JPH0557970B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to an antihypertensive agent containing a novel proline derivative and its salt as an active ingredient. Structure of the Invention The proline derivative used as an active ingredient in the antihypertensive agent of the present invention is represented by the following general formula (1). [Wherein, _R1 is _C5 - _C14 alkyl, _C2 - _C6 alkenyl, _C3 - _C8 cycloalkyl- _C1 - _C6 alkyl or

[Formula] (wherein, R ₅ represents hydrogen or C ₁ -C ₁₄ alkyl, R ₆ represents hydrogen or C ₁ -C ₆ alkyl); R ₂ represents hydrogen or C ₁ -C ₆ alkyl _R3 represents _C1 - _C6 alkyl; _R4 is hydrogen, _C1 - _C6 alkyl or phenyl- _C1 - _C6 alkyl; n represents 0, 1 or 2; ] As used herein, _C5 - _C14 alkyl groups include pentyl, isopentyl, 2-methylbutyl, 1-methylbutyl, neopentyl, hexyl, 2-methylpentyl, 3-methylpentyl,
4-methylpentyl, 2,2-dimethylbutyl,
3,3-dimethylbutyl, heptyl, octyl,
Nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, 6-methylheptyl, 7-
Mention may be made of straight-chain or branched alkyl groups such as methyloctyl, 8-methylnonyl, 9-methyldecyl, 10-methylundecyl, 11-methyldodecyl, 12-methyltridecyl, 13-methyltetradecyl, etc. can. Examples of the C ₂ -C ₆ alkenyl group include vinyl,
Examples include allyl, 2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 1-methylallyl, 2-pentenyl, and 2-hexenyl groups. Examples of the _C3 - _C8 cycloalkyl- _C1 - _C6 alkyl group having 3 to 8 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. . Examples of the C ₃ -C ₈ cycloalkyl-C ₁ -C ₆ alkyl group include cyclopropylmethyl, cyclobutylmethyl,
cyclopentylmethyl, cyclohexylmethyl,
cycloheptylmethyl, cyclooctylmethyl,
1-cyclopentylethyl, 2-cyclopentylethyl, 1-cyclohexylethyl, 2-cyclohexylethyl, 1-cyclohexylpropyl,
Examples include 2-cyclohexylpropyl, 3-cyclohexylpropyl, 4-cyclohexylbutyl, 5-cyclohexylpentyl, 6-cyclohexylhexyl and the like. Examples of C ₁ -C ₆ alkyl groups include methyl,
Examples include ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, and hexyl groups. Examples of the _C1 - _C14 alkyl group represented by _R5 include those exemplified for the _C5 - _C14 alkyl group and _C1 - _C6 alkyl group. Phenyl-C ₁ -C ₆ alkyl groups include benzyl, α-phenethyl, β-phenethyl, 3-phenylpropyl, 4-phenylbutyl, 5-phenylpentyl, 6-phenylhexyl, 1,1-
Dimethyl-2-phenylethyl, 1-methyl-2
-Phenylethyl etc. can be exemplified. Furthermore, the compound of the above general formula (1) has multiple asymmetric centers in the molecule and has multiple optical isomers, but the present invention covers all of the isomers (plural).
includes. The salts of the above proline derivatives include pharmaceutically acceptable acid addition salts. Examples of acidic compounds that form the acid addition salt include hydrochloric acid, sulfuric acid, nitric acid,
Examples include inorganic acids such as 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 above proline derivatives, those having an acidic group can be made into pharmaceutically acceptable salts by the action of a base, and the present invention also includes such salts. Examples of the base include inorganic hydrochloric acid such as sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium bicarbonate, lysine, arginine, ornithine, morpholine, piperazine, piperidine, ethylamine, dimethylamine, triethylamine, dicyclohexylamine. For example, organic bases such as The above proline derivatives and salts thereof have an angiotensin converting enzyme inhibitory effect and are useful as antihypertensive agents. The antihypertensive agent of the present invention containing the above-mentioned proline derivative as an active ingredient is particularly fast-acting and has a long duration of action when administered orally.
It has low toxicity. Furthermore, the above-mentioned proline derivatives and their salts also have an immunity-enhancing effect, an remover effect, and an intraocular pressure lowering effect, and are useful as immunostimulants, remover agents, and therapeutic agents for glaucoma. Amino acid derivatives described in JP-A-55-81845 are known as compounds having angiotensin-converting enzyme inhibitory activity. Among the compounds of general formula (1) used as the active ingredients of the antihypertensive agent of the present invention, R ₁ is a branched or straight-chain alkyl group having 5 to 6 carbon atoms (preferably n-pentyl or n-hexyl group); can be,
The compound in which n is 0, R ₄ is hydrogen or C ₁ -C ₆ alkyl, and R ₂ and R ₃ are the same as above,
Although it is included in the general formula showing the amino acid derivatives of JP-A-55-81845, it is not specifically disclosed in the publication. Moreover, the general formula in the above range
Compounds belonging to (1) have superior angiotensin-converting enzyme inhibitory action compared to the compounds specifically described in JP-A No. 55-81845, and exhibit this action within a shorter period of time after administration. It has a fast-acting effect. Compounds of general formula (1) outside the above range are
This is a new compound that is not described in No. 55-81845 at all. The proline derivatives used in the present invention and intermediates for producing the derivatives can be produced, for example, by various methods shown below. <Reaction formula-1> [In the formula, R ₁ , R ₂ , R ₃ and n are the same as above. R ₇ is a hydroxyl group, a C ₁ -C ₆ alkoxy group, or

[Formula] represents a group (R ₄ is the same as above). Further, X represents a halogen atom, an alkylsulfonyloxy group or an arylsulfonyloxy group. ] In the above propionic acid derivative (3), the halogen atom represented by X includes a chlorine atom, a bromine atom,
An iodine atom, etc., an alkylsulfonyloxy group such as a methanesulfonyloxy group, an ethanesulfonyloxy group, and an arylsulfonyloxy group such as a p-toluenesulfonyloxy group, a benzenesulfonyloxy group, a mesitylenesulfonyloxy group, etc. I can give an example. According to the method shown in Reaction Formula-1 above, compound (1a) is obtained by condensing cysteine derivative (2) and propionic acid derivative (3). 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. As a dispersing agent, for example, sodium carbonate,
Alkali metal carbonates or alkali metal hydrogen carbonates such as potassium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, triethylamine, pyridine,
Organic tertiary amines such as 1,8-diazabicyclo[5,4,0]undecane-7-ene (DBU) can be used. Oxidizing agents are usually cysteine derivatives (2)
About 1 to 2 times the mole, preferably about 1 to 1.2 times
Used twice the molar amount. The propionic acid derivative (3) is usually used in an amount of at least an equimolar amount, preferably about 1 to 1.2 times the molar amount of the cysteine derivative (2).
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 described, for example, in J.Org.Chem., 16 ,
749 (1959), Helv.Chim.Acta., 32 , 866 (1949),
Synthesized with reference to J. Biol. Chem., 140 , 131 (1941), etc. <Reaction formula-2> [In the formula, R ₁ , R ₂ , R ₃ and n are the same as above. R _7a is a lower alkoxy group or

[Formula] Group (R _4a is a C ₁ -C ₆ alkyl group or phenyl-C ₁ -
C ₆ alkyl group). Also, R _7b is a hydroxyl group or

[Formula] represents a group. ] According to the method shown in Reaction Formula-2 above, compound (1a″) can be obtained by treating compound (1a′) with an acid in the presence of a scavenger such as anisole, thioanisole, dimethyl sulfide, etc. In the acid treatment reaction, examples of the acid include acetic acid,
Trifluoroacetic acid (TFA), methanesulfonic acid,
Organic acids such as trifluoromethanesulfonic acid and
Examples include inorganic acids such as hydrochloric acid, hydrobromic acid, and hydrofluoric acid. The reaction is carried out, for example, with diethyl ether,
THF, ethers such as dioxane, methyl acetate,
The reaction can be carried out in a solvent such as an ester such as ethyl acetate, a halogenated hydrocarbon such as methylene chloride, or chloroform. When using trifluoroacetic acid as the acid, no solvent is required and this method is preferred. In addition, a scavenger such as anisole is usually used in an amount of about 1 to 10 times the molar amount of compound (1a'), preferably about 3 times
~5 times the molar amount is used. 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. <Reaction formula-3> [In the formula, R ₁ , R ₂ , R ₃ and n are the same as above. ] According to Reaction Formula-3 above, compound (5) is produced by reacting cysteine derivative (2) and α-keto acid (4).
is obtained. This reaction is a reductive bond-forming reaction using a metal hydrogen complex. That is, it is carried out by reducing the Schiff base produced by the reaction between the cysteine derivative (2) and the α-keto acid (4) using a metal hydrogen complex compound. As metal hydride compounds,
For example, sodium borohydride, lithium borohydride, cyanosodium borohydride, cyanolithium borohydride, 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). In addition, α-keto acid (4) is about 1 to 1 of cysteine derivative (2).
The amount used is 10 times the molar amount, 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, diethyl ether, THF,
Ethers such as dioxane, aprotic polar solvents such as DMF, DMSO, etc. are used alone or as a mixed solvent. The reaction is usually completed at a temperature of 0 to 50°C, preferably at or around room temperature, in about 3 to 24 hours. Furthermore, cyanosodium borohydride,
When using cyanolithium boron hydride, the pH is usually
The reaction proceeds rapidly at about 6.5 to 8.5, preferably around neutrality. <Reaction formula-4> [In the formula, R ₃ and n are the same as above. R _1a is C ₅ −
_C14 alkyl, _C2 - _C6 alkenyl, _C3 - _C8 cycloalkyl- _C1 - _C6 alkyl group or group

[Formula] (R _6a represents a C ₁ -C ₆ alkyl group, R ₅ is the same as above), and R _2a is
_R4a represents _{a C1-C6 alkyl group or} a phenyl- _C1 - _C6 alkyl group. ] According to the above reaction formula-4, the above reaction formulas-1 to -
Compound (1b) is obtained by the reaction of carboxylic acid (6) obtained in Step 3 with amine (7). The reaction is carried out according to a conventional amide bond forming reaction, for example, by the following various methods. (i) A method in which carboxylic acid (6) and ammonium (7) are subjected to a dehydration condensation reaction in the presence of a condensing agent; (ii) 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); (iii) an active ester method, in which carboxylic acid (6) is reacted with, for example, p-nitrophenyl ester, N-hydroxysuccinimide ester, N- Hydroxy-
A method in which an active ester such as 5-norbornene-2,3-dicarboximide ester is reacted with an amine (7); (iv) a carboxylic acid halide method, that is, a halide of a carboxylic acid (6) is reacted with an amine (7); (v) Other methods include, for example, converting carboxylic acid (6) into an acid anhydride using a dehydrating agent such as acetic anhydride, and reacting this with amine (7); A method in which ester is reacted with amine (7) 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 (i) above. Regarding this method, specific condensing agents include N,N'-dicyclohexylcarbodiide (DCC), DCC-N-hydroxysuccinimide, DCC-N-hydroxybenzotriazole, and DCC-N-hydroxy-5- norbornene-2,3-dicarboxide, diphenylphosphoryl azide (DPPA)-triethylamine,
Diethyl phosphorus fluorocyanidate (DEPC) −
Triethylamine etc. can be used. The reaction is generally carried out in a suitable solvent, and various known solvents that do not adversely affect the reaction can be used. Specific examples include halogenated hydrocarbons such as methylene chloride, chloroform, and dichloroethane, aromatic hydrocarbons such as benzene, toluene, and xylene, diethyl ether,
THF, ethers such as dioxane, methyl acetate,
Esters such as ethyl acetate, DMF, DMSO,
Examples include aprotic polar solvents such as HMPA. The amount of amine (7) used is usually at least an equimolar amount relative to carboxylic acid (6), preferably 1 to 1.2
The amount of the condensing agent is preferably about twice the molar amount, and the amount of the condensing agent can be about 1 to 2 times, preferably about 1 to 1.2 times the molar amount of the carboxylic acid (6). The reaction is usually completed at about -20 to 30°C, preferably at about -10°C to room temperature, in about 3 to 24 hours. <Reaction formula-5> [In the above formula, R ₁ , R ₂ , R ₃ , R ₄ and n are the same as above, and R _1b is C ₅ -C ₁₄ alkyl, C ₂ -C ₆
Alkenyl, _C3 - _C8cycloalkyl - _C1 - _C6
alkyl or group

[Formula] (R ₅ is the same as above) is shown. However, in general formula (1b), R ₆ , R ₂ ,
and R ₄ are not hydrogen atoms at the same time. ] According to the above reaction formula-5, R ₆ , R ₂ in general formula (1)
and the compound (1d) in which R ₄ is a hydrogen atom, R ₆ ,
It is produced by hydrolyzing a compound (1b) in which at least one of R ₂ and R ₄ is an ester group. The above hydrolysis reaction is carried out in a suitable solvent in the presence of a basic compound. Examples of solvents that can be used include lower alcohols such as methanol and ethanol, ethers such as diethyl ether, THF, and dioxane, and mixed solvents of acetonitrile and water. As the basic compound, for example, alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, lithium hydroxide, etc. can be used. These isobasic compounds are compound (1b) in which R ₁ in general formula (1b) is _C5 - _C14 alkyl, _C2 - _C6 alkenyl, _C3 - _C8 cycloalkyl- _C1 - _C6 In the case of each alkyl group, it is usually used in an amount of about 2 to 3 times the mole, preferably 2 to 2.2 times the mole. Also, general formula (1b)
but

In the case of [Formula], the amount used is usually about 3 to 4 times the molar amount, preferably 3 to 3.3 times the molar amount.
The reaction normally proceeds suitably at about 0 to 40°C, preferably at room temperature, and is completed in about 0.5 to 12 hours. 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. Compound (1) and its salts thus obtained can be administered to humans and animals as they are or together with a conventional pharmaceutical carrier when used as a pharmaceutical, such as an antihypertensive agent. Usually, the above-mentioned compound or its salt is prepared in a usual dosage unit form depending on the route of administration. Examples of the dosage unit form include oral preparations such as tablets, capsules, granules, and oral solutions, and parenteral preparations such as injections. ,
It is administered intravenously together with conventional fluid replacement such as amino acids, or alone intramuscularly, intradermally, subcutaneously, or intraperitoneally. The above-mentioned dosage unit forms are prepared according to conventional methods, and the carriers used are not particularly different from those commonly used. For example, tablets are prepared by using the above compound or its salt as an active ingredient and mixing it with excipients such as gelatin, starch, lactose, magnesium stearate, talc, and gum arabic. Capsules are prepared by mixing the above-mentioned active ingredient with an activating pharmaceutical filler or diluent, and then filling the mixture into hard gelatin capsules, soft capsules, or the like. Parenteral preparations such as injections are prepared by dissolving or suspending the active ingredient in a sterile liquid carrier, preferably water or physiological saline. The amount of active ingredient blended into each of the above forms is determined appropriately depending on each form, but is generally about 1 to 500 mg. The dosage of each form of the preparation prepared above is determined appropriately depending on the dosage unit form, administration route, and the degree of disease, age, weight, etc. of the patient to whom it is administered, but usually, The amount of the active ingredient is preferably about 0.06 to 50 mg per kg of body weight per day, and this is administered 1 to 3 times a day. Examples Hereinafter, production examples of raw material compounds for producing the active ingredient compound of the antihypertensive agent of the present invention will be given as reference examples, and production examples of the active ingredient compound will be given as examples. Next, test examples will be given. In the following Reference Examples and Examples, 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.
Generally, among the two or four isomers of the reaction product of S-[1-ethoxycarbonylalkyl]-L-cysteine ethyl ester and 2-bromopropionic acid-t-butyl ester, silica gel chromatography ( Of the two fractions separated in the ether-n-hexane system, the leading fraction is referred to as the A-isomer and the trailing fraction is referred to as the B-isomer. Hereinafter, a compound derived using this A-isomer will be referred to as an A-isomer, and a compound derived using this B-isomer will be referred to as a B-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 subjected to silica gel chromatography (solvent: ether: n-hexane = 1:
After separation and purification in step 2), the target compound α
-isomer was obtained as a colorless oil. Yield: 1.2g. [α] ²¹ _D = +27.3° (c = 1.2, ethanol) ¹ H-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 oily substance from the afterflow. Yield: 1.1g. [α] ²¹ _D = -40.4° (c = 0.8, ethanol) ¹ H-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, 7.5 Hz), 2.92 (1H, dd, J = 13Hz, 5Hz), 3.29 (1H, q, J = 7Hz), 3.47 (1H, dd, J = 7.5Hz, 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] ²¹ _D = +28.6° (c = 1.0, ethanol) ¹ H-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 = 7Hz), 3.46 (1H, t, J = 6Hz), 4.20 (2H, q, J = 7Hz) Afterflow fraction (β-isomer) Yield: 3.6g. [α] ²¹ _D = -42.2° (c = 1.0, ethanol) ¹ H-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=8Hz, 5.5 Hz), 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.134−136℃. [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 mg) 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 N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanyl-(S)-proline-
Production of t-butyl ester/α-isomer 1.0 g of N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanine/α-isomer obtained in Reference Example 3 and (S)- A solution of 620 mg of proline-t-butyl ester in 10 ml of DMF was added under ice-cooling and stirring.
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 chromatography (solvent: chloroform:methanol = 30:1) to obtain the α-isomer of the target compound as a colorless oil. Yield 1.5g. [α] ²¹ _D = -28° (c = 0.8, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 5Hz), 1.2 to 1.5 (6H, m), 1.45, 1.46 (Total 9H, each s), 1.21-1.8 (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) ¹ H-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 in total, each s), 1.21 to 1.8 (8H, m), 1.8 to 2.2 (4H, m), 2.54 (1H, 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) ¹ H-NMR (CDCl ₃ ): δ value 0.89 (3H, t, J = 5.5Hz), 1.29 (3H, t, J = 7Hz), 1.29 (3H, d, J = 7Hz), 1.44, 1.47 (9H in total, 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-hexylthioethyl]-alanyl-(S)-proline.
Production of α-isomer and its L-arginine salt N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanyl-(S)-proline-t-butyl ester obtained in Example 1 - 670 mg of α-isomer and 0.5 ml of anisole were dissolved in 4 ml of TFA and stirred at room temperature for 2.5 hours. TFA was distilled off under reduced pressure, and the residue was dissolved in a saturated aqueous sodium bicarbonate solution to give a pH of 8. After washing with ether, the aqueous layer was diluted to 1N.
- Adjust the pH to 4 with hydrochloric acid and extract 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 reduced 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] ²¹ _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-arginine salt N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanyl-(S)-proline-t-butyl ester obtained in Example 2 - The β-isomer of the target compound was obtained as a colorless oily substance from 690 mg of the β-isomer in the same manner as in Example 4. Yield 520
mg. L-arginine 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-[(R)-1-ethoxycarbonyl-2-pentylthioethyl]-alanyl-(S)-proline-t-butyl ester obtained in Example 3 - The β-isomer of the target compound was obtained as a colorless oily substance from 660 mg of the β-isomer in the same manner as in Example 4. Yield 590
mg. 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) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 5Hz), 1.2 to 1.4 (6H, m), 1.44, 1.49 (Total 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-ethoxycarbonyl-2-hexylthioethyl]-alanyl-(S)-proline.α
-Production of isomer Add 600 mg of the N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanyl-(S)-proline α-isomer obtained in Example 4 to 8 ml of ethanol solution. While stirring on ice, 3.2 ml of 1N aqueous sodium hydroxide solution was added. After stirring at room temperature for 2.5 hours, the mixture was applied to a Dowex 50W-×8 (H ⁺ ) column. After thorough washing with water, the product was eluted with a 4% aqueous pyridine solution, and the target image was 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] Reference Example 8 Production of N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-alanine-t-butyl ester α and β-isomers S-heptyl-L-cysteine ethyl ester
3.4 ml of triethylamine was added to a 20 ml solution of HMPA containing 5.9 g and 5.5 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 = 1:3), and the α-isomer of the target compound was obtained as a colorless oil from the preceding fraction. . yield
2.8g. [α] ²⁰ _D = +26.9° (c = 0.9, ethanol) ¹ H-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 (10H, 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 oily substance from the afterflow. Yield 2.6g. [α] ²⁰ _D = -38.6° (c = 1.3, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 7Hz), 1.29 (3H, t, J = 7Hz), 1.30 (3H, d, J=7Hz), 1.41-1.8 (10H, m), 1.47 (9H, s), 2.54 (2H, t, J=7Hz), 2.70 (1H, dd, J=13Hz, 7.5 Hz), 2.92 (1H, dd, J = 13Hz, 5Hz), 3.29 (1H, q, J = 7Hz), 3.47 (1H, dd, J = 7.5Hz, 5Hz), 4.21 (2H, q , J=7Hz) Reference Examples 9 to 11 Each compound listed in Table 3 below was obtained in the same manner as in Reference Example 8 above.

【table】

[Table] Reference Example 12 Production of N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-alanine α-isomer N-[(R)-1-ethoxy obtained in Reference Example 8 carbonyl-2-heptylthioethyl]alanine-
2.7 g of α-isomer of t-butyl ester was added to TFA10.
ml 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 then 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
2.0g. mp.130−132℃. [α] ²⁴ _D = +20.5° (c = 0.8, DMF) Reference Examples 13 to 19 Each compound listed in Table 4 below was obtained in the same manner as in Reference Example 12 above.

【table】

[Table] Reference example 20 Production of N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-(R,S)-alanine 70 ml of ethanol containing 5 g of S-heptyl-L-cysteine ethyl ester hydrochloride 2.8 g of pyruvic acid was added to a mixture of 10 ml of water under ice cooling, and the pH was adjusted to 7 with 4N aqueous sodium hydroxide solution. 2.3 g of cyanosodium borohydride was gradually added, and the mixture was further stirred at room temperature for 15 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 phase was adjusted to pH 4 with 1N hydrochloric acid and extracted with ethyl acetate. The extract was washed with water, dried over anhydrous sodium sulfate, and then evaporated under reduced pressure.
The target compound was obtained. Yield: 3.9g. Example 13 N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-alanyl-(S)-proline-
Production of t-butyl ester/β-isomer 1.0 g of N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-alanine/β-isomer obtained in Reference Example 13 and (S)- Add a solution of 580 mg of proline-t-butyl ester to 14 ml of DMF under ice-cooling and stirring.
A solution of 590 mg DEPC (90% content) in 2 ml DMF was added. Furthermore, a solution of 1.43 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 the target compound as a colorless oil. Yield: 1.4g. [α] ²² _D = -92.0° (c = 0.7, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 5Hz), 1.29 (3H, t, J = 7Hz), 1.29 (3H, d, J = 6.5Hz), 1.2 to 1.8 (10H, m), 1.44, 1.46 (9H in total, each s), 1.8 to 2.3 (4H, m), 2.52 (2H, t, J = 7Hz) , 2.69 (1H, dd, J=13Hz, 7.5Hz), 2.91 (1H, dd, J=13Hz, 6Hz), 3.2~3.7 (4H, ), 4.20 (2H, q, J=7Hz) , 4.3-4.5 (1H, m) Examples 14-16 Each compound listed in Table 2 below was obtained in the same manner as in Example 13 above.

[Table] Example 17 N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-alanyl-(S)-proline.
Production of β-isomer and its L-arginine salt N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-alanyl-(S)-proline-t-butyl ester obtained in Example 13 - 750 mg of β-isomer and 0.52 ml of anisole were dissolved in 5 ml of TFA and stirred at room temperature for 2.5 hours. TFA was distilled off under reduced pressure and the residue was dissolved in a saturated aqueous sodium bicarbonate solution at pH 8.
And so. After washing with ether, the aqueous layer was washed with 1N hydrochloric acid.
The pH was adjusted to 4 and the mixture was extracted with methylene chloride. The extract was washed with water, dried over anhydrous sodium sulfate, and then evaporated under reduced pressure to obtain the β-isomer of the target compound as a colorless oil.
Yield 580mg. After dissolving 550 mg of the compound obtained above in 10 ml of ethanol and adding a solution of 230 mg of L-arginine in 4 ml of water, the solvent was distilled off under reduced pressure. Ethanol was added to the residue and the operation of distilling off under reduced pressure was performed several times. The L-arginine salt of the target compound was obtained by reprecipitation from ether. Yield 650mg. mp.64-68℃. [α] ²⁴ _D = -57.3° (c = 0.8, ethanol) Example 18 N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-alanyl-(S)-proline.
Production of β-isomer/maleate 720 mg of N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-alanyl-(S)-proline/β-isomer obtained in Example 17 was ether 10
A solution of 200 mg of maleic acid dissolved in 15 ml of ethyl acetate was added under ice cooling. The precipitated crystals were collected and recrystallized from ethyl acetate-ether to obtain the target compound.
Yield 670mg. mp.76-79℃. [α] ²⁵ _D = -62.3° (c = 0.7, ethanol) Examples 19-21 Each compound listed in Table 6 below was obtained in the same manner as in Example 17 above.

[Table] Example 22 N-[(R)-1-ethoxycarbonyl-2-2-
heptylthioethyl]-(R,S)-alanyl-(S)
-Production of proline-t-butyl ester N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-(R,S) obtained in Reference Example 20
- The target compound was obtained as a colorless oily substance from 1.0 g of alanine in the same manner as in Example 13. Yield 1.3
g. [α] ²¹ _D = −54.3° (c = 0.8, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 5Hz), 1.2 to 1.4 (6H, m), 1.44, 1.46 (Total 9H, each s), 1.21-1.8 (10H, 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) Examples 23 to 27 Each compound listed in Table 7 below was obtained in the same manner as in Example 22 above.

[Table] Example 28 N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-(R,S)-alanyl-(S)-
Production of proline and its L-arginine salt N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-(R,S) obtained in Example 22
-alanyl-(S)-proline-t-butyl ester
530 mg and 0.4 ml of anisole were dissolved in 4 ml of TFA and stirred at room temperature for 2.5 hours. TFA was distilled off under reduced pressure, and the residue was dissolved in a saturated aqueous sodium hydrogen carbonate solution to give a pH of 8. After washing with ether, the aqueous layer was PHed with 1N hydrochloric acid.
4 and extracted with methylene chloride. Wash the extract with water,
After drying over anhydrous sodium sulfate, the residue was distilled off under reduced pressure to obtain the target compound as a colorless oil. Yield 370mg. 330 mg of the compound obtained in the above reaction was added to 8 ml of ethanol.
A solution of 137 mg of L-arginine dissolved in 3 ml of water was added. The operation of evaporating the solvent under reduced pressure, adding ethanol to the residue and evaporating under reduced pressure was performed 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 370mg. mp55~64℃. [α] ²⁰ _D = -40.3° (c = 0.7, ethanol) Examples 29 to 33 Each compound listed in Table 8 below was obtained in the same manner as in Example 28 above.

[Table] Example 34 Production of N-[(R)-1-carboxy-2-heptylthioethyl]-alanyl-(S)-proline β-isomer N-[(R) obtained in Example 17 )-1-Ethoxycarbonyl-2-heptylthioethyl]-alanyl-(S)-proline β-isomer (373 mg) in 4 ml of ethanol was added with 2.0 ml of 1N aqueous sodium hydroxide solution under ice-cooling and stirring. After stirring at room temperature for 3 hours,
It was applied to a Dowex 50W-×8 (H ⁺ ) column. After thorough washing with water, the product was eluted with a 4% aqueous pyridine solution, and the desired fractions were collected and freeze-dried. The obtained powdery substance was reprecipitated from ethanol-ether to obtain the target compound. Yield 240mg. mp.75~83℃. [α] ²³ _D = -108.1° (c = 0.6, ethanol) Examples 35 to 38 Each compound listed in Table 9 below was obtained in the same manner as in Example 34 above.

【table】

【table】 Examples 39-42 In the same manner as in Example 13, the following compound is obtained.

【table】 Examples 43-44 In the same manner as in Example 17, the following compound is obtained.

[Table] Reference example 21 N-[(R)-1-ethoxycarbonyl-ethoxycarbonylmethylthioethyl]-alanine-t
-Production of butyl ester A and B-isomers 5.3 g of S-ethoxycarbonylmethyl-L-cysteine ethyl ester and 4.7 g of t-butyl 2-bromopropionic acid ester were dissolved in 10 ml of HMPA and 2.3 g of triethylamine was added. At room temperature
After stirring for 62 hours, ice water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The extract was washed with water, then with saturated brine, and dried over anhydrous sodium sulfate. The extract was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography (solvent: ether: n-hexane =
5:3), and the A-isomer of the target compound was obtained as a colorless oil from the preceding output. yield
2.1g. [α] ²⁰ _D = +26.9° (c = 1.1, methanol) ¹ H-NMR (CDCl ₃ ): δ value 1.27 (3H, d, J = 7Hz), 1.29 (6H, t, J = 7Hz), 1.44 (9H, s), 2.15 (1H, br s), 2.94 (2H, d, J=5Hz), 3.30 (3H, q, J=7Hz), 3.33 (2H, s), 3.53 (1H, t, J = 5 Hz), 4.19 (2H, q, J = 7 Hz), 4.20 (2H, q, J = 7 Hz) The B-isomer of the target compound was obtained as a colorless oily substance from the afterflow. Yield 2.24g. [α] ²⁰ _D = -43.1° (c = 1.1, methanol) ¹ H-NMR (CDCl ₃ ): δ value 1.29 (3H, d, J = 7Hz), 1.29 (6H, t, J = 7Hz), 1.46 (9H, s), 2.0 (1H, br s), 2.82 (1H, dd, J=13Hz, 7Hz), 3.50 (1H, dd, J=13Hz, 6Hz), 3.30 (1H, q, J=7Hz), 3.31 (2H, s), 3.57 (1H, dd, J=7Hz, 6Hz), 4.19 (2H, q, J=7Hz), 4.21 (2H, q, J=7Hz), Reference Example 22 Production of N-[(R)-1-ethoxycarbonyl-2-ethoxycarbonylmethylthioethyl]-alanine A-isomer N-[(R)-1-ethoxycarbonyl-2 obtained in Reference Example 21 To a solution of 1.04 g of the A-isomer of -ethoxycarbonylmethylthioethyl]-alanine-t-butyl ester in 5 ml of trifluoroacetic acid (TFA) was added 0.93 g of anisole, and the mixture was stirred at room temperature for 2 hours. TFA was distilled off under reduced pressure, saturated aqueous sodium hydrogen carbonate solution was added to adjust the pH to 8, and the mixture was washed with ether. Citric acid was added to the aqueous layer to adjust the pH to 4, followed by extraction with methylene chloride. The extract was dried over anhydrous sodium sulfate and then evaporated under reduced pressure to obtain the A-isomer of the target compound as an amorphous substance. Yield 0.50g. [α] ²⁰ _D = +17.3° (c = 1.0, methanol) ¹ H-NMR (CDCl ₃ ): δ value 1.29 (6H, t, J = 7Hz), 1.43 (3H, d, J = 7Hz), 2.90 (1H, dd, J = 13Hz, 7Hz), 3.16 (1H, dd, J = 13Hz, 6Hz), 3.30 (2H, s), 3.44 (1H, q, J = 7Hz), 3.65 ( 1H, dd, J=7Hz, 6Hz), 4.20 (2H, q, J=7Hz), 4.23 (2H, q, J=7Hz), Reference example 23 N-[(R)-1-ethoxycarbonyl- Production of 2-ethoxycarbonylmethylthioethyl]-alanine B-isomer B of N-[(R)-1-ethoxycarbonyl2-ethoxycarbonylmethylthioethyl]-alanine-t-butyl ester obtained in Reference Example 21 0.97 g of anisole was added to a solution of 1.09 g of the isomer in 5 ml of TFA, and the reaction was carried out in the same manner as in Reference Example 22 to obtain the B-isomer of the target compound as an amorphous substance. Yield 0.43g. [α] ²⁰ _D = -32.0° (c = 0.8, methanol) ¹ H-NMR (CDCl ₃ ): δ value 1.29, 1.30 (6H, t, J = 7Hz), 1.46 (3H, d, J = 7Hz) , 2.79 (1H, dd, J=13Hz, 9Hz), 3.12 (1H, dd, J=13Hz, 5Hz), 3.27 (2H, s), 3.35 (1H, q, J=7Hz), 3.48 (1H, dd, J=9Hz, 5Hz), 4.20 (2H, q, J=7Hz), 4.21 (2H, q, J=7Hz) Example 45 N-[(R)-1-ethoxycarbonyl- Production of 2-ethoxycarbonylmethylthioethyl]-alanyl-(S)-proline-t-butyl ester/A-isomer N-[(R)-1-ethoxycarbonyl-2-ethoxycarbonyl obtained in Reference Example 22 To a solution of 520 mg of the A-isomer of methylthioethyl alanine, 319 mg of (S)-proline-t-butyl ester, and 304 mg of diethyl cyanophosphate in 5 ml of DMF, was stirred under ice-cooling.
A solution of 188 mg of triethylamine in 3 ml of DMF was slowly added dropwise. After stirring for 12 hours while gradually returning the temperature to room temperature, ice water was added to the reaction mixture, and the mixture was made slightly alkaline with a saturated aqueous sodium bicarbonate solution, followed by extraction with ethyl acetate. The extract was washed with water, then with saturated brine, and dried over anhydrous sodium sulfate.
The extract was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (elution: chloroform:methanol = 10:1) to obtain the A-isomer of the target compound as a colorless oil. Yield 400mg. [α] ²⁰ _D = -46.2° (c = 1.0, methanol) ¹ H-NMR (CDCl ₃ ): δ value 1.29 (3H, t, J = 7Hz), 1.33 (3H, t, J = 7Hz), 1.3 ~1.5 (3H, m), 1.44, 1.46 (total 9H, each s), 1.8 ~ 3.8 (13H, m), 4.08 (4H, q, J = 7Hz) Example 46 N-[(R)-1- Production of ethoxycarbonyl-2-ethoxycarbonylmethylthioethyl]-alanyl-(S)-proline-t-butyl ester/B-isomer N-[(R)-1-ethoxycarbonyl-2 obtained in Reference Example 23 Example from 450 mg of B-isomer of -ethoxycarbonylmethylthioethyl]-alanine
The B-isomer of the target compound was obtained as a colorless oil in the same manner as in 45. Yield 560mg. [α] ²⁰ _D = -93.6° (c = 1.0, methanol) ¹ H-NMR (CDCl ₃ ): δ value 1.29 (3H, t, J = 7Hz), 1.33 (3H, t, J = 7Hz), 1.3 ~1.5 (3H, m), 1.44, 1.46 (total 9H, each s) 1.8 ~ 3.8 (13H, m) 4.08 (4H, q, J = 7Hz) Example 47 N-[(R)-1-ethoxycarbonyl Production of A-isomer of -2-ethoxycarbonylmethylthioethyl]-alanyl-(S)-proline and its maleate salt N-[(R)-1-ethoxycarbonyl-2-ethoxy obtained in Example 45 To a solution of 340 mg of the A-isomer of carbonylmethylthioethyl]-alanyl-(S)-proline-t-butyl ester in 3 ml of TFA, 240 mg of anisole was added, and the reaction was carried out in the same manner as in Reference Example 22 to obtain the target compound A. -isomer was obtained as a colorless oil. Yield 80mg. 2 ml of ether containing 80 mg of A-isomer obtained in the above reaction
A solution of 23 mg of maleic acid in 3 ml of ether is added to the solution under stirring to precipitate. The precipitate was collected and dried to obtain the maleate salt of the target compound.
Yield 30mg. [α] ²⁰ _D = -3.2° (c = 0.4, methanol) ¹ H-NMR (CD ₃ OD): δ value 1.28 (3H, t, J = 7Hz), 1.33 (3H, t, J = 7Hz), 1.57 (3H, t, J = 7Hz), 1.9 ~ 2.3 (4H, m), 3.1 ~ 3.9 (5H, m), 3.46 (2H, s), 4.19 (2H, q, J = 7Hz), 4.32 (1H , q, J=7Hz), 4.3-4.5 (1H, m), 6.29 (2H, s) Example 48 N-[(R)-1-ethoxycarbonyl-2-ethoxycarbonylmethylthioethyl]-alanyl-(S )-Preparation of B-isomer of proline and its maleate salt N-[(R)-1-ethoxycarbonyl-2-ethoxycarbonylmethylthioethyl]-alanyl-(S)-proline- obtained in Example 46 From 520 mg of the B-isomer of t-butyl ester, the B-isomer of the target compound was obtained as a colorless oil in the same manner as in Reference Example 22. Yield 340mg. Example 47 from 340 mg of B-isomer obtained in the above reaction.
The maleate salt of the target compound was obtained in the same manner as above. Yield 320mg. [α] ²¹ _D = -54.6° (c = 0.9, methanol) ¹ H-NMR (CD ₃ OD): δ value 1.28 (3H, t, J = 7Hz), 1.33 (3H, t, J = 7Hz), 1.61 (3H, d, J = 7Hz), 1.9 ~ 2.4 (4H, m), 3.2 ~ 3.8 (5H, m), 3.42 (2H, s), 4.20 (4H, q, J = 7Hz), 4.32 (1H , q, J=7Hz), 4.3-4.5 (1H, m), 6.29 (2H, s) Reference example 24 N-[(R)-1-ethoxycarbonyl-2-[(R,
S)-1-ethoxycarbonylhexylthio]
Production of ethyl]-(R,S)-alanine 11.1 g of pyruvic acid in a mixture of 7.7 g of S-[(R,S)-1-ethoxycarbonylhexyl)-L-cysteine ethyl ester, 25 ml of ethanol, and 10 ml of water.
After neutralization with 4N aqueous sodium hydroxide solution, 3.2 g of cyanosodium borohydride was gradually added while stirring under ice cooling, and the reaction was carried out at room temperature for 14 hours. After the reaction, ethanol was distilled off, and a saturated aqueous sodium hydrogen carbonate solution was added to make it slightly alkaline.
After washing with ether, separate the aqueous layer and adjust the pH to 4 with 10% hydrochloric acid.
After that, the mixture was extracted with ethyl acetate, and the extract was washed with water, then with saturated brine, and dried over anhydrous sodium sulfate. The extract was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (solvent: chloroform:methanol = 10:1) to obtain the target compound as a colorless amorphous substance. Yield 7.0g. [α] ²⁰ _D = -7.4° (c = 1.1, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.89 (3H, t, J = 5Hz), 1.29 (6H, t, J = 7Hz), 1.39 (3H, d, J = 7Hz), 1.3 - 2.1 (8H, m), 2.7 - 3.8 (5H, m), 4.21 (2H, q, J = 7Hz), 4.22 (2H, q, J = 7Hz) Reference Examples 25-28 Each compound listed in Table 12 below was obtained in the same manner as in Reference Example 24 above.

[Table] Reference example 29 N-[(R)-1-ethoxycarbonyl-2-[(R,
Production of S)-1-t-butoxycarbonylhexylthio]ethyl-(R,S)-alanine S-[(R,S)-1-t-butoxycarbonylhexyl)-L-cysteine ethyl ester 5.5 g
Thus, the target compound was obtained as a colorless amorphous substance in the same manner as in Reference Example 24. Yield: 5.37g. [α] ¹⁸ _D = +11.0° (c = 1.2, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.89 (3H, t, J = 5Hz), 1.29 (3H, t, J = 7Hz), 1.48 (9H, s), 1.3-2.0 (11H, m), 2.8-3.8 (5H, m), 4.22 (2H, q, J = 7Hz) Example 49 N-[(R)-1-ethoxycarbonyl- 2-[(R,
S)-1-ethoxycarbonylhexylthio]
Production of ethyl]-(R,S)-alanyl-(S)-proline-t-butyl ester N-[(R)-1-ethoxycarbonyl-2-[(R,S) obtained in Reference Example 24 The target compound was obtained as a colorless oil from 2.0 g of -1-ethoxycarbonylhexylthio]ethyl]-(R,S)-alanine in the same manner as in Example 45. Yield 2.8g. [α] ¹⁹ _D = -46.2° (c = 0.9, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 5Hz), 1.28 (6H, t, J = 7Hz), 1.3 ~1.5 (3H, m), 1.44, 1.46 (9H, each s), 1.5 ~ 2.3 (12H, m), 2.8 ~ 3.0 (2H, m), 3.1 ~ 3.8 (6H, m), 4.19 (4H, q, J=7Hz) Examples 50 to 53 Compounds listed in Table 13 below were obtained in the same manner as in Example 49 above.

[Table] Example 54 N-[(R)-1-ethoxycarbonyl-2-[R,
Production of S)-1-t-butoxycarbonylhexylthio]ethyl]-(R,S)-alanyl-(S)-proline-t-butyl ester N-[(R)-1 obtained in Reference Example 29 -Ethoxycarbonyl-2-[(R,S)-1-t-butoxycarbonylhexylthio]ethyl]-(R,S)-alanine from 1.0 g was reacted in the same manner as in Example 45, and the residue was treated. , silica gel column chromatography (solvent; chloroform: methanol =
20:1) to obtain the target compound as a colorless oil. Yield: 1.03g. [α] ¹⁹ _D = -26.2° (c = 0.9, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 5Hz), 1.28 (3H, t, J = 7Hz), 1.44 , 1.47 (9H, s each), 1.48 (9H, s), 1.3-2.4 (15H, m), 2.8-3.0 (2H, m), 3.1-3.8 (6H, m), 1.19 (2H, q, J=7Hz) Example 55 N-[(R)-1-ethoxycarbonyl-2-[(R,
S)-1-ethoxycarbonylhexylthio]
Production of ethyl]-(R,S)-alanyl-(S)-proline and its L-arginine salt N-[(R)-1-ethoxycarbonyl-2-[(R,S) obtained in Example 49 )-1-Ethoxycarbonylhexylthio]ethyl]-(R,S)-alanyl-(S)-proline-t-butyl ester from 407 mg, the target compound was obtained as a colorless amorphous substance in the same manner as in Reference Example 22. Obtained. Yield 110mg. 300 mg of the compound obtained in the above reaction and 5 ml of ethanol
A 1.5 ml aqueous solution containing 110 mg of L-arginine was added to the solution while stirring. After water and ethanol were distilled off, the residue was dried under reduced pressure to obtain the L-arginine salt of the target compound as a colorless powder. Yield 342mg. Colorless crystals, mp 50-55°C, [α] ²⁰ _D = -33.0° (c = 0.96, ethanol) Examples 56-59 Each compound listed in Table 14 below was obtained in the same manner as in Example 55 above.

[Table] Example 60 N-[(R)-1-ethoxycarbonyl-2-[(S,
R)-1-carboxyhexylthio]ethyl]-
(R,S)-alanyl-(S)-proline and its 2.
Production of L-arginine salt N-[(R)-1-ethoxycarbonyl-2-[(R,S)-1-t-butoxycarbonylhexylthio]ethyl]-(R,S )-alanyl-(S)-proline-t-butyl ester 500
mg, the reaction was carried out in the same manner as in Reference Example 22, and the residue was purified by silica gel column chromatography (solvent: chloroform:methanol = 10:1) to obtain the target compound as a colorless oil. Yield 187mg. ^1H -NMR ( _CDCl3 ): δ value 0.88 (3H, t, J = 5Hz), 1.28 (3H, t, J = 7Hz), 1.3 ~ 2.4 (15H, m), 2.8 ~ 3.8 (8H, m) , 3.48 (3H, s), 4.21 (2H, q, J = 7Hz) From 72 mg of the compound obtained in the above reaction and 56 mg of 2 equivalents of L-arginine, 2.L- of the target compound was prepared in the same manner as in Example 55. Arginine salt was obtained as a colorless powder. Yield 110mg. Colorless crystals, mp92-97℃, [α] ²⁰ _D = -16.5° (c = 0.6, ethanol) Example 61 N-[(R)-1-carboxy-2-[(R,S)-1
-carboxyhexylthio]ethyl]-(R,
Production of S)-alanyl-(S)-proline N-[(R)-1-ethoxycarbonyl-2-[(R,S)-1-ethoxycarbonylhexylthio]ethyl]- obtained in Example 55 (R,S)-alanyl-(S)-proline in 3 ml ethanol solution of 472 mg
Add 3.3ml of 1N sodium hydroxide aqueous solution and stir at room temperature.
Stirred for 2.5 hours. A small amount of water was added to the reaction solution, and the mixture was subjected to Dowex 50W-X8 (H ⁺ ). After thorough washing with water, the product was eluted with a 4% aqueous pyridine solution.
Product fractions were lyophilized. The resulting powdery substance was collected, washed with ether, and then dried under reduced pressure to obtain the target compound as colorless crystals. Yield 106mg. Colorless crystals, mp 98-104℃, [α] ²⁰ _D = -66.8゜ (c = 0.5, ethanol) ¹ H-NMR (CD ₃ OD): δ value 0.90 (3H, t, J = 5Hz), 1.1-2.5 (15H, m), 2.9-3.9 (5H, m), 4.1-4.6 (3H, m) Reference example 30 N-[(R)-1-ethoxycarbonyl-2-[(R,
S)-1-ethoxycarbonylheptylthio]
Production of ethyl]-(R,S)-alanine S-[(R,S)-1-ethoxycarbonylheptyl]-L-cysteine ester 11.3g, pyruvic acid 15.6g, 4N aqueous sodium hydroxide solution and cyanoborohydride The target compound was obtained as a colorless amorphous substance by reacting and treating in the same manner as in Reference Example 24 using 4.5 g of sodium. Yield 6.8g. [α] ²⁰ _D = −3.1° (c = 1.2, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 5Hz), 1.2 to 1.5 (9H, m), 1.3 to 2.1 (10H, m), 2.7-3.8 (5H, m), 4.20 (4H, q, J = 7Hz) Example 62 N-[(R)-1-ethoxycarbonyl-2-[(R,
S)-1-ethoxycarbonylheptylthio]
Production of ethyl]-(R,S)-alanyl-(S)-proline-t-butyl ester N-[(R)-1-ethoxycarbonyl-2-[(R,S) obtained in Reference Example 30 -1-ethoxycarbonyl-2-[(R,S)-1-ethoxycarbonylheptylthio]ethyl]-(R,S)-alanine 900
mg, the target compound was obtained as a colorless oil in the same manner as in Example 45. Yield: 1.1g. [α] ²⁵ _D = −28.6° (c = 0.7, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 5Hz), 1.2 to 1.4 (9H, m), 1.44, 1.47 (Total 9H, each s), 1.5-2.3 (14H, m), 2.7-3.0 (2H, m), 3.1-3.8 (5H, m), 4.19 (4H, q, J = 7Hz) 4.0-4.3 (1H , m), Example 63 N-[(R)-1-ethoxycarbonyl-2-[(R,
S)-1-ethoxycarbonylheptylthio]
Preparation of ethyl]-(R,S)-alanyl-(S)-proline and its maleate salt N-[(R)-1-ethoxycarbonyl-2-[(R,S) obtained in Example 62 -1-ethoxycarbonylheptylthio]ethyl]-(R,S)-alanyl-(S)-proline and its maleic acid salt N-[(R)-1-ethoxycarbonyl- obtained in Example 62 From 900 mg of 2-[(R,S)-1-ethoxycarbonylheptylthio]ethyl]-(R,S)-alanyl-(S)-proline-t-butyl ester, the target compound was obtained in the same manner as in Reference Example 22. was obtained as a colorless amorphous substance. Yield 650mg. From 230 mg of the compound obtained in the above reaction, the maleate salt of the target compound was obtained in the same manner as in Example 47. Yield 250mg. Yield 250mg. [α] ²⁵ _D = -25.1° (c = 0.6, ethanol) ¹ H-NMR (CD ₃ 0D): δ value 0.9 (3H, t, J = 5Hz) 1.1 to 1.6 (9H, m), 1.6 to 2.4 (14H, m), 3.1~3.9 (7H, m), 4.21 (4H, q, J=7Hz) 4.2~4.5 (1H, m), 6.28 (2H, s) Reference example 31 N- [(R)- 1-ethoxycarbonyl-2--(1
-ethoxycarbonylhexylthio)ethyl]
-Production of alanine-t-butyl ester/A and B isomers S-[(R,S)-1-ethoxycarbonylhexyl]-L-cysteine ethyl ester 6.1g and 2-bromopropionic acid-t-butyl ester
4.2g Triethylamine 2.0g dissolved in 20ml HMPA
added. After stirring at room temperature for 72 hours, ice water was added to the reaction solution and extracted with ethyl acetate. The extract was washed with water and then with saturated brine, and 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, 1:3), and the A isomer of the target compound was obtained as a colorless oil from the preceding fraction. Yield 2.2g. [α] ¹⁸ _D = +13.0° (c = 1.0, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 7Hz), 1.27 (3H, d, J = 7Hz), 1.28 (6H, t, J=7Hz), 1.45 (9H, s), 1.3~1.9 (8H, m), 2.23 (1H, s), 2.7~3.0 (2H, m), 3.2~3.6 (3H, m) ) 4.20 (4H, q, J=7Hz) The B-isomer of the target compound was obtained as a colorless oily substance from the afterflow. Yield: 1.7g. [α] ¹⁸ _D = -30.8° (c = 1.2, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 7Hz), 1.28 (3H, d, J = 7Hz), 1.28 (6H, t, J=7Hz), 1.3-1.9 (8H, m), 2.04 (1H, s), 3.6-3.0 (2H, m), 3.1-3.6 (3H, m) 4.20 (4H, q, J =7Hz) Reference example 32 N-[(R)-1-ethoxycarbonyl-2-(1-
Ethoxycarbonylhexylthio)ethyl]-
Production of alanine A-isomer N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonylhexylthio)ethyl]-alanine-t-butyl ester A-isomer obtained in Reference Example 31 Add 5ml of TFA to 1.71g of body and mix at room temperature.
Stir for hours. TFA was distilled off under reduced pressure, and a saturated aqueous sodium bicarbonate solution was added to adjust the pH to 8, followed by washing with ether. After adding 10% hydrochloric acid to the aqueous layer to adjust the pH to 4, the mixture was extracted with methylene chloride. After drying the extract over anhydrous sodium sulfate, it was distilled off under reduced pressure to obtain the target compound A.
-obtained isomers. Yield: 1.37g. [α] ²⁰ _D = +6.9° (c = 0.7, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 7Hz), 1.2 ~ 1.5 (9H, m), 1.3 ~ 2.0 (8H, m), 2.9 to 3.9 (5H, m), 4.21 (2H, q, J = 7Hz), 4.23 (2H, q, J = 7Hz), 6.8 (1H, br s) Reference example 33 N- [(R)-1-ethoxycarbonyl-2-(1-
Ethoxycarbonylhexylthio)ethyl]-
Production of alanine B-isomer N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonylhexylthio)ethyl]-alanine-t-butyl ester B-isomer obtained in Reference Example 31 5 ml of TFA was added to 1.40 g of the compound, and the reaction was carried out in the same manner as in Reference Example 32 to obtain the B-isomer of the target compound. Yield 1.25g. [α] ²⁰ _D = −16.2° (c = 0.9, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 7Hz), 1.2 to 1.5 (9H, m), 1.3 to 2.0 (8H, m), 2.6-3.8 (5H, m), 4.20 (2H, q, J=7Hz), 4.24 (2H, q, J=7Hz), 5.8 (1H, br s) Example 64 N-[ (R)-1-ethoxycarbonyl-2-(1-
Ethoxycarbonylhexylthio)ethyl]-
Production of alanine-(S)-proline-t-butyl ester/A-isomer N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonylhexylthio)ethyl] obtained in Reference Example 32 -Alanine A isomer 1.26g,
629 mg of (S)-proline-t-butyl ester and 666 mg of diethyl cyanophosphate (DEPC) in 5 ml of DMF
Add 371 mg of triethylamine to the solution while stirring on ice.
5 ml of DMF solution was slowly added dropwise. After stirring for 12 hours while gradually returning the temperature to room temperature, ice water was added to the reaction mixture, and the mixture was made slightly alkaline with a saturated aqueous sodium bicarbonate solution, followed by extraction with ethyl acetate. The extract was washed with water and then with saturated brine, 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 = 20:1) to obtain the A-isomer of the target compound as a colorless oil. Yield 1.60g. [α] ²⁰ _D = -23.1° (c = 1.2, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 7Hz), 1.29 (3H, d, J = 7Hz), 1.29 (6H, t, J=7Hz), 1.44, 1.47 (total 9H, each s), 1.3 to 2.7 (12H, m), 2.27 (1H, br s), 2.8 to 3.0 (2H, m), 3.1 to 3.8 (5H, m), 4.20 (4H, q, J = 7Hz) 4.4-4.7 (1H, m) Example 65 N-[(R)-1-ethoxycarbonyl-2-(1-
Ethoxycarbonylhexylthio)ethyl]-
Production of alanyl-(S)-proline-t-butyl ester/B isomer N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonylhexylthio)ethyl]- obtained in Reference Example 33 The B-isomer of the target compound was obtained as a colorless oil from 1.08 g of alanine B-isomer in the same manner as in Example 64. Yield: 1.56g. [α] ²⁰ _D = -63.7° (c = 1.0, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 7Hz), 1.1 to 1.5 (9H, m), 1.44, 1.46 (9H, each s), 1.3-2.5 (12H, m), 2.26 (1H, br s), 2.6-3.0 (2H, m), 3.1-3.8 (5H, m), 4.20 (4H, q, J =7Hz), 4.3-4.5 (1H, m) Example 66 N-[(R)-1-ethoxycarbonyl-2-(1-
Ethoxycarbonylhexylthio)ethyl]-
Production of alanyl-(S)-proline A-isomer and its L-arginine salt N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonylhexylthio)ethyl obtained in Example 64 ]-alanyl-(S)-proline-t
-25% HBr in 530mg of butyl ester A isomer-
3 ml of acetic acid was added and stirred at room temperature for 40 minutes. The solvent was distilled off under reduced pressure, a saturated aqueous sodium hydrogen carbonate solution was added to adjust the pH to 8, and the mixture was washed with ether. 10 in the water layer
% hydrochloric acid was added to adjust the pH to 4, followed by extraction with methylene chloride. The extract was dried over anhydrous sodium sulfate and then evaporated under reduced pressure to obtain the A-isomer of the target compound as a colorless oil. Yield 452mg. To a solution of 242 mg of the A-isomer obtained in the above reaction in 4 ml of ethanol, a 1.5 ml aqueous solution of 89 mg of L-arginine was added with stirring. After distilling off water and ethanol,
It was dried under reduced pressure to obtain the L-arginine salt of the target compound as a pale yellow powder. Yield 143mg. [α] ²⁵ _D = -12.5° (c = 0.3, ethanol) ¹ H-NMR (CD ₃ OD): δ value 0.90 (3H, t, J = 7Hz), 1.2 ~ 1.5 (9H, m), 1.5 ~ 2.5 (16H, m), 2.6-3.8 (10H, m), 4.19 (4H, q, J=7Hz) 4.3-4.5 (1H, m) Example 67 N-[(R)-1-ethoxycarbonyl-2 -(1-
Ethoxycarbonylhexylthio)ethyl]-
Production of alanyl-(S)-proline B-isomer and its L-arginine salt, maleate salt N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonyl) obtained in Example 65 hexylthio)ethyl]-alanyl-(S)-proline-t
Example from 522 mg of -butyl ester B-isomer
The B-isomer of the target compound was obtained as a colorless oil in the same manner as in 66. Yield 230mg. Example 66 from 230 mg of B-isomer obtained in the above reaction.
In the same manner as above, the L-arginine salt of the target compound was obtained as a colorless powder. Yield 270mg. [α] ²⁵ _D = -27.7° (c = 0.3, ethanol) ¹ H-NMR (CD ₃ OD): δ value 0.90 (3H, t, J = 7Hz), 1.2 ~ 1.5 (9H, m), 1.5 ~ 2.3 (16, m), 2.7-3.8 (10H, m), 4.19 (1H, q, J = 7Hz) 4.3-4.5 (1H, m) In addition, maleate was prepared from 380 mg of B-isomer using the same procedure. Obtained as a colorless oil. Yield 382mg. [α] ²⁵ _D = -38.3° (c = 0.7, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 7Hz), 1.2 to 1.5 (9H, m), 1.5 to 2.5 (12H, m), 3.0~3.9 (5H, m), 4.20 (4H, q, J=7Hz) 4.3~4.5 (1H, m) 6.32 (2H, s) Reference example 34 N-[(R)-1 -ethoxycarbonyl-2-(1-
Ethoxycarbonylheptylthio)ethyl]-
Production of alanine-t-butyl ester/A and B-isomers S[(R,S)-1-ethoxycarbonylheptyl]-L-cysteine ethyl ester 5.5g and 2
-bromopropionic acid-t-butyl ester 3.6
g, 16 ml of HMPA using 2.4 ml of triethylamine
By reacting and treating in the same manner as in Reference Example 31, the A-isomer (previous fraction in silica gel column chromatography) and B-isomer (later fraction in silica gel column chromatography) of the target compound were converted to colorless Obtained as an oil. A isomer; yield 1.8 g [α] ²³ _D = +18.6° (c = 0.7, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.87 (3H, t, J = 5Hz), 1.28 (3H, d, J=7Hz), 1.29 (6H, t, J=7Hz), 1.44 (9H, s), 1.3~1.8 (10H, m), 2.8~3.0 (2H, m), 3.2~3.6 (3H, m ), 4.20 (4H, q, J = 7Hz) B-isomer; yield 1.9g [α] ²³ _D = -26.0° (c = 0.7, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H , t, J=5Hz), 1.28 (3H, d, J=7Hz), 1.29 (6H, t, J=7Hz), 1.47 (9H, s), 1.3~2.0 (10H, m), 2.6~3.2 ( 2H, m), 3.2-3.6 (3H, m), 4.20 (4H, q, J=7Hz) Reference example 35 N-[(R)-1-ethoxycarbonyl-2-(1-
Ethoxycarbonylheptylthio)ethyl]-
Production of alanyl/A-isomer N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonylhexylthio)ethyl]-alanyl-t-butyl ester/A-isomer obtained in Reference Example 34 The A-isomer of the target compound was obtained by reacting and treating 1.8 g of the compound in the same manner as in Reference Example 32. Yield 1.5g. [α] ²⁵ _D = +16.5° (c = 0.7, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 7Hz), 1.29 (6H, t, J = 7Hz), 1.40 (3H, d, J = 7Hz), 1.5 - 2.1 (10H, m), 2.7 - 3.2 (2H, m), 3.2 - 3.7 (3H, m), 4.20 (4H, q, J = 7Hz) 6.41 ( 1H, br s) Reference example 36 N-[(R)-1-ethoxycarbonyl-2-(1-
Ethoxycarbonylheptylthio)ethyl]-
Production of alanyl B-isomer N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonylheptylthio)ethyl]-alanyl-t-butyl ester B-isomer obtained in Reference Example 34 The B-isomer of the target compound was obtained by reacting and treating 1.8 g of the compound in the same manner as in Reference Example 32. Yield: 1.3g. [α] ²⁵ _D = -12.3° (c = 0.6, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 7Hz), 1.30 (6H, t, J = 7Hz), 1.43 (3H, d, J=7Hz), 1.5-2.1 (10H, m), 2.5-3.1 (2H, m), 3.1-3.5 (3H, m), 4.20 (2H, q, J=7Hz), 4.24 ( 2H, q, J = 7Hz), 5.59 (1H, br s) Example 68 N-[(R)-1-ethoxycarbonyl-2-(1-
Ethoxycarbonylheptylthio)ethyl]-
Production of alanyl-(S)-proline-t-butyl ester/A-isomer N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonylheptylthio)ethyl] obtained in Reference Example 35 -Alanine A-isomer 1.2g,
(S)-proline-t-butyl ester 530 mg,
The A-isomer of the target compound was obtained as a colorless oil by reacting and treating in the same manner as in Example 64 using 550 mg of DEPC and 0.43 ml of triethylamine. Yield: 1.36g. [α] ²⁵ _D = -12.4° (c = 0.6, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.87 (3H, t, J = 7Hz), 1.29 (6H, t, J = 7Hz), 1.31 (3H, d, J = 7Hz), 1.44, 1.47 (9H in total, each s), 1.5 to 2.2 (14H, m), 2.7 to 3.0 (2H, m), 3.1 to 3.9 (5H, m), 4.20 ( 4H, q, J=7Hz), 4.0-4.5 (1H, m) Example 69 N-[(R)-1-ethoxycarbonyl-2-(1-
Ethoxycarbonylheptylthio)ethyl]-
Production of alanyl-(S)-proline-t-butyl ester/B-isomer N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonylhexylthio)ethyl] obtained in Reference Example 36 -alanyl B-isomer 1.05g
The B-isomer of the target compound was obtained as a colorless oil in the same manner as in Example 64. Yield: 1.3g. [α] ²⁵ _D = -67.5° (c = 1.0, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 7Hz), 1.2 to 1.5 (9H, m), 1.44, 1.46 (Total 9H, each s), 1.6-2.5 (14H, m), 2.6-3.8 (7H, m), 4.19 (4H, q, J = 7Hz), 4.2-4.5 (1H, m) Example 70 N- [(R)-1-ethoxycarbonyl-2-(1-
Ethoxycarbonylheptylthio)ethyl]-
Production of alanyl-(S)-proline A-isomer and its maleate salt N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonylheptylthio)ethyl] obtained in Example 68 -alanyl-(S)-proline-t
-Reference example from 398mg of Butyl ester A-isomer
By reacting and treating in the same manner as 22, the A-isomer of the target compound was obtained as a colorless oil. yield
270mg. From 270 mg of the compound obtained in the above reaction, the maleate salt of the target compound was obtained in the same manner as in Example 47. Yield 310mg. [α] ²⁵ _D = -28.3゜ (c = 0.6, ethanol) ¹ H-NMR (CD ₃ OD): δ value 0.90 (3H, t, J = 7Hz), 1.1 ~ 1.6 (9H, m), 1.5 ~ 2.4 (14H, m), 3.0-3.9 (7H, m), 4.21 (4H, q, J=7Hz), 6.28 (2H, s) Example 71 N-[(R)-1-ethoxycarbonyl-2- (1-
Ethoxycarbonylheptylthio)ethyl]-
Production of alanyl-(S)-proline B-isomer and its L-arginine salt, maleate salt N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonyl) obtained in Example 69 heptylthio)ethyl]-alanyl-(S)-proline-t
Example from 450 mg of -butyl ester B-isomer
The L-arginine salt of the target compound was obtained as a colorless (non-crystalline) powder in the same manner as in Example 66. Yield 516
mg. [α] ²⁵ _D = -36.9° (c = 0.5, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.87 (3H, t, J = 7Hz), 1.2 to 1.5 (9H, m), 1.5 to 2.5 (18H, m), 2.7-3.8 (10H, m), 4.20 (4H, q, J = 7Hz), 4.5-4.6 (1H, m) N-[(R)-1-ethoxycarbonyl-2-(1 −
Ethoxycarbonylheptylthio)ethyl]-alanyl-(S)-proline-t-butyl ester B
- The maleate salt of the target compound was obtained from 430 mg of the isomer in the same manner as in Example 47. Yield 457mg. [α] ²⁵ _D = -38.3° (c = 0.8, ethanol) ¹ H-NMR (CD ₃ OD): δ value 0.90 (3H, t, J = 7Hz), 1.2 ~ 1.5 (9H, m), 1.6 ~ 2.5 (14H, m), 2.8~3.8 (7H, m), 4.22 (4H, q, J=7Hz), 4.5~4.6 (1H, m) 6.28 (2H, s) Reference example 37 N-[(R) -1-ethoxycarbonyl-2-(1-
Ethoxycarbonyloctylthio)ethyl]-
Production of alanyl-t-butyl ester/A and B-isomers S-[(R,S)-1-ethoxycarbonyloctyl]-1-cysteine ethyl ester 10 g and 2-bromopropionic acid-t-butyl ester
Dissolve 6.9g in HMPA16ml and triethylamine 4.6
Added ml. After stirring at room temperature for 48 hours, ice water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. Add the extract to water,
Then, it was washed with saturated brine and dried over anhydrous sodium sulfate. The extract was distilled off under reduced pressure and the residue was subjected to silica gel column chromatography (solvent: ether:
The mixture was separated and purified using n-hexane (1:3), and the A-isomer of the target compound was obtained as a colorless oil from the preceding fraction. Yield 2.7g. [α] ²² _D = +13.2° (c = 1.2, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 7Hz), 1.2-1.4 (9H, m), 1.45 ( 9H, s), 1.2~2.1 (12H, m), 2.24 (1H, br s), 2.8~3.1 (2H, m), 3.1~3.6 (3H, m), 4.20 (4H, q, J=7Hz) The B-isomer of the target compound was obtained as a colorless oil from the afterflow. Yield 2.2g. [α] ²² _D = -25.9° (c = 0.9, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 7Hz), 1.2 to 1.4 (9H, m), 1.47 (9H , s), 1.2~2.2 (13H, m), 2.76 (1H, dd, J=13Hz, 8Hz), 3.03 (1H, dd, J=13Hz, 5Hz), 3.1~3.6 (3H, m ) 4.20 (4H, q, J=7Hz) Reference Examples 38 and 39 Each compound listed in Table 15 below was obtained in the same manner as in Reference Example 37 above.

【table】

[Table] Reference example 40 N-[(R)-1-ethoxycarbonyl-2-(1-
Ethoxycarbonyloctylthio)ethyl]-
Production of alanyl B-isomer N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonyloctylthio)ethyl]-alanyl-t-butyl ester B-isomer obtained in Reference Example 37 2.2 g of the compound was dissolved in 6 ml of 25% HBr-acetic acid and stirred at room temperature for 40 minutes. HBr-acetic acid was distilled off under reduced pressure, saturated aqueous sodium bicarbonate solution was added to the residue to adjust the pH to 8, and the mixture was washed with ether. Citric acid was added to the aqueous layer to adjust the pH to 4, followed by extraction with methylene chloride. The extract was dried over anhydrous sodium sulfate and then evaporated under reduced pressure to obtain the target compound. Yield: 1.8g. [α] ²⁰ _D = -15.7° (c = 0.8, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 7Hz), 1.30 (6H, t, J = 7Hz), 1.43 (3H, d, J = 7Hz), 1.2 ~ 2.1 (13H, m), 2.3 ~ 3.5 (5H, m), 4.20 (2H, q, J = 7Hz), 4.23 (2H, q, J = 7Hz), 5.25 (1H, br s) Reference Examples 41 and 42 Each compound listed in Table 16 below was obtained in the same manner as in Reference Example 40 above.

[Table] Example 72 N-[(R)-1-ethoxycarbonyl-2-(1-
Ethoxycarbonyloctylthio)ethyl]-
Production of alanyl-(S)-proline-t-butyl ester/B-isomer N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonyloctylthio)ethyl] obtained in Reference Example 40 -alanyl B-isomer 1.52g,
(S)-proline-t-butyl ester 705mg and
A solution of 417 mg of triethylamine in 5 ml of DMF was slowly added dropwise to a solution of 747 mg of DEPC (90% content) in 30 mg of DMF under ice-cooling and stirring. 12 while gradually returning to room temperature
After stirring for an hour, the mixture was poured into ice water and extracted with ethyl acetate. After washing the oil extract with water, it was 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 = 35:1) to obtain the target compound as a colorless oil. Yield 2.08g. [α] ²⁰ _D = -60.6° (c = 1.5, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 7Hz), 1.28 (6H, t, J = 7Hz), 1.30 (3H, d, J = 7Hz), 1.44, 1.46 (9H in total, each s), 1.2 to 2.5 (17H, m), 2.6 to 3.7 (7H, m), 4.19 (4H, q, J = 7Hz), 4.4-4.5 (1H, m), Examples 73 and 74 Each compound listed in Table 17 below was obtained in the same manner as in Example 72 above.

【table】

[Table] Example 75 N-[(R)-1-ethoxycarbonyl-2-(1-
Ethoxycarbonyloctylthio)ethyl]-
Production of alanyl-(S)-proline B-isomer and its L-arginine salt N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonyloctylthio)ethyl obtained in Example 72 ]-alanyl-(S)-proline-t
-Reference example from 2.1g of butyl ester B-isomer
The target compound was obtained as a colorless oil in the same manner as in 40. Yield 1.0g. 20 ml of ethanol containing 1.0 g of the compound obtained in the above reaction
A solution of 650 mg of L-arginine in 10 ml of water was added to the solution, and the mixture was distilled off under reduced pressure. Ethanol was added to the residue and evaporated under reduced pressure several times, and the residue was recrystallized from ethyl acetate-n-hexane to obtain the L-arginine salt of the target compound.
Yield 400mg. [α] ²² _D = -42.0° (c = 0.6, ethanol) ¹ H-NMR (CD ₃ OD): δ value 0.89 (3H, t, J = 7Hz), 1.2 ~ 1.4 (9H, m), 1.2 ~ 2.2 (20H, m), 2.5-3.7 (10H, m), 4.18 (4H, q, J = 7Hz) 4.3-4.5 (1H, m) Examples 76 and 77 In the same manner as Example 75 above, the following No. 18 Each compound listed in the table was obtained.

【table】 Examples 78 and 79 The following compound was obtained in the same manner as in Example 65.

[Table] Example 80 In the same manner as in Example 13, using appropriate starting materials,
The following compound was obtained. N-[(R)-1-ethoxycarbonyl-2-octylthioethyl)-alanyl-(S)-proline-
Production of t-butyl ester/β-isomer [α] ²⁵ _D = -85.7° (c = 0.7, ethanol) ¹ H-NMR (CDCl ₃ ): δ value 0.88 (3H, t, J = 5Hz), 1.28 (3H, t, J = 7Hz), 1.29 (3H, d, J = 6Hz), 1.2 to 1.8 (12H, m), 1.44, 1.46 (9H in total, each s), 1.8 to 2.3 (4H, m), 2.52 (2H, t, J=7Hz), 2.70 (1H, dd, J=13Hz, 7.5Hz), 2.89 (1H, dd, J=13Hz, 6Hz), 3.2~3.7 (4H, m) , 4.20 (2H, q, J=7Hz), 4.3-4.5 (1H, m) Example 81 The following compound was obtained in the same manner as in Example 17 using appropriate starting materials. N-[(R)-1-ethoxycarbonyl-2-octylthioethyl]-alanyl-(S)-proline.
β-isomer/L-arginine salt [α] ²⁵ _D = -56.5° (c = 0.5, ethanol) mp.70-81°C Reference Examples 43 and 44 Compounds listed in Table 20 below in the same manner as Reference Example 8 I got it.

[Table] Reference Examples 45 and 46 In the same manner as in Reference Example 13, the compounds listed in Table 21 below were obtained.

[Table] Examples 82 and 83 Compounds listed in Table 22 below were obtained in the same manner as in Example 13.

[Table] Examples 84 and 85 Compounds listed in Table 23 below were obtained in the same manner as in Example 18.

[Table] Reference Examples 47 and 48 Compounds listed in Table 24 below were obtained in the same manner as in Reference Example 8.

[Table] Reference Example 49 The following compound was obtained in the same manner as in Reference Example 13. β-isomer [α] ²⁵ _D = -23.2° (c = 0.6, DMF) mp.125-127°C Formulation example 1 Preparation of tablets 5 mg each of N-[(R)-1-ethoxycarbonyl-2-heptyl Thioethyl]-alanyl-(S)
- 1000 tablets for oral use containing proline β-isomer maleate are prepared according to the following formulation. Blend amount (g) N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-alanyl-(S)-proline, β-isomer, maleate 5 Lactose (Japanese Pharmacopoeia product) 50 Corn starch (Japanese Pharmacopoeia) 25 Crystalline cellulose (Japanese Pharmacopoeia) 25 Methylcellulose (Japanese Pharmacopoeia) 1.5 Magnesium stearate (Japanese Pharmacopoeia) 1 N-[(R)-1-ethoxycarbonyl-2- heptylthioethyl]-alanyl-(S)-proline β
- The isomer maleate, lactose, cornstarch and crystalline cellulose are thoroughly mixed, granulated with a 5% aqueous solution of methylcellulose, passed through a 200 mesh sieve, mixed with magnesium stearate and pressed into tablets. 10 mg each of N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-alanyl-(S)
- 1000 hard gelatin capsules for oral use containing proline β-isomer maleate are prepared according to the following formulation. Blend amount (g) N-[(R)-1ethoxycarbonyl-2-heptylthioethyl]-alanyl-(S)-proline, β-isomer, maleate 10 Lactose (Japanese Pharmacopoeia product) 80 Starch (Japanese Pharmacopoeia product) 30 Talc (Japanese Pharmacopoeia product) 5 Magnesium stearate (Japanese Pharmacopoeia product) 1 The above ingredients are finely powdered, stirred thoroughly to form a uniform mixture, and then prepared for oral administration with desired dimensions. Fill into gelatin capsules. Prescription Example 3 Preparation of Injection A sterile aqueous solution suitable for parenteral administration is prepared according to the following recipe. Blend amount (g) N-[(R)-1ethoxycarbonyl-2-heptylthioethyl]-alanyl-(S)-proline, β-isomer, maleate 1 Polyethylene glycol (Japanese Pharmacopoeia product) Molecular weight :4000 0.3 Sodium chloride (Japanese Pharmacopoeia) 0.9 Polyoxyethylene sorbitan monooleate (Japanese Pharmacopoeia) 0.4 Sodium metabisulfite 0.1 Methyl-paraben (Japanese Pharmacopoeia) 0.18 Propyl-paraben (Japanese Pharmacopoeia) 0.02 Distilled water for injection 100 (ml) Dissolve the above parabens, sodium metabisulfite, and sodium chloride in about half the amount of distilled water above at 80°C while stirring. The resulting solution was cooled to 40°C, and N-[(R)-1-ethoxycarbonyl-2
-heptylthioethyl]-alanyl-(S)-proline β-isomer maleate, then polyethylene glycol and polyoxyethylene sorbitan monooleate are dissolved in the solution. The solution is then adjusted to the final volume by adding distilled water for injection, sterilized by sterilization using a suitable filter paper, and filled into sterile containers (1 ml). Formulation example 4 Preparation of tablets 5 mg each of N-[(R)-1-ethoxycarbonyl-2-hexylthioethyl]-alanyl-(S)
1000 tablets for oral use containing proline-tert-butyl ester β-isomer are prepared according to the following formulation. Blend amount (g) N-[(R)-1ethoxycarbonyl-2-hexylthioethyl]-alanyl-(S)-proline-t-butyl ester/β-isomer 5 Lactose (Japanese Pharmacopoeia product) 50 Corn starch (Japanese Pharmacopoeia) 25 Crystalline cellulose (Japanese Pharmacopoeia) 25 Methyl cellulose (Japanese Pharmacopoeia) 1.5 Magnesium stearate (Japanese Pharmacopoeia) 1 N-[(R)-1-ethoxycarbonyl-2- hexylthioethyl]-alanyl-(S)-proline-t
-Butyl ester β-isomer, lactose, corn starch and crystalline cellulose are thoroughly mixed, granulated with a 5% aqueous solution of methylcellulose, passed through a 200 mesh sieve, mixed with magnesium stearate and pressed into tablets. Formulation example 5 Preparation of tablets 5 mg each of N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-alanyl-(S)
1000 tablets for oral use containing -proline-methyl ester β-isomer maleate salt are prepared according to the following formulation. Blend amount (g) N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-alanyl-(S)-proline. Methyl ester/β-isomer/maleate 5 Lactose (Japanese Pharmacopoeia) 50 Corn starch (Japanese Pharmacopoeia) 25 Crystalline cellulose (Japanese Pharmacopoeia) 25 Methyl cellulose (Japanese Pharmacopoeia) 1.5 Magnesium stearate (Japanese Pharmacopoeia) Japanese Pharmacopoeia product) 1 N-[(R)-1-ethoxycarbonyl-2-heptylthioethyl]-alanyl-(S)-proline-methyl ester, β-isomer, maleate, lactose, corn starch and crystals Mix the cellulose thoroughly and granulate it with a 5% aqueous solution of methylcellulose.
Passed through a 200 mesh sieve, mixed with magnesium stearate and pressed into tablets. Formulation example 6 Preparation of tablets 5 mg each of N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonylhexylthio)ethyl]-alanyl-(S)-proline, B-isomer, L-arginine 1000 tablets for oral use containing salt are prepared according to the following formulation. Blend amount (g) N-[(R)-1-ethoxycarbonyl-2-(1-ethoxycarbonylhexylthio)ethyl]-alanyl-(S)-proline, B-isomer, L-arginine salt 5 Lactose (Japanese Pharmacopoeia) 50 Cornstarch (Japanese Pharmacopoeia) 25 Crystalline Cellulose (Japanese Pharmacopoeia) 25 Methylcellulose (Japanese Pharmacopoeia) 1.5 Magnesium Stearate (Japanese Pharmacopoeia) 1 N-[(R)- 1-ethoxycarbonyl-2-(1-
Ethoxycarbonylhexylthio)ethyl]-alanyl-(S)-proline, B-isomer, L-arginine salt, lactose, cornstarch, and crystalline cellulose were thoroughly mixed, granulated with a 5% aqueous solution of methylcellulose, and sieved through a 200-mesh sieve. mixed with magnesium stearate and pressed into tablets. Test Example 1 Inhibitory activity against angiotensin-converted oxygen (ACE) 100μ of an enzyme solution purified from rabbit lung acetone powder (manufactured by Sigma) and 100μ of a sample solution were mixed, gently shaken at 37°C for 10 minutes, and then Add 100μ of 6.99mM Hypril-Histidyl-Leucine (manufactured by Protein Promotion Association) solution as a substrate, react with shaking at 37℃ for 45 minutes, and then add 1N sulfuric acid solution.
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.
Using 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 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 were as follows, and the results of the above tests are shown in Table 25 below. Test compound No. Compound name 1 N-[(R)-1-carboxyl-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-carboxyl-2
-pentylthioethyl]-alanyl-(S)-proline β-isomer (compound of Example 12) 4 N-[(S)-1-carboxyl-3
-methylthiopropyl]-(R,
S)-alanyl-(S)-proline (corresponds to the compound of Example 17 in JP-A-55-81845) 5 N-[(R)-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] According to Table 25, the active ingredient compound group of the present invention is about 24 times more active (test compound No. 1) and about 9.4 times more active than the known compound (test compound No. 4), which has the highest activity (test compound No. 4), respectively. (Test Compound No. 2) and approximately 122 times (Test Compound No.
3) It is found that it exhibits strong inhibitory activity. Test Example 2 ACE inhibitory effect in normotensive rats Using male Wistar rats (200-400 g), the rats were fixed in a supine position under anesthesia by intraperitoneal administration of 1.25 g/Kg of urethane, and the ACE inhibitory effect was investigated using angiotensin (A). ) was measured from the suppressive effect on the increase in blood pressure. Blood pressure was measured using a transducer (MPU−0.5−290−0−,
(Multipurpose Polygraph, RM) via a carrier amplifier (PR-5, manufactured by Nihon Kohden Co., Ltd.).
−85, manufactured by Nihon Kohden Co., Ltd.) as the mean blood pressure. After fixing the rats in the experimental apparatus, they were allowed to stand for about 1 hour to stabilize their hemodynamics, and then the experiment was started.
Since the pressure increase due to A boost varies with time,
The suppressive effect on A was determined using the ratio of A pressure increase to A pressure increase (A/A ratio), which shows a more constant value. That is, A from the caval vein cannula.
(300ng/Kg) and 5 minutes later, A
(300ng/Kg) was administered and the A/A ratio was measured.
Then, 30 minutes after the first A administration, A
(300ng/Kg) and 5 minutes later, A
(300ng/Kg) was administered, and the A/A ratio was measured. This operation was repeated until the A/A ratio became constant. The average value of the A/A ratio measured in these operations is defined as "c". 15 minutes after the last administration of A, 1 mg/Kg of each test compound was administered as an aqueous solution directly into the stomach through a vinyl tube, and 15 minutes after the administration of the test compound, A (300ng/Kg) was administered. A was administered 5 minutes after administration, and A
/A ratio was measured. Let this be E. ACE
The inhibition rate was calculated according to the formula below. ACE inhibition rate = C-E/C x 100 Then, at 60 minutes, 120 minutes and 240 minutes after administration of the test compound, A
(300ng/Kg) and 5 minutes later, A
(00ng/Kg) was administered, and the ACE inhibition rate was determined. Table 26 shows the ACE inhibition rate of each test compound.
Each test compound in Table 26 was produced in the example shown in the table.

[Table] From Table 26 above, the active ingredient compounds of the present invention
It can be seen that the ACE inhibitory activity is high. Test Example 3 Acute toxicity test 3-week-old ICR male mice (body weight 27-37g, n
= 5) as a test animal, a suspension of the compound obtained in Example 39 in a 0.1% ruboxymethyl cellulose solution was orally administered once, and its acute toxicity value was determined. As a result, the LD ₅₀ value is 3500 ~
It was 5000mg/Kg.

Claims (1)

[Claims] 1. General formula [In the formula, R ₁ is C ₅ - C ₁₄ alkyl, C ₂ - C ₆ alkenyl, C ₃ - C ₈ cycloalkyl-C ₁ - C ₆ alkyl, or [Formula] (In the formula, R ₅ is hydrogen or C ₁ - represents _C14 alkyl, _R6 represents hydrogen or _C1 - _C6 alkyl), _R2 represents hydrogen or _C1 - _C6 alkyl,
_R3 represents _C1 - _C6 alkyl, _R4 is hydrogen, _C1-
It represents _C6 alkyl or phenyl- _C1 - _C6 alkyl, and n represents 0, 1 or 2. ] It is characterized by containing a proline derivative represented by the following or a pharmacologically acceptable salt thereof as an active ingredient.