NL8003423A - PROCESS FOR THE PREPARATION OF NEW ENKEPHALINE DERIVATIVES, ENKEPHALINE DERIVATIVES AND MEDICINAL PRODUCT WITH SUCH AN ENKEPHALINE DERIVATIVE THEREIN. - Google Patents

Description

N / 29711-Kp / vdM.

- 1 -

Process for the preparation of new enkephalin derivatives, enkephalin derivatives and medicament with such an enkephalin derivative therein.

The invention relates to a process for the preparation of new enkephalin derivatives, to such enkephalin derivatives, as well as to medicaments containing such an anephalin derivative.

The invention includes new enkephalin derivatives, their salts, and a process for their preparation. The new compounds have the following formula: H2N-C (NH) -Tyr-A-Gly-Phe-B, wherein 10 Tyr, Gly and Phe L-tyrosine, glycine, respectively. L-phenylalanine, A is an alkyl group with 1-4 carbon atoms or thioalkyl-D-α-amino acid with 1-4 carbon atoms, while B represents an amino group or L-prolinamide group.

The meanings of Tyr, Gly and Phe are in the literature / e.g. J. Biol. Cnem. 247, 977 (1972 ^ common to indicate amino acids.

It is known that the morphine-like, ie, the opiate activity of the naturally occurring methionine and leucine anephales / Hughes et al .: Nature 258, 577 (1975) _ / (peptide of the structure Tyr-G ^ yG ^ y-pêe-Met, respectively -Leu) by suitable substitution of the amino acid residues 2-Gly 25 and 5-Met, respectively. 5-Leu, can be reinforced. By incorporating the D-methionine groups in the 2-position and the L-prolinamido group in the 5-position, one of the most effective erikephalin analogues, the pentapeptidamide with the structure Tyr-D-Met-Gly-Phe-Pro-NH2, is obtained ( see Belgian patent specification 858,453).

An object of the invention is the preparation of new enkephalin derivatives, the opiate activity of which exceeds that of the known compounds.

It has now been found that the opioid activity of the amino acid exchange-derived strong active Enkefa 800 3 4 23-2 line analogs can be further enhanced when an amidino group is built on the terminal amino group, ie, a peptide analog is prepared which produces a guanidino terminal group contains. The greater opiate activity of the compounds of the above-mentioned formula prepared in this way can be demonstrated in all conventional tests, for example, on the ileum of the Guinea pig (guinea pig ileum, GPI) / Kosterlitz and Watt: Br. J. Pharmacol. 33/266 (1968); Kosterlitz and colleagues. : Br. J. Pharmacol. ^ 9. / 398 (1970) __ 7 and 10 to the mouse spermatic cord preparation / Hughes et al .:

Br. J. Pharmacol. 53, 371 (1975) J. The table lists the opiate activities of four containing a terminal guanidino group and their 1N-terminal analogues. The values stated relate to the relative activities related to normorphine (activity of normorphine = 1). For the sake of better clarity, the unsubstituted peptide is indicated below the compound according to the invention.

TABLE

20 connection activity

GPia GPIb GPiab · MVDC

HNC (NH) -Tyr-D-Met-Gly-Phe-Pro- NH2 823 173 25 H-Tyr-D-Met-Gly-Phe-Pro-NH2 8 8 25 H2N-C (NH) -Tyr-D- Met-Gly-Phe-NH2 211 79 21 H-Tyr-D-Met-Gly-Phe-NH2 17 4 H2N-C (NH) -Tyr-D-Nle-Gly-Phe-NH2 126 79 18 H-Tyr- D-Nle-Gly-Phe-NH2 22 8 HNC (NH) -Tyr-D-Nle-Gly-Phe-Pro-30 NH2 79 21 17 H-Tyr-D-Nle-Gly-Phe-Pro-NH2 12 10 GPIa: according to Kosterlitz and Watt / Br. J. Pharmacol. J3y 266 (1968) 7 GPIÜ: according to Kosterlitz et al. / Br. J. Pharmacol. 39_, 398 35 (1970) _7 MVDC: according to Hughes et al. / Br. J. Pharmacol. 5, 371 (1975) 7.

800 3 423 * i - 3 -

The result obtained by the introduction of the N-terminated guanidiogroup is surprising, since it is precisely this part of the single-phalin molecules which is the most sensitive and any intervention on the terminal amino group (eg its removal, acylation or dialkylation) generally up to attenuation or loss of opioid activity / e.g. Morgan et al .: Communications, J. Pharm. Pharmac.

28, 660 (1976) _7

The compounds of the aforementioned formula, wherein B represents an amino group, have systematic data in the test according to D1Amour-Smith / J. Pharm.

Ther. 72, 74 (194l) _7 has a stronger analgesic effect than morphine.

Our own research has shown that the compounds of the above formula selectively alter the catecholamine content of the nuclei of amygdala centralis. Since the core group has a high single calf content and a large opiate receptor thickness and is not part of the systems specifically affecting the sensation of pain, it is expected that the compounds of the above formula will specifically influence the following functions: food intake, emotionality, social relationship, learning and thought processes.

The object of the invention is therefore a process for the preparation of new enkephalin derivatives of the above-mentioned formula, in which the meaning of Tyr, Gly,

Phe, A and B are the same as previously mentioned, from the amino group-containing peptides of the corresponding amino acid series. It is known for the present process that the terminal amino group is converted into a guanidino group in a manner known per se and that a salt is optionally formed from the product obtained.

According to a preferred embodiment of the invention, the starting peptide is reacted at 20-80 ° C with 1.1-2.0 equivalents of 1-amidino-3,5-dimethylpyrazole acetate in the presence of 1.1-2.0 equivalents of trialkylamine, the reaction medium is an aqueous or anhydrous lower alcohol, while the concentration in the reaction medium is 1-1.1 mMol / ml 800 34 23 Γ j - 4 -. The solution is then evaporated, the N-terminated peptide containing guanidino group and, if desired, converted into a salt.

The invention is further elucidated by means of the following 5 examples.

The R 1 values indicated in the examples were determined using silica gel G (Reanal, Budapest), low chromatography, using the following solvent mixtures: 10 1. ethyl acetate-pyridine-acetic acid water 480: 20: 6: 11 2 ethyl acetate-pyridine-acetic acid-water 240: 20: 6: 11 3. ethyl acetate-pyridine-acetic acid-water 15 60: 20: 6: 11 4. chloroform-methanol 9: 1 5. methylene chloride-25% aqueous ammonia-methanol 12: 3: 8.

EXAMPLE I

Amidino-L-tyrosyl-D-methionyl-glycyl-L-phenylalanyl-L-prolinamide (compound of the aforementioned formula, wherein A = D-methionine residue and B = L-prolinamide residue).

Step 1: 1-amidino-3,5-dimethyl-pyrazole acetate.

20.1 g (100 mmol) of 1-amidino-3,5-dimethyl-pyrazole nitrate, Thiele and Dralle: Ann. 302, 294 (1898) J

dissolved in a mixture of 110 ml of caustic soda and 200 ml of methylene chloride with stirring. The organic phase was separated, dried over sodium sulfate, 6 ml of acetic acid was added and then evaporated under reduced pressure. The crystalline residue was suspended in diethyl ether, then filtered off, then washed with diethyl ether and finally dried. 18.25 g (92%) of the product were obtained. Melting point: 114-116 ° C.

Analysis for = 198.2) Calculated: C = 48.47% H = 7.12% N = 28.27% Found: C = 48.6% H = 7.1% N = 28.5%.

Step 2: amidino-L-tyrosyl-D-methionyl-glycyl-L-phenylalanyl-L-prolinamide acetate hydrate.

800 34 23 - 5 - Λ

1.45 g (2 mmol.) Of L-tyrosyl-D-methionyl-glycyl-L-phenylalanyl-L-prolinamide acetate trihydrate (Belgian Patent 858,453) and 0.5 g (2.52 mmol) of 1-amidino- 3,5-dimethylpyrazole acetate (Example I, step 1) dissolved in 2 ml of ethanol. The solution was mixed with 0.4 ml (2.86 mmol) of triethylamine and left to stand at 60-70 ° C for 4 hours. Then the reaction mixture was diluted with 30 ml of ethyl acetate, the precipitate formed was filtered off, washed with ethyl acetate and dried in vacuo. 0.58 g (80%) of the product were obtained.

R ^: 0.63-0.68; (α_7 ^ = + 4 ° (c = 1; n acetic acid) C33H4809N8s "732.844.

EXAMPLE II

Amidino-L-tyrosyl-D-methionyl-glycyl-L-phenyl-15-alaninamide (compound of the aforementioned formula, where A = D-methionine residue and B = amino group).

Step 1: tert-butyloxycarbonyl-D-methionyl-glycyl-L-phenylalaninamide.

5.35 g (15 mmol.) Of benzyloxycarbonyl-20 glycyl-L-phenylalaninamide. Frutones Bermann: J. Biol. Chem. 145, 253 (1942) 7 dissolved in 100 ml of methanol and then hydrated in the presence of palladium activated carbon. After the completion of the reaction, the catalyst was filtered off, washed with methanol, the washing methanol was combined with the filtrate, and the whole was evaporated under reduced pressure. After evaporation, the glycyl-L-phenylalanine amide (R1: 0.19-0.29) remained, which substance together with 6.43 g (15 mmol.) Of tert-butyloxycarbonyl-D-methionine-2, 4,5-trichlorophenyl ester / Broadbent et al .; J. Chem. Soc., 1967, 2632/30 was dissolved in 30 ml of pyridine. The solution was kept overnight and then evaporated under reduced pressure.

The residue was mixed with 15% mercaptan ethanol-containing diethyl ether, the mixture was filtered off, washed with diethyl ether and dried. 5.1 g (75%) of the product are obtained. R ^: 0.70-0.77.

C21H32 ° 5N4S 45452.564.

Step 2: D-methionyl-glycyl-L-phenylalaninamide hydrochloride.

800 34 23 - 6 -

4.52 g (10 mmol) of the protected tripeptidamide (step 1} was suspended in 20 ml of ethyl acetate, after which the suspension was added to 40 ml of 11-15% hydrochloric acid-containing ethyl acetate with stirring and cooling with ice. The reaction mixture was stirred for an hour, then diluted with 60 ml of ethyl acetate, the resulting precipitate was filtered off, washed with ethyl acetate and dried under vacuum over potassium hydroxide, yielding 3.7 g of 3 (95%) of the product. R ^: 0.50-0.55.

10 C16H25 ° 3N4SC1 "388'915 *

Step 3: tert-butyloxycarbonyl-L-tyrosyl-D-methionyl-glycyl-L-phenylalaninamide.

1.4 g (5 mmol) of tert-butyloxycarbonyl-L-tyrosine Anderson and McGregor: J. Am. Chem. Soc. 79 ^ 6180 (1957) 7 dissolved in 10 ml of dimethylformamide. The solution was cooled to -15 ° C. At this temperature, 0.6 ml (5 mmole) of N-methylmorpholine and 0.7 ml (5 mmole) of chlorocarbonic isobutyl ester were added with stirring. After stirring for 10 min., The reaction mixture was cooled to -15 ° C, suspension of 20.95 g (5 mmol.) Tripeptidamide hydrochloride (step 2) and 0.7 ml (5 mmol.) Trietnylamine in 10 ml dimethylformamide added. The reaction mixture was stirred at -15 ° C for 2 hours and at 0 ° C for one hour and then evaporated under reduced pressure. The residue left after evaporation was mixed with water, filtered, washed with water, dried and then recrystallized from 30 ml of boiling ethyl acetate. 2.15 g (70%) of the product were obtained.

R ^: 0.64-0.68.

C30H41 ° 7N5S "615'734" Step 4: L-tyrosyl-D-methionyl-glycyl-L-phenylalaninamide acetate hydrate.

1.85 g (3 mmol.) Of the protected tetra-peptidamide (Example II, step 3) was suspended in 5 ml of ethyl acetate and the resulting suspension was stirred with ice and 15 ml of 11-15% hydrochloric acid with stirring -containing ethyl acetate was added. The reaction mixture was stirred for an hour and then diluted with 20 ml of ethyl acetate. The precipitate formed was filtered off, washed with ethyl acetate 800 3 4 23-7 - and then dried. The product obtained was dissolved in 10 ml of water, after which the solution was applied to 15 ml of acetate anion exchange resin (AG 1-X2, Bio-Rad, Richmond, California). The resin was washed with 80 ml of water. Then the aqueous solutions were freeze dried. 1.07 g 3 (60%) of the product were obtained. R1: 0.55-0.60; R ^: 0.89-0.97; = + 80 ° (c = 1 n acetic acid).

C27H39 ° 8N5S + 593'688

Step 5: amidino-L-tyrosyl-D-methionyl-glycyl-L-10 phenylalaninamide acetate hydrate.

0.6 g (1 mmol) of tetrapeptide amide hydrate (Example II, step 4) and 0.24 g (1.2 mmol) of pyrazole derivative (Example I, step 1) were dissolved in 2 ml of ethanol. 0.2 ml (1.2 mmol.) Of triethylamine was added to the solution, after which the whole was stored for 5 hours at 60 ° C. The solution was then evaporated under reduced pressure, after which the solution was subsequently diluted with about 20 ml of ethyl acetate. The precipitated product was filtered off, washed with ethyl acetate, dried and then dissolved in 10 ml of water. The aqueous solution was freeze dried. 0.51 g 3 (80%) of the product were obtained. R4: 0.54-0.59; R1: 0.65-0.70; / q_7 ^ = 40.5 ° (c = 1; n acetic acid) C28H41 ° 8N7S ”635.730.

EXAMPLE III

Amidino-L-tyrosyl-D-norleucyl-glycyl-L-phenyl-alaninamide (compound of the aforementioned formula, wherein A = D-norleucyl residue and B = amino group.

Step 1: benzyloxycarbonyl-D-norleucyl-glycyl-L-phenylalaninamide.

3.98 g (15 mmol.) Of benzyloxycarbonyl-D-norleucine / Izumiya and co .: J. Chem. Soc. Japan 79. ' 420 (1958) 7 dissolved in 15 ml of dimethylformamide. The solution was cooled to -15 ° C. 1.7 ml (15 mmole) of N-methylmorpholine and 2.0 ml (15 mmole) of chlorocarboxylic acid isobutyl ester were added with stirring at this temperature. After stirring for 10 min, a solution prepared in the following manner was added to the reaction mixture: 5.35 g (15 mmol.) Of benzyloxycarbonyl-800 3 4 23-8-glycyl-L-phenylalaninamide / Fruton and Bergmann: J. Biol. Chem. 145, 253 (1942)] in 100 ml of methanol in the presence of palladium activated carbon, then the catalyst is filtered off, the methanol is evaporated under reduced pressure and the residue is dissolved in 15 ml of dimethylformamide.

The reaction mixture mixed with this solution was cooled to -15 ° C and stored at this temperature for 2 hours. Then the reaction mixture was stored at 0 ° C for one hour and then evaporated under reduced pressure. The residue was mixed with water, filtered and dried.

The product was recrystallized from a mixture of ethyl acetate and petroleum ether. Yield: 5.0 g (70%). ϊφ 0.75-0.80.

15 C25H3205N4 - 468.538.

Step 2: D-norleucyl-glycyl-L-phenylalaninamide hydrochloride.

4.7 g (10 mmol) of the protected tripeptidamide (step 1) were dissolved in a mixture of 50 ml ethanol and 50 ml dimethylformamide. The solution was mixed with 10 ml of hydrochloric acid and then hydrated in the presence of palladium activated carbon. After the completion of the reaction, the catalyst was filtered off and washed with dimethylformamide.

The filtrate was combined with the washing liquid and evaporated.

The residue left after evaporation was mixed with ethyl acetate, filtered and then washed with ethyl acetate. 3.05 g (82%) of the product were obtained, φ: 0.45-0.55.

C17H2703N4C1-370.875.

Step 3: tert-butyloxycarbonyl-L-tyrosyl-D-norleucyl-glycyl-L-phenylalaninamide.

1.4 g (5 mmol) of tert-butyloxycarbonyl-L-tyrosine and 1.85 g (5 mmol) of tripeptidamide hydrochloride (step 2) were condensed in the manner described in step 3 of Example II. 1.94 g (65%) of the product were obtained. ίφ 0.70-0.75.

C31H4307N5 - 597.694.

Step 4: L-tyrosyl-D-norleucyl-glycyl-L-phenyl-800 3 4 23-9 - alaninamide acetate hydrate.

1.8 g (3 mmol.) Of the protected tetrapeptide (step 3) were converted in the manner described in step 4 of Example II. 1.12 g (65%) of the product were obtained. ίφ 0.60-0.65; ίφ 0.9-0.95; 80.5 ° (c = 1 n acetic acid).

C28H41 ° 8N5 "575'648 ·

Step 5: amidino-L-tyrosyl-D-norleucyl-glycyl-L-phenylalaninamide acetate hydrate.

0.58 g (1 mmol) of the tetrapeptidamide acetate hydrates obtained according to step 4 were reacted in the step 5 of example II described. 1.39 g (75%) of product were obtained, φ: 0.62-0.70; (q.7 ^ = + 40.5 ° (c = 1; n acetic acid).

15 C29H43OgN7-617,690.

EXAMPLE IV

Amidino-L-tyrosyl-D-norleucyl-glycyl-L-phenylalanyl-L-prolinamide (compound of the aforementioned formula, where A = D-norleucyl and B = L-prolinamide group).

Step 1: benzyloxycarbonyl-D-norleucyl-glycyl-L-phenylalanyl-L-proline.

7.95 g (30 mmol) of benzyloxycarbonyl-D-norleucine / ozomiya and co .: J. Chem. Soc. Japan 79, 420 (1958) 7 and 5.95 g (30 mmol.) Of 2,4,5-trichlorophenol dissolved in 60 ml of ethyl acetate and the solution was cooled in ice water. 5.8 g (28 mmol) of dicyclohexylcarbondiimide were added to the cooled solution. After standing for two hours, the precipitated dicyclohexyl carbamide was filtered off and the solution was evaporated under reduced pressure. The residue was recrystallized from n-hexane, filtered, washed with n-hexane and air dried.

The active ester obtained in this way (9.35 g = 75%; 4 R1: 0.82-0.92) was dissolved in 40 ml of pyridine. To the solution, 6.4 g (20 mmole) of glycyl-L-phenylalanyl-L-proline 35 (Belgian patent 858,453) and 2.8 ml (20 mmole) of triethylamine were added. The reaction mixture was stirred at room temperature overnight. Then the mixture was evaporated, after which the residue was dissolved in a mixture of 800 34 23 - 10 - 50 ml diethyl ether and 50 ml 5% sodium hydrogen carbonate solution. The water phase was washed twice with 30 ml diethyl ether and then acidified with n-sulfuric acid to a pH of 3. The precipitated product was shaken three times with 50 ml of ethyl acetate. The combined organic phases were washed twice with 20 ml of water, then dried over sodium sulfate and then evaporated under reduced pressure. The residue was mixed with cyclohexane, filtered, washed with cyclohexane and then dried. In this way 9.97 g (88%) of product 10 was obtained. Rd: 0.28-0.38.

C30H38 ° 7N4 "566 '636

Step 2: benzyloxycarbonyl-D-norleucyl-glycyl-L-phenylalanyl-L-prolinamide.

8.5 g (15 mmol) of the protected tetrapeptide prepared according to step 1 were dissolved in 30 ml of tetrahydrofuran. The solution was cooled to -15 ° C. At this temperature, 1.7 g (15 mmol.) Of N-methylmorpholine and 2.0 ml (15 mmol.) Of chlorocarbonic isobutyl ester were added with stirring, after which, after 10 min. 20-50% aqueous ammonia solution was added. The reaction mixture was stirred for a further hour at a temperature below 0 ° C, after which the tetrahydrofuran was distilled off from the reaction mixture under reduced pressure. 20 ml of water and 50 ml of ethyl acetate were added to the residue, after which the phases were separated. The water phase was washed with 20 ml of ethyl acetate. The organic phases were combined and washed three times with 20 ml of water. After drying over sodium sulfate, the solution was evaporated under reduced pressure. The residue was stirred with diethyl ether, filtered, washed with diethyl ether and then dried.

2

9.3 g (91%) of product were obtained. R ^: 0.4-0.5 C30H39 ° 6N5 "565'652 ·

Step 3: D-norleucyl-glycyl-L-phenylalanyl-L-prolinamide.

Of the protected tetrappeptidamide obtained in step 2, 5.66 g (10 mmol.) Were dissolved in 10 ml of methanol and hydrogenated in the presence of palladium activated carbon. After the completion of the reaction, the catalyst was filtered off, washed with methanol, and the washing liquid was combined with the filtrate. The methanolic solution was evaporated under reduced pressure. The residue was mixed with diethyl ether, filtered off, washed with diethyl ether and finally dried. 3.7 g (85%) of product were obtained, ίφ 0.38-0.48.

C22H33 ° 4N5 "431.524.

Step 4: tert-butyloxycarbonyl-L-tyrosyl-D-nor-leucyl-glycyl-L-phenylalanyl-L-prolinamide.

1.4 g (5 mmole) of tert-butyloxycarbonyl-L-tyrosine and 2.16 g (5 mmole) of the tetra-peptidamide obtained according to step 3 were condensed with this in the manner described in step 3 of Example II. difference, that no triethylamine was added to the solution of the amine component prepared with dimethylformamide. 3.0 g (86%) of product were obtained. R *: 0.4-0.5.

C36H50 ° 8N6 - 694.808.

Step 5: L-tyrosyl-D-norleucyl-glycyl-L-phenyl-alanyl-L-prolinamide acetate hydrate.

2.1 g (3 mmol) of the protected pentapeptidamide obtained according to step 4 were reacted in the manner described in step 4 of example II. 1.45 g (72%) of product was obtained. : 0.47-0.52; R ^: 0.92-0.98; l \ 7 ^ ° = + 26 ° (c = 1; n acetic acid).

25 DEG C.O.N.O.-672.762.

33 48 9 6

Step 6: amidino-L-tyrosyl-D-norleucyl-glycyl-L-phenylalanyl-L-prolinamide acetate hydrate.

0.7 g (1 mmol.) Of the pentapeptidamide acetate hydrate obtained according to step 5 were reacted in the manner described in step 5 of example II. 0.57 g (80%) of product was obtained. R1: 0.33-0.43; R1: 0.65-0.75; / o / qq = + 1 ° (c = 1; n acetic acid).

C34H50 ° 9N8 - 714.804.

8003423 35

Claims (8)

1. Process for the preparation of new enkephalin derivatives of the formula H2N-C (NH) -Tyr-A-Gly-PHe-B, in which Tyr, Gly and Phe L-tyrosine, glycine, resp. L-phenylalanine, A represents an alkyl group of 1-4 carbon atoms or thioalkyl-Da-amino acid of 1-4 carbon atoms and B is an amino group or L-prolinamide, as well as the salts of these compounds from the terminal amino group-containing peptide of the corresponding amino acid series, characterized in that the final amino group of the peptide is converted into a guanidino group in a manner known per se and, if desired, the product obtained is converted into a salt. 2. A process for the preparation of pharmaceutical preparations particularly acting on the central nervous system, characterized in that one or more compounds of the formula shown in claim 1 have the meanings for Tyr, Gly, Phe, A and B stated therein. , or a salt thereof, is mixed with the carriers and / or excipients customary in pharmacy and then processed into a pharmaceutical preparation in a manner known per se, eg injection solutions, tablets, dragees or capsules. 3. Singlephaline derivatives of the formula H2N-C (NH) -Tyr-A-Gly-Phe-B, wherein Tyr, Gly and Phe L-tyrosine, glycine, respectively. L-phenylalanine, A represents an alkyl group of 1-4 carbon atoms or thioalkyl-D-α-amino acid of 1-4 carbon atoms, while 35. is an amino group or L-prolinamide, as well as salts of these compounds. 4. Amidino-L-tyrosyl-D-methionyl-glycyl-L-phenylalanyl-L-prolinamide and its salts. 5. Amidino-L-tyrosyl-D-methionyl-glycyl-L-800 34 23 Λ - 13 - • - Λ phenylalaninamide and its salts. 6. Amidino-L-tyrosyl-D-norleucyl-glycyl-L-phenylalaninamide and its salts. 7. Amidino-L-tyrosyl-D-norleucyl-glycyl-L-5-phenylalanyl-L-prolinamide and salts thereof. Pharmaceutical preparation, characterized in that it contains, as active ingredient, a enkephalin derivative of the formula shown in claim 1, with the meanings for Tyr, Gly, Phe, A and 10 stated therein, or the salts thereof. 15 800 34 23