NO131170B - - Google Patents

Description

Analogy method for the production of a new,

therapeutically active octadecapeptide.

The present invention relates to an analogue method for the production of the new therapeutically active octadecapeptide (3-alanyl-L-tyrosyl-L-seryl-L-methionyl-L-glutamyl-L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophyll -glycyl-L-lysyl-L-prolyl-L-valyl-glycyl-L-lysyl-L-lysyl-L-arginyl-L-argininamide, non-toxic acid addition salts thereof, or complexes thereof with zinc, polyglutamic acid, polyaspartic acid, copoly- (glutamyl-tyrosine) or mixtures thereof, and the peculiarity of the method according to the invention is that the amino acids (3-

alanine, L-tyrosine, L-serine, L-methionine, L-glutamic acid, L-histidine, L-phenylalanine, L-tryptophan, glycine, L-lysine, L-proline, L-valine and L-arginine in the desired sequence and/or peptide fragments containing these amino acids in the desired sequence are condensed to the indicated octadecapeptide using methods known in peptide chemistry, whereby a terminal carboxyl group and/or amino group is activated for coupling and the other reactive groups are intermediately protected,

and the resulting octadecapeptide is optionally transferred into the salts or complexes in a manner known per se.

The octadecapeptide, acid addition salts, and complex compounds are useful as therapeutically active compounds because of their strong and long-lasting action with respect to adrenaline-stimulating activity, lipotropic activity, and melanocyte-stimulating activity. All amino acids in the following description have L configuration unless otherwise stated. The abbreviated designations for amino acids, peptides and their derivatives correspond to the proposal for the IUPAC-IUB Commission Nomenclature.

Synthesis of a large number of corticotropin (ACTH) peptides has been reported from a number of laboratories. The peptide analogues are those in which individual amino acids in the natural sequence have been exchanged with other amino acids, e.g. the first amino acid serine with glycine or D-serine, or the fourth amino acid methionine with valine or norleucine, or the fifth amino acid glutamic acid with glutamine, or the seventeenth or eighteenth amino acid arginine with lysine or ornithine, or the twenty-fifth amino acid aspartic acid with valine. Although many corticotropic peptides with a slightly modified amino acid sequence have been synthesized to this day, none showed an activity greater than that of native corticotropin, with very few exceptions. Among these peptides, it is of particular interest that the peptide D-Ser''-Nlelf-Val25-ACTH(1-25)-I'ffl2, which contains the D-serine residue at the amino terminus instead of native L-serine, a valinamide residue at its carboxylating instead of an aspartic acid residue and, instead of the methionine residue in the ^-position in the corticotropin contains a norleucine residue, has a stronger biological effect than the native

corticotropin, as reported in the literature: Experientia.,

22,526 (1966). It seems most likely that the stronger effect demonstrated for D-Ser<1->Nle^-Val<2>5/ACTH(1-2?)-MH2 is a consequence of the greater stability of the peptide with respect to the action of aminopeptidase and carboxypeptidase due to the presence of the D-amino acid at the amino end and the valinamide at the carboxyl end and this is due to the fact that the oxidation of methionine in the h-position to methionine sulfoxide, which would result in inactivation, does not take place during the turnover as a consequence of the replacement of the methionine residue with the norleucine residue. Although D-Ser<1->Nle<If->Val<2>5-ACTH(1-25)-NH2 is an excellent corticotropic peptide with very strong biological activity, this peptide is not necessarily ideal due to the fact that some problems need to be solved for practical use for treating humans and also from an industrial point of view. Since D-Ser<1->Nle<Lf->Val<2>5-ACTH(l-25)-NH2 contains three modified amino acids, namely D-serine, norleucine and valine residues, it is feared that an anaphylactic reaction may occur when the compound is administered to humans due to the difference in amino acid sequence between human corticotropin and the synthetic peptide. It is well known that the anaphylactic reaction often occurs when commercially available corticotropin, which is secreted from animal glands from pigs, cattle, sheep, etc., is administered to humans. This undesirable phenomenon may be a consequence of the presence of species differences, i.e. structural differences at positions 25 and 33 in the amino acid sequences of human corticotropin and animal corticotropin,

and may also be due to certain protein-like impurities that cannot be completely removed from natural corticotropin from the animals mentioned.

Furthermore, in the preparation of serine-containing peptides, it is known

that the reactivity of the hydroxyl group in the saine as well as

the lability of seryl-peptide bonds under alkaline conditions, the tendency for (3-eliminating reactions that form the dehydropeptide and the N > 0 acyl shift make it very difficult to prepare serine-containing peptides which are often followed by racemizations due to the lability against alkaline conditions. Furthermore, as many syntheses , working time and labor are needed to obtain a long-chain peptide, during the production of a corticotropic highly active peptide it is desirable that the amino acid sequence is as short as possible.

In order to obtain a corticotropic highly active peptide whose amino acid sequence is as short as possible, the octadecapeptidamine corresponding to the first eighteen amino acid residues of the corticotropin, with the exception that the carboxyl end was converted in the amide, was synthesized as reported in the literature, J.Am.Chem. Soc., 8£ 2696 (196>5)-.and.MJ... Biochem. (Tokyo)., , 58 51 2, (1965) . Furthermore, it has previously succeeded in synthesizing an octadecapeptidamide in which the amino-end serine in corticotropin is replaced by the glycine residue and the carboxyl end is converted to the amide /Bull.Chem.Soc. Japan, i+3. 196 (1970)_7. Both peptides are very active with regard to corticotropic activity. In vivo steroidogenic activity when administered intravenously to rats is similar to that of ovine corticotropin. However, the in vitro steroidogenesis activity is somewhat less than that of ovine corticotropin.

During the investigations of corticotropic peptides to solve the technical problems mentioned above, it was discovered that an octadecapeptide of the formula (3-alanyl-tyrosyl-seryl-methionyl-glutamyl-histidyl-phenylalanyl-arginyl-tryptophyll-glycyl-lysyl-pr .olyl-valyl-glycyl-lysyl-lysyl-arginyl-argininamide, non-toxic acid addition salts thereof and complex compounds with zinc, polyamino acids or mixtures thereof show marked biological properties, adrenal-stimulating action, lipotropic activity and melanocyte-stimulating activity, all of which are higher than for natural corticotropin and for the synthetic corticotropin peptides known to date. It was also discovered that the octadecapeptide has prolonged corticotropic activity, probably due to its resistance to the action of aminopeptidase. It was also found that its complexes have a prolonged action. The derivatives have a more marked biological effect and excellent properties from an industrial and biological point of view compared to na turbid corticotropin and related known peptides with corticotropic activity and can be prepared by the present method in a pure state without inactivation, side reactions or racemization.

Polypeptides are produced using a number of methods, viz.

that the amino acid residues in a specified sequence are linked together one by one

or in the form of pre-synthesized peptide fragments or fragments. The amino acid or peptide unit can thus be joined with a further amino acid or peptide fragments by methods known in peptide chemistry.

When preparing the present octadecapeptide and the starting compounds, any free functional groups that do not participate in the reaction can therefore be advantageously protected, in particular by means of groups that can be easily eliminated by methods such as acidolysis, hydrogenolysis or hydrolysis. The carboxyl group is advantageously protected by esterification, e.g. with a lower alkanol such as methanol, ethanol, propanol, isopropanol, tertiary butanol or a lower aralkanol such as e.g. benzyl alcohol, p-nitrobenzyl alcohol, p-methoxybenzyl alcohol or by amide formation. The amino group is preferably protected by introducing a group such as the t-butyloxycarbonyl group, the t-amyloxycarbonyl group, the p-methoxybenzyloxycarbonyl group, the benzyloxycarbonyl group, the p-nitrobenzyloxycarbonyl group, the trityl group, the 2-(p-diphenyl)-isopropyloxycarbonyl group or the formyl group. To protect the guanidino group in arginine, the nitro group or the tosyl group is preferably used, but it is not always necessary to protect the guanidino group in arginine during the reaction. Also the £-amino group in lysine can advantageously be protected with the t-butyloxycarbonyl group, the t-amyloxycarbonyl group, the benzyloxycarbonyl group or the 2-(p-diphenylj-isopropyloxycarbonyl group. Furthermore, the X<*->carboxyl group in giutamic acid is preferably protected in the form of a lower alkyl ester - such as the methyl, ethyl, propyl or t-butyl ester or lower aralkyl ester such as the benzyl, p-nitrobenzyl or p-methoxybenzy ester or by amide formation, and the imino group in histidine can be protected with the benzyloxycarbonyl group or the benzyl group.

The condensation method for forming peptide bonds includes e.g. the dicyclohexylcarbodiimide method, the azide method, the reactive ester method with e.g. cyanomethyl ester, p-nitrophenyl thiol ester, p-nitrophenyl ester, N-hydroxysuccinic acid ester, tt-quinolyl ester, 1-piperidyl ester, as well as the mixed anhydride method, the N-carboxylic anhydride method, the tetraethyl pyrophosphite method, the ethyl chlorophosphite method or combinations thereof and the usual methods used in peptide chemistry. The desired peptides can also be produced by so-called solid-phase synthesis, in which the peptide is synthesized by starting from the carboxyl end which is linked in an ester-like manner to a polymer, and the amino acids are condensed on, one after the other. All of the aforementioned methods can be used for the formation of any peptide bonds for the production of the present octadecapeptide and derivatives thereof.

As mentioned above, there are several possible routes for the production of the octadecapeptide from the amino acids or peptide units. In one method, the octadecapeptide of formula (3-alanyl-tyrosyl-seryl-methionyl-glutamyl-histidyl-phenylalanyl-arginyl-tryptophyll-glycyl-lysyl-prolyl-valyl-glycyl-lysyl-lysyl-arginyl-argininamide can be prepared by condensing a decapeptide of formula R^-p-alanyl-tyrosyl-seryl-methionyl-Y-R2-glutamyl-histidyl-phenylalanyl-arginyl-tryptophyll-glycine in which R.^ is an amino protecting group and R2 is a carboxyl protecting group, with an octapeptide with formula Nf - R^-lysyl-prolyl-valyl-glycyl-N*" -R^-lysyl-N<£> -R^-lysyl-arginyl-argininamide in which R^ is an amino protecting group in an inert solvent such as eg dimethylformamide, dimethylsulfoxide, pyridine, dimethoxyethane, hexamethylphosphoric triamide, dioxane or mixtures thereof at a temperature of from -20 to +50°C for a period of from 2 to 96 hours, and deprotecting the resulting protected octadecapeptide of formula R ^-6-alanyl-tyrosyl-seryl-methionyl-Y-R2-glutamyl-histidyl-phenyl-alanyl-arginyl-tryptophyll-glycyl-N<£> -R^ -lysyl-prolyl-valyl-glycyl-N<£> _R^-iysyi-N<£ >R^-lysyl-arginyl-argininamide in which R^, R2 and R^ have the previously stated meaning, in and of themselves known ways, e.g. by acidolysis, hydrogenolysis or hydrolysis.

The most preferred method for producing the octadecapeptide is shown in the following synthesis scheme.

In the previous form, the following abbreviations have been used:

KM = carboxyl methyl cellulose, DCKI = N,N'-dicyclohexyl-

diimide, NHR = N-hydroxysuccinimide, BOK = t-butyloxycarbonyl, OBu* = t-butyl ester, R' = NH 2 or OH, Z = benzyloxycarbonyl residue, and ONF = p-nitrophenyl ester.

As shown in the synthesis scheme, the typical N-terminal decapeptide, t-butyloxycarbonyl-3-alanyl-tyrosyl-seryl-methionyl-'Y-t-butyl-glutamyl-histidyl-phenylalanyl-arginyl-tryptophyll-glyein,

which is useful as an intermediate for the preparation of the present octadecapeptide, is prepared by reacting t-butyloxycarbonyl-B-alanyl-tyrosyl-seryl-methionine hydrazide with γ-t-butyl-glutamyl-histidyl-phenylalanyl-arginyl-tryptophyll-glycine using the azLd method. The tetrapeptide hydrazide, t-butyloxycarbonyl-B-alanyl-tyrosyl-seryl-methionine hydrazide,

is useful as an intermediate for

preparation of the octaPeptide and can be prepared by introducing the t-butyloxycarbonyl group into B-alanine by means of a t-butyloxy-carbonylating agent such as e.g. t-butyl azide formate, t-butyl p-nitrophenyl carbonate or t-butyl chloroformate or the like, condense the resulting t-butyloxycarbonyl-B-alanine with tyrosine methyl ester, convert the resulting dipeptide methyl ester to the corresponding hydrazide with hydrazide hydrate, react t-butyloxycarbonyl-B -alanyl-tyrosine hydrazide with seryl methionine methyl ester using the azide method and converting the tetrapeptide ester to the corresponding hydrazide with hydrazine hydrate. The corresponding t-butyloxycarbonyl-B-alanyl-tyrosyl-seryl-methionine can be obtained in a similar manner as described here.

The hexapeptide, f-t-butyl-glutamyl-histidyl-phenylalanyl-arginyl-tryptophyll-glycine is a known compound and is described in the literature, Bull. Chem. Soc.Japan, 38 11^-8 (1965), but it has been successful to synthesize the hexapeptide by means of an improved method which contributes to the production of the octadecapeptide in a purer state and in a higher yield than before. The improved process for the preparation of the hexapeptide involves the use of formic acid to remove the t-butyloxycarbonyl group from the protected tryptophan peptides without cleavage of the tryptophan residue and the use of hydrogen fluoride to remove the nitro group from the nitroarginine residue without affecting the acid-labile tryptophan residue. It is well known that tryptophan-containing peptides treated in an acidic medium are often obtained in low yields due to the formation of by-products, /Reiv.Chim.Acta., k± 1867 (1958), J.Am.Chem.Soc., 82 2062 (196017-It is also known that the reduction of a nitroarginine residue in a peptide chain often requires a long hydrogenation time £Can.J.Chem., 38 19^6 (196017) and that the catalytic reduction often stops at the level of aminoguanidine Æelv.Chim.Acta . , }+ j± 20k2 (196117-

When producing the present hexapeptide, which contains the aforementioned amino acid residues, the hexapeptide can be obtained with a high yield and without side reactions by using the improved method. This means that Y"-t-butyl-glutamyl-histidyl-phenylalanyl-arginyl-tryptophyl-glycine can be prepared by removing the t-butyloxycarbonyl group from Na<->t-butyloxycarbonyl-NG<->nitro-arginyl-tryptophyll -glycine methyl ester with formic acid, reacting the resulting tripeptide with t-butyloxycarbonyl-phenylalanine using the dicyclohexylcarbodiimide method, removing the t-butyloxycarbonyl group from the resulting tetrapeptide, t-butyloxycarbonyl-phenylalanyl-N-nitro-arginyl-tryptophyll-glycine methyl ester with formic acid, reacting the resulting tetrapeptide with Na<->N"'"<m->dibenzyloxycarbonyl-histidine p-nitrophenyl ester to give the corresponding pentapeptide, N , N Im-dibenzyloxycarbonyl-histidyl-phenylalanyl-N Q-nitro-arginyl-tryptophyll- glycine methyl ester, which is hydrolyzed, is treated with hydrogen fluoride and reacted with Na-benzyloxycarbonyl-y-t-butyl-glutamic acid p-nitrophenyl ester, and the resulting N<oc->benzyloxycarbonyl-Y-t-glutamyl-histidyl-phenylalanyl-arginyl-tryptophyll-glycine is hydrogenated.

In the above-mentioned reaction, the cleavage of the t-butyloxycarbonyl group is carried out with formic acid at a temperature of from 20 - 60 C for from 2 to h hours using approximately a five- to fifteenfold amount of formic acid in relation to the t-butyloxycarbonyl peptide ( calculated on the scale). The nitro group is removed by dissolving the nitroarginyl peptide in hydrogen fluoride (approximately five to ten times the weight of the peptide) and the solution is allowed to stand at a temperature of from 0 to 25°C in

30 to 60 min.

The typical C-terminal octapeptide of the formula N<£->t-butyloxycarbonyl-lysyl-prolyl-valyl-glycyl-N^-t-butyloxycarbonyl-lysyl--t-butyloxycarbonyl-lysyl-arginyl-Y wherein Y is an arginine residue or an argininamide residue, can be prepared using the method described in the literature: Bull.Chem.Soc.Japan, 3Ji $82

(1966). The desired octadecapeptide can thus be prepared by

to react t-butyloxycarbonyl-B-alanyl-tyrosyl-seryl-methionyl-^-t-butyl-glutamyl-histidyl-phenylalanyl-arginyl-tryptophyll-glyein with -t-butyloxycarbonyl-lysyl-prolyl-valyl-glycyl-Ne-t -butyloxycarbonyl-lysyl-N^-t-butyloxycarbonyl-lysyl-arginyl-Y in which Y has the above meaning, by means of the dicyclohexylcarbodiimide method, or the N-hydroxysuccinimide ester method or a combination method of these at a temperature of from -20

to +50°C for from 2 to 96 hours and to remove all the protecting groups from the resulting protected octadecapeptide of t-but yloxycarbonyl-B-alanyl-tyrosyl-seryl-meth ionyl-^-t-butyl-glutamyl-histidyl -f enylalanyl-arginyl-tryptophyl-gly cyl-N -t-butyloxycarbonyl-lysyl-prolyl-valyl-glycyl-N<£->t-butyloxycarbonyl-lysyl-N^-t-butyloxycarbonyl-lysyl-arginyl-Y wherein Y

has the meaning stated above, in an acidic medium such as e.g. hydrogen halide, hydrohalic acid, trifluoroacetic acid, formic acid, acetic acid or mixtures thereof at a temperature from

-10 to +lfO°C for from 10 to 90 min.

In another method, the octadecapeptide can be prepared by

to condense the tetrapeptide, t-butyloxycarbonyl-B-alanyl-tyrosyl-seryl-methionine, with a tetradecapeptide, Y-t-butyl-glutamyl (or glutaminyl)-histidyl-phenylalanyl-arginyl-tryptophyll-glycyl-N<£->t-butyloxycarbonyl -lysyl-prolyl-valyl-glycyl-N<£->t-butyloxycarbonyl -lysyl -Nf -t-butyloxycarbonyl-lysyl-arginyl-Y wherein Y

has the meaning stated above, in a manner known per se.

The octadecapeptide can also be prepared by condensing t-butyloxycarbonyl-B-alanyl-tyrosyl-seryl-methionyl-Y-t-butyl-glutamyl-histidyl-phenylalanyl-arginyl-tryptophyll-glycyl-Ne<->t-butyloxycarbonyl-lysyl-prolyl-valyl -glycine with Ne<->t-butyloxycarbonyl-lysyl-N-t-butyloxycarbonyl-lysyl-arginyl-argininamide,

in a manner known in and of itself.

All protective groups used in these processes can be removed by catalytic reduction, using sodium in liquid ammonia, hydrolysis or treatment with an acidic medium such as e.g. trifluoroacetic acid, acetic acid, formic acid, hydrogen halide (e.g. hydrogen fluoride, hydrogen bromide or hydrogen chloride). hydrohalic acid such as e.g. hydrofluoric acid, hydrobromic acid, hydrochloric acid or mixtures thereof.

The obtained octadecapeptide can be purified by methods known per se such as e.g. column chromatography with ion-exchange resin, ion-exchange cellulose or the countercurrent distribution method.

The octadecapeptide is thus produced in the form of bases or pharmaceutically acceptable non-toxic acid addition salts thereof, depending on the reaction conditions used. The salts can be prepared by treating the peptides with inorganic acids such as hydrohalic acids, hydrogen halides, sulfuric acid or phosphoric acid, or with organic acids such as acetic acid, formic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, methanesulfonic acid, benzenesulfonic acid, or toluenesulfonic acid in a manner known per se.

The thus produced octadecapeptide shows marked biological effects such as adrenal-stimulating activity, lipotropic activity and melanocyte-stimulating activity which are superior to those corresponding to natural corticotropin and related known peptides, and is therefore applicable as medicine, especially for the treatment of acute or chronic articular rheumatism and allergic diseases in different organs in humans and animals.

The various pharmacological properties of the octadecapeptide will be shown below together with the biological data.

Tests for corticotropic activity of the octadecapeptide were carried out using five different methods, in comparison with the corresponding ones for native ovine corticotropin (as-ACTH), ACTH(1-18)-NH2, and Gly1 -ÅCTH(1-18)-NR "2. The adrenal ascorbic acid-depleting activity in the hypophysectomized rat was determined by the method according to United States Pharmacopeia XVII, 1^7 (1965). In vitro steroidogenic activity was determined by the method of Saffran and Schally ^Endocrinol., _ £6

523 (195517. In vivo steroidogenic activity by intravenous administration to hypophysectomized rats was determined by the method of Lipscomb and Nelson /JEndocrinol. , 21 13 (196217 with a minor modification <£å. Tanaka and C.H.Li. Endocrinol.Japonica., 13

180 (196617- In vivo steroidogenic activity was also determined in dexamethasone-pentobarbital-pretreated mice £k. Tanaka and N. Nakamura, "Integrative mechanism of neuroendocrine system", Hokkaido University Medical Library Series, Vol. 1_ >+9 (196817-

In addition, steroidogenic activity was determined by intramuscular administration to hypophysectomized rats, where a preparation (0.05 ml/rat) was injected into the thigh muscle and a blood sample was taken from the abdominal aorta 30 min. after the injection. During the experiment, the Third USP Corticotropin Reference Standard was used as a standard and the production of 11-hydroxycorticosteroids (11-OHCS) was determined by the fluorophotometric method of Peterson Biol. Chem. , 22J> 25 (195717- For each test method several determinations were carried out and the independently obtained data were subjected to statistical treatment by means of Sheps and Moore's method. £5. Pharmacol.Extl. Therap. , 128 99 (196017-The results for these tests on the present octadecapeptide

are given in the following table, in comparison with known corticotropin peptides and natural corticotropin.

Note:

The activities are expressed in USP units/mg, relative to Third

USP Corticotropin Reference Standard.., The abbreviations are as

follows: Ia = ACTH(1-1 8)-NH2, Ib = Gly -ACTH(1-18)-NH2,

I = (B-Ala1 )-ACTH(1-18)-NH9. a = ovine corticotropin.

As shown in Table 1, the peptide I prepared by the present method is highly active in all respects in terms of adrenal-stimulating activity. In vivo steroidogenic activity for I has the same order of magnitude as for Ia and Ib and ccg-ACTH when administered by intravenous injection. Peptide I

however, has a strikingly greater activity than peptide Ia and Ib with regard to in vitro steroidogenesis and adrenal ascorbic acid depletion test. Additionally, it is worth noting that the potency ratios for subcutaneous or in vitro vs. intravenous administration is 1 to 0.5 for peptide I, while the ratios for peptides Ia and Ib are 1/7 to 1/8. In this respect, peptide I is much more closely related than compounds Ia and Ib to the natural hormone ag-ACTH, which has a corresponding numerical ratio of 1/1.

Figure 1 shows that (B-Ala1)-ACTH(1-18)-NH2 (I) remains active for a longer time than Gly1-ACTH(1-18)-NH2 (lb) and natural ACTH when the peptides I

and Ib (5 µg/rat) (1 mg peptide/ml) and natural ACTH (125 mU) are administered to the thigh muscle in hypophysectomized rats by intramuscular injection, where the steroidogenic activity is expressed calculated as the 11-hydroxycorticosteroid (11-0HCS) level in plasma .

The octadecapeptide, (B-Ala1)-ACTH(1-18)-WH2 (I) exhibits a

markedly high lipotropic activity which in the following table is compared with the activities of synthetic peptides; ACTH(1-18)-IIH2 (Ia), Gly<1->ACTH(1-18)-NH2 (Ib) and ovine corticotropin (a -ACTH) .

Note:

The lipotropic activity was determined using the method described by Tanaka et al (Arch.Biochem.Biophys., 22. 29^

(1962), with rat epidymal adipose tissue and rabbit perirenal adipose tissue. The increase in the concentration of non-esterified fatty acid in both medium and tissue is the parameter. The activity is expressed as the minimum effective dose per 50 mg tissue.

It is clear from the table that the octadecapeptide I produced by the present method is approximately ten times more active with regard to lipotropic activity than Ia, Ib and α-ACTH when tested in rats. A smaller amount of the compound I can exert activity in rabbits compared to activity in rats. The same relationship is observed with the other peptides Ia and Ib. However, it should be noted that the ratio between the minimum effective dose in rat fat and the equivalent in rabbit fat is smaller for I than for Ib and Ia. This ratio means that the peptide I produced according to the present method has a much greater similarity to natural hormone than the peptides Ia and Ib.

Figures 2 and 3 show the inactivation patterns for I and Ib as lipotropic agents in fresh plasma respectively. in buffer containing rat muscle fragments. There was no substantial difference between I and Ib in the case of plasma, while on the other hand the inactivation for I was markedly delayed compared to Ib in the case of tissue. Inactivation of lipotropic activity of plasma and tissue was carried out as follows. A peptide sample is dissolved in the fresh plasma from anesthetized rats or in Krebs-Ringer bicarbonate buffer containing bovine serum albumin, rat thigh muscle fragments, and glucose. The mixture is then incubated at 37°C and samples are taken from the incubation mixtures and tested for lipotropic activity using the above-mentioned method after 0.5, 15 2 and k hour incubation times.

The biological half-life of (6-Ala1)-ACTH(1 -18)-NH2 (I) injected intravenously into hypophysectomized rats weighing 200 - 250 g was measured. Blood samples were taken from the abdominal aorta at various times after the injection. The withdrawn plasma was adjusted to pH h - 5 with N hydrochloric acid and the parameter for the biological activity remaining in the plasma was in vitro lipotropic activity, using the aforementioned rat epidymal fat. The results are shown in the following table in comparison with lipotropic activity for natural corticotropin (ACTH) and Gly1-ACTH(1-18)-NH2 (Ib).

From the data in the above table, the half-life of natural ACTH, Ib and I with respect to in vitro lipotropic activity was calculated to be 266 sec., 117 sec. and 188 sec., and the lipotropic activity samples for the peptides are given in fig. k. It can be said that (P-Ala<1>)-ACTH(1-18)-NH2 (I) is retained in the plasma for a significantly longer period of time than Gly<1->ACTH(1-18)-NH2 (Ib) .

Similarly, the biological half-life of I injected intramuscularly in hypophysectomized rats was measured.

The results are given in the following table and fig.

Note: In the above columns, the numbers in parentheses are relative lipotropic activities for the values obtained at 10 min. after injection of each hormone.

The half-life for ACTH, Ib and I was calculated to be 1389 sec., 6 sec. and ^516 sec. It is noted that the half-life of I prepared by the present method is approximately three times as long as that of natural corticotropin, which is clearly evident from fig. 5-

The stability of (B-Ala<1>)-ACTH(1-18)-NH2 (I) against the action of porcine kidney leucine aminopeptidase (LAP) was compared with that of ACTH(1-18)-NH2 (Ia) and Gly<1->ACTH(1-18)-NHg (Ib). Fig. 6 shows the effect of diisopropylfluorophosphate (DFF)-treated aminopeptidase on peptides I, Ia and Ib. It can be seen from the figure that the octadecapeptide produced by the present method can be preserved for a longer time than the known peptides Ia and Ib without rapid inactivation by an attack by aminopeptidases in the tissue, and the peptide I is therefore very useful for therapeutic purposes. The stability tests for the synthetic peptides will be described in detail in the following.

For the sample, a commercially available LAP preparation (Product

of Washington Biochemical Co., Freehold, New Jersey, USA) found to hydrolyze (B-Ala<1>)-ACTH(1-10)-OH (prepared from the corresponding protected decapeptide t-butyloxycarbonyl-B-alanyl-tyrosyl- seryl-methionyl-γ-t-butyl-glutamyl-histidyl-phenylalanyl-arginyl-tryptophyll-glycine (XVI) by treatment with formic acid) and (3-ala1 )-ACTH(1-l8)-NH2 (I), but not p- alanyl-tyrosyl-seryl-methionine hydrazide (prepared from t-butyloxycarbonyl-B-alanyl-tyrosyl-seryl-methionine hydrazide (VII) by treatment with formic acid). Since no 8-alanine could be detected in the enzymatic samples using the amino acid analyzer, it must be observed that hydrolysis is due to the presence of an endopeptidase that contaminates the LAP preparation. This apparent lack of stability of B-alanyl peptides towards the LAP preparation completely disappeared when the enzyme preparation was treated with diisopropylfluorophosphate (DFF). With the DFF-treated LAP, Ser 1 peptide (Ia) was hydrolyzed faster than Gly 1 peptide (Ib) and no hydrolysis was observed with B-Ala 1-

peptide (I) within measurement accuracy.

The DFF-treated LAP was obtained by dialyzing a suspension

of enzyme in 75% saturated ammonium sulfate at <1>+°C for use against 0.005 M

magnesium chloride, dilution of LAP suspension (5 mg enzyme) to 1/5 concentration with 0.0005 M Tris buffer, addition to the diluted enzyme solution of 0.08 ml M diisopropylfluorophosphate in isopropyl alcohol, the mixture is incubated at 37 C for M-5 min . and then the mixture is dialysed against 0.0005 M Tris buffer at h°C.

The LAP activity of the enzyme solution He determined at pH 8.3 and 25°C according to Tuppy et al Z2.Physiol.Chem., ^29_ 278 (196217 with some modifications using L-leucine p-nitro-anilide (LNA ) as substrate.One LAP unit was defined for this purpose as the amount of enzyme which produced an increase in adsorption at <!>+00 rn^i of 0.001 per min over the control sample, using 1-cm cells in a Hitachi photoelectric spectrometer.

For the measurements of the cleavage of synthetic substrate by LAP, a mixture comprising one part 0.1 mM substrate in water, one part 0.05 M Tris buffer (pH 8.3) and one half part enzyme (^.88 units/ml, LNA as substrate) incubated at 37°C. From the incubated mixture, 0.5 ml of samples were withdrawn and immediately mixed with 0.5 ml of ninhydrin reagent and the mixture was then heated in a boiling water bath for 15 min. After cooling and appropriate dilution with 50% ethanol, adsorption at 570 mp was determined in a Hitachi spectrophotometer. For each sample, control incubation mixtures without substrate or without enzyme were analyzed. In the riLnhydrin reaction, a standard solution of L-leucine was also checked each time as a reference. The results for the enzymatic cleavage of peptides 1, Ia and Ib using DFF-treated LAP are shown in fig. 6 as mentioned above, where the release of amino acids is indicated on the basis of the leucine concentration.

The test for in vitro melanocyte-stimulating activity of (6-Ala<1>)-ACTH(1-18)-NH2 was carried out by a method developed by K. Shizume, A.B. Lerner and T.B. Fitzpatrick (Endocrinol., $ h 553, 195^) using Rana pipiens as experimental animal, in comparison with Gly<1->ACTH(1-18)-NHg and ACTH(1- 2h)-OH ("Synacthen", manufactured by Ciba Co.). Natural pure α-melanocyte-stimulating hormone (oc-MSH) was used as a reference standard. The results are shown in the following table.

The melanocyte-stimulating activity of (B-Ala1)-ACTH(1 -18)-

NB.2 is strikingly higher than for Gly 1-ACTH( 1 -18)-NH2 and ACTH(1-2^-)-OH. In addition, since natural porcine corticotropin exhibits melanocyte-stimulating activity of 1.7 x 10^ units/g as reported by R. G. Shepherd et al., J.Am.Chem.Soc., 23. 5051

(1956)? the melanocyte-stimulating activity of (B-Ala^)-ACTH(1-18)NH2 produced by the present method is about 50 times as high as that of natural porcine corticotropin.

The octadecapeptide produced by the present method can be converted into corresponding complexes with compounds such as zinc chloride, zinc hydroxide, zinc acetate and zinc sulfate, or with the poly-amino acids poly-L-glutamic acid, poly-D-glutamic acid, poly-DL-glutamic acid, poly-L- aspartic acid, poly-D-aspartic acid, poly-DL-aspartic acid, co-poly-L-glutamyl-tyrosine or mixtures thereof, which show excellent properties with respect to prolonged activity in the blood compared to the single octadecapeptide. When e.g. a zinc complex of (8-Ala<1>)-ACTH(1-18)-NH2, comprising the peptide (0.5 mg), kO mM zinc chloride (0.25 ml), sodium chloride (^t-.5 mg) and ^-0 mM disodium hydrogen phosphate (0.25 ml) administered to rats by intramuscular injection (5 µl/rat), the amount of 11-hydroxycorticosteroid in plasma (2 hours after the injection) was found to be 80 µg/ml, but in the case of the preparation lacking the zinc chloride, the amount of corticosteroid was 6 µg/ml. It is therefore advantageous to use the complexes for therapeutic purposes. The complexes can be obtained by treating the octadecapeptide with a zinc-

compound, a polyamino acid or mixtures thereof in a weight ratio of about 1:0.1-20 under slightly acidic conditions,

especially pH 6.5 to 7?0. When using the polyamino acid, the preferred molecular weight for the polyamino acid is from 1,000 to 100,000, especially 2,000 to 6,000. When preparing the complexes, other suitable carriers, preservatives, buffers, stabilizing agents and isotonizing agents can be added if required.

The sample data described above are exemplary. The compounds produced by the method according to the invention are generally very useful and advantageous for therapeutic purposes, e.g. treatment of acute or chronic articular rheumatism and allergic disorders in various organs in animals and humans.

The subject octadecapeptide, acid addition salts and complex compounds thereof can thus be administered orally or parenterally in ways known per se, e.g. in the form of injections, liquids, suspensions, emulsions, aerosols or tablets, or in admixture with suitable carriers, stabilizers, emulsifiers, preservatives, buffers, isotonizing and/or humectants, only the preparations contain a therapeutically active amount of the active constituent.

The effective dose can be easily determined by medics on the basis of

the data described herein. E.g. is a typical clinical dose of

0.2 U/kg to 0.8 U/kg for normal adults. The octadecapeptide is advantageously administered in dose form by injection and the administration is repeated as often as required in accordance with the doctor's instructions.

The following examples illustrate the invention. Weight parts i

these examples have the same ratio to parts by volume as g to ml.

Example 1

Preparation of Na-t-butyloxycarbonyl-8-alanyl-tyrosyl-seryl-methioninehydrazine (VII)

1) Na-t-butyloxycarbonyl-B-alanine (I).

8-Alanine (8.91 g) is dissolved in N sodium hydroxide (100 ml) and sodium bicarbonate (21.0 g) and dioxane (70 ml) are added to this solution. The solution is stirred at 50°C and a solution of t-butylazide formate (7.2 g) in dioxane (30 ml) is added dropwise thereto. The mixture is stirred for 1.5 hours at the same temperature and concentrated under reduced pressure to approximately 100 ml vol.um. The concentrate is cooled with ice and adjusted to pH 2 with VN hydrochloric acid (180 ml). The solution is extracted with ice-cold ethyl acetate (about 100 ml), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a syrupy residue. The residue is crystallized from ethyl acetate/petroleum ether to give the desired product (12.6 g) with a melting point of 77 to 78°C.

Analysis calcd for CgH^NO^: C, 50.78; H, 7.99; N, 7.^0.

Found: C, 50.99; H, 8.06; N, 7.1*7.

2) Na<->t-butyloxycarbonyl-6-alanyl-4-yrosine methyl ester (II)

Tyrosine methyl ester hydrochloride (3.8 g) is dissolved in water (15 ml).

50% potassium carbonate (5 ml) is added to the solution while cooling with ice and the mixture is allowed to stand at •+ C for *f0 min. The crystals formed are collected by filtration, washed with cold water and dried in vacuo to give tyrosine methyl ester (2.55 g).

On the other hand, N OL-t-butyloxycarbonyl-|3-alanine dissolves

(1.89 g) obtained as above in anhydrous tetrahydrofuran and the solution cooled to -10°C. A t-butylamine (2.0*f g) and ethyl chloroformate (1.19 g) are slowly added to the solution with stirring. After 10 min. is added a suspension of tyrosine methyl ester (1.95 g) obtained in relation to the preceding in anhydrous

tetrahydrofuran (20 ml). The mixture is stirred for 30 min. at -10°C and for 2-g-hour at room temperature and the solvent is removed by evaporation under reduced pressure. The resulting residue is dissolved in ethyl acetate, washed first with N hydrochloric acid, water, 5% > sodium bicarbonate and finally with water and dried over sodium sulfate. After removal of the solvent by evaporation under reduced pressure, ether is added to the residue obtained to precipitate crystals. After standing overnight, the crystals formed are collected by filtration and concentrated in vacuo to give the desired dipeptide ester (2.99 g). Recrystallization from methanol/ether gives crystals melting at 1^-1 to 1<i>f2°C and having an optical rotation of Za7p<3> = +8.2*0.5° (c = 1.020 in methanol).

Analysis calculated for C~i8H26N2°6: C, 59.00; H, 7.15; N, 7.65-Found: G, 59.03; H, 7.12; N, 7.63. 3) Na<->t-butyloxycarbonylB-alanyl-tyrosine hydrazide (IV). Na-t-butyloxycarbonyl-8-alanyl-tyrosine methyl ester (2.56 g) is dissolved in anhydrous ethanol (26 ml). Hydrazine hydrate (1.7 ml) is added to the solution and the mixture is kept at room temperature for 5 hours and at h°C overnight. The crystals formed are collected by filtration, washed with ethanol and ether and dried to give the desired dipeptide hydrazide (2.5<*>+ g). Recrystallization from hot water gives crystallites with melting point 213.5 to 215°C with optical rotation Za7^<3>= +3.6oio.6° (c= 0.978 in 50% acetic acid).

Analysis calcd for C 2 H 2 N 2 O 2 : C, 55.72; H, 7.15; N, 15,29.

Found: C, 55.7^; H, 7.11; N, 15.30.

■+) Na<->t-butyloxycarbonyl-B-alanyl-tyrosyl-seryl-methionine methyl ester (VI).

Na-t-but<y>lox<y>carbon<y>l-B-alanyl-tyrosine hydrazide (1.10 g)

dissolve in cold N hydrochloric acid (7.5 ml). The solution is kept at about -10°C and cold 2M sodium nitrite (3.6 mL) is added to the solution. After resting the mixture for k min. be extracted

the azide produced with ether, washed with cold M sodium bicarbonate and dried over anhydrous sodium sulfate. After removal of the solvent by evaporation under reduced pressure at a bath temperature of approximately 10°C, the obtained residue is dissolved in cold acetonitrile. To the solution is added seryl methionine methyl ester (0.756 g)' (prepared according to the method described in the literature: Bull. Chem. Soc. Japan, 2. 11 71 (1966) and the mixture is discarded for ^8 hours. The solvent is removed by evaporation under reduced pressure and the residue is dissolved by the addition of ethyl acetate and parts of water. The solution is washed successively with cold N hydrochloric acid, water, 5% sodium bicarbonate and water and dried over anhydrous sodium sulfate. After removal of the solvent by evaporation under reduced pressure, the resulting gelatinous product is collected by filtration, washed with cold ethyl acetate and ether and dried to give the desired tetrapeptide methyl ester (1.20 g). The product is purified by renewed precipitation from ethyl acetate.

Melting point: 121.5 to 123°C.

/o7p<3> = -13.0 - 0.6° 0.997 in methanol)

RF 0.53 (by thin-layer chromatography in the solvent system methanol:acetic acid = 15:85)

Analysis calculated for C^H^iy^S: C, 52.^-1; H, 6.90; N, 9.58;

S, 5,^8.

Found: C, 52.9^; H, 6.85; N, 9.65; S, 5,if8.

5) Na<->t-butyloxycarbonyl-B-alanyl-tyrosyl-seryl-methionine-

hydrazide (VII).

Na<->t-butyloxycarbonyl-8-alanyl-tyrosyl-seryl-methionine methyl ester (0.97 g) is dissolved in dimethylformamide (6 ml). To the solution is added hydrazide hydrate (0.5 ml) and the mixture is set aside at k°C for approximately ^3 hours. The solution is added to ethyl acetate and forms a gelatinous material which is collected by filtration, washed with cold ethyl acetate and dried to give the desired tetrapeptide hydrazide (1.01 g). Recrystallization from water yields crystals melting at 186° to 188°C and having an optical rotation of

fqj^ > = -20.1<i>o.5° (c = 1.3^6 in 50% methanol).

Analysis calculated for C^H^OgNgS-HgO: C, ^-9.82; H, 7.02; N, 13.9^5

S, 5.32.

Found: C, M-9.72; H, 7.05; .N, lM-.50-, .S, 5.15- ■

Preparation of")^ - t- butyl- glutamyl- histidyl- phenylalanyl- arginyl- tryptophyll- glycine ( XV). 1) nitroarginyl- tryptophyll- glycine methyl ester formate (IX). t- butyloxycarbonyl- N - nitro- arginyl- tryptophyll- glycine methyl ester ( 5.77 g) is dissolved in formic acid (98 -'100%, 50 ml) and the solution is allowed to stand for 3^ hours. The formic acid is removed by evaporation at a bath temperature of ^0 - ^5°C and the residue is dissolved in water. The solution is washed with ether and lyophilized to give the desired product (^.86 g) with a melting point of 107 to 111°C and with an optical rotation of Z«7^ = +13.0-0.5° (c = 2.062 in methanol).

Analysis calculated for C20H2gNg06.HC00H-iH20: C, k?,^5; H, 5.88;

N, 21.08.■

Found: C, ^7.25; H, 6.03; N, 20.90. 2) t-butyloxycarbonyl-phenylalanyl-N Q -nitro-arginyl-tryptophyll-glycine methyl ester (X).

This compound is synthesized in exactly the same way as described in the literature: Bull. Chem. Soc. Japan, 3_8 11^8 (1965) with the exception of the use of the tripeptide ester formate obtained above in place of the trifluoroacetate. The desired compound is obtained in a yield of <*>+.06 parts by weight by reacting N r*-nitro-arginyl-tryptophyll-glycine methyl ester formate (^-.81 parts by weight) with t-butyloxycarbonyl-phenylalanine dicyclohexylamine salt ( h,]+ 2 parts by weight) in acetonitrile using the dicyclohexylcarbodiimide method. The product melts at 176 to 177°C and has an optical rotation of 23

&B = -20.k-2° (c = 2 in methanol).

Analysis calculated for C^H^N^Cy C, 56,+2; H, 6.27; N, 17,^2.

Found: C, 56.3+; H, 6.22; N, 17.58.

3) F(XenIy)l.alanyl-N Q-nitro-arginyl-tryptophyll-glycine methyl ester formate

t-Butyloxycarbonyl-phenylalanyl-N Q -nitro-arginyl-tryptophyll-glycine methyl ester (1.81 g) is dissolved in formic acid (18 ml) and the solution is kept at room temperature for 3-h. After removing the formic acid, the residue is crystallized from N-butanol. The crystalline product is filtered off, washed with ether and dried in vacuum. Yield (1.71 g). The product melts at 12+ to 125.5°C and has an optical rotation

At /«T^<3> = -9.0^0.5° (c =.0.977'' in methanol).

Analysis calculated for C^<H><0> -HCOOH-iHgO: C, 5^,3<8>; H, 6.28;

N, 1 7.85+.

Found: C, 53.00; H,6,10; N, 17.79.

+) N<a>,N<Im->dibenzyloxycarbonyl-histidyl-phenylalanyl-NG<->nitro-arginyl-tryptophyll-glycine methyl ester (XII).

This peptide is obtained in a similar manner by the method described in Bull.Chem. Soc. Japan. , 3ji 11 ^8 (1965), with the exception that the tetrapeptide ester formate obtained above is used instead of the corresponding trifluoroacetate. That is that the pentapeptide ester is prepared from phenylalanyl-N Q-nitro-arginyl-tryptophyll-glycine methyl ester formate (2.60 g) and N<a>,N<Im->dibenzyloxycarbonyl-histidine p-nitrophenyl ester (1, 91 parts by weight). Yield 2.70 g. Optical rotation of the product is = -22.3^0.3° (c = 2.08+ in dimethylformamide).

Analysis calculated for C^H^N., 2'H2°: <C>' ?8'?°5 H> 5>58> N? 16.05.

Found: C, 58.63; H, 5.70; N, 15.89.

5) Histidyl-phenylalanyl-arginyl-tryptophyll-glycine monoacetate (XIII)

Not,N"'"m-dibenzyloxycarbonyl -histidyl-phenylalanyl-N^-nitro-arginyl-tryptophyll-glycine methyl ester (1.++ g) is hydrolyzed in 90% dioxane and lyophilized from acetic acid to berj.zyloxycarbonylhistidyl- ferryl-alanyl-N-nitro-arginyl-tryptophyll-glycine (1.35 g) by the method described in Bull. Chem. Sog ., Japan, 3J3 11+8

(1965).

The pentapeptide (0.72 g) obtained as above and anisole

(0.72 mL) is dissolved in anhydrous hydrogen fluoride (7 to 8 mL) at a carbon dioxide ice/acetone bath temperature and the mixture is stirred at 0°C

for 30 min. After removing the hydrogen fluoride by evaporation, the residue is washed over sodium hydroxide beads overnight in a vacuum. The residue is dissolved in water (20 ml) and the solution is washed thoroughly with ethyl acetate and then passed through a small column of "Amberlite" CG-+00 (the acetate form). The column is washed with quantities of water. The aqueous effluents are combined/lyophilized. Yield (0.6+ g,

^Six"<HC1> = 279 W <^cm 66.2), 288 m Ce]<*>, 53.5) and ^<0.1>s» <Na>OH = 280 ^ (B<*> 65)0))238 m (e,Jb 5S5).

Paper chromatography (n-butanol:acetic acid ipyridine:water = 30:6: 20:2+ parts by volume as solvent) shows the presence of a major component (Rf = 0,+7 to 0.53) °g two trace amounts which were all reactive towards ninhydrin, Pauly and Ehrlich reagents. Only the main part is reactive towards Sakaguchi's reagent. 6) Benzyloxycarbonyl-V-t-butyl-glutamyl-histidyl-phenylalanyl-arginyl-tryptophyll-glycine monoacetate (XIV).

A solution of compound (XIII) (0.83 parts by weight) and triethylamine (0.28 ml) in 90% dimethylformamide (10 ml) is stirred at 0°C and benzyloxycarbonyl-Y-t-butyl-glutamic acid p-nitrophenyl ester (0,+ 6 g) and the mixture is stirred at +°C overnight. A further amount (0,+6 g) of the active ester is introduced and the mixture is kept at +°C until the pentapeptide (XIII) can no longer be detected by thin layer chromatography. The reaction mixture is then added dropwise to ice-cold ethyl acetate (100 ml) and gives a gelatinous precipitate which is filtered off, washed with ethyl acetate and dried in vacuum. The product is precipitated again from acetic acid-water and gives the desired hexapeptide. The yield was 0.8+ parts by weight,

/*7jp = -26.6-0.5° (c = 1.377 in 50% acetic<y>re).

Analysis calculated for C^H^^ ^ 1 • CH^OOH^THgO : C, 52.86;

H, 6.7>+; N, 13.98.

Found: C, 52.73; H, 6.85; N, 13.92. 7) Y-t-butyl-glutamyl-histidyl-phenylalanyl-arginyl-tryptophyll-glycine monoacetate (XV).

The compound (XIV) (0.265 g) in 50% acetic acid (10 ml) is hydrogenolyzed in the presence of palladium black for 2 g-hour. After the catalyst has been removed by filtration, the filtrate is evaporated under reduced pressure at a bath temperature of +5 to 50°C. The residue is lyophilized from acetic acid and dried over sodium hydroxide beads in vacuum. Yield was 0.22 g. The product behaves as a single component by paper chromatography (n-butanol:acetic acid: water = 1+:1:2 parts by volume as solvent).

Analysis calculated for C^H^g^gO^CH^COOH^HgO: C, +9.53; H, 7.21;

N, 15.^0.

Found: C, +9, +6; H, 6.77; N, 15,^9.

Preparation of t- butyloxycarbonyl- 3- alanyl- tyrosyl- seryl- methionyl- 7- t- butyl- glutamyl- histidyl- phenylalanyl- arginyl- tryptophyll- glycine ( XVI)

Ice-cold N hydrochloric acid (2.0 ml) and ice-cold M sodium nitrite (0 .83 ml)

and the mixture is stirred for + min. Ice-cold saturated sodium chloride (20 ml) and ice-cold ethyl acetate are then introduced. The aqueous layer which separates is extracted twice with ethyl acetate. The organic solutions are combined, washed with cold 5% sodium bicarbonate, dried over sodium sulfate and added to a solution of XV (0.50 g) and triethylamine (0.21 ml) in dimethylformamide (15 ml) and the mixture is evaporated at a bath temperature of 10 to 15 C under reduced pressure to remove the ethyl acetate. The resulting clear solution is kept at +°C overnight. A further amount of the azide (prepared from 0.23 g of VII as described above) is introduced and the reaction mixture is kept at +°C for a further dbgn. It

is then added dropwise to ice-cold ethyl acetate (200 ml) to give an amorphous precipitate which is filtered off, washed with ethyl acetate and dried in vacuo with a yield of 0.69 g. Renewed precipitation from dimethylformamide methanol (2:5) gives the pure decapeptide derivative with optical drainage > on A/Jp = -19.^-0.7° (c = 0.882 in dimethylformamide). The product behaves as a single component (Rf = 0.5*+ to 0.57) with thin layer chromatography (dimethylformamide:ethyl acetate:acetic acid = 15:10:2 parts by volume as solvent).

Analysis calculated for C.gH^N.j ^ S*8H 2 O: C, 51.57; H, 7.00;

N, 1+.15; S, 2.02.

Found: C, 51.62; H, 6.66; N, 1+.06; S, 2.6+.

Preparation of 3- alanyl- tyrosyl- seryl- methionyl- glutamyl- histidyl- phenylalanyl- arginyl- tryptophyll- glycyl- lysyl- prolyl- valyl- glycyl- lysyl- lysyl- arginyl- argininamide ( XX)

To a solution of compound XVI (0.37g) in dimethylformamide (10ml) is added ice-cold N hydrochloric acid (0.25ml) at 0°C and the solution is added at once to ice-cold ethyl acetate/ether (1:1), (200 ml). The resulting precipitates are filtered off, washed with ether and dried in vacuo with a yield of 0.36 g. These precipitates were dissolved in dimethylformamide (5 ml) and N-hydroxysuccinimide (0.115 g) and N,N'-dicyclohexylcarbodiimide (0 .21 g)

in turn and the mixture is allowed to stand at +°C overnight. After removal of excreted urine, the filtrate is introduced into ice-cold ethyl acetate-ether (1:1, 200 ml). The precipitates are filtered off, washed with ether and dried in vacuo to give the active decapeptide ester with a yield of 0.39 g.

Triacetate of i'-t-butyloxycarbonyl-lysyl-prolyl-valyl-glycyl-N<£> -t-butyloxycarbonyl-lysyl-N<e> -t-butyloxycarbonyl-lysyl-arginyl-argininamide (0.23 g) is dissolved in dimethylformamide (3 ml)

and triethylamine (0.2+ mL) is added. To this is added a solution of the active decapeptide ester obtained above (0.39 g) in dimethylformamide and the reaction mixture (total volume approx. 5 ml) is allowed to stand at +°C for 60 hours. The crude protected octadecapeptide is obtained as an amorphous solid when the reaction mixture is introduced.

in cold ethyl acetate (100 ml) with a yield of 0.55 g.

The protected peptide obtained above (0.55 g) is dissolved in

to remove the protection in 90% trifluoroacetic acid (6 ml) and the solution is kept at room temperature for 60 min. after which the solvent is removed by evaporation under reduced pressure. The residue is dissolved in water and the solution is passed through a column (2.2 x 7 cm) of "Amberlite" CG-+00 (the acetate form). The column is washed several times with water. The aqueous solutions are combined and concentrated to 10 to 15 mL under reduced pressure. M mercaptoethanol (1 ml) is added to the concentrate and the mixture is incubated at 37°C overnight and lyophilized. The crude deblocked peptide (0.51 g) thus obtained is subjected for purification to chromatography on a column (2.7 x 12 cm) of carboxymethyl cellulose (CMC, "Serva" 0.6 meq/g) using an ammonium acetate buffer (pH 6.8, +000 parts by volume) with a linear concentration gradient of 0.05 to 0.<*>+ M. The fractions (15 ml/ test tube) corresponding to a main peak (samples 166-2+5) are combined and the main part of solvents removed under reduced pressure. The residue is repeatedly lyophilized to constant weight and yield

the partially purified octadecapeptide with a yield of 0.22 g.

The product (0.2 g) is chromatographed for further purification on a column (2.2 x 18 cm) of CMC ("Serva" 0.6 meq/g) using an ammonium acetate buffer (pH 6.8, 2000 ml) with a linear concentration

-gradient of 0.05 to 0.8 M). The fractions (7.5 ml/sample) corresponding to a major peak (samples 125 to 160) are combined, concentrated and lyophilized to give the pure peptide, B-alanyl-tyrosyl-seryl-methionyl-glutamyl-histidyl-phenylalanyl-arginyl-tryptophyl- glycyl-lysyl-prolyl -valyl-glycyl-lysyl-lysyl-arginyl-argininamide (0.17 g) Dtff = -5^,0-1.8° (c = 0.515, 0.1 N acetic-

(E /o 17.5) and ?\0'1 N NaOH= 281.5 mM (E /o 2+.9), 288 mM

1 cm ' max ' 1 cm ' '

^1 1 9c^m 2*+,3). The peptide behaves as a single component towards ninhydrin, Pauly, Ehrlich, Sakaguchi and methionine-(Ptlg") reagents by paper chromatography (n-butanol:acetic acid:pyridine:water = 30:6:20:2+ as solvent ) and in paper electrophoresis (600 V/36 cm in 2N acetic acid). The amino acid ratios in the acid hydrolyzate are as follows: Ser 0.75, Glu 0.90, Pro 0.91,

Gly 1.89; Val 1.00, Met 0.96, Tyr 1.01, Phe 0.97, P-Ala 1.00,

Lys 2.83, His 0.92, Arg 2.82, Trp 0.55, NH3 1.1+2. The Trp/Tyr ratio in intact octadecapeptide is determined spectrophotometrically to be 1.16.

Example 2

(B-Ala1)-ACTH(1-18)NH2acetate (5 g) is dissolved in 10 mM zinc chloride (2.5 ml). The solution is added to a solution (2.5 ml) of h0 mM disodium hydrogen phosphate containing sodium chloride (*+5 g)

to prepare a suspension of the desired complex.

The mixture is administered to hypophysectomized rats by intramuscular injection (5 .ul/rat). After two hours, an amount of 11-hydroxycorticosteroid in plasma was determined to be 80 µg/dl.

In contrast, when supplying a preparation obtained on

in the same manner, but with the exclusion of zinc chloride, the amount of 11-hydroxycorticosteroid was 6 /ag/dl.

The test was carried out according to the method described

in Endocrinol. Japanica., 23, 180 (1966).

Example

(B-Ala1)-ACTH(1-18)-NH2-acetate (10 g) is dissolved in distilled water, (2 ml). To the solution is added with stirring a solution of poly-L-aspartic acid (20 g molecular weight of approximately 3-000) which has been neutralized with 0.1 N sodium hydroxide (3 ml) for use and a suspension of a given precipitate is formed in this way . The desired suspension of the complex is obtained by further addition to the suspension obtained above of a solution (5 ml) at pH 6.8 of M/15 disodium hydrogen phosphate/dipotassium hydrogen phosphate containing sodium chloride (90 g). The preparation is administered in hypophysectomized rats by intramuscular injection (5 µl/rat). Two hours after the injection, the amount of 11-hydroxycorticosteroid

steroid in plasma determined to be +5 P- g/ eel, but the amount that could be detected when a preparation without poly-L-aspartic acid produced on

the same way was used, was found to be 6 jag/dl.

Example h

([3-ala1 )-ACTH(1-18)-NH2 acetate (5g) is dissolved in distilled water (1.5 ml). A solution (1 ml) of poly-L-glutamic acid (5 g) with a molecular weight of approximately 1,500-2,000 is added to the solution

which has been adjusted with 0.1 N sodium hydroxide. A suspension of the precipitated complex is formed. By adding a solution of M/30 phosphate buffer (pH 7.0) (2.5 ml) containing sodium chloride (90 parts by weight) to the suspension, the desired preparation form of the complex is obtained.

Example

(3-ala1)-ACTH (1-18)-NH2 acetate (5 g) is dissolved in 100 mM zinc chloride (15 ml). On the other hand, poly-L-glutamic acid (5

g) with molecular weight 1,500 to 2,000 with 0.1 sodium hydroxide (1 ml)

and sodium chloride (*+5 g) and ho mM are added to the solution

disodium hydrogen phosphate (25 ml). The thus obtained solution of the octadecapeptide and a solution of the polyglutamic acid are combined to form a suspension of the desired complex.

Claims (1)

Analogy method for the production of the new, therapeutic active octadecapeptide 3-alanyl-L-tyrosyl-L-seryl-L-methionyl-L-glutamyl-L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophyll-glycyl-L-lysyl-L-prolyl-L- valyl-glycyl-L-lysyl-L-lysyl-L-arginyl-L-argininamide, non-toxic acid addition salts thereof, or complexes thereof with zinc, polyglutamic acid, polyaspartic acid, copoly-(glutamyl-tyrosine) or mixtures thereof characterized in that the amino acids 3-alanine, L-tyrosine, L-serine, L-methionine, L-glutamlic acid, L-histidine. L-phenylalanine, L-tryptophan, glycine, L-lysine, L-proline, L-valine and L-arginine in the desired sequence and/or peptide fragments containing keeping these amino acids in the desired sequence, is condensed to the indicated octadecapeptide using methods known in peptide chemistry, with a terminal carboxyl group and/or amino group being activated for coupling and the other reactive groups are intermediately protected, and the resulting octadecapeptide is optionally transferred into the salts or complexes in a manner known per se.