JPS6143352B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to epoxysuccinylamino acid compounds, and more particularly to epoxysuccinylamino acid compounds whose thiol group inhibits the activity of proteases involved in the expression of the activity. As a result of various studies, the present inventors discovered that the thiol group of trans-epoxysuccinyl amino acid compounds strongly inhibits the activity of proteolytic enzymes involved in the expression of the activity, and has extremely low toxicity. completed. The present invention will be explained in detail below. The target compound of the present invention has the general formula [In the formula, R' is a hydrogen atom, an alkali metal atom,
Lower alkyl group, lower alkenyl group, lower alkynyl group or formula

[Formula] (in the formula, B represents an alkylene group having 0 to 3 carbon atoms, and X represents a halogen atom or a methyl group), and R ² is an amine group or the formula -O-R ³ (in the formula, , R ³ represents a hydrogen atom, an alkali metal atom, a lower alkyl group, or a benzyl group), and A is substituted with an amino group, a guanidyl group, an amino group with a protecting group, a guanidyl group with a protecting group, or an imidazole group. represents an alkylene group having 2 to 5 carbon atoms. ] is an epoxysuccinyl amino acid compound represented by Here, the lower alkyl group refers to a linear or branched alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, etc., and the lower alkenyl group refers to vinyl, allyl, etc. , butenyl, etc. with 2 or more carbon atoms
The lower alkynyl group is an alkynyl group having 2 to 4 carbon atoms, such as ethynyl, propargyl, butynyl. A halogen atom is fluorine, chlorine, bromine or iodine. The term "protecting group" refers to a protecting group commonly used in the field of amino acid chemistry, such as carbobenzoxy group, methylbenzyloxycarbonyl group, butoxycarbonyl group, amyloxycarbonyl group, tosyl group, benzyl group, acetyl group. , formi group or nitro group. Also, a compound represented by the general formula () [hereinafter abbreviated as compound ()]. ] are all trans isomers. Compound () can be produced by the following method. That is, the general formula (In the formula, R′ is the same as above, and R′ is a hydrogen atom or an alkali metal atom.) The starting material is an epoxysuccinic acid monoester represented by R ⁴ is a hydrogen atom or an alkali metal atom. In this case, the acid chloride is converted into an acid chloride using a halogenating agent such as oxalyl chloride or thionyl chloride, and is represented by the general formula H ₂ NA—COR ² () (wherein A and R ² are the same as above). React with amino acid ester to form compound (). A compound represented by the general formula () [hereinafter abbreviated as compound ()]. ] is an acid addition salt,
Alkali hydroxide, triethylamine, pyridine,
Remove the acid with a base such as methylmorpholine. A compound represented by the general formula () [hereinafter abbreviated as compound ()]. ] The reaction between the acid chloride of the compound () and the compound () is carried out by dropping the acid chloride of the compound () under ice-cooling into a solution of the compound () in which a base such as triethylamine, pyridine, or methylmorpholine coexists. . Alternatively, when R ⁴ of compound () is a hydrogen atom, compound () can be obtained by reacting compound () with compound () in the presence of a condensing agent such as N,N'-dicyclohexylcarbodiimide. can. At this time, N-hydroxysuccinimide, 1-
It is desirable to coexist an N-hydroxy compound such as hydroxybenzotriazole. A new ester of compound () can also be obtained by dissolving the ester of compound () in a suitable alcohol and adding a catalytic amount of sulfuric acid, an alkali metal, an alkali metal alcoholate, or an alkali hydroxide for transesterification. . A compound () in which R ² , R ² and R ³ do not represent a hydrogen atom, an alkali metal atom or an amino group is hydrolyzed with an alkali hydroxide such as sodium hydroxide or potassium hydroxide, and optionally ethanol, acetone, Alternatively, a compound () in which both or one of R ¹ and R ³ are an alkali metal atom can also be obtained by adding an organic solvent such as ether and causing precipitation. It is also possible to obtain a compound () in which both or one of R ¹ and R ³ are hydrogen atoms by making this further acidic with a mineral acid, etc., and then extracting with a suitable organic solvent such as ethyl acetate, ether, or benzene. . When the compound () has an amino group protected with a carbobenzoxy group or a nitro group, or a carboxyl group protected with a benzyl group, the protecting group can be removed by catalytic reduction using palladium-carbon or palladium black. can. Compound (2), which is the object compound of the present invention, strongly inhibits the activity of proteolytic enzymes such as papain, huicin, bromelain, bromelain, and cathepsin B, in which thiol groups are thought to be involved in the expression of their activities. There is no increased permeability, and its toxicity is extremely low. In order to demonstrate that compound () strongly inhibits the activity of these enzymes, a test example using papain is shown below. Test example: 0.25 ml of a cysteine solution (PH6.8) adjusted to a 40 mmol concentration with a 20 mmol disodium ethylenediaminetetraacetic acid solution and an aqueous solution of compound () or a 10% dimethyl sulfoxide aqueous solution were mixed with a papain aqueous solution (80 μg/ ml, twice-crystallized sample, manufactured by Sigma) and heated to 40℃ for 15 minutes. .8)] was added, and the reaction was further heated to 40°C for 10 minutes, followed by the addition of 5 ml of a 440 mmol trichloroacetic acid solution to stop the reaction. After that, the absorbance A at 280 nm of the liquid was measured, and at the same time, the absorbance B was measured using only water or 10% dimethyl sulfoxide aqueous solution instead of the compound () as a control, and the inhibitory activity rate B - A / B × 100 was determined. Calculated. The amount of the substance showing 50% inhibition of activity by this method is shown in Table 1 as _ID50 .

【table】

【table】

【table】

[Table] Compound () does not inhibit the casei-degrading activity of serine proteases typified by trypsin and acidic proteases typified by pepsin. That is, compound () specifically inhibits the activity of a proteolytic enzyme whose activity involves a thiol group. Next, the present invention will be explained with reference to Examples. Example 1 Monobenzylepoxysuccinate potassium salt (i.e., benzylpotassium oxirane-2,
3-dicarboxylate) was suspended in 30 ml of ethyl ether, a solution of 1.75 g of oxalyl chloride in ethyl ether was added dropwise with stirring under ice cooling, and the mixture was stirred at room temperature for 3 hours. The resulting precipitate was removed and ethyl ether was distilled off to obtain epoxysuccinic acid monobenzyl ester chloride. This was dissolved in 20 ml of benzene and added dropwise to 100 ml of a benzene solution containing 4.85 g of N-carbobenzoxy-L-ornithine benzyl ester and 1.5 g of triethylamine under ice-cooling and stirring, followed by stirring at room temperature for 2 hours. Ethyl acetate
After adding 100 ml, the mixture was washed with 5% hydrochloric acid, saturated sodium bicarbonate, and saturated water, dried over magnesium sulfate, and concentrated to dryness. The obtained crude crystals were dissolved in chloroform.
Recrystallization from ether gave white needle-like crystals of N-(DL-3-benzyloxycarbonyloxirane-2-carbonyl)-N-carbobenzoxy-L-ornithine benzine ester (Compound 1).
6.0g was obtained. (Yield 80%). mp 124-125℃. The following compounds were obtained in a similar manner to Example 1 using the appropriate starting materials. N〓-(DL-3-ethoxycarbonyloxirane-2-carbonyl)-N〓-carbobenzoxy-L-lysine benzyl ester (compound 2). mp 70-73℃. N〓-(DL-3-allyloxycarbonyloxirane-2-carbonyl)-N〓-carbobenzoxy-L-lysine benzyl ester (compound 3). mp 114-116℃. Example 2 Benzyl hydrogen epoxy succinate (i.e., benzyl hydrogen oxirane-2,
3-dicarboxylate) 1.7g, N ^G -Nitro-
L-arginine benzyl ester ditosyrena
5.1 g, N-hydroxysuccinimide 1.3 g and N-methylmorpholine 1.6 g were dissolved in 30 ml of dimethylformamide and heated for 20 minutes while cooling to -10°C.
N,N′- dissolved in ml of dimethylformamide
3.0 g of dicyclohexylcarbodiimide was added dropwise. The mixture was stirred at −10° C. for 1 hour, then at room temperature for 1 hour, and then left at room temperature overnight. Separate the resulting precipitate, add 200 ml of ethyl acetate to the solution, wash with saturated sodium bicarbonate water and then saturated saline, dry and concentrate the organic layer, and perform silica gel column chromatography (chloroform:
methanol = 25:1), and then crystallized from n-hexane-acetone to obtain N〓(DL-3-benzyloxycarbonyloxirane-2-carbonyl)-N ^G -nitro-L-arginine benzyl ester (compound 4). 2.4 g of white needle-like crystals of ) were obtained. (yield 61%). mp 141-142℃. The following compound was obtained in a similar manner to Example 2 using the appropriate starting materials. N〓-(DL-3-isopropyloxycarbonyloxirane-2-carbonyl)-N〓-carbobenzoxy-L-lysine benzyl ester (compound 5). mp 89-91℃. N〓-(DL-3-ethoxycarbonyloxirane-2-carbonyl)-N〓-carbobenzoxy-L-ornithine benzyl ester (compound 6). mp 107-109℃. N〓-(DL-3-propargyloxycarbonyloxirane-2-carbonyl)-N〓-carbobenzoxy-L-ornithineamide (compound 7). mp 177-179℃. N〓-(DL-3-n-butyloxycarbonyloxirane-2-carbonyl)-N〓-carbobenzoxy-L-ornithine benzyl ester (compound 8). mp 73-74℃. Example 3 p-Methylbenzyl hydrogen epoxy succinate (i.e. p-methylbenzyl hydrogen oxirane-2,3-dicarboxylate)
1.6 g of N,N'-dicyclohexylcarbodiimide was added to a solution of 1.2 g, N-carbobenzoxy-L-lysine benzyl ester tosylate 2.7 g, 1-hydroxybenzotriazole 0.81 g and N-methylmorpholine 0.6 g in tetrahydrofuran in 50 ml.
10 ml of a tetrahydrofuran solution was added dropwise under ice-cooling and stirring. After stirring at the same temperature for 1 hour, the mixture was stirred at room temperature for 1 hour, the resulting precipitate was removed, and the resulting residue was dissolved in 100 ml of ethyl acetate, washed with 5% aqueous hydrochloric acid, saturated aqueous sodium bicarbonate, and then saturated brine. The organic layer was dried, concentrated, purified by silica gel column chromatography (chloroform:acetone = 50:1), and then crystallized from n-hexane-chloroform.
2.1 g of white needle-like crystals of 〓-(DL-3-P-methylbenzyloxycarbonyloxirane-2-carbonyl)-N〓-carbobenzoxy-L-lysine benzyl ester (Compound 9) were obtained. (yield
71.4%). mp 117-118℃. The following compound was obtained in a similar manner to Example 3 using the appropriate starting materials. N〓-(DL-3-benzyloxycarbonyloxirane-2-carbonyl)-N ^G -nitro-
L-arginine methyl ester (compound 10). mp 71-73℃. N〓-(DL-3-phenyloxycarbonyloxirane-2-carbonyl)-N〓-carbobenzoxy-L-lysine benzyl ester (compound
11). mp 100-102℃. N〓-(DL-3-m-fluorobenzyloxycarbonyloxirane-2-carbonyl)-N
-Carbobenzoxy-L-lysine benzyl ester (compound 12). mp 108-110℃. N〓-[DL-3-(3'phenylpropyloxy)carbonyloxirane-2-carbonyl]-
N-carbobenzoxy-L-lysine benzyl ester (compound 13). mp 80-82℃. N〓-(DL-3-0-iodobenzyloxycarbonyloxirane-2-carbonyl)-N〓
-Carbobenzoxy-L-lysine benzyl ester (compound 14). mp 138-139℃. N〓(DL-3-ethoxycarbonyloxirane-2-carbonyl)-L-histidine methyl ester (compound 15). mp 69-73℃. Example 4 56 mg of potassium hydroxide was added to a solution of 512 mg of N-(DL-3-ethoxycarbonyloxirane-2-carbonyl)-N-carbobenzoxy-L-lysine benzyl ester dissolved in 10 ml of ethanol.
A solution prepared by dissolving 10ml of ethanol in 10ml of ethanol was added dropwise to the mixture under ice-cooling and stirring, followed by stirring at room temperature for 2 hours. The resulting precipitate was collected and reprecipitated from water-ethanol.
(DL-3-carboxyoxirane-2-carbonyl)-N〓-carbobenzoxy-L-lysine ethyl ster potassium salt (compound 16) white powder 320
I got mg. (yield 70%). mp 148-149℃. The following compound was obtained in the same manner as in the example using the appropriate starting material and solvent. N〓-(DL-3-carboxyoxirane-2
-carbonyl)-N〓-carbobenzoxy-L-
Lysine benzyl ester potassium salt (compound
17). mp 178-180℃ (decomposition). N〓-(DL-3-carboxylan-2-carbonyl)-N〓-carbobenzoxy-L-lysine methyl ester potassium salt (compound 18). mp 163-165℃ (decomposed). N〓-(DL-3-carboxyoxirane-2
-carbonyl)-N〓-carbobenzoxy-L-
Ornithine benzyl ester potassium salt (compound
19). mp 142-145℃ (decomposed). N〓-(DL-3-carboxyoxirane-2
-carbonyl)-N〓-carbobenzoxy-L-
Ornithine methyl ester potassium salt (compound
20). mp 137-138℃ (decomposed). N〓-(DL-3-carboxyoxirane-2
-carbonyl)-N〓-carbobenzoxy-L-
Ornithine ethyl ester potassium salt (compound
twenty one). mp 159-162℃ (decomposed). N〓-(DL-3-carboxyoxirane-2
-carbonyl)-N〓-carbobenzoxy-L-
Ornithine-n-butyl ester potassium salt (compound 22). mp 145-148℃ (decomposed). N〓-(DL-3-carboxyoxirane-2
-carbonyl)-N ^G -nitro-L-arginine methyl ester potassium salt (compound 23). mp 158-160℃ (decomposition). Example 5 342 mg of N-(DL-3-ethoxycarbonyloxirane-2-carbonyl)-N-carbobenzoxy-L-lysine benzyl ester was dissolved in a mixture of 10 ml of ethanol and 1 ml of water, and 75 mg of potassium hydroxide was dissolved. Add 0.5 ml of the dissolved aqueous solution under ice-cooling, stir at room temperature for 2 hours, concentrate until just before dryness, add ethanol and remove the resulting precipitate.
204 mg of white powder of (DL-3-carboxyoxirane-2-carbonyl)-N-carbobenzoxy-L-lysine dipotassium salt (Compound 24) was obtained.
(yield 65%). mp 218-220℃. The following compound was obtained in the same manner as in Example 5 using the appropriate starting materials. N〓-(DL-3-carboxyoxirane-2
-carbonyl)-N ^G -nitro-L-arginine dipotassium salt (compound 25). mp 184-186℃ (decomposed). Example 6 N〓-(DL-3-benzyloxycarbonyloxirane-2-carbonyl)-N ^G -nitro-
Dissolve 400 mg of L-arginine methyl ester in a mixture of 16 ml of methanol, 4 ml of acetic acid, and 2 ml of water.
% palladium-carbon was added thereto, and the mixture was stirred at room temperature under a hydrogen stream for 4 hours. After removing the catalyst, the solvent was concentrated, water was added, and the mixture was washed with ethyl acetate. After concentrating the aqueous layer to dryness, Sephadex LH20 column chromatography (Pharmacia, 5% aqueous methanol)
to produce a white glassy N〓-(DL-3-
210 mg of carboxylan-2-carbonyl)-L-arginine methyl ester (Compound 26) was obtained.
(yield 76%). mp 109-111℃. The following compound was obtained in the same manner as in Example 6 using the appropriate starting materials. N〓-(DL-3-ethoxycarbonyloxirane-2-carbonyl)-L-lysine (compound
27). mp 196-197℃ (decomposed). N〓-(DL-3-carboxyoxirane-2
-carbonyl)-L-arginyl (compound 28). mp 175-177℃ (decomposed). Example 7 1 mg of N〓-(DL-3-ethoxycarbonyloxirane-2-carbonyl)-N〓-carbobenzoxy-L-ornithine benzyl ester to 10 mg
dry methanol containing sodium methylate 50%
ml and stirred at room temperature for 2 hours. After most of the methanol was distilled off, it was diluted with 100 ml of ethyl acetate, washed with 5% hydrochloric acid, saturated sodium bicarbonate and then saturated saline, dried and concentrated to dryness. Ether was added to the residue to obtain crude crystals. Then, it was recrystallized from chloroform-ether to obtain 0.8 mg of white needle-like crystals of N-(DL-3-methoxycarbonyloxirane-2-carbonyl)-N-carbobenzoxy-L-ornithine methyl ester (Compound 29). Ta. (99%).
mp 111-112℃. Example 8 N〓-(DL-3-benzyloxycarbonyloxirane-2-carbonyl)-N〓-carbobenzoxy-L-ornithine benzyl ester 0.7
mg was dissolved in 30 ml of methanol containing 0.03 ml of concentrated sulfuric acid and left at room temperature for 2 hours. After distilling off most of the methanol, it was diluted with 100 ml of ethyl acetate, washed with saturated sodium bicarbonate solution and then saturated saline, dried, concentrated, and then subjected to silica gel column chromatography (ethyl acetate: n-hexane = 1:2). It was purified and crystallized from ethyl acetate-n-hexane to give N-3-methoxycarbonyloxirane-2-carbonyl)-N.
0.6 g of white needle-like crystals of -carbobenzoxy-L-ornithine benzyl ester (compound 30) was obtained. (99% yield). mp 111-113℃.

Claims (1)

[Claims] 1. General formula [In the formula, R' is a hydrogen atom, an alkali metal atom,
Lower alkyl group, lower alkenyl group, lower alkynyl group or formula [Formula] (wherein, B represents an alkylene group having 0 to 3 carbon atoms, and X represents a halogen atom or a methyl group), and R ² represents an amino group or the formula -O-R ³ (in the formula, R ³ represents a hydrogen atom, an alkali metal atom, a lower alkyl group, or a benzyl group), and A represents an amine group, a guanidyl group, or an amine with a protective group. group, a guanidyl group with a protecting group, or an alkylene group having 2 to 5 carbon atoms substituted with an imidazole group. ] An epoxysuccinyl amino acid compound represented by