JPH0696563B2 - Acyl amino acid derivative, production method and use thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) (In the formula, n represents 0 or an integer of 1 to 6, R ¹ represents a saturated or unsaturated fatty acid residue having 5 to 25 carbon atoms,
R ² represents a hydrogen atom, R ³ represents a hydrogen atom and has 3 to 10 carbon atoms.
5 represents a branched alkyl group, a phenyl group, a hydroxyphenyl group, an aralkyloxyphenyl group, an indolyl group, an imidazolyl group, a methylthio group, or R ² and R ³
Relates to a novel acylamino acid derivative having a carbon-nitrogen bond), a process for producing the same, and a use thereof.

More specifically, the novel 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 ameliorating 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 vasopressin, which is considered to be related to substance P, TRH (thyroid stimulating hormone) and neurotensin, which are considered to be neurotransmitters, and vasopressin. It is known to activate. 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 acylamino acid 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 cut off, the energy metabolism disorder generally progresses in stages, and the cells lose their function over time and eventually collapse organically. However, 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 acylamino acid derivative represented by the general formula (1) has antiprolyl endopeptidase activity, and the novel acylamino acid derivative is The present invention was completed by obtaining completely new knowledge that it also has an antiamnestic effect on experimental model animals.

That is, the novel acylamino acid 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 significantly different from the conventionally well-known piracetam derivative-based anti-amnestic agent in that it contains an amino acid residue and a fatty acid residue. Therefore, the toxicity to the living body is extremely low.

Among the compounds of formula (1), the following compounds are preferable because of their high antiprolyl endopeptidase activity.
In the following, these compounds may be referred to by the numbers in the cutlet.

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

L-Pro: L-proline residue L-Val: L-valine residue L-Phe: L-phenylalanine residue L-Tyr: L-tyrosine residue L-Leu: L-leucine residue L-Tyr (OBzl): L-tyrosine-O-benzyl ether residue L-nLeu: L-norleucine residue WSCD: N-ethyl-N, N'-dimethylaminopropylcarbodiimide Z: benzyloxycarbonyl group Boc: tertiary butyloxy Carbonyl group TEA: triethylamine The compound of the general formula (1) of the present invention can be synthesized as follows.

That is, the general formula (2a) or (2b) (Wherein R ¹ represents a saturated or unsaturated fatty acid residue having 5 to 25 carbon atoms, and A represents a hydroxyl group or a halogen atom), and a general formula (3) (In the formula, n represents 0 or an integer of 1 to 6, R ² represents a hydrogen atom, R ³ represents a hydrogen atom, a branched alkyl group having 3 to 5 carbon atoms, a phenyl group, a hydroxyphenyl group, an alkyl group. Reaction with an amino acid amide derivative having an alkyloxyphenyl group, an indolyl group, an imidazolyl group, a methylthio group, or R ² and R ³ always represents a carbon-nitrogen bond) in the presence of a base. Can be obtained.

This synthetic method is a commonly used acylation reaction of an amino group, and the base used depends on whether the starting component having the formula (2a) or (2b) is a carboxylic acid, an acid halide or an acid anhydride. Etc. are different reagents. For example, when an acid halide is used, a trialkylamine such as triethylamine is preferable as a base, but an aqueous solution of an alkali metal hydroxide, an alkali metal carbonate, or pyridine 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.

In this reaction, the reaction temperature is preferably room temperature or lower, and the solvent may be one that dissolves the above base.

In the present synthetic method, when R ³ of the starting material of the formula (3) is a hydroxyphenyl group, the steps of protecting and deprotecting the hydroxyl group are carried out by a conventional method.

There are many other methods for producing the compound of the present invention, but in general, for example, the compound represented by the general formula (4) An N-acyl carboxylic acid having the formula (wherein R ¹ , R ² and R ³ are as defined above) and the formula (5) The compound of the present invention can also be obtained by condensing the pyrrolidine of the above with a condensing agent.

Examples of the condensing agent include reagents commonly used in peptide synthesis, such as N, N′-dicyclohexylcarbodiimide, WSCD, etc., but other methods such as the acid chloride method, mixed acid anhydride method, active ester method, etc. A commonly used method may be used.

Next, the present invention will be described in more detail based on Examples and Reference Examples.

Reference Example: Synthesis of Starting Material Represented by Formula (3) (a) Synthesis of HL-Phe-Pyrrolidine Z-Phe-OH (1 equivalent) and pyrrolidine (1 equivalent) were dissolved in dry methylene chloride and iced. WSCD (1 eq) was added under cold conditions. After stirring at room temperature for 20 hours, the reaction solution was 1N hydrochloric acid,
The extract was washed with water, saturated aqueous sodium hydrogen carbonate, water, and saturated brine, and dried over anhydrous magnesium sulfate. The residue obtained by distilling off the solvent under reduced pressure was purified by column chromatography using silica gel. The obtained Z-L-Phe-pyrrolidine was dissolved in ethanol (1 equivalent), boron trifluoride-ether complex (1 equivalent) and palladium carbon (small amount) were added to remove the Z group by catalytic reduction, and the solvent was added. Was distilled off under reduced pressure to obtain the desired product.

(B) H-L-Val-pyrrolidine was obtained by using Z-L-Val-OH instead of Z-L-Phe-OH in (a).

Similarly, (c) HL-nLeu-pyrrolidine and (d) HL-Leu-pyrrolidine were obtained.

(E) Synthesis of HL-Pro-pyrrolidine Z-L-Pro-OH (1 equivalent) and pyrrolidine (1 equivalent) were dissolved in dry methylene chloride, and WSCD (1 equivalent) was added under ice cooling, and the mixture was cooled to room temperature. After stirring for 20 hours at room temperature, the reaction solution was mixed with 1N hydrochloric acid,
The extract was washed with water, saturated aqueous sodium hydrogen carbonate, water, and saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure and the resulting residue was purified by medium pressure column chromatography using silica gel. The obtained Z-L-Pro-pyrrolidine was dissolved in ethanol (1 equivalent), boron trifluoride-ether complex (1 equivalent) and palladium carbon (small amount) were added, and the Z group was removed by catalytic reduction in a hydrogen atmosphere. Then, the solvent was distilled off under reduced pressure to obtain the desired product as an oily compound. All the desired compounds can be obtained in the form of oil.

(F) Synthesis of HL-Tyr (OBzL) -pyrrolidine trifluoroacetate Boc-L-Tyr (OBzL) -OH (1 equivalent) and pyrrolidine (1 equivalent) were dissolved in dry methylene chloride and cooled with ice. WSCD (1 eq) was added below. After stirring at room temperature for 20 hours, add 1N reaction solution.
It was washed with hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate, water, and saturated brine, and dried over anhydrous magnesium sulfate. The residue obtained by distilling off the solvent under reduced pressure was purified by column chromatography using silica gel. The obtained Boc-L-Tyr (OBzl) -pyrrolidine (1 equivalent) was dissolved in dry methylene chloride, excess trifluoroacetic acid was added and stirred (about 6 hours), and the solvent was distilled off under reduced pressure to obtain the desired product. Got (Oil compound) As a result of investigating the efficacy of the compound of the present invention to prevent the degradation by prolyl endopeptidase, it showed a very strong anti-prolyl endopeptidase activity as shown in the test examples described below, and showed papain, bromelain, trypsin, chymotrypsin. , It showed no inhibitory activity against proteinases such as thermolysin and pepsin.

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

Example 1 (a) N-oleoyl-L-phenylalanylpyrrolidine (SUAM1283) HL-Phe-pyrrolidine (1 equivalent) and TEA (1 equivalent) were dissolved in dry tetrahydrofuran, and oleic acid chloride (1 equivalent) was added dropwise under ice cooling. The mixture was stirred at room temperature for 6 hours, and the precipitated TEA hydrochloride was excessively removed. The solvent was distilled off under reduced pressure,
It was dissolved in a small amount of ether, washed with 1N hydrochloric acid, saturated saline, saturated aqueous sodium hydrogen carbonate and saturated brine, and dried over anhydrous magnesium sulfate. After concentration under reduced pressure, an ether solution of diazomethane was added to (excess) unreacted oleic acid to form a methyl ester. The solvent was distilled off under reduced pressure and the obtained residue was purified by medium pressure column chromatography using silica gel to obtain the target compound. (Oil Compound) By using HL-Val-pyrrolidine instead of HL-Phe-pyrrolidine in the above (a), (b) N-oleoyl-L-valylpyrrolidine (SUAM12
82) by using HL-nLeu-pyrrolidine, (c) N-oleoyl-L-norleucylpyrrolidine (SUAM1284), by using HL-Leu-pyrrolidine, (d) N-oleoyl-L-leucylpyrrolidine (SUAM
1276) was obtained.

Example 2 (a) N-oleoyl-L-prolylpyrrolidine (SUAM
1280) HL-Pro-Pyrrolidine (1 equivalent) and TEA (1 equivalent) were dissolved in dry tetrahydrofuran, and oleic acid chloride (1 equivalent) was added dropwise under ice cooling. The mixture was stirred at room temperature for 6 hours, and the precipitated TEA hydrochloride was excessively removed. The solvent was evaporated under reduced pressure, the residue was dissolved in a small amount of ether, washed with 1N hydrochloric acid, saturated saline, saturated aqueous sodium hydrogen carbonate and saturated brine, and dried over anhydrous magnesium sulfate. After concentration under reduced pressure, an ether solution of diazomethane was added to methylate the (excess) unreacted oleic acid. The solvent was distilled off under reduced pressure and the obtained residue was purified by medium pressure column chromatography using silica gel to obtain the target compound. (Oil compound) By using palmitic acid chloride in place of oleic acid chloride in (a) above, (b) N-palmitoyl-L-prolylpyrrolidine (SU
AM1391) uses lauric acid chloride to give (c) N-lauroyl-L-prolylpyrrolidine (SUAM
1392) uses (d) N-linoleoyl-L-prolylpyrrolidine (SU) by using linoleic acid chloride.
AM1393) was obtained.

Example 3 N-oleoyl-L- (O-benzyl) tyrosylpyrrolidine (SUAM1275) HL-Tyr (OBzL) -pyrrolidine trifluoroacetate (1 eq) and TEA (2 eq) were dissolved in dry tetrahydrofuran and oleic chloride (1 eq) was added dropwise under ice cooling. The mixture was stirred at room temperature for 6 hours, and the precipitated TEA hydrochloride was excessively removed. The solvent was evaporated under reduced pressure, the residue was dissolved in a small amount of ether, washed with 1N hydrochloric acid, saturated saline, saturated sodium hydrogen carbonate solution and saturated saline, and dried over anhydrous magnesium sulfate. After concentration under reduced pressure, an ether solution of diazomethane was added to (excess) unreacted oleic acid to form a methyl ester. The solvent was distilled off under reduced pressure and the obtained residue was purified by medium pressure column chromatography using silica gel to obtain the target compound. (Oil compound) Example 4 N-stearoyl-L-tyrosylpyrrolidine (SUAM128
6) 1 equivalent of the compound of Example 3 (SUAM1275) was dissolved in ethanol, palladium / carbon (catalytic amount) was added, and catalytic reduction was carried out under a hydrogen stream under normal pressure. The solvent was evaporated under reduced pressure, and the residue was subjected to column chromatography using silica gel to give the title compound.

Table 1 shows the physical properties of the compounds obtained in Examples 1 to 4.

Example: 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 2.

Example 1 Measurement of Experimental Preventive Effect of Scopolamine on Rats Using Rats (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 20 mg / kg, 1 mg / kg
Each of the above-mentioned physiological saline solutions containing 1 is administered to the abdominal cavity of a Wistar male rat (100 to 120 g) once
After a while, perform passive avoidance learning by electric shock,
Immediately after that, 3 mg / kg of scopolamine 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 3.

Example Acute Toxicity Test Using Mice CDF-1 male mice (body weight 27.2 to 30.1 g) (purchased from Awaz experimental animals) were used to examine the acute toxicity of the compounds 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 SUAM1282 615.4 No.2 SUAM1284 557.7 No.3 SUAM1280 334.4 As a result, at the above dose, each group was healthy and showed no toxicity after 24 and 48 hours. It was not recognized.

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
Oral administration at a dose of 10-4000 mg, preferably 100-1000 mg, or 1-2000 mg, preferably 50-50
It is given parenterally at a dose of 0 mg. 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.

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

Preparation 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 were mixed by heating and sterilized to obtain 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 500 mg / kg / mouse is not toxic, and the safety margin is sufficiently wide, and it is useful as a drug for the prevention and treatment of amnesia.

 ──────────────────────────────────────────────―― ─── Continued Front Page (1) 1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka Prefecture 1-1-1 Wakayamadai, Suntory Limited (72) Inventor Ryuji Tanaka Wakayama, Shimamoto-cho, Mishima-gun, Osaka Prefecture No. 1-11-1, Suntory Ltd.

Claims (7)

[Claims] 1. A general formula (In the formula, n represents 0 or an integer of 1 to 6, R ¹ represents a saturated or unsaturated fatty acid residue having 5 to 25 carbon atoms,
R ² represents a hydrogen atom, R ³ represents a hydrogen atom and has 3 to 10 carbon atoms.
5 represents a branched alkyl group, a phenyl group, a hydroxyphenyl group, an aralkyloxyphenyl group, an indolyl group, an imidazolyl group, a methylthio group, or R ² and R ³
Represents a bond between a carbon and a nitrogen). 2. General formula (In the formula, n represents 0 or an integer of 1 to 6, R ¹ represents a saturated or unsaturated fatty acid residue having 5 to 25 carbon atoms,
R ³ represents a hydrogen atom, a branched alkyl group having 3 to 5 carbon atoms, a phenyl group, a hydroxyphenyl group, a benzyloxyphenyl group, an indolyl group, an imidazolyl group or a methylthio group). The compound according to the item. 3. General formula The compound according to claim ¹ , wherein R ¹ represents a saturated or unsaturated fatty acid residue having 5 to 25 carbon atoms. During wherein general formula R ¹ -A or R ¹ -O-R ¹ (wherein, R ¹ represents a saturated or unsaturated fatty acid residue of 5 to 25 carbon atoms, A a represents a hydroxyl group or a halogen atom Represents a carboxylic acid, an acid halide or an acid anhydride having the general formula (In the formula, n represents 0 or an integer of 1 to 6, R ² represents a hydrogen atom, R ³ represents a hydrogen atom, a branched alkyl group having 3 to 5 carbon atoms, a phenyl group, a hydroxyphenyl group, an alkyl group. Reaction with an amino acid amide derivative having an alkyloxyphenyl group, an indolyl group, an imidazolyl group, a methylthio group, or R ² and R ³ always represents a carbon-nitrogen bond) in the presence of a base. A general formula characterized by (Wherein n, R ¹ , R ² and R ³ are as defined above). 5. The production method according to claim 4, wherein the base is a trialkylamine or a pyridine base. 6. A general formula (In the formula, n represents 0 or an integer of 1 to 6, R ¹ represents a saturated or unsaturated fatty acid residue having 5 to 25 carbon atoms,
R ² represents a hydrogen atom, R ³ represents a hydrogen atom and has 3 to 10 carbon atoms.
5 represents a branched alkyl group, a phenyl group, a hydroxyphenyl group, an aralkyloxyphenyl group, an indolyl group, an imidazolyl group, a methylthio group, or R ² and R ³
A prolyl endopeptidase activity inhibitor comprising an acylamino acid derivative having a carbon-nitrogen bond) as an active ingredient. 7. General formula (In the formula, n represents 0 or an integer of 1 to 6, R ¹ represents a saturated or unsaturated fatty acid residue having 5 to 25 carbon atoms,
R ² represents a hydrogen atom, R ³ represents a hydrogen atom and has 3 to 10 carbon atoms.
5 represents a branched alkyl group, a phenyl group, a hydroxyphenyl group, an aralkyloxyphenyl group, an indolyl group, an imidazolyl group, a methylthio group, or R ² and R ³
A cerebral function-improving agent containing an acylamino acid derivative having a carbon-nitrogen bond) as an active ingredient.