Amino acid derivatives

(74) Agent

Cand mag. Johan Η. Gørbitz, Bryn & Aarflot A / S, Oslo.

(561 Publications cited

No.

This invention relates to a process for the preparation of novel substituted acyl derivatives of amino acids of the general formula

R. - NH

I «7^H2> m AB

III

R₃ - S - (CH₂)_n - CH - CO - N - CH - CO - RI * * and its salts, where

R is hydroxy or lower alkoxy;

R 2 is hydrogen or lower alkanoyl;

R₃ is hydrogen or lower alkanoyl;

A and B together form a (CH-) bridge which forms a ring on

Z or 6 atoms with the nitrogen and carbon to which they are attached, n is 0 or 1;

m is 0, 1, 2, 3 or 4;

wherein at least one of m and n is different from 0; and p is 3 or 4.

The stars denote asymmetry centers.

Compounds of formula I derived from or containing the amino acids glycine, alanine, methionine, cysteine ​​or proline are generally preferred. Preferred modifications are compounds of formula I wherein R is hydroxy;

R₁ is hydrogen or acetyl; is hydrogen or acetyl;

A and B complete a 5- or 6-membered ring; m is 0, 3 or 4, and n is 0 or 1, but not both m and n are 0.

Particularly preferred are those compounds of formula I which are derived from proline and have the formula

Rj-NH w »O

R₇-S- (CH₉) -CH-CO-N-CH-CO-R II jzn * *

The symbols have the above-mentioned preferred meanings.

The lower alkoxy groups have 1 to 7 carbon atoms, e.g. methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, t-butoxy and the like. The lower alkanoyl groups are the acyl radicals of the lower (up to 7 carbon atoms) fatty acids, e.g. acetyl, propionyl, butyryl and the like, with acetyl being preferred.

The compounds of formula I and the preferred subgroups can be prepared by various synthetic methods. According to the invention, the amino acid is acylated. with the formula:

ABII

HN - CH - COR III wherein A, B and R are as above, with an acid of the formula:

R .'- NH ¹ I (CH-)

I 2 ΓΠ

R 1 '- S- (CH 2) -CH-COOH 3 2 n where' is a protecting group or a lower alkanoyl group, R 1 'is a protecting group or, and R 1, m and n have the meanings given above, by a of the known methods in which the acid IV, prior to reaction with the amino acid III, is converted to an activated carboxylic acid derivative, such as a mixed anhydride, symmetric anhydride, acid chloride, active ester, Woodward reagent K,

N, N'-carbonyl bisimidazole, EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) or the like. When R is lower alkoxy, this or other known coupling methods for such groups can be used [an overview of these methods can be found in the Method of Organizational Chemistry (Houben-Weyl), vol. XV, Parts 1 and 2 (1974)]. Optionally, R-j_ 'and / or R' are converted to hydrogen.

When the product obtained is an ester, e.g. R is t-butoxy, the ester group can be converted to the free carboxyl group (R is hydroxy) by cleavage with acids, e.g. trifluoroacetic. Conversely, the free acid can be esterified in the usual way.

Starting materials of formula IV wherein m is 0 are derivatives of the amino acid cysteine ​​which can be prepared by known methods.

The acids of formula IV where n is 2, 3 or 4 can be synthesized according to a preferred method by adding a thiol acid to the substituted acrylic acid of the formula:

CH

CO₂H

The latter is obtained by hydrolysis, e.g. with 6N hydrochloric acid, of the methylene lactams of formula (CH.) - NH 1² IN

H₂c - C - C = 0 VI [J. Org. Chem. 39, 893 (1974)].

The compounds of formula I have two asymmetric carbon atoms. These carbon atoms are denoted by a star of formula I. These compounds thus exist in diastereoisomeric forms or in racemic mixtures thereof. All of these fall into general formula I. The syntheses described above may utilize the racemate or one of the enantiomers as starting material. When the racemic starting material is used in the synthesis, the stereoisomers obtained in the product can be separated by chromatography or fractional crystallization by conventional methods. In general, the L isomer with respect to the carbon atom of the amino acid is the preferred isomeric form.

The compounds of formula I form salts which are also prepared according to the invention. The salts include acid addition salts which are formed by reaction with various inorganic and organic acids to give acid addition salts such as e.g. includes hydrohalides (especially hydrochloride and hydrobromide), sulfate, nitrate, borate, phosphate, oxalate, tartrate, maleate, citrate, acetate, ascorbate, succinate, benzenesulfonate, methanesulfonate, cyclohexane sulfamate and toluene sulfonate.

The salts are formed in the usual manner by reacting the product in free acid form with one or more equivalents of the appropriate base to obtain the desired anion or cation in a solvent or medium in which the salt is insoluble, or in water and removing the water by freeze-drying. . By neutralizing the salt with an insoluble acid such as a hydrogen cation exchange resin [e.g. polystyrene sulfonic acid resin Dowex 50 (Mikes, Laboratory Handbook of Chromatographic Methods (Van Nostrand, 1961), page 256], elution with a volatile buffer (e.g. pyridine acetic acid) and extraction with an organic solvent, the free form can be obtained, and optionally another salt is formed.

Further experimental details can be found in the examples which represent preferred embodiments and which also serve as a model for the preparation of other members of the group.

The novel compounds of the invention inhibit the conversion of the decapeptide angiotensin I to angiotensin II and are therefore useful in reducing or alleviating angiotensin-associated hypertension. The effect of the enzyme renin on angiotensin, a blood plasma pseudoglobulin, produces angiotensin I. Angiotensin I is converted by angiotensin converting enzyme (ACE) to angiotensin II. The latter is an active pressor substance which has been suggested to be the substance that induces different forms of hypertension in different mammalian species, e.g. rats, dogs, etc. The compounds of the invention interfere with the angiotensinogen - "angiotensin I -" angiotensin II course by inhibiting the angiotensin-converting enzyme and reducing or eliminating the formation of the angiotensin II pressor substance.

Inhibition of detangiotensin-converting enzyme with compounds of formula I can be measu red in vitro with isolated angiotensin-converting enzyme from rabbit lungs following the procedure described by Cushman and Cheung (Biochem. Pharmacol., 20, 1637 (1971)) excised smooth muscle (E. O'Keefe et al., Federation Proc. 31, (1972)), where these compounds have been shown to be potent inhibitors of the contractile action of angiotensin I and enhancers of the contractile activity of bradykinin.

Administration of a preparation containing one or a combination of the compounds of formula I or a physiologically acceptable salt thereof from various species of hypertensive mammals relieves or reduces angiotensin-associated hypertension. A single dose, or preferably two to four doses daily, given on the basis of approx. 5 to 1000 mg per day kg per day, preferably approx. 10 to 500 mg per day kg per Nowadays, you are appropriate to reduce your blood pressure. The animal model experiments described by SL Angel,

TR Schaeffer, MH Waugh, and B. Rubin, Proc. Soc. Exp. Biol. With. 143, 483 (1973) serves as a useful guide.

The active compound is preferably administered orally, but parenteral administration, such as subcutaneous, intramuscular, intravenous or intraperitoneal, may also be used.

The compounds of the invention can be used to achieve blood pressure reduction by preparing in preparations such as tablets, capsules or elixirs for oral administration or in sterile solution or suspension for parenteral administration. About. 10 to 500 mg of an active compound or a mixture of compounds of formula I or a physiologically acceptable salt thereof are admixed with a physiologically acceptable carrier, excipient, binder, preservative, stabilizer, flavoring, etc., in unit dosage form as required by presumed pharmaceuticals. practice. The amount of active substance in these preparations is such that a suitable dose is obtained in the specified range.

Test Results

The activity of angiotensin-converting enzyme inhibitors is expressed in the following table as the amount of inhibitor in pg / ml required to inhibit by 50% the activity of angiotensin-converting enzyme as measured by spectrophotometric measurement. ΕΟ, -θ is the concentration of

<td>the test compound as contractual effect.</td><td>generates</td><td>50% inhibition of</td><td>angiotensin I * s</td>

<td>Connection</td><td></td><td>^Σ5Ο in pg / ml</td><td>^EC50 in μΜ / 1</td>

<td>s π HS-CH "-CH-CN^L COOH</td><td></td><td> 0,0180</td><td> 0,470</td>

<td>(Cn₂)₃</td><td></td><td></td><td></td>

<td>^NH2-</td><td></td><td></td><td></td>

<td>^{0 0}II II 1 Ί CH_O-CS-CH.-CH-CNL. J £ I</td><td>- COOH</td><td> 0,120</td><td> 2,60</td>

<td>&lt;ch₂&gt;₃</td><td></td><td></td><td></td>

<td>nh₂</td><td></td><td></td><td></td>

<td>Γ² Π HS-CH_O-HC-CN-1- COOH</td><td></td><td> 0,130</td><td> 50,0</td>

<td>v-2 n HS-CH_n-CH-CN LcOOH in</td><td></td><td> 8,0</td><td> 23,0</td>

<td>in? ί η CH.-CS-CH.-CH-CNL 3 2. i</td><td>COOH</td><td> 22,0</td><td> 23,0</td>

C = 0

IN

The following examples are intended to further illustrate the invention. All temperatures are in ° C.

Example 1

N-tert-butyloxycarbonyl-Sp-methoxybenzyl-D-cysteinylL-proline tert-butyl ester

To a solution of L-proline tert -butyl ester (0.85 g) and hydroxybenzotriazole (0.67 g) in methylene chloride (10 ml) cooled in an ice bath, add dicyclohexylcarbodiimide (1.03 g) and N-tert-butyloxycarbonyl -Sp-methoxybenzyl-D-cysteine ​​(1.7 g) in this order. After 15 minutes, the ice bath is removed and the mixture is stirred at room temperature overnight. The precipitate is filtered off and the filtrate is washed with 10% potassium bisulfate, water, saturated sodium bicarbonate and water. The organic phase is dried and concentrated to dryness in vacuo to give N-tert-butyloxycarbonyl-Sp-methoxybenzyl-D-cysteinyl-L-prolonged-butyl ester as an oil. R f = 0.2 (silica gel, chloroform). Example 2

D-cysteinyl-L-proline

To a solution of N-tert-butyloxycarbonyl-Sp-methoxy-benzyl-D-cysteinyl-L-proline-tert-butyl ester (1.8 g) and anisole (4.4 ml) in dichloromethane (8 ml) cooled in a trifluoromethanesulfonic acid (6.0 g) is added. The ice bath is removed and the mixture is stirred at room temperature for 30 minutes. The dichloromethane is removed in vacuo and the residue is triturated with hexane (2 x 200 ml). The residue is dissolved in water and extracted twice with ether. The aqueous phase is added to a column of 200 ml of cation exchange resin [Dowex 50] in hydrogen form. The column is washed with water until no more acidic material is evaporated. D-cysteinyl L-proline acetate is eluted with a pyridine-acetic acid buffer, pH 6.5, yield 0.66 g. Rf = 0.38 (silica gel, chloroform: methanol-acetic acid: water).

Example 3

N, S-diacetyl-DL-cysteinyl-L-proline tert-butyl ester

By using N, S-diacetyl-DL-cysteine ​​instead of N-tert-butyloxycarbonyl-Sp-methoxybenzyl-D-cysteine ​​in the procedure of Example 1, N, S-diacetyl-DL-cysteinylL-proline tert-butyl ester is obtained. R f = 0.25 (silica gel, ethyl acetate).

Example 4

N, S-diacetyl-DL-cysteinyl-L-proline

N, S ~ diacetyl-DL-cysteinyl-L-proline tert-butyl ester (1.9 g) is dissolved in a mixture of anisole (6 ml) and trifluoroacetic acid (12 ml), and the solution is stored at room temperature for 1 hour. The solvents are removed in vacuo and the residue is precipitated from ethyl acetate-ether-hexane to give N, S-diacetyl-DLcysteinyl-L-proline, yield 1.08 g, m.p. 80-140 °.

Example 5

N-acetyl-DL-cysteinyl-L-proline

N, S-diacetyl-DL-cysteinyl-L-proline (0.3 g) is dissolved in a mixture of water (4 ml) and concentrated ammonia (4 ml) under a blanket of argon. The solution is stored for 30 minutes at room temperature, saturated with sodium chloride and extracted with ethyl acetate and chloroform. The organic layers are collected and concentrated to dryness in vacuo to give N-acetyl-DL-cysteinyl-L-proline, yield 0.1 g, = 0.25 (silica gel; benzene: acetic acid, 75:25).

Preparation of a starting material of formula IV in the form of an active ester

A

Methyl-N- (p-methoxybenzyl) nipekotat hydrochloride

A mixture of 23 g of methyl nipecotate, 24.3 g of potassium carbonate and 52 g of p-methoxybenzyl trichloroacetate in 800 ml of toluene is refluxed under nitrogen for 72 hours. The mixture is cooled, the toluene is removed in vacuo, the residue is dissolved in chloroform, and this solution is washed once with 400 ml of aqueous potassium carbonate and then with 400 ml of 10% hydrochloric acid. The chloroform solution is dried and concentrated in vacuo to give a viscous brown oil. Trituration of this oil with ethyl acetate affords 30.7 g of methyl N- (p-methoxybenzyl) nipecotate hydrochloride as an off-white crystalline solid. Recrystallization from ethyl acetate gives an analytical sample, m.p. 150-154 °.

f

B

1- (p-methoxybenzyl) -3-methylene-2-piperidone

A solution of methyl N- (p-methoxybenzyl) nipecotate hydrochloride (30.7 g) and 8.4 g of sodium hydroxide in 900 ml of methanol and 45 ml of water is stirred at room temperature for 17 hours. The solution is evaporated to dryness in vacuo, the residue is diluted with toluene and this is evaporated again to dryness in vacuo. To the residue is added 1 liter of acetic anhydride and 140 ml of triethylamine, and the resulting mixture is heated under reflux for 4 hours. The reaction mixture is evaporated to dryness in vacuo, the residue is taken up in chloroform, washed with water, dried and concentrated in vacuo. The residual oil is chromatographed on silica gel using 1: 1 hexane-ethyl acetate as eluant to yield 16.9 g of 1- (p-methoxybenzyl) -3-methylene-2-piperidone as a chromatographically pure yellow oil. Alternatively, the oil can be distilled to give analytically pure 1- (p-methoxybenzyl) -3-methylene2-piperidone, bp 145-155 ° / 0.1 mm.

C

3-methylene-2-piperidone

A solution of 1- (p-methoxybenzyl) -3-methylene-2piperidone (16.9 g) and 21.3 g of anisole in 400 ml of trifluoroacetic acid is refluxed under nitrogen for 48 hours. The solution is evaporated to dryness in vacuo and the residue is chromatographed on 900 g of silica gel using ethyl acetate as eluant to yield 6.5 g of 3-methylene-2-piperidone, as a crystalline solid.

D

2-methylene-5-aminopentanoic acid hydrochloride

A solution of 2.6 g of 3-methylene-2-piperidone in 150 ml of 6N hydrochloric acid is refluxed for 24 hours. The cooled solution is extracted with chloroform and the aqueous layer is concentrated in vacuo to 3.8 g of glassy foam. The foam is heated with methanol, filtered through Celite (diatomaceous earth clarifier) ​​to remove a small amount of insoluble material, and the filtrate is evaporated to dryness in vacuo to give 2.5 g of 2-methylene-5-aminopentanoic acid hydrochloride as a light brown crystalline , solid. Recrystallization from isopropanol gives an analytical sample, m.p. 138-144 °.

E

2-methylene-5- (p-methoxybenzyloxycarbonyl) aminopentanoic

To a solution of 8.8 g of 2-methylene-5-aminopentanoic acid hydrochloride in 100 ml of water is added with stirring 6.36 g of magnesium oxide, followed by a solution of 12.2 g of p-methoxybenzyloxycarbonyl azide in 100 ml of dioxane, and the resulting mixture Stir at room temperature for 2 days. The reaction mixture is filtered and the filtrate is diluted with 200 ml of ethyl acetate, two equivalents of Dowex 50 ion exchange resin are added and the mixture is stirred at room temperature for 2 hours. The resin is then filtered off and washed with water. The layers of the filtrate are separated and the aqueous layer is extracted twice with ethyl acetate.

The combined organic layers are dried and concentrated in vacuo to give 18.2 g of 2-methylene-5- (p-methoxybenzyloxycarbonyl) aminopentanoic acid as an amber oil which crystallizes on standing. This is used without further purification.

F 2 2-Acetylthiomethyl-5- (p-methoxybenzyloxycarbonyl) aminopentanoic acid

A solution of 2-methylene-5- (p-methoxybenzyloxycarbonyl) amino pentanoic acid (53 mmol) in 50 ml of thiol acetic acid is allowed to stand at room temperature for 48 hours. The solution is evaporated to dryness in vacuo and the residue is taken up in chloroform and added to a silica gel column (700 g). Elution with 5% methanol in chloroform gives 14.2 g of 2-acetylthiomethyl-5- (p-methoxybenzyloxycarbonyl) aminopentanoic acid as an oil. Treatment of this oil with an equivalent of dicyclohexylamine in ether followed by recrystallization from ethyl acetate gives the corresponding dicyclohexylamine salt, m.p. 112-114 °.

G

2-Acetylthiomethyl-5- (p-methoxybenzyloxycarbonyl) amino pentanoic acid-hydroxysuccinimide ester

To a solution of 3.7 g of 2-acetylthiomethyl-5 (p-methoxybenzyloxycarbonyl) aminopentanoic acid and 1.21 g of N-hydroxysuccinimide in 60 ml of dichloromethane at 0-5 ° is added 2.16 g of N, N'-dicyclohexylcarbodiimide for 20 minutes. with stirring. The resulting mixture is stirred overnight at 0-5 °. The precipitated dicyclohexylurea is filtered off, the filtrate is concentrated in vacuo and the residue is taken up in ethyl acetate and washed through a silica gel column to give 4.6 g of 2-acetylthiomethyl-5- (p-methoxybenzyloxycarbonyl) amino-pentanoic acid-N-hydroxysuccinimide crystallizes upon trituration with ether. Recrystallization from ethyl acetate-hexane gives an analytical sample, m.p. 85-87 °.