GB1590645A - Process for preparing hentriacontapeptide having activity in reducing blood calcium levels - Google Patents

Abstract

The novel polypeptide of the formula <IMAGE> and the pharmaceutically acceptable acid-addition salts thereof reduce the levels of serum calcium. The compound is built up from the amino acids and/or peptides shown in the formula in the amino acid sequence shown in the formula, and the peptide unit, which contains the <IMAGE> group, in which R is a reactive ester, is subjected to a ring-closure reaction at any desired step of the reaction.

Description

(54) PROCESS FOR PREPARING HENTRIACONTAPEPTIDE HAVING ACTIVITY IN REDUCING BLOOD CALCIUM LEVELS (71) We, TOYO JOZO KABUSHIKI KAISHA, a Japanese Body Corporate of 632-1 Mifuku, Ohito-cho, Tagata-gun, Shizuoka-ken, Japan, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention relates to a process for preparing a polypeptide of the formula:

and pharmaceutically acceptable acid addition salts and complexes thereof. This polypeptide is active in reducing blood calcium levels.

The abbreviations employed throughout this specification are as follows: BOC: t-butoxycarbonyl Cbz: benzyloxycarbonyl, Bzl: benzyl, Bzl(CI2): 2,6-dichlorobenzyl, OEt: ethyl ester, OBzl: benzyl ester, OSU: N-hydroxysuccinylimide ester, Ser: L-serine, Leu: L-leucine, Val: L-valine Arg: L-arginine, Ala: L-alanine, AOC: t-amyloxycarbonyl, ClCbz: o-chlorobenzyloxycarbonyl, Tos: tosyl, OBu: t-butyl ester, ONP: p-nitrophenyl ester Asn: L-asparagine, Thr: L-threonine, Pro: L-proline, Asp: L-aspartic acid, Gly: glycine, Gln: L-glutamine, His: L-histidine, Lys: L-lysine, Glu: L-glutamic acid, Tyr: L-tyrosine, Phe: L-phenylalanine, TFA: trifluoroacetic acid, CHA: cyclohexylamine, THF: tetrahydrofuran, AcOEt: ethyl acetate, WSC: N-ethyl-N'-dimethylaminopropyl-carbodiim HOSU: N-hydroxysuccinylimide, MeOH: methanol, AcOH: acetic acid, HMPA: hexamethyl phosphoric triamide, Met: L-methionine, Ile: L-Isoleucine, TosOH: p-toluenesulfonic acid DCHA: dicyclohexylamine, DMF: dimethylformamide, DCC: Dicyclohexylcarbodiimide, HOBT: I-hydroxybenzotriazole, EtOH: ethanol, BuOH: n-butanol.

Calcitonin is a well known polypeptide which has the ability to reduce blood calcium levels. It may be isolated from the thyroid gland of mammals or from the ultimobranchial gland in birds and fish. The amino acid sequence of the calcitonin depends on the species from which it has been obtained, and synthetic calcitonins having the same chemical structure as those of natural origin have recently been synthesised.

Calcitonins of natural origin, such as eel-, salmon- or human-calcitonin, are polypeptides consisting of 32 amino acids, in which the first and 7th amino acids are L-cysteine, whose mercapto groups are bonded to each other to form a disulfide bridge, and the terminal carboxy group is prolinamide. These calcitonins have differing amino acid sequences and differing activities.

The amino acid sequence of human calcitonin is as follows:

The disulfide bond of this calcitonin is chemically very unstable, and is thus easily destroyed or cleaved, this latter reaction causing the polymerisation of calcitonin molecules. The chemical synthesis of human calcitonin is consequently very difficult. Also the stability of human calcitonin for pharmaceutical purposes is very low.

The polypeptide produced by the process of the present invention has neither the first nor 7th amino acid, L-cysteine, which amino acids are replaced by aaminosuberic acid of the formula:

The carboxyl group at o-position of a-aminosuberic acid is bonded with glycine (the second amino acid in the sequence for human calcitonin) to form a ring structure. Thus it will be seen that the polypeptide of formula (I) has, including a- aminosuberic acid, 31 amino acid residues. Viewed from the N-terminal group, glycine, which as noted above is the second amino acid in the sequence for human calcitonin, is now the first amino acid for the polypeptide of formula (I). The | --NHCHCO-portion of a-aminosuberic acid constitutes the 6th amino acid residue of the polypeptide of formula (I).

Polypeptide (I) has ten times higher biological activity as compared with natural human calcitonin, and is chemically very stable. Polypeptide (I) has also the same amino acid sequence as human calcitonin from 11th to 32nd amino acid residue from the N-terminal. This sequence is an antigenic determinant for reaction with antiserum of calcitonin. [Europ. J. Clin. Invest., 4, 213-222 (1974)].

The polypeptide (I) is therefore immunologically identical to natural human calcitonin, and hence is pharmaceutically very safe, with no cross-immunity with pig or salmon calcitonin.

According to the present invention there is provided a process for the preparation of a polypeptide of the formula (I):

the symbols being as hereinbefore defined, or a pharmaceutically acceptable acid addition salt or complex thereof, which process comprises condensing in known manner acids or peptides or combinations thereof in an appropriate number of steps in the arrangement of the amino acids in the compound of formula (I), in so doing cyclising a peptide comprising a group of formula:

wherein R is a reactive ester residue, to form a peptide comprising a group of formula:

at a suitable stage of the process, and optionally converting the resulting compound of formula (I) into a pharmaceutically acceptable acid addition salt or complex thereof.

Preferably peptides containing 2, 3 or 4 amino acid residues are condensed, and said peptide comprising the group of formula:

in which R is as defined above is cyclised after the 1st to 6th amino acids of the polypeptide of formula (I) have been condensed together, but before the final condensation reaction producing the polypeptide of formula (I) is effected.

Generally, each amino acid or peptide involved in a particular condensation reaction will have its cr-amino group or terminal carboxyl group, which it is intended should not participate in that reaction, protected. The protecting groups are subsequently removed as desired. The use of protecting groups is dependent upon, for example, the condensation technique that is employed. Also, one our more of the y-carboxyl group of L-glutamic acid, the hydroxyl group of L-serine Lthreonine and L-tyrosine, the E-amino group of L-lysine and the imine group of Lhistidine may be protected during the production of polypeptide of formula (I), and the protecting groups subsequently removed.

The protecting groups employed in the synthesis of the starting or intermediate combinations of amino acids are conventional protecting groups used for peptide synthesis, and should be easily removable by hydrolysis, acid decomposition, reduction aminolysis or hydrazinolysis.

For example, the amino group may be protected in conventional manner by an acyl group such as formyl, trifluoroacetyl, phthaloyl, benzensulfonyl, ptoluenesulfonyl, o-nitrophenylsulfenyl or 2,4-dinitrophenylsulfenyl group; an aralkyl group such as benzyl, diphenylmethyl or triphenylmethyl (these groups may optionally be substituted with a C1-C4 alkoxy group such as o-methoxy or pmethoxy); a benzyloxy-carbonyl group such as benzyloxycarbonyl, o- bromobenzyloxy-carbonyl, p-bromobenzyloxycarbonyl, o- chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, pmethoxybenzyloxycarbonyl, p-phenylazo-benzyloxycarbonyl or methoxyphenylazo)-benzyloxycarbonyl; an aliphatic oxycarbonyl group such as cyclopentyloxycarbonyl, trichloroethyloxycarbonyl, t-amyloxycarbonyl, tbutoxycarbonyl or diisopropylmethoxycarbonyl, or an aralkyloxycarbonyl group such as 2-phenylisopropoxycarbonyl, 2-torylisopropoxycarbonyl or 2-p-diphenylisopropoxycarbonyl. The amino groups can also be protected by the formation of an enamine when reacted with a 1,3-diketone, such as benzoyl acetone, acetylacetone or dimedone.

The carboxyl group of starting or intermediate combinations of amino acids may be protected by amide formation, hydrazide formation or esterification. The amide group is substituted at its N atom by, for example, a 3,4-dimethoxybenzyl or bis-(p-methoxyphenyl)-methyl group. The hydrazide group is substituted at its terminal N-atom by, for example, a benzyloxycarbonyl, 2,2,2trichloroethyloxycarbonyl, trifluoroacetyl, t-butoxycarbonyl, trityl or 2-p-diphenylisopropoxycarbonyl group. The ester group is formed by reaction with an alkanol such as methanol, ethanol, t-butanol or cyanomethylalcohol; an aralkanol such as benzyl alcohol, p-bromobenzyl alcohol, p-chlorobenzyl alcohol, p-methoxybenzyl alcohol, p-nitrobenzyl alcohol, 2,4,6-trimethylbenzyl alcohol, benzhydryl alcohol, benzoylmethyl alcohol, p-bromobenzoylmethyl alcohol or p-chlorobenzoylmethyl alcohol; a phenol such as 2,4,6-trichlorophenol, 2,4,5-trichlorophenol, pentachlorophenol, p-nitrophenol, 2,4-dinitrophenol, p-cyanophenol or pmethanesulfonylphenol; or a thiophenol such as thiophenol, thiocresol or pnitrothiophenol.

The hydroxy group in L-serine, L-threonine or L-tyrosine may optionally be protected by esterification or etherification. When protected by esterification, the hydrogen atom of the hydroxy group is replaced by, for example, a C1-C4 alkanoyl group such as an acetyl group; an aroyl group such as a benzoyl group; or a group containing a carbonyl group such as benzyloxycarbonyl or ethyloxycarbonyl. When protected by etherification the hydrogen atom of the hydroxy group is replaced by, for example, a benzyl, tetrahydropyranyl or t-butyl group. Protection of the hydroxy group may also be effected by a 2,2,2-trifluoro - I - t butyloxycarbonyl - aminoethyl or 2,2,2 - trifluoro - 1 benzyloxycarbonylaminoethyl group. However, it is not always necessary to protect the hydroxy groups of L-serine, L-threonine or L-tyrosine.

The imino group in L-histidine may be protected by a benzyl, trityl, benzyloxycarbonyl, tosyl, adamantyloxycarbonyl, 2,2,2 - trifluoro - 1 - t - butyloxycarbonylaminoethyl or 2,2,2 - trifluoro - 1 benzyloxycarbonylaminoethyl group, although the imino group does not always need to be so protected.

The peptides of the starting materials or intermediates are synthesized by condensation of amino acids, or peptides which preferably have from two to four amino acids, in the order of the amino acid sequence of formula (I). For example, an amino acid or peptide having a protected a-amino group and activated terminal carboxyl group is reacted with an amino acid or peptide having free a-amino group and protected terminal carboxyl group. On the other hand, an amino acid or peptide having activated a-amino group and protected terminal carboxyl group can be reacted with amino acid or peptide having free terminal carboxyl group and protected a-amino group.

The carboxyl group may be activated in a conventional manner by, for example, an acid azide, acid anhydride, carbodiimide or an acid imidazolide (in the latter case forming an N,N'-carbonyldiimidazole or an isoxazolium salt such as Woodward reagent). The carboxyl group may also be activated by the formation of an active ester, such as by converting it to a cyanomethyl ester, thiophenylester, pnitrophenyl ester, p-nitrothiophenyl ester, p-methanesulfonylphenyl ester, 2,4dinitrophenyl ester, 2,4, 5-trichlorophenyl ester, 2,4,6-trichlorophenyl ester, pentachlorophenyl ester, N-hydroxysuccinimide ester, N-hydroxyphthalimindo ester, 8-hydroxyquinoline ester or N-hydroxypiperidine ester group.

Preferred condensation reactions are the Wunsch method. (Z. Naturforsch, 21b, 426 (1966)), azide, active ester or Geiger methods (W. Knig and R. Geiger, "Peptide", Ed. E. Scoffone, page 17, North-Holland Publishing Co., Amsterdam (1969)). In the condensation reaction, racemization should carefully be avoided.

A construction unit for the polypeptide of formula (I) which includes a peptide sequence, formed using techniques indicated above, of the formula:

wherein R is as defined above, is provided for the ring formation reaction. This reaction is performed by a condensation reaction employing an activated a,- carboxyl group of a-aminosuberic acid and a free amino group in the N-terminal amino acid (glycine). In this condensation reaction the hydroxyl groups of serine and threonine are preferably protected.

In a preferred process of the present invention the N-terminal peptide fragment consisting of the Ist to the nth amino acid residue, which has already undergone cyclisation and in which the serine and threonine hydroxy groups are optionally protected, is condensed with the residual peptide (C-terminal fragment) of the total sequence of the (n+l)th to 31st amino acid residue, wherein n is 6,7,8 or 9. To promote the reactivity of both fragments and to prevent racemisation, glycine (the 9th amino acid residue of the polypeptide of the invention) is preferably used as the C-terminal amino acid of the N-terminal polypeptide fragment. Consequently, it is particularly preferred for a peptide consisting of 19th amino acid sequence (N-terminal fragment) to be condensed with a peptide consisting of 10-31st amino acid sequence (C-terminal fragment).

Such a condensation reaction may be effected by starting from the peptide having free terminal carboxyl group (the Wunsch method) or by active ester methods. It can also preferably be effected by the azide method starting from the peptide having an azide or hydrazide terminal or by the mixed anhydride method.

The synthesis of an N-terminal fragment, such as a nonapeptide consisting of the Ist to 9th amino acid sequence, will now be explained in detail below by way of example. However, a hexapeptide 1-6, heptapeptide 1-7, or octapeptide 1-8 may also be produced by substantially the same process. Generally, the N-terminal peptide fragment may be produced by condensing amino acids and/or peptides consisting of from 2 to 4 amino acid residues in the order of the amino acid sequence from the C-terminal amino acid of said fragment and then cyclising said fragment. With regard to the nonapeptide fragment, individual amino acids, such as glycine, leucine, a-aminosuberic acid, threonine, methionine, serine or asparagine, are preferably condensed by the active ester method. Lower peptides containing from 2 to 6 amino acid residues, such as the tripeptide of the 2nd to the 4th amino acid residues are preferably condensed by the Geiger method or by the Wiinsch method.

When effecting the condensation reaction by the azide, active ester or acid anhydride method, it is not necessary to protect the terminal carboxyl group of the nonapeptide. The carboxyl group may, however, be protected by esterification by alcohols such as methanol or benzyl alcohol. The ester group thus formed, such as methyl ester, may be removed by dilute sodium hydroxide solution or by conversion to hydrazide, and a benzyl ester group can be removed by catalytic hydrogenation. The amino group of any intermediate may be protected by a conventional protecting group, such as a benzyloxycarbonyl, trityl, tbutoxycarbonyl or 2-p-diphenyl-isopropoxycarbonyl group. As noted above, the carboxyl group may be protected, if required, by conventional esterification. The hydroxyl group is serine and threonine may be protected, if necessary, by esterification using t-butanol or benzyl alcohol.

Protecting groups may be removed, or converted into reactive hydrazide derivatives, as follows: the benzyloxycarbonyl, p-nitrobenzyl ester and benzyl ester groups are split off by catalytic hydrogenation in the presence of palladium carbon; the N-trityl group is split off by aqueous acetic acid; the t-butoxycarbonyl group may be decomposed and thus removed by the action of trifluoroacetic acid; the o-nitrophenylsulfenyl group is split off by hydrogen chloride, hydrogen cyanide or sulfurous acid in organic solvent; the diphenylisopropoxycarbonyl group is split off by a mixture of acetic acid-formic acid-water (7:1:2 by volume), methyl ester, ethyl ester or pnitrobenzyl ester groups are changed to hydrazide by using hydrazine hydrate; the methyl ester group may be split off by the action off dilute sodium hydroxide solution, and the t-butyl ester group is split off by trifluoroacetic acid.

The C-terminal polypeptide fragment consisting of the amino acid sequence from the 7th, 8th, 9th, or 10th to the 31st residue, which is subsequently condensed with the N-terminal peptide fragment described above, is preferably synthesized by connecting its C-terminal amino acid (amino acid No. 31) or C-terminal fragment, such as a peptide of amino acid sequence 30-31st, 28-31st, 25-31st, 24-31st or 23 31 sot, to individual amino acids and/or peptides consisting of from two to four amino acid residues in the order of the amino acid sequence from the C-terminal end of the polypeptide of formula (I).

For example, the C-terminal fragment of the 10th-31st amino acid residues may be produced by condensation of amino acids and/or lower peptides containing from 2 to 6 amino acid residues in the order of amino acid sequence from the Cterminal of the polypeptide of formula (I), for example, by condensing dipeptide 30-31st, 28-29th, tripeptide 25-27th, dipeptide 23-24th, tripeptide 21-23rd, tetrapeptide 16-19th and hexapeptide 10-15th in the order listed. Preferred protective groups are: a-amino group by t-butoxycarbonyl group; side chain carboxyl group of L-glutamic acid by benzyl ester group; E-amino group of L-lysine by o-chlorobenzyloxycarbonyl group; hydroxyl group of L-serine and L-threonine by benzyl group; hydroxy group of L-tyrosine by 2,6-dichlorobenzyl group and imino group of L-histidine by tosyl group.

The protecting group of the N-terminal a-amino group of the C-terminal fragment consisting of the 7-10th to 31st amino acid residues, for example docosapeptide amide of 10-31st sequence, is removed by any suitable method. For example, a trityl group may be split off by aqueous acetic acid; and diphenyl isopropoxycarbonyl, benzyloxycarbonyl and t-butoxycarbonyl groups may be removed by a mixture of glacial acetic acid, formic acid and water; hydrogenation; and trifluoroacetic acid, respectively.

Thus a hentriaconta peptideamide having protected a-amino and E-amino groups, optionally with protected side chain carboxyl and/or hydroxyl groups, can be obtained. These protective groups are split off by the processes hereinbefore described, preferably by acid decomposition such as by hydrogen fluoride, and finally the polypeptide (I) can be obtained.

In the synthesis of the polypeptide of formula (I) by the Merrifield solid phase peptide synthetic method, the hydroxy group in L-serine and L-threonine may be protected by, for example, benzyl, and the hydroxy group in L-tyrosine protected by 2,6-dichlorobenzyl group; the imino group in L-histidine may be protected, for example, by l-benzyloxycarbonylamino - 2,2,2 - trifluoroethyl group; and the ycarboxyl group in L-glutamic acid may be protected, for example, by a benzyl ester group. The protective group for the a-amino group is, for example, a tbutyloxycarbonyl, o-chlorobenzyloxycarbonyl or o-bromobenzyloxycarbonyl group.

The protected peptide is removed from the carrier resin and the protective group is removed by anhydrous hydrogen fluoride.

One-step removal of all protecting groups in the resulting polypeptide of formula (I) by hydrolysis using trifluoroacetic acid can be achieved when tbutoxycarbonyl group is used for amino group protection; t-butyl ester for the protection of the y-carboxyl group of L-glutamic acid, t-butyl ether for hydroxyl group protection in L-serine, L-threonine and L-tyrosine; and 2,2,2 - trifluoro 1 - t - butoxycarbonylaminoethyl group for imino group protection in L-histidine.

The polypeptide of formula (I) may be obtained in the form of the base or a salt thereof. The base may conventionally be obtained from its salt. The base may be changed to its pharmacologically acceptable acid addition salt by reaction with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid, or an organic acid such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, citric acid, tartaric acid, benzoic acid, salicyclic acid, (C1-C4 alkane)sulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid.

The polypeptide of formula (I) can be converted into a complex thereof by addition of various inorganic or organic substances. Such a complex may be produced by adding an inorganic or organic substance to the polypeptide, such that the resulting complex has a long term activity when administered to a patient.

Examples of such complex-forming substances are inorganic compounds derived from metals such as calcium, magnesium or zinc, especially phosphate, pyrophosphate or polyphosphate of the said metals, and organic substances such as non-antigenic gelatine, carboxymethylcellulose, sulfonic acid ester or phosphoric acid ester of alginic acid, dextran, polyalcohol, polyglutamic acid and protamine.

The invention also includes within its scope a pharmaceutical preparation comprising, as active ingredient, a polypeptide or a pharmaceutically acceptable acid addition salt or complex thereof produced by the process of the present invention, together with a pharmaceutically acceptable carrier or diluent.

The following Examples illustrate the present invention.

The assay method for blood calcium level reducing activity, and conditions for amino acid analysis are as described immediately below. The carrier and developing system for thin layer chromatography are as described in Example 1.

Assay Method A sample is diluted adequately with 0.1 N sodium acetate--0.1 /,, w/v albumin solution. Male rats are subjected individually to intravenous injection with 0.2 ml of the respective diluted solutions. After one hour, all the rats are sacrificed to obtain their blood. The serum calcium value of each blood sample is determined by atomic absorption spectrophotomery.

Standard salmon calcitonin, which is distributed from National Institute for Biological Standard and Control, is used for standard curve. From the potency of the corresponding Research Standard, the potency of the calcitonin in the sample is determined.

Amino Acid Analysis A sample is hydrolyzed with 6 N HCI (with addition of a few drops of anisole) at 110 C, for 45 hours, and evaporated to dryness under reduced pressure, then is subjected to amino acid analysis.

EXAMPLE 1 Production of:

2.0 g (0.6 m mole) of BOC-Thr(Bzl)-Tyr[Bzl(Cl2)] -Thr(Bzl)-Gln-Asp(OBzl)-Phe-As-Lys(ClCbz)- Phe-His-Thr(Bzl)-Phe-Pro-Gln-Thr(Bzl)-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 was dissolved in 10 ml of TFA at -50C. After stirring for 30 minutes at room temperature, this solution was concentrated in vacuo and diethyl ether was added thereto to form a precipitate. The precipitate was dried over sodium hydroxide.

The dried precipitate was dissolved in 10 ml of DMF and adjusted to pH 9 with addition of triethylamine under cooling. To that solution was added water to form a precipitate. The precipitate was dried over P205 to obtain the corresponding de BOC compound as a base.

0.8 g (0.71 m mole) of

was dissolved in dissolved in 5 ml of DMF: N-methylpyrrolidone (1:1 v/v) HOSU (122 mg) was added thereto and further DCC (146 mg) dissolved in DMF (3 ml) was added under cooling. This solution was stirred overnight. After reaction the resulting precipitate was removed, and to the thus obtained solution was added the de-BOC base obtained hereinabove. HOBT (100 mg) was added. This solution was stirred for 5 days at 300 C. After reaction, water was added thereto. The thusformed precipitate was collected by filtration, washed with water, then dried to obtain 2.6 g of

as a crude product.

1.0 g of this crude material was treated with hydrogen fluoride (25 ml), methionine (0.1 g) and anisole (2 ml) at -50C for 60 minutes with cooling in dry icemethanol under reduced pressure in the presence of dimethylthioether. After distillation off of the hydrogen fluoride, the residue was washed with diethyl ether and the precipitate collected by decantation (repeated three times). The precipitate was dissolved in a mixed solution of acetic acid (30 ml) and water (10 ml). This solution was passed through a column of "Dowex" lx2 (acetate form, 2.5x8 cm, "Dowex" is a Registered Trademark), washed with water (200 ml) and the eluate was again passed through a column of HP-20 (2.5x7 cm). 80 /" v/v ethanol solution was passed through the column and after distilling the eluate to remove ethanol, the solution was lyophilised to obtain 440 mg of the powder. 440 mg of this powder dissolved in 0.01 M aqueous ammonium acetate were poured into the top of a column packed with carboxymethylcellulose (2.2x25 cm) and eluted with linear gradient elution by 0.01--0.2 mole ammonium acetate (each 750 ml) (pH 4.5). Each 10 g of fraction was collected, and the active fractions (fraction tube Nos. 67-70) were pooled and freeze dried to obtain the active powder. The lyophilizate was dissolved in 1 M acetic acid and chromatographed through a column of "Sephadex" LH-20 (2.2x137 cm "Sephadex" is a Registered Trademark) by elution with 1 M AcOH. The eluate (6 g) was fractionated, then the active fractions (Nos. 28-37) were collected and lyophilized. The lyophilizate was dissolved in the upper layer of mixture of n-butanol:acetic acid:water (4:1:5 v/v/v). This solution was charged on a column of"Sephadex" G-25 (2.7x52 cm) which was packed with the lower layer of the above solvent mixture and thereafter replaced by the upper layer of the said mixed solvent. Elution was carried with the same upper layer solvent to fractionate each 6 g fraction and the active fractions (5-14th tubes) were collected and lyophilized. The thus-obtained power was rechromatographed under the same conditions on "Sephadex" G-25 and lyophilized. The lyophilized active powder was dissolved in I mole acetic acid, poured onto a column of "Sephadex" LH-20 (2.2x137 cm), eluted with 1 mole acetic acid for 5 g of the fractions. The active fractions were pooled and lyophilized to obtain 29.7 mg of the product (1000 MRC u/mg).

Rf=0.82 [carrier: "Merck" cellulose, developer ("Merck" is a Registered Trade Mark); n-butanol-acetic acid-water-pyridine (15:3:12:10 v/v/v/v)] Rf=0;43 carrier: "Merck" cellulose, developer, n-butanol:acetic acid:water (upper layer) (4:1:5 v/v/v)] [a]26=69.6 (c=0.72, 1 M acetic acid).

Amino acid analysis: Lys 1.15(1), His 1.01(1), Asp 2.94(3), Thr 4.80(5), Ser 1.06(1), Glu 2.14(2), Pro 1.96(2), Gly 4.00(4), Ala 2.00(2), Val 0.95(1), Met 0.87(1), Ile 0.96(1), Leu 1.84(2), Tyr 0.96(1), Phe 3.18(3), a-amino suberic acid 1.02(1).

EXAMPLE 2 The starting materials are produced as follows: (A) Preparation of amino acid sequence No. 1--9:

is produced as follows.

(1) Preparation of BOC-Leu-Gly-OBzl: A solution of WSC (34 g) in dichloromethane (50 ml) was added dropwise to a suspension of BOC-Leu-OH (46.2 g), HOBT (1 g) and H-Gly-OBzl . TosOH (74 g) in DMF (100 ml) and dichloromethane (200 ml) with stirring at -50C for 1 hour.

After one hour, the solution was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo to remove dichloromethane. Water was added to the DMF layer. The mixture was extracted with ethyl acetate (I litre), and again with the same solvent (500 ml). The ethyl acetate layer was washed with I N HCI, water, 5% w/v sodium bicarbonate and water, in this order, dried over anhydrous sodium sulfate and then concentrated in vacuo to obtain oily BOC-Leu Gly-OBzl (82 g).

(2) Preparation of BOC-Met-Leu-Gly-OBzl: Ethyl acetate (1 litre) was added to BOC-Met-OH . DCHA (150 g), washed twice with I N H2SO4 (600 ml) and water (500 ml), dried by anhydrous sodium sulfate and concentrated in vacuo. The residue was dissolved in dichloromethane (100 ml) and THF (100 ml.) HOBT (41 g) was added at -50C followed by the dropwise addition of DCC (62 g) in dichloromethane.

TFA (300 ml) was added to BOC-Leu-Gly-OBzl (137 g) at -50C, stirred for 30 minutes and concentrated in vacuo. The residue was dissolved in THF (75 ml) and pH was adjusted to 6.7-7 by addition of triethylamine (155 ml) under cooling. This solution was added to the BOC-Met-OH in dichloromethane at -5"C, stirred for I hour at -5"C and overnight at room temperature. The reaction mixture was adjusted to pH 6 by adding triethylamine and after 6 hours a soluti

(5) Preparation of

(oil, 56 g.) was dissolved in methanol (300 ml.) and water (150 ml). Active charcoal was added to this solution and, atter stirring tor I hour, palladium-carbon was added thereto. Hydrogenation was effected for 10 hours. The catalyst was removed and the solution was concentrated in vacuo down to 100 ml. Dioxane (200 ml), triethylamine (21 ml) under cooling and BOC Thr(Bzl)-OSU (80 g) were added to the concentrate. After stirring for 3 days at room temperature, N,N-dimethylamino - 1,3 - propanediamine was added, stirred for 3 hours, then concentrated down to 100 ml. This second concentrate was extracted with ethyl acetate. The ethyl acetate layer was washed with 1 N HCI and water, and was distilled in vacuo. The thus obtained oily product was dissolved in diethyl ether. This solution was poured into a 5% w/v sodium bicarbonate solution.

After carefully washing the aqueous layer with diethyl ether, the product was extracted again with ethyl acetate, washed with water, 1 N HCI and water in that order, and dried with anhydrous sodium sulfate. The ethyl acetate was distilled off to obtain an oily product (57 g).

(6) Preparation of

(50 g) was dissolved in TFA (150 ml) under cooling. After 30 minutes at room temperature, the TFA was removed under reduced pressure and the oily residue was dried over NaOH in vacuo overnight. The oily material was dissolved in DMF (100 ml), and the pH was adjusted to 6 by addition of triethylamine (40 ml) under cooling. HOBT (5 g) and BOC-Ser(Bzl) OSU (50 g) were added thereto. After suitably working-up the reaction product cyclohexylamine was added thereto and the desired product was obtained.

(7) Preparation of BOC-Asn-Leu-OBzl: BOC-Asn-OH (926.6 g) and H-Leu-OBzl . TosOH (43.3 g) were dissolved in DMF (150 ml). HOBT (2 g) was added to this solution. WSC (16.3 g) was added dropwise over a period of 30 minutes at --100C. The resulting solution was stirred overnight at room temperature. Water was added to the reaction mixture thus obtained to precipitate a product which was extracted with ethyl acetate. The extract was washed with 1 N HCI, water, 5% w/v aqueous sodium carbonate and water in this order, dried with anhydrous magnesium sulfate and concentrated in vacuo. The residue was recrystallized from ethyl acetate-n-hexane. 44.3 g of product was obtained.

m.p.: 146--1470C. [a]D5=26.5 (c=2, DMF).

(8) Preparation of BOC-Gly-Asn-Leu-OBzl: TFA (30 ml) was added to BOC-Asn-Leu-OBzl (15 g) under cooling and left to react for 1 hour at room temperature. The TFA was then distilled off and the oily residue was dried over NaOH. The residue was dissolved in DMF (50 ml) and this solution was adjusted to pH 6.5 by adding triethylamine under cooling. BOC-Gly OSU (11.3 g) and HOBT (1 g) were then added and the reaction mixture was stirred overnight at room temperature. N,N-dimethylaminopropylamine was added and after stirring for 30 minutes water was added thereto, then extracted twice with chloroform. The chloroform layer was washed with 1 N HCI and water, dried with anhydrous magnesium sulfate, and the chloroform was distilled off. The oily residue was crystallized from ethyl acetate-n-hexane to obtain the product (13.0 g, yield: 76.5%). M.P.: 86--88"C.

(9) Preparation of BOC-Gly-Asn-Leu-NHNH2: BOC-Gly-Asn-Leu-OBzl (5 g) was dissolved in methanol (20 ml). To that solution was added 80% w/v NH2NH2 . H2O (20 ml). The mixture was left to stand overnight at room temperature. Diethyl ether was added thereto to precipitate the product completely. The precipitate was washed with diethyl ether, then recrystallized from MeOH-diethyl ether to obtain the product. (3.9 g), (yield: 93.7 /"), m.p.: 204-2070C, (decomp.).

(10) Preparation of

was treated with 1 N HCI in ethyl acetate to prepare the free acid which was thereafter dried with anhydrous sodium sulfate and concentrated in vacuo. TFA (30 ml) was added to the oily residue under cooling. After stirring for 30 minutes at room temperature, the TFA was removed under reduced pressure. The residue was dried over NaOH in vacuo, dissolved in DMF (10 ml) and neutralized by adding triethylamine.

BOC-Gly-Asn-Leu-NHNH2 (4.2 g) was dissolved in DMF (15 ml), and dioxane (3.5 ml) containing 6 N HCI and isoamyl nitrite (2.0 ml) were added. This mixture was reacted for 10 minutes to form the azide.

The solution containing azide was cooled to -500C and the DMF solution containing the free acid obtained above was slowly added. The pH was adjusted to 7 by adding triethylamine, and after stirring the mixture for 1 hour at -5 to --100C, it was stirred overnight under ice-cooling. The pH was readjusted to 7.0 by adding N-methylmorpholine and the mixture was stirred for 3 days.

The reaction mixture thus obtained was slowly added at -50C to 0.5 N HCI (300 ml). The filtered precipitate was washed with water, refluxed with MeOH (100 ml)-water (200 ml) for 10 minutes and the solution was filtered after cooling. The precipitate was again refluxed with chloroform (200 methyl acetate (100 ml) and the solution filtered after cooling. The residue was re-precipitated by adding MeOH-diethyl ether to obtain the product. (6.1 g, yield: 63.8%). m.p.: 192--194"C (decomp.).

[a]020=-6.330 (c=1.5, DMF).

Elemental analysis (C47HsgO14N7. bH2O): C% H% N% found: 58.40 7.30 10.19 calculated: 58.48 7.31 10.16 (11) Preparation of

(2.8 g) dissolved in dry pyridine (30 ml) was added TFA-ONP (5 g). This mixture was stirred for 3 hours at 45"C. After the reaction product had been concentrated in vacuo, diethyl ether was added to the residue. The precipitate was filtered off and washed with diethyl ether to obtain a yellowish brown powder (2.8 g).

TFA (15 ml) was added to this powder under cooling. This mixture was stirred for 30 minutes at room temperature and the TFA was distilled. The residue was dissolved in DMF (20 ml). This solution was added dropwise to dry pyridine (3.0 1) at 45"C with stirring over a period of 1 hour. Thereafter the reaction mixture was stirred for 8 hours at 500 C, then stirred overnight at 400C. The reaction mixture thus-obtained was concentrated in vacuo down to 200 ml, stirred for 3 hours at 40"C, then concentrated down to 50 ml under reduced pressure. The resulting concentrate was dissolved in chloroform (600 ml). This solution was washed with saturated sodium chloride. The chloroform layer was concentrated in vacuo down to 10 ml. Diethyl ether was added to this concentrate. The resulting precipitate was filtered off, washed with diethyl ether, and dried to obtain the product. (1.5 g, yield: 60%).

(12) Preparation of

1.4 g was dissolved in DMF (5 ml) and methanol (30 ml). 80% w/v NH2NH2. H2O (20 ml) was added to this solution and the mixture was stirred overnight at room temperature. After reaction, water was added thereto. The precipitate was filtered and washed with water. Methanol (50 ml)-ethyl acetate (50 ml) was added and the mixture was refluxed. The refluxed mixture was cooled to ambient temperature, and the precipitate was filtered and dried to obtain the product. (1.1 g, yield: 78.6%). m.p.: 21 20C (softening)-236-250"C (decomp.).

[&alpha;]D25=-15.5 (c=0.2, DMF).

Elemental analysis (C4,H59OloN9): C /n H /n N /n found: 58.40 7.00 15.21 calculated: 58.75 7. I I 15.04 (13) Preparation of

To BOC-Met-Leu-Gly-OH (2 g) obtained in (3) hereinbefore was added dimethylthioether (0.5 ml) and TFA (10 ml) under cooling. The mixture was stirred for 20 minutes at room temperature. The TFA was then distilled off. Diethyl ether was added to the residue and the resulting precipitate was collected by decantation.

The precipitate was dried over NaOH and dissolved in DMF (2 ml).

(840 mg) was suspended in DMF (5 ml). Dioxane (1 ml) containing 6 N HCI was added at -50C to this suspension, and completely dissolved therein at 100C.

Isoamyl nitrite (0.2 ml) was added at -50C and stirred for 10 minutes to prepare the azide. After the azide reaction, the DMF solution previous prepared was slowly added thereto at --500C. The pH was adjusted to 7 by adding triethylamine. The mixture was stirred for 1 hour at -5"C and for a further 3 days in an ice bath. The resulting reaction mixture was slowly added to 0.5 N HCl (200 ml) under cooling.

The precipitate was collected and boiled with MeOH (200 ml) after adding water, then cooled and filtered to obtain the product. (1.1 g). m.p.:243-2470C (decomp.).

[a]D5=10.7 (c=0.31, DMF).

Elemental analysis (Cs4HaoNi0Oi4S . 3/2H2O): C% H% found: 56.17 7.07 12.29 calculated: 56.27 7.27 12.15 Amino acid analysis: Asp 0.96(1), Thr 0.94(1), Ser 0.88 (1), Gly 2.00(2), Met 0.99(1), Leu 1.88(2), a-amino suberic acid 1.02 (1), NH3 1.08 (B) Preparation of amino acids sequence 1031: BOC-Thr(Bzl)-Tyr[Bzl(Cl2)]-Thr(Bzl)-Gln-Asp(OBzl)-Phe-Asn-Lys(ClCbz)- Phe-His-Thr(Bzl)-Phe-Pro-Gln-Thr(Bzl)-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2: (1) Preparation of Cbz-Ala-Pro-NH2: Cbz-Ala-OH (74.1 g), H-Pro-NH2 . HCl (50 g) and HOBT (50.5 g) were added to DMF (300 ml). WSC (61 ml) was added thereto under cooling at 0 to -5 C. This mixture was urrea tor X nour at u to Dvt ana tnen overnignt at room temperature. The reaction mixture was concentrated in vacuo and chloroform was added to the residue, which was then washed with 1 N HCI, water, 5% w/v sodium bicarbonate solution and water. After drying over anhydrous magnesium sulfate, the organic layer was concentrated in vacuo. The oily material was treated with diethyl ether and filtered. The residue was twice recrystallized to obtain the product. (71 g, yield: 67.6%). m.p.: 171.3--171.8"C.

[a]D9=94.2 (c=2, MeOH).

Elemental analysis (CX6H2,O4N3): C% H% N% found: 60.15 6.66 13.31 calculated: 60.17 6.63 13.16 (2) Preparation of BOC-Val-Gly-OEt: BOC-Val-DCHA (80 g) was treated with I N HCI in ethyl acetate to obtain an oily free acid. This oily material and H-Gly-OEt . HCI (28 g) were dissolved in chloroform (150 ml). THF (150 ml) and then triethylamine (28 ml) and DCC (41.2 ml) were added thereto after cooling. The mixture was stirred for 1 hour at 0 to -2"C and overnight at room temperature. The reaction mixture, which had been filtered, was concentrated in vacuo and the residue was dissolved in ethyl acetate.

The solution was washed with 1 N HCI, water, twice with 5% w/v sodium bicarbonate solution and water, in this order, and, after drying with anhydrous magnesium sulfate, concentrated in vacuo. The residue was treated with n-hexane to obtain crude material. Recrystallization was carried out with ethyl acetatediethyl ether-n-hexane to obtain the product. (54 g, yield: 89.2%), m.p.:94--960C.

(3) Preparation of BOC-Val-Gly-OH: BOC-Val-Gly-OEt (10 g) was dissolved in MeOH (20 ml). 1 N NaOH (40 ml) was added under cooling. After stirring for 15 minutes under cooling and for 45 minutes at room temperature, the reaction mixture was adjusted with 1 N HCI to pH 7 and concentrated in vacuo. Water was added water to the residue and the solution was extracted with diethyl ether. The pH of the aqueous layer was adjusted to pH 2 by adding 1 N HCI and then extracted with ethyl acetate. The ethyl acetate layer was washed with water, dried with magnesium sulfate and then concentrated in vacuo. The residue was solidified by treatment with n-hexane and was recrystallized from ethyl acetate-n-hexane to obtain the product. (8.0 g, yield:89%), m.p.: 101-1040C.

(4) Preparation of BOC-Val-Gly-Ala-Pro-NH2: Cbz-Ala-Pro-NH2 (30 g) was dissolved in 25% w/v HBr/AcOH (200 ml). The solution was stirred for 30 minutes at room temperature. The residue was treated with diethyl ether, filtered, washed with diethyl ether and dried over sodium hydroxide. The residue was dissolved in DMF (200 ml), and BOC-Val-Gly-OH (26 g) and HOBT were added thereto, followed by WSC (17.4 ml). After stirring for 1 hour at room temperature, the mixture was adjusted to pH 4 by adding Nmethylmorpholine (5 ml) and stirred at room temperature overnight. The DMF was distilled off and to the residue was added water which was saturated with sodium chloride. The solution extracted with chloroform. Chloroform layer was washed with 1 N HCI saturated with sodium chloride, water and 5% w/v aqueous sodium bicarbonate. The solution was dried over magnesium sulfate and concentrated in vacuo. Residue was recrystallized from ethanol to obtain the product. (36 g, yield: 85.9%).

[ai30=-79.90 (c=1.0, AcOH).

(5) Preparation of BOC-Ile-Gly-OEt: BOC-Ile-OH . XH2O (120 g) was added to chloroform (200 ml) and toluene (200 ml) and the mixture was concentrated in vacuo. The oily residue thus obtained was dissolved in dichloromethane (300 ml), and H-Gly-OEt . HCl (77 g) was added thereto. The reaction mixture was stirred under cooling and neutralized with triethylamine. DCC (114 g) in chloroform was added thereto under cooling over a period of I hour and the mixture was stirred for 18 hours at room temperature.

Acetic acid (4 ml) was added to the reaction mixture, which was then stirred for 2 hours and filtered. The filtrate was dissolved in chloroform, washed with water, 1 N HCI, water and 5% w/v sodium bicarbonate solution, and after drying with anhydrous sodium sulfate, concentrated in vacuo. The residue was recrystallized from diethyl ether-n-hexane to obtain the product. (126.1 g). m.p.: 107--1080C.

[a]19=12.6 (c=l, DMF).

Elemental analysis (C5H28O5N2): C% H% found: 56.77 9.13 8.95 calculated: 56.94 8.92 8.85 (6) Preparation of BOC-Ala-Ile-Gly-OEt: TFA (20 ml) was added dropwise at -SOC to BOC-lle-Gly-OEt (8.0 g). The resulting solution was stirred for 40 minutes. After stirring, the TFA was distilled off and diethyl ether was added to that solution. The precipitate was collected by decantation and dried over NaOH. The residue was dissolved in DMF (40 ml) and neutralized with triethylamine. BOC-Ala-ONSU (8.7 g) was added thereto and the mixture was left for 2 days. N,N'-dimethylamino propylamine (2 ml) was then added, and reaction was allowed to take place for 1 hour. The DMF was distilled off in vacuo and chloroform was added. The resulting solution was washed with water, 1 N HCI, water, 5% w/v sodium carbonate solution and water, in this order, and then dried with magnesium sulfate. Recrystallization carried out with ethylacetate to obtain the product. 8.8 g, yield: 84.9%). m.p.: 190--190.50C, [a]D7=-26.7 (c=l, DMF).

Elemental analysis (C1sH33O6N3): C% H% N ,4 found: 55.70 8.70 10.96 calculated: 55.79 8.58 10.85 (7) Preparation of BOC-Ala-Ile-Gly-OH: Aqueous IN sodium hydroxide (25 ml) was added dropwise at -5 C to BOC Ala-Ile-Gly-OEt (8.5 g) dissolved in MeOH. After 5 hours the reaction mixture was adjusted to pH 4--5 by adding 1 N HCI. The MeOH was distilled off, water was added, and thereafter the pH was adjusted to 2 by addition of 1 N HCI. The resulting solution was extracted twice with ethyl acetate. The ethyl acetate layer was dried over magnesium sulfate, concentrated in vacuo, and recrystallized three times from ethyl acetate to obtain the product. (6.8 g, yield: 84.8 /").

m.p.: 205.6--207.50C, [a]17=34.70 (c=0.7, DMF).

Elemental analysis (C10H29O8N3): C% H% N /,, found: 53.31 8.24 11.91 calculated: 53.46 8.13 11.69 (8) Preparation of B OC-Gln-Thr(Bzl)-OBzl: 1 M sodium carbonate (100 ml) was added to H-Thr(Bzl)-OBzl. (COOH)2 (21.4 g) suspended in ethyl acetate (300 ml) and shaken well. After filtering, the ethyl acetate layer was extracted with water and saturated sodium chloride solution, and thereafter dried with magnesium sulfate and concentrated in vacuo. To the oily residue dissolved in THF (40 ml) was added BOC-Gln-OH (12.3 g) and HOBT (6.8 g). DCC (10.3 g) in THF (10 ml) was added dropwise thereto over a period of 5 minutes. The mixture was stirred for 2 hours at the same temperature and for 21 hours at room temperature.

After removing the insoluble material and extracting with THF, the filtered THF layer was evaporated to dryness in vacuo. The oily residue thus obtained was dissolved in ethyl acetate and stood for 1 hour in the cold. The resulting precipitate was removed and the ethyl acetate layer was washed with water, I N HCI, water, 5% w/v sodium bicarbonate (three times) and water, in this order, a further three times with 1 N HC1 and three times with aqueous sodium chloride, then dried with magnesium sulfate. After concentrating in vacuo, the residue was crystallized from a volume consisting of a small amount of n-hexane and a large amount of diethyl ether to obtain the product. (22.3 g, yield: 84.5%). m.p.: 88-89.50C, [a]030=-25.l0 (c=l.l, MeOH).

Elemental analysis (C28H37O7N3): C% H% N% found: 63.74 7.07 7.96 calculated: 63.37 6.97 8.09 (9) Preparation of BOC-Gln-Thr(Bzl)-NHNH2: BOC-Gln-Thr(Bzl)-OBzl (16.4 g) was dissolved in MeOH (20 ml) and 80 /a w/v hydrazine hydrate (20 g) was added thereto. After stirring for 44 hours, water was added to break up the crystals thus obtained. After filtration, the crystals were washed with water and with diethyl ether, then recrystallized from hot methanol diethyl ether to obtain the product. (8.2 g yield: 58.5%).

[Q:]30=9.9 (c=1.0, MeOH).

Elemental analysis (C21H33O6N5): found: 55.86 7.37 15.51 calculated: 55.89 7.52 15.17 (10) Preparation of BOC-Gln-Thr(Bzl)-Ala-Ile-Gly-OH: TFA (15 ml) was added to BOC-Ala-Ile-Gly-OH (3.6 g) at -50C and allowed to react for 40 minutes at room temperature. After distilling off the TFA, diethyl ether was added to the residue. The precipitate thus obtained was collected by decantation and dried over NaOH. The dried precipitate was dissolved in water, neutralized by adding 1 N sodium bicarbonate solution, further dissolved in water (10 ml) after concentration in vacuo, and then triethylamine (1.4 ml) and DMF (10 ml) were added under cooling, and the solution was further cooled at --150C.

BOC-Gln-Thr(Bzl)-NHNH2 (5.87 g) was dissolved in DMF (40 ml) at -200C. 6 N HCI in dioxane (9 ml) was added to this solution. Isoamylnitrite (2.28 ml) was added slowly thereto and the resulting mixture was stirred for 50 minutes at --100C.

After adding triethylamine (7.56 ml) at -200C, the DMF solution prepared previously was added. The resulting mixture was stirred for 1 hour at -100C and for 6 days under ice cooling. 1% w/v acetic acid (200 ml) was added thereto and the precipitate was filtered, washed with water and diethyl ether, then recrystallized from MeOH-diethyl ether to obtain the product. (3.3 g, yield: 48.7%). m.p.: 23 232"C. (decomp.).

[a]023=-32.70 (c=1.0, AcOH).

(11) Preparation of BOC-Gln-Thr(Bzl)-Ala-Ile-Gly-Val-Gly-A1aProNH2: BOC-Val-Gly-Ala-Pro-NH2 (26.5 g) was added to TFA (10 ml) under cooling, and after stirring for 50 minutes, concentrated in vacuo to remove the TFA. The residue was treated with diethyl ether, and the precipitated material was collected by decantation and dried in vacuo over sodium hydroxide. DMF (20 ml) and HMPA (20 ml) were added thereto. BOC-Gln-Thr(Bzl)-Ala-Ile-Gly-OH (3.87 g) and HOBT (0.82 g) were added to the resulting solution with stirring at below 0 C.

WSC (1.1 ml) was added to this mixture, which was then stirred at the same temperature. After 3 days, 2% w/v AcOH (200 ml) was added and the thus formed precipitate was filtered, and then washed with water and diethyl ether. Methanol was added to the precipitate refluxed, cooled, and then ethyl acetate (50 ml) and diethyl ether (50 ml) were added. After cooling the precipitate was collected, and washed with ethyl acetate to obtain the product. (4.1 g, yield: 71.8%). m.p.: 25 260"C (decomp.).

[a]028=-57.00 (c=1.0, AcOH).

Elemental analysis (C47H7N 1013. 2/3H2O): H% H N% found: 55.18 7.60 14.54 calculated: 54.85 7.64 14.97 (12) Preparation of Cbz-Phe-His-OMe: - H-His-OMe.2HC1 (126 g) and Cbz-Phe-ONSU (213 g) were suspended in chloroform (600 ml) with stirring under cooling. Triethylamine (145.6 ml) was added dropwise to this suspension over a period of 40 minutes. Thereafter the mixture was stirred for 4 days at room temperature. Chloroform ( I litre) was added thereto. The resulting mixture was washed twice with 5% w/v sodium carbonate solution, 4 times with water and twice with saturated sodium chloride solution, then dried with magnesium sulfate. The solution was concentrated in vacuo and diethyl ether:n-hexane (1:2 v/v) was added to the residue. The thus formed crystals were washed with the same solvent mixture and recrystallized from MeOH-ethyl acetate. The resulting crystals were washed with diethyl ether to obtain the product. (216.5 g, yield:92.5%). m.p.:135--140"C.

(13) Preparation of BOC-Lys(C1Cbz)-Phe-His-OMe: To Cbz-Phe-His-OMe (27 g) was added 26 w/v HBr in acetic acid (110 ml) under cooling. After 5 minutes, this mixture was stirred for a further 60 minutes at room temperature. Dry diethyl ether was added to precipitate a product, which was then washed with 3 times with the diethyl ether. The thus obtained powder was dried over NaOH. The HBr salt was dissolved in DMF (100 ml) and neutralized with triethylamine (16.8 ml) under cooling. BOC-Lys(ClCbz)-ONP (32.2 g) was added then added and the mixture was stirred for 30 minutes under cooling, and then for a further period at room temperature. After 70 hours, the reaction mixture was evaporated to dryness in vacuo. Ethyl acetate (350 ml), water (150 ml) and aqueous sodium carbonate (to adjust pH of the aqueous layer to pH 8-9) were added to the residue. The ethyl acetate layer was washed 5 times with 5% w/v sodium bicarbonate (150 ml) and twice with saturated sodium chloride (150 ml).

Organic layer was dried with magnesium sulfate, then concentrated in vacuo.

Diethyl ether was added to the crystalline residue, which was then filtered, recrystallized from ethyl acetate (700 ml) and MeOH (50 ml), then washed with ethyl acetate to obtain the product. (27.5 g, yield: 64.2%). m.p.: 134--136"C, [a]32--19.80 (c=1.0, DMF).

Elemental analysis (C3sH4sO8N6CI . AH2O): C% H% N% found: 58.36 6.48 11.64 calculated: 58.20 6.42 11.69 (14) Preparation of BOC-Asn-Lys(CICbz)-Phe-His-OMe: BOC-Lys(C1Cbz)-Phe-His-OMe (27.1 g) was treated with TFA (60 ml) under cooling for 10 minutes and for 65 minutes at room temperature, and thereafter evaporated to dryness in vacuo. Diethyl ether was added to the residue, and the precipitate was collected by decantation and dried over NaOH. This was dissolved in DMF (50 ml), neutralized with triethylamine, and BOC-Asn-ONP (16.1 g) was added thereto. The resulting solution was stirred for 1 hour under cooling and 72 hours at room temperature. The reaction mixture was concentrated in vacuo. Water was added to the residue, followed by saturated sodium carbonate to adjust to pH 9. The thus precipitated material was washed with water and diethyl ether and then recrystallized from MeOH-ethyl acetate and MeOH-diethyl ether. (15.6 g, yield: 49%), m.p.:175--176"C), [a]D2=44.2 (c=1.0, DMF).

Elemental analysis (C39Hs,O,oNaCI . 4/3H2O): C /O H% found: 55.22 6.14 12.79 calculated: 55.02 6.35 13.16 (15) Preparation of BOC-Asn-Lys(ClCbz)-Phe-His-NHNH2: 80% w/v hydrazine hydrate (6.3 ml) was added with stirring to BOC-Asn Lys(C1Cbz)-Phe-His-OMe (8.3 g) dissolved in MeOH (20 ml) and DMF (10 ml.

After 5 days water was added and the resulting precipitate filtered off and washed with water. The reaction mixture was refluxed with MeOH (150 ml).

Diethyl ether (200 ml) was added after cooling. The thus formed precipitate was filtered and washed with diethyl ether. (6.9 g, yield: 84.5%). m.p.: 20-2020C (decomp.), [lx]D5=43.0 (c=l.l, DMF).

Elemental analysis (C38HsaOgN10CI ,H2O): C% H% found: 54.36 6.28 16.90 calculated: 54.57 6.27 16.75 (16) Preparation of BOC-Phe-Pro-OBzl: Triethylamine (70 ml) was added dropwise with stirring to H-Pro-OBzl . HCI (121 g) in THF (400 ml) under cooling. THF (100 ml) and chloroform (50 ml) were added to this mixture, followed by BOC-Phe-OH (132 g). DCC (113 g) in chloroform was added thereto with stirring under cooling over a period of 1 hour.

Stirring was continued below -10"C. After 16 hours, acetic acid (5 ml) was added under cooling. The mixture was further stirred for 2 hours. The resulting precipitate was filtered off. The filtrate was washed with THF and concentrated in vacuo. The oily residue thus obtained was dissolved in ethyl ether (I litre), the precipitate was filtered off and the filtrate was washed with water, I N HCI, water, 5% w/v sodium bicarbonate and water, in this order, dried with magnesium sulfate and concentrated in vacuo. The resulting oily residue was dissolved in diethyl ether, crystallized by adding n-hexane and recrystallized from ethyl acetate-n-hexane.

(169 g, yield: 74.8%), m.p.: 104--1060C.

(17) Preparation of BOC-Thr(Bzl)-Phe-Pro-OBzl: TFA (150 ml) was added under cooling to BOC-Phe-Pro-OBzl (67.9 g). This mixture was stirred for 40 minutes and concentrated in vacuo. The oily residue thus obtained, after adding dry diethyl ether thereto and reconcentration, was dried over NaOH. DMF (140 ml) was added and neutralized by addition of triethylamine.

BOC-Thr(Bzl)-OSU (67.1 g) was added and the mixture stirred under cooling for 1 hour at room temperature. Whilst being stirred, the reaction mixture was maintained at pH 7 by adding triethylamine. After 72 hours, N-methylmorpholine was added to continue the reaction at 250C. After 117 hours, N,Ndimethylaminopropylamine (2 ml) was added and the mixture was stirred f (20) Preparation of B OC-Asn-Lys(ClCbz)-Phe-H is-TH r(B zl)-Phe-Pro- Gln-Thr(Bzl)-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2: BOC-Gln-Thr(Bzl)Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 (3.9 g) was treated wtih TFA (10 ml) for 5 minutes under cooling and for 40 minutes at room temperature, and evaporated to dryness in vacuo. 6 M HCl/dioxane (0.65 ml) was added to the residue, followed by diethyl ether. The resulting precipitate was washed twice by decantation and dried overNaOH. The dried residue was dissolved in DMF(IOml) and HMPA (15 ml). HOBT (545 mg) and BOC-Asn-Lys(C1Cbz)-Phe-His-Thr(Bzl)- Phe-Pro-OH (5.0 g) were added to this solution which was stirred below 0 C. WSC (0.75 ml) was then added.

After 24 hours the reaction mixture was concentrated in vacuo. Ethyl acetate was added to the residue. The resulting precipitate was filtered off, and washed with ethyl acetate and diethyl ether. The precipitate thus obtained was broken up in water, which was then adjusted to pH 9 by adding aqueous sodium carbonate, filtered, washed with water and diethyl ether. Methanol (100 ml) was added to the precipitate and this solution was refluxed. After cooling, diethyl ether was added and the precipitate was filtered off and washed with MeOH. (7.1 g, yield: 85.3%), m.p.: 228-2310C (decomp.).

[a]027=-43.20 (c=1.0, AcOH).

Elemental analysis (CiosH 143O24N22Cl . 3/2H2O): C% H% found: 58.34 6.87 14.35 calculated: 58.39 6.81 14.27 Amino acid analysis: Lys 0.90(1), His 0.99(1), Asp 0.99(1), Thr 1.72(2), Glu 1.11(1), Pro 2.18(2), Gly 2.00(2), Ala 2.04(2), Val 1.02(1), Ile 1.13(1), Phe 2.12(2).

(21) Preparation of B OC-A sp(OB zl)-Phe-OH: Triethylamine (2.8 ml) was added with stirring to L-phenylalanine (3.3 g) suspended in DMF (10 ml). Then water and BOC-Asp(OBzl)-ONP (8.9 g) were added with stirring. After 44 hours further BOC-Asp(OBzl)-ONP (4.4 g) was added.

The mixture was stirred for 3 days, concentrated in vacuo, adjusted to pH 2 by the addition of water and 1 N HCI and extracted with ethyl acetate. The ethyl acetate layer was washed with I N HCI, water and aqueous sodium chloride, dried over sodium sulfate and concentrated in vacuo. The resulting oily residue was dissolved in diethyl ether. This solution was concentrated and the precipitate was collected by filtration. After washing with diethyl ether, the product was obtained by recrystallization with ethyl acetate-diethyl ether and n-hexane. (3.7 g). m.p.: 146 147"C.

[a]20-+3.90 (c=1.0, MeOH).

Elemental analysis (C25H30O7N2): C% H% N% found: 63.55 6.48 6.04 calculated: 63.81 6.43 5.95 (22) Preparation of BOC-Gln-Asp(OBzl)-Phe-OH: BOC-Asp(OBzl)-Phe-OH (3.3 g) in TFA (10 ml) was reacted for 20 minutes under cooling and for a further 40 minutes at room temperature, before being concentrated in vacuo. Crystals were formed by the addition of diethyl ether. The crystals was filtered, washed with diethyl ether and dried over NaOH. The dried crystals were dissolved in DMF (10 ml). This solution was neutralised by the addition of triethylamine (1.96 ml) under cooling with stirring BOC-Gln-ONP (3.34 g) was added thereto. After 42 hours, the DMF was distilled off and ethyl acetate and 1 N HCI were added. The aqueous layer was re-extracted with ethyl acetate.

The ethyl acetate layer was washed with 1 N HCI and water, then diethyl ether was added. The thus precipitated crystals were broken up in 0.5 N HCI, filtered and recrystallized with MeOH-diethyl ether. (3.5 g, yield: 83.5%). m.p.: 161--1630C (decomp.).

[a]26--l7.00 (c=l.l, DMF).

Elemental analysis (C30M3sO9N4. H2O): C% H% N% found: 58.03 6.23 9.37 calculated: 58.43 6.54 9.09 (23) Preparation of B OC-Thr(Bzl)-Gln-Asp(OBzl)-Phe-OH: TFA (100 ml) was added to BOC-Gln-Asp(OBzl)-Phe-OH (45 g) under cooling. This mixture was stirred for 50 minutes and concentrated in vacuo. The resulting precipitate was collected by the addition of dry diethyl ether and washed with the same solvent, then dried over NaOH. DMF (200 ml) was added to this powder and DMF-water (1:1 v/v) (200 ml) was added with stirring under cooling.

Triethylamine (21 ml) was then added thereto dropwise, followed by the addition of BOC-Thr(Bzl)-ONSU (33.7 g) with stirring. After 20 hours N-methylmorpholine (5 ml) was added. Stirring was continued for a further 22 hours and the mixture was then concentrated in vacuo. 1 N HCI (I litre) was added to the oily residue. The thus-formed crystals were broken up, filtered, washed with water and diethyl ether and recrystallized from MeOH-diethyl ether. (46.5 g, yield: 78.5%) m.p.: 172 173.5"C (decomp.).

[a]026=-3.70 (c=1.0, DMF).

Elemental analysis (C41M51O11N5 . +M2O): found: 61.61 6.57 9.00 calculated: 61.64 6.56 8.77 (24) Preparation of BOC-Tyr[Bzl(CI2)]-Thr(Bzl)-Gln-Asp(OBzl)-Phe-OH: TFA (100 ml) was added to BOC-Thr(Bzl)-Gln-Asp(OBzl)-Phe-OH (45 g) under cooling. This mixture was stirred for 10 minutes under cooling and for a further 60 minutes at room temperature before being evaporated to dryness in vacuo. Diethyl ether was added to the residue. This mixture was filtered. The residue remaining on the filter paper was washed with diethyl ether, then dried over NaOH. This dried powder was dissolved in DMF (100 ml). This solution was neutralized by the addition of triethylamine under cooling. BOC-Tyr[Bzl(CI2)]- ONSU (32.2 g) was then added and the mixture was stirred for I hour under cooling followed by a further 46 hours at room temperature before being concentrated in vacuo. Cold 0.5 N HCI was added to the residue under cooling. The thus formed crystals were broken up, filtered, then washed with water, diethyl ether and ethyl acetate. The resulting residue was refluxed with MeOH (150 ml) and ethyl acetate (150 ml). After cooling diethyl ether was added and the precipitate was filtered off and washed with diethyl ether. (54.5 g, yield: 86.0%). m.p.: 198-1990C (decomp.), [Ct]28=09 (c=l.0, DMF).

Elemental analysis (C57M64O13N6Cl2 . +M2O): N% found: 60.92 5.78 7.85 calculated: 61.07 5.84 7.50 (25) Preparation of B OC-Thr(Bzl)-TyrlB zl(CI2)]-Thr(Bzl)-Gln-Asp(OBzl)-Phe-OH TFA (130 ml) was added to BOC-Tyr[Bzl(CI2)]-Thr(Bzl)-Gln-Asp(OBzl)-Phe- OH (51 g) under cooling. This mixture was stirred for 5 minutes under cooling and for a further 50 minutes at room temperature before being evaporated to dryness in vacuo. Diethyl ether was added to the residue and the precipitate was filtered off.

This precipitate was washed with diethyl ether and dried over NaOH. The thus obtained powder was dissolved in DMF (120 ml). This solution was neutralized by the addition of triethylamine, and BOC-Thr(Bzl)-ONSU (19.6 g) was added thereto. After 20 hours stirring at room temperature, triethylamine was added to adjust pH to 7-8. The mixture was stirred for a further 20 hours and then concentrated in vacuo. Cold 0.5 N HCI (1 litre) was added to the residue under cooling. The resulting crystals were broken up, filtered and washed with water and diethyl ether. After reflux with MeOH (200 ml) for 20 minutes, ethyl acetate (100 ml) was added thereto. After cooling, diethyl ether (600 ml) was added and .this mixture kept in an ice-box. The precipitated crystals were filtered and washed with diethyl ether. (53 g, yield: 88.9%). m.p.: 196--198"C (decomp.).

[a]26-+5 90 (c=1.0, DMF).

Elemental analysis (C66H7,Ol5N,Cl2: 3/2H2O): C% H% N% found: 61.43 5.88 7.62 calculated: 61.39 6.06 7.37 Amino acid analysis: Asp 1.00, Thr 1.68, Glu 1.08, Tyr 0.91, Phe 0.85.

(26) Preparation of BOC-Thr(Bzl)Tyr[Bzl(C12)l-Thr(BzI)-Gln-Asp(OBzl) - Phe Asn - Lys(ClCbz) - Phe - His - Thr(Bzl) - Phe - Pro - GIn - Thr(Bzl) Ala - lIe - Gly - Val - Gly - Ala - Pro - NH2: TFA (20 ml) was added to BOC-Asn-Lys(ClCbz)-Phe-His-Thr(Bzl)-Phe-Pro- Gln-Thr(Bzl)-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2. 3/2H2O (6.70 g) under cooling.

This mixture was stirred for 5 minutes under cooling and for a further 50 minutes at room temperature before being concentrated in vacuo. During concentration, 8 M HCI in dioxane (0.5 ml) was added and the resulting mixture was evaporated to dryness in vacuo. Diethyl ether was added to the residue and the precipitate was isolated by decantation. The dried powder was dissolved in DMF (30 ml), to which solution BOC-Thr(Bzl)-Tyr[Bzl(C12)]-Thr(Bzl)-Gln-Asp(OBzl)-Phe-OH . 3/2M2O (4.44 g) and N - methyl - 2 - pyrrolidone (20 ml) were added. The resulting solution was cooled below 0 C and WSC (0.62 ml) was added. The mixture was stirred for about 5 hours and then for a further period of time at room temperature.

After 3 days, ethyl acetate (400 ml) was added. The precipitate was filtered off, washed with ethyl acetate, broken up in 0.5 M acetic acid, washed with water and filtered. The precipitate was then washed with 0.5 M acetic acid and water, then refluxed with MeOH (50 ml).

Ethyl acetate and diethyl ether were added to that mixture, which was then allowed to cool, before being filtered. The crystals thus obtained were washed with ethyl acetate. (9.2 g, yield: 89.4 /"). m.p.: 228-2340C (decomp.).

[a],30=-27.00 (c=0.5, AcOH).

Amino acid analysis: Asp 1.90(2), Thr 3.40(4), Glu 2.16(2), Pro 1.70(2), Gly 2.00(2), Ala 2.12(2), Val 0.93(1), Ile 1.03(1), Tyr 1.15(1), Phe 3.06(3).

WHAT WE CLAIM IS: 1. A process for the preparation of a polypeptide of the formula (1):

the symbols being as hereinbefore defined, or a pharmaceutically acceptable acid addition salt or complex thereof, which process comprises condensing in known manner amino acids or peptides or combinations thereof in an appropriate number of steps in the arrangement of the amino acids in the compound of formula (I), in so doing cyclising a peptide comprising a group of formula:

wherein R is a reactive ester residue, to form a peptide, comprising a group of formula:

at a suitable stage of the process, and optionally converting the resulting compound of formula (I) into a pharmaceutically acceptable acid addition salt or complex thereof.

2. A process according to claim I wherein peptides containing 2, 3 or 4 amino acid residues are condensed, and peptide comprising the group of formula:

in which R is as defined in claim 1, is cyclised after the 1st to 6th amino acids of the polypeptide of formula (1) have been condensed together, but before the final condensation reaction producing the polypeptide of formula (I) is effected.

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

Claims (9)

**WARNING** start of CLMS field may overlap end of DESC **. (26) Preparation of BOC-Thr(Bzl)Tyr[Bzl(C12)l-Thr(BzI)-Gln-Asp(OBzl) - Phe Asn - Lys(ClCbz) - Phe - His - Thr(Bzl) - Phe - Pro - GIn - Thr(Bzl) Ala - lIe - Gly - Val - Gly - Ala - Pro - NH2: TFA (20 ml) was added to BOC-Asn-Lys(ClCbz)-Phe-His-Thr(Bzl)-Phe-Pro- Gln-Thr(Bzl)-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2. 3/2H2O (6.70 g) under cooling. This mixture was stirred for 5 minutes under cooling and for a further 50 minutes at room temperature before being concentrated in vacuo. During concentration, 8 M HCI in dioxane (0.5 ml) was added and the resulting mixture was evaporated to dryness in vacuo. Diethyl ether was added to the residue and the precipitate was isolated by decantation. The dried powder was dissolved in DMF (30 ml), to which solution BOC-Thr(Bzl)-Tyr[Bzl(C12)]-Thr(Bzl)-Gln-Asp(OBzl)-Phe-OH . 3/2M2O (4.44 g) and N - methyl - 2 - pyrrolidone (20 ml) were added. The resulting solution was cooled below 0 C and WSC (0.62 ml) was added. The mixture was stirred for about 5 hours and then for a further period of time at room temperature. After 3 days, ethyl acetate (400 ml) was added. The precipitate was filtered off, washed with ethyl acetate, broken up in 0.5 M acetic acid, washed with water and filtered. The precipitate was then washed with 0.5 M acetic acid and water, then refluxed with MeOH (50 ml). Ethyl acetate and diethyl ether were added to that mixture, which was then allowed to cool, before being filtered. The crystals thus obtained were washed with ethyl acetate. (9.2 g, yield: 89.4 /"). m.p.: 228-2340C (decomp.). [a],30=-27.00 (c=0.5, AcOH). Amino acid analysis: Asp 1.90(2), Thr 3.40(4), Glu 2.16(2), Pro 1.70(2), Gly 2.00(2), Ala 2.12(2), Val 0.93(1), Ile 1.03(1), Tyr 1.15(1), Phe 3.06(3). WHAT WE CLAIM IS:

1. A process for the preparation of a polypeptide of the formula (1):

the symbols being as hereinbefore defined, or a pharmaceutically acceptable acid addition salt or complex thereof, which process comprises condensing in known manner amino acids or peptides or combinations thereof in an appropriate number of steps in the arrangement of the amino acids in the compound of formula (I), in so doing cyclising a peptide comprising a group of formula:

wherein R is a reactive ester residue, to form a peptide, comprising a group of formula:

at a suitable stage of the process, and optionally converting the resulting compound of formula (I) into a pharmaceutically acceptable acid addition salt or complex thereof.

2. A process according to claim I wherein peptides containing 2, 3 or 4 amino acid residues are condensed, and peptide comprising the group of formula:

in which R is as defined in claim 1, is cyclised after the 1st to 6th amino acids of the polypeptide of formula (1) have been condensed together, but before the final condensation reaction producing the polypeptide of formula (I) is effected.

3. A process according to claim 1 or 2 wherein an N-terminal peptide fragment

consisting of the 1st to the nth amino acid residues of the polypeptide of formula (I), which has already undergone cyclisation, is condensed with the C-terminal peptide fragment consisting of the (n+l)th to the 31st amino acid residue of the polypeptide of formula (I), wherein n is 6, 7, 8 or 9.

4. A process according to claim 3 wherein said N-terminal peptide fragment consists of the 1st to the 9th amino acid residues, and said C-terminal peptide fragment consists of the 10th to the 31st amino acid residues.

5. A process according to claim 3 or 4 wherein said N-terminal peptide fragment is produced by condensing amino acids and/or peptides of from 2 to 4 amino acid residues in the order of the amino acid sequence from the C-terminal end of said fragment and then cyclising said fragment, and said C-terminal polypeptide fragment is produced by condensing amino acids and/or peptides of from 2 to 4 amino acid residues in the order of the amino acid sequence from the Cterminal end of said fragment.

6. A process according to any one of the preceding claims wherein one or more of: the y-carboxyl group of L-glutamic acid, the hydroxyl group of L-serine, L-threonine and L-tyrosine, the E-amino group of L-lysine and the imine group of L-histidine are protected during the production of the polypeptide of formula (I), and the protecting groups are subsequently removed.

7. A process according to claim I substantially as described in Example 1 and in which the C-terminal and N-terminal polypeptide fragments employed are produced substantially as described in Example 2.

8. A polypeptide of the formula (I) or a pharmaceutically acceptable acid addition salt or complex thereof which has been produced by a process as claimed in any one of the preceding claims.

9. A pharmaceutical preparation comprising, as active ingredient, a polypeptide or a pharmaceutically acceptable acid addition salt or complex thereof as claimed in claim 8, together with a pharmaceutically acceptable carrier or diluent.