JPH0699472B2 - Novel compound having peptidase inhibitory activity, its production method and use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to prolyl endopeptidase (EC, 3.4.21.2).
6, Prolyl-endopeptidase), a novel compound showing enzyme inhibitory activity, further its chemical synthesis method, and a prolyl endopeptidase activity inhibitor and drug containing it as an active ingredient, especially as an antiamnestic agent Is related to the use of.

(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 Dr. Yoshimoto, Faculty of Pharmaceutical Sciences, 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.

(Technical Problems to be Solved by the Invention) The inventors of the present invention have been based on the above findings.
Studies were conducted to find compounds having strong peptidase inhibitory activity and anti-amnestic activity. Further, in order to find a novel compound with 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 represented by the following general formula (I). The present invention has been completed by finding that a novel compound having an anti-prolyl endopeptidase activity has a remarkably excellent action on amnesia.

(Structure of the Invention) The N-acylpyrrolidine derivative of the present invention has the general formula (I): (In the formula, n represents a number of 0 to 2, R ₁ represents a saturated or unsaturated linear organic group having 11 to 25 carbon atoms, and the unsaturated carbon chain represents a plurality of double bonds. R ₂ may include Or a —CH ₃ group, and R ₃ represents a lower alkyl ester group, a —CH ₂ OH group, or an aldehyde group. ) Is represented.

The compound of the formula (I) 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 chain. Since it is a peptide derivative, its toxicity to the living body is extremely low.

Among the compounds of formula (I), the following compounds are preferable because of their high anti-prolyl 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 synthesis method, but is conveniently synthesized by the synthetic method of the present invention described below. The abbreviations have the following meanings.

Z: Benzyloxycarbonyl group Ala: Alanine residue Pro: Proline residue Val: valine residue Leu: leucine residue OMe: methyl ester group WSCD: N-ethyl-N ', N'-dimethylaminopropylcarbodiimide TEA: triethylamine The compound of the general formula (I) of the present invention is as follows. Can be synthesized.

(1) First, in the general formula (III), R ₃ is a lower alkyl ester group in the general formula (I): (In the formula, n represents a number of 0 to 2, R ₁ represents a saturated or unsaturated linear organic group having 11 to 25 carbon atoms, and the unsaturated carbon chain represents a plurality of double bonds. R ₂ may include Alternatively, it represents a —CH ₃ group, and R ₄ represents a lower alkyl group. ) Is represented by the general formula: R ₁ —COCl (wherein R ₁ represents a saturated or unsaturated linear organic group having 11 to 25 carbon atoms, where an unsaturated carbon chain is used). May contain a plurality of double bonds) and a carboxylic acid chloride represented by the general formula (II): (In the formula, n represents a number of 0 to 2, and R ₂ is Alternatively, it represents a —CH ₃ group, and R ₃ represents a lower alkyl ester group. The peptide having a lower alkylesterified proline residue represented by the formula (1) or a lower alkylesterified proline hydrochloride can be easily synthesized by a conventional method.

(2) Next, in the general formula (I), the formula (IV) in which R ₃ is a —CH ₂ OH group: (In the formula, n represents a number of 0 to 2, R ₁ represents a saturated or unsaturated linear organic group having 11 to 25 carbon atoms, and the unsaturated carbon chain represents a plurality of double bonds. R ₂ may include Alternatively, it represents a —CH ₃ group. In order to obtain the compound represented by the formula (1), the following reaction is performed. Methanol was added dropwise to a suspension of the compound represented by the general formula (III) and sodium borohydride in tert-butyl alcohol or tetrahydrofuran to give the general formula (IV): (In the formula, n represents a number of 0 to 2, R ₁ represents a saturated or unsaturated linear organic group having 11 to 25 carbon atoms, and the unsaturated carbon chain represents a plurality of double bonds. R ₂ may include Alternatively, it represents a —CH ₃ group. ) The alcoholic compound according to the present invention represented by

(3) Further, using a sulfur trioxide-pyridine complex from the compound represented by the general formula (IV), the general formula (V): (In the formula, n represents a number of 0 to 2, R ₁ represents a saturated or unsaturated linear organic group having 11 to 25 carbon atoms, and the unsaturated carbon chain represents a plurality of double bonds. R ₂ may include Alternatively, it represents a —CH ₃ group. ) The compound of the present invention represented by Dimethyl sulfoxide is used as the solvent, the reaction temperature is room temperature, and the reaction time is about 1 hour.

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 (II) When n = 0 in Formula (II), methyl ester hydrochloride of proline is used.

When n = 1 in the formula (II), (a) H-Ala-Pro-OMe Z-Ala-OH (1 equivalent), Pro-OMe hydrochloride (1 equivalent) and T
Dissolve EA (1 eq) in dry methylene chloride and WS under ice cooling.
Add CD (1 equivalent). After stirring at room temperature for 20 hours, the reaction mixture is washed with 1N 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 is purified by medium pressure column chromatography using silica gel. The obtained Z-Ala-Pro-
OMe was dissolved in (1 equivalent) ethanol, boron trifluoride-ether complex (1 equivalent) and palladium carbon (small amount) were added to remove Z group by catalytic reduction, and the solvent was distilled off under reduced pressure to obtain the desired product. obtain.

Instead of Z-Ala-OH in (a) above, (A) Z-Val-OH,
By using (a) Z-Leu-OH, (a ') Z-Val-Pro-OMe and (a') H-Leu-Pro-OMe can be obtained, respectively. (Oil compound) When n = 2 in the formula (II), (b) H-Leu-Leu-Pro-OMe Z-Leu-OH (1 equivalent), H-Leu-Pro synthesized in (a) -
OMe (1 eq) and TEA (1 eq) are dissolved in dry methylene chloride and WSCD (1 eq) is added under ice cooling. After stirring at room temperature for 20 hours, the reaction solution was 1N hydrochloric acid, water, saturated sodium hydrogen carbonate solution,
Wash with water and saturated saline, and dry over anhydrous magnesium sulfate. The residue obtained by distilling off the solvent under reduced pressure is purified by medium pressure column chromatography using silica gel. The obtained Z-Leu-Leu-Pro-OMe was dissolved in ethanol (1 equivalent), boron trifluoride-ether complex (1 equivalent) and palladium carbon (small amount) were added, and Z group was formed by catalytic reduction in a hydrogen atmosphere. And the solvent is 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.

As a result of investigating the efficacy of the present compound for inhibiting the decomposition of Z-glycyl-prolyl-β-naphthylamide by prolyl endopeptidase, it showed a very strong anti-prolyl endopeptidase activity as shown in the test examples described below, and papain , Proteinases such as bromelain, trypsin, chymotrypsin, thermolysin and pepsin showed no inhibitory activity.

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

Example 1 a) N-oleoyl-Pro-OMe (SUAM 1131) Pro-OMe hydrochloride (1 eq) and TEA (2 eq) were dissolved in dry tetrahydrofuran and oleic chloride (1) under ice cooling.
Equivalent) was added dropwise. TEA precipitated after stirring for 6 hours at room temperature
Was over-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) (a) Palmitoyl-Pro-OMe (SUAM 1135) was obtained by using (a) palmitic acid chloride in place of oleic acid chloride in the above (a).

(B) N-oleoyl-Leu-Pro-OMe (SUAM 1136) Leu-Pro-OMe (1 equivalent) and TEA (1 equivalent) were dissolved in dry tetrahydrofuran, and oleic acid chloride (1 equivalent) was added under ice cooling. Dropped. 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 (a) palmitic acid chloride, (a) stearic acid chloride, (u) linoleic acid chloride in place of oleic acid chloride in (b) above,
(A ') Palmitoyl-Leu-Pro-OMe (SUAM 11
41), (a ') stearoyl-Leu-Pro-OMe (SUAM 114
2), (U ') Linole oil-Leu-Pro-OMe (SUAM 114
3) got. Also, in (b) above, instead of Leu-Pro-OMe, (E) Val-Pro-OMe, (E) Ala-Pro-OMe, (F) Le
By using u-Leu-Pro-OMe,
Oleoyl-Val-Pro-OMe (SUAM 1147), (O ') Oleoyl-Ala-Pro-OMe (SUAM 1076), (C') Oleoyl-Leu-Leu-Pro-OMe (SUAM 1159) It was

Example 2 (a) oleoyl _{-Leu-Pro-CH 2 OH (} SUAM 1138) oleoyl obtained in Example 1 -Leu-Pro-OMe (SUAM 1136)
Dissolve (2g) and sodium borohydride (600mg) in tert-butyl alcohol (60ml) and stir with heating (80
C). Then, anhydrous methanol (50 ml) was added dropwise under reflux,
After completion of dropping, the mixture was heated and refluxed with stirring for 2 hours. The reaction solution was returned to room temperature and water (several ml) was added under ice cooling to inactivate 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 saline and dried over anhydrous magnesium sulfate. 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 (1 g). (Oil compound) In the above (a), the starting compound is oleoyl-Leu-
Instead of Pro-OMe (A) Oleoyl-Pro-OMe (SUAM 1
131), (a) Palmitoyl-Pro-OMe (SUAM 1135),
(U) Palmitoyl-Leu-Pro-OMe (SUAM 1141) (D)
Stearoyl-Leu-Pro-OMe (SUAM 1142), (e) Linoleoyl-Leu-Pro-OMe (SUAM 1143) (mosquito) Oleoyl-Ala-Pro-OMe (SUAM 1076), (ki) Oleoyl-Val-
Pro-OMe (SUAM 1147), (ku) oleoyl-Leu-Leu-Pr
o-OMe (SUAM 1159) was used as the target compound (a ′) oleoyl-Pro-CH ₂ OH (SUAM 1133),
(A ′) Palmitoyl-Pro-CH ₂ OH (SUAM 1137),
(U ') Palmitoyl-Leu-Pro-CH ₂ OH (SUAM 1144),
(D ') Stearoyl-Leu-Pro-CH ₂ OH (SUAM 1145),
(O ') Linole oil-Leu-Pro-CH ₂ OH (SUAM 1153),
(K ') oleoyl-Ala-Pro-CH ₂ OH (SUAM 1077),
(Ki ′) oleoyl-Val-Pro-CH ₂ OH (SUAM 1151),
(H ') oleoyl _{-Leu-Leu-Pro-CH 2} OH (SUAM 1152)
Got

Example 3 (a) oleoyl -Leu-Pro-CHO (SUAM 1140 ) oleoyl obtained in Example _{2 -Leu-Pro-CH 2 OH} (SUAM 113
8) (1 g) and TEA (800 mg) were dissolved in anhydrous dimethylsulfoxide (8 ml), and a solution of sulfur trioxide-pyridine complex (700 mg) in dimethylsulfoxide (8 ml) was added with stirring. After stirring at room temperature for about 1 hour, 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 over anhydrous magnesium sulfate. 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. (700 mg) (Oil compound) In the above (a), oleoyl-Le was used as a raw material compound.
instead of the _{u-Pro-CH 2 OH (} SUAM 1138), ( a) oleoyl _{-Pro-CH 2 OH (SUAM 1133} ), ( b) palmitoyl _{-Pro-CH 2 OH (SUAM 1137} ), ( c) palmitoyl −Leu
-Pro-CH ₂ OH (SUAM 1144) (E) Stearoyl-Leu-Pr
o-CH ₂ OH (SUAM 1145), (o) Linole oil-Leu-Pro
-CH ₂ OH (SUAM 1153) (f) oleoyl -Ala-Pro-CH ₂
OH (SUAM 1077), (g) oleoyl -Val-Pro-CH ₂ OH
(SUAM 1151), (h) oleoyl _{-Leu-Leu-Pro-CH 2} O
H (SUAM 1152) was used as the target compound (a ') oleoyl-Pro-CHO (SUAM 1134), (a'), respectively.
Palmitoyl-Pro-CHO (SUAM 1139), (U ') Palmitoyl-Leu-Pro-CHO (SUAM 1155), (E') Stearoyl-Leu-Pro-CHO (SUAM 1154), (O ') Linoleoyl-Leu- Pro-CHO (SUAM 1157), (Ka ') ole oil-
Ala-Pro-CHO (SUAM 1092), (ki ') oleoyl-Val-
Pro-CHO (SUAM 1156), (ku ') oleoyl-Leu-Leu-
Pro-CHO (SUAM 1158), was obtained. Physicochemical data of the obtained compound are shown in Table 1.

Example 4 Measurement of anti-prolyl endopeptidase activity The measurement of anti-prolyl endopeptidase activity was carried out 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 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. The absorbance (a) at 410 nm was measured.

At the same time, a blind absorbance (b) was measured using only a buffer solution instead of the solution of the anti-prolyl endopeptidase compound,
The prolyl endopeptidase inhibition rate was calculated by the following formula: [(ba) / b] × 100, and the amount required for 50% inhibition [IC ₅₀ ] was determined.
The test results are shown in Table 2.

Example 5 Measurement of experimental amnestic preventive effect of scopolamine using rat (intraperitoneal administration) The antiprolyl 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 1mg / kg, 0.25mg / kg
After adjusting to an amount equivalent to 0.1 mg / kg 0.025 mg / kg 0.010 mg / kg and dissolving in 0.3 ml of physiological saline, each Wistar (W
Sister male rat (100-120g) is administered once into the abdominal cavity, and 1 hour after administration, passive avoidance learning is performed by electric shock (1.7mA). Immediately afterwards, an amount equivalent to 3 mg / kg of scopolamine is intraperitoneally administered. did.

Efficacy was determined by a passive avoidance test at 24 hours and 48 hours after administration of scopolamine and physiological saline in the control animals without administration of the test compound and the test compound and scopolamine. This was done by comparing the numbers of amnestic and non-amnestic rats for each group of animals that were run together. The test results are shown in Table 3.

SUAM 1156 and 1140 showed no remarkable effect at the doses of 1 mg / kg, but showed extremely excellent results at the doses of 0.1 mg / kg, 0.025 mg / kg, and 0.010 mg / kg of the compound. The action of all these compounds is dose-dependent.

Claims (5)

[Claims] 1. General formula (I): (In the formula, n represents a number of 0 to 2, R ₁ represents a saturated or unsaturated linear organic group having 11 to 25 carbon atoms, and the unsaturated carbon chain represents a plurality of double bonds. R ₂ may include Or a —CH ₃ group, and R ₃ represents a lower alkyl ester group, a —CH ₂ OH group or an aldehyde group. ) The compound represented by. 2. A general formula: R ₁ --COOH, (In the formula, R ₁ represents a saturated or unsaturated linear organic group having 11 to 25 carbon atoms, and the unsaturated carbon chain may include a plurality of double bonds.). carboxylic acid,
Carboxylic anhydride or carboxylic acid chloride and the general formula (II): (In the formula, n represents a number of 0 to 2, and R ₂ is Alternatively, it represents a —CH ₃ group, and R ₃ represents a lower alkyl ester group. ), A peptide containing a proline residue in which a carboxyl group is lower alkyl esterified, or a lower alkyl esterified proline hydrochloride is reacted to give a compound represented by the general formula (III): (In the formula, n represents a number of 0 to 2, R ₁ represents a saturated or unsaturated linear organic group having 11 to 25 carbon atoms, and the unsaturated carbon chain represents a plurality of double bonds. R ₂ may include Alternatively, it represents a —CH ₃ group, and R ₄ represents a lower alkyl group. ) The method of manufacturing the N-acyl pyrrolidine derivative represented by these. 3. A general formula: R ₁ --COOH, (In the formula, R ₁ represents a saturated or unsaturated linear organic group having 11 to 25 carbon atoms, and the unsaturated carbon chain may include a plurality of double bonds.). carboxylic acid,
Carboxylic anhydride or carboxylic acid chloride and the general formula (II): (In the formula, n represents a number of 0 to 2, and R ₂ is Alternatively, it represents a —CH ₃ group, and R ₃ represents a lower alkyl ester group. ) Is reacted with a peptide containing a lower alkyl esterified proline residue represented by the formula (1) or a lower alkyl esterified proline hydrochloride.
I): (In the formula, n represents a number of 0 to 2, R ₁ represents a saturated or unsaturated linear organic group having 11 to 25 carbon atoms, and the unsaturated carbon chain represents a plurality of double bonds. R ₂ may include Alternatively, it represents a —CH ₃ group, and R ₄ represents a lower alkyl group. ), An N-acylpyrrolidine derivative represented by the formula: (In the formula, n represents a number of 0 to 2, R ₁ represents a saturated or unsaturated linear organic group having 11 to 25 carbon atoms, and the unsaturated carbon chain represents a plurality of double bonds. R ₂ may include Alternatively, it represents a —CH ₃ group. ) The compound represented by the general formula (V): (In the formula, n represents a number of 0 to 2, R ₁ represents a saturated or unsaturated linear organic group having 11 to 25 carbon atoms, and the unsaturated carbon chain represents a plurality of double bonds. R ₂ may include Alternatively, it represents a —CH ₃ group. ) A method for producing an N-acylpyrrolidine derivative represented by 4. General formula (I): (In the formula, n represents a number of 0 to 2, R ₁ represents a saturated or unsaturated linear organic group having 11 to 25 carbon atoms, and the unsaturated carbon chain represents a plurality of double bonds. R ₂ may include Alternatively, it represents a —CH ₃ group, and R ₃ represents an aldehyde group. ) A prolyl endopeptidase activity inhibitor comprising an N-acylpyrrolidine derivative represented by: 5. General formula (I): (In the formula, n represents a number of 0 to 2, R ₁ represents a saturated or unsaturated linear organic group having 11 to 25 carbon atoms, and the unsaturated carbon chain represents a plurality of double bonds. R ₂ may include Alternatively, it represents a —CH ₃ group, and R ₃ represents an aldehyde group. ) An anti-amnestic agent containing an N-acylpyrrolidine derivative represented by the following as an active ingredient.