JPS61103897A - Preparation of anti-carpaine - Google Patents

Abstract

NEW MATERIAL:The compound of formula I (R is -CH2-S-CH3, -CH2-CH2-CH3 or group of formula II). EXAMPLE:The compound of formula III. USE:An anticarpaine compound. It exhibits strong enzymeinhibiting activity against carpaine, and little inhibiting activity to serine protease. PREPARATION:The objective compound of formula I can be prepared by suspending the ester of formula IV (R' is methyl or ethyl) in a tert-alcohol, adding a reducing agent such as sodium borohydride, adding anhydrous methanol to the mixture under refluxing in an inert gas atmosphere, and oxidizing the resultant alcohol of formula V with sulfur trioxide-pyridine complex in dimethyl sulfoxide. The starting compound of formula IV is synthesized by conventional peptide synthesis using corresponding amino acids or peptides.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to an anti-calpain compound that exhibits strong enzymatic inhibitory activity against calpain, and a method for synthesizing the compound.

(Prior art) Calpain (E, C, 5, 4, 22, 171 Calpa
in ) ht.

Calpain, a type of dystingulothiase, is thought to be the cause of the intractable disease muscular dystrophy. Therefore, is calpain active as a therapeutic agent for muscular dystrophy? The development of drugs that specifically inhibit this is desired. In addition, drugs that specifically inhibit cysteine pcotease activity %, such as calpain and hahain,
Its development is also desired for its use as an anti-disaster drug.

As a calpain field-damaging substance, leupeptin, which was previously synthesized by Shimizu et al.

J, Antib iotics l vol. 25, 5
15 (1972) 1 and Strevin p-1 ('￥9 Gansho 58-
ii6616 Specification V) is known, which is calpain.

It lacks specificity because it inhibits not only distingufacturing proteases such as Paipan but also serine proteases such as trypsin and plasmin.

It is not fully satisfactory as a therapeutic agent for muscular dystrophy, etc.

(Purpose of the invention) Therefore, the present inventors developed distingufacturing prothiase.

This is the result of extensive synthetic research aimed at finding a compound that has a particularly strong inhibitory effect on calpain, but has no inhibitory effect on serine proteases.

The invention has been completed.

(Structure of the Invention) The compound having anti-carbaine activity of the present invention is as follows.
General formula I of f: (wherein R is -CH2-8-CH3, -CH2-CH2
-CH3 or less For convenience, 5R in the above formula I is 10H2-8-
The CH3 one is SUAM312. -CH2-CH2-C
The H3 one is called SUAM314.

The compounds of the present invention are similar to leupeptin of Shimizu et al.
It is structurally different in that the aldehyde end is not made of arginine.

Although the compounds of the present invention can be synthesized by general peptide synthesis methods, they are conveniently synthesized by the synthetic methods of the present invention described below. In addition, each abbreviation has the meaning 2
Represents: Z: penzylox7carbonyl group, Leu: leucine residue.

OEt: ethyl ester group, M, et: methionine residue nLeu: Norleucine residue.

OMe: methyl ester group, Ac Niacetyl group.

WSCI: N-ethyl-N'.N'-dimethylaminopropylcarpodiimide.

TEA nitriethylamine, BF-0Et2: boron trifluoride ether complex.

In the synthesis examples, the N-terminal amine group is protected by a careacetyl group, but it may be protected by other suitable acyl groups or urethane-type protecting groups.

To prepare compound Y of formula I by the synthetic method of the present invention,
The following general formula 11: In the formula 1, the meaning given in the RI formula I is represented by 2, R
' represents a methyl group or an ethyl group,
(filtered ester)) Dissolved in a trialcohol and added dropwise to anhydrous methanol under reflux in an inert gas atmosphere to convert it into an alcohol represented by the following general formula III: -oxidize with pyridine complexes.

The starting material represented by 2 is obtained by appropriately reacting a corresponding amino acid or peptide with the amino terminal A protected with a protecting group such as a Z group, and a corresponding amino acid or peptide with the carboxy terminal protected with an ester group, etc. using a conventional method. Let's get what we can.

Hereinafter, the present invention will be explained in more detail with reference to Examples. Reference Example (Synthesis of starting material represented by formula U)! 11
Synthesis of Ac-Leu-Leu-Met-OMe a) Z
-Leu-Leu-OEt Z-Leu-OH (1 unit), Leu-OEt
Dissolve eHct (1 eq.) and TEA (1 eq.) in dry methylene chloride and add WSCT (1 eq.) under water cooling.

After stirring at room temperature for 16 hours, the reaction solution was mixed with 1N hydrochloric acid, water,
Wash with saturated sodium bicarbonate solution and saturated saline solution, and dry with anhydrous magnesium sulfate. The # solvent was distilled off under reduced pressure to obtain crystals of the target compound 7-5 b) Z-Leu-Leu -0H z-Leu-Leu-OEt (1 equivalent)? 5 Reactions: Add 1N sodium hydroxide (2 equivalents) fjx to methanol, and stir at room temperature for 2 hours. After decompression concessions,
Acidify by adding 1N weir rilY and extract with ethyl acetate. After washing the extract with saturated saline and drying over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure! and obtain crystals of the target compound.

c) Z-Leu-Leu-Met-OMeZ-Le
u-Leu-OH (1 equivalent), M e t -Qp, 1
6@)] C6 (1 eq.) and TEA (1 eq.) were dissolved in dry methylene chloride, and WSCI (1 eq.) was added under water cooling.
After stirring at room temperature for 16 hours, add 1N to the reaction solution.
Wash PVC, water, saturated sodium bicarbonate solution, and saturated salt solution.

Dry with anhydrous magnesium sulfate. The solvent is distilled off under reduced pressure to obtain crystals of the target compound.

d) Ac Leu-Leu-Met-OMeZ
-Leu-Leu-Met -OMe (1 equivalent) was dissolved in dry methanol, palladium carbon (a small amount) and BF ・0Et2 (approximately 3 equivalents) Y: In addition, Z group ? 5 After filtering the reaction solution to be removed.

The residue obtained by distillation under reduced pressure is suspended in dry diluted methylene, and then acetic anhydride is added. After stirring at room temperature for 16 hours, the reaction solution was reduced to EE! The residue obtained by shrinking l8rF
, dissolved in ethyl acid and washed with water, saturated sodium bicarbonate solution and saturated brine. Dry with anhydrous value 1 set magnesium and swamp 1? Distill under reduced pressure to obtain crystals of the target compound.

(Synthesis a of 21Ac Leu-Leu-nLeu-OEt) Z-Leu-Leu-nLeu-OEtZ-
Leu-Leu-OH (1 equivalent), nLeu OEt・
HC/, (per i, TEA (1 equivalent)? Dissolved in dry methylene chloride, add WSCI (1 equivalent) under ice cooling. After stirring at room temperature for 16 hours, the reaction solution was t1N hydrochloric acid 1*,
Wash with saturated sodium bicarbonate solution and saturated saline solution.

Dry with anhydrous magnesium chloride. Remove the solvent under reduced pressure,
Crystals of the target compound? obtain.

b) Ac-Leu -Leu-nLeu-OEtZ-
Leu-Leu-nLeu-OEt Y was dissolved in methanol, in addition to water, acetic acid, and palladium on carbon.
5 reaction solutions for removing Z group by catalytic reduction? The residue obtained after filtration and distillation under reduced pressure? Bath in dry chloroform
4, add dry benzene, and acetic anhydride, and stir at room temperature for 16 hours.

Reaction liquid? The residue obtained by concentration under reduced pressure was dissolved in ethyl diacetate, washed with water, 5 saturated sodium bicarbonate solution and saturated brine, dried over anhydrous transferred magnesium, and the solvent # was distilled off under reduced pressure to obtain crystals of the target compound. .

t31 Synthesis of Ac-Leu-, T, eu-OEta
) Z-Leu-Leu-Leu-OEtZ-Leu
-Leu -OH (1t), Leu-OEt
・HC4 (1 equivalent), TEA (1 equivalent)'l:! : Pyrophorize dry methylene chloride, add WSCI (1 equivalent) under ice cooling, stir at room temperature for 16 hours, and then add the reaction solution. Wash with 1N hydrochloric acid, water, water, and saturated saline, and dry with anhydrous #C magnesium. The 6 medium is distilled off under reduced pressure and crystals of the target substance are obtained? obtain.

b) Ac-Leu-Leu-Lcu-OEtZ-Leu
-Leu Leu-OEty methanol vc (g.
Water, acetic acid, and palladium on carbon were added, and the reaction residue was filtered to remove the Z group by evaporation, and then distilled under reduced pressure to leave the resulting residue in dry chloroform.
Akira, Eibenzene and acetic anhydride added 7:). 16 at room temperature
After stirring for an hour, the reaction/! Gu? Discard the residue obtained by reducing the EE daughter and obtain it as a crystal of the desired compound with cold vinegar and ethyl chloride.

Real J4 Shio 1.1 (SUAM312ρ synthesis) 4
a) 8c-Leu7Leu-Met-oL (SUAM3
Intermediate having -CH20H instead of -CI-10 in 12) Ac-Leu-Leu-Met obtained in Reference Example (1)
-OMe (866 mg, 2 mmol) and sodium borohydride (190/Q, 5 ml J mol) were added to tJ! The mixture was made cloudy, heated and stirred under a nitrogen stream, and then refluxed with anhydrous methanol (2.4 ml).
I dripped it. After completion of the dropwise addition, the mixture was stirred under reflux for 1 hour, then returned to room temperature, and water (10+nl') Y was added thereto while cooling with ice.

After methanol and tert-butyl alcohol were distilled off under reduced pressure,
It was extracted three times with ethyl acetate, washed with saturated brine, and dried over anhydrous magnesium sulfate. Ethyl acetate? The target compound (520rny, 64ml) was obtained by crystallizing p5 from the crude ethyl crystals obtained by distillation under reduced pressure.

b) SUAM312 Ac -Leu-Leu-Met -oL (404m
? , 1 mmol) and triethylamine (0,56 mA
, 4 mmol) Kaoru anhydrous dimethyl sulfoxide (4,2d
) and stirred with sulfur trioxide-pyridine complex (6
67! n9・4 mmol) of dimethyl sulfoxy
f-do(4m/) solution? added. After stirring at room temperature for 10 minutes, the mixture was poured into ice water (30d) and extracted three times with ethyl acetate.

It was washed with an aqueous solution of 10 citric acid, water, a saturated aqueous sodium bicarbonate solution and a saturated saline solution, 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 (300m91
75%)Y obtained.

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

Example 2 (SUAM3.13 combination M,) a) Ac
-Leu-Leu-nLeu-oLc SUAM313
Ac-Leu-Leu-nLeu70E obtained in Reference Example (2)
t (556η11.6 mmol) and sodium borohydride (123In9.3.25 mmol) were suspended in tert-butyl alcohol (12 m7), heated and stirred under a nitrogen stream, and then refluxed with anhydrous methanol (1.8 d ) was added dropwise. After the dropwise addition was completed, the mixture was refluxed and stirred for 1 hour, then returned to 1 temperature, and water (10 mA) was added while cooling with water.

Methanol and tertiary butyl alcohol? After distilling off under reduced pressure,
It 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% 1, 42 ml). Obtained.

b) SUAM313 Ac-Leu-Leu-nLeu-ot (193m9
+ 0.5 mmol) and triethylamine (0.2 M
, 2.0 mmol) in anhydrous dimethyl sulfoxide (2.0 mmol)
d) and add the sulfur trioxide-pyridine complex (
A solution of 520/n9, 2.0 mmol) in dimethyl sulfoxide (21 nl) was added. Stir for 10 minutes at room temperature, pour into ice water (30 mA), extract 3 times with ethyl acetate,
It was washed with an aqueous LS citric acid solution, water, a saturated aqueous sodium bicarbonate solution, and a saturated saline solution, 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 SUAM315 (901n9,4
7%)Y obtained.

H3 The physical properties of the obtained compound are shown in Table 2 below.

Example 3 (Synthesis of SUAM314) a) Ac-L
eu-Leu-Leu-ol (SUAM 3 j 4
Intermediate with -CH20Hw instead of -CHO of ) Ac-Leu-Leu-Leu- obtained in reference eiIJ+31
OEt (8551W, 1.3 mmol) and sodium borohydride (19QIn 9.5 mmol) were suspended in tert-butyl alcohol (16m), heated and stirred under a nitrogen stream, and then dissolved in anhydrous methanol (2,5 mmol) under reflux. 4IILl)
2 hilts were lowered. After the dropwise addition was completed, the mixture was stirred under reflux for 1 hour, then returned to room temperature, and 10 ml of water was added while cooling with water.

After distilling methanol and tert-butyl alcohol χ under reduced pressure,
The extract 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 (490rn9.64%).

b) SUAM314 Ac-Leu-Leu-Leu -ol (386m?
+ 1 mmol) and triethylamine (Q, 5 (5 m
1.4 mmol) in anhydrous dimethyl sulfoxide (4,2
A solution of sulfur trioxide-pyridine complex (637In 9.4 mmol) in dimethyl sulfoxide (4M) was added under stirring. After stirring for 10 minutes at room temperature.

Pour into ice water (30a/ml), extract 6 times with ethyl acetate,
It was washed with a 10% citric acid aqueous solution, water, saturated sodium bicarbonate solution and saturated brine, and dried over anhydrous magnesium sulfate. The crude crystals obtained by distilling off ethyl acetate under reduced EE were recrystallized from ethyl acetate to obtain SUAM314 (260 Da.
68chi)? Obtained.

SUAM314 The physical properties of the obtained compound are shown in Table 6 below.

=79 Example 4 Physiological activity of the substance of the present invention The enzyme inhibitory activity of the substance of the present invention was measured as follows.

The substance of the present invention and the enzyme solution are mixed, pre-incubated at 60°C for 5 minutes, and then the substrate bath solution is mixed and reacted. Let it start. 0.5mhA casein was used as a substrate, and 5mhA calcium chloride, 10mM cysteine, and 50mHA calcium chloride were used as substrates.
mM) IJ-HCl buffer rpH7,5)'? Add and react at 30°C for 60 minutes.

Next, reaction WLlfC6.5% trichloroacetic acid was added to stop the reaction, and the amount of protein in the trichloroacetic acid soluble fraction of casein that had been hydrolyzed by the enzyme was determined by low concentration.
It is measured by the Lowry-Folin method, and the harmful activity of 1slI is determined from comparison with a control solution.

The inhibitory effects of the substances of the present invention on the thus obtained calpain Ⅰ, calpain a and papain are shown in Table 4 and the figure in comparison with the effects of leupeptin and strepin P1.

The substance of the present invention is 0.05 to 10μ for calpain L and calvine U, respectively. It was found that the inhibitory activity was more than 50%.

Furthermore, by the above method, the inhibitory effect Tf of the substance of the present invention on serine protease (trypsin/plasmin):
When W++ was determined, no inhibition of % hardening was observed.・

[Brief explanation of drawings]

Figure 3 is a graph showing the inhibitory effect of the substance of the present invention and leupeptin on the activity of calpain U. Patent Applicant: Suntory Limited (5 others) Amount of Inhibition S Percentage (p (1/Test Tube)) Procedure Amendment Written August 2, 1985 1, Incident Indication 1982 Patent Application @ 225486 No. 2, Name of the invention Process for producing anti-calpain 3, Relationship with the case of the person making the amendment Patent applicant's address 亦 (190) Suntory Ltd. 4 Agent Person 5: [Claims of the specification to be amended] ] and [Detailed Description of the Invention] column (attachment) (1) The scope of the claims is amended as follows: "(1) The following general formula I: (wherein, R(z = CH2-S -CHa, -CH2-C
A compound represented by H2-CHa. (2) The following general formula ■: (wherein, R represents the meaning given by the following formula I; R/ represents a methyl group or an ethyl group) to a tertiary alcohol. By adding suspended methanol, it is converted into an alcohol represented by the following general formula (1), and then the alcohol is oxidized with dimethyl sulfoxide (A) and a sulfur trioxide-pyridine complex. Formula I: (wherein R+z-CH2-8-CH3, -CH2-CH
A method for producing a compound represented by 2-CH3. (2) Amended from the last line of page 6 of Book A. (3) “(3) Ac-Le” on page 10, line 18 of the same
j (3J Ac-L
eu-Leu-Leu-OEt”. (4) Amend the same as on page 17, line 1. (5) On page 7, lines 3 and 4, amend "suspended in, in an inert gas" to "suspended in, added a reducing agent such as sodium boroboronate, and in an inert gas." .

Claims (2)

[Claims] (1) The following general formula I: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (In the formula, R is -CH_2-S-CH_3, -CH_2-
Represents the group CH_2-CH_3 or ▲, which has mathematical formulas, chemical formulas, tables, etc. ) A compound represented by (2) The following general formula II: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) (In the formula, R represents the meaning given in the following formula I, and R
' represents a methyl group or an ethyl group. ) is suspended in a tertiary alcohol and anhydrous methanol is added under reflux in an inert gas to form the alcohol represented by the following general formula III: and then oxidizing the alcohol with a sulfur trioxide-pyridine complex in dimethyl sulfoxide according to the following general formula I: ▲Mathematical formulas, chemical formulas, tables, etc.▼(I) (wherein R is -CH_2-S-CH_3, -CH_2-
Represents the group CH_2-CH_3 or ▲, which has mathematical formulas, chemical formulas, tables, etc. ) A method for producing a compound represented by