JPH0774193B2 - Dipeptide derivative, production method and use thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention provides a compound represented by the general formula (1): (In the formula, m represents an integer of 1 to 8, and n is 0 to 6
R ¹ represents a hydrogen atom, R ² represents a hydrogen atom, a branched alkyl group having 3 to 5 carbon atoms, a phenyl group, a hydroxyphenyl group, or a methylthio group, or
R ¹ and R ² together represent a bond between carbon and nitrogen, X represents an aralkyl group, an aryl-alkenyl group, or an aryloxy group, provided that when n is 0, R ² is other than a hydrogen atom. Express meaning. And a method for producing the same and use thereof.

More specifically, the compound of the present invention having the general formula (1) is a prolyl endopeptidase (EC, 3.4.21.
26. Prolyl-endopeptidase) is a compound that not only exhibits enzyme inhibitory activity, but is also effective in improving and treating symptoms due to organic disorders in the brain.

Here, "organic disorder in the brain" is a cerebral infarction sequelae, sequelae of cerebral hemorrhage, various symptoms derived from cerebral ischemic disorders such as sequelae of cerebral arteriosclerosis and senile dementia, presenile dementia, amnesia, sequelae of head trauma, It means various organic disorders caused by aftereffects of brain surgery.

(Prior Art) Prolyl endopeptidase acts on substance P, TRH (thyroid stimulating hormone) and neurotensin, which are considered to be neurotransmitters, and vasopressin which is considered to be related to memory, and inactivates these. It is known to change. On the other hand, Dr. Tsuru and Mr. Yoshimoto of Nagasaki University found that compounds that inhibit prolyl endopeptidase activity prevent experimental amnesia caused by rat scopolamine, and reasoned that prolyl endopeptidase inhibitors are involved in memory consolidation. . The results also suggest that prolyl endopeptidase inhibitors could be used for the prevention and treatment of amnesia.

In fact, the effect of the novel dipeptide derivative represented by the general formula (1) of the present invention on amnesia was confirmed by experiments using experimental animals.

Brain cells maintain an intracellular environment that is completely different from the surrounding environment (extracellular fluid), maintain the difference and live, but in order to do so, they must continually generate and supply energy . Most of the energy required by the nerve cells in the brain is supplied by oxygen and glucose, and these energy sources are scarcely stored in the brain, so that they are constantly supplied from blood.

If the brain is damaged and the supply of oxygen and glucose is fierce, energy metabolism disorders generally progress in stages, and over time, cells lose their function and eventually organically collapse. , The function cannot be normally performed.

Therefore, in order to stably supply the energy source of the brain tissue and keep the external environment of the cerebral nerve cells constant, a mechanism for adjusting the cerebral blood flow of the cerebral blood vessels themselves is well developed.

When treating cerebrovascular disorders medically, various cerebral circulation improving agents, cerebral vasodilators, cerebral metabolism improving agents and the like have been used so far. However, although these drugs improve the subjective symptoms, they hardly improve the neurological symptoms at present.

(Problems to be Solved by the Invention) The inventors of the present invention have a prolyl endopeptidase inhibitory activity which is considered to be closely involved in the improvement / treatment of symptoms caused by various disorders in the brain. We have conducted intensive research to find compounds having antiamnestic activity. Further, in order to find a novel compound having sufficiently low toxicity, a compound similar to a natural product is synthesized by combining a highly safe fatty acid as a natural compound, an amino acid and a peptide compound, and is represented by the general formula (1). It was found to have anti-prolyl endopeptidase activity.

(Means for Solving Problems) As a result of earnest research by the present inventor, the novel dipeptide derivative represented by the general formula (1) has antiproline endopeptidase activity, and the novel dipeptide derivative is an experimental model animal. On the other hand, a completely new finding that it also has an antiamnestic effect was obtained, and the present invention was completed.

That is, the novel dipeptide derivative represented by the formula (1) of the present invention is effective for improving / treating mental functional symptoms caused by organic disorders in the brain, and is particularly effective for amnesia.

The compound of the formula (1) of the present invention is greatly different from the conventionally well-known anti-amnestic agent of the piracetam derivative type in that it contains a proline residue and a linear fatty acid, and further, an amino acid or peptide. Since it is a derivative, its toxicity to the living body is extremely low.

The compound of the present invention can be synthesized by a general peptide synthesis method, but is conveniently synthesized by the synthetic method of the present invention described below. The abbreviations have the following meanings.

Pro: Proline residue Phe: phenylalanine residue Val: valine residue Leu: leucine residue OMe: methyl ester residue WSCD: N-ethyl-N ', N'-dimethylaminopropylcarbodiimide The compound of the general formula (1) of the present invention is , Can be synthesized as follows.

General formula (2): [In the formula, m and X have the same meanings as described above, and A is a hydroxyl group, a halogen atom or a group: (Where m and X have the same meanings as described above). ] The carboxylic acid, acid halide or acid anhydride having the general formula (3): (Wherein, n, R ¹ and R ² have the above-mentioned meanings, and B represents a lower alkyl group.) In the presence of a base, and then the ester group is hydrolyzed. According to the general formula (4) (Wherein, m, n, R ¹ , R ² and X have the same meanings as described above). Further, by reacting this compound (4) with proline alkyl ester hydrochloride in the presence of a base, a compound of the general formula (1a): (Wherein, m, n, R ¹ , R ² and X have the same meanings as described above, and B represents a lower alkyl group), and the compound is reduced to give a compound of the general formula (1
b): (Wherein, m, n, R ¹ , R ² and X have the same meanings as described above), and then the hydroxymethyl group on the pyrrolidine ring of the compound is oxidized to an aldehyde group. By conversion, the target compound of the general formula (1) can be obtained.

The above synthetic method of the compound of the general formula (1) is a commonly used amino group acylation reaction and peptide synthesis reaction, and the starting component having the formula (3) is a carboxylic acid, an acid halide or an acid anhydride. The reagents used, such as bases, differ depending on which is used. For example, when an acid halide is used, a trialkylamine such as triethylamine is preferable as the base, but an aqueous solution of an alkali metal hydroxide, an alkali metal carbonate or the like may be used.

When using an acid anhydride, an aqueous solution of the above-mentioned alkali metal hydroxide, for example, sodium hydroxide, potassium hydroxide or the like, or an alkali metal carbonate, such as sodium carbonate,
Examples include potassium carbonate.

When using a carboxylic acid, a condensing agent such as WSCD or dicyclohexylcarbodiimide is used in an organic solvent that does not participate in the reaction.

Next, in the above reaction, the compound (1a) was replaced with the compound (1a).
To reduce to b), methanol is added dropwise to a suspension of tert-butyl alcohol or tetrahydrofuran containing the compound (1a) together with sodium borohydride to reduce the alcohol (1b) in good yield. Can be converted to.

As a general method including the method exemplified here, a reducing agent such as sodium borohydride, lithium borohydride, zinc borohydride, potassium borohydride and the like is used as a compound (1
The alcohol compound (1b) can also be obtained by adding it to the alcohol solution of a).

Further, the step of converting the alcohol compound (1b) into the target compound of the general formula (1) is performed by converting the compound (1b) into an organic solvent
It may be treated with an oxidant. The solvent used in this oxidation reaction may be one that does not participate in the reaction, but dimethyl sulfoxide is the best. Examples of the oxidizing agent include sulfur trioxide-pyridine complex, dimethyl sulfoxide, dimethyl sulfoxide-dicyclohexylcarbodiimide-phosphoric acid, silver oxide, manganese dioxide and the like.

The present invention is further described in detail by the following preparation examples and examples. Preparative examples illustrate the synthetic methods of the starting compounds used in Examples 1-9.

Preparation Example (a) N- [o- (E) -stilbenyloxybutanoyl] -Pro-OH Orthohydroxy- (E) -stilbene (10 mmol) and potassium hydroxide (15 mmol) were dried over dimethyl sulfone. Dissolve in oxide (10 ml). 4-Bromobutyric acid ethyl ester (11 mmol) is added to this solution, and the mixture is stirred at room temperature overnight. After completion of the reaction, the mixture is partitioned with water and benzene, the organic layer is dried over anhydrous magnesium sulfate, and the solvent is distilled off to obtain orthohydroxy- (E) -stilbenyloxybutyric acid ethyl ester. This is dissolved in a small amount of ethanol, about 100 ml of 1N sodium hydroxide aqueous solution is added, and the mixture is stirred at room temperature for about 3 hours. After the reaction is complete, acidify the reaction solution with 10N hydrochloric acid (pH ~
2) and extract with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and the solvent was distilled off to give o-
(E) -Stilbenyloxybutyric acid is obtained.

Next, this ortho- (E) -stilbenyloxybutyric acid (5m
Mol) and L-proline methyl ester hydrochloride (5 mmol)
Is dissolved in about 100 ml of dry methylene chloride and equimolar triethylamine is added. To this solution, 1.2 equivalents of WSCD is added and stirred overnight at room temperature. After completion of the reaction, add 1N
It is washed with hydrochloric acid saturated saline solution, saturated sodium hydrogen carbonate solution and saturated saline solution in this order, and the organic layer is dried over anhydrous sodium sulfate, and then the solvent is distilled off. The residue is purified by medium pressure column chromatography on silica gel to give ortho- (E) -stilbenyloxybutanoyl-L-proline methyl ester. This is dissolved in a small amount of methanol, 100 ml of a 1N aqueous sodium hydroxide solution is added, and the mixture is stirred at room temperature for about 3 hours. After completion of the reaction, the reaction solution is acidified with 10N hydrochloric acid (pH ~ 2) and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and the solvent was distilled off to give ortho- (E) -stilbenyloxybutanoyl-
L-proline is obtained.

In the above (a), instead of proline methyl ester hydrochloride, (a) phenylalanine ethyl ester hydrochloride,
By using (a) leucine ethyl ester hydrochloride and (u) valine ethyl ester hydrochloride,
(A ') N- [o- (E) -stilbenyloxybutanoyl] -Phe-OH, (a') N- [o- (E) -stilbenyloxybutanoyl] -Leu-OH, (U ') N-
[O- (E) -Stilbenyloxybutanoyl] -Val
-OH was obtained.

(B) N- (p-benzylphenoxybutanoyl)-
Pro-OH In (a) above, the target compound is obtained by using p-benzylphenol instead of orthohydroxy- (E) -stilbene.

Instead of proline methyl ester hydrochloride in (b) above, (a) phenylalanine ethyl ester hydrochloride,
By using (a) leucine ethyl ester hydrochloride and (u) valine ethyl ester hydrochloride,
(A ') N- (p-benzylphenoxybutanoyl)-
Phe-OH, (I ') N- (p-benzylphenoxybutanoyl) -Leu-OH, (U') N- (p-benzylphenoxybutanoyl) -Val-OH can be obtained It was

(C) N- (p-phenoxy-phenoxypentanoyl) -Pro-OH In the above (a), p-phenoxyphenol was used instead of orthohydroxy- (E) -stilbene. Obtain the compound.

Example 1 N- (o- (E) -stilbenyloxybutanoyl)-
Pro-Pro-CHO (SUAM-1420) N- (o- (E) -stilbenyloxybutanoyl)-
Pro-OH (1 eq), Pro-OMe hydrochloride (1 eq), triethylamine (1 eq) and WSCD hydrochloride (1 eq) were dissolved in dry methylene chloride and after stirring at room temperature overnight.
The reaction mixture was washed with 1N hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate, water and saturated brine in this order, and dried over anhydrous magnesium sulfate. The residue obtained by evaporating the solvent under reduced pressure was purified by column chromatography using silica gel, and N- (o- (E) -stilbenyloxybutanoyl) -Pro-Pro-OMe (reference example number) 1a) was obtained.

Next, the above compound (1.0 g) and sodium borohydride (330 mg) were dissolved in tert-butyl alcohol (10 ml),
Heat and stir (70-80 ℃). Next, dry methanol (2.4 ml) was added dropwise under reflux, and after completion of dropping, the mixture was heated under reflux with stirring for 1 hour. The reaction solution was returned to room temperature, and water (several ml) was added under ice cooling to decompose unreacted sodium borohydride. After distilling off methanol and tert-butyl alcohol under reduced pressure, the mixture was extracted with ethyl acetate, the organic layer was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure and the obtained residue was purified by column chromatography using silica gel to obtain N- (o- (E) -stilbenyloxybutanoyl) -Pro-Pro-CH ₂ OH (reference). Example number 1b) (920mg)
Got

Furthermore, the above compound (900 mg) and triethylamine (1.1
ml) in anhydrous dimethylsulfoxide (5 ml),
A solution of sulfur trioxide-pyridine complex (1.2 g) in dimethyl sulfoxide (5 ml) was added with stirring. After stirring at room temperature for about 20 minutes, the mixture was poured into ice water (100 ml), extracted with ethyl acetate, washed with 10% citric acid solution, saturated saline solution, saturated sodium bicarbonate solution and saturated saline solution, and dried with anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the obtained residue was purified by column chromatography using silica gel to obtain the target compound (640 mg).

Examples 2 to 9 Instead of the above N- [o- (E) -stilbenyloxybutanoyl] -Pro-OH, N- [o- (E) -stilbenyloxybutanoyl]-
Phe-OH, N- [o- (E) -stilbenyloxybutanoyl]-
Leu-OH, N- [o- (E) -stilbenyloxybutanoyl]-
Val-OH, N- (p-benzylphenoxybutanoyl) -Pro-O
H, N- (p-benzylphenoxybutanoyl) -Phe-O
H, N- (p-benzylphenoxybutanoyl) -Leu-O
H, N- (p-benzylphenoxybutanoyl) -Val-O
H, N- (p-phenoxy-phenoxypentanoyl) -Pr
N- [o- (E) -stilbenyloxybutanoyl]-by performing Examples 2 to 9 in the same manner as in Example 1 using o-OH.
Phe-Pro-CHO (SUAM 1421), N- [o- (E) -stilbenyloxybutanoyl]-
Leu-Pro-CHO (SUAM 1422), N- [o- (E) -stilbenyloxybutanoyl]-
Val-Pro-CHO (SUAM 1423), N- (p-benzylphenoxybutanoyl) -Pro-Pro
-CHO (SUAM 1424), N- (p-benzylphenoxybutanoyl) -Phe-Pro
-CHO (SUAM 1425), N- (p-benzylphenoxybutanoyl) -Leu-Pro
-CHO (SUAM 1426), N- (p-benzylphenoxybutanoyl) -Val-Pro
-CHO (SUAM 1427), N- (p-phenoxy-phenoxypentanoyl) -Pr
o-Pro-CHO (SUAM 14462) was obtained.

Physicochemical data of the obtained compound are shown in Table 1. In addition, data on the (a) ester form and (b) alcohol form obtained as intermediates are shown in Table 2 as a reference example.

Each of the compounds in Table 1 is oily and soluble in carbon tetrachloride, ether, chloroform, methylene chloride, ethyl acetate and methanol.

As a result of investigating the efficacy of the present compound for inhibiting the degradation of Z-glycyl-prolyl-β-naphthylamide by prolyl endopeptidase, it showed a very strong antiprolyl endopeptidase activity as shown in Example 10 described later, It showed no inhibitory activity against proteinases such as papain, bromelain, trypsin, chymotrypsin, thermolysin and pepsin.

Further, the present compound thus obtained is novel and has an antiamnestic effect as shown in Example 11.

Example 10 Measurement of anti-prolyl endopeptidase activity Anti-prolyl endopeptidase activity was measured by Yoshimoto (T.
Yoshimoto and D. Tsuru. Agr. Biol. Chem. 42, 2417, 197
8) and so on. That is, 0.0025M Z-glycyl-
A mixed solution containing 0.25 ml of proline-β-naphthylamide, 0.99 ml of 0.1 M phosphate buffer (pH 7.0) and 0.01 ml of the solution of the anti-prolyl endopeptidase compound of the present invention was placed in a test tube at 37 ° C for 3 minutes. After heating, 0.1 ml of prolyl endopeptidase solution (0.2 unit / ml) was added and reacted at 35 ° C for 10 minutes. Then, 2.0 ml of Triton X-100 (Triton X-100) solution in 1 M acetate buffer (pH 4.0) was added so that the final concentration of the surfactant would be 10%, and the mixture was allowed to stand at room temperature for 15 minutes and then 410 nm. Absorbance (a) was measured.

At the same time, the blind absorbance (b) was measured using only the buffer solution instead of the solution of the anti-prolyl peptidase compound, and the prolyl endopeptidase inhibition rate was calculated by the following formula: [(ba) / b] × 100. The amount required for 50% inhibition [IC ₅₀ ] was calculated.
The test results are shown in Table 3.

Example 11 Measurement of experimental amnesia preventive effect of scopolamine using rat (intraperitoneal administration) With respect to the anti-prolyl endopeptidase compound of the present invention, the effect of preventing long-term memory fixation inhibition by scopolamine was examined. That is, the compound of the present invention 0.2 mg / kg or
Wister containing physiological saline containing 1 mg / kg each
A male rat (100 to 120 g) was intraperitoneally administered once, and 1 hour after the administration, passive avoidance learning by electric shock was performed, and immediately after that, scopolamine 3 mg / kg was intraperitoneally administered.

The effect was determined by a passive avoidance test after 24 hours, in which both the test compound and scopolamine were administered to a group of control animals that received intraperitoneal administration of scopolamine and physiological saline without administration of the test compound. For each animal group, the number of amnestic rats and non-amnestic rats was compared. The test results are shown in Table 4.

Example 12 Acute toxicity test using mouse CDF-1 male mice (body weight: 27.2 to 30.1 g) (purchased from Awaz experimental animal) were used to examine the acute toxicity of the compound of the present invention.

The drug solution was prepared by dissolving each compound in DMSO and was adjusted to 0 per mouse.
1 ml was administered intraperitoneally. Five mice were used for each administration group and observed at 24 hours and 48 hours after administration. The average dose of each compound is shown in Table 4.

Table 4 Average dose of compound mg / kg No.1 SUAM 1423 ＞ 400 No.2 SUAM 1424 ＞ 400 No.3 SUAM 1427 ＞ 400 As a result, at the above dose, each group was healthy after 24 and 48 hours. No toxicity was observed.

The present invention also includes a pharmaceutical composition containing the compound of the present invention which is effective for ameliorating / treating the symptoms of organic disorders in the brain and a pharmaceutically acceptable adjuvant.

These active ingredients and pharmaceutical compositions are orally administered in solid dosage forms such as capsules, tablets and powders, or in liquid dosage forms such as elixirs, syrups and suspensions. It is also used parenterally, for example as an injection and a suppository.

Auxiliary agents for solid dosage contained in the pharmaceutical composition include, for example, solid powder carriers, lactose, sucrose, dextrose, mannitol, sorbit, cellulose,
Examples include glycine.

Examples of lubricants include silicon dioxide, talc, magnesium stearate, polyethylene glycol, and binders such as starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone. Disintegrators include starch and agar.

The dose of the compound of the present invention is usually
Orally administered at a dose of 10 to 4000 mg, preferably 100 to 1000 mg, or 1 to 2000 mg, preferably 50 to 500 m
Administer parenterally at a dose of g. It is clear that the dose varies depending on the type of disease to be administered, the age of the patient, the body weight, the degree of symptoms, and the administration form.

Formulation Example 1. Active substance 10 parts Lactose 75 parts Heavy magnesium oxide 15 parts were uniformly mixed to give tablets and capsules.

Formulation Example 2. 45 parts of active substance, 15 parts of starch and 40 parts of lactose were uniformly mixed to obtain a powder or granules.

Formulation Example 3. Active substance 1 part Surfactant 5 parts Physiological saline 94 parts was mixed by heating and sterilized to prepare an injection.

(Effects of the Invention) As described above, the compound according to the present invention exhibits remarkable anti-endoprolyl peptidase activity and anti-amnestic effect.
Further, the results of the acute toxicity test show that even 400 mg / kg / mouse has no toxicity, and the safety margin is sufficiently wide, and it is useful as a drug for the prevention and treatment of amnesia.

Claims (8)

[Claims] 1. General formula (1): (In the formula, m represents an integer of 1 to 8, and n is 0 to 6
R ¹ represents a hydrogen atom, R ² represents a hydrogen atom, a branched alkyl group having 3 to 5 carbon atoms, a phenyl group, a hydroxyphenyl group, or a methylthio group, or
R ¹ and R ² together represent a bond between carbon and nitrogen, X represents an aralkyl group, an aryl-alkenyl group, or an aryloxy group, provided that when n is 0, R ² is other than a hydrogen atom. Express meaning. ). 2. A general formula: (In the formula, m represents an integer of 1 to 8, and n is 0 to 6
R ⁴ represents a hydrogen atom, a branched alkyl group having 3 to 5 carbon atoms, a phenyl group, a hydroxyphenyl group, or a methylthio group, and X represents an aralkyl group or an aryl- group.
It represents an alkenyl group or an aryloxy group, provided that when n is 0, R ⁴ has a meaning other than a hydrogen atom. ) The compound according to claim 1, wherein 3. A general formula: (In the formula, m represents an integer of 1 to 8, and X represents an aralkyl group, an aryl-alkenyl group, or an aryloxy group.) The compound according to claim 1. 4. The compound according to claim 1, wherein X is a styryl group, a benzyl group or a phenoxy group. 5. A general formula (2) [In the formula, m represents an integer of 1 to 8, X represents an aralkyl group, an aryl-alkenyl group, or an aryloxy group, and A is a hydroxyl group, a halogen atom or a group: (Where m and X are as defined above). ] The carboxylic acid, acid halide or acid anhydride having the general formula (3): (In the formula, n represents an integer of 0 to 6, R ¹ represents a hydrogen atom, R ² represents a hydrogen atom, a branched alkyl group having 3 to 5 carbon atoms, a phenyl group, a hydroxy group or a methylthio group, Or R ¹ and R ² together represent a bond between carbon and nitrogen, B represents a lower alkyl group, provided that when n is 0, R ² represents a meaning other than a hydrogen atom. The compound is reacted with an ester derivative in the presence of a base, and then the ester group is hydrolyzed to give a compound represented by the general formula (4): (Wherein m, n, R ¹ , R ² and X are the same as defined above), and the compound is reacted with proline alkyl ester hydrochloride in the presence of a base to give a compound. Of the general formula (1), characterized in that the alkyl ester substituent on the pyrrolidine ring of is reduced and then oxidized. (Wherein m, n, R ¹ , R ² and X are the same as defined above). 6. The method according to claim 5, wherein the base is a trialkylamine, an alkali metal hydroxide or an alkali metal carbonate. 7. General formula (1): (In the formula, m represents an integer of 1 to 8, and n is 0 to 6
R ¹ represents a hydrogen atom, R ² represents a hydrogen atom, a branched alkyl group having 3 to 5 carbon atoms, a phenyl group, a hydroxyphenyl group, or a methylthio group, or
R ¹ and R ² together represent a bond between carbon and nitrogen, X represents an aralkyl group, an aryl-alkenyl group, or an aryloxy group, provided that when n is 0, R ² is other than a hydrogen atom. Express meaning. The prolyl endopeptidase activity inhibitor containing the dipeptide derivative which has these. 8. General formula (1): (In the formula, m represents an integer of 1 to 8, and n is 0 to 6
R ¹ represents a hydrogen atom, R ² represents a hydrogen atom, a branched alkyl group having 3 to 5 carbon atoms, a phenyl group, a hydroxyphenyl group, or a methylthio group, or
R ¹ and R ² together represent a bond between carbon and nitrogen, X represents an aralkyl group, an aryl-alkenyl group, or an aryloxy group, provided that when n is 0, R ² is other than a hydrogen atom. Express meaning. An anti-amnestic agent containing a dipeptide derivative having