SE446093B - LONG-CHAIN FAT ACID AMOIDS AND HYDRAZIDES AND CYCLOPROPAN ANALYSIS THEREOF FOR USE AS ANTIATEROSCLEROTIC AGENT AND PHARMACEUTICAL COMPOSITION - Google Patents

Description

10 15 25 50 7900142-6 2 CQOR7 -CH-CHZ I __ \ Il l "" RL; Wherein R 4 is as defined above, wherein R 7 is (C 1-8] alkyl or benzyl, and R 8 is hydrogen, (C 1-8) alkyl or benzyl, provided that when R 2 is a group of formula II or III A has below (ii) the definition given above.

In a group of the compounds I according to the invention, A is as indicated under (i) (A is hereinafter referred to as A ') a residue of a long-chain fatty acid, this residue having 7-25 carbon atoms and having 1 to 4 ethylenically unsaturated groups. These residues have thus been obtained from long-chain fatty acids of the formula A'-COOH having 8 to ZH carbon atoms and 1 to N ethylenically unsaturated groups. These acids are preferably unbranched and preferably of the natural fatty acid type, i.e. those containing an even number of carbon atoms. A 'is thus preferably unbranched and preferably contains an odd number of carbon atoms.

Particularly preferred residues A 'comprise those of formula IX (residues A1) and formula X (residues Aa), i.e. c fl3- (Cn¿) n- (cn: cnàb (cn2) Ix P where n is l to 10, o is l to Å and p is 3 to 9, provided that n + 2xo + p does not exceed 22, and CH3- (CH2) r- (cH = cn-cH2) s- (cn2) t- x wherein r is 1 to H, s is 2 to 4 and t is 1 to 7, provided that r + jxs + t does not exceed 22.

Preferred residues of formula IX are those in which n + 2xo + p is an even number, in particular such a residue in which n = 5 or 7, o = 1 and p = 7. Preferred residues of formula X are those in which 1 = is 1 or N, s is 2 to 4, and t is 2 or 6.

It is to be understood that compounds in which A is A 'exist in isomeric forms depending on the presence of one to several unsaturated groups. While the invention is not intended to be limited to any particularly isomeric form, those compounds are preferred in which the hydrogen atoms in each pair of ethylenically unsaturated carbon atoms are present in the cis configuration.

Particularly preferred residues A 'comprise those derived from the following acids, namely palmitoleic, olein, petroselen, vaccine, punicin (or eleostearin), parinarin, gadolein, cetolein, linol, linolenic and arachidonic acid, and in particular oleic, linoleic, linolenic, arachidonic and palmitoleic acids.

In another group of compounds I according to the invention, A has the meaning given under (ii) (wherein A is hereinafter referred to as A "), i.e. consists of cyclopropanyl derivatives with A 'residues, wherein each ethylene residue -CH = CH ~ is replaced by a cyclopropanyl group -C fi- H-. seen cyclopropanol grouperl. meln XI (wherein A "below is denoted Aï), i.e.

It is preferable that the residues A "are unbranched {from- Special groups A" are those with for-! lf / “HA cl; (cH2) ñ - LtH-- ~ cH-cnz V (CH2) w- XI wherein u is l to 15, v is l or 2, and w is l to 13, provided that when v is l, then u + w 3 to 19 and when v is 2, then u + w is 2 to 16. Particularly preferred residues A1 of formula XI are as follows, i.e. (i) when v is 1, then u + w = 7 to 19, when v is 2, then u + w = U to 16; (ii) when v is 1, then u + w = an odd number, when v is 2, then u + w = an even number; (iii) when v is 1, then u = 5 or 7 and w = 6, and when v is 2, then u = H and w = 6.

In view of these preferred alternatives, it should be understood that the preferred residues A "are derived from mono- or diunsaturated fatty acids of the kind found in nature, eg palmitoleic or oleic acid (v = 1) and linoleic acid ( v = 2).

It should also be understood that compounds in which A is A "exist in isomeric forms. Although the invention does not relate to a limitation to any particular isomeric form, it is that prefer that each pair of hydrogen atoms bonded to tertiary fi KOIHCONGT in cyclopropyl groups has a cis configuration.

When residues A "having a single cyclopropyl moiety have a smaller number of asymmetric carbon atoms than those with a larger number, the former are generally easier to purify and are therefore preferable in cases where the ease of carrying out a purification is an important factor.

In a group of compounds (hereinafter referred to as compounds Ia), A A 'and R 2 are a grouping of formula IV. When in these compounds R 7 is an alkyl, it is preferably unbranched and is preferably ethyl. When R 4 is alkyl, it is preferably methyl, when this symbol is alkoxy, it is preferably methoxy, and when this symbol is halogen, it is preferably fluorine or chlorine. When R 4 has a meaning other than hydrogen, this symbol is preferably present in the 5-position of the indole nucleus. R4, however, is preferably hydrogen. R 8 is also preferably hydrogen.

In further groups of compounds, A is A "and R 2 is a grouping of formula II (compounds Ih); or R 2 is a grouping of formula III (compounds Ii); or Rzen is a grouping of formula IV (compounds Ij);

The preferred alternatives of these compounds Ih to Ij are the same as those given above for compounds wherein A is A '.

The preferred compounds of the invention are compounds Ia.

The compounds of formula I can be prepared by acylation of a compound of formula n 1 I HN - R 2 XII wherein R 1 and R 2 have the meaning given above, with an acid of formula A - COOH XIII wherein A has the meaning given above, or with a reactive derivative thereof.

The acylation can be carried out in a manner known per se for transferring an amine function to its corresponding amide. The process can be carried out, for example, using the technique of mixed anhydrides, wherein the compound of formula XII is treated with a compound of formula O 0 -a-o-å-oala xIIIa wherein A has the above meaning, and R 1a is n (Cl_6) alKY1- This embodiment can be conveniently carried out at a temperature from -10 to +} 5 ° C and in an inert organic medium, e.g. a chlorinated hydrocarbon such as methylene chloride. Alternatively, the compound of formula XII may be treated with an acyl halide of formula O A - 8 - Hal XIIIb wherein A has the meaning given above and Hal is chlorine or bromine.

This embodiment is suitably carried out at a temperature of from 10 to 50 ° C, preferably 20-30 ° C, in an inert medium and in the presence of an acidic accent. In the acylation process, the compound of formula XII may be used in the form of an acid addition salt and the reagents may be used in excess, when liquid under the reaction conditions, for use as a reaction medium.

It should be noted that the acylation process for the preparation of the compounds of the invention does not affect the isomeric form of the starting compounds, so that the final products are obtained with the same isomeric form as the starting materials.

The final products of formula I can be isolated and purified using techniques known per se.

The compounds of formula XII are either known or can be prepared in a manner known per se from available starting materials.

The compounds of the formula A '- COOH XIIIG wherein A' has the meaning given above, are also known or can be prepared in a manner known per se from available starting materials.

The compounds of formula A "- COOH XIIId wherein A" has the meaning given above, are also known and can be prepared, for example, by converting ethylenically unsaturated groups in corresponding compounds of formula XIIIc to cyclopropanyl groups. . This can be done in a manner known per se, for example by reaction with methylene diiodide by the method of Simmons-smith (JAcS gi, 4256 (1959)}. For the preparation of compounds with a single cyclopropanyl group the starting acids may be present in or trans-form and the product obtained Together, mixtures of cis- and trans-forms give corresponding product mixtures When a cyclopropanoic acid (or derivative) is reacted with a compound of formula XII having asymmetric carbon atoms, it should be considered that the final product of formula I formed is obtained as a mixture of diastereoisomers, which, if desired, can be separated in a known manner, for example by chromatography or crystallization. Alternatively, if desired, the starting material in the form of cyclopropane the acid is divided into its antipodes and then reacted to form the corresponding isomeric product in a relatively pure form.Also, for the production of cyclopropanoic acids with two or more your cyclopropanyl units, the olefinic acids used as starting materials with a corresponding number of double bonds and the Simmons-Smith reaction a mixture of diastereoisomers, which, if desired, are separated for a further reaction.

The compounds of formula XIIIa and formula XIIIb may be prepared from the free acids in a manner known per se, suitably obtained in the same form. in situ. .

The compounds of formula I of the present invention have pharmacological activity. The compounds of formula I are particularly useful for controlling the cholesterol ester content of mammalian artery walls and are therefore particularly suitable for use as anti-atherosclerotic agents, i.e. useful as agents for the prophylactic treatment of atherosclerosis and in the control of atherosclerotic symptoms due to the accumulation of cholesterol esters in the artery walls. Such an ability of the compounds of formula I is apparent from known test procedures, in which the total cholesterol ester content of cultured cells is found to be reduced by means of a test compound, as compared with untreated cells, by, for example, the following procedure.

A) Cell culture. Cells of smooth muscle (from artery wall, eg aorta) from rhesus monkey, obtained according to the method of K. Jischer 10 15 25 30 35 7 7900142-6 K. Fischer-Dzoga et al. (Experimental and Molecular Pathology, 162-176 (1975)) were grown periodically in 75 ml tissue culture flasks using "Minimum Essential Medium (Eagle)" with the addition of 10 percent bovine fetal serum. For testing, a 75 ml vial with an almost confluent cell growth was selected. The cells were removed from the flask wall by a mild enzymatic treatment with pronase. After centrifugation and decantation of the enzyme solution, the cell bead is resuspended in an appropriate amount of medium to inoculate the desired number of 60 mm tissue culture dishes. the diluted cell suspension is pipetted into each dish. the dishes are labeled with data on cell type dates and numbers of origin and incubated at 3700 in an atmosphere of about 5 ml of After inoculation 5% CO 2 in a high humidity incubator. When the cultures are confluent, testing of the pharmacological action of the compounds of formula I begins. Test compounds I are usually dissolved in An equivalent amount of ethanol is also added to the control groups. The dishes with the tissue cultures are divided. To one group was added hyperlipemic rabbit. To the other 100% ethanol. randomly in groups. room (HHS) in a volume of 5 percent (control). groups are added 5 percent HRS and 1 mg per 100 ml of medium of test compound I. The dishes are reinserted in the incubator for another 2 h. All operations until the completion of the last incubation are performed using sterile technique in a laminated cover. After incubation, the dishes are examined microscopically by means of "Zeiss Axiomat" with phase contrast optics and the nature of the cultures is recorded, in particular with regard to the size, number and configuration of cytoplasmic inclusions and with regard to cellular morphology. The medium is separated from the cultures and 0.9 percent sodium chloride solution is added. The cells are removed from the dishes by means of a scraper and transferred to a conformally graded centrifuge tube. The cells are washed three times by suspending in an isotonic saline solution, centrifuging at 800 x g for 10 minutes and aspirating the supernatant.

B) Cell extraction procedure An appropriate volume of isopropyl alcohol (about 1 ml / mg) protein) was then added to the cell bead and the sample was sonicated with a microsample (10N x 3 mm) for 10 seconds with "LO" control of 50 in a "Bronwell Biosonic IV". After centrifugation for 15 minutes at 800 x g, the clear supernatant solution is decanted and an aliquot is taken for cholesterol analysis. The residue is dissolved in 0.1 n sodium hydroxide solution and an aliquot is taken for protein determination by the method of Lowry et al., J. Biol. Chem. lââ, 265 (1951).

Analysis Eri cholesterol; Standard solutions containing isopropyl alcohol, samples and solvents (isopropyl alcohol only) are treated as indicated above. An aliquot of 0.1 ml of free reagent (reagent Å, Table 1 below) is added to a 10 x 75 mm disposable glass test tube to which 20 μl of the isopropyl alcohol solution was added and mixed. After standing for about 5 minutes at room temperature, 0.8 ml of 0.5 N sodium hydroxide solution (Reagent C, Table 1 below) are added and mixed. The fluorescence is measured with an "Aminoo-Bowman" spectrophotofluorometer with an excitation wavelength of 325 nm and an emission wavelength of H15 nm. A 1 cm light path cuvette was used with a xenon lamp, an "IP28" photomultiplier tube and 2 mm slits.

Qotal cholesterol; The same procedure as described above for free cholesterol is followed in the determination of total cholesterol except that the total cholesterol reagent (reagent B, Table 1 below) is used instead of the free cholesterol reagent and that the samples are incubated for 20 minutes at 37 ° C before the addition of ( L5 sodium hydroxide solution (Reagent C, Table 1).

Alternatively, the determination of cholesterol, i.e. (above) obtained by steps A and B, is carried out by the method of Ishikava and co. J. Lipid Res. lä, 286 (l97U).

The amount of cholesterol ester is obtained by subtracting the amount of free cholesterol from the amount of total cholesterol in the cells obtained in the assays. Upon obtaining a lower amount of cholesterol ester in the group of cells to which the test compound was added, in comparison with the control group (untreated) shows that the test compound has activity for reducing the cholesterol ester in the cells. step C Table 1 Composition of cholesterol determination reagents A. Free cholesterol determination reagent Sodium phosphate buffer pH 7.0 0.05 M Cholesterol oxidase 0.08 U / ml Horseradish peroxidase 30.0 U / ml p-hydroxyphenylacetic acid 0.15 mg / ml 10 15 20 25 30 35 9 7900142-6 B. Reagent for determination of total cholesterol Sodium phosphate buffer pH 7.0 0.05 M Cholesterol ester hydrolase 0.08 U / ml Cholesterol oxidase 0.08 U / ml Horseradish peroxidase 30.0 U / Sodium taurocolate 5.0 mM Carbowax-6000 0.17 mM p-hydroxyphenylacetic acid 0.15 mg / ml C. Sodium hydroxide solution 0.5r For the above therapeutic use of compounds I, the administered dose is from about 100 mg to about 5000 mg to be preferred, suitably administered in divided doses from 25 to 2500 mg two to four times daily or in retarded form.

The compounds I can be worked up with conventional pharmaceutically acceptable diluents and carriers as well as any other useful additives and administered in the form of, for example, tablets or capsules.

Compounds I are preferably administered orally.

The following examples further illustrate the invention.

Example 1. u - {(1-oxo-9β2-cis, cis-octadecadienylamino)} - 1H-indole-3-propanoic acid ethyl ester To a solution of 7.0 g of linoleic acid in 150 ml of methylene chloride cooled to -2000 is added 2, 5 g of triethylamine and then 2.7 g of ethyl chloro. The reaction mixture is stirred for 2 hours and the temperature is allowed to rise gradually to room temperature. Then 2.5 g of triethylamine are added and then 6.7 g of DL-tryptophanethyl ester hydrochloride and the reaction mixture is stirred for 18 hours. Then the mixture is extracted several times with 2 n aqueous sodium hydroxide, washed with saturated aqueous sodium chloride solution and the organic phase is dried over anhydrous sodium sulfate. , is filtered and the filtrate is evaporated in vacuo to dryness. the residue is then filtered over silica gel using chloroform as eluent to give the title product in the solvent which is then evaporated to give the title product as roformate. a waxy solid.

Example 2. In a manner analogous to Example 1 and using the corresponding starting material in approximately equivalent amounts, the following substances are obtained: rt, ...._..._....._. P., _ .. v .. .--- \ ----...-._ .. -. 10 15- 25 30 35 a) b) c) d). e) f) S) n) i) J) 1 :) 1) m) n) o) D) 8.3. - {(1-oxc-9-cis-octadecenylamino)} -1H-indole-3-propanoic acid ethyl ester (waxy solid); u - {(1-oxo-9,12,2,5-cis, cis, cis-octadecatrienylamino)} - 1H-indole-3-propanoic acid ethyl ester; Q - {(1-oxo-9-cis-hexadecenylamino)} - 1H-indole-3-propanoic acid ethyl ester; u - {(1-oxo-5,3,11,14-cis, cis, cis, cis-eicosatetraenylamino)} - 1H-indole-3-propanoic acid ethyl ester; 2- (1-oxo-9-cis-cctadecenylamino) -3- (1-methylindolyl) -propanoic acid ethyl ester; 2- (1-oxo-9-cis-octadecenylamino) -3- (1H-5-methoxyindolyl) -propanoic acid ethyl ester 2- (1-oxo-9-cis-octadecenylamino) -3- (1H-5-chloroindolyl) -propanoic acid ethyl ester; 2- (1-oxo-9-cis-octadecenylamino) -3- (1-benzylindolyl) -propanoic acid ethyl ester; 0 - {(1-oxc-9,12-cis, cis-octadecadienylamino)} - 1H-indole-5-propanoic acid n-propyl ester; α-f (1-oxo-9,12-cis, cis-octadecadienylamino)} -1H-indole-5-propanoic acid n-butyl ester; ) α - {(1-oxo-9,12-cis, cis-octadecadienylamino)} -1H-indole-3-propanoic acid n-octyl ester; mp 66-68 ° C; m = {(1-oxo-9,12-cis, cis-octadecadienylamino)} -1H-indole-5-propanoic acid benzyl ester; 'a - {(1-oxo-9-cis-octadecenylamino)} - 1H-indole-3-propanoic acid-n-propyl ester-{(1-oxo-9-cis-octadecenylaminoX-1H-indole-3- propanoic acid n-butyl ester; u - {(1-oxo-9-cis-octadecenylamino) -1-1H-indole-3-propanoic acid n-octyl ester; u - {(1-oxo-9-cis-ocpadecenylamino3- 1H-indole-3-propanoic acid benzyl ester;) N- (β-methylbenzyl) -cis-2-octyl-cyclopropanooctanamide; {m.p. 44 DEG-56 DEG C.) (prepared from d (+) - α-methylbenzylamine); - {(1-oxo-cis-2-octyl-cyclopropanooctylamino)} - [1H] -indole-3-propanoic acid ethyl ester; ) N- (α-methylbenzyl) -cis-2-octyl-cyclopropanooctanamide (mixture of two racemates); melting point 30-32 ° C; (prepared from d, l-α-methylbenzylamine); L-n 10 15 20 30 35 ae) af) ag) ah) ai) ai) akaman) ao) ap) 11 79ÛÛ142-6 N- (o-methylphenyl) -cis-2-octyl-cyclopropanooctanamide; N-ia- (p-methylbenzyl) -benzyl} -cis-2-octyl-cyclopropanooctanamide; mp 78-8100; N- (α-methylbenzyl) -cis-2-hexyl-cyclopropanooctanamide; N- (α-methylbenzyl) -trans-2-octyl-cyclopropanooctanamide; N- (Q-methylbenzyl) -cis-2-tetradecylcyclopropanubanamide; N- {u- (p-methylbenzyl) -p-methyl-phenylethyl} -cis-2-octyl-cyclopropanooctanamide; mp 60-65 ° C; N- (q-methylbenzyl-cis, cis-2 - {(2-pentylcyclopropyl) -methyl} cyclopropanooctanamide; {mixture of diastereoisomers - oil - prepared from d (+) - Q-methylbenzylamine Q-cia, cis- { (1-oxo-2-nentylcyclopropyl) -methyl-cyclopropanooctanamino} -1-indole-3-propanoic acid ethyl ester3 (of DL-tryptophan-ethyl ester hydrochloride »N- (u-methylbenzyl) -cis, cis-2- {2 -pentylcyclopropyl) -methyl} -cyclopropanooctanamide (mixture of diastereoisomers - prepared from (d, 1) -Q-methylbenzylamine); N- (o-mecylphenyl) -cis, cis-2 - {(2-pentylcyclopropyl) -methyl } -cyclopropanooctanamide; N- {q- (benzyl) -β- (phenyl) ethyl} -cis-2-octylcyclopropanooctanamide; mp NO-45 ° C; aaao) 2- (1-oxo-9-cis-ok fi adecenylamino ) -3- (1H-5-fluoroindolyl) -pro- given examples. pionic acid ethyl ester (wax).

The following table shows NMR data for compounds according to NMR determined in CDCl3, the signals are given in ppm and the numbers in parentheses show the number of protons; S = singlet, d = doublet, t = triplet, b = width. 10 15 20 25 30 35 79ÛÛ142 ° 6 I U Ex. NMR data l t 5.3 (2), d 6.2 (l), s 9.2 (1) 2a t 5.3 (4) ¿d 6.l (l), s a.8 (1) ze f; s.35 (2), d so (1), s 3.1 (3) (oil 2f 5.4 (2), d 6.l (1), s 8.4 (l), S 4.8S (3) {VaX} 21 5.35 (4), d 6.l (l), S 8.S (l), S SJJZ) {oil} 2ac -0.36 (l), 'd 6.l (l), 5 8.8 (l) s 0.6 (3) +0.7 (5), b -0.3 (2), d 6.2 (l), s 8.5 (1) {Vax} ttb 2ak b +0.7 (5), b -0.3 (2), d 6.0 (l ), s 7.3 (5) 2am bb 2an +0.65 (5), b -0.3 (2), d 5.8 (l), s 7.3 (5) {01j fi} Zaaao t 5.3 (2), d 6.l (l ), p 8.7 (l) w M4 The compounds of formula I and in particular the compound prepared according to Example 1 are also suitable for use as agents for reducing the levels of serum cholesterol and cholesterol ester as documented by oral administration of a dose of 200 mg / kg of the test compound_per day for 9 weeks to rabbits in combination with a high cholesterol diet, which results, compared to control experiments, a reduction in serum cholesterol and cholesterol ester levels as well as reduced formation or absence of nlaques in the artery walls.

For this use, the dose administered is the same as indicated above.

Claims (2)

1. 3 7900142-6 PATENTKRAy 1. Compound of formula (1): O l | A - C - NH - R 2 wherein A is (1) a residue of a long chain, unsaturated fatty acid (minus the carboxylic acid function), which residue has 7 to 23 carbon atoms and has from 1 to 4 ethylenically unsaturated groups, or (ii) a corresponding residue, in which each ethylene group (-CH = CH-) is CH 2 2 replaced by a cyclopropanyl group (~ CH-CH-), and R 2 is a group of formula II, III or IV, i.e. R 4 is -cn- (cxaz), - <3 in 11 n, J wherein g is 0 or 1, R 4 is hydrogen, fluorine, chlorine, bromine, or LCl-3) alkyl or alkoxy, R 5 is hydrogen, fluorine, chlorine or (C 1-3) alkyl or n-alkoxy, and R 3 is hydrogen, (C 1-8) alkyl or a group of the formula Y VI - (CH 2). -J Y 1 wherein j is 0 or 1, Y is hydrogen, fluorine, chlorine, bromine or (C 1-3) alkyl or alkoxy, and Y 1 is hydrogen, fluorine, chlorine or (C 1-3) alkyl or alkoxy; wherein R 5 is as defined above and R 6 is hydrogen, fluorine, chlorine, bromine. ] - 3) alkyl or -alkoxy, or a group having the formula - wherein B is -CH 2 - or -O-, f is 0 or 1, and R 4 has the above the meaning; 7900142-6 14 COOR7 -c1 ~ 1-cH., ______ \ _ "I: 0- a" f N / 'R8 wherein R4 has the meaning given above, wherein R7 is (C1-8) alkyl or benzyl, and Rs is hydrogen, (C 1-8] alkyl or benzyl; provided that when R 1 is a group of formula II or III A has the definition given in (ii) above. A compound according to claim 1, characterized in that A has the meaning given under (i). Α - {(1-oxo-9-cis-octadecenylamino)} -1H-indole-3-propanoic acid ethyl ester. Compounds according to claim 1, characterized in that A has it under (ii) A compound according to claim 4, characterized in that R 2 is a group of formula II as stated in claim 1. 6. N- {u- (benzyl) -β- (phenyl) ethyl} -cis-Z -Octylcyclopropane-octanamide A compound according to any one of the preceding claims for use as an anti-atherosclerotic agent A pharmaceutical composition containing a compound I according to any one of the preceding claims, together with a pharmaceutically acceptable excipient or carrier.