IE71192B1 - 2,4-pyrimidinedione derivatives and pharmaceutical compositions containing them - Google Patents

Abstract

These derivatives correspond to the formula <IMAGE> in which one of the symbols R1, R2, R3 or R4 denotes a C7-C20 alkyl group and each of the others denotes H or a C1-C3 alkyl group and each of Z1 and Z2 denotes H or together they denote the carbonyl or thiocarbonyl group, and their addition salts with pharmaceutically acceptable acids. Medications.

Description

The present invention relates to 2,4-pyrimidinedione derivatives and medicaments containing them.

These compounds in particular have an anti-radical activity and inhibit the peroxidation of lipids of the biological membranes.

It is known that uric acid and some of its N-methyl derivatives trap free radicals in vitro and it was envisaged in Pro. Natl. Acad. Sci. USA 78 6858 (1981) that uric acid might have a protective effect against the toxicity of oxygenated radicals in humans. However, - it has been found that uric acid is only active on some of these reactive species because it does not inhibit the peroxidation of the membrane lipids, unlike the compounds of the present invention.

FR-A-2 232 320 describes amino-uracils with anticholesterolaemic properties which do not have a long-chained alkyl substituent. Chem. Abs. Vol. 112 (17), (1990), 688 No. 157937 describes the use of uric acid derivatives, which are similar to the compounds of the invention, for the purposes of studying hostresistor interactions.

EP-A-0 092 398 relates to 8-phenylxanthines prepared from 5, 6-diaminouracils similar to the compounds of the invention but having no long-chained alkyl substituent.

FR-A-2 531 085 similarly illustrates the preparation of xanthine derivatives from 5,630 diaminouracils which do not have any long-chained alkyl groups either.

Chem. Abs. Q4 (1976) 576 No. 105545u describes the synthesis of 1-hexyltheobromine derivatives substituted in the 8-position.

Recueil des travaux chimiques des Pays-Bas, vol. 83, (1964) 1215-1229 relates to the preparation of xanthine derivatives having diuretic properties. 711 92 - 2 US-A-2 781 344 proposes 4,5-diaminouracil derivatives as intermediates for synthesising purine derivatives.

The compounds of the invention correspond to the formula wherein Rx denotes H or (C7.20)alkyl, R2 denotes H or (C7.20)alkyl, R3 denotes H, CH3 or (C7.20) alkyl, R4 denotes H, CH3 or (C7_20)alkyl, Zx and Z2 each denote H or together represent the carbonyl or thiocarbonyl group, on the understanding that only one of the substituents Rx to R4 denotes a (C7.20) alkyl group and either R3 or R4 is other than H when Rx denotes n-dodecyl, and the addition salts of these compounds with pharmaceutically acceptable organic or inorganic acids. The disclaimer was introduced in order to eliminate the compound of formula I wherein Rx = n-C12H25, R2 = R3 = R4 = H and Zx. . . Z2 = xC = O, described in J. Amer. Chem.

Soc. 109, (1987), 6549-6551. This publication discloses a study of the host-receptor interactions in the case of uric acid derivatives.

The compounds of formula I may be prepared as follows: 1°) an NRXZX amino group is a compound of formula introduced into position 5 of O H N' XNHR.

I V - either by reduction of an NO or NO2 group introduced into the 5-position, respectively, by nitrosation or nitration of this compound (V) in order to obtain compounds wherein Zx = Rx = H - or by reacting HNRXZX with the derivative brominated in position 5, obtained by bromination of this compound (V), in order to obtain compounds wherein Zx = H and Rx denotes (C7_20) alkyl and optionally, in order to obtain compounds wherein Zx and Z2 together represent the carbonyl or thiocarbonyl group, 2°) urea or thiourea, respectively, is reacted with the compound of formula II optionally via the carbamate in position 5 when Rx denotes (C7.20)alkyl and R2 denotes H.

This process is explained in more detail hereinafter.

The compounds of formula II wherein Rx represents H and R2, R3 and R4 are as in formula I may be prepared from derivatives of formula III, either via the formamide (IV) according to reaction plan (a) either directly by catalytic hydrogenation or by the action of an alkyl metal dithionite in an aqueous medium or in formamide.

The formamide (IV) is obtained, for example, by the action of zinc or alkyl metal dithionite and formic acid.

Some derivatives of formula III are known; the others may be prepared, in a manner known per se, by the action of an alkyl nitrite, such as isoamyl nitrite, or an alkali metal nitrite with the pyrimidinediones of formula V wherein R2, R3 and R4 are defined as in formula I.

The compounds of formula II wherein Rx represents H may also be prepared from 6-chloro-2,4-pyrimidinedione according to reaction plan (b) O using conventional methods, such as the one described in Liebigs Ann. Chem. 677 p. 113 (1964) .

The compounds of formula II wherein Rx is a C7_20-alkyl group are prepared by reacting the RXNH2 amine with the bromo derivative of formula VI wherein R2, R3 and R4 are as in formula I. Derivative (VI) is obtained by reacting bromine in acetic acid or N-bromosuccinimide in a mixture of acid and acetic anhydride with compound (V), as described in Liebigs Ann. Chem. p. 1847-54 (1979); when R3 denotes H it is preferable, before bromination is carried out, to protect the nitrogen, for example, with a benzyl group which will be eliminated by catalytic hydrogenation after the reaction with R3NH2.

The compounds of formula VII wherein Rx to R4 are as in formula I and Z is 0 or S, may be prepared by reacting urea or thiourea with the diaminopyrimidinediones of formula II using the method described in Liebigs Ann. Chem. p. 2030 (1974) for products wherein Rlf R2 = H and R3, R4 = H or CH3.

When Rx represents a C7.20-alkyl group, the compound of formula VII is preferably prepared using a process described in Liebigs Ann. Chem. p. 1847-54 (1979) for a compound II wherein Rx = CH3. For the compound in which R2 = R3 = R4 = H, it is necessary to block the nitrogen which carries R3 and the reaction plan is as follows: The carbamate is prepared in conventional manner by reacting an alkyl chloroformate with the compound (II) wherein R2 = H, R3 = CH2CeHs and R4 = H, in the presence of a base, whilst cyclisation is carried out by prolonged heating in a basic medium in a polar solvent such as an alcohol or without a solvent in a sealed tube in the presence of sodium alkoxide, as described in Liebigs Ann. Chem. p. 1847 (1979).

The starting 6-amino-pyrimidinediones (V) may be prepared by methods which are known in principle and which have been described in particular for compounds wherein R2 = H and R3 or R4, which may be identical or different, represent H or CH3. Thus the compounds of formula V wherein R3 is other than H and R2 = R4 = H are prepared by the method described in J. Am. Chem. Soc. 63 2567 (1941) in an alcohol according to reaction plan (c) : V bis C) R3NHCONH2 + Ο R00C-CH2-CN + R’ONa wherein R, R' denote a Cx_4-alkyl group.

In order to obtain the compounds (V) wherein R3 and R4 are other than H, the compound of formula V bis is treated, in a basic medium, with a compound R4X wherein X denotes a halogen atom or, when R4 represents CH3, preferably with (CH3)2SO4.

The compounds of formula V wherein R3 and R4 represent H are prepared by reacting 6-chloropyrimidine dione, a known compound, with R2NH2 in an acidic medium according to reaction plan (d): as described, for example, in J. Med. Chem. 20 p. 1186 (1977) or by fusion of compound (V) wherein R2 = R3 = R4 = H with R2NH2 in the presence of R2NH2, HCl.

The compounds of formula V wherein R2 and R3 represent H are prepared by the process described in Liebigs Ann. Chem. 631 p. 168 (1960) according to reaction plan (e) wherein R and R' each represent a Cx_4-alkyl group, using the method described in Liebigs Ann. Chem. 612 p. 158 (1958) .

The salts of the compounds of formula I which carry amino functions are prepared by the action of at least one acid equivalent with the amine in solution, then they are isolated by precipitation or by evaporation of the solvent and purified by crystallisation if required.

The invention further relates to the pharmaceutical compositions and medicaments containing as active principle at least one of the compounds of formula I wherein Rj denotes H or (C7.20)alkyl, R2 denotes H or (C7.20) alkyl, R3 denotes H, CH3 or (C7.20)alkyl, R4 denotes H, CH3 or (C7.20)alkyl, Z3 and Z2 each denote H or together represent the carbonyl or thiocarbonyl group, on the understanding that only one of the substituents Rx to R4 denotes a (C7.20) alkyl group and either R3 or R4 is other than H when R3 denotes n-dodecyl, or one of the pharmaceutically acceptable acid addition salts thereof; the compounds wherein one of the symbols Rlf R2, R3 or R4 denotes a C12 or C20-alkyl group are particularly preferred.

The presence of excessive quantities of free radicals has been demonstrated in numerous pathological conditions and it has now been practically demonstrated that the tissue damage observed in rheumatoid arthritis, autoimmune diseases, iron overloads, the effects of cerebral and cardiac ischaemias and certain eye diseases depend largely on the formation of free radicals. Thus, it is possible to envisage administering to humans compounds which inhibit the formation of these highly reactive species as a preventive or curative agent in the treatment of the diseases mentioned above and probably in diseases of old age in which free radicals have been associated with the degenerative phenomena observed.

It is known that, of the free radicals, those derived from oxygen and particularly the hydroxyl radical OH·, have an important role,· although it is not possible at present to measure the concentration of these radicals directly in vivo, it is possible to estimate them by studying the neutralisation of various free radicals in vitro and especially the peroxidation of membrane lipids, the consequence of which, in vivo is the formation of cytotoxic aldehydes and the destruction of cell membranes leading to an increase in their permeability.

The anti-radical action of an XH product is currently determined in vitro by measuring the neutralisation of the 1,1-diphenyl, 2-picryl-hydrazyl (DPPH·) radical, stable at 20’C, by the product XH in the reaction DPPH· + XH - DPPH2 + X· The disappearance of the DPPH* radical is studied by spectrophotometry at 517 nm, the wavelength at which the radical strongly absorbs radiation unlike the hydrazine molecule DPPH2. This method is described in particular in Free Radical in Biology & Medicine i P- 251-257 (1987) with regard to uric acid and the N-methyl derivatives thereof.

In this study, the compounds of the invention are active at doses comparable to those of uric acid.

Inhibition of lipid peroxidation caused by oxygenated free radicals, a peroxidation which is particularly inportant in the presence of iron, was determined in vitro by measuring the quantity of malonic aldehyde formed in mitochondrial suspensions brought into contact with a superoxide ion generator such as dihydroxyfumaric acid.

Uric acid is inactive in this experiment, whereas the compounds according to the invention have an activity similar to that of the 2-amino steroids described in Drugs of the future vol. 14. (2), 143-152, (1989) and especially 21-[4-(3,6-bis (diethylamino)-2pyridinyl) -1-piperazinyl] -16of-methylpregna-l, 4,9 (ll) triene-3,20-dione (compound A).

The pharmaceutical compositions according to the invention comprising as active principle at least one of the compounds of formula I or one of its salts with a pharmaceutically acceptable acid will be administered for the preventative or curative treatment of degenerative phenomena linked to the presence of free radicals, by oral, parenteral, transmucous or percutaneous route. The single and daily doses will depend on the compound, the nature and severity of the disease, the body weight and condition of the patient and the route of administration; generally, by oral route, the single dose will be 1 mg to 500 mg in adults, whereas by intravenous route it will be from 0.1 to 25 mg.

The compounds of formula I and the salts thereof with pharmaceutically acceptable acids are also used in cosmetic compositions to counteract ageing of the skin.

Some examples of compounds according to the invention, a process for preparing them and the results obtained in studies of their anti-radical and antilipoperoxidising activities as well as those of the reference compound A mentioned above are described hereinafter.

The results of percentage analyses of the isolated compounds are compatible with their crude formulae; the melting points given are instantaneous. The NMRXH spectra were plotted in solution in deuterised dimethylsulphoxide using tetramethylsilane as the internal standard.

First of all, some examples of the preparation of 6-amino-2,4-pyrimidinediones of formula V will be described.

A) 6-Alkylamino-2,4-pyrimidinedione i) formula V : R2 = C12H25, R3 = H, R4 = H A mixture of 7.5 g of 6-amino-2,4-pyrimidinedione, 12 g of dodecylamine and 12 g of dodecylamine hydrochloride is maintained at 160C for 3 hours. The reaction medium is then brought back to ambient temperature before 100 ml of ethanol are added. The solid is isolated by filtration and then suspended for several minutes in 100 ml of a 2N aqueous NaOH solution. The remaining solid is isolated and recrystallised from acetic acid. In this way, 7 g of the expected product are obtained melting at above 250eC.

NMR (300 MHz) δ : 0.85(t,3H); 1.25(m,18H); 1.50(m,2H); 2.98(q,2H); 4.37(S,1H); 5.98(t,lH); 9.78(s,lH); 10.07(s,lH). ii) The derivative of formula V wherein R2 = C16H33, R3 = H, R4 = H, melting at above 250’C, is prepared in the same way from hexadecylamine.

B) 1 - Alkyl - 6 - amino- 2,4 -pyrimidinedione Formula V : R3 = C12H25, R2 = H, R4 = H 17.6 g of ethyl cyano acetate and 35 g of dodecylurea are added to a solution of sodium ethoxide prepared with 4 g of sodium in 100 ml of ethanol. The medium is maintained at reflux tenperature for 7 hours and then, after returning to ambient temperature, a concentrated aqueous HCI solution is added until the pH is acidic and then 200 ml of water are added. The precipitate formed is isolated by filtration, then recrystallised from ethanol. In this way, 29 g of the desired product are isolated melting at more than 250’C. NMR (300 MHz): δ 0.87(t,3H); 1.26(m,18H); 1.52(m,2H); 3.73(t,2H); 4.58(s,lH); 6.69(m,2H).

C) 1-Alkyl-3-alkyl-6-amino-2,4-pyrimidinedione Formula V : R3 = C12H2S, R2 = H, R4 = CH3. 2.95 g of 6-amino-1-dodecyl-2,4-pyrimidinedione prepared in B are put into suspension in 10 ml of an aqueous NaOH solution and 10 ml of ethanol and then 1.3 g of (CH3)2S04 are added at 60"C. Heating is continued until the pH of the medium is substantially neutral; then a IN aqueous NaOH solution is added until the pH is basic and, after cooling, the solution is extracted with chloroform. The organic phase is washed with water and dried and brought to dryness. In this way, 2.35 g of the expected product are obtained, melting at 74*C.

D) 3-Alkyl-6-amino-2,4-pyrimidinedione Formula V R2 — H, R3 — H, R4 — C32H25 · a) 35 g of dodecylurea and 24.3 g of ethyl malonate are added to 150 ml of a sodium ethoxide solution prepared with 3.5 g of sodium; the mixture is kept at reflux temperature for 8 hours before the addition, at ambient temperature, of a concentrated aqueous hydrochloric acid solution until an acid pH is obtained. The precipitate is then isolated by filtration, washed with water and recrystallised from ethanol. In this way, 30 g of 1-dodecyl-2,4,6-pyrimidine-trione are obtained. Mp = 129-130". b) 30 g of the product obtained in a) are suspended in 13.5 ml of water and 102 ml of POC13 are added dropwise. The mixture is then maintained at reflux temperature for one hour, then the excess POC13 is distilled under reduced pressure. The residue is poured onto crushed ice and the precipitate formed is isolated. After washing with water and recrystallisation from a . water/methanol mixture, 20.5 g of 6-chloro-3-dodecyl2,4-pyrimidine-dione are obtained, melting at 156-157"C. c) A mixture of 30 g of the chloro derivative obtained in b) and 150 g of benzylamine is maintained at 170’C for 1 hour. After the medium has been brought back to ambient temperature, 100 ml of water are added and the precipitate formed is separated off. After it has been washed with methanol and ethyl ether, 25.13 g of 6-benzylamino-3-dodecyl-2,4-pyrimidine-dione are obtained, melting at 191’C. d) 25 g of the compound obtained in c) and 4 g of 10% palladium on charcoal are suspended in 400 ml of absolute ethanol in a hydrogenation apparatus in which a hydrogen pressure of 3 MPa and a temperature of 80C are maintained for 6 hours. The catalyst is eliminated from the hot solution by filtration before the solvent is evaporated off under reduced pressure and the residual solid is washed with ethyl ether. In this way 17.02 g of 6-amino-3-dodecyl-2,4-pyrimidine-dione are isolated, melting at 242eC.

The following is a description of the preparation of compounds of formula III.

E) 6-Amino-1-dodecyl-5-nitroso-2,4-pyrimidine-dione (Formula III : R2 = H; R3 = C12H2S; R4 = H) . ml of isoamyl nitrite are added dropwise to 120 ml of ethanol in which are suspended 5 g of 6-amino1-dodecyl-2,4-pyrimidine-dione, and then 2 drops of a concentrated aqueous HCl solution are added. After 3 hours stirring at ambient temperature, the violet crystals formed are isolated and recrystallised from ethanol. In this way, 4.35 g of the expected product are obtained, melting at 206"C.

This can also be represented by the tautomeric formula: C12H25 I Using the method described in E, the compounds whose features are shown in Table 1 were prepared, of the formula: r2 r3 r4 Mp’C F H 012^25 ch3 158 G C16H33 H H 168 H ^12^25 H H 173 I H H C12H25 230 BXAMELE. .1 , 6 -Diamino-1 -dodecyl - 3 -methyl -2,4 - pyrimidine-dione (Formula I : Zx — Z2= H; Rx = k2 — H; R3 — CX2H25; R4 = CH3) . a) 4 g of powdered zinc are added in small batches to a solution, heated to reflux temperature of 4.35 g of the compound of Example C in 100 ml of formic acid; after 1 hours' refluxing, the mixture is filtered hot and the solvents are evaporated from the filtrate under reduced pressure. 30 ml of CH3OH are poured onto the residue and the precipitate is isolated.

In this way, 3.85 g of 6-amino-l-dodecyl-5formylamino-3-methyl-2,4-pyrimidine-dione are obtained, melting at 215’C. b) 3.63 g of the compound obtained in a) are dissolved in 100 ml of CH3OH, into which gaseous hydrochloric acid is introduced by gentle bubbling for 15 minutes. The solution is then maintained for 3 hours at reflux tenperature, then cooled to O’C. The precipitate is then filtered to isolate 3.21 g of the monohydrochloride of the end product which melts at 195’C.

The diamine may be prepared by treating a solution of its hydrochloride in CH3OH with a concentrated aqueous solution of NH4OH.

EXAMPLES 2 to .7 The compounds of formula I wherein Z1=Z2=H, the characteristics of which are shown in Table 2, are prepared using the process of Example l.

Example Rx r2 r3 r4 Mp’C (salt) 2 H H ^12^25 H 237 3 H H H c12h25 >250 (HC1) 4 H Ci6H33 H H 242 (HC1) 5 H C12H2S H H >200 (HC1) 6 H H ^10^21 H >250 (HC1) 7 H H c8H17 H >200 (HC1) NMR EX 6 : (500 MHz) δ : 0.84(t,3H); 1.25(m,14H); I. 51(m,2H); 3.81(t,2H); 7.72(s,2H); 9.75(S,3H); II. 10(s,lH).

EX 7 : (250 MHz) δ : 0.85(t,3H); 1.25(m,10H); I. 49(m,2H); 3.80(t,2H); 7.81(s,2H); 9.46(S,3H); II. 13(s,1H).

EXAMPLES 8 and 9 3-Dodecyl-1-methyl [1H, 3H, 9H] 2,6,8-purine-trione (formula I * Z3 and Z2= CO; R3 = R2 = H; R3 — C^2H2g; R4 = CH3) .

A mixture of 800 mg of the confound obtained in Example l with 1 g of urea is maintained at 180’C for 1 hour. At ambient temperature, 20 ml of water are poured into the medium,- the solid is isolated, washed with ethanol and recrystallised from acetic acid. In this way, 680 mg of the desired product are isolated, melting at 266’C.

NMR (250MHz) - δ : 0.86(t,3H); 1.24 (m, 18H) ; 1.56 (m, 2H) ; 3.19(S,3H); 3.77(t,2H); 10.79(s,lH); 11.89(S,1H).

Using the same method with thiourea, 3octyl [1H, 3H, 9H] 2,6-purine-dione-8-thione are obtained: melting point >250’C.

NMR (90MHz) δ : 0.85(t,3H); 1.24(m,12H); 3.87(t,2H); 11.24(S,1H); 12.98(S,1H); 13.47(s,lH).

EXAMPLES IQ tQ 15 The compounds of formula I wherein Zx and Z2 represent C=0, the characteristics of which are shown in Table 3, were prepared using the method described in Example 8.

Example Ri r2 r3 r4 Mp"C 10 H H H C12H25 >250 11 H ^12^25 H H >250 12 H ^16^33 H H >250 13 H H ^12^25 H >250 14 H H C8H17 H >250 15 H H ^10^21 H >250 the NMR data of which are shown in Table 4. 15 TABLE 4 Example Frequency δ 10 300 MHz 0.86(t,3H); 1.25(m,18H); 1.52 (m, 2H) ,- 3.70(t,2H); 10.77(s,lH); 11.03(S,lH); 11.83(S,1H). 11 300 MHz 0.85(t,3H); 1.25(m,18H); 1.51 (m,2H); 3.63(t,2H); 10.82 (s, 1H) ; 10.86(s,lH); 12.00(s,lH). 12 250 MHz 0.84(t,3H); 1.22(m,26H); 1.51 (m, 2H); 3.62(t,2H); 10.78(s,lH); 10.8l(s,lH); 11.98(s,lH). 13 250 MHz 0.85(t,3H); 1.22(m,18H); 1.48 (m,2H); 3.73(t,2H); 10.54(s,lH); 11.35(S,1H); 12.03(s,lH). 14 250 MHz 0.85(t,3H); 1.25(m,10H); 1.53 (m,2H); 3.71(t,2H); 10.76(s,lH); 11.0l(s,lH); 11.85(s,lH). 15 500 MHz 0.84(t,3H); 1.25(m,14H); 1.51 (m,2H); 3.81(t,2H); 10.71(s,lH); 1O.98(S,1H); 11.82(s,lH).

Example 16 -Amino- 6 -octadecyl amino- 3 -methyl -2,4 -pyrimidine-dione (formula I : Zx and Z2 = H; Rx = H; R2 = C18H37; R3 = H; R4 = CH3) a) 2.05 g of 6-chloro-3-methyl-5-nitro-2,4pyrimidine-dione resulting from nitration of 6-chloro-3methyl-2,4-pyrimidine-dione using a process described in Liebigs Ann. Chem. 677 113 (1964), are added in small batches to a solution of 3 g of octadecylamine and 1 g of triethylamine in 20 ml of ethanol. The solution is maintained at reflux temperature for 20 minutes, then neutralised by the addition of acetic acid. The precipitate formed is washed with water and recrystallised from ethanol.

In this way, 2.11 g of 6-octadecylamino-3-methyl-5nitro-2,4-pyrimidine-dione are isolated, melting at 130C. b) The product thus obtained is dissolved in 50 ml of formic acid and 2 g of powdered zinc are added to the medium in small amounts; after 1 hour of refluxing the mixture is filtered hot and the solvents are evaporated from the filtrate under reduced pressure. The residue is crystallised with 15 ml of methanol. In this way, 1.8 g of 5-formamido-6-octadecylamino-3-methyl-2,4pyrimidine-dione are obtained, melting at 196’C. c) The formamide obtained above is treated with HCl in methanol as in Example 1-b.

The hydrochloride of the desired product is thus obtained, melting at 187"C.

NMR (250MHz) δ : 0.89(t,3H); 1.27(m,30H); 1.55(m,2H); 3.15(s,3H); 3.32(m,2H); 7.86(s,lH); 10.40(S,3H).

The anti-radical activity of these compounds was determined as follows: 20 μΐ of a solution of DPPH· lmM and 10 μΐ of a solution of the product to be tested in dimethylsulphoxide were added to 970 μΐ of a mixture of methanol and aqueous solution buffered by TRIS (tromethamine), HCl (10 mM-pH 7.4) at a rate of 1 volume of methanol to 2 volumes of buffer. The- mixture was kept at 20’C for 20 minutes before the optical density at 517 nm was determined by comparison with a control mixture for which the test product had not been added in the dimethylsulphoxide.

The percentage P of trapping of the free radical is defined as a ratio of optical densities D D(product) - D(control) P =---------------------------x iQo D(control) The effective concentration 50 (ECS0) , for which P = » 50, is calculated on the basis of the results of measurements made with 4 different concentrations in a range of 10 around the EC50.

Table 5 shows the EC50 for compounds according to the invention and for uric acid and its N-methyl derivatives and for product A.

TABLE 5 PRODUCT EC50 (μΜ) PRODUCT ECS0 (μΜ) uric acid 4.5 Example 10 5.2 uric acid CH3-1 4.5 11 6.2 CH3-3 3.2 12 4.5 CH3-7 20 13 3.2 CH3-9 5.2 A 4 14 4 example 1 5 15 3.4 2 7 3 5.5 4 6.4 16 6.2 5 6.4 6 5.6 7 6.5 8 2.7 9 6.5 The inhibiting activity of the compounds on the peroxidation of membrane lipids was determined as follows: a) preparation of the mitochondria An ox heart is taken immediately after slaughter of the animal and cooled to 4°C, the temperature at which all the following operations will be carried out. The heart is washed with an aqueous buffer solution at pH 7.4 (TRIS, HCI 10 mM, EDTA 2 mM, KC1 20 mM, saccharose 250 mM) and then the connective tissue and fat are removed. The muscle is then chopped and the chopped meat is suspended in an aqueous buffer solution at pH 7.4 (TRIS, HCl 10 mM, saccharose 250 mM, EDTA 1 mM) in a quantity of 700 g of muscle to 3 litres. The pH of the suspension is regularly readjusted to 7.4 before the chopped meat is isolated by filtering through gauze. It is then resuspended in 3 litres of the second buffer and the suspension is ground before being centrifuged for 20 minutes at 1000 g, then the supernatant is centrifuged for 15 minutes at 17,000 g. The centrifugation residue is washed several times with an aqueous buffer solution pH 4 (TRIS, HCl 15 mM, saccharose 250 mM) with separation, by centrifuging, of the washing solution and finally a homogeneous mitochondrial suspension is prepared in this same buffer containing 50 mg of protein/ml, which will be stored frozen in droplets in liquid nitrogen. After thawing, the respiratory checks carried out by measuring the rate of oxygen consumption with or without ADP present for the succinate and glutamate/malate substrates are 80% of those obtained before freezing, several months after the preparation of the mitochondria. b) Lipoperoxidation At 4C a thawed mitochondrial fraction is dispersed in a buffered aqueous solution at pH 7.4 (TRIS, HCl 30 mM containing KC1 140 mM), to obtain a protein concentration of 0.37 mg/ml; 10 μΐ of a 100 mM dihydroxyfumaric acid solution, the pH of which has been adjusted to 7.4, 2 μΐ of a solution of the product to be tested in solution in dimethyl sulphoxide (DMSO) and 30 μΐ of a solution of FeSO4, (NH4)2SO4 ImM, stored away from oxygen and light, are added to 960 μΐ of the suspension previously obtained. The control sample contains DMSO without the test product whilst the blank contains neither dihydroxyfumaric acid nor iron.

After a first sample of 100 μΐ has been taken, the samples are kept at 37*C with gentle stirring and samples of 100 μΐ are taken after 20 and 40 minutes.

Each sample is added immediately to 0.1 ml of a 13.5% (weight/volume) solution of trichloroacetic acid and 0.33% (weight/volume) thiobarbituric acid in a 0.85 N aqueous hydrochloric acid solution and the containers are sealed and then heated to 100"C for 15 minutes before being cooled to ambient temperature in 5 minutes. Then 1 ml of a 70% (weight/volume) aqueous trichloroacetic acid solution are added before spectrofluorimetric measurements are carried out with an excitation wavelength of 515 nm and an emission wavelength of 553 nm.

A standard range of dilute bis-(diethylacetal)malonaldehyde in a 0.1 N aqueous solution of HCl makes it possible to calculate the aldehyde concentration corresponding to each fluorescence intensity i.

Under the experimental conditions, i - 1.89 c (nmole) = -----------35.5 was determined and consequently the percentage inhibition of peroxidation I may be calculated by the formula (i - 1.89)control - (i - 1.89)experimental χ ---------------------------------------------X 100 (i - 1.89)control - (i - 1.89)blank The concentration which inhibits 50% of the signal, IC50, i.e. the concentration at which I = 50, is calculated from the measurements carried out with 4 different concentrations of the test product in a range of 10 around the IC50.

The results obtained are indicated in Table 6.

Uric acid does not inhibit the formation of malonaldehyde at a concentration below 100 μΜ,- its Nmethyl derivatives are not very active, for the most active I = 35 at a concentration of 100 μΜ.

TABLE 6 Example ic50 Example ic50 A 0.7 10 1.25 1 0.24 11 0.3 2 0.27 12 0.9 3 0.26 13 1.6 10 4 0.9 5 0.3 14 28 6 0.28 15 3.7 7 0.62 8 0.85 16 20 15 9 25

Claims (14)

1. Compound of formula 0 1 f 1 '3 Λ{ I ^N*2 1 1 R 3 *2 wherein R x denotes H or (C 7 . 20 ) alkyl, R 2 denotes H or (C 7 _ 20 ) alkyl, R 3 denotes H, CH 3 or (C 7 . 20 ) alkyl, R 4 denotes H, CH 3 or (C 7 . 20 )alkyl, Z x and Z 2 each denote H or together represent the carbonyl or thiocarbonyl group, on the understanding that only one of the substituents R x to R 4 denotes a (C 7 . 20 ) alkyl group and either R 3 or R4 is other than H when R x denotes n-dodecyl, and the addition salts thereof with a pharmaceutically acceptable acid.

2. Compound according to claim 1, characterised in that one of R x , R 2 , R 3 or R 4 denotes a C 12 . 20 -alkyl group.

3. Compound according to one of claims 1 and 2, characterised in that Z x and Z 2 each denote H.

4. Conpound according to one of claims 1 and 2, characterised in that Z x and Z 2 together denote a carbonyl or thiocarbonyl group.

5. Compound according to one of claims 1 and 2, characterised in that Z x and Z 2 together denote the carbonyl group.

6. Medicament containing as active principle a compound according to claim 1.

7. Medicament according to claim 6, characterised in that R x , R 2 , R 3 or R 4 denotes a C 12 _ 20 -alkyl group.

8. Medicament according to’one of claims 6 and 7, characterised in that Z x and Z 2 each denote H.

9. Medicament according to one of claims 6 and 7, characterised in that Z x and Z 2 taken together denote a carbonyl or thiocarbonyl group.

10. Process for preparing compounds according to one of claims 1 to 5, characterised in that l’) an NR 1 Z 1 amino group is introduced into position 5 of a compound of formula - either by reduction of an NO or N0 2 group introduced into the 5-position, respectively, by nitrosation or nitration of this compound (V) in order to obtain compounds wherein Z x = R x = H - or by reacting HNR 1 Z 1 with the derivative brominated in position 5, obtained by bromination of this compound (V), in order to obtain compounds wherein Z x = H and R x denotes (C 7 _ 20 )alkyl and optionally, in order to obtain compounds wherein Z x and z 2 together represent the carbonyl or thiocarbonyl group, 2. ) urea or thiourea, respectively, is reacted with the compound of formula

11.II optionally via the carbamate ih position 5 when R x denotes (C 7 . 20 ) alkyl and R 2 denotes H. -2511. A compound as claimed in claim 1 or an addition salt thereof with a pharmaceutically acceptable acid, substantially as hereinbefore described and exemplified. 5

12. A process for the preparation of a compound as claimed in claim 1 or an addition salt thereof with a pharmaceutically acceptable acid, substantially as hereinbefore described and exemplified. 10

13. A compound as claimed in claim 1 or an addition salt thereof with a pharmaceutically acceptable acid, whenever prepared by a process claimed in claim 12.

14. A medicament as claimed in claim 6, substantially as 15 hereinbefore described.