Sil").
Such composi-tions may also contain~other excipients in finely divided solid form such as preservatives, buffers,or surface active agonts. 1;
For sublinguai or vaginal administration it is preferred to formulate the agent in solid dosage forms such as sublingual tablets or vaginal inserts or suppositories with sufficient quantitites of solid excipients such as starch, lac~lose) certain types of clay, buffers, and lubricating, disintegrating, or surface-,-- .

~379L~35 active agents, or witn semi-solid excipients commonly used in the formulation of suppositories~ Examples of such excipients are found in standard pharmaceutical texts, e.g. in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 1970.
The dosage of the LH- and FSH-releasing agent obtained by the process of this invention will vary with the form of administration and with the particular subject under treatment. Generally, treatment is initiated with small dosages substantially less than the optimum dose of the hormone. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reachedO
In general, the agent obtained by the process of tnis invention is most desirably administered at a concentration level that will generally afford effective release of LH and of FSH without causing any harmful or deleterious side effects, and preferably at a level that is in a range of from about 10 ~9 to about 2000 ~9 per kilogram body weight, although as aforementioned variations will occur. However, a dosage level that is in the range of from about 50 ~9 to a~out IOOO ug per kilo-gram body weight is most desirably employed in order to achieve effective results.
; 20 It is often deslrabl~ 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 pharmaceuti- ¦
cal Iy acceptable salt of the agent having a low degree of solubility in body fluids, for example one of those salts described below, or they ~5 may contain the agent in the form of a water-soluble salt together with a protective carrier which prevents rapid releaseO In the latter case, for example, the agent may be formulated with a non-antigenic partially hydrolyzed gelatin in the form of a viscous -liquid; or the agent may be adsorbed on a pharmaceutically acceptable . .

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solid carrier, for example zi llC hydroxide, and may be administered in suspension in a pharmaceutically acceptable liquid vehicle; or the agent may be formulated in gels or suspensions with a protective non-antigenic hydrocolloid, for example sodium carboxymethylcellulose, polyvinylpyrrolidone, sodium 31ginate, gelatine, polygalacturonic acids, for example pectin, or certain mucopolysaccharides, together with aqueous or non-aqueous pharmaceutically liquid vehicles, pre-servaTives, or surfactants. Examples of such formulations are found in standard pharmaceutical texts, e.g. in Remington's Pharmaceutical Sciences cited above. Long-acting, slow-release preparations of the agent produced according to the process of this invention may also be obtained by microencapsulation in a pharmaceutically acceptable coating material, for example gelatine, polyvinyl alcohol or ethyl cellulose. Further examples of coating materials and of the pro-1~ cesses 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 agent which are only sparingly soluble in body fluids, for example salts with pamoic,alginic or tannic acid are designed to r^elease from about I ~9 to about 1000 ug of the hormone per krlogram body weight per day, and are preferably administered by intramuscular injection. Alternatively, some of the solid dosage forms list`ed above~ for example certain sparingly water-soluble salts or dispersions in or adsorbates on solid carriers of salts ~5 of the agent, for example dispersions in a neutral hydrogel of a polymer of ethylene glycol mothacrylate or similar monomers cross-linked as described in U.S. Patent 3,5519556 may also be fot^mulated in the form of pellets releasing about the same amounts as shown above and may be implanted subcutaneously or intramuscularly.

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7~'7~3~
Alternatively, slow-release effec-~s over prolonged periods of time may also be obtained by administering the agent obtained by the process of thi~ invention as an acid addition salt i an intra-vaginal device or in a temporary implant, for example a container made of a non-irritating sil7cone polymer such as 3 polysiloxane, e.g. "Silastic", or of a neutral hydrogel of a polymer as described above, possessing the required degree of permeability to release from about I ~9 to about 1000 ~9 per kilogram body weight per day. Such intra-vaginal or implant dosage forms for prolonged administration have the advan~age that they may be removed when it is desired to interrupt or to terminate treatment.
Process The process of this invention is carried out in the following manner.
A solution of an amino protected and hydroxyl protected activated ester of tyrosine, preferably benzyloxycarbonyl-0-benzyl-tyrosine 2,4,5-trichlorophenyl ester, prepared as described by J~S~ Morley, J. ~hem. Soc., (C), 2410, (1947~, in an inert anhydrous solvent, preferably dimethylformamide, is added to a solution containing substantially one molar equivalent of a protected hydrazide of D-alanine, preferably D-alanine carboxyhydrazide t butyl ester (prepared as described for the cor~responding '~-isomer by M. Felix and R.B. Merrifield, J. Amer. Chem. Soc., '~,2, 1385 (1970), in an inert anhydrous solvent, preferably dimethylformamide, cooled to a temperature of from about -10C to about 5C. The mixture is kept at~a temperature of from about -10C to about 5C for about one to three days and evaporated under reduced pressure. The residue is taken up in an inert anhydrous solvent, preferably ethyl acetate and ; -i8-.

addea to an anhydrous non-polar solvent, preferably diethyl ether. The precipitate is collected by -fiItration ancl crystallization yields the corresponding fully protected dipeptide tyrosyl-D-alanine, preferably benzyloxycarbonyl-O-benzyl-tyrosyl-D-alanine carboxyhydrazide _-butyl ester, Said last-named compound is dissolved in an inert solvent system, preferably a mixture of methanol and dimethylformamide, a noble metal catalyst, e.g. palladium on charcoal~ is added and the mixture is agitated in an atmosphere of hydrogen at room temperature until substantially two molar equivalents of hydrogen have been taken up, FiItration of the catalyst, evaporation of the fiItrate, trituration of tne residue with a non-polar solvent, preferably diethyl ether, yields the corresponding protected hydrazide of the dipeptide tyrosyl-D-alanine, preferably tyrosyl-D~alanine carboxyhydrazide t-butyl ester.
Said last-named compound is dissolved in an inert solvent, preferably dimethylformamide, cooled to a temperature of about 0C
and mixed with a molar excess, preferably 1.1 to 1.4 molar equivalents, of an activated ester of a protected serine, preferably benzyloxy-carbonyl-serine pentachlorophenyl ester, prepared as described by J. Kovacs et al., J. Org. Chem., 32, 3696 (19~7), in an inert solvent, preferably dimethylformamide. The mixture is kept at about 0C for one to three days and the soi~ent is evaporated under reduced pressure.
The residue is taken up in a substantially water-immiscible organic solvent, preferably ethyl acetate and the solution is dried and evaporated. The residue is taken up in a mixture of organic solvents of suitable polarity and an organic base, preferably methanol-ethyl acetate-pyridine, and the solution îs subjected to chromatography, I
preferab!y on silica. Elution, evaporation of the eluates, and crystallization yields the corresponding protected hydrazide \

.. ,, --19--.

~7~ AHP-6497 of the tripeptjde seryl-tryrosyl-D-alanine in which the terminal amino ~rou~ is a!so protected, preferably benzyloxy-carbonyl-seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester.
Said last-named compound is dissolved in an inert solvent, preferably methanol, a noble metal catalyst, e.g~ palladium on charcoal, is added and the mixture i5 agitated in an atmosphere of hydrogen at room temperature until hydrogen is no longer absorbed, usually -~wo to five hours. FiItration of the catalyst, andevaporation of the fiItrate yields the corresponding protected hydrazide of the tr;peptide seryl-ty~osyl-D-alanine, preferably seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester (Il), A solution of 5-oxoprolyl-histidyl-tryptophan hydrazide, described by H. Immer et al., J. Med. Chem., 17, 1060 (1974), in an inert anhydrous solvent, preferably a mixture of dimethylformamide tS and dimethyl sulfoxide, is cooled to a temperature of from about -15C to about 5C, mixed with a solution of about two to five molar equivalents, preferably three molar equivalent of a strong mineral acid, preferably hydrogen chloride, in an anhydrous organic solvent, preferably ethyl acetate. The mixture is cooled to about t -12C and an organic nitrite, preferably t-butyl ~itrîte (1.0 to 1.5 molar equivalents, preferably 1.3 molar equivalents), is added to the stirred solution. After about 10 to 20 minutes at -15 to -10C
the mixture is rendered alkaline by the addition of an organic base, preferably three to five equivalents of N-ethyldiisopropylamine, 2S followed by the dropwise addition of substantially one molar equivalent of a protected hydrazide of the tripeptide seryl-tyrosyl-D-alaniner preferably seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester, prepared as described above, in an inert organic solvent, preferably dimethylformamide. Stirring is continued for 30 to 90 minutes at about '~ .

r . _ .

.-: .-..... : . . :

-~ - A~IP-6497 ~7~7~5 0C, and finally for 20 to 30 hours at about 20 lo 30 C. Evaporation of the solvent, taking up the residue in a lowor alkanol, preferably methanol, and addition to a non-polar solvent, preferably diethyl ether or petroleum ether, collection OT the precipitate, ~ollowed by crystalliza-tion yields the corresponding protected hydra~ide of the hexapeptide5-oxoprolyl-histidyl-tryptophyl-scryl-tyrosyl-D-alanine carboxyhydra~ide t-butyl ester (111). Said last-named compound is dissolved in tri-tluoroacetic acid at a temperature of about 0 to 5C, and the solution Is stirred at about 0 to 5C, for 20 to ~0 minutes and then at 20 to 30C for another 20 to 40 minutes. Evaporation of the solvent, taking up the residue in a lowor alkanol, preferably methanol, addltlon to a non-polar solvent, preferably diethyl ether, and collection of the precipitate yields the hexapeptide 5-oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl- ~ alanine hydrazide (IV) as the trifluoroacetic acid addltion salt. Said last-nar~d compound Is Isolated In the form of the free base by lon exchange chromatography on a strongly baslc anionic resih; preferred resins are cross-linked polystryene resins substituted with strongly bas7c groups, for example Amberlite* IRA-400 or IRA-410.
A lowcr alkyl ester of arglnyl~prollne In wl~lch tho tormlnal amlno and the guanldTno groups are protected, preferably t-butyloxy-carbonyl-nitroarginylproline methyl ester (prepared as described in U.S. Patent No. 3,835,108, issued September 10, 197~) is dissolvad in trifluoroacetic acid and the soiution is allowed to stand at room temperature for 20 to 40 minutes. Evaporation of the solvent, taking up the residue in a lower alkanol, preferably methanol, addition to a non-polar solvent, preferably dTethyl ether, and collection of the preclpitate ylelds the guanldlne proteclod lower alkyl es~er o~ arglnyl-proline, preferably nitroarginyl-proline methyl ester as the trlfluoro-acetic acid addition salt. Said last-narned coMpound is dissolved in an inert organic solvent, preferably dimcthylformamide, containing a : substantially equlmolar amount of an organic base, preferably ~Trade Mark ~3' . ~ -21-A~IP-6497 7~7~5 triethylamine, cooled to a temperature of about 0C and mixed with a solution containing a substantially equimolar amount of a protected activated ester of !eucine, preferably benzyloxycarbonyl-leucine 2,4,5-trichlorophenyl ester, in an inert organic solvent, preferably dimethylformamide, cooled to a temperature of about 0 to 10C. The mixture is allowed to stand at 0 to 5C for two to four days and evaporated. The residue is taken up in a substantially water immiscible organic solvent, preferably ethyl acetate, washed, dried, and evaporated. The residue is taken up in a solvent of suitable polarity, preferably a mixture of methanol and ethyl acetate, and sub-jected to chromatography on silica. Evaporation of the eluate, dissolving the residue in a lower alkanol, preferably methanol, addition to a non-polar solvent, preferably diethyl ether, and collection of the precipitate yields the corresponding protected lower alkyl ester of leucyl-arginyl-proline, preferably benzyloxycarbonyl-leucyl-nitroarginyl-proline methyl ester. Said last-named compound is taken up in a lower alkanol or alkoxyalkanol, preferably methoxy-ethanol~ an excess (1.1 to 1.5 molar equivalents preferably 1~2 equivalents) of an aqueous alkali metal hydroxide, preferably sodium hydroxide or potassium hydroxide, is added and tne mixture is stirred at room temperature for one-half to two hours, preferably for about one hour. A substantially equimolar amount of the aqueous alkali metal hydroxide is added and the mixture stirred at 20 to 30C for 20 to 30 hours. The mixture is cooled to a temperature of 2S about 0C, acidified with a strong mineral acid, preferably hydro- il chloric acid, and evaporated almost to dryness. After addit70n of water the residue is collected and dried to yield the corresponding pro-tected tripeptide leucyl-arginyl-proline, preferably benzyloxycarbonyl-.

AHP-64s7 ~7~

leucyl-nitroarginyl-proline, Said las-~-named compound and a compound having a hydroxyl group which is capable of -rorming an activated ester, pre.'erably l-hydroxybenzotriazole, (0.1 to 2.5 molar equivalents, preferably about two molar equivalents) in an inert organic solvent, preferably dimethylformamide is cooled to a temperature of about 0C
and treated with an excess,;preferably of about 1.2 molar equivalents, of dicyclohexylcarbodiimide, The mixture is stirred at a temperature of about 0C for about 45 to 75 minutes, then at about 20 to 30C for about 45 to 75 minutes, and cooled to a temperature of about 0C. Sub-stantially two molar equivalents of a lower alkylamine, preferably ethylamine, is added and the mixture is stirred at 20 to 30C for 20 to 30 hours. After removal of tne precipitate, the fiItrate is evaporated.
The residue is dissolved in a substantially water-immiscible organic solvent, preferably ethyl acetate, washedJ dried and evaporated. The residue is taken up in an organic solvent of suitable polari~y, pre-ferably a mixture of methanol and ethyl acetate, and subjected to chromatography on silica gel. After evaporation of the eluate, the residue is taken up in a lower alkanol, preferably methanol, and added to a non-polar solvent9 preferably diethyl ether. The pre-cipitate is collected and dried to yield the corresponding protected lower alkylamide of leucyl-arginyl-proline9 preferably benzyloxy-carbonyl-leucyl-nitroarginyl-proli~ne ethylamide (the latl-er compound has also been prepared in a different manner as described in the UOS.
Patent No. 3,853,837, issued December 10, 1974). Said last-named compound is dissolved in an inert solvent, preferably a mixture of methanol and acetic acid, a noble metal catalyst, e.g. palladium on charcoal, is added and the mixture is agitated in an atmosphere of hydrogen at room temperature until hydrogen is no longer absorbed, preferably for about 15 to 30 hours. FiItration of the catalyst AHP-64g7 3~7~

and evaporation of the filtrate yields a residue of the corresponding lower alkylamide of the tripeptide leucyl-arginyl-proline, pre-ferably leucyl-arginyl-proline ethylamide (V), isolated as the , acetic acid addition salt.
A solution of 5-oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine hydrazide trifluoroacetate (IV), obtained as described above, in an inert anhydrous solvent, prefèrably a mixture of dimethylformamide and dimethyl sulfoxide, is cooled to a temperature of from about -20C to about -10C, mixed with a solution of about three to seven molar equivalents, preferably five molar equivalents of a strong mineral acid, preferably hydrogen chloride, in an inert organic solvent, preferably ethyl acatate, and the mixture is cooled to a temperature of from about -30C to about -20C. An organic nitrite, preferably t-butyl nitrite or isoamyl nitrite (1.0 to 1.5 molar equivalents, preferably 1.2 molar equivalents), is added with stirring and the mixture is stirred for 15 to 30 minutes, preferably for about 20 minutes at a temperature of from about -25C to about -10C. Sufficient quantities of an organic base, preferably N-ethyldiisopropylamine, are added with stirring to make the mixture alkaline, preferably pH 8-9. Keeping the mixture at a temperature of from about -30C to about -15C, a solution of leucyl-arginyl-proline ethylamjde ~ diacetate (V) (1~0 to 1.5 molar equivalents, preferably 1.1 molar equivalents), prepared as described above.and substantially 2 molar equivalents of an organic base9 preferably N-ethyldiisopropylamine, in an inert solvent, preferably dimethylformamide,is added with stirring. The mixture is agitated at a temperature of from about -25C to about -10C for ~5 to 75 minutes, at a temperature of about 0 to 5C for about ~5 to 75 2~

AI~P-6~97 ~7~7~3~

minutes, and at a temperature of about 20 to 30C for about 20 to 30 hours. After evaporation, the residue is dissolved in an organic solvent, preferably methanol, added to a non-polar solvent, preferably diethyl ether, and the precipitate is collected. The precipitate is purified by column chromatography in which a variety of supports may be used, for example, carboxymethylcellulose or chemically modified cross-linked dextrans such as "Sephadex-~G-25 or L~1-20". Dependlng upon tl)e support used, the above desTred compound is obtained in the form of an acid addition salt or as the free base. The preferred method comprises purlfication of the precipitate by gel tiItration chromato-graphy on a chemically modified cross-linked dextran ("Sephadex LH-20") usTng a suitable solvent, preferably methanol, and yields the sub-stantially pure nonapeptide 5-oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanyl-leucyl-arginyl-proline ethylamide (1), isolated as the hydrochlortde sal-t. If the acetic acid addition salt of the nonapeptida is desired, the precipitate is purified by ion exchange chromatography, for example on carboxymethylcellulose, using an aqueous solution of ammonium acetate as eluant. Purlficatlon by partition chromatography on a chemically modified cross-linked dextran ( "Sephadex G-25") using acetic acid as the eluting solvent gives ~he substantially pure nonapeptido as the acetic acid addition salt. The free base of the above nonapepttde is obtained by subjecting an acid addition salt to ion exchange chromalography on a strongly basic anionic resin, for example one of those listed in Greenstein and Winit2,"Chemistry of the Amino Acids", John Wiley and Sons, Inc., New York and London 1961, Vol. 2, p 1456. Preferred anion exchange resins are cross-linked polystyrene resins substituted with strongly basic groups such as Amberlite*lRA - 400 or IRA - 410.

..
c~ -25 AHP-6~97 ~74~

It it is desired to obtain a pharmaceutically acceptable acid 1 addition salt ofthe nonapeptide of formula 1, the free base of the non-apeptide is conYer~ed to pharmaceutlcally accep~ab~a ~cld addltlon satts by reacting it with acids which are pharmaceutically acceptable.
Examples of such acids are inorganic acids, for example hydrochloric acid, sulfuric acid or phosphoric acid and organic acids, for example formtc acid, acetic acid, propionic acid,maleic acid or lactic acid.
If it is desired to obtain a salt of the nonapeptide of formula I which is sparingly soluble in water or in body fluids, the base or an acid addition salt tnereof as obtained by the process of this invention is treated in aqueous solution with a pharmaceutically acceptable sparingly water-soluble acid, for example tannic, alginic, or pamoic acid, preferably in the form of one of their salts, for example the alkali metal salts. The hormone precipitates as the salt with the respective sparingly water-soluble acid and is isolated, for example by filtration or centrifugation.
The above sequence of reactions constituting an embodiment of the process of this invention is shown in Fig. 2, using the con-ventional abbreviations for the various amino acids and protective groups.
The followirg E~amples illustrate this invertion.

AHP- 6fi97 S

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

_ Z
--_r ~ I ~

A ~

~1^ ~

e ~. j~
~ ~ ~ ~ / _. . ~

I_ N N N N N

_ . . _ __ O
. . 3 . C ~ 3 3 3 I

O
O
I

~7~7B~

EXAMPL~ I
Banzyloxycarbonyl-O-benzvl-tyrosvl-D-alan;ne Carboxvhydrazide-t-Butyl Ester (~-Tyr-D-Ala-NHNH-Boc) ~ Bzl To a cooled (ice bath), stirred solution of D-alanine carboxyhydrazide t-butyl ester (6 9, 29.6 mmoles) in dimethylformamide (50 ml) is added a solution of benzyloxycarbonyl--benzyl-tyrosine 2,4,5-trichlorophenyl ester (17 9, 28.3 mmoles) in dimethyl-formamide (50 ml). The mixture is kept in an ice bath fôr 2 days. The solvent is evaporated, the residue dissolved in e-~hyl acetate and precipitated with ether. The precipitate is crystallized from ethyl acetate giving the title compound: mp 154 - 156, [~]D5 ~9-9 (c - 1%,dimethylformamide).

-2a-S

Tyrosyl-D-alanine Carboxyhydrazide t-BUtYl Ester (I-l-Tyr-D-Ala-N~INH-Boc Benzyloxycarbonyl- ~benzyl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester ~described in Example 1, 12 9, 20.4 mmoles) is dissolved in methanol (250 ml~ and dimethylformamide (25 ml) and 5% palladium-charcoal ca~-alyst (2.5 9) is added unclsr nitrogcn. The hydrogcnation is carrisd out With th~ hydrog~nation vessel connected to a stirred solution of sodium hydroxide to absorb carbon dioxide. The mixture is hydrogenated overnight. The catalyst is removed by filtration through diatomaceous earth ~ Celite ~, the fiItrate evaporated under reduced pressure, and the oily residue triturated with ether. The solid is collected and dried under reduced pressure to give the title compound.

*Celite .

~37~

~XAMP~E 3 Benzyloxvcarbonyl-seryl-tyrosyl-n-alanjne Carboxyhydrazide t- Butyl Ester (Z-Ser-Tyr-D-Ala-NHNH-Boc To a cold (ice bath) solution of tyrosyl-D-alanine carboxy-hydrazide ~-butyl ester (described in Example 2, 20.4 mmoles in dimethyl-formamide (50 ml) is added with stirring a solution of benzyloxycarbonyl-serine pentachlorophenyl ester (11.6 g, 23.8 mmoles) in dimethylformamide (40 ml). The mixture is kept in an ice bath for 2 days. The solvent is evaporated, the residue dissolved in ethyl acetate and the solution washed with 10% citric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution. After drying over anhydrous magnesium sulfate the solvent is evaporated and the residue subjccted to column chromatography on silica gel using 1% methanol 1~ and 1% pyridine in ethyl acetate. Crystallization from ethyl acetate-methanol-benzene-hexane gives the title compound mp 178, [~]D5 -18.4 (c = ~, dimethylformamide1.

~0 -_ ., AHP-6~97 ~7~7~5 Seryi-tyrosyi-D-alanine Carboxyhydra~ide t-Butvl-Ester (ll) (H-Ser-Tyr-D-Ala-NHNH-Boc Benzylocycarbonyl-5eryl-tyrosyl-D-alarline carboxyhydra.zTde t-butyl ester (described in Example 3, 5 9, 8.52 mmoles) is dissolved in methanol (250 ml), 5% palladium-charcoal catalyst (600 mg) is added and the mixture is agitated under hydroyen in tne same manner as described in Example 2 for 3 hr. The reaction mixture is filtered through diatomaceous earth ("Celite")and the flltrate Is evaporated to dryness under reduced pressure to give the title compound;
amino acid analysis: Ser, 0.92; Tyr, 1.05; Ala, 1~00.

*Trade Mark 7~S

5-Oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine Carboxyhydrazide t-Butyl-Ester tlll) (H-Pyr-His-Trp-Ser-Tyr-D-Ala-NHNH-Boc) To a stirred solution of 5-oxoprolyl-histidyl-tryptophane hydrazide (3.71 9, 8.0 mmoles) in dimethyl sulfoxide (24 ml) is added dimethylformamide (50 ml) followed by a 2.04 N solution of hydrogen chloride in ethyl acetate (9.5 ml, 19.4 mmoles) at -10C. The mixture is cooled to -12C and t-butyl nitrite (1.2 ml, 10.4 mmoles) is added. The solution is stirred at -10C f~r 17 min, cooled to -15C and neutralized with N-ethyldiisopropylamine (3.32 ml, 19 mmoles). To the mixture is added dropwise a solution of seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester (described in Example 4, 3.5 9, 7.75 mmoles) in dimethyl-formamide (45 ml). The solution is stirred at -10 for I hr., at 0C
for I hr. and at room temperature overnight. The solvent is evaporated under reduced press~re, the oily residue dissolved in methanol (40 ml) and tne product precipitated with ether (1000 ml). The precipltate is cry-stallized from methanol giving the t7tle compound mp. 178C
(dec.), [~]D25 _9.4o (c = ~, dimethylformamide), amino acid analysis:
His 1.04, NH3 0.34, Ser 0.90, Glu 1.04, Ala 1.0, Tyr 1.02.

~7~5 ~XAMPLE 6 5-Oxoprolyl-histidyl-tryptophy-l-servl-tyrosyl-D-alanine HYdrazide Trif!uoroacetate (IV) rH-Pyr-His-Trp-Ser-Tyr-~-Ala-NllNH ~ ,CO~H (IV~
S-Oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester ~described in Example 5, 1.07 9, 1.14 mmole) is dissolved in cold (ice bath)trifluoroacetic acid (30 ml) with stirring, The solution is stirred at 0C for ~0 min and then at room tcmperature for 30 min. The solvent is evaporaled at reduced pressure, the residue is dTssolved in methanol and the product is precipitated with ether. The precipitate is collected by fiItration and dried under reduced pressure to yield the title compound; amino acid analysis: His, 0.9~;
Ser, 0.84; Glu, 1.05; Ala, 1.00; Tyr, 0.97; thin layer chromato9raphy on slllca gel from n-butanol-water-ethyl acetate-acetic acid (1:1:1:1) gives Rf 0.55; single spot with ~ and Cl-tolidine reagentsO The tTtle compound as the trtfluoroacetic acid salt is subjected to ion exchange chromatography on a strongly baslc anionic resin ("Amberlite*lRA-400") using methanol-water as the eluant to yield the tftle compound as the free base, amino acid analysTs: His, 1.01; Ser, 0.81; Glu, 1.03; Ala, 1.00; Tyr, 1.06.

* Trade Mark . . .. . - . , . . :

7~

Nitroarqinvl-proline methyl ester trifluoroacetate,(H-Arlq-Pro-OMe-CF3C02H) t-Butyloxycarbonyl-nitroarglnyl-proline methyl ester (10.7 9, 25 mmoles) is dissolved in trifluoroacetic acid (125 ml) and the solution is S left to stand at room temperature for 30 min. Trifluoroacetic acid is evaporated and the residue is dissolved in methanol (75 ml). The methanolic solution is added to ether (3000 ml) dropwise under stirring. The pre-cipitate is collected by fiitration and dried under reduced pressure to give the title compound.

. 34 47~S

Benzyloxvcarbon~l-leucyl-nitroarqinyl-proline Methyl Ester (;~-Leu-AIrq-Pro-CMe To a cooled (0~) stirred solution of nitroarginyl-proline methyl ester (described in Example 7, 2.3 9, 5 mmoles) and dry triethylamine ~0.8 ml) in dry dimethylformamide (10 ml) is added a cold solution of benzyloxycarbonyl-leucine 2,4,5-trichlorophenyl ester (2.3 9, 5 mmoles) in dry dimethylformamide (10 ml). The mixture is left to stand at 0C for 3 days. The solvent is evaporated under reduced pressure and th~ residue dissolved in ethyl acetate (150 ml).
The solution is washed with water (3 x 30 ml), IN hydrochloric acid ~2 x 30 ml), 5g sodium bicarbonate solution (2 x 30 ml) and s~turated sodium chloride solution (2 x 30 ml). After drying over anhydrous magnesium sulfate, the soivent is evaporated under reduced pressure, and the residue is subjected to chromatography on silica gel using 9~ methanol in ethyl acetate as the eluant. After evaporation of the eluate, the residue is taken Up in methanol and added to ether. The precipitate is collected by filtration and dried under reduced pressure toyieId the title compound, nmr (CDC13) ~ 0.90 (d J = 5Hz, 6H), 3.70 (s, 3H), 5.13 (s, 2H), 7.40 (s, 5H). ~-.

.. . .

- : ~

7~

Benzyloxycarbonyl-leucyl-nitroarqinyl-proline (Z-leu-AIrq-Pro-OH) To a solution of benzyloxycarbonyl-nitroarginyl-proline methyl ester ~described in Example 8, 2 9, 3.46 mmoles) in methoxyethanol (20 ml) is added under vigorous stirring IN sodium hydroxide solution ~ ml). After I hr more IN sodium hydroxide (3 ml) is added and the mixture is stirred overnight. The mixture is cooled in an ice bath and IN hydrochloric acid solution is added (7O5 ml). The solvent is evaporated under reduced pressure and the residue precipitated by addition of water. The precipitate is collected by fiItration and dried under reduced pressure to give the title compound, nmr (CDC13) A 0.95 ~d,J = 5Hz, 6H), 5.05 (s, 2H), 7.35 (s, 5H).

- ,, . : , ~7~7~35 Benzyloxycarbonvl-leucyl-nitroarqinyl-proline Ethylamide (~-Leu-A~q-Pro-NHEt) To a cooled (ice bath) stirred soiution of benzyloxycarbonyl~
leucyl-nitroarginyl-proline (described in Example 9, 7.0 9, 12.4 mmoles) and l-hydroxybenzotriazole (3.3 9, 24.5 mmoles) in dry, distilled dimethylformamide is added dicyclohexylcarbodiimide (3.07 9, 14.9 mmoles).
The solution is stirred for I hr a~ 0C and for I hr at room temperature.

The mix1ure is cooled to 0~ and ethylamine ~I.l 9, 1.6 ml, 24.5 mmoles) is added. The mixture is stirred overnight at room temperature. The pr~cipitate is removed by fiItration, the fiItrate evaporated and the r~sidue dissolved in ethyl acetate. The solution is washed with 10%
sodium carbonate solution, water, IN aqueous hydrochloric acid and saturated sodium chloride solution. The solution is dried over magnesium sulfate and the solvent evaporated. The residue is subjected to chromatography on silica gel using 9% metharol in ethyl acetate.
After evaporation of tne eluate, the residue is taken up in methanol and added to ether. The residue is collected by filtration and dried under reduced pressure to yield the title compound: [~]2D5 = -33.8~ (c = I~, dimethylformamide).

AHP-6~97 ~t7~Lt7~

Leucyi-arqinyl-pr-o-!-ine Ethylamide Diacetate (V) [H-Leu-Arq-Pro-NHEt~2 CH ~ ~H (V)l Benzyloxycarbonyl-leucyl-nitroarginyl-proline ethylamide S (described in Example 10, 2.76 9, ~.67 mmoles~ is dissolved in methanol (69 ml) and glacial acetic acid (69 ml) followed by hydrogenation over 5 palladium-charcoal catalyst (276 mg) in the same manner as de~cribed in Example 2. After 24 hr., the ca~alyst is removed by filtration through diatomaceous earth ("Celite") and the filtrate evaporated under reduced pressure to give the title compound.

Trade Mark ,~ .

'1~7~7B5 5-Oxoprolyl-histidyl-tryptophyl-seryl-tvrosvl-D-alanYl-leucYl-ar-linYl-proline EthYlamide (I) [H-PYr-His-Trp-Ser-Tyr-D-Ala-Leu-Arq-Pro-NHEt (l) 5-Oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine hydrazide trifluoroacetate (IV) (descrlbed in Example 6, 4.3 9, 4.25 mmolesl is dissolved in dimethyl sulfoxide t21 ml), dimethylformamide (21 ml) is added and the mixture is cooled to -15C. To this mixture is added a solutio of hydrogen chloride in ethyl acetate (2.69 N, 7.9 ml, 21.2 me~). The mixture is cooled to -20oc and t-butylnit~ite (527 mg, 0.6 ml), S.l mmoles) is added. The mixture is stirred at -15C for 20 min, cooled to -20C and N-ethyldiisopropylamine (5 ml, pH 8) is added. Leucyl-arginyl-proline ethylamide diacetate (V . described in Example 11, 4.67 mmoles) is dissolved in dimethylformamide (15 ml) and N-ethyldiisopropylamine (2.8 ml pH 8), the solution is subjected to reduced pressure in a rotary evaporator for 30 min and added to the solution of the azide prepared above at -20Co The mixture is kept at -15C for I hr., at 0C for I hr and at room temperature overnightO The solvent is evaporated, ~he residue dissolved in methanol and the crude product precipitated from 4000 ml ether. The precipitate is separated by filtration and dried under reduced pressure. The precipitate is purified by gel fiItration chromatography on a chemically modified cross-linked dextran ("Sephadex*
LH-20") using methanol as the eluant. The combtned fracttcnsare concentrated to dryness under reduced pressure to yield the title compound as the hydrochloric acid addition salt, ~max 289 (~ 4530)~ 279 (~ 5920)J
220 nm (~ 40,500), amino acid analysis His, 1.02; Arg, 1.15; Ser, 0~89; Glu, 1.06; Pro, 1.24; Ala, 1.00; Leu, 1.01; Tyr, 1.03.
Alternatively, the precipitate is purified by ion exchange chromatography on carboxymethylcellulose ("Whatman~-CM-23") using * Trade Mark A~IP-6~97 ~7~7~3~

aqueous ammonium acetate as eluant. After removal of the solvent trom the eiuates the ti-tle compound is obtained as the acetic acid addition sall, amino acid analysis: Ilis, 1.05; Arg, 1.10; Ser, O.B5; GIU~ 1.04;
Pro, 1.19; Ala, 1.00; Leu, 0.95; Tyr, 1.01.
All-ernatively, an acid addition salt is subjected to ion exchange chromatography on a strongly basic anionic resin (''AmberliteX
I RA - 400~) to yield the title compound as the free base, arnino a^id analys;s: His, 1.03; Arg, 1.06; Ser, 0.91; Glu, 1.01; Pro, 1.22;
Ala, 1.00; Leu, 1.01; Tyr, 0.93.

Tracle Mark .

Claims (27)

We claim:

1. A process for preparing a nonapeptide of the formula I
H-Pyr-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt (I) in the form of an acid addition salt thereof, which comprises treating the hexapeptide 5-oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine hydrazide trifluoroacetate in an anhydrous inert organic solvent with a reagent which furnishes nitrous acid in situ in the presence of a strong acid at a temperature within the range of from about -30°C to about 0°C making the mixture alkaline by addition of a strong organic base, adding a solution of leucyl-arginyl-proline ethylamide diacetate in an anhydrous inert organic solvent, agitating the mixture at a temperature within the range from about -30°C to about 0°C, and isolating the nonapeptide of formula I as an acid addition salt thereof.

2. A process as claimed in Claim I in which the salt of the nonapeptide of formula I is treated with an ion exchange resin in the form of its salt with a pharmaceutically acceptable acid and the corresponding salt of the nonapeptide is isolated.

3. A process as claimed in Claim I in which the salt of the nonapeptide of formula I is treated with a strongly basic anion exchange resin and the nonapeptide is isolated as the free base.

4. A process as claimed in Claim I in which the salt of the nonapeptide of formula I is treated with tannic, alginic or pamoic acid and the corresponding salt of the nonapeptide is isolated.

5. A process as claimed in Claim I in which the anhydrous inert solvent is dimethylformamide or a mixture of dimethylformamide and dimethyl sulfoxide.

6. A process as claimed in Claim I in which the nitrous acid is furnished by t-butyl nitrite and the strong acid is hydrogen chloride.

7. A process as claimed in Claim I in which the strong organic base is N-ethyl-diisopropylamine.

8. A process as claimed in Claim I in which the hexa-peptide5-oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-allanine hydrazide trifluoroacetate is prepared by treating a solution of 5-oxoprolyl-histidyl-tryptophane hydrazide in an anhydrous inert organic solvent with a reagent which furnishes nitrous acid in situ in the presence of a strong acid at a temperature within the range from about -30°C to about 0°C, making the mixture alkaline by addition of a strong organic base, adding a solution of seryl-tyrosyl-D-alanine carboxyhydrazide -t-butyl ester in an anhydrous inert organic solvent, agitating the mixture at a temperature within the range from about -30°C to about 0°C, to obtain 5-oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester; treating said last-named compound with trifluoro-acetic acid, and isolating the hexapeptide named above.

9. A process as claimed in Claim 8 in which the anhydrous inert organic solvent is dimethylformamide or a mixture of dimethyl-formamide and dimethyl sulfoxide.

10. A process as claimed in Claim 8 in which the nitrous acid is furnished by t-butyl nitrite and the strong acid is hydrogen chloride.

11. A process as claimed in Claim 8 in which the strong base is N-ethyldiisopropylamine.

12. A process as claimed in Claim 8 in which the seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester is prepared by treating D-alanine carboxyhydrazide t-butyl ester in solution in an inert organic solvent with an activated ester of benzyloxy-carbonyl-O-benzyl-tyrosine, keeping the mixture at about 0°C for several days and isolating benzyloxycarbonyl-O-benzyl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester; treating said last-named compound with hydrogen and a noble metal catalyst and isolating tyrosyl-D-alanine carboxyhydrazide t-butyl ester; treating said last-named compound in solution in an inert organic solvent with an activated ester of benzyloxycarbonyl-serine, keeping the mixture at about 0°C for several days and isolating benzyloxycarbonyl-seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester; treating said last-named compound with hydrogen and a noble metal catalyst, and isolating seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester.

13. A process as claimed in Claim 12 in which the inert organic solvent is dimethylformamide.

14. A process as claimed in Claim 12 in which the noble metal catalyst is palladium.

15. A process as claimed in Claim 12 in which the activated ester of benzyloxycarbonyl-O-benzyl-tyrosine is the 2,4,5-trichloro-phenyl ester.

16. A process as claimed in Claim 12 in which the activated ester of benzyloxycarbonyl-serine is the pentachlorophenyl ester.

17. A process as claimed in Claim I in which the leucyl-arginyl-proline ethylamide diacetate is prepared by treating benzyloxycarbonyl-leucyl-nitroarginyl-proline ethylamide with hydrogen and a noble metal catalyst in the presence of acetic acid and isolating the tripeptide named above.

18. A process as claimed in Claim 17 in which the noble metal catalyst is palladium.

19. A process for preparing a nonapeptide of formula H-Pyr-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHE+ which comprises:
reacting S-oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D alanine hydrazide wtth a reagent whlch furnishes nitrous acid in situ in the presence of a strong acid at a temperature within the range of from -30°C to 20°C to obtain the corresponding azide, rendering the resulting mixture containing said azide alkaline by the addition of a strong base, and reacting said azide with leucyl-arginyi-proline ethylamide at a temparature within the range of -30° to 30°C to obtain the nonapeptide.

20. A process for preparing a peptide of formula 5-oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine hydrazide trifluoroacetate, leucyl-arginyl-proline ethylamida diacetate or seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester, which comprises selecting a process from the group consisting of:
(a) when the peptide of formula 5-oxoprolyl-histidyl tryptophyl-seryl-tyrosyl-D alanine hydrazide trifluoroacetate is required, treating a solution of 5-oxoprolyl-histidyl-tryptophane hydrazide in an anhydrous inert organic solvent with a reagent which furnishes nitrous acid in situ in the presence of a strong acid at a temperature within the range from about -30°C to about 0°C, making the mixture alkaline by addition of a strong organic base, adding a sol.ution of seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester in an anhydrous inert organic solvent, agitating the mixture at a temperature within the range from about -30°C

to about 0°C, to obtain 5-oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester and treating said last-named compound with trifluoroacetic acid;
(b) when the peptide of formula Leucyl-arginyl-proline ethyl-amide diacetate is required, treating benzyloxycarbonyl-leucyl-nitro-arginyl-proline ethylamide with hydrogen and a noble metal catalyst in the presence of acetic acid; and (c) when the peptide of formula seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester is required, treating D-alanine carboxy-hydrazide t-butyl ester in solution in an inert organic solvent with an activated ester of benzyloxycarbonyl-O-benzyl tyrosine, keeping the mixture at about 0°C for several days and isolating benzyloxycarbonyl-O-benzyl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester; treating said last-named compound with hydrogen and a noble metal catalyst and isolating tyrosyl-D-alanine carboxyhydrazide t-butyl ester; treating said last-named compound in solution in an inert organic solvent with an activated ester of benzyloxycarbonyl-serine, keeptng the mixture at about 0°C for several days and isolating benzyloxycarbonyl-seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester; and treating said last-named compound with hydrogen and a noble metal catalyst.

21. A process as claimed in Claim 20 in which the peptide 5-oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine hydrazide trifluoroacetate is prepared by treating a solution of 5-oxoprolyl-histidyl-tryptophane hydrazicle in an anhydrous inert organic solvent with a reagent which furnishes nitrous acid in situ in the presence of a strong acid at a temperature within the range from about -30°C
to about 0°C, making the mixlure alkaline by addition of a strong organic base, adding a solution of seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester in an anhydrous inert organic solvent, agitating the mixture at a temperature within the range from about -30°C
to about 0°C, to obtain 5-oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester and treating said last-named compound with trifluoroacetic acid.

22. A process as claimed in Claim 20 in which the peptide leucyl-arginyl-proline ethylamide diacetate is prepared by treating benzyloxycarbonyl-leucyl-nitro-arginyl-proline ethylamide with hydrogen and a noble metal catalyst in the presence of acetic acid.

23. A process as claimed in Claim 20 in which the peptide seryl-tyrosyl-D-alanine carboxyhydrazide t-bulyl ester is prepared by treating D-alanine carboxyhydrazide t-butyl ester in solution in an inert organic solvent with an activated ester of benzyloxycarbonyl-O-benzyl-lyrosine, keeping the mixture at about 0°C for several days and isolating benzyloxycarbonyl-O-benzyl-tyrosyl-D-alanine carboxyhydra-zide t-butyl ester; treating said last-named compound with hydrogen and a noble metal catalyst and isolating tyrosyl-D-alanine carboxy-hydrazide t-butyl ester; treating said last-named compound in solution in an inert organic solvent with an activated ester of benzyloxy-carbonyl-serine, keeping the mixture at about 0°C for several days and isolating benzyloxycarbonyl-seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester; and treating said last-named compound with hydrogen and a noble metal catalyst.

24. A peptide of formula 5-oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine hydrazide trifluoroacetate, leucyl-arginyl-proline ethylamide diacetate or seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester, when prepared by the process of Claim 20 or an obvious chemical equivalent thereof.

25. 5-Oxoprolyl-histidyl-tryptophyl-seryl-tyrosyl-D-alanine hydrazide trifluoroacetate when prepared by the process of Claim 21, or an obvious chemical equivalent thereof.

26. Leucyl-arginyl-proline ethylamide diacetate when pre-pared by the process of Claim 22, or an obvious chemical equivalent thereof.

27. Seryl-tyrosyl-D-alanine carboxyhydrazide t-butyl ester when prepared by the process of Claim 23, or an obvious chemical equiv-alent thereof.