CH645093A5 - ENKEPHALINE DERIVATIVES AND METHOD FOR THE PRODUCTION THEREOF. - Google Patents

Description

The invention relates to new enkephalin derivatives, their salts and a process for their preparation. The new compounds correspond to formula I.

s H2-C (NH) -T yr-A-Gly-Phe-B (I),

wherein

Tyr, Gly and Phe represent L-tyrosine, glycine or L-phenyl-alanine group,

A is a D-a-amino acid group with an alkyl or thioalkyl side chain containing 1 to 4 carbon atoms and

B means amino group or L-prolinamide group.

The terms Tyr, Gly and Phe are used in the specialist literature [e.g. J. Biol. Chem. 247,977 (1972)] for labeling the amino acids.

It is known that the morphine-like, i.e. the opiate activity of naturally occurring methionine and leucine enkephalin [Hughes et al .: Nature 258, 577 (1975)]

20 (peptides of the structure Tyr-Gly-Gly-Phe-Met or -Leu) can be increased by suitable substitution of the amino acid residues 2-Gly and 5-Met or 5-Leu. By incorporating the D-methionine group in the 2-position and the L-prolinamide group in the 5-position, one of the 25 most effective enkephalin analogues, the pentapeptidamide of the structure Tyr-D-Met-Gly-Phe-Pro-NH2, is obtained (see Belgian patent no . 858 453).

The aim of the invention is to produce new enkephalin derivatives whose opiate activity is greater than that of the known ones.

It has now been found that the opiate activity of the highly effective enkephalin analogs obtained by amino acid exchange can be increased even further if an amidino group is attached to the terminal amino group, i.e. Prepares peptide analogs containing a terminal guanidino group. The greater opiate activity of the compounds of the general formula I prepared in this way can be demonstrated by all the tests customary for this, for example on the guinea pig ileum (gui-4i nea pig ileum, GPI) [Kosterlitz and Watt: Br. J. Pharmacol. 33, 266 (1968); Kosterlitz et al .: Br. J. Pharmacol. 39, 398 (1970)] and on the vas deferens of the mouse [Hughes et al .: Br. J. Pharmacol. 53,371 (1975)]. Table 1 shows the opiate activities of four analogues containing a terminal guanidino group 45 and their H2N-terminal analogs. The numerical values mean relative activities related to normorphin (activity of normorphin = 1). For the sake of clarity, the unsubstituted peptide is given below the compound according to the invention.

Table I

Connection activity

GPP GPI "GPIab MVDC

H2N-C (NH) -Tyr-D-Met-Gly-Phe-Pro-NH2

823

173

25th

H-Tyr-D-Met-Gly-Phe-Pro-NH2

8th

8th

H2N-C (NH) -Tyr-D-Met-Gly-Phe-NH2

211

79

21st

H-Tyr-D-Met-Gly-Phe-NH2

17th

4th

H2N-C (NH) -Tyr-D-Nle-Gly-Phe-NH2

126

79

18th

H-Tyr-D-Nle-Gly-Phe-NH2

22

8th

H2N-C (NH) -Tyr-D-Nle-Gly-Phe-Pro-NH2

79

21st

17th

H-Tyr-D-Nle-Gly-Phe-Pro-NH2

12

10th

GPIa: according to Kosterlitz and Watt [Br. J. Pharmacol 33, 266 (1968)] GPIb: according to Kosterlitz et al. [Br. J. Pharmacol. 39, 398 (1970)] MVDC: after Hughes et al. [Br. J. Pharmacol. 53, 371 (1975)].

The result obtained by introducing the N-terminal guanidino group is surprising, since this part of the enkephalin molecule is the most sensitive and any intervention on the terminal amino group (for example its removal, acylation or dialkylation) generally leads to a decrease or loss of opioid activity [e.g. Morgan et al .: Communications, J. Pharm. Pharmac. 28, 660 (1976)].

Those compounds of the general formula I in which B represents an amino group have, when administered systemically, in the D'Amour-Smith test [J. Pharm. Ther. 72, 74 (1941)] has a stronger analgesic effect than morphine.

According to our own investigations, the compounds of general formula I selectively change the catecholamine content of the core of amygdala centralis. Since this core group has a high enkephalin content and a high opiate receptor density and is not part of the system that specifically affects the feeling of pain, it can be expected that the compounds of general formula I will specifically influence the following functions: food intake, emotionality, social behavior, learning and memory processes .

The invention accordingly relates to a process for the preparation of new enkephalin derivatives of the general formula I, in which the meaning of Tyr, Gly, Phe, A and B is the same as above, from the peptides of a corresponding amino acid sequence containing a terminal amino group. It is characteristic of the process that the terminal amino group is converted into a guanidino group in a manner known per se by reaction with a guanidating agent and a salt is optionally formed from the product obtained.

According to a preferred embodiment of the process according to the invention, the starting peptides are set at 20-80 ° C. with 1.1-2.0 equivalents of l-amidino-3,5-dimethyl-pyrazole acetate in the presence of 1.1-2. 0 equivalents of trialkylamine, the reaction medium being an aqueous or anhydrous lower alcohol and the concentration in the reaction medium being 1-1.1 mmol / ml. The solution is then evaporated, the peptide containing a guanidino group at the N-terminal is separated off and, if desired, converted to a salt.

The invention is illustrated by the following examples.

The Rf values given in the examples were determined on silica gel G (Reanal, Budapest) by layer chromatography using the following solvent mixtures:

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 60: 20: 6: 11

4. Chloroform-methanol 9: 1

5. Methylene chloride - 25% aqueous

Ammonia-methanol 12: 3: 8

example 1

Amidino-L-tyrosyl-D-methionyl-glycyl-L-phenylalanyl-L-prolinamide (compound of the formula I with A = D-methionine and B = L-prolinamide)

Step I: l-amidino-3,5-dimethyl-pyrazole acetate 20.1 g (100 mmol) l-amidino-3,5-dimethyl-pyrazole nitrate [Thiele and Dralle: Ann. 302, 294 (1898)] are dissolved with stirring in a mixture of 110 ml of sodium hydroxide solution and 200 ml of methylene chloride. The organic phase is separated off, dried over sodium sulfate, mixed with 6 ml of acetic acid and then evaporated under reduced pressure. The crystalline residue is suspended in diethyl ether.

3,645,093

oscillated, filtered off, washed with diethyl ether and then dried. 18.25 g (92%) of product are obtained. Melting point: 114-116 ° C.

Analysis for C8Hi202N4 (M = 198.2)

s 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

1.45 g (2 mmol) of L-tyrosyl-D-methionyl-glycyl-L-phenyl-io alanyl-L-prolinamide acetate trihydrate (Belgian Patent No. 858 453) and 0.5 g (2.52 mmol) ) l-Amidino-3,5-dimethyl-pyrazole acetate (Example 1, step 1) are dissolved in 2 ml of ethanol. 0.4 ml (2.86 mmol) of triethylamine are added to the solution and the mixture is kept at 60-70 ° C. for 4 hours. Then the reaction mixture is diluted with 30 ml of ethyl acetate, the precipitate is filtered off, washed with ethyl acetate and dried in vacuo. 1.17 g (80%) of product are obtained. Rf5: 0.63-0.68; [ci] d20 = + 4 ° (c = 1; n acetic acid) 20 C33H + s09N8S-732.844

Example 2

Amidino-L-tyrosyl-D-methionyl-glycyl-L-phenylalani-namide (compound of the formula I with A = D-methionine residue 25 and B = amino group)

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

5.35 g (15 mmol) of benzyloxycarbonyl-glycyl-L-phenyl-alaninamide [Fruton and Bergmann: J. Biol. Chem. 145, 253 30 (1942)] are dissolved in 100 ml of methanol and hydrogenated in the presence of palladium-activated carbon. After the reaction has ended, the catalyst is filtered off, washed with methanol, the washing methanol is combined with the filtrate and the latter is evaporated under reduced pressure. The glycyl-L-phenyl-alaninamide (Rf3: 0.19-0.29) and 6.43 g (15 mmol) of tert-butyloxycarbonyl-D-methionine-2,4,5 obtained as a vapor residue trichlorophenyl ester [Broadbent and others: J. Chem. Soc., 1967, 2632] are dissolved in 30 ml of pyridine. The solution is left overnight and then evaporated under reduced pressure. The residue is triturated with diethyl ether containing 15% mercaptoethanol, then filtered off, washed with diethyl ether and dried. 5.1 g (75%) of product are obtained. Rf2: 0.70-0.77.

45C2IH32OsN4S-452,564

Step 2: D-methionyl-glyeyl-L-phenylalanine amidhydrochloride

4.52 g (10 mmol) of the protected tripeptide amide (step 1) are suspended in 20 ml of ethyl acetate and 40 ml of ethyl acetate containing 11-15% hydrochloric acid are added to the suspension with stirring and ice-cooling. The reaction mixture is stirred for one hour, then diluted with 60 ml of ethyl acetate, the precipitate which has separated out is filtered off, washed with ethyl acetate and dried in vacuo over 55% potassium hydroxide. 3.7 g (95%) of product are obtained. Rf3: 0.50-0.55.

Ci6H25 03N4SC1- 388.915

Step 3: tert-butyloxycarbonyl-L-tyrosyl-D-methionyl-glycyl-L-phenylalaninamide 60 1.4 g (5 mmol) of tert-butyloxycarbonyl-L-tyrosine [Anderson and McGregor: J. Am. Chem. Soc. 79, 6180 (1957)] are dissolved in 10 ml of dimethylformamide. The solution is cooled to -15 ° C. At this temperature, 0.6 ml (5 mmol) of N-methylmorpholine and 0.7 ml of 65 (5 mmol) isobutyl chlorocarbonate are added with stirring. After stirring for 10 minutes, the reaction mixture is added to the suspension of 1.95 g (5 mmol) tripeptide amide hydrochloride (step 2) and 0.7 ml (5 mmol) cooled to −15 ° C.

093

Triethylamine in 10 ml of dimethylformamide. The reaction mixture is stirred at -15 ° C for two hours, at 0 ° C for one hour and then evaporated under reduced pressure. The evaporation residue is triturated with water, filtered, washed with water, dried and then recrystallized from 30 ml of boiling ethyl acetate. 2.15 g (70%) product are obtained. Rf1: 0.64-0.68.

C30H41O7N5S- 615.734

Step 4: L-Tyrosyl-D-methionyl-glycyl-L-phenylalanine amide acetate hydrate

1.85 g (3 mmol) of the protected tetrapeptide amide (Example 2, step 3) are suspended in 5 ml of ethyl acetate and, with stirring and ice-cooling, 15 ml of ethyl acetate containing 11-15% hydrochloric acid are added. The reaction mixture is stirred for one hour and then diluted with 20 ml of ethyl acetate. The precipitate is filtered off, washed with ethyl acetate and then dried. The product obtained is dissolved in 10 ml of water and the solution is applied to 15 ml of acetate anion exchange resin (AG 1-X2, Bio-Rad, Richmond, California). The resin is washed with 80 ml of water. Then the aqueous solutions are lyophilized. 1.07 g (60%) of product are obtained. Rf3: 0.55-0.60; Rf5: 0.89-0.97; [<x] D20 = + 82 ° (c = 1; n acetic acid).

C27H3908N5S-593,688

Step 5: Amidino-L-tyrosyl-D-methionyl-glycyl-L-phenylalaninamide acetate hydrate

0.6 g (1 mmol) of tetrapeptide amide hydrate (example 2, step 4) and 0.24 g (1.2 mmol) of pyrazole derivative (example 1, step 1) are dissolved in 2 ml of ethanol. The solution is mixed with 0.2 ml (1.43 mmol) of triethylamine and then kept at 60 ° C. for 5 hours. The solution is then concentrated under reduced pressure and then diluted with about 20 ml of ethyl acetate. The precipitated product is filtered off, washed with ethyl acetate, dried, then dissolved in 10 ml of water and the solution is lyophilized. 0.51 g (80%) product are obtained. Rf3: 0.54-0.59; Rf5: 0.65-0.70; [o] d20 = 40.5 ° (c = 1; n acetic acid). C28H4108N7S- 635.730

Example 3

Amidino-L-tyrosyl-D-norleucyl-glycyl-L-phenylalanine amide (compound of the formula I with A = D-norleucyl radical and B = amino group)

Step 1: Benzyloxycarbonyl-D-norleucyl-glycyl-L-phenylalaninamide

3.98 g (15 mmol) of benzyloxycarbonyl-D-norleucine [Izu-miya et al .: J. Chem. Soc. Japan 79, 420 (1958)] are dissolved in 15 ml of dimethylformamide. The solution is cooled to -15 °. 1.7 ml (15 mmol) of N-methylmorpholine and 2.0 ml (15 mmol) of isobutyl chlorocarbonate are added with stirring at this temperature. After stirring for 10 minutes, a solution prepared as follows is added to the reaction mixture:

5.35 g (15 mmol) of benzyloxycarbonyl-glycyl-L-phenyl-alaninamide [Fruton et al. Bergmann: J. Biol. Chem. 145, 253 (1942)] are hydrogenated 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 is cooled to −15 ° C. and stirred at this temperature for two hours, then at 0 ° C. for one hour and finally evaporated under reduced pressure. The residue is triturated with water, filtered and dried. The product is recrystallized from a mixture of ethyl acetate and petroleum ether. Yield: 5.0 g (70%). Rf2: 0.75-0.80. C25H3205N4-468.538

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

4.7 g (10 mmol) of the protected tripeptide amide (step 1) are dissolved in a mixture of 50 ml of ethanol and 50 ml of dimethylformamide. The solution is mixed with 10 ml of hydrochloric acid and hydrogenated in the presence of palladium-active carbon. After the reaction has ended, the catalyst is filtered off and washed with dimethylformamide. The filtrate is combined with the washing liquid and evaporated. The evaporation residue is triturated with ethyl acetate, filtered and washed with ethyl acetate. 3.05 g (82%) of product are obtained. Rf3: 0.45-0.55. CX7H2703N4C1-370.875

Step 3: tert-Butyloxycarbonyl-L-tyrosyl-D-norleucyl-15 glycyl-L-phenylalaninamide

1.4 g (5 mmol) of tert-butyloxycarbonyl-L-tyrosine and 1.85 g (5 mmol) of tripeptide amide hydrochloride (step 2) are condensed in the manner described in step 3 of example 2. 1.94 g (65%) of product are obtained. Rf1: 2o 0.70-0.75.

C31H4307Ns-597.694

Step 4: L-Tyrosyl-D-norleucyl-glycyl-L-phenyl-alanine amide acetate hydrate

1.8 g (3 mmol) of the protected tetrapeptide (step 3) 25 are reacted in the manner described in step 4 of example 2. 1.12 g (65%) of product are obtained. Rf3: 0.60-0.65; Rfs: 0.9-0.95; [a] d20 = 80.5 ° (c = 1; n acetic acid)

C28H41OsNs- 575,648 30 Step 5: Amidino-L-tyrosyl-D-norleucyl-glycyl-L-phenylalaninamide acetate hydrate

0.58 g (1 mmol) of the tetra-peptide amide acetate hydrate obtained in step 4 are reacted in the manner described in step 5 of example 2. 0.46 g (75%) of product 35 are obtained. Rf5: 0.62-0.70; [a] d20 = + 40.5 ° (c = 1; n acetic acid).

C29H43OsN7-617,690

Example 4

40 Amidino-L-tyrosyl-D-norleucyl-glycyl-L-phenylalanyl-L-prolinamide (compound of the formula with A = D-Norleucyl group and B = L-prolinamide group)

Step 1: Benzyloxycarbonyl-D-norleucyl-glycyl-L-phenylaianyl-L-proline 45 7.95 g (30 mmol) of benzyloxycarbonyl-D-norleucine [Izu-miya et al .: J. Chem. Soc. Japan 79.420 (1958)] and 5.95 g (30 mmol) of 2,4,5-trichlorophenol are dissolved in 60 ml of ethyl acetate and cooled in ice water. 5.8 g (28 mmol) of dicyclohexylcarbodiimide are added to the cooled solution. 50 After two hours, the precipitated dicyclohexyl carbamide is filtered off and the solution is evaporated under reduced pressure. The residue is recrystallized from n-hexane, filtered off, washed with n-hexane and air-dried. The active ester 55 obtained in this way (9.35 g = 75%; Rf4: 0.82-0.92) is dissolved in 40 ml of pyridine. 6.4 g (20 mmol) of glycyl-L-phenylala-nyl-L-prolin (Belgian Patent No. 858 453) and 2.8 ml (20 mmol) of triethylamine are added to the solution. The reaction mixture is stirred at room temperature overnight. The mixture is then evaporated and the residue is dissolved in a mixture of 50 ml of diethyl ether and 50 ml of 5% sodium hydrogen carbonate solution. The aqueous phase is washed twice with 30 ml of diethyl ether and then acidified to pH 3 with n sulfuric acid. The product which separates out is shaken out three times with 50 ml of ethyl acetate. The combined organic phases are washed twice with 20 ml of water, dried over sodium sulfate and then evaporated under reduced pressure. The

The residue is triturated with cyclohexane, filtered, washed with cyclohexane and then dried. 9.97 g (88%) of product are obtained. Rf2: 0.28-0.38.

C30H38O7N4- 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 are dissolved in 30 ml of tetrahydrofuran. The solution is cooled to -15 ° C. At this temperature, 1.7 g (15 mmol) of N-methylmorpholine and 2.0 ml (15 mmol) of isobutyl chlorocarbonate are added, after stirring for 10 minutes, 8 ml of 20-25% aqueous ammonia solution cooled to below 5 ° C. are added. The reaction mixture is stirred at temperatures below 0 ° C. for a further hour, then the tetrahydrofuran is distilled off from the reaction mixture under reduced pressure. 20 ml of water and 50 ml of ethyl acetate are added to the residue and the phases are separated from one another. The aqueous phase is washed with 20 ml of ethyl acetate. The organic phases are combined and washed three times with 20 ml of water. After drying over sodium sulfate, the solution is evaporated under reduced pressure. The residue is triturated with diethyl ether, filtered, washed with diethyl ether and then dried. 9.3 g (91%) of product are obtained. Rf2: 0.4-0.5.

C30H39O6N5-565.652

Step 3: D-norleucyl-glycyl-L-phenylalanyl-L-prolin amide

5.66 g (10 mmol) of the protected tetrapeptide amide obtained in step 2 are dissolved in 100 ml of methanol and hydrogenated in the presence of palladium-activated carbon. After the reaction has ended, the catalyst is filtered off.

5 645 093

trated, washed with methanol and the washing liquid combined with the filtrate. The methanolic solution is evaporated under reduced pressure. The residue is triturated with diethyl ether, filtered, washed with diethyl ether and then dried. 3.7 g (85%) of product are obtained. Rf3: 0.38-0.48.

C22H3304Ns-431.524

Step 4: tert-butyloxycarbonyl-L-tyrosyl-D-norleucyl-glycyl-L-phenylalanyl-L-prolinamide io 1.4 g (5 mmol) of tert-butyloxycarbonyl-L-tyrosine and 2.16 g (5 mmol ) of the tetrapeptide amide obtained in step 3 are condensed in the manner described in step 3 of example 2, with the difference that no triethylamine is added to the solution of the amine component prepared with dimethylformamide. 3.0 g (86%) of product are obtained. Rf ': 0.4-0.5.

C36H50O8N6-694.808

Step 5: L-Tyrosyl-D-norleucyl-glycyl-L-phenyl-alanyl-L-prolinamide acetate hydrate 20 2.1 g (3 mmol) of the protected pentapeptide amide obtained according to Step 4 are added to those in Step 4 of the example 2 implemented way described. 1.45 g (72%) of product are obtained. Rf3: 0.47-0.52; Rf5: 0.92-0.98; [cc] d20 = + 26 ° (c = 1; n acetic acid).

25 C33H4809N6 - 672.762

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 in step 5 are reacted in the manner described in step 5 of example 2. 0.57 g (80%) product are obtained. Rf3: 0.33-0.43; Rf5: 0.65-0.75; [a] d20 = + 1 ° (c = 1; n acetic acid).

C34H50O9N8- 714.804.

Claims (8)

645 093 1. Enkephalin derivatives of the formula I. H2N-C (NH) -Tyr-A-Gly-Phe-B (I), wherein Tyr, Gly and Phe represent L-tyrosine, glycine or L-phenyl-alanine group, A is a D-a amino acid group with an alkyl or thioalkyl side chain containing 1 to 4 carbon atoms and B means amino group or L-prolinamide group, and the salts of these compounds. 2. Amidino-L-tyrosyl-D-methionyl-glycyl-L-phenyl-ala-nyl-L-prolinamide and its salts as compounds according to claim 1. 2nd PATENT CLAIMS 3. Amidino-L-tyrosyl-D-methionyl-glycyl-L-phenyl-alaninamide and its salts as compounds according to claim 1. 4. Amidino-L-tyrosyl-D-norleucyl-glycyl-L-phenyl-ala-ninamide and its salts as compounds according to claim 1. 5. Amidino-L-tyrosyl-D-norleucyl-glycyl-L-phenyl-alanyl-L-prolinamide and its salts as compounds according to claim 1. 6. Process for the preparation of new enkephalin derivatives of the formula I. H2N-C (NH) -Tyr-A-Gly-Phe-B (I), wherein Tyr, Gly and Phe represent L-tyrosine, glycine or L-phenyl-alanine group, A is a D-a amino acid group with an alkyl or thioalkyl side chain containing 1 to 4 carbon atoms and B denotes amino group or L-prolinamide group, and the salts of these compounds from the peptides of the corresponding amino acid sequence containing a terminal amino group, characterized in that the terminal amino group of the peptide is converted into the guanidino group by reaction with a guanidating agent and, if desired, from the product obtained Salt forms. 7. The method according to claim 1, characterized in that l-amidino-3,5-dimethyl-pyrazole is used as guanidating agent. 8. pharmaceutical preparations, characterized by a content of enkephalin derivatives of the formula I according to claim 1.