FI71553B - ANALOGIFICATE FARAMENT FRAMSTAELLNING AV NYA INHIBITORER FOERETT ANGIOTENSIN OMVANDLANDE ENZYM - Google Patents

Description

71553

Analog method for the preparation of new inhibitors of angiotensin converting enzyme

Separated by division of application 770437

The invention relates to an analogous process for the preparation of new inhibitors of the angiotensin-converting enzyme of the formula 10 R3 I4 I1 ί] (I)

15 HS— (CH) CH —CO — N- * 1 — COR

* n * wherein R 1 is hydroxy or lower alkoxy, R 1 and R 2 independently represent hydrogen, lower alkyl or phenyl-lower alkyl and R 1 may further be phenyl, R 1 is hydrogen, hydroxy or lower alkyl, and n is 0, 1 or 2, and for the preparation of their basic salts.

Novel inhibitors can be prepared from a compound of formula R3

30 J

R4 R1 I ^

i i I

Y - S (CH) —CH —CO — N __ * L_ COR

* n * 35 2 71553 wherein R, R 1, R 2 / R 2 and n are as defined above and Y is lower alkanoyl, phenyl-lower alkanoyl, benzoyl, benzyl, methoxybenzyl, triphenylmethyl, tetrahydropyranyl or acetamidomethyl, excluded A sulfur protecting group Y is introduced to regenerate the hydrogen atom.

The asterisks in the formulas indicate asymmetric carbon atoms. Each of the carbons having a substituent R 1, R 2 or R 2 is asymmetric when said substituent 10 is other than hydrogen.

Stereoisomers in which proline is in the L-form are particularly preferred.

Alkyl groups lower than the above substituents are straight or branched chain from methyl to heptyl, e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, iso-pentyl and the like. Lower alkoxy groups are of the corresponding type containing 1 to 7 carbon atoms attached to oxygen, e.g. ethoxy, propoxy, isopropoxy.

The compounds of formula I and its preferred subgroups can be prepared by a variety of synthetic methods.

Compounds of formula I are generally prepared by acylation of a compound of formula (II) 25 - Λ HN_1-COR (II) 35 3 71553 with mercaptoalkanoic acid of formula?

5 I I

Y - S - (CH) n - CH - COR (IHd)

Removal of the protecting group Y can be carried out in a manner known per se, such as by treatment with hot trifluoroacetic acid, cold trifluoromethanesulfonic acid, mercuric acetate, sodium in liquid ammonia, zinc and hydrochloric acid or the like. These methods are described in Methoden der Organischen Chemie (Houben-Weyl), Vol. XV, Part I, starting on page 736 (174).

The protecting group Y is preferably lower alkanoyl or phenyl-lower alkanoyl and is removed by standard hydrolysis, for example by ammonolysis with alcoholic ammonia or concentrated ammonium hydroxide solution or by alkaline hydrolysis with aqueous metal hydroxide such as sodium hydroxide.

The compounds of formula I have one or more asymmetric carbon atoms. When R 1, R 2 or other than hydrogen, the carbon to which they are attached is asymmetric. These carbon atoms are marked with an asterisk in Formula I. The compounds are thus in stereoisomeric forms or as racemic mixtures thereof.

4,71553

All of these forms are within the scope of the invention. The racemate or enantiomer can be used as starting materials in the methods described above. When a racemic starting material is used in the synthesis described above, the stereoisomers contained in the compound can be separated by known chromatographic or fractional crystallization methods. In general, the L-isomer with respect to the carbon of the amino acid comprises the preferred isomeric form. The D-isomer is also preferred over the α-carbon of the acyl side chain (carbon having).

The compounds of the invention form base salts with various inorganic and organic bases and are also within the scope of the invention. Such salts include ammonium salts, early metal salts such as sodium and potassium salts (which are preferred), alkaline earth metal salts such as calcium and magnesium salts, salts with inorganic bases, e.g. dicyclohexylamine salt, benzazine, N-methyl -glucamine, hydrabamine salts, salts with amino acids such as arginine and lysine. Non-toxic, physiologically acceptable salts are preferred, although other salts are also useful, e.g., in isolating or purifying the product as described in connection with the dicyclohexylamine salt in the working examples.

Salts are formed in a manner known per se by reacting the free acid form of the product with an equivalent of a base to give the desired cation, in a solvent or medium in which the salt is soluble, or in water and then removing the water by freeze-drying. Neutralization of the salt with an insoluble acid such as a cation exchange resin in hydrogen form (e.g. polystyrene sulfonic acid resin such as Dowex 50) or aqueous acid and extraction as an organic solvent, e.g. ethyl acetate, dichloromethane or the like can give the free acid and the like free acid and the like.

5,71553

The compounds of the invention inhibit the conversion of decapeptide angiotensin I to angiotensin II and are therefore useful in alleviating or relieving angiotensin-induced hypertension. The effect of the enzyme renin on angiotensinogen / plasma pseudoglobulin produces angiotensin I. Angiotensin I is converted to angiotensin II by angiotensin-producing enzyme (ACE). The latter is an active antihypertensive agent that has been found to cause various forms of hypertension in various mammals, e.g., rats and dogs. The compounds of the invention act on a series of transformations angiotensin (resin) angiotensin I-> angiotensin II by inhibiting the action of an angiotensin converting enzyme and reducing or eliminating the formation of pressure-inducing angiotensin II. Administration of a composition comprising one or more compounds of formula I, or a physiologically acceptable salt thereof, improves angiotensin-dependent hypertension in mammals. A single dose, or preferably 2 to 4 divided daily doses in an amount of about 0.1 to 100 mg per kg per day, preferably about 1 to 50 mg per kg per day, is suitable for lowering blood pressure, as demonstrated in animal experiments described in: S.L. Engel, T.R. Schaeffer, M.H. Waugh and B. Rubin, Proc. Soc. Exp. Bio !. Med 143, 483 (1973). The agent is preferably administered orally, but parenteral administration, such as intravenous, intramuscular, subcutaneous or intraperitoneal administration, may also be used.

One of the elevating systems that regulates blood pressure in the human body is the renin-angiotensin-aldosterone system. Renin, an enzyme, is formed in and released from myoepithelial-35 cells of the glomerular organ group adjacent to the kidney. The mechanism of regulation of renin recruitment is primarily controlled by blood salinity and 6 71553 water content and pressure at which the heart pumps blood through the kidney. As these factors diminish, renii-ni enters directly into the bloodstream. In it, it encounters a substrate d 2-globulin, angiotensinogen (active protein-5). Renin breaks down angiotensinogen into two

also as an inactive component to a tetrapeptide and decapeptide called angiotensin I (A-I). As it circulates in the capillaries (primarily the lung capillaries), A-I encounters angiotensin-converting enzyme 10 (ACE), which is present in the endothelial cells of the capillaries. ACE

breaks down A-I into a dipeptide and an octapeptide, or angiotensin II (A-II), which is the most potent antihypertensive agent in the human body.

The antihypertensive effect of A-II occurs locally in the muscles and also in the central nervous system; The posterior region of the brain, after being stimulated by A-II, sends impulse-raising impulses to the rest of the body. In addition, A-II produces another effect, e.g. physiological stimulation of the secretion of aldosterone by the adrenal cortex-20 of a mineralocorticoid. Aldosterone increases the resorption of sodium ions from the renal tubules, increasing the blood volume so that blood pressure still rises as a result of this second mechanism.

25 Apart from the conversion of A-I to A-II, ACE has an additional function. The blood contains an octapeptide bradykinin with vasodilatory properties; ACE converts bradykinins to inactive components. In relation to this function, ACE is sometimes known as kininase II.

In people with hypertension, the renin-angiotensin-aldosterone system is disrupted, which adversely raises the patient's high blood pressure. A drug that inhibits ACE produces the following effects: 7 71553 1. Inhibition of A-II formation and thus: a) direct inhibition of vasoconstriction b) removal of aldosterone from the adrenal cortex (i.e., water and inhibition of excessive salt resorption 5) and 2. inhibition of ACE results in inhibition of the degradation of bradykinin so that its vasodilatory (antihypertensive) effect is maintained.

None of the conventional antihypertensive drugs is an ACE inhibitor.

The compounds of formula I are potent inhibitors of ACE. They prevent the conversion of the decapeptide angiotensin I to angiotensin II and thus minimize and eliminate angiotensin I-induced hypertension. The compounds of the invention lack the conversion of angiotensinogen (renin) -angiotensin I to angiotensin II by blocking ACE and thus minimizing and eliminating the formation of angiotensin II, which is the compound that causes the increase in ve-20 pressure. The effective site on the molecule for the action of the compounds of formula I is completely different from the effective site on the molecule for commonly used antihypertensive drugs. The compounds according to the invention make it possible, in contrast to conventional antihypertensive drugs, to address the factors which cause hypertension.

As already mentioned above, the pharmacological utility of the compounds of formula I is based on their ability to inhibit the action of the angiotensin-modifying enzyme-30 min. Inhibition is determined in vitro by measuring the effect of an angiotensin-converting enzyme isolated from rabbit lung and determining the inhibitory effect of angiotensin I-induced contractions in the guinea pig ileum.

35 8 71553

Inhibition of angiotensin converting enzyme isolated from rabbit lung____

The activity of angiotensin-converting enzyme in cell-free extract -5 isolated from rabbit lung is measured spectrophotometrically by determining the hippuric acid released from hippuryl-L-histidyl-L-lenin (HHL) by the method described by Cushem & Cheung, Bioch. Pharmacol.

20, 1637 (1971).

/ (^) \ -Gly-His-Leu -> / - + His-Leu '-' HHL \ - / hippuric acid 15 In this spectrophotometric test, the following components must be incubated at 37 ° C in a volume of 0,25 ml: 100 mM phosphorus buffer (pH 8.3) 300 mM sodium chloride 0.001-1000 yug / ml inhibitor or without 5-10 mUnits of enzyme

After incubation of the above components at various concentrations of inhibitor (with or without inhibitor) for 30 min, the enzyme reaction is stopped by the addition of 0.25 ml of hydrochloric acid. The check is performed by inactivating the enzyme at time zero by adding acid before the enzyme. Ethyl acetate (1.5 ml) is added to each test tube to extract the hippuric acid formed by the enzyme. After vortexing for 20 seconds and separating the ethyl acetate layer by centrifugation for 1a, take a 1.0 ml batch and evaporate to dryness at 100 ° C and redissolve the hippuric acid in 1.0 ml of water and its quantitative amount is calculated from the absorbance at 228 nm.

The inhibitory capacity of the angiotensin converting enzyme is expressed as I, which is the amount of inhibitor 71553 at 9 μg / ml or μM required to inhibit 50% of the activity of the angiotensin converting enzyme as measured by spectrophotometry. This value is obtained by plotting the percentage inhibition produced by the inhibitor concentration from 2 to 3 times in the range of 0.001 ug / ml to 1000 ug / ml. Percent inhibition is calculated from the equation% inhibition = (A-A °) - (B-B °) (A-A °) 10 A = absorbance of hippuric acid for 30 min in the experiment without inhibitor A ° = background absorbance in the experiment incubated for 0 min.

15 B = absorbance of hippuric acid for 30 min in the assay at the inhibitor concentration examined to determine the percentage inhibition B ° = background absorbance in the assay containing inhibitor but with an incubation time of 0 min.

20 Method of angiotensin I-converting enzyme (ACE) to investigate the inhibitor activity of guinea pig ileum in vitro__ This method is used to declare whether a compound of angiotensin I-converting enzyme inhibitors other-25 rotor and the assay is based on the activity of angiotensin test compound I-induced against guinea-pig ileal contractions . ACE inhibitors generally inhibit angiotensin I-induced contractions.

Feeded Hartley guinea pigs of both sexes weighing 300-400 g were killed by striking the head. The ileum was removed and 2-3 cm samples were prepared by the Vane method. Each strip of ileum was suspended in a 10 ml tissue bath 2 filled with Krebs' solution at 37 ° C and aerated with 95% 0 £ and 5% CO 2 gas, muscle activity was recorded isotonically at 10 71 553 1 g: n using a Harvard ^ 356 cardiac / smooth muscle transducer connected to a Backman Type R Dynograph. After equilibration of the tissues for about 90 minutes, 2-minute control responses were obtained every 10 minutes with the following operator:

Antiotensin I (A-J) 0.025 μg / ml FBC (FBC = final bath concentration). The contractions caused by this A-I concentration represent 50-75% of the maximum value.

The test compound is usually dissolved in water or saline and diluted 10-fold with 1 x 0: 11. The operator is pretreated with the test compound for 2 min and the percentage change in contraction responses is calculated. Highly active compounds, i.e.

Compounds that are likely to be ACE inhibitors usually inhibit angiotensin I-induced contractions.

The EC50 is sometimes referred to as IC50 (the concentration of test compound that provides 50% inhibition of the contractile potency of A-I) is assessed graphically or biometrically.

Literature 1. Vane, J.R. Brit. J Pharmacol. 23: 360, 1964 2. Handschumacher, R.C. and J.R. Vane. Brit. J.

25 Pharmacol. 29: 105, 1967.

3. Rubin, B., E. O'Keefe, D.G. Kotler, D.A.

DeMaio and D.W. Cushman. Fed. Proc. 34: 770, 1976.

4. O'Keefe, E.H., Kotler, D.G., Waugh, M.H. & Rubin, B. Fed. Proc. 31: 511, 1972 30 5. Rubin, B., Laffan, R.J., Kotler, D.G., O'Keefe, E.H., DeMaio; D. A., & Goldberg, M.E. J. Pharmacol.

Exper. Therap. 204: 271, 1978.

HA-B χ 100 A = A-I contractions (mms) before addition of test compound A.

35 B = A-I contractions (mms) after addition of test compound.

n 71553

The following Table I shows the values of 1 and IC 1 -q (EC 2 q) of the compounds of formula I.

12 71 553 s en r- <£> · * τ cm ro o ro m r- .h

W £> ΓΟ VO O CM U ~) O VOrHOO

3 ooooo o r- cm i o o \ V fc, V fc * S ^ ^ ^ w

w OOOOO OfHOOOO

g

Ph o \ m tn oo »h CN

,, ^ 21 <T \ ro ro in r-ι o <N co rH r-4 o

I H 3 r-H (N O Ο <N O LO Ή O O O

\ ^ S V w w l »^« k I »0 '-' ooooo oooooo oT-g'5 *" * Ί I C ΟΟΟιΗιΗ r-lp-I r-l i—! R — I * —i o u - s

M CM

3 κ i

Ή O

2 u <0

H I

«.Ns S <N <NCM <N <N <N <N <N <N <NCM

c pfig

I ro w ΖΠ iC

Sg ps κ κ κ κ o κ κ κ κ o o • ♦

pf. > IKK K K K K K K

H QJQJ ^ Q

* ffi ^ ~ ^ - 'Q ^ - roro ro ro ro' - · ro ro

υυκκ δ δ S S δ S

H K K K K K K K K ^ 1 KKK

PS ooooo oozooou 71 553 S 'ä m vo r) nF h ΙΟ Ifi Γ "O <N lT> H 7 ?, o ^ rovooo ^ ro Γ'ΙΠΊ · ooo ^ oooo ^ ^ (NOO I oooooooo • ä a>, -, -3r σ> ^ n Γ 'Φ

^ [n fTi rovo OVOOCNOrHrHO

iM g <h cn m «noooooooo

0 (NOO (NOOOOOOOO

0

rflglB

| _ | ϋ | g (N rHrH i-Hr-lr — IrHr-li— | r — IrHtH

9 4j * 2 tn i

iH O

a u (0

H I

sT-g E <N (N (N <N (N (N (N (N (N (N (N (N

m ofig ^

Jj oT aaa k! EkkkSV °° «« ä

pT4 eg ^ sg aaciEKKKKffiK

g

_ I

et 3 s § - 5 g s § S S u in m fo * '-' s— '^ S ro ro B ffi U ro ro ro ro

g 8 S ifiTi S ϊ B S

ro af1 ^ «g g g Sgggggggg g i4 71 5 5 3 cf Ϊ = 0 örri vomm o m vo vo ro σ \ 3 · —i o cn m h o ld r-t vo

^ ^ Fei ^ ^ ^ ^ V

- OOOIOOOOOO

r-i g cc o \ m ^ o u-ityi voo'J 'vocNLnmro I H 3 r-HvOCvirNCOCNOOro ^

O - · OOOCOOOOOOrH

u _ε 'oT-S-0

M '1 C »Hr — li — li — Ι» Η »Η» ΗιΗι — li — I

Q U -Z

. £ CN

2 κ i

»H O

3 u <0

E-ι I

ηϋδ E <N (NrM (NCN | (N (NCNCN (N

I »H

c 8 cr w οΓ-S t g pT ffiffiffiKKKZZK53 '^

J -H

Ω rH

> 1 -H tn S rH

H Jj i> i -H

«TC 33 ffi K ffi K r ~~ S tn> i 3 3 s y lii QQQQQ SS. * C g .g«

'- - ^ · - in in H

ro ro ro ro ro iH h e ii ii ii ^ s s s s s a a Ä

S C C i K

aT a ^ ΐ: £ a01 “· I

nkU "^ 8 I hit I Ö 33 33 33 33 3

CC oooooooooo K

15 71 553

The compounds of the invention may be used to lower blood pressure by formulating compositions such as tablets, capsules or liquids for oral administration or sterile solutions or suspensions for parenteral administration. About 10 to 500 mg of a compound of formula I or a mixture of compounds or a physiologically acceptable salt thereof is combined with a physiologically acceptable medium, carrier, additive, binder, preservative, stabilizer, flavoring agent, etc. in unit dosage form according to accepted pharmaceutical practice. The amount of active ingredient in these compositions or preparations is such that a suitable amount will be obtained within the range indicated.

Exemplary additives that can be used with tablets, capsules, and the like include: a binder such as tragacanth, acacia, corn starch, or gelatin; an additive such as dicalcium phosphate; a disintegrant such as corn starch, potato starch, alginic acid and the like; a lubricating agent such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; a flavoring agent such as peppermint, wintergreen oil, or cherry.

When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. Various other agents may be present as coatings or for other modification of the physical form of the unit dose. For example, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, a precipitate-rose as a sweetening agent, methyl and propyl paraben as a preservative, a coloring agent and a sweetening agent such as cherry or pomegranate essence.

Suitable compositions for injection are obtained in accordance with standard pharmaceutical practice by dissolving or suspending the active ingredient in a vehicle such as water for injection in naturally occurring vegetable oils such as sesame oil, peanut oil, coconut oil, cottonseed oil, etc. such as ethyl oleate or the like. Buffers, preservatives, antioxidants and the like may be added as needed.

The following examples illustrate the method according to the invention and particularly preferred embodiments. All temperatures are in degrees Celsius.

Example 1 1- (2-Mercaptoacetyl) -L-proline 1- (2-Benzoylthioacetyl) -L-proline (3.4 g) is dissolved in a mixture of water (10.5 ml) and concentrated ammonia (6.4 ml). After 1 hour, the reaction mixture is diluted with water and filtered. The filtrate is extracted with ethyl acetate and acidified with concentrated hydrochloric acid, saturated with sodium chloride and extracted twice with ethyl acetate. The ethyl acetate extracts are washed with saturated sodium chloride and concentrated to dryness, yield 1.5 g. The product 1- (2-mercaptoacetyl) -L-proline is crystallized from ethyl acetate (mp 133-135 °).

Example 2 1- (2-Mercaptopropanoyl) -L-proline 1- (2-Benzoylthiopropanoyl) -L-proline (5.7 g) is dissolved in a mixture of water (12 ml) and concentrated ammonium hydroxide (9 ml) with stirring. After 1 hour, the mixture is diluted with water (10 mL) and filtered. The filtrate is extracted twice with ethyl acetate, concentrated to 1/3 volume, acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The organic phase is washed with saturated sodium chloride, dried and concentrated to dryness in vacuo. The residue 1- (2-mercaptopropanoyl) -L-proline is crystallized from ethyl acetate / hexane, yield 3 g, m.p. (105) 116-120 ° C.

17 71553

Example 3 1- (3-mercaptopropanoyl) -L-proline

Method A

1- (3-Benzoylthiopropanoyl) -L-proline (4.9 g) is dissolved in a mixture of water (8 ml) and concentrated ammonium hydroxide (5.6 ml), and the solution is kept under stirring under an argon atmosphere for 1 hour. The reaction mixture is diluted with water, filtered and the filtrate is extracted with ethyl acetate. The aqueous phase is acidified with concentrated hydrochloric acid, saturated with sodium chloride and extracted with ethyl acetate. The organic layers are washed with saturated sodium chloride, dried over magnesium sulfate and concentrated to dryness in vacuo. The residue 1- (3-mercaptopropanoyl) -L-proline is crystallized from ethyl acetate / hexane, yield 2.5 g, m.p. 68-70 °.

Method B

1- (3-Acetylthiopropanoyl) -L-proline (0.8 g) is dissolved in 5.5 N methanolic ammonia (5 ml) and the solution is kept under argon at room temperature. After 2 hours, the solvent is removed in vacuo, the residue is dissolved in water and passed to an ion exchange column in the H + step (Dowex 50 (analytical grade)) and eluted with water. The fractions giving a thiol-positive reaction are collected and concentrated to dryness, yield 0.6 g. This product is crystallized from ethyl acetate / hexane according to Method A to give 1- (3-mercaptopropanoyl) -L-proline.

salts

Sodium 1- (3-mercaptopropanoyl) -L-proline (500 mg) is dissolved in a mixture of water (2.5 ml) and N sodium hydroxide (2.5 ml). The solution is lyophilized to give the sodium salt.

Magnesium 35 1- (3-mercaptopropanoyl) -L-proline (500 mg), magnesium oxide (49.5 mg) and water (10 ml) are mixed with 18 71 55 3 while gently heating until a complete solution is formed. The solvent is then removed by freeze-drying to give the magnesium salt.

Potassium 5 1- (3-mercaptopropanoyl) -2-proline (500 mg) is dissolved in a mixture of calcium hydroxide (91 mg) and water (10 ml), and the solution is lyophilized to give the calcium salt.

Potassium 10 1- (3-mercaptopropanoyl) -L-proline (500 mg) is dissolved in a mixture of potassium bicarbonate (246 mg) and water (10 ml) and lyophilized to give the potassium salt.

N-methyl-D-glucamine 15- (3-Mercaptopropanoyl) -L-proline (500 mg) and N-methyl-D-glucamine (480 mg) are dissolved in water (10 ml) and freeze-dried N-methyl-D-glucamine to obtain the salt.

Example 4 1- (3-Mercaptopropanoyl) -L-hydroxyproline Using L-hydroxyproline and treating the product according to Method A of Example 3, 1- (3-benzoylthiopropanoyl) -L-hydroxyproline and 1- (3-mercaptopropanoyl) -L-hydroxyproline are obtained. respectively 25 (m.p. 193.5-195 ° C of the latter as the dicyclohexylamine salt).

Example 5 1- (3-Mercaptopropanoyl) -D-proline Using D-proline and treating the product according to Method A of Example 3, 1- (3-benzoylthiopropanoyl) -D-proline and 1- (3-mercaptopropanoyl) -D are obtained. -proline, m.p. 68-70 °.

19 71 55 3

Example 6 1- (3-Mercapto-2-D-methylpropanoyl) -L-proline 0.85 g of 1- (3-acetylthio-2-methylpropanoyl) -L-proline is dissolved in 5.5 N methanolic ammonia and a solution kept at room temperature for 2 hours.

The solvent is removed in vacuo and the residue is dissolved in water, fed to an ion exchange column in the H + phase (Dowex 50, analytical grade) and eluted with water. The fractions which give a positive thiol reaction are collected and freeze-dried. The residue is crystallized from ethyl acetate / hexane, yield 0.3 g. 1- (3-mercapto-2-D-methylpropanoyl-L-proline melts at 103-104 °, (o <fO-131 (C, 2, EtOH).

Example 7 1- (3-Mercapto-2-benzylpropanoyl) -L-proline 1- (3-acetylthio-2-benzylpropanoyl) -L-proline is treated with methanolic ammonia according to Example 6 to give 1- (3-mercapto -2-benzylpropanoyl) -L-proline as an oil, Rf = 0.47 (silica gel, benzene / acetic acid 75:25).

Example 8 1- (3-Mercapto-2-methylpropanoyl) -L-hydroxypropyl Treatment of 1- (3-acetylthio-2-methylpropanoyl) -L-hydroxyproline tert-butyl ester according to Example 6 gives 1- ( 3-mercapto-2-methylpropan-2-yl) -L-hydroxyproline as an amorphous substance -61.90 (c = 2, methanol).

Example 9 1- (4-Mercaptobutanoyl) -L-proline 1- (4-Benzylthiobutanoyl) -L-proline (1.08 g) is dissolved in a mixture of water (4 ml) and concentrated ammonia (2.7 ml). After stirring for 1 hour at room temperature, the mixture is diluted with water, filtered, extracted with ethyl acetate and the aqueous phase is concentrated in vacuo. The ammonium salt of 1- (4-mercaptobutanoyl) -L-proline is purified by ion exchange chromatography using a diethylaminoethyl-Sephadex (cross-linked with dextran) column and eluting with ammonium bicarbonate, yield 0.7 g. The airanonium salt is dissolved in water (2 ml) and applied to a column of Dowex 50 sulfonic acid resin (analytical grade) in the form of hydrogen and the free acid is eluted with water. Fractions containing the desired substance (sulfhydryl reagent and carboxyl reagent positive) are collected and lyophilized to give 1- (4-10 mercaptobutanoyl) -L-proline. The dicyclohexylammonium salt is obtained by dissolving the compound obtained above in ethyl acetate and adding dicyclohexylamine. The crystalline salt is filtered and recrystallized from isopropanol, m.p. 157-158 °.

Example 10 1- (3-Mercaptobutanoyl) -L-proline 1- (3-Acetylthiobutanoyl) -L-proline (0.86 g) is dissolved in 5.5 N methanolic ammonia (20 ml) and the reaction mixture is kept at room temperature for 2 hours.

The solvent is removed in vacuo and the residue is chromatographed on an ion exchange column (Dowex 50) using water as eluent. Fractions containing the desired 1- (3-mercaptobutanoyl) -L-proline are collected and lyophilized, yield 0.6 g. The dicyclohexylamine salt 25 m.p. 183-184 ° C.

Example 11 1- (3-Mercapto-2-phenylpropanoyl) -L-proline According to Example 6, 1- (3-acetylthio-2-phenylpropanoyl) -L-proline and 1- (3-mercapto-2-phenyl-lipropanoyl) are obtained. ) -L-proline. + 2.5 ° (c = 1 / ethanol). The final product m.p. 49-52 ° C.

Example 12 1- (3-Mercapto-2-methylpropanoyl) -L-proline 1- [3- (4-methoxyphenyl) methylthio] -2-methylpropanyl-L-proline tert-butyl ester (1.2 g , 0.003 mol), anisole (5 ml) and trifluoromethanesulfonic acid (0.5 ml) are dissolved in 20 ml of trifluoroacetic acid under a nitrogen atmosphere and the resulting red solution is allowed to stand for 1 hour at room temperature. The solution is evaporated in vacuo to a red residue which is taken up in ethyl acetate and washed with water, brine, then dried (MgSO 4) and evaporated. The residue is stirred several times with hexane and the remaining hexane is evaporated; the oily residue is 0.4 g. A portion of this material (180 mg) is subjected to preparative thin layer chromatography using 2 mm silica gel plates and benzene / acetic acid 75:25 as eluent. The main nitroprusside-positive band (Rf = 0.40) is recovered to give 135 mg of 1- (3-mercapto-2-methylpropanyl) -L-proline as an oil. Thin layer chromatography using Bentene / acetic acid 75:25 (Rf = 0.40) and chloroform / meta-15 ol / acetic acid 50:40:10 (Rf = 0.62).

Example 13 1- (3-Mercapto-2-D-methylpropanoyl) -L-proline

Under an argon atmosphere, 1- [3- (acetylthio) -2-D-methylpropanoyl] -L-proline (10.0 g) is slurried in 150 ml of water at 10 °. To this mixture is added 5N sodium hydroxide and the pH of the solution is maintained at 13 for 1.5 hours. After consumption of sodium hydroxide is complete, the solution is acidified to pH 2.0 with concentrated sulfuric acid.

The aqueous solution is then extracted three times with methylene chloride (3 x 150 mL) and the combined methylene chloride fractions are concentrated to an oil. The concentrate is taken up in ethyl acetate, filtered and the filtrate is diluted with hexane (30 mL). After more than 1/2 hour 30, more hexane is added and the mixture is cooled to 10 ° for 1 hour.

The crystals are filtered and washed with hexane (2 x 25 ml) and dried to constant weight to give 6.26 g of 1- (3-mercapto-2-D-methylpropanoyl) -L-proline as white crystals, m.p. 100-102 °.

35 22 71 5 5 3

Example 14 1- (3-Mercaptopropanoyl) -L-proline To a solution of 75 mg (0.27 mmol) of N - (ethylamino) carbonyl / thio7-propanoyl-N-L-proline in 1 ml of concentrated ammonium hydroxide and 1 The solution is diluted with a small amount of water and extracted with ether, the aqueous layer is acidified with cold concentrated hydrochloric acid 10 and extracted with ethyl acetate. identical to thin layer thin layer chromatography (silica gel, benzene: acetic acid 7: 3) Rf = 0.4.

Example 15 1- (3-Mercapto-2-methylpropanoyl) -L-proline Zinc dust (10.0 g) is added to a slurry of 1- [E (dithiobis- (2-methyl-3-propanoyl)] -bis- proline (5.0 g) in 100 ml of 1.0 N sulfuric acid, and the mixture is stirred at 18 ° for 4 hours under a nitrogen atmosphere, the solution is filtered, the zinc is washed with water (20 ml) and the combined filtrates are extracted with methylene chloride ( 3 x 75 ml) The methylene chloride washings are extracted with water (25 ml) and then the organic solution is concentrated to an oil.

This oil is taken up in ethyl acetate (20 ml) and filtered. Hexane (15 mL) is added to the filtrate and the mixture is stirred for 15 minutes. Additional hexane (30 mL) is then added and the solution is cooled to 5 ° for 1 hour. The mixture is then filtered and the product is washed with hexane (2 x 10 ml) and dried to give 4.17 g of 1- (3-mercapto-2-methylpropanoyl) -L-proline as white crystals. TLC, = 0.60 (solvent system: benzene / acetic acid 75:25).

2λ 71553

Example 16 1- (3-Mercapto-2-methylpropanoyl) -L-proline 1- [3- (benzylthio) -2-methylpropanoyl] -L-proline (0.1 g) is suspended in boiling liquid ammonia (10 g). ml) and add small pieces of sodium while stirring until a permanent blue color. The color is removed by adding a few crystals of ammonium sulfate and the ammonia is allowed to evaporate under a stream of nitrogen. The residue is dissolved in a mixture of dilute hydrochloric acid and ethyl acetate. The organic layer is dried and concentrated to dryness in vacuo to give 1- (3-mercapto-2-methylpropanoyl) -L-proline. Rf: 0.5 (silica gel; benzene / acetic acid 3: 1); Rf 0.5 (silica gel; methyl ethyl ketone / acetic acid / pyridine / water 14: 1: 2: 1), which is identical to the compound of Example 6.

Example 17 1- (3-Mercapto-2-D-methyl-1-oxopropyl) -4-cis-methyl-L-proline

To a solution of 1- (3-mercapto-2-D-20 methyl-1-oxopropyl) -4-cis-methyl-L-proline (1.67 g, 6.1 mmol) and methanol (10 mL) , aqueous NaOH (1N, 15.25 mL) is added and the mixture is allowed to stand under argon for 15 min. I drive. The solution is centrifuged to separate a small amount of suspended solid and the methanol is removed from the supernatant in vacuo. The remaining aqueous solution is acidified with HCl (3 N, 5 mL) and extracted thoroughly with ethyl acetate. The ethyl acetate is removed in vacuo and the residue is chromatographed on 2 silica gels (E. Merck, 70 g) at 1.4 kp / cm 3 using a mixture of benzene and acetic acid (7: 3) as eluent. The product from the column is dissolved in chloroform, the chloroform is removed in vacuo to give the desired product as a viscous oil. Yield 950 mg (67%), m.p. = -139.7 ° (c = 1.26, CHCl3). Rf = 0.54 35 (silica gel, benzene: acetic acid 7: 3). The dicyclohexylamine salt of 24 71553 is obtained by using ethyl acetate as a solvent. Mp = -62.6 ° (c = 1.07, CHCl 3), m.p. (175 °) 177-179 ° C.

Analysis: cxoH17NO3S dicyclohexylamine 5 Calculated C, 64.04, H, 9.77, N, 6.79, S, 7.77 Found C, 63.67, H, 9.98, N, 6.74, S, 7.74 HS: 99.4%

Example 18 1- (3-Mercapto-2-D-methyl-1-oxopropyl) -4-cis-heptyl-L-proline ___

The compound 1- [3- (acetylthio) -2-D-methyl-1-oxopropyl] -4-cis-heptyl-L-proline (3.3 g) is treated under argon with a cold solution of 10 ml of concentrated ammonia and 25 ml of concentrated ammonia. ml of water. When the mixture is stirred for several 15 minutes, it turns into a solution. The solution is cooled and extracted with 20 mL of EtOAc (2 times). The aqueous phase is stirred, 20 ml of EtOAc are poured on it, it is acidified with 6N hydrochloric acid and the layers are separated. The aqueous phase is extracted with EtOAc (2 times). The organic phases are combined, dried over MgSO 4, filtered and the solvent is separated off to give 2.87 g (98%) of an almost colorless syrup, = 74 ° (1% in EtOH), 0.22 (85% toluene). - 15% acetic acid on silica gel,

Rf 0.72 (95: 5: 5 CH 2 Cl 2 - MeOH - AcOH on silica gel).

Analysis: c16H29NO3S · 0.25 H2O:

Calculated C, 50.05, H, 9.45, N, 4.38, S, 10.02, SH, 10 Found C, 60.44, H, 9.36, N, 4.48, S, 10 , 12, SH, 10.

Example 19 1- [3-Mercapto-2-methylpropanoyl] -L-proline 1- [3- (triphenylmethylthio) -2-methylpropanoyl] -L-proline tert-butyl ester (5 g) is dissolved in sni-Sol (55 g). ml) and trifluoroacetic acid (110 ml).

35 After 1 hour at room temperature.

The solvent is removed in vacuo and the residue is applied to a silica gel column equilibrated with benzene / acetic acid. (75:25) and eluted at the same time as solvent. Fractions corresponding to component Rf 0.40 (TLC on silica gel 5 with the same system) are collected and concentrated to dryness to give 1- (N-mercapto-2-methylpropanoyl) -7-proline. Rf 0.62 (silica gel, chloroform / methanol / acetic acid / water 50:40:10 identical to the compound of Example 6).

Example 20 1- (3-Mercapto-2-methylpropanoyl) -L-proline A solution of 1- (3- (tetrahydropyran-2-ylthio) -2-methylpropanoyl) -7-L-proline (1 g ) in a mixture of methanol (25 ml) and concentrated hydrochloric acid (25 ml) is kept at room temperature for 30 minutes. The solvents are removed in vacuo to give 1- (3-mercapto-2-methylpropanoyl) -L-proline. Rf: 0.35 (silica gel, benzene / acetic acid, 3: 1), Rf 0.5 (silica gel, methyl ethyl ketone / acetic acid / pyridine / water 14: 1: 2: 1), identical to Example 6 with a compliant product.

Example 21 1- (3-Mercapto-2-methylpropanoyl) -L-proline 1- [3- (acetamidomethylthio) -2-methylpropanoyl] -L-proline (1.4 g) and mercuric acetate (1.93 g) solvent-25 to a mixture of acetic acid (25 ml) and water (25 ml). After stirring for 1 hour on a steam bath, hydrogen sulfide is bubbled through until no more mercury sulfide precipitate is observed. The mixture is filtered, the precipitate is washed with ethanol and the filtrate is concentrated to dryness in vacuo to give 1- (3-mercapto-2-methyl-propanoyl) -L-proline. Rf: 0.35 (silica gel, benzene / acetic acid 3: 1), Rf: 0.5 (silica gel, methyl ethyl ketone / acetic acid / pyridine / water 14: 1: 2: 1), identical to the compound of Example 6 with.

35 26 71 S 5 3

Example 22 1- (2- (Mercaptomethyl) -1-oxo-9-phenylnonyl] -L-proline____________

The compound 1 - [* 2- (acetylthiomethyl) -1-oxo-®t-5-phenylnonyl) -L-proline is taken up in 15 cm3 of cold methanol under argon. To this is added 15 cm3 of cold concentrated ammonium hydroxide. 30 min. after which the reaction mixture is acidified with concentrated HCl and extracted with ethyl acetate to give 4 g of a clear oil (m.p. 104 DEG-110 DEG C. as an adamantylamine salt prepared from ether - hexane).

Example 23 1- (2-Mercaptomethyl) -1-oxononyl-7-L-proline 1.7 g of 1- (2- (acetylthiomethyl) -1-15 oxononyl) -L-proline are taken up in 8 ml of cold methanol under argon and this is treated with 8 ml of cold concentrated ammonia After 30 minutes the reaction mixture is cooled, acidified with concentrated HCl and extracted with ethyl acetate to give, after evaporation to dryness, 1.5 g of product as an oil (m.p. 159-162 ° as the adamantyl amine salt).

Example 24 1- (3-Mercapto-1-oxodecyl) -L-proline

15 g (0.043 mol) of (+) - 1- (3-acetylthio) -1-oxode-25-yl / -L-proline from position B

3 160 cm water 3 65 cm concentrated ΝΗ.0Η 4

The mixture of components is stirred under nitrogen at room temperature for about 3 h. The alkali-insoluble matter is extracted with ethyl acetate. The pale yellow aqueous layer is covered with ethyl acetate and strongly acidified with 20% HCl. Layers 3 are separated and the aqueous layer is extracted with a further 200 cm of ethyl acetate. The combined organic layers are washed with saturated NaCl and dried over MgSO 4. The solvent is removed to give 8.7 g (67%) of a residue which gives a strong SH test. The residue is dissolved in 200 cm of benzene and lyophilized to give 8.4 g (62%) of the title compound as a viscous liquid.

Analysis: C ^ j-I ^^ NO ^ S. 0.5. Ν 0: 5 Calculated: C, 58.02, H, 9.09, S, 10.33, N, 4.51, SH, 100% Found C, 57.77, H, 9.37, S, 10 , 24, M, 4.47, SH, 98.9%.

Claims (9)

28 71 553 1. An analogous method for the preparation of new angiotensin converting enzyme inhibitors of the formula I3 R> 1 R, I4 i1 I HS- (CH) -CH-CO-N - & - COR * n * c wherein R is hydroxy or lower alkoxy, R1 and R1 independently of one another are hydrogen, lower alkyl or phenyl-lower alkyl and R R 1 may further be phenyl, R 1 is hydrogen, hydroxy or lower alkyl, and n is 0, 1 or 2, and for the preparation of their basic salts, characterized in that the compound of formula 20; - (CH) -CH-CO-N - ^ - COR * n * 25 wherein R, R1, R1, R1 and n are as defined above and Y is lower alkanoyl, phenyl-lower alkanoyl, benzoyl, benzyl, methoxybenzyl, triphenylmethyl, tetrahydropyranyl or acetamidomethyl are removed with a sulfur protecting group Y to regenerate the hydrogen atom. Process according to Claim 1, characterized in that the protecting group Y is lower alkanoyl, phenyl-lower alkanoyl and that the protecting group is removed by hydrolysis. 35 29 71 553 Process according to Claim 2, characterized in that the protecting group Y is removed by ammonolysis. Process according to Claim 2, characterized in that the protecting group Y is removed by alkali hydrolysis with a metal hydroxide. Process according to one of Claims 1 to 4, characterized in that a compound of the formula 10 f · O is prepared HS - (CH ~) —CH — CO — N -— COR 2 'n * wherein R, R 1 and n are as defined in claim 1. Process according to one of Claims 1 to 5, characterized in that R is hydroxy. Process according to Claim 6, characterized in that R 1 is methyl. Process according to Claim 7, characterized in that 1- (3-mercapto- 2-D-methylpropanoyl) -L-proline. 9. Compounds which are intermediates in the preparation of therapeutically useful proline derivatives, characterized in that they have the formula 30 * 3 RR I4 I1 Γ Y-S - (CH) -CH-CO-N-COR * n * 35 wherein R 1 is hydroxy or lower alkoxy, R 1 and R 2 independently of one another are hydrogen, lower alkyl 71553 or phenyl-lower alkyl and may in addition be phenyl, R 1 is hydrogen, hydroxy or lower alkyl, the protecting group Y is lower alkanoyl, phenyl-lower alkanoyl, benzoyl, benzyl, methoxybenzyl, triphenylmethyl, tetrahydropyranyl or acetamidomethyl and n is 0, 1 or 2. Intermediates according to Claim 9, characterized in that they have the formula OY-S- (CH-) - CH-CO-N-1 COR 2 n. * 15 in which R, R 1, Y and n have the same meaning as in the claim 9. Intermediates according to Claim 10, characterized in that R is hydroxy. Intermediates according to Claim 11, characterized in that R 1 is methyl. Intermediate according to Claim 12, characterized in that it is 1- (3-acetylthio- 2-D-methylpropanoyl) -L-proline. 31 71553