JPH0548237B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to an anti-calpain compound that exhibits strong oxygen inhibitory activity against calpain, and a method for synthesizing the compound. (Prior Art) Calpain (EC3.4.22.17, Calpain) is a type of cysteine protease, and activation of this calpain is thought to be the cause of muscular dystrophy, which is an incurable disease. Therefore, there is a desire to develop a drug that specifically inhibits the activity of calpain as a therapeutic agent for muscle cysttrophy. Furthermore, there is a desire to develop drugs that specifically inhibit cysteine protease activity, such as calpain and papain, for use as anti-disaster drugs. As a calpain inhibitor, leupeptin, which was previously synthesized by Shimizu et al. [Shimizu, B. et al., J.
Antibiotics, Vol. 25, 515 Mitsugu (1972)] and Strepin p-1, which the present inventors extracted and purified from the culture solution of actinomycetes (Japanese Patent Application No. 116616/1982 (Japanese Patent Application No. 28999/1989)
However, since it inhibits the oxygen activity of not only cysteine proteases such as calpain and papain, but also serine proteases such as trypsin and plasmin, it lacks specificity and is not suitable for the treatment of muscular dystrophy, etc. It is not completely satisfactory as a medicine. (Purpose of the Invention) Therefore, the present inventors conducted various synthetic studies in order to find a compound that has a strong inhibitory effect on cysteine proteases, particularly calpain, and has no inhibitory effect on serine proteases, and as a result, the present invention was completed. did. (Structure of the Invention) The compound having anti-calpain activity of the present invention has the following general formula: (In the formula, R is -CH ₂ -S-CH ₃ , -CH ₂ -CH ₂ -
CH ₃ or

Indicates the group of [Formula]. ). For convenience, in the above formula, R is -CH ₂ -S-
SUAM312 for CH ₃ , SUAM313 for −CH ₂ −CH ₂ −CH ₃ ,

[Formula] is called SUAM314. The compound of the present invention is similar to leupeptin of Shimizu et al., but differs structurally in that the altehyde terminus is not derived from arginine. Although the compounds of the present invention can be synthesized by general peptide synthesis methods, they are conveniently synthesized by the synthetic method of the present invention described below. In addition, each abbreviation represents the following meaning: Z: benzyloxycarbonyl group, Leu: leucine residue, OEt: ethyl ester group, Met: methionine residue, nLeu: norleucine residue, OMe: methyl ester group, Ac: acetyl group, WSCI: N-ethyl-N',N'-dimethylaminopropylcarbodiimide, TEA: triethylamine, _BF3 · _OEt2 : boron trifluoride ether complex. In addition, in the synthesis example, the N-terminal amino group is protected with an acetyl group, but it may be protected with another suitable acyl group or urethane type protecting group. The synthetic method of the present invention can be used to prepare compounds of the following general formula: (In the formula, R represents the meaning given in the above formula, and R' represents a methyl group or an ethyl group.) The ester represented by is suspended in a tertiary alcohol, and a reducing agent such as sodium borohydride is added. ,
By adding dropwise anhydrous methanol under reflux in an inert gas, the following general formula: The alcohol is then oxidized with a sulfur trioxide-pyridine complex in dimethyl sulfoxide. The starting material represented by the formula is obtained by appropriately reacting a corresponding amino acid or peptide whose amino terminal is protected with a protecting group such as a Z group, and a corresponding amino acid or peptide whose carboxy terminal is protected with an ester group, etc. by a conventional method. Obtainable. Hereinafter, the present invention will be explained in more detail with reference to Examples. Reference example (synthesis of starting material represented by the formula) (1) Synthesis of Ac-Leu-Leu-Met-OMe (a) Z-Leu-Leu-OEt Z-Leu-OH (1 equivalent), Leu-OEt・HCl
(1 equivalent) and TEA (1 equivalent) were dissolved in dry methylene chloride, and WSCT (1 equivalent) was added under ice cooling.
Add. After stirring at room temperature for 16 hours, the reaction solution was washed with 1N hydrochloric acid, water, saturated aqueous sodium bicarbonate 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) Z-Leu-Leu-OH Dissolve Z-Leu-Leu-OEt (1 equivalent) in methanol and add 1N sodium hydroxide (2 equivalents).
and stirred at room temperature for 2 hours. After concentrating the reaction solution under reduced pressure, 1N hydrochloric acid was added to make it acidic.
Extract with ethyl acetate. After washing the extract with saturated brine and drying over anhydrous magnesium sulfate, the solvent is distilled off under reduced pressure to obtain crystals of the target compound. (c) Z-Leu-Leu-Met-OMe Z-Leu-Leu-OH (1 equivalent), Met-
OMe・HCl (1 equivalent) and TEA (1 equivalent) were dissolved in dry methylene chloride, and WSCI was performed under ice cooling.
(1 equivalent) is added. After stirring at room temperature for 16 hours, the reaction solution was washed with 1N hydrochloric acid, water, saturated aqueous sodium bicarbonate and saturated brine, and dried over anhydrous magnesium sulfate. The solvent is distilled off under reduced pressure to obtain crystals of the target compound. (d) Ac−Leu−Leu−Met−OMe Z−Leu−Leu−Met−OMe (1 equivalent) was dissolved in dry methanol, and palladium carbon (a small amount) and BF ₃ · OEt ₂ (about 3 equivalents) were added. Then, the Z group is removed by catalytic reduction. After filtering the reaction solution, the residue obtained by distillation under reduced pressure is suspended in dry methylene chloride, and acetic anhydride is added. After stirring at room temperature for 16 hours, the reaction solution was concentrated under reduced pressure, and the resulting residue was dissolved in ethyl acetate, and washed with water, saturated aqueous sodium bicarbonate, and saturated brine. Dry over anhydrous magnesium sulfate, and remove the solvent under reduced pressure to obtain crystals of the desired compound. (2) Synthesis of Ac-Leu-Leu-nLeu-OEt (a) Z-Leu-Leu-nLeu-OEt Z-Leu-Leu-OH (1 equivalent), nLeu-
OEt・HCl (1 equivalent) and TEA (1 equivalent) were dissolved in dry methylene chloride, and WSCI (1 equivalent) was added under ice cooling.
equivalent amount). After stirring at room temperature for 16 hours, the reaction solution was washed with 1N hydrochloric acid, water, saturated aqueous sodium bicarbonate 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-Leu-Leu-nLeu-OEt Z-Leu-Leu-nLeu-OEt is dissolved in methanol, water, acetic acid, and palladium on carbon are added to remove the Z group by catalytic reduction. After filtering the reaction solution, the residue obtained by distillation under reduced pressure was dissolved in dry chloroform,
Add dry benzene and acetic anhydride. at room temperature
After stirring for 16 hours, the reaction solution was concentrated under reduced pressure, and the resulting residue was dissolved in ethyl acetate, water,
Wash with saturated sodium bicarbonate solution and saturated saline solution. Dry over anhydrous magnesium sulfate and remove the solvent under reduced pressure to obtain crystals of the desired compound. (3) Synthesis of Ac-Leu-Leu-Leu-OEt (a) Z-Leu-Leu-Leu-OEt Z-Leu-Leu-OH (1 equivalent), Leu-
OEt・HCl (1 equivalent) and TEA (1 equivalent) were dissolved in dry methylene chloride, and WSCI (1 equivalent) was added under ice cooling.
equivalent amount). After stirring at room temperature for 16 hours, the reaction solution was washed with 1N hydrochloric acid, water, saturated aqueous sodium bicarbonate 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-Leu-Leu-Leu-OEt Z-Leu-Leu-Leu-OEt is dissolved in methanol, water, acetic acid, and palladium on carbon are added to remove the Z group by catalytic reduction. After filtering the reaction solution, the residue obtained by distillation under reduced pressure was dissolved in dry chloroform,
Add dry benzene and acetic anhydride. at room temperature
After stirring for 16 hours, the reaction solution was concentrated under reduced pressure to obtain a residue, which was washed with cold ethyl acetate to obtain the desired compound as crystals. Example 1 (Synthesis of SUAM312) (a) Ac−Leu−Leu−Met−ol (− of SUAM312)
Intermediate with -CH ₂ OH instead of CHO) Ac-Leu-Leu-Met-OMe obtained in Reference Example (1)
(863 mg, 2 mmol) and sodium borohydride (190 mg, 5 mmol) were suspended in tert-butyl alcohol (16 ml), heated and stirred under a nitrogen stream, and then added to anhydrous methanol (2.4 ml) under reflux.
was dripped. After the dropwise addition was completed, the mixture was stirred under reflux for 1 hour, then returned to room temperature, and water (10 ml) was added under ice-cooling. After methanol and tert-butyl alcohol were distilled off under reduced pressure, the residue was extracted three 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 recrystallized from ethyl acetate to obtain the target compound (520
mg, 64%). (b) SUAM312 Ac-Leu-Leu-Met-ol (404 mg, 1 mmol) and triethylamine (0.56 ml, 4 mmol) were dissolved in anhydrous dimethyl sulfoxide (4.2 ml) and the sulfur trioxide-pyridine complex (637 mg , 4 mmol) in dimethyl sulfoxide (4 ml) was added. After stirring at room temperature for 10 minutes, it was poured into ice water (30 ml) and extracted three times with ethyl acetate.
It was washed with a 10% aqueous citric acid solution, water, saturated aqueous sodium bicarbonate and saturated brine, and dried over anhydrous magnesium sulfate. The crude crystals obtained by distilling off ethyl acetate under reduced pressure were recrystallized from ethyl acetate to obtain SUAM312 (300 mg, 75%) represented by the following formula. The physical properties of the obtained compound are shown in Table 1 below. Example 2 (Synthesis of SUAM313) (a) Ac−Leu−Leu−nLen−ol (− of SUAM313)
Intermediate with -CH ₂ OH instead of CHO) Ac-Leu-Leu-nLeu-OEt obtained in Reference Example (2)
(556 mg, 1.3 mmol) and sodium borohydride (123 mg, 3.25 mmol) were suspended in tert-butyl alcohol (12 ml), heated and stirred under a nitrogen stream, and then anhydrous methanol (1.8
ml) was added dropwise. After the dropwise addition was completed, the mixture was stirred under reflux for 1 hour, then returned to room temperature, and water (10 ml) was added under ice-cooling. After methanol and tert-butyl alcohol were distilled off under reduced pressure, the residue was extracted three 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 recrystallized from ethyl acetate to obtain the target compound (210
mg, 42%). (b) SUAM313 Ac-Leu-Leu-nLeu-ol (193 mg, 0.5 mmol) and triethylamine (0.28 ml, 2.0 mmol) were dissolved in anhydrous dimethyl sulfoxide (2 ml) and sulfur trioxide-pyridine complex (320 mg, 2.0 mmol) in dimethyl sulfoxide (2 ml) was added. After stirring at room temperature for 10 minutes, the mixture was poured into ice water (30 ml), extracted three times with ethyl acetate, washed with a 10% aqueous citric acid solution, water, saturated aqueous sodium bicarbonate and saturated brine, and dried over anhydrous magnesium sulfate. The crude crystals obtained by distilling off ethyl acetate under reduced pressure were recrystallized from ethyl acetate to obtain SUAM313 (90 mg, 47%) represented by the following formula. The physical properties of the obtained compound are shown in Table 2 below. Example 3 (Synthesis of SUAM314) (a) Ac−Leu−Leu−Leu−ol (− of SUAM314)
Intermediate with -CH ₂ OH instead of CHO) Ac-Leu-Leu-Leu-OEt obtained in Reference Example (3)
(855 mg, 1.3 mmol) and sodium borohydride (190 mg, 5 mmol) were suspended in tert-butyl alcohol (16 ml), heated and stirred under a nitrogen stream, and then added to anhydrous methanol (2.4 ml) under reflux.
was dripped. After the dropwise addition was completed, the mixture was stirred under reflux for 1 hour, then returned to room temperature, and water (30 ml) was added while cooling with ice. After methanol and tert-butyl alcohol were distilled off under reduced pressure, the residue was extracted three 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 recrystallized from ethyl acetate to obtain the target compound (490
mg, 64%). (b) SUAM314 Ac-Leu-Leu-Leu-ol (386 mg, 1 mmol) and triethylamine (0.56 ml, 4 mmol) were dissolved in anhydrous dimethyl sulfoxide (4.2 ml) and the sulfur trioxide-pyridine complex (637 mg , 4 mmol) in dimethyl sulfoxide (4 ml) was added. After stirring for 10 minutes at room temperature,
The mixture was poured into ice water (30 ml), extracted three times with ethyl acetate, washed with a 10% aqueous citric acid solution, water, saturated aqueous sodium bicarbonate and saturated brine, and dried over anhydrous magnesium sulfate. The crude crystals obtained by distilling off ethyl acetate under reduced pressure were recrystallized from ethyl acetate to obtain SUAM314 (260 mg, 68%) represented by the following formula. SUAM314 The physical properties of the obtained compound are shown in Table 3 below.

【table】

【table】

[Table] Example 4 Physiological activity of the substance of the present invention The oxygen inhibitory activity of the substance of the present invention was measured as follows. The substance of the present invention and an oxygen solution are mixed, brain-cubated at 30°C for 5 minutes, and then a substrate solution is mixed to start the reaction. 0.5% casein was used as a substrate, and 5mM calcium chloride and 10mM
Cysteine and 50mM Tris-HCl buffer (PH)
7.5) and react at 30℃ for 30 minutes. Next, 6.5% trichloroacetic acid was added to the reaction solution to stop the reaction, and the amount of protein in the trichloroacetic acid soluble fraction of enzyme-hydrolyzed casein was measured by the Lowry-Folin method.
Determine the inhibitory ability by comparing with the control solution. The inhibitory effects of the substances of the present invention on calpain, calpain, and papain thus obtained are shown in Table 4 and the figures in comparison with the effects of leupeptin and strepin P-1. It was found that the substance of the present invention exhibits 50% or more inhibitory activity against calpain and calpain at doses of 0.05 to 10 μg each. Furthermore, when the inhibitory effect of the substance of the present invention on serine protease (trypsin/plasmin) was measured by the above method, almost no inhibitory effect was observed.

【table】 [Brief explanation of the drawing]

The figure is a graph showing the inhibitory effect of the substance of the present invention and leupeptin on calpain activity.

Claims (1)

[Claims] 1. The following general formula: (In the formula, R is -CH ₂ -S-CH ₃ , -CH ₂ -CH ₂ -
Represents CH ₃ or a group of [Formula]. ) A compound represented by 2 The following general formula: (In the formula, R represents the meaning given in the following formula, and R' represents a methyl group or an ethyl group.) The ester represented by is suspended in a tertiary alcohol, a reducing agent is added, and the mixture is refluxed in an inert gas. By adding anhydrous methanol under the following general formula: and then oxidation of the alcohol with a sulfur trioxide-pyridine complex in dimethyl sulfoxide: (In the formula, R is -CH ₂ -S-CH ₃ , -CH ₂ -CH ₂ -
Represents CH ₃ or a group of [Formula]. ) A method for producing a compound represented by