CH654841A5 - APPETIZING TRIPEPTIDES AND METHOD FOR THE PRODUCTION THEREOF. - Google Patents

Description

The invention relates to appetite suppressing tripeptides of the general formula I.

X-Y-Pro-OH

(I),

wherein

X for the amino acid groups pyroglutamyl, L-2-keto-imidazolidine-4-carbonyl, L-thiazolidine-4-carbonyl,

Y for the amino acid groups glycyl, alanyl, valyl, iso-leucyl, prolyl, L-2-aminobutyryl, norvalyl, norleucyl, L-2-aminodecanoyl, L-cyclohexylalanyl, phenylalanyl, tryptophyl, tyrosyl, seryl, threonyl, methionyl Glutamyl, lysyl, arginyl and leucyl are available, and their acids can be formed using a method customary in peptide chemistry, e.g. with the method described in Hungarian Patent No. 168 431. According to the invention, the procedure is such that proline or a derivative thereof protected on the carboxyl group is linked with the linking methods customary in peptide chemistry with the amino acids X and Y or the protected derivatives thereof and the protective groups on the tripeptide obtained in protected form be split off in a known manner.

According to an advantageous variant of the method according to the invention, proline methyl ester is acylated with the protected tert-butyloxycarbonyl-Y-OH amino acid, the dipeptide methyl ester obtained is saponified with dioxane-containing sodium hydroxide solution, and the tert-butyloxycarbonyl group is passed through Treatment with hydrochloric acid ethyl-45 tat removed. The dipeptide salt Y-Pro-OH-HCl thus obtained is then acylated with benzyloxycarbonyl-pyroglutamic acid pen-tafluorophenyl ester and the benzyloxycarbonyl group is removed from the protected tripeptide by catalytic hydrogenolysis.

50 The compounds of the general formula (I) are purified by methods known per se, expediently by column chromatography or simple envelope. The compounds are obtained in the form of either lyophilized powders or solid powders which are suitable for the preparation of different forms of medication.

The anorexic activity of the compounds of the general formula (I) was measured in rats in such a way that the animals starving for 96 hours were given intracerebroventricular (icv.) Physiological sodium chloride (control) or the 60 substances to be examined and after 1 or 5 hours the food intake by the test animals was measured. Table 1 contains the data of some representatives of the compounds of the general formula (I), the TRH and the tripeptide Glp-His-Gly-OH.

65

654 841

Table 1

Control Glp-His-Pr o-N H2

Glp-His-Gly-OH

Glp-Leu-Pro-OH

Glp-Abu-Pro-OH

Dose (Hg)

300 300 300 300 300 300 300

Food supply after the injection (hours)

5 1 5 1 5 1 5 5

Method of administration icv. icv. icv. icv. icv. icv. icv. icv.

Food (g) consumed within 24 hours in rats hungry for 96 hours

22.16 ± 0.88 19.81 ± 1.52 17.88 ± 2.94 19.33 ± 1.08 18.38 ± 1.80 6.10 ± 1.99 9.23 ± 0.92 8 , 31 ± 1.50

From the information in the table it can be seen that under the given experimental conditions, neither the TRH nor the Glp-His-Gly-OH significantly reduced the food intake of the test animals, while the individual representatives of the compounds of the general formula (I) led to a considerable reduction in the food intake led.

The peptides according to the invention and their salts can be used in therapy in the form of the usual medications. Such medicaments contain the compounds according to the invention together with inorganic or organic carriers which are suitable for enteral or parenteral dosing. The drugs can also be solid lyophilisates if substances which do not react with the peptides, e.g. Carbohydrates. Thin or concentrated suspensions or emulsions, which can also contain different preservatives and stabilizers, are also suitable.

The practical implementation of the method according to the invention will be illustrated using two general examples. The connections made with one of these methods are summarized with their physical constants in a table.

The abbreviations used in the examples correspond to the abbreviations accepted in the literature [J. Biol. Chem. 247,977 (1972)]. Other abbreviations: Abu = L-2-aminobutyric acid; Ada = L-2-aminodecanoic acid; Cha = L-cyclohexylalanine; Kic = L-2-keto-imidazolidine-4-carboxylic acid; Tea = L-thiazolidine-4-carboxylic acid.

When the connections were made, evaporation was always carried out in the Rotavapor «R» apparatus according to Büchi. The melting point was determined in an apparatus according to Dr. Tottoli (Büchi) determined. The thin-layer chromatograms were produced on a silica gel layer of the type silica gel G (Merck) made according to steel. The following solvent mixtures were used to develop the chromatograms:

(1) Ethyl acetate: PAW = 3: 2

(2) Ethyl acetate: PAW = 1: 1

(3) n-Butanol: acetic acid: water = 3: 1: 1

PAW = pyridine: acetic acid: water = 20: 6: 11

The thin-layer chromatograms are developed on the one hand with ninhydrin, on the other hand with the chlor-to-lidin-KJ method.

Columns filled with silica gel G type with a grain size of 0.062-0.2 mm were used to clean the individual substances.

Example 1 (method «A»)

L-pyroglutamyl-L-leucyl-L-prolin

Step 1

L-leucyl-L-prolinHCl

5.5 g (33 mmol) Pro-OMe-HCl are dissolved in 80 ml dichloro-

dissolved methane, the solution is cooled to 0 ° C, and with stirring 4.52 ml (33 mmol) of triethylamine, then 6.93 g (30 mmol) of Boc-Leu-OH are added. After the reagents have dissolved, the solution of 6.18 g (30 mmol) of dicyclohexylcarbodiimide containing 40 ml of dichloromethane 20 is added. The reaction mixture is stirred at 0 ° C for 2 hours, then the dicyclohexylcarbamide is filtered off. The filtrate is shaken out three times with 30 ml of hydrochloric acid solution, three times with 30 ml of sodium hydrogen carbonate solution 25 and once with 30 ml of water. The dichloromethane-containing solution is dried over dry sodium sulfate, then evaporated. The 9.7 g of oil-like Boc-Leu-Pro-OMe thus obtained are dissolved in 43 ml of dioxane and 43 ml of sodium hydroxide solution are added to the solution with stirring. The reaction mixture is stirred at room temperature for 1 hour, then neutralized by adding n hydrochloric acid solution. After the dioxane has been distilled off, the aqueous solution obtained is acidified to a pH of 2 in the presence of 60 ml of chloroform with concentrated hydrochloric acid solution. After separation, the aqueous solution is shaken twice with 30 ml of chloroform and dried over dry sodium sulfate. The 9% oily Boc-Leu-Pro-OH obtained after removal of the solvent are dissolved in 25 ml of ethyl acetate and 25 ml of 5N hydrochloric acid ethyl acetate are poured into 40 of the solution. The reaction mixture is left to stand at room temperature for 1 hour, then, after dilution with ether, the precipitate which has separated out is filtered off, washed with ether and dried. 6.48 g (81.5%, calculated on Boc-45 Leu-OH) are thus obtained H-Leu-Pro-OHHCl, which is a chromatographically uniform, white, water-absorbing

Is fabric. R ^ = 0.20; R ^ = 0.41.

50

2nd step

Benzyloxycarbonyl-L-pyroglutamyl-L-leucyl-L-proline 2.09 g (7.87 mmol) H-Leu-Pro-OHHCl, 2.2 ml triethylamine and 3.38 g (7.87 mmol) benzyloxycarbonyl-L pentafluorophenyl py-roglutamic acid are dissolved in 50 ml of chloroform and after 10 minutes, 1.1 ml (7.87 mmol) of triethylamine are added and the solution is left to stand at 60 room temperature overnight. Then the reaction mixture is shaken three times with 10 ml of hydrochloric acid solution and twice with 10 ml of water, dried over dry sodium sulfate and evaporated. The oily evaporation residue is crystallized with ether and 3.15 g of crude 65 product are obtained, which is recrystallized from 10 ml of ethyl acetate. In this way, 2.7 g (73.5%) of chromatographically uniform Z-Glp-Leu-Pro-OH are obtained. Melting point: 98-100 ° C.

55

654 841

3rd step

4.13 g (8.7 mmol) of Z-Glp-Leu-Pro-OH are dissolved in 80 ml of ethanol and, after the addition of 0.8 g of 10% bone carbon-palladium catalyst, the solution becomes hydrogen gas for 1 hour blown. After filtering off the catalyst, the filtrate is evaporated and the amorphous residue is triturated with ether. The 2.8 g of crude product are dissolved in 20 ml of water, clarified with bone carbon, and the solution is lyophilized. 2.6 g (91%) of amorphous, chromatographically uniform Glp-

Get Leu-Pro-OH. R ^ = 0.33.

Example 2 (method «B») L-pyroglutamyl-glycyl-L-prolin 1st step Glycyl-L-prolin

5.33 g (22 mmol) of H-Pro-OBzlHCl and 4.2 g (20 mmol) of Z-Gly-OH are dissolved in 50 ml of dichloromethane, 3.08 ml of triethylamine are added, the mixture is cooled to 0 ° C. and a solution of 4.12 g (20 mmol) of dicyclohexylcarbodiimide containing 30 ml of dichloromethane is added with stirring. The reaction mixture is stirred at 0 ° C for 1 hour, then placed in the refrigerator overnight. Then the dicyclohexylcarbamide is filtered off and the filtrate is shaken twice with 30 ml of hydrochloric acid solution, twice with 30 ml of sodium hydrogen carbonate solution and 30 ml of water. The dichloromethane-containing solution is dried over dry sodium sulfate and evaporated. The 8.5 g of oily Z-Gly-Pro-OBzl thus obtained are dissolved in 170 ml of ethanol and hydrogen is blown through the solution by adding 1.5 g of 10% bone carbon palladium. After the reaction has ended, the reaction mixture is diluted with 100 ml of water, the catalyst is filtered off, washed with water, and the filtrate is then 10

steamed. This gives 2.12 g (62%, calculated on Z-Gly-OH) of H-Gly-Pro-OH, which is chromatographically uniform. Rf = 0.19.

2nd step

Benzyloxycarbonyl-L-pyroglutamyl-glycyl-L-proline 2.15 g (5 mmol) of Z-Glp-OPfp and 0.86 g (5 mmol) of H-Gly-Pro-OH are suspended in 20 ml of dimethylformamide. 0.7 ml of triethylamine is added to the suspension, and another 0.7 ml of triethylamine is added after half an hour. After one hour, the reaction mixture is evaporated, the residue is dissolved in 30 ml of chloroform and the solution is extracted twice with 7 ml of hydrochloric acid solution, twice with 7 ml of sodium hydrogen carbonate solution and with water. After drying, the solution is evaporated. The oily residue is crystallized with ether, filtered and the crude product (1.4 g) recrystallized from the mixture of ethanol-ethyl acetate 20. This gives 1.23 g (53%) of chromatographically uniform Z-Glp-Gly-Pro-OH.

3rd step

0.35 g (0.84 mmol) of Z-Glp-Gly-Pro-OH are dissolved in 30 ml of 2S ethanol and hydrogen gas is blown through the solution by adding 0.05 g of 10% bone carbon-palladium catalyst. After filtering off the catalyst, the filtrate is evaporated and the residue is triturated with ether. This gives 0.22 g of product, which is treated with ethyl acetate and filtered. Yield: 0.17 g (71.5%) chromatographically uniform amorphous substance. Rf = 0.06; Rf = 0.25.

New compounds made using the above two detailed Me-3S methods are shown in Table 2.

l aoeuez tripeptides

method

Melting point ° C

Rf

Rf

Rf

Glp-Ala-Pro-OH

A

234-236 (decomposed)

0.12

0.31

Glp-Val-Pro-OH

A

amorphous

0.20

Glp-Ile-Pro-OH

A

amoprh

0.26

Glp-Pro-Pro-OH

A

188-191 (decomposed)

0.07

0.25

Glp-Abu-Pro-OH

A

248-250 (decomposed)

0.11

Glp-Nva-Pro-OH

A

183-186

0.19

Glp-Nle-Pro-OH

A

amorphous

0.26

Glp-Ada-Pro-OH

A

amorphous

0.35

Glp-Cha-Pro-OH

A

amorphous

0.30

Glp-Phe-Pro-OH

A

amorphous

0.22

Glp-Trp-Pro-OH

A

204-206 (decomposed)

0.27

Glp-Tyr-Pro-OH (a)

A

132-134 (decomposed)

0.22

Glp-Ser-Pro-OH (b)

A

144-146 (decomposed)

0.07

0.25

Glp-Thr-Pro-OH (c)

A

179-181 (decomposed)

0.12

0.31

Glp-Met-Pro-OH (d)

A

amorphous

0.22

Glp-Gln-Pro-OH (e)

A

amorphous

0.07

0.23

Glp-Lys-Pro-OHra

A

165-167 (decomposed)

0.16

Glp-Arg-Pro-OH (s)

A

297-298 (decomposed)

0.17

Glp-Gly-Pro-OH

B

amorphous

0.06

0.25

Kic-Leu-Pro-OH

A

amorphous

0.37

Tca-Leu-Pro-OH (h>

A

157-160

0.48

(a) using Tyr (But);

<b) using Ser (Bzl);

(c) using Thr (Bzl);

(d) the protective group Z is removed using TFA;

(e) purified in the form of a protected tripeptide using column chromatography;

(f) using Lys (Z);

(g) using Arg (NO2);

(h) using Boc-Tca.

C.

Claims (4)

654 841 PATENT CLAIMS 1. Appetite suppressing tripeptides of general formula I X-Y-Pro-OH (I), wherein X for the amino acid groups pyroglutamyl, L-2-keto-imidazolidine-4-carbonyl, L-thiazolidine-4-carbonyl, Y for the amino acid groups glycyl, alanyl, valyl, iso-leucyl, prolyl, L-2-aminobutyryl, norvalyl, norleucyl, L-2-aminodecanoyl, L-cyclohexylalanyl, phenylalanyl, tryp-tophyl, tyrosyl, seryl, threonyl, methionyl Glutamyl, lysyl, arginyl, and leucyl are available, as well as their acid addition salts. 2. Process for the preparation of appetite suppressing tripeptides of the general formula I X-Y-Pro-OH (I), wherein X for the amino acid groups pyroglutamyl, L-2-keto-imidazolidine-4-carbonyl, L-thiazolidine-4-carbonyl, Y for the amino acid groups glycyl, alanyl, valyl, iso-leucyl, prolyl, L-2-aminobutyryl, norvalyl, norleucyl, L-2-aminodecanoyl, L-cyclohexylalanyl, phenylalanyl, tryp-tophyl, tyrosyl, seryl, threonyl, methionyl Glutamyl, Lysyl, arginyl and leucyl and their acid addition salts, characterized in that proline or a derivative thereof protected on the carboxyl group is linked to the amino acids X and Y or the protected derivatives thereof and the protective groups are cleaved off from the tripeptide obtained in protected form. 3. The method according to claim 2, characterized in that proline methyl ester is acylated with the protected tert-butyloxycarbonyl-Y-OH-amino acid, the dipeptide methyl ester obtained, suitably saponified with dioxane-containing sodium hydroxide solution, the tert.- Butyloxycarbonyl protective group, expediently removed by treatment with hydrochloric acid ethyl acetate, the dipeptide thus obtained or its acid addition salt acylated with pentafluorophenyl benzyloxycarbonyl pyroglutamine and the protective group removed from the protected tripeptide obtained, expediently by catalytic hydrogenolysis. 4. A process for the preparation of tripeptides of the general formula I, wherein X and Y are as defined in claim 1, containing appetite suppressing preparations, characterized in that the active ingredient of the formula I mixed with pharmaceutical carriers and / or auxiliaries is converted to a pharmaceutical preparation . addition salts, a process for the preparation of these compounds or of appetite suppressing preparations containing these compounds. Reichelt et al. (Neuroscience 3, 1207 (1978)) succeeded in isolating 5 from the urine of patients suffering from anorexia nervosa the tripeptide Glp-His-Gly-OH (gypsum = pyroglutamyl), which had an anorexic effect in animal experiments. This finding provided another clue for the thesis that certain peptides can play an important role in the previously unknown mechanism of the development of the feeling of hunger or satiety. Taking into account the structural similarity of the above tripeptide to TRH (thyrotropin releasing hormone) (Glp-His-Pro-NH2), it is conceivable that both arise from the same precursor molecule. _ It was found that the free carboxyl group of the C-terminal is necessary for the anorexic effect, since compounds which contain a carboxamido group at this point have no anorexic effect. It also emerged in 2o that central histidine is not necessary to achieve the anorexic effect. The compounds which had the best effect were those where the central amino acid has an aliphatic side chain, while the C-terminal amino acid is a proline containing a free carboxyl group. 25 The compounds of the general formula (I) corresponding to the above determination X-Y-Pro-OH (I)