JPH0680077B2 - Novel peptide compound having proline endopeptidase inhibitor activity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to proline endopeptidase (EC, 3.4.21.26).
Prolyl endopeptidase), a novel compound showing enzyme inhibitory activity, and a chemical synthesis method thereof, and a proline endopeptidase activity inhibitor containing the compound as an active ingredient, and a drug, particularly the use as an antiamnestic agent. is there.

(Prior Art) Proline endopeptidase acts on and inactivates substance P, TRH (thyroid stimulating hormone) and neurotensin, which are neurotransmitters, and vasopressin, which is considered to be related to memory. Is known to do. On the other hand, Mr. Tsuru and Mr. Yoshimoto of Nagasaki University Faculty of Pharmacy
Compounds that inhibit proline endopeptidase activity
We found that rat scopolamine prevents experimental amnesia and suggested the possibility of applying proline endopeptidase activity inhibitor to anti-amnestic agent.

(Technical problem to be solved by the invention) The present inventors have conducted research to find out a novel compound having strong antiamnestic activity and sufficiently low toxicity, based on the above findings, and the following general formula (I) It was found that the peptide derivative having an anti-proline endopeptidase activity represented by the formula (1) has a remarkably excellent effect on experimental retrograde amnesia caused by scopolamine, and completed the present invention.

(Structure of the Invention) The peptide derivative of the present invention has the general formula (I): (In the formula, A represents a methyl group or a benzyloxy group, R represents an isopropyl group or an isobutyl group provided that they have the same meaning in one formula I, and n represents a number of 2 or 3.) It is represented by.

The compound of formula (I) of the present invention is a peptidomimetic compound, which is significantly different from the conventionally well-known piracetam derivative anti-amnestic agent, and is also a peptidic compound.
The toxicity to the living body is extremely low.

Among the compounds of formula (I), the following compounds are preferable because of their high antiproline endopeptidase activity. In addition, hereinafter, these may be referred to by the numbers in Katsuko.

The compound of the general formula (I) of the present invention can be synthesized as follows. In the following description, each abbreviation has the following meaning.

Z: benzyloxycarbonyl group Ac: acetyl group Boc: t-butoxycarbonyl group Val: valine residue Leu: leucine residue Pro: proline residue OMe: methyl ester group WSCI: N-ethyl-N ', N'-dimethyl Aminopropylcarbodiimide TEA: triethylamine TFA: trifluoroacetic acid To prepare the compound of formula (I) by the synthetic method of the present invention, the following general formula (II): (In the formula, A, R and n have the meanings shown in the above formula I, and R'represents a methyl group or an ester group.) The ester represented by the formula (3) is suspended in tert-butyl alcohol and hydrogenated. Sodium boron was added, and methanol was added dropwise under reflux in an inert gas to give the following general formula (III): The compound represented by the above general formula (I) can be obtained by converting it into an alcohol represented by the formula (1) and then oxidizing the alcohol with a sulfur trioxide-pyridine chain in dimethyl sulfoxide.

The starting material represented by the general formula (II) is a corresponding amino acid or peptide whose amino terminal is protected by a protecting group such as Boc group by a conventional method, and a corresponding amino acid whose carboxy terminal is protected by an ester group or the like, or It can be obtained by appropriately reacting with a peptide.

Hereinafter, the present invention will be described in more detail with reference to Examples and Reference Examples.

Reference example Synthesis of starting material represented by formula (II) (1) Synthesis of Ac-Val-Val-Pro-OMe a) Boc-Val-Val-OMe Boc-Val-OH (1 equivalent), Val- Dissolve OMe.Hcl (1 eq) and TEA (1 eq) in dry methylene chloride and add WSCI (1 eq) under ice cooling. After stirring at room temperature for 16 hours, the reaction mixture is washed with 1N hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate and saturated brine, and dried over anhydrous magnesium sulfate. The solvent is distilled off under reduced pressure to obtain crystals of the target compound.

b) Ac-Val-Val-OMe Boc-Val-Val-OMe (1 equivalent) is dissolved in methylene chloride, TFA (10 equivalents) is added under ice cooling, and the mixture is stirred at room temperature for 5 hours. The reaction solution is concentrated under reduced pressure, anhydrous ether is added, the precipitated crystals are collected, suspended in dry methylene chloride, and acetic anhydride is added. After stirring for 16 hours at room temperature,
The residue obtained by concentrating the reaction solution under reduced pressure is dissolved in ethyl acetate and washed with water, saturated aqueous sodium hydrogen carbonate and saturated brine. It is dried over anhydrous magnesium sulfate and the solvent is distilled off under reduced pressure to obtain the target compound.

c) Ac-Val-Val-OH Ac-Val-Val-OMe (1 equivalent) was dissolved in methanol to give 1
Add N sodium hydroxide (2 eq) and stir at room temperature for 2 hours. The reaction mixture is concentrated under reduced pressure, acidified with 1N hydrochloric acid, and extracted with ethyl acetate. The extract is washed with saturated brine and dried over anhydrous magnesium sulfate, and the solvent is evaporated under reduced pressure to give the target compound crystals.

d) Ac-Val-Val-Pro-OMe Ac-Val-Val-OH (1 eq), Pro-OMe.HCl (1 eq), TEA (1 eq) were dissolved in dry methylene chloride and cooled on ice. Add WSCI (1 equivalent). After stirring at room temperature for 16 hours, the reaction mixture is washed with 1N hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate and saturated brine, and dried over anhydrous magnesium sulfate. The solvent is distilled off under reduced pressure to obtain the target compound.

Example 1 (Synthesis of SUAM 1004) a) Ac-Val-Val-Pro-Ol Ac-Val-Val-Pro-OMe (570 mg, 1.54 mmol) obtained in Reference Example and sodium borohydride (150 mg, 3.86 mmol) was suspended in tert-butyl alcohol (12 ml), and the mixture was heated with stirring under a nitrogen stream and then refluxed under anhydrous methanol (2 m
l) was added dropwise. After completion of the dropwise addition, the mixture was refluxed and stirred for 1 hour, then returned to room temperature, and water (10 ml) was added under ice cooling.

After distilling off methanol and tertiary butyl alcohol under reduced pressure,
It was extracted 3 times with ethyl acetate, washed with saturated brine, and dried over anhydrous magnesium sulfate. The crude crystals obtained by distilling off ethyl acetate under reduced pressure were purified by silica gel medium pressure column chromatography (solvent system chloroform) to obtain the target compound (480 mg, 91%).

b) SUAM 1004 Ac-Val-Val-Pro-ol (440 mg, 1.3 mmol) and triethyleneamine (0.72 ml, 5.2 mmol) were dissolved in anhydrous dimethylsulfoxide (5.5 ml), and sulfur trioxide-pyridine complex was stirred. A solution of (820 mg, 5.2 mmol) in dimethyl sulfoxide (5 ml) was added. After stirring at room temperature for 10 minutes, it was poured into ice water (20 ml), extracted 3 times with ethyl acetate, washed with 10% aqueous citric acid solution, water, saturated aqueous sodium hydrogen carbonate and saturated brine,
It was dried over anhydrous magnesium sulfate. The oily crude product obtained by distilling off ethyl acetate under reduced pressure was purified by medium pressure column chromatography (solvent system chloroform) on silica gel,
SUAM 1004 (330 mg, 75%) was obtained.

Similarly, SUAM 1000 (Ac-Leu-Leu-Pro-CHO) can be obtained.

The physical properties of the obtained compound are shown in Table 1 below.

Example 2 (Synthesis of SUAM 1001) a) Z-Val-Val-Pro-ol Z-Val-Val-Pro-OMe (1.3 g, 2.8 mmol) and sodium borohydride (267 mg, 7.1 mmol) were added. It was suspended in tert-butyl alcohol (12 ml), heated and stirred under a nitrogen stream, and anhydrous methanol (1.1 ml) was added dropwise under reflux. After the completion of the dropping, the mixture was refluxed and stirred for 1 hour and then returned to room temperature,
Water (10 ml) was added under ice cooling. After distilling off methanol and tert-butyl alcohol under reduced pressure, the mixture was extracted 3 times with ethyl acetate, washed with saturated brine and dried over anhydrous magnesium sulfate. The crude product obtained by distilling off ethyl acetate under reduced pressure was purified by silica gel medium pressure column chromatography (solvent system chloroform) to obtain the target compound (1.0 g, 82%).

b) SUAM 1001 Z-Val-Val-Pro-ol (1.0 g, 2.3 mmol) and triethylamine (1 ml, 7.0 mmol) were dissolved in anhydrous dimethyl sulfoxide (7 ml), and the mixture was mixed with sulfur trioxide-pyridine (1.1 ml). g, 7.0 mmol) in dimethylsulfoxide (7 ml) was added. After stirring for 10 minutes at room temperature, ice water (30m
l), extracted three times with ethyl acetate, washed with 10% aqueous citric acid solution, water, saturated aqueous sodium hydrogen carbonate and saturated brine, and dried over anhydrous magnesium sulfate. The crude product obtained by distilling off ethyl acetate under reduced pressure was purified by silica gel medium pressure column chromatography (solvent system chloroform).
(800 mg, 80%) was obtained.

Similarly, SUAM 1002 (Z-Leu-Leu-Pro-CHO) and SUAM 1003 (Z-Val-Val-Val-Pro-CHO) were obtained.

The physical properties of the obtained compound are shown in Table 1 below.

Example 2 Measurement of anti-proline endopeptidase activity The anti-proline 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 mixture 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-proline endopeptidase compound of the present invention was heated in a test tube at 37 ° C for 3 minutes. After that, 0.1 ml of proline 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 1M 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-proline endopeptidase compound, and the proline endopeptidase inhibition rate was calculated by the following formula: [(b-a) / b] × 100 The amount required for 50% inhibition [IC ₅₀ ] was calculated.
The test results are shown in Table 2.

Example 3 Measurement of Experimental Preventive Effect of Scopolamine on Rats Using Rats (Intraperitoneal Administration) The antiproline endopeptidase compound of the present invention was examined for its effect of preventing long-term memory fixation inhibition by scopolamine. That is, the compound of the present invention is 20 mg / kg, 2 mg / k
g, 0.2 mg / kg and 0.02 mg / kg of physiological saline were administered once to each abdominal cavity of Wistar male rat (100-120 g), and 1 hour after administration, passive avoidance learning by electric shock was performed. Immediately after that, 3 mg / kg of scopolamine was intraperitoneally administered.

The effect was determined by a passive avoidance test after 24 hours and 48 hours, in a group of control animals that received intraperitoneal administration of scopolamine and saline without administration of the test compound, administration of the test compound and administration of scopolamine. Was performed by comparing the numbers of amnestic rats and non-amnestic rats for each group of animals. The test results are shown in Table 3.

Claims (1)

[Claims] 1. General formula I: (In the formula, A represents a methyl group or a benzyloxy group, R represents an isopropyl group or an isobutyl group provided that they have the same meaning in one formula I, and n represents a number of 2 or 3.) A compound represented by.