DK149272B - METHOD OF ANALOGUE FOR THE PREPARATION OF LHRH ANALOGUE NONA OR DECAPEPTIDES OR PHARMACEUTICAL USE OF ACID ADDITION SALTS THEREOF - Google Patents

Description

149272

The present invention relates to an ahaalog process for the preparation of novel LHRH analogous nona or decapeptides having luliberin agonist properties, luliberin being the internationally recognized trivial name for LH-RF (luteinizing hormone release factor) (J. Biol Chem., 1975, 250, 3215), or pharmaceutically useful acid addition salts thereof.

It is known (Dutta, Furr, Giles, and Morley, Clinical Endocrinology, 1976, 5, Supplement, pages 291s-298s) that substitution of cc-aza-amino = acids at positions 6 or 10 of luliberin produces compounds that are less potent than the parent molecule in their ability to release Luteinizing Hormone (LH) from the pituitary gland, and from Biochem. Biophys.

tes. Commun., 49 (1972) 863-869, and 67 (1975) 576-581, substituted for Gly10 with ethylamino in LHRH and in some of its analogs wherein

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shelter has also been replaced with other amino acids can result. either greater or less ability to release LH compared to the corresponding Gly ^ parent compound. It has now been found that substitution of azaglycine at position 10 combined with substitution of various Da amino acids at position 6 in luliberin or substitution of azaglycine or azalanine at position 6 combined with replacement of 5 terminated glycinamide with an ethylamino radical in luliberin produces compounds , which are more active than luliberin and, in part, also than luliberin, in which Gly is replaced with various Da amino acids, in their ability to release luteinizing hormone.

According to the invention, an analogous method for preparing LHRH analogous nona or decapeptides of the general formula is disclosed:

^ -Glu-His-Trp-Ser-Tyr-A-B-Arg-Pro-E-F I

where A is D-Tyr, D-Tyr (Me), D-Ser (Bu **) or D-Phe, B is Leu or Meleu, E is Azgly, and F is an amino radical, or A is Azgly or

Azala, B is Leu, E is the direct bond, and F is an ethyl amino radical, or pharmaceutically useful acid addition salts thereof.

In the above formula I and throughout the present disclosure, the amino acid residues are denoted by their standard abbreviations (Pure and Applied Chemistry, 1974, 40, 317-331). An α-aza amino acid residue is one in which a-CH in an amino acid has been replaced with nitrogen. The abbreviation for an α-aza amino acid is derived from the abbreviation for the corresponding amino acid by inserting the prefix "Az". Azgly is thus aza-glycine, and Azala is azalanine. Where the configuration of a given amino acid is not indicated, this amino acid (other than the α-aza amino acids, which contains no asymmetric center adjacent to the carboxy group) has the natural L configuration.

A preferred group of compounds that can be prepared by the process of the invention are those wherein A is D-Tyr (Me), D-Ser (But) or D-Phe, B is Leu or MeLeu, E is Azgly, and F is an amino radical.

The three preferred compounds which can be prepared by the process of the invention have the following structures:

Qnu-His-Trp-Ser-Tyr-D-Phe-Leu-Arg-Pro-Azgly-NH2 3 149272

Glu-His-Trp-Ser-Tyr-D-Tyr (Me) -Leu-Arg-Pro-Azgly-NH2 Glu-His-Trp-Ser-Tyr-D-Ser- (But) -Leu-Arg-Pro Azgly-NH2 A particularly pharmaceutically useful acid addition salt prepared according to the invention is e.g. a hydrochloride, phosphate, citrate or acetate.

The process of the invention is characterized by, (a) removing the protecting group or groups from a compound of formula I wherein one or more amino acid residues are protected in the usual manner, or (b). reaction of Cfilu-OHj .Elu-His-OH, Glu-His-Trp-OH, 15 i-Glu-His-Trp-Ser-OH. · —Glu-His-Trp-Ser-Tyr-OH, L-Glu-His-Trp-Ser-Tyr-A-OH, - Glu-His-Trp-Ser-Tyr-AB-OH, Qsiu-His Trp-Ser-Tyr-AB-Arg-OH or CJlu-His-Trp-Ser-Tyr-AB-Arg-Pro-OH or a suitably activated derivative thereof with 2o H-His-Trp-Ser-Tyr-AB, respectively. -Arg-Pro-EF, H-Trp-Ser-Tyr-AB-Arg-Pro-EF, H-Ser-Tyr-AB-Arg-Pro-EF, H-Tyr-AB-Arg-Pro-EF, HAB -Arg-Pro-EF, HB-Arg-Pro-EF, H-Arg-Pro-EF, H-Pro-EF or H-Azgly-NH2 or a suitable activated derivative thereof by a peptide-forming condensation reaction commonly used in peptide chemistry , whereupon the resulting compounds are converted, if desired, into pharmaceutically useful acid addition salts.

30

In process (a), there may be as many protecting groups in the starting material as there are radicals that may require protection, e.g. some or all of the radicals that exist in the product as free OH radicals or basic NH radicals.

In process (a), the protecting group (s) may be those described in standard peptide chemistry textbooks, e.g. M. Bodansky and M.A. Ondetti, "Peptide Synthesis", Interscience, New York, 1966, 35 4 149272 Capital IV, F.M. Finn and K. Hofmann, "The Proteins," Volume II, edited by H. Neurath and R.L. Hill, Academic Press Inc., New York, 1976, page 106, "Amino Acids, Peptides and Proteins" (Specialist Periodical Reports), The Chemical Society, London, Vols 1 to 8. Various methods for removing the protective groups are also described in these books.

In process (a), a particularly useful NH protecting group is the benzyloxycarbonyl radical and a particularly useful OH protecting group 10 is the benzyl radical. Both of these groups can be easily removed by hydrogenolysis, e.g. in the presence of a palladium catalyst on charcoal.

In process (a), another particularly useful NH protecting group is the t-butoxycarbonyl radical and another particularly useful OH protecting group is the t-butyl radical. Both of these groups can be easily removed by treatment with an acid such as hydrochloric or trifluoroacetic acid.

In process (a), another particularly useful NH protecting group is the benzyloxycarbonyl or t-butoxycarbonyl radical, and a particularly useful 20 OH OH protecting group is the t-butyl radical. These protecting groups can be easily removed by treatment with HBr in acetic acid.

For the method (b), any standard peptide-forming chemical reaction can be used, e.g. those described in a standard textbook on pep = 25 time chemistry, e.g. the above textbook by Bodansly and Ondetti, Chapter V, and the above volumes 1 to 8 by the Specialist Periodical Reports of the Chemical Society.

In process (b), a particular coupling reaction is an azide coupling, an active ester coupling or a coupling containing N, N'-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole. A preferred coupling reaction is an azide coupling, and in particular such coupling which forms the His-Trp or Ser-Tyr peptide bond. 1 40

The starting materials for use in the method of the invention can be prepared from known compounds by standard peptide coupling reactions, standard peptide protection reactions, and standard peptide removal reactions well known to those skilled in the art, e.g. as shown in Examples 1 to 10.

As mentioned above, the compounds which can be prepared by the inventive process have luliberinagonist properties, i.e. the 5 mimic the effects of luliberin, a natural hormone secreted by the hypothalamus that acts on the pituitary gland and releases it to release luteinizing hormone (LH) and follicle stimulating hormone (FSH). These two pituitary hormones are involved in the regulation of reproductive processes, the latter FSH acting on the ovaries to promote maturation of peoples, and the former LH to induce ovulation. The compounds of formula I are unexpectedly more potent than luliberin in its ability to release LH and are therefore useful for regulating and / or enhancing pure production in humans. It can also be useful for improving infertility conditions in men and women.

15

The luliberin agonist effect of the compounds can be demonstrated, e.g. by their ability to induce ovulation in androgen-sterilized rats in constant oestrus or by their ability to release LH and FSH as measured by double antibody radioimmunoassay in immature 20 male rats or in blood plasma of female oestrus or diestrus.

The above test on androgen sterilized rats is performed as follows:

Androgen-sterilized female rats produced by the treatment of 3, 4 and 25 5-day-old rats with 100 µg testosterone propionate exhibit an elaborate vaginal composition with persistent oestrus and numerous preovulatory follicles in the ovaries. Administration of luliberin and active analogs results in the release of an ovulatory wave of LH and FSH, which can be judged by the presence of eggs in the fallopian tubes and fresh corpora in 30 lutea in the ovaries.

All of the compounds exemplified in the present disclosure are more active than luliberin in their ability to induce ovulation in rats with constant estrus, and additionally exhibit no toxic effects when dosed at least four times their minimum active dose. Specifically, the preferred compounds of the invention described in Examples 4, 6 and 7 are approx. one hundred times as active as luliberin, and they exhibit no toxic effects when dosed at a hundred times their minimally effective dose. The activity level of the compounds of the invention is shown in the following B.

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It is known that luliberin agonists have an effect on dimethylbenz = anthrene-induced tumors in rats and are therefore useful in the treatment of certain hormone-dependent tumors in humans. (Cancer Research, 1976, 36, 3830-3833). The compounds of the invention 5 are active on the same animal model and therefore have the same utility in humans.

The compounds which can be prepared by the process of the invention can be used in the form of a pharmaceutical preparation comprising as active ingredient the compounds together with a pharmaceutically useful solvent or diluent.

The agent may e.g. can be in a form suitable for oral or cheek administration, e.g. a tablet, capsule, solution or suspension for nasal administration, e.g. snuff, nasal spray or nasal drops for vaginal or rectal administration, e.g. a suppository, or parenteral administration, e.g. a sterile injectable solution or suspension.

2Q The agent is usually administered such that a daily oral dose is from 50 µg per day. kg to 20 mg per kg, and a daily parenteral dose, e.g. by intravenous subcutaneous or intramuscular injection or infusion, is from 0.2 µg per ml. kg to 100 µg per kg.

For humans, these doses are equivalent to a total daily dose of 3.5 mg to 1.4 g administered orally and a total daily dose of 14 µg to 7 mg administered parenterally. When administered via the mucous membranes 25, the doses will be between those listed above for oral and parenteral administration.

The invention is illustrated by the following examples.

In the examples, Rf refers to ascending thin-layer chromatography (t.l.c.) on silica gel plates (silica gel G). The solvent systems used for this chromatography were butan-1-ol / acetic acid / water (4: 1: 5 v / v) (R 2A), butan-1-ol / acetic acid / water / pyridine (15: 3: 12: 10 v / v) (RfB), butan-2-ol / 3% w / v aqueous ammonia (3: 1 v / v) (RfC), acetonitrile / water (3: 1 v / v) 3 3 (R |. D), acetone / chloroform (1: 1 v / v) (RfE), chloroform / ethanol (1: 4 v / v) (Rf), cyclohexane / ethyl acetate (1: 1 v / v) (R ), cyclohexane / ethyl acetate / methanol (1: 1: 1 v / v) (R 2 H), chloroform / methanol / water '(11: 8: 2 v / v) (R 2 K), chloroform / methanol' ( 19: 1 v / v) (R 1 P) and chloroform / methanol (9: 1 v / v) 40 (R 2, Q). In all cases, plates were examined under U, V, light, and treated with fluorescamine, ninhydrin and chlorine / starch / iodide reagents. Unless otherwise stated, mention of an Rf implies that a single spot was revealed by these methods.

Acid hydrolysates of all the products described in the present specification were prepared by heating the peptide or protected peptide with 6N hydrochloric acid containing 1 $ w / v phenol in a closed evacuated tube for 16 hours to 100 ° C. The amino acid composition of each hydrolyzate was determined with the LoCarte amino acid analyzer and in every 10 cases it was in accordance with the expected composition. The term "worked up in the usual manner" used in the Examples implies that after the reaction, any solid residue was removed by filtration, the filtrate evaporated to dryness below °3 ° 0, the residue in ethyl = 15 acetate washed with a 20 $ citric acid solution, water, saturated sodium = bicarbonate solution and water, dried over anhydrous sodium sulfate and the ethyl acetate evaporated in vacuo to leave the compound.

Examples 1 to .7.

20

Synthesis of L · -PVΓoglutamyl-L · -histidyl-L · -tryptophyl-L · -servl-1 · -tvΓOsyl-A-L-leucyl-L-arginyl-L-prolyl-E-F. General Procedure (m) (Schemes 1 and 11. 1 2 3 4 5 a cooled (0 ° C) and stirred suspension of L-pyroglutamyl-L-histidine = 2 hydrazide (0.2 mmol) in 0.9 ml of dime thylformamide and 0.7 ml of dimethyl sulphide oxide was added with 5 µl of hydrogen chloride in dioxane (0.8 mmol). A clear solution was obtained after 5 minutes of vigorous stirring. The solution was cooled to -20 ° C, 0.22 mmol t butyl nitrite was added and stirring 3q continued for 2 minutes, then the temperature was lowered to -30 ° C and the solution neutralized by the addition of 0.8 mmol of triethyl = amine. A pre-cooled (-20 ° C) mixture of L-tryptophyl-L-seryl-L-tryrosyl-AL-leucyl-L-arginyl-L-prolyl-EF dihydrochloride (0.1 mmol, obtained by hydrogenolysis of the N-benzyloxycarbonyl derivative in 80 $ 4 35 v / v aqueous methanol containing two equivalents of hydrogen chloride over 5% w / w palladium on charcoal for 16 hours), and 0.1 mmol of triethylamine in 1 ml of dimethylformamide was added and the reaction mixture was stirred for 2b hours at 50 ° C. in vacuo and the residue was chromatographed on Sephadex LH-20 using dimethyl = 5-formamide as eluant. The peptide hydrochloride was further purified by separating chromatography on Sephadex G-25 using the solvent system n-butanol / acetic acid / water / pyridine (5: 1: 5: 1 v / v).

9 149272

Synthesis of L-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-A-B-L-arginyl-L-prolyl-azaglycinamide. General Procedure (s) (Schemes 3, 4 and 5).

5 0.2 mmol of L-pyroglutamyl-L-histidyl-L-tryptophyl-L-serine hydrazide was dissolved in dimethylformamide (4 ml) and converted to the azide as described in process (m). It was coupled, as described in process (m), with L-tyrosyl-ABL-arginyl-L-prolyl-azaglycinamide hydrochloride (0.15 mmol), prepared by catalytic reduction of N-benzyloxycarbonyl-O-benzyl -L-tyrosyl-AL-leucyl-N + -nitro-L-arginyl-L-prolylazaglycinamide in 80% w / w aqueous methanol for 20 hours over 5% w / w palladium on charcoal and the final product, like the hydrochloride, was purified as above.

Synthesis of L-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-A-B-L-arginyl-L-prolyl-azaglycinamide. General Procedure (o) (Schedules 4 and 6).

A solution of the protected decapeptide derivative (with D-Ser (Bu 2) at position 6) (50 mg) was dissolved in 90% v / v aqueous trifluoroacetic acid (5 ml). Three drops of β-mercaptoethanol were added and the solution was allowed to stand at room temperature for 45 minutes. The solvent was removed in vacuo and the residue was lyophilized once with water and twice with t-butanol.

25

The compounds of the invention by one of these three general methods are listed in Examples 1 to 7 of the following table. 1 10 149272 OO O Un Un U \ J- cj CM ΓΟ CM CM CM ro m η j r · * c »es r o» v r * aj ooooooo d • ri d © fo

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The starting materials for use in the above processes can be obtained as set out in Schemes 1 and 2 (Method (m)), Schemes 3, 2 4 and 5 (Method (s)) and Schemes 4 and 6 (Method (o)).

. The following abbreviations are used in these schemes: OCp = 2,4,5 trichlorophenyl ester, 10 Bzl = benzyl, Z = benzyloxycarbonyl,

Boc = t.-butoxycarbonyl, and DMF = dimethylformamide.

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Step (1). N-benzyloxycarbonyl-L-proline (19.94 g, 80 mmol) and N-methyl-morpholine (8.8 ml, 80 mmol) were dissolved in dry tetrahydrofuran (200 ml) and the solution cooled to -20 ° C. . Ethyl chloroformate (7.15 ml, 76 mmol) was added dropwise and after 2 minutes of stirring, a pre-cooled (-20 ° C) 70% aqueous solution of ethylamine (20 ml, 300 ml) was added.

mmol) was added and stirring was continued for 18 hours at 4 ° C. Reaction S

The reaction mixture was worked up in the usual manner and the residue was crystallized by ethyl acetate / petroleum ether (bp 60-80 ° C). Yield 12.97 g (58.7%), m.p. 107-108 ° C, ZT <xJ + 5'5-43.88 ° (c = 1 in methanol), R f D 0.69, R f E 0 , 53, R f P 0.67, R f H 0.62, R f P 0.57, R f Q 0.66.

Step (2h Catalytic reduction above 5% Pd / C in aqueous ethanol containing an equivalent of hydrogen chloride for 5 hours at room temperature.

Step Q. A solution of Na-t-butoxycarbonyl-Ne * i-nitro-L-arbinine (13.5 g, 42.3 mmol), L-prolinethylamide hydrochloride (7.15 g, 47 mmol), 1-hydroxybenzotriazole (11.5 g 85 mmol) and triethylamine (6.58 ml, 47 mmol) in DMF were cooled to 0 ° C and dicyclohexylcarbodiimide (9.13 g, 44.4 mmol) was added. The reaction mixture was stirred overnight at 4 ° C, filtered to remove the solid, and the filtrate was evaporated to dryness in vacuo. The residue was partitioned between ethyl acetate and water by a countercurrent distribution (4 transfers). The aqueous phases were combined, evaporated to dryness, and the residue was partitioned between n-butanol and 5% v / v aqueous acetic acid by countercurrent distribution (12 transfer). The crude peptide obtained by evaporation of the combined n-butanol phases was purified by column chromatography on silica gel using 5% v / v methanol in chloroform and 10% v / v methanol in chloroform as eluents. The product-containing fractions were combined, evaporated to dryness, and an aqueous solution of the residue was passed through an anion exchanger (AG 1-X2) column to remove Na-t-butoxycarbonyl-N '+ - nitroarginine. The column was then washed with water, and the combined aqueous phases and washings were freeze-dried to form the azapeptide derivative, yield 16.67 g (89%), mp 109-111 ° C, decomposed, r1 Jl -39.0 ° (c = 1 in methanol), RfA 0.62, RfB 0.74, RfC 0.59, RfD 0.70, RfE 0.20, RfF 0.60, RfH 0.61, RfK 0.85, RfQ 0, 13th

Step (Γ). N-t-butoxycarbonyl derivative was dissolved in ethyl acetate and treated with 3N HCl in ethyl acetate solution (4 equivalents) for 1 hour at room temperature.

Step © (R = H). A solution of t-butoxycarbonylhydrazide (2.90 g, 22 mmol) and N-benzyloxycarbonyl-O-benzyl-L-tyrosine-2,4,5-trichloro-phenyl ester (11.71 g, 20 mmol) in dimethylformamide ( 40 ml) was kept overnight at room temperature. Working up in the usual manner followed by recrystallization of the ether / petroleum ether residue (bp 60-80 ° C) gave the protected hydrazide as a white powder 3.46 g (67%), mp 126-127 ° C, 13.2 ° (c = 1 in methanol), RfD 0.82, RfE 0.65, RfF 0.63, RfH 0.70.

Step (D (R = H). 1- (N-Benzyloxycarbonyl-O-benzyl-L-tyrosyl) -2-t-but = oxycarbonylhydrazide (5.19 g, 10 mmol) was dissolved in ethyl acetate (50 µl) and was treated with 5N hydrochloric acid in ethyl acetate (8 ml, 40 mmol) for one hour at room temperature, ethyl acetate was removed in vacuo and the hydrochloride filtered with ether and dried.

Step few) (R = H). The above hydrochloride was taken up in tetrahydrofuran 2Q (75 ml) and triethylamine (1.15 g, 8 mmol) was added followed by N-carbonyl-L-leucine methyl ester (1.36 g, 8 mmol). After 16 hours at room temperature, the reaction mixture was worked up in the usual manner and the residue was recrystallized from ethyl acetate / petroleum = ether (bp 60-80 ° C) to give the azatripeptide derivative, 4.57 g (77.7%), m.p. 157 °, (1 ^ 7 ^ 4 -10.3 ° (c = 1 in metha = nol), RfD 0.81, RfE 0.45, RfP 0.26, RfQ 0.47.

Step (|). Hydrazine hydrate (5 mL, 100 mmol) was added to a solution of N-benzyloxycarbonyl-O-benzyl-L-tyrosylazaglycyl-L-leucine methyl = 3Q ester (2.95 g, 5 mmol) in methanol (50 mL). After 2 hours at room temperature, the hydrazide was precipitated with water and recrystallized from methanol / ether, yield 2.74 g (92.8%), m.p. 169-170 ° C, / α α D D -9.05 ° (c = 1 in dimethylformamide), R f A 0.76, R f B 0.75, R f C 0.73, R f D 0.63, RfF 0.60, RfH 0.55.

Step ® and (Π) (R = H). N-benzyloxycarbonyl-O-benzyl-L-tyrosyl-aza = glycyl-L-leucine hydrazide (1.18 g, 2.0 mmol) was dissolved in dimethyl form = amide (10 ml) and, after cooling the solution to -20 ° C was added a 5.49M solution of hydrogen chloride in dioxane (1.46 mL, 8 mmol) followed by t-butyl nitrite (0.25 mL, 2.2 mmol). After 5 minutes, the solution was cooled to -30 ° C and a pre-cooled mixture of N-nitro-L-arginyl-L-prolinethylamide hydrochloride (0.836 g, 2.2 mmol) and triethylamine (1, 43 ml, 10.2 mmol) in dimethylformamide (10 ml) was added. The reaction mixture was stirred at -10 ° C for 1 hour and at 4 ° C for 48 hours. It was worked up in the usual manner and the pentapeptide = derivative was purified by column chromatography on silica gel using chloroform and 3% v / v methanol in chloroform as eluent. agents, yield, 695 g (38.5%), RfA 0.71, RfB 0.72, RfC 0.84.

10 Λ

Step fl.4 (R = H). Catalytic reduction with 5% w / w palladium on charcoal in 80% v / v aqueous acetic acid containing two equivalents of chlorine = hydrogen.

Step Get _. To a vigorously stirred and cooled (-20 ° C) solution of N-benzyloxycarbonyl-L-tryptophan (33.84 g, 100 mmol) and N-methyl = morpholine (11.0 ml, 100 mmol) in tetrahydrofuran (200 ml ), ethyl chloroformate (9.0 ml, 95 mmol) was added. After 2 minutes, a pre-cooled (-20 ° C) solution of L-serine methyl ester hydrochloride (17.10 g, 110 mmol) and N-methylmorpholine (12.1 ml, 110 'mmol) in dimethylformamide (150 ml) was added. ) and stirring was continued at -20 ° C for 30 minutes and at room temperature for 3 hours. Usual reprocessing gave an oil. Two crystallisations of ethyl acetate / petroleum ether (bp 60-80 ° C) gave the dipeptide derivative (30.53 g, 69.5%), mp 140.5-141 ° C, / 7a27 + -22.13 ° (c = 1, 4 in dimethylformamide).

25 D

Step Θ. Prior ester (30.53 g, 69.5 mmol) was dissolved in methanol (1 liter) and a 62% w / v solution of hydrazine = hydrate (15 ml) was added. After 16 hours, the hydrazide was collected, washed with methanol and ether and recrystallized from hot ethanol (23.18 g, 75.8%) mp 178-179 ° C, α-7ρ -25.27 ° (c = 1 in dimethylformamide)

RfD 0.65, RfE 0.20, RfF 0.43, RfH 0.50.

Step jQL and ¢ 5). 6.02 N chlorine hydrogen in dioxane (0.77 ml, 4.64 mmol) was added to a cooled (-20 ° C) and stirred solution of N-benzyloxy = carbonyl-L-tryptophyl-L-serine hydrazide (0.502 g, 1 (16 mmol) in dimethyl = formamide (5 ml) followed by t-butyl nitrite (0.14 ml, 1.22 mmol). After 30 minutes, the solution was cooled to -30 ° C and neutralized by the addition of triethylamine (0.65 mL, 4.65 mmol). A pre-cooled (-20 ° C) mixture of L-tyrosylazaglycyl-L-leucyl-L-arginyl-L-proline = 40 ethylamide dihydrochloride (0.547 g, 0.77 mmol) and triethylamine (0.108 ml, 0.77 mmol) in dimethylformamide (5 ml) was added and stirring was continued for 1 hour at -20 ° C and for 48 hours at 4 ° C. The reaction mixture was filtered and the filtrate evaporated to dryness in vacuo.

The crude peptide was purified by column chromatography on silica gel using chloroform, 10% v / v methanol in chloroform, and a mixture of chloroform, methanol and water (11: 8: 2 v / v) as eluents, yield: 424 g (52.9%) J ~ α 7 25 -i c. qa ° / „η c.

yr 'y' i-aj D -16.84 (c = 1.5 in methanol), R A

0.61, RfC 0.36, RfD 0.67 RfK 0.90. f

Step Q (R = Me). As Step ©, yield 66%, mp 102-104 ° C,

rmm 24 O

a_ / D -15.5 (c = 1 in methanol), RfD 0.76, RfE 0.68, RfP 0.76,

RfH 0.74.

Step @ (R = Me). As a step ©.

Step (gg) (R = Me). As Step ©, Yield 93%, Melting point 145-146 ° C, 27 [α] D + 8.7 ° (c = 1.2 in methanol), RfA 0.88, RfB 0.88, RfC 0.83 .

RfD 0.80, RfE 0.59, RfF 0.78, RfH 0.73.

Step. 1N sodium hydroxide (12 mL, 12 mmo]) was added to a stirred 2Q solution of N-benzyloxycarbonyl-O-benzyl-L-tyrosyl-azalanyl-L-leucine = methyl ester (2.41 g, 4 mmol) in methanol (36 ml) at room temperature and stirring was continued for 3 hours. Methanol was removed in vacuo and an aqueous solution (40 ml) of the residue was acidified with citric acid (pH 3) and extracted with ethyl acetate. After washing the ethyl acetate = 25 extract with water and drying (Na 2 SO 4), the solvent was evaporated and the residue in a mixture of dimethylformamide and water (3: 2 v / v, 200 ml) was passed onto a column of AG 1X-2 resin ( 100 ml). The column was washed with the above solvent (50 ml) and the tripeptide eluted with 0.2M acetic acid in dimethylformamide water (3: 2 v / v).

Thirty tripeptide-containing fractions were combined, evaporated in vacuo, and the residue triturated with ether and collected, 1.22 g (51.7%), mp 195 ° C, decomposing 25 ° C to 25.4 ° (c = 1 in dimethyl = formamide). 1

Step (Q) (R = Me). As a step, yield 43% "α 2--25.9 ° (c = 1 in methanol), RfA 0.72, RfB 0.76, RfC 0.85.

Step Θ _ (R = Me). As a step. IN

Step (24) (R = Me). The wounds · as a step except that the final product was also purified by gel filtration on Sephadex LH-20 in di = methylformamide after column chromatography on silica gel, yield 63%, 0. a_J + -24.76 ° (c = 0 , 8 in methanol), R 2 A 58, R 2 -C 0.42, R 2 D 0.65,

R-K 0.95.

5 f

Step (gj). For a stirred and cooled (0 ° C) suspension of N-benzyloxy = carbonyl-L-proline (24.9 g, 100 mmol), semicarbazide hydrochloride (11.2 g, 100 mmol) and triethylamine (14.5 ml, 100 mmol) ) in dimethylformamide (200 ml), dicyclohexylcarbodiimide (20.6 g, 100 mmol) was added and stirring was continued for 16 hours at 4 ° C. Dicyclohexylurea was removed by filtration and the filtrate was evaporated to a small volume. 200 ml of water was added and the solution was extracted with ethyl acetate (3 x 50 ml). The product precipitated from the aqueous solution of ca. 1 hour. Recrystallization from aqueous methanol gave the dipeptide = ^ amide (16.5 g, 53.9%), m.p. 189-190 ° C, 77 DEG 17 DEG-43.6 DEG (c = 1.4 in dimethylformamide), R , 54, RfF 0.52, RfH 0.38, Rf K 0.78.

Step JSL. Catalytic reduction over 5% w / w palladium on charcoal in 80% v / v aqueous dimethylformamide for six hours at room temperature in the presence of two equivalents of hydrogen chloride.

Step (27). Ethyl chloroformate (2.83 ml, 29.5 mmol) was added to a solution of N-benzyloxycarbonyl-L-leucine (8.24 g, 31 mmol) and triethylamine (4.55 ml, 32.5 mmol) in tetrahydrofuran (100 ml) at -10 to 25 o

-15 C. The reaction mixture was stirred for 3 minutes at this temperature and then poured into a vigorously stirred solution of N 2 -nitro-L-arginine (5.79 g, 31 mmol) in 2N sodium hydroxide (15.5 mL, 31 mL). mmol) and dimethylformamide (50 ml) at -10 ° C. Stirring was continued at -10 ° C

for 30 minutes and then at room temperature for 1 hour. The solvents were removed in vacuo and the residue partitioned between ethyl acetate (50 ml) and water (50 ml). The aqueous phase was separated and extracted with two additional portions of ethyl acetate. The combined organic phases were washed once more with water (25 ml) and discarded.

The combined aqueous phases were acidified with saturated citric acid solution 35 and extracted with ethyl acetate (3 x 100 ml). The ethyl acetate extracts were combined, washed with water, dried (Na 2 SO 4) and evaporated to dryness. Recrystallization of the residue of ethyl acetate / petroleum ether (bp 60-80 ° C) afforded the dipeptide (8.98 g, 62%), mp 150-165 ° C during decomposition.

40

Step (£ δ). A solution of N-benzyloxycarbony (1-L-leucyl-N-nitro) - "L-arginine (9.2 g, 20 mmol), L-prolylazaglycinamide hydrochloride (4.2 g, 20 mmol), 1-hydroxybenzotriazole ( 5.4 g, 40 mmol) and triethylamine (3 ml, 20 mmol) in dimethylformamide (200 ml) were cooled to 0 ° C and dicyclohexylcarbodiimide (8.2 g, 40 mmol) was added. The reaction mixture was stirred overnight at room temperature. Dicyclohexylurine = 5 substance was removed by filtration and the filtrate was evaporated to dryness. Recrystallization of the residue of methanol-ether gave the tetrapeptide derivative (12.2 g, 98.3%), m.p. 88-90 ° £ 7α -30.2 ° (c = 1.6 in dimethylformamide), RfD 0.57 , RfF 0.40, RfH 0.26, Rf K 0.63.

10 Steps (2§). Hydrogenation over 5% w / w palladium on charcoal in aqueous ethanol for 16 hours in the presence of two equivalents of hydrogen chloride.

Step. N-Benzyloxycarbonyl-O-benzyl-L-tyrosine-2,4,5-trichlorophenyl ester (6.484 g, 11.0 mmol) and D-alanine methyl ester hydrochloride (1.396 g, 10 mmol) were dissolved in dimethylformamide (50 ml). and triethylamine (1.4 ml, 10.0 mmol) was added to the solution, which was stirred overnight at room temperature. The reaction mixture was worked up in the usual manner and the residue was recrystallized from hot ethyl acetate to give 3.782 g, 77.2% of the protected dipeptide methyl ester, mp 163 ° C, / J α® -12.84 ° (c = 1, 1 in. Dimethylpharmamide).

Step JiL. N-benzyloxycarbonyl-O-benzyl-L-tyrosyl-D-alanine methyl ester I

(3.435 g, 7.0 mmol) was dissolved in hot methanol (400 ml) and the solution was treated with 62% w / v hydrazine hydrate (10 ml, 120 mmol) and the mixture was allowed to stand at 25 ° C overnight. The hydrazide was filtered off, washed with methanol and ether and crystallized twice by boiling methanol, yield 3.068 g, 89.2%, mp 217 ° C, -20.44 ° (c = 1.1 in dimethylformamide) R 73, R f B 0.75, R f C 0.67 R, D 0.70, R Ξ 0.50, RF 0.54, RH 0.67, R_K 0.85, RQ 0.25.

30 1 1 f t 1 1 t

Steps @ and 5¾). To a cooled (-20 ° C) and stirred solution of N-benzyloxycarbonyl-O-benzyl-L-tyrosyl-D-alanine hydrazide (1.18 g, 2.4 mmol) was added a 6.02 M solution of hydrogen chloride in dioxane (1.6 mL, 9.6 mmol) followed by t-butyl nitrite (0.29 mL, 2.52 mmol).

35 o

After 15 minutes, a pre-cooled (-20 ° C) solution of L-leucyl-L-arginyl-L-prolylazaglycinamide dihydrochloride (1.03 g, 2.0 mmol) and triethylamine (1.62 mL, 11.6 mmol) was added. ) in dimethylformamide 115 ml). Stirring was continued at 4 ° C for 24 hours. Triethylamine = hydrochloride was removed by filtration and the filtrate was evaporated to dryness in vacuo. The residue was charged to a silica gel column and the column was eluted with 5% v / v methanol in chloroform, 10% v / v methanol in chloroform and a mixture of chloroform, methanol and water (11: 8: 2 v / v). . The product-containing fractions were combined and evaporated and the peptide was chromatographed again on a silica gel column using acetonitrile / water (3: 1 v / v) as eluent, yield 890 mg (46.4%), ΖΓ α -45.7 ° (c = 1.1 in methanol), R f A 0.54, R f B 0.69, R f C 0.41.

Step 61). As a step.

Step 65). As step (Q), yield 43% δ α * 5ρ5 -41.4 ° (c = 1.3 in methanol), RfA 0.80, RfC 0.47, RfD 0.65, RfK 0.95.

Step (3). As Step (3), yield 69%, mp 135 ° C, RfA 0.49,

RfB 0.65, RfC 0.46, RfD 0.64, RfF 0.35, RfH 0.19, RfK 0.86. .

Step few). As step Φ 20 ^

Step (4Q) (A = D-Phe). A solution of N-benzyloxycarbonyl-D-phenyl = alanine (7.41 g, 24.8 mmol) and L-leucine methyl ester (3.62 g, 25 mmol) in 100 ml of ethyl acetate was cooled to 0 ° C, and added dicyc = lohexylcarbodiimide (5.15 g, 25 mmol). The reaction mixture was stirred overnight at 4 ° C. The usual work-up followed by recrystallization of the residue of ethyl acetate / petroleum ether (bp 60-80 ° C) gave the dipeptide (9.1 g, 86%), m.p. 123-124 ° C, Γ_aJ7p6 -18 / 7 ° (c = 2 , 1 in methanol), RfD 0.76, RfE 0.65, RfF 0.74,

RfH 0.73.

Step (jl) (A = D-Phe). Catalytic reduction over 5% w / w palladium on charcoal in ethanol containing an equivalent of hydrogen chloride for 5 hours.

Step (few (A = D-Phe). To a stirred solution of N-benzyloxycarbonyl-0-benzyl-L-tyrosine-2,4,5-trichlorophenyl ester (4.89 g, 8.36 mmol) and 35. D -phenylalanyl-L-leucine methyl ester hydrochloride (2.5 g, 7.6 mmol) in dimethylformamide, triethylamine (1.1 ml, 7.6 mmol) was added and stirring was continued overnight at room temperature. Triethylamine = hydrochloride was filtered off and the filtrate was evaporated to dryness. Recrystallization of the residue of aqueous methanol gave the tripeptide derivative, 3.6 g (69.7%), mp 183-184 ° C, R 2 D 0.82, R 0.69, RfH 0.78, RfP 0.71, RfQ 0.82.

25 149272

Step (Q) (A = D-Phe). A solution of the previous methyl ester (3.42 g, 5.04 mmol) and hydrazine hydrate (60 mmol) in dimethylformamide (30 ml) was stirred at room temperature for 4 hours, concentrated to a small volume, and the hydrazide precipitated by addition of water (500 ml).

5

It was collected, washed with water, ethanol / ether (1: 4 v / v) and ether and dried. Yield 2.94 g (85.9%), m.p. 179-180 ° C, R f A 0.81, RfB 0.79, RfC 0.88, RfD 0.69, RfE 0.49, RfF 0.65 , RfH 0.67,

RfP 0.25, RfQ 0.57.

10 steps j2L and ^ 5) (A = D-Phe). A solution of 6.02 M hydrogen chloride in dioxane (1.83 ml, 11 mmol) was added to a solution of N-benzyloxy = carbonyl-O-benzyl-L-tyrosyl-D-phenylalanyl-L-leucine hydrazide (1.86 g, 2.75 mmol) in dimethylformamide (5 mL) at -20 ° C followed by t-butyl = nitrite (0.33 mL, 2.89 mmol). After 2 minutes, a pre-cooled (-20 ° C) solution of triethylamine (1.89 ml, 13.5 mmol) and N-nitro-L-arginyl-L-prolylazaglycinamide hydrochloride (1.02 g, 2.5) was added. mmol) in dimethylformamide (10 mL), and the reaction mixture was stirred overnight at 4 ° C, and usual workup gave the hexapeptide derivative, which was further purified by column chromatography on silica gel 20 (120 g) using 5% v / v methanol. in chloroform, 10% v / v methanol in chloroform and a mixture of chloroform / methanol / water (11: 8: 2 v / v) as eluents, yield 0.74 g (29.3%), m.p.

137-139 ° C, RfA 0.68, RfB 0.72, RfC 0.58, RfD 0.62, RfH 0.39, RfK

0.95.

25

Steps, 7) and Cg). L-pyroglutamyl-L-histidine hydrazide (10 mmol) was converted to the azide as described in the general procedure (m) and coupled with L-tryptophyl-L-serine methyl ester (11 mmol prepared by hydrogenation of the N-benzyloxycarbonyl derivative over 5% w / (palladium on charcoal in dimethylformamide) at -10 ° C for 30 minutes and at 4 C for 24 hours. The triethylamine hydrochloride was removed by filtration and the filtrate was evaporated to dryness. The crude peptide was purified by column chromatography on silica gel using 10% v / v methanol in chloroform, 20% v / v methanol in chloroform and a mixture of chloro = form / methanol / water (11: 8: 2 v / v) as eluents, yield 70%, m.p. 142-145 ° C during decomposition, R f A 0.39, R f B 0.72, R f C 0.45, RfD 0.48, RfK 0.61.

Step C) L-pyroglutamyl-L-histidyl-L-tryptophyl-L-serine methyl ester (5.4 mmol) was dissolved in dimethylformamide (70 ml) and treated with hydrazine hydrate (100 mmol) for 4 hours. Dimethylformamide was removed in vacuo and the residue was triturated with ethanol, collected, washed with ethanol and ether and dried (88.2%), mp 184 -189 ° C, RfA 0.18, RfB 0.55, RfC 39, RfD 0.27, RfK 0.58.

Step (go) (A = D-Trp). Dicyclohexylcarbodiimide (4.87 g, 23.6 mmol) was added to a solution of N-benzyloxycarbonyl-D-tryptophan (7.27 g, 21.5 mmol), leucine methyl ester (3.12 g, 21.5 mmol) and 1-hydroxy = benzotriazole (5.8 g, 43 mmol) in dimethylformamide (50 ml) at 0 ° C. The reaction mixture was stirred overnight at room temperature and worked up in the usual manner. Recrystallization of ethyl acetate / petroleum ether (bp 60-80 ° C) gave the dipeptide derivative (9.55 g), which showed traces of impurities by thin layer chromatography. It was purified by column chromatography on silica gel (300 g) using chloroform and 5% v / v methanol in chloroform as eluents. Yield 9.18 g (91.7%), mp 151-153 ° C, R f A 0.84, R f B 0.80,

RfC 0.86, RfD 0.78, RfE 0.61, RfF 0.68, RfH 0.73, RfP 0.55, RfQ 0.73.

20 r \

Step (A = D-Trp). Catalytic reduction in 80% v / v aqueous dimethyl = formamide over 5% w / w palladium on charcoal for 5 hours.

Step (52) (A = D-Trp). A solution of N-benzyloxycarbonyl-O-benzyl-L-tyrosine-2,4,5-trichlorophenyl ester (11.69 g, 20 mmol), D-tryptophyl-L-leucine methyl ester (6.28 g, 19 mmol) in dimethylformamide (100 ml) was stirred at room temperature for 60 hours. The reaction mixture was worked up in the usual manner and the residue was crystallized from ethyl = acetate / petroleum ether (bp 60-80 ° C) to give the tripep = 50 time derivative, 8.52 g (62.5%), mp 165-166 ° C , R f A 0.78, R f B 0.73, RfC 0.84, RfD 0.80, RfE 0.62, RfF 0.70, RfH 0.76, RfP 0.58,

RfQ 0.68.

Step Θ (A = D-Trp). A solution of N-benzyloxycarbonyl-O-benzyl-35 L-tyrosyl-D-tryptophyl-L-leucine methyl ester (7.26 g, 10.1 mmol) in a mixture of methanol (200 ml) and dimethylformamide (50 ml) was treated with hydrazine hydrate (100 mmol) at room temperature. After 24 hours, the solution was concentrated (about 30 ml) and 500 ml of water was added. The tripeptide hydrazide was collected, washed with water, 40 methanol / ether (1: 4 v / v) and ether and dried, 6.86 g (94.6%), mp 200-202 ° C, RfA 0.90, RfB 0 , 95, RfC 0.90, RfD 0.74, RfQ 0.59.

27 149272

Step E1) and (A = D-Trp). An unstirred and cooled (-20 ° C) solution of N-benzyloxycarbonyl-O-benzyl-L-tyrosyl-D-tryptophyl-L-leucine = hydrazide (1.97 g, 2.75 mmol) in dimethylformamide (10 mL) was treated with a 6.02M solution of hydrogen chloride in dioxane (1.83 mL, 2 11 mmol) followed by t-butyl nitrite (0.33 mL, 2.89 mmol). After 2 minutes, a pre-cooled (-20 ° C) solution of N + nitro-L-arginyl-L-prolylazaglycinamide hydrochloride (1.02 g, 2.5 mmol) and triethylamine (1.89 mL, 13.5) was added. mmol) in dimethylformamide (10 ml) and the reaction mixture was stirred overnight at 4 ° C. It was prepared in the usual manner and the residue (1.27 g) was applied to a column of silica gel (230 g) and the column was eluted with chloroform and 5% v / v methanol in chloroform. Yield 91.9 g (34.4%), mp 139-140 ° C during decomposition, RfA 0.67, RfB 0.72, RfC 0.58, RfD 0.62, RfH 0.34, RfK 0.95 .

Step (s6 ^ (A = D-Tyr (Me)). A solution of ZD-Tyr (Me) -OH (3.17 g, 9.64 mmol), H-Leu-QMe, HCl (1.92 g , 10.6 mmol), 1-hydroxybenzotriazole (2.6 g, 19.2 mmol) and triethylamine (1.6 mL, 11 mmol) in dimethylformamide (30 mL) were cooled to 0 ° C and added N, N'-dicyclo = 20 hexylcarbodiimide (2.29 g, 11.1 mmol) The reaction mixture was stirred overnight at 4 ° C and then worked up in the usual manner Recrystallization of hot cyclohexane yielded the protected dipeptide derivative. 1.41 g (95.2%), RfD 0.83, RfE 0.69, RfP 0.72, RfQ 0.76.

(C = D-Tyr (Me)). Catalytic reduction over 5% w / w palladium on charcoal in methanol / dimethylformamide / water (8: 1: 1) containing 1.2 equivalents of hydrogen chloride for three hours.

Step (^ 8) (A = D-Tyr (Me)). A solution of Z-Tyr (Bzl) -OCp (8.2 mmol), 3q HD-Tyr (Me) -Leu-QMe, HCl (8.2 mmol) and triethylamine (8.2 irrole) in dimethyl = formamide ( 60 ml) was stirred overnight at room temperature and the reaction mixture then worked up in the usual manner. The product was filtered with ether, washed with ether and dried. Yield 81.2%, mp 191-192 ° C, RfD 0.85, RfE 0.73, RfF 0.72, RfQ 0.78.

35 ZT \

Step ^ 9) (A = D-Tyr (Me)). Hydrazine hydrate (12.9 mmol) was added to a solution of Z-Tyr (Bzl) -D-Tyr (Me) -Leu-OMe (4.59 g, 6.4 mmol) in di = methylformamide (25 ml) and methanol (50 ml) and the reaction mixture was allowed to stand overnight at room temperature. Methanol was removed in vacuo and the product was precipitated with water, collected, washed with water and dried, mp 212-213 ° C, R f 0.49, RfF 0.66, R f H 0.69, R f Q 0.70.

28 149272

Steps (βθ) and (βΐ) (A = D-Tyr (Me)). The hydrazide from step (5 ^) (3.5 ^, 5.0 mmol) was dissolved in EMF (10 mL) and the stirred solution was cooled to -20 ° C. 5.92M HCl in dioxane (3 »38 ml, 20 mmol) was added followed by t-butyl nitrite (0.6 ml, 5.25 mmol). After 2 minutes 5, a pre-cooled solution of H-Arg (NO2) -Pro-Azgly-NH2, HCl (2.0 µg, 5 mmol) and triethylamine (3.55 ml, 25 mmol) in DMF ( 10 ml) and stirring was continued overnight at 1 ° C. The reaction mixture was worked up in the usual manner and the crude product was purified by column chromatography on silica gel using cbloro = -q form, 5% v / v methanol in chloroform and 10% v / v methanol in chloroform as eluents, Yield 3.72 g (70.9%), R f A 0.6 * +, R f B 0.72,

BfC 0.55, RfD 0.66, RfF 0.1 * 0, RfH 0.52.

Step (ό2) (A = D-Ser (Bu ^)). As step (^ β). The product was crystallized from aqueous methanol. Yield 9 ° Λ%, mp 107-108 ° C, R

RfE 0.68, RfF 0.73, RfH 0.72, RfP 0.72, RfQ 0.7 * K

Step (63) (A = D-Ser (Bu 2)). Catalytic reduction over 5% w / w palladium on charcoal in DMF water (8: 2) for five hours.

20 _

Step @ (A = D-Ser (Bu ^)). A solution of Z-Tyr (Bzl) -OCp (19.17 g, 32.7 mmol) and HD-Ser (But) -Leu-Ofe (32.7 mmol) in DMF (100 ml) was allowed to stand at room temperature for 1 h. 72 hours. Usual work-up gave a solid which was collected, washed with ether and dried. Yield 25 17.6 g (79>%), mp 135-137 ° C, RfD 0.80, RfH 0.77, RfQ 0.8l.

Step (5¾) (A = D-Ser (Bu ^)). As step (^). Recrystallized from aqueous methanol. Yield 56.2%, m.p. 13 ^ 136 ^, RfD 0.66, RfH 0.6 ^,

RfQ 0.6¼.

Step ^ 6) and (A = D-Ser (Bu ^)). As steps (60) and (6), the product was purified by column chromatography on silica gel using chloroform and 5% v / v methanol in chloroform as eluent. Yield 38.5%, m.p. -100 ° C, RfA 0.6 **, RfB 0.71, RfC 0.55, RfD 0.65, RfF 0, lt6, RfH-0, lf3, RfQ 0.16.

Step @ (A = D-Tyr (Me)). Dicyclohexylcarbodiimide (5.13 g, 2l *, 9 mmol) was added to a cooled (0 ° C) and stirred solution of ZD-Tyr (Me) -OH (22.6 mmol), H-MeLeu-OMe, HBr ( 5.98 g, 2 µ, 9 mmol), triethylamine (3.5 ml, 2.1 g, 9 mmol) and 1-hydroxybenzotriazole (6.12 g, 5.2 mmol) in DMF (50 ml) and stirring was continued overnight at k-° C. Reaction 29 149272

the mixture was worked up in the usual manner and the product was purified by column chromatography on silica gel using chloroform as solvent. Yield 55.2%, oil, RfD 0.83, R f E 0.78, R f H

0.79, RfP 0.80, RfQ 0.79.

Step (A = D-Tyr Me)). Catalytic reduction over 5% w / w palladium on charcoal in methanol / water (8: 2 v / v) containing an equivalent hydrogen chloride for six hours.

10 ~ _

Step (tq) (A = D-Tyr (Me)). As a step. The product was purified by column chromatography on silica gel using ether as solvent.

Step (A = D-Tyr (Me)). A solution of Z-Tyr (Bzl) -D-Tyr (Me) -MeLeu-

OMe (4.85 g, 6.69 mmol) and hydrazine hydrate (120.7 mmol) in methanol (150 ml) were allowed to stand overnight at room temperature. The hydrazide was precipitated by the addition of water, collected and crystallized by methanol / water. Yield 91.1%, m.p. 129-131 ° C, R f D 0.79, R f E

0.60, RfF 0.68, RfH 0.73, RfQ 0.77.

Step (S) and (A = D-Tyr (Me)). As steps ^ o) and. Recrystallized from methanol / ether, yield 23.8%, mp 152-154 ° C, R f A 0.67, RfB 0.68, RfC 0.58, RfD 0.59, RfH 0.50, RfK 0, 94, RfQ 0.35.

25

Step (74) Ethyl chloroformate (1.8 ml, 18 mmol) was added to a cooled (-15 ° C) and stirred solution of ZD-Phe-OH (5.99 g, 20 µmol) and N-methyl = morpholine (2.2 ml, 20 mmol) in DMF (60 ml). After 2 minutes, a pre-cooled (-15 ° C) solution of H-MeLeu-OMe, HBr (4.8 g, 30 mmol) and triethylamine (2.8 ml, 20 mmol) in DMF (20 ml) was added. and the reaction mixture was stirred for 30 minutes at 0 ° C and overnight at room temperature. It was worked up in the usual way. Yield 7.54 (90%), oil.

Step (75). Catalytic reduction over 5% w / w palladium on charcoal in methanol containing an equivalent of hydrogen chloride for three hours.

Step, (76). Prepared by coupling Z-Tyr (But) -OH (16.02 g, 43.2 mmol) and H-D-Phe-MeLeu-OMe, HCl (13.15 g, 40.0 mmol) as in step 74.

The product was purified by column chromatography on silica gel using chloroform and 5% v / v methanol in chloroform as solvent. Yield 60%, oil, R f 0.48, R f P 71, R f 0.73.

149272 30

Step »A solution of Z-Tyr (Bu *) - D-Phe-MeLeu-OMe (6.52 g, 9.76 mmol) and hydrazine hydrate (97.6 mmol) in methanol (50 ml) was allowed to stand overnight. Methanol was removed in vacuo, and the hydrazide was crystallized by methanol / ether, washed with methanol / water (1: 1 v / v) and ether and dried to yield 5.2 g (80%), m.p. 135 ° C, RfD 0.75, RfE 0.69, RfF 0.66, RfH o, 79, RfQ o, 73.

Step and JzL. During steps, the product was precipitated by ethyl acetate, filtered off, washed with ethyl acetate and ether and dried to yield 58.5%, mp 145-148 ° C.

RfD 0.72, RfF 0.40, RfH 0.53, RfQ 0.18.

Example 8.

15 t

The compound of formula I wherein A is D-Ser (Bu), B is Leu, E

is Azgly, and F is N 1 / β was prepared as shown in Scheme 7 using reaction conditions and work-up methods analogous to those described for the corresponding coupling reactions in the previous examples. The product had the same analytical properties as that in Example 5. Yield 42.6%.

31 149272

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Si a> 1 rH —__________ _ t7> ----

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P id "id e H rH a Q)

N N M

ffl fflv: W

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m _ H ———— -1 · «1 ffi 0 a