IE910853A1 - 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 to pharmaceutical compositions containing them.

These compounds, in particular, have free radical-inhibitory activity and are inhibitors of the peroxidation of lipids of biological membranes.

Uric acid and some of its N-methyl derivatives are known to trap free radicals in vitro, and it has been described in Pro. Natl. Acad. Sci. USA 78 6858 ( 1981) that uric acid could perform a protective role in man against the toxicity of oxygen-containing radicals. However, it has been shown that uric acid is active only against some of these reactive species, since it does not inhibit the peroxidation of membrane lipids, in contrast to the compounds of the present invention.

The compounds of the invention have the formula in which one of the symbols Rt, R2, R3 or R« represents a C7 to C20 alkyl group while the others each represent H or a Cx to C3 alkyl group, and Zx and Z2 each represent H or together cepresent a carbonyl or thiocarbonyl group, with the proviso that one of R3 or R4 is other than H when Rr represents n-dodecyl, as well as the addition salts of these compounds with pharmaceutically acceptable inorganic and organic acids.

The compounds of formula I may be prepared in the following manner : An amino group N R^ is introduced in the 5 position of a compound of the formula 1853 __: by reduction of a group NO or NC^ introduced in the position respectively by nitrosation or nitration of this compound V for obtaining compounds in which Z^= R1=H - or by reaction of HNR^ Z^ with the derivative brominated at the 5 position, obtained by bromination of the compound V, for obtaining compounds in which Z^=H and is alkyl, and possibly, for obtaining compounds in which Z and Z2 represent together the group carbonyl or thiocarbonyl, respectively urea or thiourea are reacted with the compound NHRX nhr2 II possibly by the intermediate of carbamate in the 5 position when R^ is alkyl and R2 is H.

This process is explained in a more detailed manner hereafter.

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

The formamide (IV) is obtained, for example, by the action of zinc or alkali 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 of an alkali metal nitrite on the pyrimidinediones of formula V -H nhr2 in which R2, R3 and R4 are as in the formula I.

The compounds of formula II in which R2 represents H may also be prepared from 6-chloro-2,4-pyrimidinedione according to the reaction scheme (b) b) applying conventional methods such as that described in Liebigs Ann. Chem. 677 p. 113 (1964).

The compounds of formula II in which Rx is an alkyl group are prepared by the action of the amine RjNH2 on the brominated derivative of formula VI in which R2, R3 and R4 are as in the formula I. The derivative (VI) is obtained by the action of bromine in acetic acid or of N-bromosuccinimide in a mixture of acetic acid and acetic anhydride on the compound (V), as described in Liebigs Ann. Chem. p. 1847-54 (1979); when R3 represents H, it is preferable, before performing the bromination, to protect the nitrogen, for example with a benzyl group, which will be removed by catalytic hydrogenation after the reaction with R1NH2.

The compounds of formula VII in which R1 to R4 are as in the formula I and Z is 0 or S, maybe prepared by the action of urea or thiourea on the diaminopyrimidinediones of formula II, applying the method described in Liebigs Ann. Chem. p. 2030 (1974) for products in which Rlr R2, R3 and R4 = H or CH3.

When Rx represents an alkyl group, the compound of formula VII is preferably prepared by applying a process described in Liebigs Ann. Chem. p. 1847-54 (1979) for a compound II in which Rr = CH3. For the compound in which R2 = R3 = R4 = H, it is necessary to block the nitrogen bearing R3, and the reaction scheme is as follows: Η H conventional ο /-Wy LJ CH2C6H5 ch2c6h5 The carbamate is prepared in a manner by the action of an alkyl chloroformate on the compound (II) in which R2 = H, R3 = CH2C6H5 and R4 = H, in the presence of a base, while the cyclisation is performed 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 alcoholate, as described in Liebigs Ann. Chem. p. 1847 (1979).

The starting 6-aminopyrimidinediones (V) may be prepared by processes whose principles are known, which have been, in particular, described for compounds in which R2, R3 and R4, which may be identical or different, represent H or CH3. Thus, the compounds of formula V in which R3 is other than H and R2 = R4 = H are prepared by the process described in J. Am. Chem. Soc. 63 2567 (1941), in an alcohol according to the reaction scheme (c): c) R3NHCONH2 + ROOC-CH2-CN + R'ONa Va in which R and R' represent a Cx to C4 alkyl group.

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

The compounds of formula V in which R3 and R4 represent H are prepared by the action of R2NH2 in an acid medium on 6-chloropyrimidinedione, a known compound, according to the reaction scheme (d): d) Q H ii H + R->NH~ o^^ci I · H as described, for example, in J. Med. Chem. 20 p. 1186 10 (1977), or by fusion of the compound (V) in which R2 = R3 = R4 = H with R2NH2 in the presence of R2NH2.HC1.

The compounds of formula V in which R2 and R3 represent H are prepared by the process described in Liebigs Ann. Chem.631 p. 168 (1960) according to the reaction scheme (e) θ) R.NHCONH, in which R and R' each represent a Cx to C4 alkyl group, applying the procedure described in Liebigs Ann. Chem. 612 p. 158 (1958).

The salts of the compounds of formula I which bear amine functions are prepared by the action of at least one equivalent of the acid on the amine in solution. They are isolated by precipitation or by evapora25 tion of the solvent and purified by recrystallisation if - 7 ' necessary.

The pharmaceutical compositions and the medicinal products having as active principle at least one of the compounds of formula I, in which one of R , R^, r3 or R^ represents a C? to C2Q alkyl and others represent H or a to C3 alkyl, aM and represent H or together represent a carbonyl or thiocarbonyl, or one of its salts with a pharmaceutically acceptable acid, are another subject of the invention; the compounds in which one of the symbols R^, R^, R3 or represents a C^2 to 02θ alkyl group are especially preferred.

The presence of excessive amounts of free radicals has been detected in many pathological states and it i s now demonstrated that the tissue destruction observed in rheumatoid arthritis, autoimmune diseases, iron overload, the sequelae of cerebral and cardiac ischaemias and some ocular pathologies is greatly dependent on free radical formation. Thus, it is possible to consider administering to humans, compounds which inhibit the formation of these very reactive species, preventively or curatively in the treatment of the diseases mentioned above as well as, probably, in the diseases of middle age for which free radicals have been associated with the degenerative phenomena observed.

It is known that, among free radicals, those derived from oxygen, and especially the hydroxyl radical OH', have an important role; while it is not yet possible at the present time to measure the concentration of these radicals directly in vivo, this concentration can be estimated by studying the neutralisation of various freo radicals in vitro, and especially the peroxidation of membrane lipids, the consequence of which in vivo is the formation o: cytotoxic aldehydes and the destruction of cell membranes leading to an increase in their permeabi1i ty, The free radical-inhibitory action of a product XH is commonly determined in vitro by measuring the neutralisation of the 1,l-diphenyl-2-picrylhydrazyl radical (DPPII), stable at 20°C, by the product XII in the reaction DPPH' + XH -> DPPH2 + X'.

The disappearance of the DPPII' radical is studied by spectrophotometry at 517 run, at which wavelength the radical strongly absorbs the radiation in contrast to the hydrazine molecule DPPH2. This method is, in particular, described in Free Radicals in Biology and Medicine 3 p. 251-257 (1987) for uric acid and its N-methyl derivatives .

The compounds of the invention are, in this test, active at doses comparable to that of uric acid.

The inhibition of lipid peroxidation due to 10 oxygen-containing free radicals, a peroxidation of special importance in the presence of iron, has been determined in vitro by measuring the quantity of malonaldehyde formed in suspensions of mitochondria brought into contact with a superoxide ion generator such as dihydroxyfumaric acid, Uric acid is inactive in this test, 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 in particular 21-(4-[3,6-bis(diethylamino)-2-pyridin]1-piperaz inyl}-16a-methylpregna-1,4,9(ll)-triene3,20-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 he administered orally, parenterally, transinucosally or percutaneously for the preventive or curative treatment of degenerative phenomena linked to the presence of free radicals. The single and daily doses will depend on the compound, the nature and severity of the disease, the patient weight and condition and also the administration route; in general, the orally administered single dose in adults will be from 1 mg to 500 mg, while intravenously it will be from 0.1 to 25 mg.

The compounds of the formula I and their salts with pharmaceutically acceptable acids may be used in addition in cosmetic compositions for opposing the skin ageing.

In what follows, a description is given of examples of compounds of the invention, their process of preparation and the results obtained during the study of their free radical-inhibitory and lipid peroxidationinhibitory activities as well as those of the reference compound A mentioned above.

The results of the elementary analysis of the compounds isolated are compatible with their empirical formulae; the melting points stated are instantaneous.

The NMR spectra were plotted in solution in deuterated dimethyl sulphoxide with tetramethylsilane as an internal standard.

Examples of preparation of the 6-amino-2,4-pyrimidinediones of formula V are described first.

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 160°C for 3 hours. The reaction mixture is then brought back to room temperature before the introduction of 100 ml of ethanol. The solid is isolated by filtration and then suspended for a few minutes in 100 ml of 2N aqueous NaOH solution. The remaining solid is isolated and recrystallised in acetic acid. 7 g of the expected product, melting point above 250°C, are thereby obtained.

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, 1H); 9.78 (s, 1H); 10.07 (s, 1H). ii) The derivative of formula V in which R2 = C16H33, R3 = H and R4 = H, melting point above 250°C, is prepared in the same manner from hexadecylamine. Β) 1-Alkyl-6-amino-2,4-pyrimidinedione Formula V: R3 = C12H25, R2 = H, R4 = H. 17.6 g of ethyl cyanoacetate and 35 g of dodecylurea are introduced into a solution of sodium ethylate prepared with 4 g of sodium in 100 ml of ethanol. The medium is maintained at its refluxing temperature for 7 hours and then, after return of the mixture to room tempera35 ture, concentrated aqueous HCl solution is introduced to an acid pH, followed by 200 ml of water. The precipitate formed is isolated by filtration and then recrystallised in ethanol. 29 g of the desired product, melting point above 250°C, are thereby isolated.

NMR (300 MHz) 5: 0.87 (t, 3H) ; 1.26 (m, 18H); 1.52 (m, 2H); 3.73 (t, 2H); 4.58 (s, IH) ; 6.69 (m, 2H).

C) l-Alkyl-3-alkyl-6-amino-2,4-pyrimidinedione Formula V: R3 = C12H25, R2 = H, R4 = CH3. 2.95 g of 6-amino-l-dodecyl-2,4-pyrimidinedione, prepared in B, are suspended in 10 ml of aqueous NaOH solution and 10 ml of ethanol, and 1.3 g of (CH3)2SO4 are introduced at 60°C. Heating is maintained until the pH of the medium is almost neutral; N aqueous NaOH solution is then introduced to a basic pH and, after cooling, the solution is extracted with chloroform. The organic phase, washed with water and dried, is taken to dryness. 2.35 g of the expected product, melting point 74°C, are thereby obtained.

D) 3-Alkyl-6-amino-2,4-pyrimidinedione Formula V: R2 = H, R3 = H, R4 = C12H25. a) 35 g of dodecylurea and 24.3 g of ethyl malonate are introduced into 150 ml of a solution of sodium ethylate prepared with 3.5 g of sodium; the mixture is maintained for 8 hours at its refluxing temperature before the addition, at room temperature, of concentrated aqueous hydrochloric acid solution to an acid pH. The precipitate is then isolated by filtration, washed with water and thereafter recrystallised in ethanol. 30 g of l-dodecyl-2,4,6-pyrimidinetrione are thereby obtained. M.p. 129-130°C. b) 30 g of the product obtained according to a) are suspended in 13.5 ml of water, and 102 ml of POCl3 are added dropwise. The mixture is then maintained at its refluxing temperature for one hour and the excess POC13 is thereafter distilled off under reduced pressure. The residue is poured onto crushed ice and the precipitate formed is isolated. After washing with water and recrystallisation in a water/methanol mixture, 20.5 g of 6-chloro-3-dodecyl-2,4-pyrimidinedione, melting point 156-157°C, are obtained. c) A mixture of 30 g of the chlorinated derivative obtained according to b) and 150 g of benzylamine is maintained at 170eC for 1 hour. After the medium has been brought back to room temperature, 100 ml of water are introduced and the precipitate which has appeared is isolated. After the latter has been washed with methanol and ethyl ether, 25.13 g of 6-benzylamino-3-dodecyl2.4- pyrimidinedione, melting point 191 °C, are recovered. d) 25 g of the compound obtained according to c) and 4 g of palladium on charcoal (10% Pd) are suspended in 400 ml of absolute ethanol in a hydrogenation apparatus in which a hydrogen pressure of 3 MPa and a temperature of 80°C are maintained for 6 hours. The catalyst is removed by filtration of the solution while the latter is still hot before evaporating off the solvent under reduced pressure, and the residual solid is washed with ethyl ether. 17.02 g of 6-amino-3-dodecyl-2,4-pyrimidinedione, melting point 242°C, are thereby isolated.

In what follows, the preparation of compounds of formula III is described.

E) 6-Amino-l-dodecyl-5-nitroso-2,4-pyrimidinedione (Formula III: R2 = H; R3 = C12H25; R4 = H) . ml of isoamyl nitrite are introduced dropwise in 120 ml of ethanol in which 5 g of 6-amino-1-dodecyl2.4- pyrimidinedione are suspended, and 2 drops of concentrated aqueous HCl solution are then introduced. After 3 hours' stirring at room temperature, the violet crystals formed are isolated and recrystallised in ethanol. 4.35 g of the expected product, melting point 206°C, are thereby obtained.

The latter product may also be represented by the tautomeric formula: Applying the procedure described in E, the compounds were prepared whose characteristics appear in Table 1 of formula: R2R3 R4 m.p. °C F H ^12^25 ch3 158 5 G Cl6R33 H H 168 H C 12^25 H H 173 I H H Cl2R25 230 EXAMPLE 1 ,6-Diamino-l-dodecyl-3-methyl-2,4-pyrimidinedione (formula I: Zx = Z2 = H; Rx = R2 = H; R3 = C12H25; R4 = CH3) . a) 4 g of zinc powder is added in small portions to a solution, maintained at its refluxing temperature, of 4.35 g of the compound of Example C in 100 ml of formic acid; after 1 hour under reflux, the mixture is filtered while hot and the solvents are evaporated off from the filtrate under reduced pressure. 30 ml of CH3OH are poured onto the residue and the precipitate is isolated. 3.85 g of 6-amino-l-dodecyl-5-formylamino20 3-methyl-2,4-pyrimidinedione, melting point 215°C, are thereby obtained. 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 maintained for 15 minutes. The solution is then maintained for 3 hours at its refluxing temperature and thereafter cooled to 0°C. The precipitate is then filtered off to isolate 3.21 g of monohydrochloride of the final product, melting point 195°C.

The diamine may be prepared by treating a suspension of its hydrochloride in CH3OH with concentrated aqueous NH40H solution.

EXAMPLES 2 to 8 The compounds of formula I in which Z2 = Z2 = H, the characteristics of which appear in Table 2, are prepared by applying the process of Example 1.

ExampleRi r2 r3 r4 M.p. °C (salt) 2 H H ^12^25 H 237 3 H H H C 12^25 >250 (HCI) 4 H ^16^33 H H 242 (HCI) 5 H ^12^25 H H >200 (HCI) 6 H H ^•6^13 H >200 (HCI) 7 H H *-10^21 H >250 (HCI) 8 H H H >200 (HCI) NMR EX 7: (500 MHz) Ss 0.84 (t, 3H); 1.25 (m, 14H) ; 1.51 (m, 2H); 3.81 (t, 2H); 7.72 (s, 2H); 9.75 (θ/ 3H); 11.10 (s, 1H) . EX 8: (250 MHz) 5: 0.85 (t, 3H); 1.25 (m, 10H) ; 1.49 (m, 2H); 3.80 (t, 2H); 7.81 (Sr 2H); 9.46 (s, 3H); 11.13 (s, 1H).

EXAMPLES 9 and 10 3-Dodecyl-1-methyl-2,6,8(1H,3H,9H)-purinetrlone (formula I: Zx and Z2 = CO; Rx = R2 = H; R3 = CX2H25; R« = CH3) .

A mixture of 800 mg of the compound obtained in Example 1 with 1 g of urea is maintained at 180°C for 1 hour. 20 ml of water are poured into the mixture at room temperature; the solid is isolated, washed with ethanol and recrystallised in acetic acid. 680 mg of the desired product, melting point 266 °C, are thereby isolated.

NMR (250 MHz) 5: 0.86 (t, 3H) ; 1.24 (m, 18H) ; 1.56 (m, 2H); 3.19 (s, 3H); 3.77 (t, 2H); 10.79 (s, 1H); 11.89 (s, 1H).

Applying the same procedure with thiourea, 3-octyl-2,6(lH,3H,9H)-purinedione-8-thione, melting point >250°C, is obtained.

NMR (90 MHz) is 0.85 (t, 3H) ; 1.24 (m, 12H); 3.87 (t, 2H); 11.24 (s, 1H); 12.98 (s, 1H); 13.47 (s, 1H). EXAMPLES 11 to 16 The compounds of formula I in which Zx and Z2 represent C=0, the characteristics of which appear in Table 3, were prepared by applying the process described in Example 9.

ExampleR1 r2r3 r4 M.p. °C 11 H H H ^12^25 >250 12 H Cl2R25 H H >250 13 H Cl6R33 H H >250 10 14 H H Ci2R25 H >250 15 H H CgHi7 H >250 16 H H C10H2I H >250 The NMR data for these appear in Table 4. 15 TABLE 4 Example Frequency δ 11 300 MHz 0.86 (t, 3H); 1.25 (m, 18H); 1.52 (m, 2H); 3.70 (t, 2H); 10.77 (s, 1H); 11.03 (s, 1H); 11.83 (s, 1H) . 12 300 MHz 0.85 (t, 3H); 1.25 (m, 18H)? 1.51 (m, 2H); 3.63 (t, 2H); 10.82 (s, 1H); 10.86 (s, 1H); 12.00 (s, 1H) . 13 250 MHz 0.84 (t, 3H)ί 1.22 (m, 26H); 1.51 (m, 2H); 3.62 (t, 2H); 10.78 (s, 1H); 10.81 (s, 1H); 11.98 (s, 1H) . 14 250 MHz 0.85 (t, 3H); 1.22 (m, 18H); 1.48 (m, 2H); 3.73 (t, 2H); 10.54 (s, 1H); 11.35 (s, 1H); 12.03 (s, 1H) . 15 250 MHz 0.85 (t, 3H); 1.25 (m, 10H); 1.53 (m, 2H); 3.71 (t, 2H); 10.76 (s, 1H); 11.01 (s, 1H); 11.85 (s, 1H). 16 500 MHz 0.84 (t, 3H); 1.25 (m, 14H); 1.51 (m, 2H); 3.81 (t, 2H); 10.71 (s, 1H); 10.98 (s, 1H); 11.82 (s, 1H).

EXAMPLE 17 7-Dodecyl-2,6,8(1H,3H,9H)-purinetrione (formula I: Zx and Z2 = CO; Rx = C12H25; R2 = R3 = RA = H) . a) A suspension of 5.12 g of 6-amino-l-benzyl5 5-bromo->-2,4-pyrimidinedione, 11.1 g of dodecylamine and 100 ml of n-butanol is maintained for 3 hours at its refluxing temperature. The solvent is then removed under reduced pressure, 50 ml of ethanol are thereafter poured onto the residue and the precipitate formed is isolated. 4.2 g of 6-amino-l-benzyl-5-dodecylamino-2,4-pyrimidinedione, melting point 161°C, are thereby obtained. b) 2 g of the product obtained according to a), 1 g of dry K2CO3 and 1 g of ethyl chloroformate in 20 ml of dimethylformamide are kept stirring for 2 hours at room temperature before 200 ml of chloroform are introduced. The organic phase is washed with water at pH 5-6, then dried and concentrated. The residue is chromatographied on a column of silica gel, eluting with a CH3OH/CHC13 (5% v/v) mixture. 1.64 g of 6-amino-l-benzyl20 5-(N-dodecyl-N-ethoxycarbonylamino)-2,4-pyrimidinedione, melting point 196°C, are thereby obtained. c) 100 ml of ethanol, 6 g of the product obtained according to b), 10 ml of concentrated aqueous NHAOH solution and 0.3 g of palladium on charcoal (10% Pd) are introduced into a hydrogenation apparatus. Hydrogenation is performed at atmospheric pressure and at 50°C. When the theoretical quantity of hydrogen has been consumed, 400 ml of ethanol are added at about 50°C and the catalyst is filtered off at about this temperature. The solvent is removed until a residual volume of 20 ml is left. The precipitate which has appeared on cooling is isolated. 3.84 g of 6-amino-5-(N-dodecyl-N-ethoxycarbonylamino)-2,4-pyrimidinedione, melting point above 250°C, are thereby obtained. d) 3.5 g of the product obtained according to c) are introduced into 100 ml of a solution of sodium ethylate prepared with 2.3 g of sodium, and the solution is maintained for 4 days at its refluxing temperature.

The solvent is then distilled off and the residue suspended in 20 ml of water. The aqueous phase is acidified by adding concentrated aqueous hydrochloric acid solution before the precipitate formed is filtered off.

The latter is recrystallised in acetic acid. 5 1.56 g of the desired purinetrione, melting point above 280°C, are thereby obtained.

NMR (300 MHz) 5: 0.86 (t, 3H) ; 1.27 (m, 18H) ; 1.53 (m, 2H)? 3.78 (t, 2H); 10.43 (s, IH); 11.28 (s, 2H). EXAMPLE 18 1.0 6-Amino-5-dodecylamino-2,4-pyrimidinedione (formula I: Zx and Z2 = H; Rx = C12H25; R2 = R3 = R4 = H) . g of the compound obtained in Example 17 a) is dissolved in 50 ml of ethanol. 100 mg of Pd/C (10% Pd) are added and a hydrogenation is performed with H2 at 50°C at atmospheric pressure until the theoretical quantity of hydrogen has been consumed. The catalyst is separated by filtration while the mixture is hot and the solvent removed under reduced pressure to a volume of approximately 15 ml. The precipitate formed is recrystallised in ethanol. 510 mg of the desired product, melting point above 250°C, are thereby isolated.

NMR (500 MHz) δ: 0.86 (t, 3H) ,· 1.27 (m, 18H) ; 1.48 (m, 2H); 2.62 (t, 2H); 3.32 (m, IH); 5.67 (s, 2H); 9.80 (s, IH); 10.09 (s, IH).

EXAMPLE 19 -Amino-6-octadecylamino-3-methyl-2,4-pyrimidinedione (formula Is Zx and Z2 = H; Rx = H; R2 = C18H37; R3 = H; R4 = CH3). a) 2.05 g of 6-chloro-3-methyl-5-nitro-2,4-pyri30 midinedione, resulting from the nitration of 6-chloro3-methyl-2,4-pyrimidinedione according to a process described in Liebigs Ann. Chem. 677 113 (1964), are introduced portionwise into a solution of 3 g of octadecylamine and 1 g of triethylamine in 20 ml of ethanol.

The solution is maintained for 20 minutes at its refluxing temperature and then neutralised by adding acetic acid. The precipitate formed is washed with water and recrystallised in ethanol. 2.11 g of 6-octadecylamino-3-methyl-5-nitro_ 17 _ 2,4-pyrimidinedione, melting point 130°C, are thereby isolated. b) The product thereby obtained is dissolved in 50 ml of formic acid, and 2 g of zinc powder are intro5 duced into the medium in small portions; after 1 hour under reflux, the mixture is filtered while hot and the solvents are evaporated off from the filtrate under reduced pressure. The residue is crystallised with 15 ml of methanol. 1.8 g of 5-formamido-6-octadecylamino10 3-methyl-2,4-pyrimidinedione, melting point 196°C, are thereby obtained. c) The formamide obtained above is treated with HCl in methanol as in Example 1-b.

The hydrochloride of the desired product, melting 15 point 187°C, is thereby obtained.

NMR (250 MHz) 6: 0.89 (t, 3H) ; 1.27 (m, 30H) ; 1.55 (m, 2H); 3.15 (s, 3H); 3.32 (m, 2H); 7.86 (s, IH); 10.40 (s, 3H).

The free radical-inhibitory activity of these 20 compounds was determined as follows: 20 pi of a 1 mM solution of DPPH' and 10 pi of a solution of the test product in dimethyl sulphoxide are introduced into 970 pi of a mixture of methanol and aqueous solution buffered with Tris (tromethamine)-HCl (10 mM - pH 7.4) in the proportion of 1 volume of methanol per 2 of buffer. The mixture is maintained for 20 minutes at 20°C before the optical density is determined at 517 nm against a control mixture for which the test product has not been introduced in dimethyl sulphoxide.

The percentage trapping T of the free radical is defined as a ratio of the optical densities D D (product) - D (control) T = - x 100 D (control) The median effective concentration (ECS0), that for which T = 50, is calculated on the basis of the results of measurements made with 4 different concentrations in a series of 10 around the EC50. 1&The EC50 values for compounds according to the invention, as well as for uric acid and its N-methyl derivatives and for the product A, appear in Table 5.

TABLE 5 PRODUCT ec50 (μΜ) PRODUCT ec50 (μΜ) Uric acid 4.5 Example 11 5.2 1-CH3 uric acid 4.5 12 6.2 3-CH3 11 3.2 13 4.5 7-CH3 If 20 14 3.2 9-CH3 II 5.2 A 4 15 4 Example 1 5 16 3.4 2 7 17 27 3 5.5 18 4.5 4 6.4 19 6.2 5 6.4 6 6.5 7 5.6 8 6.5 9 2.7 10 6.5 The inhibitory activity of the compounds with respect to the peroxidation of membrane lipids was determined as follows: a) Preparation of mitochondria An ox heart is removed immediately after slaughter of the animal and cooled to 4°C, at which temperature all the following operations will be performed. The heart is washed with an aqueous buffer solution at pH 7.4 (10 mM Tris-HCl, 2 mM EDTA, 20 mM KC1, 250 mM sucrose) and the connective tissues and fats are then removed. The muscle is then minced and the minced preparation is suspended in an aqueous buffer solution at pH 7.4 (10 mM Tris-HCl, 250 mM sucrose, 1 mM EDTA) on the IS basis of 700 g of muscle per 3 litres. The pH of the suspension is readjusted regularly to pH 7.4 before the minced preparation is isolated by filtration on a gauze. It is then resuspended in 3 litres of the second buffer and the suspension is ground before being centrifuged for 20 minutes at 1,000 g, and the supernatant is thereafter centrifuged for 15 minutes at 17,000 g. The centrifugation pellet is washed several times with an aqueous buffer solution, pH 7.4 (15 mM Tris-HCl, 250 mM sucrose), with separation of the washing solution by centrifugation, and a homogeneous suspension of mitochondria in this same buffer at a concentration of 50 mg of protein/ml is finally prepared, and will be stored frozen in droplets in liquid nitrogen. After thawing, respiratory checks carried out by measuring the rates of oxygen consumption in the presence or absence of ADP for the substrates succinate and glutamate/maleate are 80% of those obtained before freezing, several months after the preparation of the mitochondria. b) Lipid peroxidation A portion of thawed mitochondria is dispersed at 4°C in a buffered aqueous solution, pH 7.4 (30 mM Tris-HCl containing 140 mM KCl), to obtain a protein concentration of 0.37 mg/ml; 10 μΐ of a 100 mM solution of dihydroxyfumaric acid, the pH of which has been adjusted to 7.4, 2 μΐ of a solution of the test product dissolved in dimethyl sulphoxide (DMSO) and 30 μΐ of a 1 mM solution of FeSO4. (NH4)2SO4 stored protected from oxygen and light are introduced into 960 μΐ of this suspension. The control sample contains DMSO without the test product, while the blank contains neither dihydroxyfumaric acid nor iron.

After the removal of a first 100 μΐ aliquot, the samples are maintained at 37°C with gentle agitation and 100 μΐ aliquots are removed after 20 and 40 minutes.

Each aliquot is introduced immediately into 1.5 ml of a solution of 13.5% (w/v) trichloroacetic acid and 0.33% (w/v) thiobarbituric acid in 0.85 N aqueous hydrochloric acid solution, and the vessels are closed and then brought to 100°C for 15 minutes before being cooled to room temperature in the course of 5 minutes. 1 ml of 70% (w/v) aqueous trichloroacetic acid solution is then added before the spectrofluorimetric measurement is performed at an excitation wavelength of 515 nm and an emission wavelength of 553 nm.

A reference series of malonaldehyde bis(diethyl acetal) diluted in 0.1 N aqueous HCl solution enables the aldehyde concentration corresponding to each fluorescence intensity i to be calculated.

Under the experimental conditions, i - 1.89 C (nmol) = 35.5 was determined, and the percentage inhibition of peroxidation I can accordingly be calculated by the formula (i - 1.89) control - (i - 1.89) test I = - x 100 (i - 1.89) control - (i - 1.89) blank 20 The concentration inhibiting 50% of the signal, IC50, that is to say that for which I = 50, is calculated from the measurements performed with 4 different concentrations of the test product in a series of 10 around the IC50.

The results obtained are shown in Table 6. Uric acid does not inhibit malonaldehyde formation at a concentration below 100 pM; its N-methyl derivatives are only weakly active: for the most active, I = 35 at a concentration of 100 pM.

TABLE 6 Example IC50 (μΜ) Example IC50 (μΜ) A 0.7 11 1.25 1 0.24 12 0.3 2 0.27 13 0.9 3 0.26 14 1.6 4 0.9 5 0.3 15 28 6 3.5 16 3.7 7 0.28 17 1.6 8 0.62 18 0.57 9 0.85 19 20 10 25

Claims (17)

1. A coumpond of the formula : in which one of the symbols R^, R2 R^ or R^ represents C? to 0 2 θ alkyl while the others each represent H or C^ to C^ alkyl, and Z 1 and Z 2 each 10 represent H or together represent carbonyl or thiocarbonyl, with the proviso that, when R^ represents n-dodecyl, one of R^ or R^ is other than H, and its addition salts with a pharmaceutically acceptable acid.

2. Coumpcurd according to claim 1, in which one of R , R^ R^ or R^ represents C^ 2 to C 2 q alkyl. 15

3. Compound according to claim 1, in which Z^ and Z 2 each represent H.

4. Compound according to claim 1, wherein one of Rp R^ or R^ represents C 12 to C 2 g alkyl and Z^ and Z 2 each represent H.

5. Compound according to claim 1 in which Z^ and Z 2 together repre 20 sent a group selected from carbonyl or thiocarbonyl.

6. Compound according to claim 1, in which and Z 2 together represent a carbonyl.

7. Corrpourtd according to claim 1, in which one of or R4 represents C l2 to C 2 g alkyl and Z^ and Z 2 together represent a 2 5 group selected from carbonyl and thiocarbonyl

8. Pharmaceutical composition comprising an amount effective for inhibiting degenerative phenomena linked to the presence of free radicals,of a compound of formula I in which one of R^ R^ or R^ represents C^ to C 2Q alkyl and others represent H or C^ to C^ alkyl, and Z^ and Z 2 represent H or together represent - carbonyl or thiocarbonyl, and its addition salts together with a pharmaceutical carrier, together with a pharmaceutical carrier.

9. Pharmaceutical composition according to claim 8, in which Rj, Rj, Rj or R^ represents Cj 2 to C^q alkyl.

10. Pharmaceutical composition according to claim 8, in which Zj and Z~ each represent H. 5 2

11. Pharmaceutical ccsrposition according to claim 8, in which one of Rj, Rj, Rj or R 4 represents Cj 2 to C 2Q alkyl and Zj and Z 2 each represent H.

12. Pharmaceutical ccsrposition according to claim 8, in which Zj and 10 Z 2 taken together represent a group selected from carbonyl and thiocarbonyl.

13. Pharmaceutical composition according to claim 8, in which one of Rj, Rj, Rj and R 4 represents Cj 2 to C 2Q alkyl and Zj and Z 2 taken together represent a group selected from carbonyl and thiocarbonyl.

14. A compound as claimed in claim 1 or an addition salt thereof with a pharmaceutically acceptable acid, substantially as hereinbefore described and exemplified.

15. 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.

16. 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 15.

17. A pharmaceutical composition according to claim 8, substantially as hereinbefore described.