JPH0629229B2 - Cysteine proteinase inhibitor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) {In the formula, R ₁ is a benzyloxycarbonyl group, or 4
Represents a phenylbutyryl group, R ₂ is an isopropyl group,
Alternatively, it represents an isobutyl group, R ₃ represents a butyl group, a benzyl group, or a methylthioethyl group, and R ₄ represents a hydrogen atom or a chloromethyl group. } The present invention relates to a cysteine proteinase, particularly a cysteine proteinase inhibitor showing a strong oxygen inhibitory activity against papain and calpain.

(Prior Art) Papain (E.
C. 3.4.22.2, PAPAIN) and calpain (EC 3.4.22.217, CALPAIN) are specifically muscular dystrophies as anti-inflammatory agents, and especially with respect to calpain. Or, it is known to be effective as a therapeutic agent for cataract and the like. Various cysteine proteinase inhibitors have been found so far with the aim of developing these applications.
imiz, B .; Et al., J. Antibio / t. , 25
Volume, 515, (1972), JP-A-60-28990.
JP-A-61-106600, JP-A-61-103
897), activity, specificity, improvement in bioavailability and the like are currently strongly desired.

(Problems to be Solved by the Invention) Therefore, the present inventors have completed the present invention as a result of various synthetic investigations in order to find a compound having a strong inhibitory activity against calpain among cysteine proteinases and further having high in vivo transferability. did.

(Means for Solving Problems) According to the present invention, a novel compound having a strong calpain-inhibiting activity or papain-inhibiting activity, represented by the general formula (1) {In the formula, R ₁ is a benzyloxycarbonyl group, or 4-
It represents a phenylbutyryl group, R ₂ represents an isopropyl group or an isobutyl group, R ₃ represents a butyl group, a benzyl group or a methylthioethyl group, and R ₄ represents a hydrogen atom or a chloromethyl group. }, N
-Acyl-peptidyl-aldehyde or N-acyl-peptidyl-chloromethylketone is supplied.

The compound of the present invention can be produced as follows. First, in order to produce a compound of the present invention in which R ₄ is hydrogen in formula (1), the following general formula (2) {In the formula, R ₁ , R ₂ and R ₃ represent the meanings given in the formula (1), and R ₅ represents a lower alkyl group. } Is easily produced by reducing the compound represented by the formula (1) to an alcohol by using a reducing agent in an organic solvent and further oxidizing the compound to an aldehyde by using an oxidizing agent. Further, in the general formula (1), R ₄ is a chloromethyl group to produce the compound of the present invention, the following general formula (3) {In the formula, R ₁ , R ₂ and R ₃ represent the meanings given in the formula (1). } The carboxylic acid represented by the above is led to an active ester using ethyl chlorocarbonate or the like in an organic solvent,
It is easily produced by reacting diazomethane to give diazomethyl ketone and further treating with hydrochloric acid.

(Examples) Next, the present invention will be described in more detail with reference to Examples and inhibitory activity tests, but it goes without saying that the technical scope of the present invention is not limited to these Examples. The SUAM number is used to identify the compound in the oxygen inhibition activity test and the examples, and is described below.

Example 1 N-benzyloxycarbonyl-L-leucyl-L-phenylalaninal (SUAM-14541) L-phenylalanine ethyl ester hydrochloride (4.6 g,
20 mmol) and N-benzyloxycarbonyl-L-leucine (5.4 g, 20 mmol) in dry methylene chloride 100
It was dissolved in m and triethylamine (2.0 g, 20 mmol) was added. 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSCD) (4.
2 g, 22 mmol) was added, and the mixture was stirred overnight at room temperature. After completion of the reaction, the reaction solution was washed with 1N hydrochloric acid, saturated saline solution, saturated sodium bicarbonate solution, and saturated saline solution in this order, and dried over anhydrous sodium sulfate.

The solvent was distilled off and the residue was purified by medium pressure column chromatography using silica gel to obtain N-benzyloxycarbonyl-L-leucyl-L-phenylalanine ethyl ester (8.4 g, crystals). This N-benzyloxycarbonyl-L-leucyl-L-phenylalanine ethyl ester (2.2 g, 5 mmol) and sodium borohydride (570 mg, 15 mmol) were suspended in tert-butyl alcohol (50 m) and heated under reflux under a nitrogen atmosphere. (9
0 ° C). Then, anhydrous methanol (8 m) was added dropwise under reflux. After completion of the dropwise addition, the mixture was refluxed and stirred for 1 hour, then returned to room temperature, and water (30 m) 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 residue obtained by distilling off ethyl acetate under reduced pressure was purified by medium pressure column chromatography using silica gel to obtain N-benzyloxycarbonyl-L-leucyl-L-phenylalaninol (1.5 g, crystals). . This N-benzyloxycarbonyl-L-leucyl-L-phenylalaninol (1.2 g, 3 mmol) and triethylamine (1.2 g, 12 mmol) were dissolved in anhydrous dimethylsulfoxide (8 m), and sulfur trioxide-pyridine complex was stirred. A solution of (1.9 g, 12 mmol) in dimethyl sulfoxide (8 m) was added. After stirring at room temperature for 10 minutes, it was poured into ice water (120 m), extracted 3 times with ethyl acetate, and extracted with 10
% Aqueous citric acid solution, saturated saline solution, saturated sodium hydrogen carbonate solution, and saturated saline solution in this order, and dried over anhydrous sodium sulfate. The residue obtained by distilling off ethyl acetate under reduced pressure was purified by medium pressure column chromatography using silica gel to obtain the target compound N-benzyloxycarbonyl-L-leucyl-L-phenylalaninal (0.6
g, oily substance) was obtained.

1H-NMR; TMS standard 0.80-1.00 (6H, m), 1.22-1.72 (3H, m), 3.12 (2H, m), 4.16 (1 in CDCl ₃
H, m), 4.66 (1H, m), 5.08 (2H, s), 5.12 (1H, m), 5.64 (1H, m),
7.16-7.34 (10H, m), 9.56 (1H, s) IR spectrum; Measurement shape is film, wave number (cm ^-1 ); 33
30,3270,3030,2960,1730,1680,1650,1530,1240,1040,75
0,740,700 Example 2 N-benzyloxycarbonyl-L-leucyl-L-norleucinal (SUAM-14542) By substituting L-norleucine methyl ester hydrochloride (3.6 g, 20 mmol) in place of L-phenylalanine ethyl ester hydrochloride in Example 1, the target compound N-benzyloxycarbonyl-L-leucyl-L-
Norleucinal (0.5 g, powder) was obtained. Melting point; 9
3 ℃ 1H-NMR; in CDCI _3, TMS reference 0.80-1.00 (9H, m), 1.22-1.28 (9H, m), 4.12-4.58 (2H, m), 5.
12 (2H, s), 5.22 (1H, d, J = 8.0), 6.57 (1H, d, J = 7.0), 7.36
(5H, s), 9.54 (1H, s) IR spectrum; measurement shape is KBr, wave number (cm ^-1 ); 332
0,3030,2950,1720,1680,1640,1530,1230,1050,740,700 Example 3 N-benzyloxycarbonyl-L-leucal-L-methioninal (SUAM-14543) By substituting L-methionine methyl ester hydrochloride (4.0 g, 20 mmol) in place of L-phenylalanine ethyl ester hydrochloride in Example 1, the target compound N-benzyloxycarbonyl-L-leucyl-L-methioninal (0.5 g , Oily substance) was obtained. Refractive index; (D
Line, 25 ° C.); 1.5342 1H-NMR; DMSO-d ₆ in TMS standard 0.94 (6H, d, J = 6.0), 1.42-2.58 (5H, m), 2.06 (3H, s), 4.08-
4.62 (2H, m), 5.10 (2H, s), 5.37 (1H, d, J = 7.0), 6.95 (1H, d,
J = 6.0), 7.34 (5H, s), 9.56 (1H, d, J = 2.0) IR spectrum; measurement shape is film, wave number (cm ^-1 ); 33
00,3070,2950,1720,1700,1660,1530,1240,1040,740,700 Example 4 N- (4-phenyl) butanoyl-L-leucyl-L-
Phenylalaninal (SUAM-14544) N-benzyloxycarbonyl-L-leucyl-L-phenylalanine ethyl ester (2.2 g, 5 mmol), which is a synthetic intermediate of Example 1, was mixed with ethyl alcohol (50 m).
), A small amount of palladium carbon was added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 24 hours. After completion of the reaction, palladium carbon was filtered and ethyl alcohol was distilled off under reduced pressure. This residue was dissolved in 50 m of tetrahydrofuran, and triethylalumine (1.0 g, 10 mmol) was added. This solution was cooled with ice (4-phenyl) butanoyl chloride (0.9 g,
(5 mmol) was added dropwise, and the mixture was stirred for 1 hour. Then, the temperature was returned to room temperature and the mixture was further stirred for 1 hour. After completion of the reaction, tetrahydrofuran was distilled off under reduced pressure, and the residue was dissolved in 50 m of ethyl acetate. This solution was washed with 1N hydrochloric acid, saturated saline, saturated sodium hydrogen carbonate, and saturated saline in this order, and dried over anhydrous sodium sulfate. The residue obtained by distilling off the solvent under reduced pressure was purified by medium pressure column chromatography using silica gel to give N- (4-phenyl) butanoyl-L-leucyl-L-phenylalanine ethyl ester (2.0
g, crystals) were obtained. This N- (4-phenyl) butanoyl-L-leucyl-L-phenylalanine ethyl ester (1.4 g, 3 mmol) and sodium borohydride (34
0 mg, 9 mmol) was added to tert-butyl alcohol (30 m)
And was heated to reflux (90 ° C.) under a nitrogen atmosphere. Then, anhydrous methanol (5 m) was added dropwise under reflux. After completion of the dropwise addition, the mixture was refluxed and stirred for 1 hour, then returned to room temperature, and water (30 m) 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 residue obtained by distilling off ethyl acetate under reduced pressure was purified by medium pressure column chromatography using silica gel to give N-
(4-Phenyl) butanoyl-L-leucyl-L-phenylalaninol (1.2 g, crystals) was obtained. This N-
(4-Phenyl) butanoyl-L-leucyl-L-phenylalaninol (1.1 g, 2.5 mmol) and triethylamine (1.0 g, 10 mmol) were dissolved in anhydrous dimethyl sulfoxide (8 m), and sulfur trioxide-pyridine was added with stirring. Complex (1.6g, 10mmol) of dimethyl sulfoxide (8m
) Solution was added. After stirring at room temperature for 10 minutes, ice water (120
m), extracted 3 times with ethyl acetate, 10% aqueous citric acid solution, saturated saline solution, saturated sodium hydrogen carbonate solution,
Then, the extract was washed with saturated saline and then dried over anhydrous sodium sulfate. The residue obtained by distilling off ethyl acetate under reduced pressure was purified by medium pressure column chromatography using silica gel to obtain the target compound N- (4-phenyl) butanoyl-
L-leucyl-L-phenylalaninal (0.6 g, oily substance) was obtained.

IH-NMR; in CDCl _3, TMS reference 0.80-1.00 (6H, m), 1.52-2.26 (7H, m), 2.52-2.72 (2H, m), 3.
12 (2H, m), 4.40-4.76 (2H, m), 5.72 (1H, d, J = 7.0), 6.68 (1
H, d, J = 6.0), 7.14-7.26 (10H, m), 9.58 (1H, s) IR spectrum; measurement shape is film, wave number (cm ^-1 ); 37
20,3060,2950,1730,1630,1540,1240,740,700 Example 5 N- (4-phenyl) butanoyl-L-leucyl-L-
Norleucinal (SUAM-14545) In Example 4, instead of the synthetic intermediate N-benzyloxycarbonyl-L-leucyl-L-phenylalanine ethyl ester of Example 1, the synthetic intermediate of Example 3, N-benzyloxycarbonyl-L-leucyl-L. −
By using norleucine methyl ester (5.3 g, 12 mmol), the target compound N- (4-phenyl) butanoyl-L-leucyl-L-norleucinal (0.6
g, oily substance) was obtained.

Refractive index (D line, 25 ° C.); 1.5123 1H-NMR; CDCl ₃ in TMS standard 0.80-1.00 (6H, m), 1.24-2.32 (13H, m), 2.57-2.72 (2H, m),
4.30-4.63 (2H, m), 6.02 (1H, d, J = 8.0), 6.28 (1H, d, J = 7.
0), 7.18-7.23 (5H, m), 9.54 (1H, s) IR spectrum; measurement shape is film, wave number (cm ^-1 ); 32
70,3060,2950,1730,1630,1540,1240,740,700 Example 6 N- (4-phenyl) butanoyl-L-leucyl-L-
Methioninal (SUAM-14546) In Example 4, instead of the synthetic intermediate N-benzyloxycarbonyl-L-leucyl-L-phenylalanine ethyl ester of Example 1, the synthetic intermediate of Example 2, N-benzyloxycarbonyl-L-leucyl-L. −
The target compound N- (4-phenyl) butanoyl-L-leucyl-L-methioninal (0.5 g, oily substance) was obtained by using methionine methyl ester (2.1 g, 5 mmol).

Refractive index (D line, 25 ° C.); 1.5327 1H-NMR; in CDCl ₃ , TMS standard 0.80-1.00 (6H, m), 1.40-2.80 (11H, m), 2.03-2.06 (total-3
H, both-s), 4.39-4.60 (2H, m), 6.02 (1H, d, J = 8.0), 7.18-
7.22 (6H, m), 9.55,9.58 (total-1H, both-s), IR spectrum; measurement shape is film, wave number (cm ^-1 ); 32
70,3060,2950,1730,1630,1540,1240,740,700 Example 7 N-benzyloxycarbonyl-L-leucyl-L-phenylalanyl chloromethyl (SUAM-11705) N-Benzyloxycarbonyl-L-leucyl-L-phenylalanine ethyl ester (2.6 g, 6 mmol), which is the synthetic intermediate of Example 1, was dissolved in a small amount of methyl alcohol, and a 1N sodium hydroxide aqueous solution was added thereto for 10 m.
The suspension was stirred at room temperature until it became a clear solution. Methyl alcohol was distilled off under reduced pressure, and the residue was partitioned between water and ethyl acetate. The aqueous layer was acidified with 10N hydrochloric acid, extracted three times with ethyl acetate, and the organic layer was dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain N-benzyloxycarbonyl-L-leucyl-L-phenylalanine (2.4 g, crystals). The N-benzyloxycarbonyl-L-leucyl-L-phenylalanine (2.1 g, 5 mmol) was dissolved in dry tetrahydrofuran (20 m), triethylamine (0.5 g, 5 mmol) was added, and the mixture was cooled to -10 ° C.
Ethyl chlorocarbonate (0.6 g, 5 mmol) was added to this solution, and the mixture was stirred at -10 ° C for 20 minutes. The mixture was returned to room temperature, an excess diazomethane ether solution was added, and the mixture was further stirred for 30 minutes. Hydrochloric acid gas was blown into the reaction solution for about 10 minutes.
After completion of the reaction, the solvent was distilled off, ethyl acetate (50 m) was added, and the mixture was washed with saturated saline, saturated sodium hydrogen carbonate solution, and saturated saline in this order, and dried over anhydrous sodium 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 N- (4-phenyl) butanoyl-L-leucyl-L-phenylalanyl chloromethyl (1.5 g. ,crystal)
Got

Melting point: 140 ° C. 1H-NMR; DMSO-d ₆ in TMS standard 0.80 (6H, dd, J = 4.0, J = 7.0), 1.00-1.70 (3H, m), 2.70-3.4
0 (2H, m), 3.95 (1H, m), 4.45 (2H, dd, J = 5.0, J = 16.0), 4.50
(1H, m), 5.00 (2H, S) 7.21 (5H, s), 7.28 (5H, s), 7.41 (1H, d, J
= 8.0), 8.42 (1H, d, J = 8.0) IR spectrum; measurement shape is KBr, wave number (cm ^-1 ); 331
0,3280,2950,1730,1685,1540,1265,1240,700 Example 8 N-benzyloxycarbonyl-L-leucyl-L-norleucyl chloromethyl (SUAM-11706) In Example 7, instead of the synthetic intermediate N-benzyloxycarbonyl-L-leucyl-L-phenylalanine ethyl ester of Example 1, the synthetic intermediate of Example 2, N-benzyloxycarbonyl-L-leucyl-L. −
The target compound N- (4-phenyl) butanoyl-L-leucyl-L-norleucylchloromethyl (1.6 g, crystals) was obtained by using norleucine methyl ester (2.4 g, 6 mmol).

Melting point; 111 ° C. 1H-NMR; DMSO-d ₆ in TMS standard 0.80-1.00 (9H, m), 1.00-1.90 (9H, m), 4.04 (1H, m), 4.30 (1
H, m), 4.54 (2H, s), 5.00 (2H, S), 7.36 (5H, s), 7.46 (1H, d, J
= 8.0), 8.36 (1H, d, J = 8.0) IR spectrum; measurement shape is KBr, wave number (cm ^-1 ); 330
0,2950,1740,1680,1660,1640,1530,1280,1240,690 Example 9 N- (4-phenyl) butanoyl-L-leucyl-L-
Norleucyl chloromethyl (SUAM-11707) In Example 7, instead of the synthetic intermediate N-benzyloxycarbonyl-L-leucyl-L-phenylalanine ethyl ester of Example 1, the synthetic intermediate of Example 6, N- (4-phenyl) butanoyl-L-. Leucyl-L
-Norleucine methyl ester (2.4 g, 6 mmol) was used to obtain the target compound N- (4-phenyl) butanoyl-L-leucyl-L-norleucylchloromethyl (1.0 g, crystals).

Melting point; 114 ° C. 1H-NMR; DMSO-d ₆ in TMS standard 0.80-1.00 (9H, m), 1.00-2.40 (15H, m), 4.30 (2H, m), 4.52 (2
H, s), 7.20 (2H, s), 8.00 (1H, d, J = 8.0), 8.32 (1H, d, J = 8.
0) IR spectrum; measurement shape is KBr, wave number (cm ^-1 ); 330
0,2950,1730,1630,1530,690 Test Example Oxygen inhibitory activity of the substance of the present invention The oxygen inhibitory activity of the substance of the present invention was measured as follows.

The anti-papain activity was adjusted to various concentrations, the compound of the present invention, papain (0.015 unit), and EGTA (0.88 mg)
Of citrate buffer solution (20 mM, pH = 6.2, 1 m)
Pre-incubate for 5 minutes at 0 ° C, then add the substrate solution (1m
) Was added to start the reaction. A 1% citrate buffer solution of casein was used as a substrate and reacted at 30 ° C. for 20 minutes. Then 6.5 trichloroacetic acid (3 m) was added to the reaction solution.
Was added to stop the reaction, and the amount of protein in the trichloroacetic acid-soluble fraction of casein hydrolyzed by the enzyme was measured by the Lowry-Folin method, and the inhibitory activity was determined by comparison with the control solution. It was The anti-calpain activity is as follows for calpain I and II,
Compounds of the present invention prepared at various concentrations, calpain I or II (0.33 unit), and CaCl ₂ (0.22 mg)
Imidazole-hydrochloric acid buffer solution (50 mM, pH = 7.5, 1
m) was pre-incubated at 30 ° C. for 5 minutes and the substrate solution (1 m) was added to start the reaction. A 0.4% imidazole hydrochloric acid buffer solution of casein was used as a substrate, and 3
The reaction was carried out at 0 ° C for 30 minutes. Then, 5% trichloroacetic acid (3 m) was added to the reaction solution to stop the reaction, and the protein amount in the trichloroacetic acid-soluble fraction of casein hydrolyzed by the enzyme was measured by Ross-Schats (Roos-Schat).
z) method, and the inhibitory activity was determined by comparison with the control solution.

The activity-inhibiting effect of the compounds of the present invention thus obtained on papain, calpain I and II is shown in Tables I, II and III.

(Effects of the Invention) The novel compound of the present invention not only has extremely excellent inhibitory activity against cysteine proteinases such as papain, calpain I and calpain II, but also its synthesis is easy, and therefore, it is an anti-inflammatory agent and muscular dystrophy. Alternatively, it can be expected to be used as a therapeutic agent for cataracts.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location C07C 271/22 7188-4H 323/41 7419-4H C12N 9/99

Claims (2)

[Claims] 1. A general formula (1) {In the formula, R ₁ is a benzyloxycarbonyl group, or 4-
It represents a phenylbutyryl group, R ₂ represents an isopropyl group or an isobutyl group, R ₃ represents a butyl group, a benzyl group or a methylthioethyl group, and R ₄ represents a hydrogen atom or a chloromethyl group. } The compound represented by these. 2. General formula (1) {In the formula, R ₁ is a benzyloxycarbonyl group, or 4-
It represents a phenylbutyryl group, R ₂ represents an isopropyl group or an isobutyl group, R ₃ represents a butyl group, a benzyl group or a methylthioethyl group, and R ₄ represents a hydrogen atom or a chloromethyl group. } The cysteine proteinase inhibitor containing the compound represented by these as an active ingredient.