JPH0461878B2 - - Google Patents

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to novel proline derivatives and salts thereof. Prior Art Conventionally, amino acid derivatives described in JP-A-55-81845 are known as compounds having an angiotensin-converting enzyme inhibitory effect. However, the amino acid derivatives described in this publication are extremely wide, and only a few of them are specifically disclosed, and furthermore, according to the research of the present inventors, only a few of them are specifically disclosed in this publication. The compound is
It has been found that the above-mentioned action is insufficient, or even if the compound has an excellent action, it takes a long time from the administration of the compound to the onset of the above-mentioned action, and the immediate effect is poor. The present inventors continued to conduct research on the above amino acid derivatives, and found that a compound represented by the following general formula (1), which is included in the wide range of amino acid derivatives in the publication but is not specifically described at all. It has been discovered that the compound has an excellent angiotensin-converting enzyme inhibitory effect and can express this excellent effect within a short period of time after administration of the compound, an unexpected effect based on the above-mentioned publication. . Structure of the Invention The proline derivative of the present invention is represented by the following general formula (1). [In the formula, R ₁ represents a pentyl group or a hexyl group.
R ₂ and R ₄ are the same or different and represent a hydrogen atom or a lower alkyl group. R ₃ represents a lower alkyl group. ] In the above general formula (1), the lower alkyl group represented by R ₂ , R ₃ and R ₄ includes, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl group, etc. be able to. Further, the compound of the above general formula (1) has three asymmetric carbon atoms in the molecule and has eight optical isomers, and the present invention includes all of these isomers. The proline derivatives and salts thereof of the present invention have excellent angiotensin converting enzyme inhibitory activity and are useful as antihypertensive agents. In particular, the time required for the onset of its action is short and it is fast-acting, and also has a long duration of action and low toxicity. Furthermore, the proline derivatives and salts thereof of the present invention also have an immunity-enhancing effect, a tanning effect, and an intraocular pressure-lowering effect, and are immunostimulants,
It is useful as an expectorant and glaucoma treatment. The salts of the proline derivatives of the present invention include pharmaceutically acceptable acid addition salts. Examples of acidic compounds that form the acid addition salt include hydrochloric acid, sulfuric acid,
Examples include inorganic acids such as nitric acid, phosphoric acid, and hydrobromic acid, and organic acids such as oxalic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, benzoic acid, and picric acid. Furthermore, among the proline derivatives of the present invention, those having an acidic group can be made into salts by reacting them with pharmaceutically acceptable bases, and the present invention includes such salts. Examples of the base include inorganic bases such as sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium monoate, potassium bicarbonate, lysine, arginine, ornithine, morpholine, piperazine, piperidine, ethylamine, dimethylamine, triethylamine, dicyclohexyl. Examples include organic bases such as amines. The proline derivative of the present invention can be produced, for example, by various methods shown below. [In the formula, R ₁ , R ₂ and R ₃ are the same as above. R ₅ is a hydroxyl group, a lower alkoxy group, or

[Formula] represents a group (R ₄ is the same as above). Moreover, X represents a halogen atom, an alkylsulfonyloxy group, or an arylsulfonyloxy group. ] In the above compound (3), the halogen atom represented by as,
Examples include p-toluenesulfonyloxy group and mesitylenesulfonyloxy group. According to the method shown in Reaction Formula-1 above, by bonding cysteine derivative (2) and compound (3),
Compound (1a) is obtained. The condensation reaction is carried out in a suitable solvent in the presence of a deoxidizing agent. Examples of solvents include alcohols such as methanol, ethanol, 2-propanol, and t-butanol, ethers such as diethyl ether, tetrahydrofuran (THF), and dioxane, dimethylformamide (DMF), dimethylsulfoxide (DMSO), and hexamethylphosphorus. Aprotic polar solvents such as acid triamide (HMPA) can be used. Examples of deoxidizing agents include alkali metal carbonates or alkali metal hydrogen carbonates such as sodium carbonate, potassium carbonate, sodium hydrogen carbonate, and potassium hydrogen carbonate;
Organic tertiary amines such as triethylamine, pyridine, 1,8-diazabicyclo[5,4,0]undecane-7-ene (DBU), etc. can be used.
The deoxidizing agent usually has a ratio of about 1 to 1 to the cysteine derivative (2).
It is used in an amount of 2 times the mole, preferably about 1 to 1.2 times the mole. Compound (3) has the following properties for cysteine derivative (2):
Usually at least an equimolar amount, preferably about 1 to 1.2
Used twice the molar amount. The reaction is generally carried out at a temperature of about 0 DEG to 80 DEG C., preferably at or near room temperature, and is completed in about 3 to 72 hours. The cysteine derivative (2) used as a starting material in the above reaction is, for example, J.Org.Chem., 16 ,
749 (1959), Helv.Chim.Acta., 32 , 866 (1949)
It is synthesized with reference to etc. [In the formula, R ₁ , R ₂ and R ₃ are the same as above. R _5a is a lower alkoxy group or

[Formula] represents a group (R _4a represents a lower alkyl group). In addition, R _5b is a hydroxyl group or

[Formula] represents a group. ] According to the method shown in Reaction Formula-2 above, compound (1a') is treated with an acid in the presence of a scavenger such as anisole, thioanisole, dimethyl sulfide, etc., without producing by-products. , Compound (1a″) can be obtained. In the acid treatment reaction, examples of the acid include trifluoroacetic acid (TFA), methanesulfonic acid, trifluoromethanesulfonic acid, hydrochloric acid, hydrobromic acid, hydrofluoric acid, acetic acid, etc. For example, the reaction can be carried out in a solvent such as ethers such as diethyl ether, THF, and dioxane, esters such as methyl acetate and ethyl acetate, and halogenated hydrocarbons such as methylene chloride and chloroform. When using acetic acid, no solvent is required;
This method is preferred. In addition, scavengers such as anisole usually have about 1 to 10
It is used in twice the molar amount, preferably in about 3 to 5 times the molar amount. The reaction is carried out at a temperature of about 0 to 50°C, preferably about 0 to 25°C, for about 1 to 10 hours. [In the formula, R ₁ , R ₂ and R ₃ are the same as above] According to the above reaction-3, cysteine derivative (2) and α
- Compound (5) is obtained by reacting with keto acid (4). This reaction is a reductive bond formation reaction using a metal hydrogen complex, that is, cysteine derivative (2)
This is carried out by reducing the Schiff base produced by the reaction between the compound and the α-keto acid (4) using a metal hydrogen complex compound. As the metal hydrogen complex compound, for example, sodium borohydride, lithium borohydride, cyanosodium borohydride, cyanolithium boron hydroxide, etc. can be used. The isocomplex compound is generally used in an amount of about 2 to 6 times, preferably about 2 to 3 times, the amount of the cysteine derivative (2). Also α−
The keto acid (4) is used in an amount of about 1 to 10 times the molar amount of the cysteine derivative (2), preferably about 3 to 5 times the molar amount.
The above reaction is carried out in an inert solvent that does not adversely affect the reaction. Examples of the solvent include water, alcohols such as ethanol, methanol, and 2-propanol, ethers such as diethyl ether, THF, and dioxane, and aprotic polar solvents such as DMF and DMSO, which may be used alone or as a mixed solvent. It will be done. The reaction is generally completed at a temperature of 0 to 50°C, preferably at or around room temperature, in about 3 to 24 hours. When using cyanosodium boron hydride or cyanolithium boron hydride, the pH is usually 6.5 to 8.5.
The reaction proceeds rapidly at a neutral temperature, preferably around neutrality. [In the formula, R ₁ and R ₃ are the same as above. R _2a and R _4a each represent a lower alkyl group. ] According to the above reaction formula-4, the above reaction formulas-1 to -
The compound (1b) of the present invention is obtained by the reaction of the carboxylic acid (6) obtained in Step 3 with the amine (7).
The reaction is carried out by the following various methods according to the amide bond forming reaction. (a) A method in which carboxylic acid (6) and amine (7) are subjected to a dehydration condensation reaction in the presence of a condensing agent; (b) A mixed acid anhydride method, that is, a method in which carboxylic acid (6) is reacted with a haloformic acid alkyl ester. A method of reacting a mixed acid anhydride with an amine (7); (c) an active ester method, that is, a method of reacting the carboxylic acid (6) with, for example, p-nitrophenyl ester, N-hydroxysuccinimide ester, N- hydroxy-
Active ester such as 5-norbornene-23-dicarboximide ester, and amine (7)
(d) Carboxylic acid halide method, that is, reacting the halide of carboxylic acid (6) with amine (7); (e) Others, for example, reacting carboxylic acid (6) with a dehydrating agent such as acetic anhydride. A method of reacting an acid anhydride with an amine (7); A method of reacting an amine (7) with an ester of a carboxylic acid (6) and a lower alcohol under high pressure and high temperature. Each of the above methods is carried out under substantially the same conditions as the known methods. A particularly preferred method is the method (a) above. Specifically, the condensing agent includes N,N'-dicyclohexycarbodiimide (DCC), DCC-N-hydroxysuccinimide, DCC-N-hydroxybenzotriazole, DCC-N-hydroxy- 5-norbornene-2,3-dicarboximide, diphenylphosphoryl azide (DPPA)-triethylamine, diethylphosphorocyanidate (DEPC)-triethylamine, etc. can be used. The reaction is generally carried out in a suitable solvent,
As the solvent, various known solvents that do not adversely affect the reaction may be used. Specific examples include halogenated hydrocarbons such as methylene chloride, chloroform, and dichloroethane, aromatic hydrocarbons such as benzene, toluene, and xylene, ethers such as diethyl ether, THF, and dioxane, methyl acetate, ethyl acetate, etc. esters, DMF,
Examples include aprotic polar solvents such as DMSO and HMPA. The amount of amine (7) to be used is usually at least equimolar to the carboxylic acid (6), preferably about 1 to 1.2 times the molar amount, and the condensing agent is about 1 to 1.2 times the molar amount to the carboxylic acid (6). It can be about 1 to 2 times the molar amount, preferably about 1 to 1.2 times the molar amount.
The reaction is usually carried out at around -20 to 30°C, preferably around -10°C.
~Complete in about 3 to 24 hours at room temperature. [In the formula, R ₁ , R _2a , R ₃ and R _4a are the same as above. ] According to the above reaction formula-5, R ₂ and
The compound (1d) of the present invention, in which R ₄ is a hydrogen atom, has R ₂
and R ₄ are both lower alkyl groups, or the compound (1c) of the invention, in which R ₂ is a lower alkyl group and R ₄ is a hydrogen atom, is produced by hydrolyzing. The above hydrolysis reaction is carried out in a suitable solvent in the presence of a basic compound. As the solvent, for example, lower alcohols such as methanol and ethanol, ethers such as diethyl ether, THF and dioxane, and mixed solvents with water such as acetonitrile can be used. As the basic compound, for example, alkyl metal hydroxides such as sodium hydroxide, potassium hydroxide, lithium hydroxide, etc. can be used. The isobasic compound is the compound of the present invention (1d)
or (1c), usually about 2 to 3 times the molar amount, preferably 2 to 2.2 times the molar amount. The reaction usually proceeds suitably at about 0 to 40°C, preferably at room temperature,
It takes about 0.5 to 12 hours to complete. The target compounds obtained by the reactions shown in each of the above reaction formulas can be easily isolated and purified by conventional separation means. Examples of such means include solvent extraction, dilution, distillation, recrystallization, column chromatography, preparative thin layer chromatography, ion exchange chromatography, and gel chromatography. The compounds of the present invention can be administered to humans neat or together with conventional pharmaceutical carriers. The dosage unit form is not particularly limited and can be appropriately selected and used as required. Examples of such dosage unit forms include tablets, powders, granules, oral preparations such as oral solutions, and parenteral preparations such as injections. The amount of the active ingredient to be administered is not particularly limited and can be appropriately selected from a wide range, but in order to achieve the desired effect, it is preferably 0.06 to 10 mg per kg of body weight per day. It also contains 1 to 500 mg of active ingredient in dosage unit form.
It is better to include it. In the present invention, oral preparations such as tablets, capsules, and oral solutions are manufactured according to conventional methods. That is, tablets are prepared by mixing the compound of the present invention with pharmaceutical excipients such as gelatin, starch, lactose, magnesium cytearate, talc, gum arabic, and the like. Capsules are prepared by mixing the compound of the present invention with an inert pharmaceutical filler or diluent, and filling the mixture into hard gelatin capsules, soft capsules, and the like. Syrups or elixirs contain the compounds of the present invention, sweeteners such as sucrose, and methyl-
It is manufactured by mixing with preservatives such as propylparabens, coloring agents, seasonings, etc. Moreover, parenteral preparations are manufactured according to conventional methods. That is, a drug for parenteral administration is prepared by dissolving the compound of the present invention in a sterilized liquid carrier. Preferred carriers are water or saline. Solutions having the desired clarity, stability and suitability for parenteral use are prepared by dissolving about 1 to 500 mg of the active ingredient in water and organic solvents and in polyethylene glycol having a molecular weight of 200 to 5000. . Preferably, such a liquid agent contains a lubricant such as sodium carboxymethyl cellulose, methyl cellulose, polyvinylpyrrolidone, or polyvinyl alcohol. Furthermore, the above liquid preparation contains bactericides and fungicides such as benzyl alcohol, phenol, and thimerosal, as well as isotonic agents such as sucrose and sodium chloride, local anesthetics, stabilizers, buffers, etc. Good too. To further increase stability, parenterally administered drugs may be frozen after filling and water removed by freeze drying techniques known in the art. The lyophilized powder can then be reconstituted immediately before use. Examples Hereinafter, production agents for raw material compounds for producing the compounds of the present invention will be listed as reference examples, and production examples of the compounds of the present invention will be listed as examples. Next, test examples will be given. In each example, S-alkyl-L-cysteine ethyl ester and 2-bromopropionic acid-
Of the two isomers of the reaction product with t-butyl ester, in silica gel chromatography (ether-n-hexane system), the leading fraction is referred to as the α-isomer and the trailing fraction is referred to as the β-isomer. Hereinafter, a compound derived using this α-isomer will be referred to as an α-isomer, and a compound derived using this β-isomer will be referred to as a β-isomer. Reference Example 1 Production of N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanine-t-butyl ester/α and β-isomers S-hexyl-L-cysteine ethyl ester
1.5 ml of triethylamine was added to a 6 ml solution of HMPA containing 2.5 g and 2.3 g of t-butyl 2-bromopropionic acid, and the mixture was stirred at room temperature for 24 hours. The reaction solution was poured into ice water and extracted with ethyl acetate. The extract was thoroughly washed with water and then dried over anhydrous sodium sulfate.
The extract was distilled off under reduced pressure, and the residue was separated and purified by silica gel column chromatography (solvent: ether: n-hexane = 1.2), and the α-isomer of the target compound was obtained as a colorless oil from the preceding fraction. yield
1.2g. [α] ²¹ _D = +27.3° (c = 1.2, ethanol) NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 5Hz), 1.27 (3H, d, J = 7Hz), 1.29 (3H , t, J=7Hz), 1.4-1.8 (8H, m), 1.45 (9H, s), 2.55 (2H, t, J=7Hz), 2.80 (2H, d, J=6Hz), 3.32 (1H, q, J = 7 Hz), 3.46 (1H, t, J = 7 Hz), 4.20 (2H, q, J = 7 Hz). The β-isomer of the target compound was obtained as a colorless oil from the afterflow. Yield: 1.1g. [α] ²¹ _D = -40.4° (c = 0.8, ethanol) NMR (CDCl ₃ ): δ value 0.89 (3H, t, J = 5Hz), 1.29 (3H, t, J = 7Hz), 1.29 (3H, d, J=7Hz), 1.4-1.8 (8H, m), 1.47 (9H, s), 2.54 (2H, t, J=7Hz), 2.70 (1H, dd, J=13Hz, 7.5Hz), 2.92 (1H, dd, J=13Hz, 5Hz), 3.29 (1H, q, J=7Hz), 3.47 (1H, dd, J=5Hz, 7.5Hz), 4.21 (2H, q, J= 7Hz), Reference Example 2 Production of N-[(R)-1-ethoxycarbonyl-2-pentylthioethyl]-alanine-t-butyl ester/α and β-isomers S-pentyl-L-cysteine ethyl ester
The desired compound as a colorless oil was obtained by treating in the same manner as in Reference Example 1 using 8.6 g of 2-bromopropionic acid-t-butyl ester, 8.2 g of triethylamine, and 5.5 ml of triethylamine. Preliminary fraction (α-isomer) Yield: 3.9 g. [α] ²⁵ _D = -28.6° (c = 1.0, ethanol) NMR (CDCl ₃ ): δ value 0.90 (3H, t, J = 5.5Hz), 1.28 (3H, d, J = 7Hz), 1.29 (3H , t, J=7Hz), 1.45 (9H, s), 1.3-1.8 (6H, m), 2.56 (2H, t, J=7Hz), 2.81 (2H, d, J=6Hz), 3.32 (1H, q, J = 7 Hz), 3.46 (1H, t, J = 6 Hz), 4.20 (2H, q, J = 7 Hz), After-flow fraction (β-isomer) Yield: 3.6 g. [α] ²⁵ _D = -42.2° (c = 1.0, ethanol) NMR (CDCl ₃ ): δ value 0.90 (3H, t, J = 5.5Hz), 1.30 (3H, d, J = 7Hz), 1.30 (3H , t, J=7Hz), 1.47 (9H, s), 1.3-1.8 (6H, m), 2.23 (1H, br, s), 2.54 (2H, t, J=7Hz), 2.70 (1H, d- d, J=13Hz, 8Hz), 2.92 (1H, dd, J=13Hz, 5.5Hz), 3.29 (1H, q, J=7Hz), 3.45 (1H, dd, J=5.5Hz, 8Hz) ), 4.21 (2H, q, J = 7Hz), Reference Example 3 Production of N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanine α-isomer Obtained in Reference Example 1 800 mg of α-isomer of N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanine-t-butyl ester
It was dissolved in 5 ml of TFA and stirred at room temperature for 3 hours. TFA
was distilled off under reduced pressure, and the residue was poured into ice water, adjusted to pH 4 with a saturated aqueous sodium bicarbonate solution, and extracted with methylene chloride. The extract was washed with water and dried over anhydrous sodium sulfate. The extract was distilled off under reduced pressure, and the residue was recrystallized from methylene chloride to obtain the α-isomer of the target compound. Yield 580mg. mp.1347−136℃. [α] ²¹ _D = -20.9° (c = 0.6, DMF) Reference Examples 4 to 6 Each compound listed in Table 1 below was obtained in the same manner as in Reference Example 3 above.

[Table] Reference Example 7 Production of N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-(R,S)-alanine S-hexyl-L-cysteine ethyl ester
2.6 g of pyruvic acid was added to a mixture of 1.4 g of ethanol (25 ml) and water (10 ml) under ice cooling, and the pH was adjusted to 7 with a 4N aqueous sodium hydroxide solution. Gradually add 750 mg of cyanosodium borohydride, and then add 750 mg of cyanosodium borohydride to
Stir for hours. The solvent was distilled off under reduced pressure, and the residue was dissolved in a saturated aqueous sodium bicarbonate solution to make it slightly alkaline. After washing with ether, the aqueous layer was diluted to pH4 with 1N hydrochloric acid.
The mixture was extracted with ethyl acetate. Wash the extract thoroughly with water,
After drying over anhydrous sodium sulfate, the residue was distilled off under reduced pressure to obtain the target compound. Yield 1.5g. Example 1 Production of N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanyl-(S)-proline-t-butyl ester/α-isomer N- obtained in Reference Example 3 A solution of 1.0 g of [(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanine α isomer and 620 mg of (S)-proline-t-butyl ester in 10 ml of DMF was stirred under ice cooling.
A solution of 650 mg DEPC (90% content) in 2 ml DMF was added. Furthermore, a solution of 0.5 ml of triethylamine in 2 ml of DMF was slowly added dropwise. After stirring for 2 hours under ice-cooling, the mixture was further stirred at room temperature for 10 hours. The reaction solution was poured into ice water, made weakly alkaline by adding a saturated aqueous sodium bicarbonate solution, and then extracted with ethyl acetate. The extract was thoroughly washed with water and dried over anhydrous sodium sulfate. The extract was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (solvent: chloroform:methanol = 30:1) to obtain α-
The isomer was obtained as a colorless oil. Yield 1.5g. [α] ²¹ _D = −28.2° (c = 0.8, ethanol) NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 5Hz), 1.2 to 1.5 (6H, CH ₃ × 2), 1.45, 1.46 (9H, each s), 1.2-1.8 (8H, m), 1.8-2.5 (4H, m), 2.5-3.0 (4H, m), 3.4-3.9 (4H, m), 4.1-4.5 (3H, m) ) Example 2 N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanyl-(S)-proline-
Production of t-butyl ester/β-isomer Example 1 was prepared from 1.0 g of N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanine/β-isomer obtained in Reference Example 4. In the same manner as above, the β-isomer of the target compound was obtained as a colorless oil. Yield: 1.4g. [α] ²¹ _D = -97.2° (c = 0.9, ethanol) NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 5Hz), 1.29 (3H, t, J = 7Hz), 1.29 (3H, d, J = 6.5Hz), 1.44, 1.46 (9H, each s), 1.2 to 1.8 (8H, m), 1.8 to 2.2 (4H, m), 2.54 (2H, t, J = 7Hz), 2.70 (1H , dd, J=13Hz, 7.5Hz), 2.92 (1H, dd, J=13Hz, 5Hz), 3.2~3.7 (4H, m), 4.20 (2H, q, J=7Hz) 4.3~4.5 (1H, m), Example 3 N-[(R)-1-ethoxycarbonyl-2-pentylthioethyl]-alanyl-(S)-proline-
Production of t-butyl ester/β-isomer From 1.5 g of N-[(R)-1-ethoxycarbonyl-2-pentylthioethyl]-alanine/β-isomer obtained in Reference Example 6, Example 1 and Similarly, the β-isomer of the target compound was obtained as a colorless oil. Yield 2.2g. [α] ²¹ _D = -94.3° (c = 1.0, ethanol) NMR (CDCl ₃ ): δ value 0.89 (3H, t, J = 5.5Hz), 1.29 (3H, d, J = 7Hz), 1.29 (3H , d, J = 7Hz), 1.44, 1.47 (9H, each s), 1.3 to 1.8 (6H, m), 1.8 to 2.3 (4H, m), 2.52 (2H, t, J = 7Hz), 2.68 (1H , dd, J=13Hz, 7Hz), 2.90 (1H, dd, J=13Hz, 6Hz), 3.31 (1H, q, J=7Hz), 3.58 (1H, dd, J=7Hz, 6Hz), 3.4-3.7 (2H, m), 4.20 (2H, q, J = 7Hz) 4.3-4.5 (1H, m), Example 4 N-[(R)-1-ethoxycarbonyl-2-hexylthio Ethyl]-alanyl-(S)-proline
Production of α-isomer and its L-arginine salt N-(R)-1-ethoxycarbonyl-2-exylthioethyl]-alanyl- obtained in Example 1
(S)-Proline-t-butyl ester α-isomer
Dissolve 670 mg and 0.5 ml of anisole in 4 ml of TFA,
The mixture was stirred at room temperature for 2.5 hours. TFA was distilled off under reduced pressure,
Dissolve the residue in saturated aqueous sodium bicarbonate solution and adjust the pH
It was set as 8. After washing with ether, the aqueous layer was adjusted to pH 4 with 1N hydrochloric acid and extracted with methylene chloride. The extract was washed with water, dried over anhydrous sodium sulfate, and then evaporated under reduced pressure to obtain the α-isomer of the target compound as a colorless oil.
Yield 540mg. Add 500 mg of the α-isomer obtained above to 10 ml of ethanol.
A solution of 217 mg of L-arginine in 4 ml of water was added. The solvent was distilled off under reduced pressure, ethanol was added to the residue, and the mixture was distilled off under pressure several times. Anhydrous ether was added to the residue and the precipitated crystals were collected to obtain the L-arginine salt of the target compound. Yield 580mg. mp.63~65℃. [α] ²⁰ _D = -32.7° (c = 0.8, ethanol) Example 5 N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanyl-(S)-proline.
Production of β-isomer and its L-arginun salt N-[1(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanyl-(S)-proline-t-butyl obtained in Example 2 The β-isomer of the target compound was obtained as a colorless oily substance from 690 mg of the ester β-isomer in the same manner as in Example 4. Yield 520mg. L-araginine salt of the target compound. mp.73-76℃. [α] ²⁰ _D = -61.7° (c = 0.8, ethanol) Example 6 N-[(R)-1-ethoxycarbonyl-2-pentylthioethyl]-alanyl-(S)-proline.
Production of β-isomer and its L-arginine salt N-4[(R)-1-ethoxycarbonyl-2-pentylthioethyl]-alanyl-(S)-proline-t-butyl obtained in Example 3 The β-isomer of the target compound was obtained as a colorless oil from 660 mg of the ester β-isomer in the same manner as in Example 4. Yield 590mg. L-arginine salt of the target compound. mp.72~80℃. [α] ²¹ _D = -38.2° (c = 0.8, ethanol) Example 7 N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-(R,S)-alanyl-(S)-
Production of proline-t-butyl ester N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-(R,S) obtained in Reference Example 7
- The target compound was obtained as a colorless oily substance from 1.0 g of alanine in the same manner as in Example 1. Yield 1.4
g. [α] ²¹ _D = −61.0° (c = 1.0, ethanol) NMR (CDCl ³ ): δ value 0.88 (3H, t, J = 5Hz), 1.2 to 1.4 (6H, CH ₃ × 2), 1.44, 1.49 (9H, each s), 1.2-1.8 (8H, m), 1.8-2.3 (4H, m), 2.3-2.9 (4H, m), 3.3-3.8 (4H, m), 4.0-4.5 (3H, m) ) Example 8 N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-(R,S)-alanyl-(S)-
Production of proline and its L-arginine salt N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-(R,S) obtained in Example 7
-alanyl-(S)-proline-t-butyl ester
The target compound was obtained as a colorless oily substance from 600 mg of filtrate, 0.41 ml of anisole, and 4 ml of TFA in the same manner as in Example 4. Yield 480mg. L-arginine salt of the target compound. mp.55~65℃. [α] ²¹ _D = -39.5° (c = 0.7, ethanol) Example 9 N-[(R)-1-carboxy-2-hexylthioethyl]-alanyl-(S)-proline α-isomer Production 600 mg of N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanyl-(S)-proline α-isomer obtained in Example 4 was dissolved in 8 ml of ethanol under ice-cooling and stirring. 3.2 ml of 1N aqueous sodium hydroxide solution was added. Example of stirring at room temperature for 2.5 hours,
It was applied to a Dowex 50W-×8 (H ⁺ ) column. After sufficient water washing, the product was eluted with a 4% aqueous pyridine solution, and the target product fractions were collected and freeze-dried. The obtained powdery substance was reprecipitated from ethanol-ether to obtain the target compound. Yield 270mg. mp.109-112℃. [α] ²¹ _D = -45.6° (c = 0.6, ethanol) Examples 10 to 12 Each compound listed in Table 2 below was obtained in the same manner as in Example 9 above.

[Table] Test Example Inhibitory activity against angiotensin converting enzyme (ACE) 100μ of the enzyme solution purified from rabbit lung acetone powder (manufactured by Sigma) and 100μ of the sample solution were mixed and gently shaken at 37°C for 10 minutes. To this was added 100μ of 6.99mM hipryl-histidyl-leucine (manufactured by Protein Promotion Association) as a substrate, and after reacting at 37°C for 45 minutes with shaking, a 1N sulfuric acid solution was added.
The reaction was stopped by adding 200μ. In order to extract the hippuric acid produced by the reaction, a saturated amount of sodium chloride and 2 ml of diethyl ether were added to the reaction solution, and after shaking vigorously for 15 minutes, centrifugation was performed (2000 rpm, 5 minutes) to obtain a 1.5 ml ether layer. was fractionated. After distilling off the solvent, the ether layer has a concentration of 1.5
It was dissolved in 1 ml of distilled water and the absorbance at 228 nm was measured. As a control, distilled water was used instead of the sample solution.
After adding 100μ, the same operation as above was performed. Inhibitory activity is determined by subtracting the absorbance when the sample solution is added from the absorbance of the control, and then dividing this value by the absorbance of the control to calculate the inhibition rate as a percentage. It was expressed as the concentration of the sample in the reaction solution (IC ₅₀ ). The feed compounds are as follows, and the results of the above tests are shown in Table 3 below. Test compound No. Compound name 1 N-[(R)-1-carboxy-2-
Hexylthioethyl]-alanyl-(S)-proline β-isomer (compound of Example 10) 2 N-[(R)-1-carboxy-2-
hexylthioethyl]-(R,S)
-alanyl-(S)-proline (compound of Example 11) 3 N-[(R)-1-carboxy-2-
Pentylthioethyl]-alanyl-(S)-proline β-isomer (compound of Example 12) 4 N-[(S)-1-carboxy-3-
Methylthiopropyl]-(R,S)
-alanyl-(S)-proline (Example 17 in JP-A-55-81845)
) 5 N-[(S)-1-methoxycarbonyl-3-methylthiopropyl]-
(R,S)-alanyl-(S)-proline hydrochloride (corresponds to the hydrochloride of the compound of Example 16 in JP-A-55-81845)

[Table] From Table 3, the compound group of the present invention shows that even when compared with the known compound with the highest activity (test compound No. 4),
It can be seen that the inhibitory activity is about 24 times (Test Compound No. 1), about 9.4 times (Test Compound No. 2) and about 122 times (Test Compound No. 3) stronger, respectively.

Claims (1)

[Claims] 1. General formula [In the formula, ₁ represents a pentyl group or a hexyl group. _R2
and R ₃ are the same or different and represent a hydrogen atom or a lower alkyl group. R ₃ represents a lower alkyl group. ] A proline derivative represented by and its salt.