HU196226B - Process for producing new tripeptide derivatives of antiamnesic activity and pharmaceutical compositions containing them as active components - Google Patents

Abstract

Tripeptide derivs. of formula A-X-Y-Gly-B have an antiamnesic effect on the central nervous system. Medically acceptable acid adduct salts are also prepd.. A is benzyloxycarbonyl-, or tert.-butyl oxycarbonyl-, or 9 fluorenyl-methyl-oxy-carbonyl gps. or hydrogen atom. X is L-Pro; D-Pro; L-Pep-, D-Pep-, L-HPro-, or D-Lle gps., B is amino, methoxy or methyl gps..

Description

The present invention relates to a process for the preparation of

A-X-Y-Gly-B (I) is a novel antiamnesic tripeptide derivative acting on the central nervous system, where

H, or a Boc, Z or Fmoc protecting group,

X is a GABA-L-Pro group, and Y is a L-Leuco group, and B is an amino group, or

X is a D-Pip group and then

Y represents an L-Leu or L-Val group and B represents an amino or methoxy group, or

X is L-Pip and then

Y represents a D or L-Ile group and B represents an amino group, or

X is HP6, and then. Y represents an L-Leu group and B represents an amino or methoxy group, or

X is D-Pro and then

Y is D-Leu or L-Leu, and B is methyl or methoxy, or

X is L-Pro and then

Y is D-Val, L-Ile or L-Leu and B is methyl, amino or methoxy, but for Y L-Leu B can be only methyl, or

X is L-Glp and then

Y is L-Leu or D-Ile, and B is methyl or amino - and pharmaceutical compositions containing them as active ingredients.

It is known [Proc. Nat. Acad. Sci. USA 68, 1428. (1971)] that the known peptide hormone of the pituitary dorsal lobe, oxytocin, when treated with a microsomal preparation of the hypothalamus, is split into two parts: the so-called "tocic acid" containing the cystine ring and Pro-Leu. C-terminal tripeptide amine of formula -Gly-NH ₂ . The latter substance has a melanotropin release inhibitory effect and is therefore referred to in the literature by the acronym MIF (melanotropin inhibiting factor). Presumably, this tripeptide amide is a melanotropin-releasing hormone inhibitor.

A pentapeptide amide of Pro-His-PheArg-Gly-NH ₂ with similar activity was isolated one year later from the hypothalamus [Biochem. Biophys. Gap. Commun., 47, 1420 (1972)]. This material is identified by the acronym MSH-RIH. (Melanocyte Stimulating Hormone Release Inhibiting Hormone. Although the literature does not fully understand the use of these acronyms, we use them in this sense as described above.) MIF is generated from oxytocin but not by peptide metabolism.

The literature on the biological effects of MIF is inconsistent. Pharmacol. Biochem. Behav., 8, 93 (1978)] that MIF reduces puromycin [6-dimethylamino-9- [3- (p-methoxy-L -? - phenyl-alanyl-amino) -3- deoxy-β-D-ribofuranosyl-purine) amnesia. Later it was also reported by Kovács and Telegdy, Proceeding of the Symposium on Integrative Neurohumoral Mechanism. 1982. Edited by E. Endrőczi, Budapest] that MIF partially normalizes retrograde amnesia induced by electroconvulsive shock in rats.

For the treatment of the amnesic state, it has become important for the practice to develop MIF analogs that achieve or surpass the MIF in their anti-amnesic activity. The development of the synthesis was also important because it was only in this way that it was possible to obtain the necessary amounts of the active ingredient for practice.

Surprisingly, it has been found that the tripeptide derivatives of formula (I) have more favorable pharmacological properties than MIF. Among them, it outperforms a naturally occurring parent molecule in several potencies.

According to the invention, the tripeptide derivatives of formula (I) are prepared by:

a) For the preparation of tripeptide derivatives of the formula I wherein B, A and X are defined above and Y is as defined above, according to the rules of solid phase synthesis, a chloromethylated resin of a compound of formula II

The protected glycine derivative A'-Gly-OH (II), wherein A 'has the same meaning as defined above other than hydrogen, is reacted with the other amino acids of formula (I) after deprotection. phase synthesis, and thus obtained

The protected tripeptide resin derivative of formula A'-XY-Gly-O-Rez (IX), wherein A ', X and Y are as defined above and Copper represents the resin residue, is reacted with optionally removing a protecting group A 'from a tripeptide derivative of Formula I wherein A', X and Y are as defined above;

b) for the preparation of tripeptide derivatives of formula (I) wherein B is amino or methoxy, wherein A, X and Y are as defined above, a compound of formula (X):

_{A'-Y-Gly-O-CH3} (X) of the protected dipeptide methyl ester - after removal of a protecting group of formula (VIII) - wherein A 'A and Y are as defined above'

A'-X-OH (VIII) is reacted with the active ester or mixed anhydride of the protected amino acid A 'and X are as defined above, followed by the general formula (I) containing the protecting group A', A 'and B' methoxy. the iripeptide derivative of the formula wherein X and Y are as defined above, if desired, deprotecting A 'and / or treating it with ammonia to form B having amino group, or

c) for the preparation of tripephd derivatives of the formula I wherein B, A and X and Y are as defined above, a compound of the formula XII

Reacting a mixed anhydride of A'-X-Y-OH (XII) protected dipeptide, wherein A ', X and Y are as defined above, with 1-aminopropan-2-one,

-2196226 and then, if desired, deprotecting the tripeptide derivatives of formula (I) wherein A and A are A, and B is methyl, wherein X and Y are as defined above.

The mixed anhydride coupling steps can be accomplished by any mixed anhydride customary in peptide chemistry. It is preferred to use mixed anhydride with isobutyl chloroformate. The active esters may be those commonly used in peptide chemistry. Especially preferred are the p-chlorophenyl esters. Nitrogen bases are used as acid scavengers in these coupling reactions. Preferred examples of these are triethylamine and N-methylmorpholine. The removal of the protecting group A 'can be effected by catalytic hydrogenation (preferably palladium on activated carbon) or by acidic treatment (e.g. with glacial acetic acid).

The pharmacological assays of the tripeptide derivatives of the invention were carried out as follows. As experimental animals, male rats of CFY (LATI) weighing 160-180 g and, unless otherwise stated, male mice of CFLP (LATI) weighing 18-22 g were used.

The active compounds to be tested were dissolved in 96% (v / v) ethanol and then diluted with physiological saline to a concentration of 6% (v / v) ethanol and 200 µg / ml. The mode of administration is given separately for each test. In the anti-amnesia assay, the normal control and the ECS control group (ECS = electroconvulsive shock) received only 0.5 ml of a 6% v / v ethanol solution. The administration of s. c. injection.

1. Examination of anti-amnesia effect

The study was conducted in rats. The animals were housed in a 12-hour light cycle and were given food and water freely. Five animals per cage were housed.

The test was carried out with a metal grid bottom 40 x 40 cm dark box with a door with a lighted platform in front of its door. The animals were placed in the dark box for the first day for 2 minutes and then removed from there with their backs placed on the illuminated platform. Normal-acting rodents avoid light, so they go into the dark box after a shorter or longer period. For the experiments, we used animals that entered the box within 15 seconds. The above latency determination was repeated twice the next day, except that in the second experiment, the rats received an inevitable electric shock (0.75 mA, 3 seconds) through the metal grid upon entering the box. Within 10 seconds of the electric shock, the animals were removed from the box and treated with an electroshock at 220 V, 0.5 sec, through superficial ether narcosis, applied to their ears. As a result, the animals exhibited tonic-clonic convulsions. Animals showing no such symptoms were excluded from further experiment. The animal's latency time (passive latency time, PLI) was again determined 24 hours after the above study period with a 300 second observation time.

The experimental animals were divided into four groups for each study:

1. Normal control (without ECS):

This group underwent a second day latency determination and received unavoidable electric shock, mild ether narcosis, and electrode placement on their ears, but did not receive electroshock, and physiologically containing only 0.5 mL of 6% v / v ethanol instead of drug injection. saline solution was administered to them. c. 23 hours after the inevitable electric shock.

ECS Check:

They received the same treatment as the previous group with the exception that they received electro-shock.

MIF control:

They received treatment similar to that of the ECS control group but received 100 µg of MIF at 23 hours. c.

4. Analog-tested group:

She received a treatment similar to that of the MIF control group, but was given 100 µg of test analogue instead of MIF.

The PLI score was determined for each of the four groups. The antiamnesic effect in% was calculated using the following formula:

AH [%] = 100x (PLI ⁴ -PLI ² ) / (PLI'-PLI ² )

Table 1 breaks down the percentages so obtained.

/. spreadsheet

Test substance AH [%]

Pro-Leu-Gly-NH ₂ (MIF) + 52 Z-D-Pip-Leu-Gly-O-CHj + 132 D-Pip-Leu-Gly-NH ₂ + 119 Z-D-Pip-Val-Gly-O-CH + 90 Z-D-Leu-Pip-Gly-NFL + 69 HPro-Leu-Gly-NH ₂ + 93 BOC-H-Pro-Leu-Gly-O-CH + 81 Z-HPro-Leu-Gly-NH ₂ + 59 Z-D-Pro-D-Leu-Gly-CRJ + 60 Z D-Pro-Leu-Gly-O-CH ₃ + 54

2. Investigation of L-DOPA potentiating effect In mice at a dose of 40 mg / kg i. p. treated with pargyline [N-methyl-N- (2-propynyl) -benzylamine] and, after 1 hour, administered i. p. of the test substance, and finally after 1 hour at a dose of 100 mg / kg i.p. They are given L-DOPA [(-) - 3- (3,4-dihydroxyphenyl) -L-alanine]. The locomotor activity of the animals was recorded using a DSE-type ANlMEX (LKB, Denmark) motimeter for 2 hours at 33 minute intervals (Test description can be found in Breese, GR, et al., In The Thyroidaxis, Drugand Behavior, Ed .: J. Prague, Jr., Raven Press, NY 1974, p.

The results are shown in Table 2.

3. Measurement of analgesic effect In mice, the active ingredient i. p. following administration

After 1 hour, 0.3 mL of a 0.6% aqueous acetic acid solution was administered i. p., then record the number of abdominal wrinkles occurring at 10 minute intervals for 30 minutes. [Description of the test can be found in J. Fed. i'roc., 18, 412 (1959)

-3196226 is a measure of the analgesic effect expressed as the percentage of animals that did not respond to the pain stimulus,

4. Protection against Asphyxial Anoxia (AA)

After 16 hours of fasting, mice (22-24 g) are treated with test substance i. p., and after treatment, place the animals in 100 ml sealable vials, close the vials and measure the time to the last respiratory movement. Animals whose survival time is 30% greater than the mean survival time of the control group are considered protected. [The test is described in Life Sci., 16, 1607 (1975).] The antihypoxic effect (AA) column in Table 2 gives the number of such surviving animals.

5. Protection against Hypobaric Hypoxia (HH)

After 16 hours of fasting, 22-24 g mice were i.p.

the animals were placed in a vacuum desiccator and the pressure was reduced to 22.7 kPa (170 mmHg) within 20 seconds. From this time on, the survival time is recorded until the last respiratory movement. Those are the ones

Second

animals are considered protected with a survival time of at least 100% greater than the mean survival time of the untreated control group. The antihypoxic effect (HH) column in Table 2 shows the percentage of animals that survive. [Description of the test can be found in Proc. Soc. Exptl. Biol., 132, 629 (1969).]

6. Inhibition of tetrabenazine catalepsy

The studies were performed in rats. For animals i. p. administration of the test compound, followed by 1 mg i.v. p. tetrabenazine (1,2,3,4,6,7-hexahydro-3-isobutyl-9,10-dimethoxy-11H-benzo [a] quinolizin-2-one) is administered. Catalepsy is defined as animals that do not correct their bizarre posture within 30 seconds by placing their forelegs on a 7 cm high column. The anti-catalepsy column in Table 2 gives the number of non-catalepsy animals expressed as a percentage. [The test description can be found in Compt. Isoproterenol. Congr. Med. Alenistes Neurologistes 19, 497 (1952)

agent L-DOPA potentiating effect Antika- antihypoxic Grouse. % of control talep- effect chili. sziás effect effect 30 60 90 120 AA HH in minutes [%] [%] [%] I%]

Pro-Leu-Gly-NHI 61 92 81 117 0 40 0 0 Z-D-Pip-Leu-Gly-O-CH 96 98 140 102 0 10 0 Pip-D-Leu-Gly-CHI 112 77 78 126 10 10 10 0 Z-D-Pip-Val-Gly-O-CH 338 157 86 73 0 10 0 0 ZD-Pip-Leu-Gly-NH ₂ 235 81 56 58 10 0 0 40 H-Pro-Leu-Gly-NH 298 190 187 145 10 40 20 40 Z-H-Pro-Leu-Gly-NHI 72 97 or 95 0 0 0 0 BOC ~ HPro-Leu-Gly-NH ₂ 241 147 140 105 10 0 0 10 Z-D-Pro-Leu-Gly-O-CH 198 188 128 133 10 40 Boc-D-Pro-Leu-Gly-O-CH 313 113 57 23 20 0 0 40 Z-D-Pro-D-Leu-Gly-CHI 370 350 200 150 10 40 20 40 Z-Glp-Leu-Pro-O-CHI 467 312 86 76 10 0 0 0 Z-Pro-Leu-Gly-CHI 81 99 149 195 10 0 0 0 BOC-GABA-Pro-Leu-Gly-NH ₂ 283 81 71 53 20 0 0 20

The following conclusions can be drawn from the data in the tables:

1. The anti-amnesic and L-DOPA potentiating effects of D-pipecolic acid containing tripeptides are significantly more potent than those of MIF, but their L-DOPA potentiating effect is only significantly different in the first hour. The anti-catalepsy effect and the Z-D-PipLeu-Gly-NHI have an analgesic effect in this group of vc compounds, but the animals are less protected against hypoxia than those treated with MIF.

2. Substitution of N-terminal proline with homoproline significantly affects the anti-amnesia, L-DOPA potentiating, analgesic effect and tolerance to hypoxia. The BOC protecting group in the HPro group does not significantly alter the potentiating effect of antiamnesia and L-DOPA, while the Z protecting group reduces efficacy over unprotected tripeptidamide. Derivatives which have a Z-protecting group on the N-terminal amino acid or have a methoxy or methyl moiety on the C-terminal amino acid group are more potent or equivalent than MIF. The L-DOPA potentiating effect of these derivatives is significantly superior to that of MIF. In addition, there is a weak anti-catalepsy, antihypoxic and analgesic effect.

Thus, compounds having a D-pipecolyl group and / or having a Z-protecting group on the N-terminal amino acid or a C-terminal amino acid residue on the N-terminal amino acid exhibit stronger antiamnesic activity. methoxy or methyl. Thus, the tripeptide derivatives of formula (I) of the present invention are one or more superior in their effect to MIF, and the methods of the present invention provide the active compounds in the amounts required for practical purposes.

The tripeptide derivatives of formula (I) of the present invention are particularly useful in cases of acute or chronic memory impairment in a patient. In addition, the potentiating effect of L-DOPA is mainly used in the treatment of patients with Parkinson's disease.

The tripeptide derivatives of formula (I) are conveniently used in conventional pharmaceutical formulations. These formulations may be in solid, liquid or semi-liquid form and may be prepared using additives, excipients and excipients customarily used in the preparation of such compositions, fillers, diluents, stability enhancers, pH, osmotic pressure, flavor, odor and formulation. up.

The pharmaceutical composition is administered to the patient in an amount that contains the dose of active ingredient required to achieve the desired effect. This dose will depend on the degree of the disease, the patient's weight and sensitivity to the active ingredient, the route of administration and the number of daily treatments. The dose of active substance to be used at any given time can be determined by the attending physician, in the light of the knowledge of the patient to be treated, and based on his / her expert knowledge.

The amino acid and protecting group abbreviations used herein are the same as those widely used in the art. These are summarized in J. Bioi. Chem., 247, 977 (1972). Fmoc stands for 9-fluorenylmethyloxycarbonyl, HPro homprolyl (i.e. 2-pyrrolidinylacetyl), GABA-γ-aminobutyryl, Pip-2-piperidinecarbonyl, Glp. - denotes a pyroglutamil group.

Melting points were determined on a Boetius instrument (VEB Analytik Dresden). Thin layer chromatography was performed on Merck DC-Alufolien Kieselgel 60 sheets. The following was used as the runner. The ratios represent the volume ratio.

A: n-butanol-acetic acid-water 4: 1: 1

B: ethyl acetate 95% + (pyridine-acetic acid-water 20: 6: 11) 5%

C: ethyl acetate 80% + (pyridine-acetic acid-water 20: 6: 11) 20%

D: chloroform-methanol 9: 1.

Thin-layer sheets were developed with ninhydrin and chloro-tholidine / iodine reagent.

The amino acid composition was determined by hydrolyzing the test substance in 6N aqueous hydrochloric acid for 24 hours at 110 ° C and analyzing the resulting hydrolyzate using an AAA 881 automatic amino acid analyzer (manufactured by Mikrotechna, Prague, Czechoslovakia).

Optical rotation was measured with Polamat Type A (manufactured by Zeiss, Jena, NDK) polarimeter.

HPLC was carried out on an ODS Hypersil column (Merck, Darmstadt, Germany) with acetonitrile / 0.03m pH 7.5 phosphate buffer (6: 4 v / v) and fractions checked at 215 nm UV.

The column chromatographic purifications were also carried out on Merek's Kieselgel 60 column with eluent B and C as indicated above.

Further details of the present invention are illustrated by the following non-limiting examples.

Example I

BOC-GABA -Pro-Leu-Gly-NH ₂

a) BOC-GABA-Pro-Leu-Gly-O-resin 6g chloromethylated 99% polystyrene resin copolymerized with 1% divinylbenzene (FLUKA; chloromethylation degree 1.04 mmol / lg resin) in 25 ml ethanol suspended. To the alcohol suspension was added 1.68 mL (12 mmol) of triethylamine and 2 g (12 mmol) of BOC-Gly-OH protected glycine, and the reaction mixture was refluxed for 80 hours. After cooling, the resin was filtered off from the reaction mixture, washed twice with 30 ml each of ethanol and dichloromethane, and then dried in a vacuum desiccator over phosphorus pentoxide. The resin obtained contains 0.42 mmol of glycine per gram.

g of the BOC-Gly-O resin obtained above is weighed into a so-called Merrifield vessel fitted with a filter for use in solid phase syntheses, and 30 ml of dimethylformamide are added and swirled on a shaker for 24 hours. The solvent is then aspirated and the resin is washed three times with 30 ml each of dichloromethane, ethanol and acetic acid for three to three minutes. To the washed resin was added 30 ml of 2N hydrochloric acid in glacial acetic acid and shaken for 15 minutes. The hydrochloric acid-glacial acetic acid agitation is repeated with 30 ml of fresh solution after aspirating the glacial acetic acid solution. Subsequently, the resulting H-Gly-O resin was extracted three times with 30 ml each of 30 ml of acetic acid, ethanol and dichloromethane, twice with 30 ml each of dichloromethane containing 10% triethylamine and three times with 30 ml of dichloromethane. wash with methane.

The washed resin was aspirated with solvent, poured onto fresh dichloromethane (30 mL), then BOC-Leu-OH protected leucine (760 mg, 3.36 mmol, 4 molar equivalents) and dicyclohexyl (336 mg, 1.68 mmol, 2 equivalents). carbodiimide was added and shaken for 2 hours. Another dicyclohexylcarbodiimide (336 mg, 1.68 mmol, 2 molar equivalent) was added and shaken for a further 2 hours. The reaction mixture is aspirated from the resin, followed by 30 ml of fresh dichloromethane, 760 mg (3.36 mmol, 4 molar equivalents) of BOC-Leu-OH protected leucine and 672 mg (3.36 mmol, 4 molar equivalents) of dicyclohexylcarbodiimide. adding the resin, the reaction continued for another 24 hours.

At the end of reaction, the reaction mixture is sucked off the resulting Boc-Leu-Gly-O-resin and in all of BOC-Gly-O-of resin described _Wed: acting rnt removal of the BOC-protecting groups with glacial hydrogen chloride, and Boc-Leu- Instead of OH-protected leucine, an equivalent amount of BOC-Pro-OH protected proline (722 mg, 3.36 mmol) and 5

-5196226

Using dicyclohexylcarbodiimide (336 mg, 1.68 mmol), repeating the coupling step, the BOC-Pro-Leu-Gly-O-tripeptide chain is formed on the resin. This gives BOC-Pro-Leu-Gly-O resin.

The resulting BOC-Pro-Leu-Gly-O resin is aspirated and then deprotected with glacial acetic acid hydrochloride as described for BOC-Gly-O-resin and the resulting H-Pro-Leu-Gly-O resin is removed. resin was reacted with BOC-GABA-OH protected γ-butyric acid 682 mg (3.36 mmol, 4 molar equivalents) in the presence of 336 mg (1.68 mmol, 2 molar equivalents) of dicyclohexylcarbodiimide as described for H-Gly-O-resin. . The described iterations are also performed as described above.

At the end of the reaction, the resulting BOC-GABA-Pro-Leu-Gly-O resin is aspirated and the resin is washed three times with 30 ml each of dichloromethane and ethanol, then removed from the Merrifield vessel, washed with methanol on a filter and finally dried in a vacuum desiccator over phosphorus pentoxide in vacuo. 6.68 g of the title resin are obtained.

b) BOC-GABA-Pro-Leu-Gly-NH ₂ g of the above BOC-GABAPro-Leu-Gly-O dry resin obtained above are suspended in a pressurized vessel in 20 ml of anhydrous methanol and 40 ml at -40 ° C. conducting liquid ammonia. The vessel was hermetically sealed, the reaction mixture was stirred and allowed to warm to room temperature. After maintaining at this temperature for 48 hours, the pressure was released and the ammonia and methanol were evaporated. The residue is taken up three times with 5 ml of dimethylformamide, the resin is filtered off from the suspension, the dimethylformamide solution is concentrated in vacuo and the title crude peptide obtained as a residue is crystallized from 2.5 ml of dimethylformamide and 1.2 ml of ether. . 157 mg (80%) of the title compound are obtained.

Rf ^B = 0.1, Rf * = 0.55.1 ^ = 0.1.

96-99 ° C.

Amino acid analysis: Gly = 1.0, Leu = 0.88, Pro = 1.04, GABA = 1.03.

Example 2

Z-D-Pip-Leu-Gly-OCH₃

a) Z-Leu-Giy-OCH]

13.27 g (50 mmol) of Z-Leu-OH protected leucine are dissolved in 75 ml of anhydrous chloroform, cooled to -15 ° C and then stirred under constant stirring.

Anhydrous N-methylmorpholine (5.5 mL, 50 mmol) was added. Immediately thereafter, 6.5 ml (50 mmol) of isobutyl chloroformate was added dropwise and the reaction mixture was stirred at this temperature for an additional 5 minutes. Meanwhile, while heating, 6.2 g (50 mmol) of glycine methyl ester hydrochloride are dissolved in 10 ml of dimethylformamide, cooled and added.

5.5 ml (50 mmol) of N-methylmorpholine.

The two solutions were combined and the reaction mixture was stirred at -10 ° C for 1 hour and then at room temperature for 2 hours. The reaction mixture was then concentrated under reduced heat and the residue was dissolved in ethyl acetate (250 mL). The ethyl acetate solution was washed with 100 ml of 10% aqueous hydrochloric acid, water, 10% aqueous sodium carbonate solution and water again, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The resulting oil was crystallized by trituration with ether and the crystalline material was dissolved in ethyl acetate and precipitated by addition of petroleum ether. 10.93 g (65%) of the title compound are obtained.

M.p. 91-92 ° C.

R f ^B = 0.76, R f ^A = 0.65.

[α] 24 ^D = -28.9 ° (c = 1, in methanol).

Elemental analysis for C17H24N2O3 (M = 336.38):

Calculated: C, 60.7; H = 7.19%; N, 8.33%; Found: C, 60.1%; H = 7.08%; N, 8.25%.

b) Lea-Gly-OCH 2 -acetate

3.36 g (10 mmol) of the Z-LeuGly-OCH 3 protected dipeptide ester obtained above are dissolved in 30 ml of a 2: 1 mixture of methano'acetic acid and hydrogenated in the presence of 10% palladium on activated carbon. The progress of hydrogenation was monitored by TLC. When no starting material is present in the reaction mixture, hydrogenation is complete, the catalyst is filtered off and the filtrate is concentrated in vacuo.

The residue was evaporated to yield 2.0 g of the title dipeptide ester acetate as an oil.

Rr ^B = 0.11, Ri ^c = 0.56.

c) Z-D-Pip-Leu-Gly-OCHs

2.6 g (10 mmol) of ZD-Pip-OH protected D-pipecolic acid are dissolved in 10 ml of anhydrous chloroform, 1.1 ml (10 mmol) of anhydrous N-methylmorpholine are added and the solution is cooled to -15 ° C with constant stirring. . Immediately thereafter, 1.34 ml (10 mmol) of isobutyl chloroformate was added dropwise and stirring was continued for 5 minutes at this temperature.

Meanwhile, 2.0 g of the Leu-GlyOCHa acetate obtained above (10 mmol) are dissolved in 10 ml of anhydrous chloroform, 1.65 ml of anhydrous N-methylmorpholine are added, and the solution is cooled to 0 ° C. The latter solution was added to the mixed anhydride solution prepared above, and the reaction mixture was stirred at -15 ° C for 1 hour and at room temperature for 4 hours. The reaction mixture was concentrated in vacuo and the residue was dissolved in ethyl acetate (100 mL). The ethyl acetate solution was washed with 50 ml of 10% aqueous hydrochloric acid, 50 ml of 10% aqueous sodium carbonate solution and 50 ml of water, then dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The oily residue was crystallized from ether and recrystallized from a mixture of 10 ml of ethanol and 10 ml of water. Yield: 03 g (68%) of the title protected tripeptide.

M.p .: 129-130 ° C.

R _f ^A = 0.77, R _f ^D = 0.74.

[α] ₂₄ ^D = -5.9 ° (c = 1, in ethanol)

The purity of the product was found to be greater than 90% by HPLC.

Amino acid analysis: Gly = 1.0, Leu = 1.08, D-Pip = δ, 95.

Example 3

Z-D-Leu-Pip-Gly-GLY

Z-D-Pip-Leu-Gly-OCHa protected tripeptide ester (2.23 g, 5 mmol) was dissolved in dry methanol (20 mL), cooled to 0 ° C and bubbled with ammonia gas for 12 hours. At this time, the vessel was sealed and the reaction was allowed to stand at room temperature overnight, then concentrated in vacuo and the residue was crystallized from ether. The crystalline product was recrystallized from 15 ml of a 1: 1 mixture of methanol and ether. 1.94 g (90%) of the title protected tripeptide amide are obtained.

Mp 142-144 ° C.

R f = 0.7, R f = 0.35. [D] ₂ 4 ^d = + 9.35 ° (c = 1, ethanol).

The purity of the product was found to be greater than 90% by HPLC.

Amino acid analysis: Gly = 1.0, Leu = 1.02, D-Pip = 0.98.

Example 4

D-Pip-Leu-Gly-NHj · 1 / 2H ₂ O

Z-D-Pip-Leu-Gly-NFh protected tripeptide amide (648 mg, 1.8 mmol) was dissolved in ethanol (5 mL) and hydrogenated with 10% palladium on activated carbon in the presence of 2 molar equivalents of hydrogen chloride. After completion of the reaction, the catalyst is filtered off, the filtrate is concentrated to dryness in vacuo, the hygroscopic residue is dissolved in 25 ml of chloroform and the solution is saturated with ammonia gas. After 1 hour, the precipitated ammonium chloride salt was filtered off and the filtrate was concentrated in vacuo. The residue was crystallized with ether, filtered and dried. 331 mg (72%) of the title tripeptide amide hydrate are obtained.

Mp 150-152 ° C.

Amino acid analysis: Gly = 1.0, Leu = 1.08, D-Pip = 0.96.

R _f ^B = 0.95, R f ^A = 0.77.

Example 5

Fmoc-D-Pip-Val-Gly-OCH ₃ a) Z-Val-Gly-OCHi

Z-Val-OH protected valine (35.1 g, 150 mmol) was dissolved in anhydrous chloroform (225 mL), N-methylmorpholine (16.5 mL, 150 mmol) was added and the solution was cooled to -15 ° C with stirring. . 19.5 ml (150 mmol) of isobutyl chloroformate was added dropwise to the reaction mixture, which was stirred at this temperature for a further 5 minutes.

Glycine methyl ester hydrochloride (18.6 g, 150 mmol) was dissolved in dimethylformamide (25 mL) with heating, cooled and N-methylmorpholine (16.5 mL, 150 mmol) was added. The solution was cooled to 0 ° C and then mixed with the mixed anhydride solution prepared above. The reaction mixture was stirred at -10 ° C for 2 hours and then at room temperature for 6 hours. The reaction mixture was worked up as described in 2.a. Example 1b. 27 g (55.9%) of the title protected dipeptide ester are obtained.

157-159 ° C.

Rr * = 0.70, Rr ^B = 0.89.

[α] 24 D = -24 ° (c = 1 in methanol).

b) Val-Gly-OCH) acetate

Protected dipeptide ester Z-Val-Gly-OCH3 (4.83 g, 10 mmol) was dissolved in methanolic acetic acid 2: 1 (30 mL) and hydrogenated in the presence of 1 g of 10% palladium on activated carbon. The reaction was followed by thin layer chromatography. When complete, the catalyst was filtered off and the filtrate was concentrated in vacuo. The title free dipeptide ester acetate is obtained as an evaporation residue in the form of an oil.

Yield: 3.5 g.

Rf = 0.22, Rf ^B = 0.10.

C Fmoc-D-Pip-Val-Gly-OCHi

Val-Gly-OCHb-acetate (3.5 g, 10 mmol) and N-methylmorpholine (1.65 ml) were dissolved in dimethylformamide (25 ml) and Fmoc-D-Pip (4.45 g, 10 mmol) was added at room temperature. -OSu-protected ND-pipecolyloxy-succinimide and the reaction mixture was stirred at room temperature for 3 days. After evaporation in vacuo, the residue was dissolved in ethyl acetate (100 mL), washed with 10% aqueous hydrochloric acid (50 mL), water (3 x 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was crystallized from ether and recrystallized from ethanol-water. 3.5 g (68%) of the title tripeptide ester are obtained.

106-108 ° C.

Rf * = 0.69, Rf = 0.89.

[α] 25 ^D = -4.25 ° (c = 1 in methanol).

Amino acid analysis: Gly = 1.0, Val = 1.03, DPip = 0.85.

Example 6

BOC-H-Pro-Leu-Gly-O}

2.29 g (10 mmol) of BOC-HPro-OH protected homopoline are dissolved in 10 ml of anhydrous chloroform, 1.1 ml (10 mmol) of anhydrous N-methyl-morpholine are added and the solution is cooled to -15 ° C with constant stirring. to. Immediately thereafter, 1.34 ml (10 mmol) of chloroformic acid isobutyl ester was added dropwise and the reaction mixture was stirred at this temperature for 5 minutes.

Meanwhile, 2.0 g (10 mmol) of Leu-Gly-OCH3 acetate was dissolved in 10 ml of anhydrous chloroform, 1.65 ml of anhydrous N-methylmorpholine was added and the solution was cooled to 0 ° C. The resulting 0 ° C solution was added to the mixed anhydride solution prepared as above and the reaction mixture was stirred at -10 ° C for 2 hours and then at room temperature for 3 hours. The reaction mixture was concentrated in vacuo and the residue was dissolved in ethyl acetate (100 mL). The ethyl acetate solution was washed with 50 ml of 20% aqueous sulfuric acid solution, 50 ml of water, 50 ml of 10% aqueous sodium carbonate solution and then again with 50 ml of water, dried over anhydrous sodium sulfate, filtered and the filtrate evaporate in vacuo. The residue was crystallized from ether and recrystallized from a mixture of 15 ml of ethanol and 15 ml of water. 2.97 g (72%) of the title protected tripeptide ester are obtained.

95-97 ° C.

R _f * = 0.85, R _f ^D = 0.65.

[Α] ^D = -44.24 ° (c = 1, in ethanol).

-7196226

Purification by high performance liquid chromatography indicated that the product was greater than 90% pure.

Amino acid analysis: Gly = 1.0, Leu = 0.92, HPro = 0.9.

Example 7

BOC-H-Pro-Leu-Gly-NHI

BOC-HPro-Leu-Gly-OCH ₃ protected tripeptide ester (413 mg, 1 mmol) was dissolved in methanol (5 mL) and ammonia gas was passed at 0 ° C for 12 h. The ammonia-saturated solution was then sealed and allowed to stand at room temperature for 1 day. Carefully open the vessel and evaporate its contents under vacuum. The residue was crystallized from ether and recrystallized from methanol / ether (5 mL, 1: 1). 329 mg (90%) of the title protected tripeptide amide are obtained.

145-146 ° C.

R f ^A = -0.64, R _f ^a = 0.36.

(p] m ^D = -27.2 ° (c = 1, ethanol).

The purity of the product was found to be greater than 90% by HPLC.

Amino acid analysis: Gly = 1.0, Leu = 1.0, HPro = 0.86.

Example 8

Z-D-Pro-D-Leu-Gly-CH

a) Z-D-Pro-D-Leu-OH

D-leucine (1.17 g, 9 mmol) was dissolved in 9 mL of 1N aqueous sodium hydroxide solution and cooled to 0 ° C with constant stirring. A solution of 4.14 g (8 mmol) of Z-D-Pro-OPCP in 15 ml of dioxane is added with stirring and stirring is continued at 0-5 ° C for 48 hours. The reaction mixture was cooled to 0 ° C, the excess D-leucine was filtered off, the filtrate was concentrated to about 10 mL (dioxane removed), and the resulting aqueous solution was extracted three times with 10 mL of ethyl acetate. The aqueous phase was then adjusted to pH 3 with dilute aqueous hydrochloric acid (1: 1), extracted with ethyl acetate (3 x 50 ml), washed with water, dried (Na2SO4), filtered and evaporated. The residue was recrystallized from 20 ml of ethyl acetate-n-hexane (2: 1). 2.20 g (76%) of the title dipeptide are obtained.

Mp 128-130 ° C.

R f ^A = 0.80, R f ^c = 0.35.

[α] ₂₄ ^D = + 68 ° (c = 1, in methanol).

b) Z-D-Pro-D-Leu-Gly-CH 3

ZD-Pro-D-Leu-OH protected dipeptide (725 mg, 2 mmol) was dissolved in 5 mL of anhydrous chloroform, cooled to -15 ° C, and 0.3 mL (2.2 mmol) of triethylamine and 0.286 were added. ml (2.2 mmol) of isobutyl ester of chloroformate. After stirring at this temperature for 5 minutes, a solution of 220 mg (2 mmol) of 1-aminopropan-2-one (H2N-CH2-CO-CH3) in 1 ml of anhydrous dimethylformamide was added, followed by stirring. additional 0.3 mL (2.2 mmol) of triethylamine was added to the reaction mixture. Stirring was continued at -15 ° C for a further 120 minutes, after which the reaction mixture was allowed to stand in a refrigerator for 24 hours and then concentrated in vacuo. The residue was dissolved in ethyl acetate (50 mL), washed with 2N aqueous hydrochloric acid (25 mL), 10% aqueous sodium bicarbonate (25 mL), water (25 mL), dried over anhydrous sodium sulfate, filtered, and evaporated in vacuo. The residue was dissolved in 10 ml of ethanol and precipitated with ether to give 710 mg (85.1%) of the title protected product.

Mp 142-143 ° C.

R _f ^A = 0.65, R _r ^B = 0.49.

[Α] 25 D = + 80.82 ° (c = 1, in ethanol).

HPLC showed the product to be greater than 90% pure.

Amino acid analysis: Gly = 1.0, D-Leu = 0.91, D-Pro = 0.85.

Example 9

Z-Glp-Leu-Gly-CH}

a) ZGlp-Leu-OH

In each case, the procedure of Example 8 (a) was followed with the exception that D-leucine was replaced by L-leucine and ZD-Pro-OPCP was replaced by Z-Glp-OPCP. The oily residue obtained during work-up of the reaction mixture was recrystallized from methanol-n-hexane. This gives the title protected dipeptide in 80% yield. 184-185 ° C. R A = O, 55, _Rf = 0.7 ^c. MI4 ^D = -70.6 ° (c = 1, methanol).

b) Z-Glp-Leu-Gly-CH ₃

The protected dipeptide Z-Glp-Leu-OH (654 mg, 2 mmol) was reacted with a solution of 220 mg (2 mmol) of 1-aminopropanone in anhydrous dimethylformamide according to Example 8 (b). The reaction mixture was worked up as described in the example. The residue obtained by evaporation of the reaction mixture is triturated with ether and the precipitated solid is recrystallized from ethyl acetate-ether. 329 mg (35%) of the title compound are obtained. 181 ° C.

Rr ^A : = 0.79, R, ^B = 0.8. [α] ₂₄ ^D = -25.5 ° (c = 1, in dimethylformamide). High performance liquid chromatography indicated that the detectable smear / stays remain below 7%.

Amino acid analysis: Glu = 0.96, Leu = 1.03, Gly = 1.0.

Example 10

Z-Pro-Leu-Gly-CH, t) Z-Pro-Leu-OH

In each case, the procedure of Example 8 (a) was followed except that L-'eucine was used instead of D-leucine. The reaction mixture was worked up by crystallization of the resulting solid evaporation residue from acetone in hexane. This gives the title protected dipeptide in a yield of 82.8%. 134-136 ° C. R _f ^A = 0.67, R _f ^c = 0.8. [a] 24 = -63.8 ⁿ ° (c = 1, methanol).

b) Z-Pro-Leu-Gly-CH]

The protected dipeptide Z-Pro-Leu-OH (724 mg, 2 mmol) was reacted according to Example 8 (b) with 220 µg (2 mmol) of 1-aminopropanone in anhydrous dime-81.

136226?

til formamide solution. The reaction mixture was worked up as described in the example. The residue obtained by evaporation of the reaction mixture is triturated with ether and the precipitated solid is recrystallized from ethanol / petroleum ether. This gave 635 mg (76%) of the title compound. 140-142 ° C. Rf ^A = 0.9, Rf ^c = 0.85. [α] 24 ^D = -82.2 ° (c = 1, in ethanol). According to HPLC, detectable impurities remain below 5%.

Amino acid analysis: Pro = 0.82, Leu = 1.11, Gly = 1.0.

Example 11

Z-Pip-D-Ile-Gly-NH ₂

4.46 g (10 mmol) of Z-Pip-D-Ile-Gly-OCHI protected tripeptide ester (prepared as in Example 2 (c)) are dissolved in 40 ml of anhydrous methanol and the solution is cooled to 0 ° C. and purge it with ammonia gas for 12 hours. The vessel was then sealed and the solution was allowed to warm to room temperature. After completion of the reaction, the solution was concentrated in vacuo and the residue was crystallized with ether. The resulting crystalline product was recrystallized from methanol-ether. 3.64 g (84%) of the title protected tripeptide amide are obtained. 162-164 ° C. R _f ^A = 0.6, R _f ^c = 0.2. [α] 24 D = -12.2 DEG (c = 1 in methanol). According to HPLC, detectable impurities remain below 7%.

Amino acid analysis: Pip = 0.85, Ile = 1.02,

Gly = 1.0.

Example 12

Z-Pro-D-Val-Gly-GLY

a) Z-Pro-D-Val-Gly-O resin

In each case, the procedure of Example 1 (a) was followed except that Boc-D-Val-OH protected valine was used instead of Boc-Leu-OH protected leucine. 6.48 g of the title resin are obtained.

b) Z-Pro-D-Val-Gly-NH ₂ g of the dry Z-Pro-D-Val-Gly-O resin obtained above were further reacted in an analogous manner to that of Example 1 (b). The resulting crude peptide was recrystallized from dimethylformamide / ether. 135 g of pure title compound are obtained. Mp 206-208 ° C. R _f ^A = 0.6, R _f ^c = 0.25. [α] 24 ^D = -18 ° (c = 1, in methanol). According to HPLC, the detectable impurities are below 8%.

Amino acid analysis: Pro = 0.93, D-Val = 1.1, Gly = 1.0.

Example 13

Z-Pro-lle-Gly-NH ₂

a) Z-Pro-Ile-Gly-O resin

In each case, the procedure of Example 1 (a) was followed except that Boc-Ile-OH protected isoleucine was used instead of Boc-Leu-OH protected leucine. 6.58 g of the title resin are obtained.

b) Z-Pro-Ile-Gly-NH ₂ g of the dry Z-Pro-Ile-Gly-O resin obtained above were reacted in an analogous manner to that of Example 1 (b). The resulting crude peptide was recrystallized from dimethylformamide / ether. 147 g of pure title compound are obtained. Mp 234-236 ° C. M.p. = 0.22. [α] ₂₅ D = -4.4 ° (c = 1, in methanol). According to HPLC, the detectable impurities remain below 8%.

Amino acid analysis: Pro = 0.95, Ile = 0.92, Gly = 1.0.

The following compounds were also prepared as described in the Examples. (The letters in brackets refer to the procedure followed for the preparation of the compounds, consistent with the designation of claim 1.)

BOC-Pro-D-Val-Gly-NH 3 (b), mp 184-185 ° C; Z-Pro-D-Val-Gly-OCH ₃ , (b), mp 154-155 ° C; Z-Pip-lle-Gly-NH1, (a), mp 124-126 ° C; BOC-Pro-D-Val-Gly-NH 3 (a), mp 235-236 ° C; Fmoc-D-PIO-Leu-Gly-OCH 3, (b), m.p. 90-92 ° C; Glp-Leu-Gly-CH ₃ , (c), mp 140-141 ° C; Glp-D-Ile-Gly-NH1, (a), m.p. 229-233 ° C.

Claims (9)

Claims 1. Process according to formula I, A-X-Y-Gly-B (I) is a novel antiamnesic tripeptide derivative acting on the central nervous system, where H, or a Boc, Z or Fmoc protecting group, X is a GABA-L-Pro group and Y is a L-Leu group and B is an amino group, or X is a D-Pip group and then Y represents an L-Leu or L-Val group and B represents an amino or methoxy group, or X is L-Pip and then Y represents a D or L-Ile group and B represents an amino group, or X is HPro and then Y represents an L-Leu group and B represents an amino or methoxy group, or X is D-Pro and then Y is D-Leu or L-Leu, and B is methyl or methoxy, or X is L-Pro and then Y is D-Val, I-yl or L-Leu and B is methyl, amino or methoxy, but for Y L-Leu B can be only methyl, or X is L-Glp and then Y represents a L-Leu or D-Ile group and B represents a methyl or amino group, characterized in that a) For the preparation of tripeptide derivatives of the formula I wherein B, A and X are defined above and Y is as defined above, according to the rules of solid phase synthesis, a chloromethylated resin of a compound of formula II The protected glycine derivative of A'-Gly-OH (II), wherein A 'has the same meaning as defined above, other than hydrogen, is reacted with the additional amino acids of formula (I) after deprotection. phase synthesis, and thus obtained The protected tripeptide resin derivative of formula A'-XY-Gly-O-Rez (IX), wherein A ', X and Y are as defined above and Copper represents the resin residue, is reacted with optionally removing the protecting group A 'from the tripeptide derivative of formula I wherein A', X and Y are as defined above, wherein A 'is A; or b) for the preparation of tripeptide derivatives of formula (I) wherein B is amino or methoxy, wherein A, X and Y are as defined above, a compound of formula (X): A'-Y-Gly-O-CH3 (X) protected dipeptide methyl ester, where A 'and Y are as defined above, after removal of the protecting group A' A'-X-OH (VIII) is protected with the active ester or mixed anhydride of the protected amino acid, wherein A 'and X are as defined above, followed by the general formula (I) containing the protecting group A, A being protected, A being methoxy. the tripeptide derivative of the formula wherein X and Y are as defined above, if desired, removes the protecting group A 'and / or, by treatment with ammonia, transforms B into an amino group, or c) for the preparation of tripeptide derivatives of the formula I wherein B, A, X and Y are as defined above, a compound of the formula XII A mixed anhydride of the protected dipeptide of A'-XY-OH (XII), wherein A ', X and Y are as defined above, is reacted with 1-aminopropan-2-one and optionally protected with A', The tripeptide derivatives of formula (I) wherein B is methyl, wherein X and Y are as defined above, are deprotected. Process a) according to claim 1, characterized in that the protected glycine derivative of formula II, wherein A 'has the same meaning as defined above, other than hydrogen, and the solid phase chloromethylated resin is coupled with a tertiary nitrogen base. , preferably in the presence of triethylamine, in 1-4 carbon atoms. 3. A process according to claim 1 or 2, wherein A 'is a protected glycine derivative of the formula II having a tert -butyl-oxycarbonyl group. Process b) according to claim 1, characterized in that a mixed anhydride of the protected amino acid of the formula VIII, wherein A 'and X are as defined above, is used. 5. A process according to claim 4, wherein the mixed anhydride with the isobutyl ester of chloroformate is used as the mixed anhydride of the protected amino acid of formula VIII wherein A 'and X are as defined above. Process b) according to claim 1, characterized in that the active ester of the protected amino acid of the formula (VIII), wherein A 'and X are as defined above, is used. 7. The process according to claim 6, wherein the active ester of the protected amino acid of formula VIII, wherein A 'and X are as defined above, is p-chlorophenol or N-hydroxysuccinimide. . A method according to any one of the preceding claims. characterized in that the resulting chemical compounds of formula (I) wherein A ', X, Y and B are A The protecting group A 'as defined in claim 1 is removed with hydrogen chloride in glacial acetic acid. 9. Process according to any one of claims 1 to 3, characterized in that the resulting compounds of formula (I) wherein A ', X, Y and B are as defined in claim 1, are deprotected by catalytic hydrogenation. . IC. A process for the manufacture of a medicament for the treatment of central nervous system, preferably anti-amnesic, anti-Parkinson's, analgesic and anti-oxygen deficient, characterized in that one or more of the compounds of any one of claims 1-9. A tripeptide compound according to any one of claims 1 to 3, wherein A, X, Y and B are as defined in claim 1, which are conventionally used in the preparation of pharmaceutical compositions as diluents, fillers, stability enhancers, pH, osmotic pressure, taste, perfume, and adjuvants and adjuvants which make it easier to formulate and to make a pharmaceutical composition. Without illustration