New derivatives of the catéchol, a process for their preparation and compositions therapeutic containing these derivatives.

The present invention relates to novel catechol derivatives, a process for their preparation and therapeutic compositions containing them.

The invention relates, more particularly, catechol derivatives having the general formula I

I

in which R is an acyclic hydrocarbon radical straight or branched chain containing 1 to 8 carbon atoms, one of the carbon atoms optionally being asymmetric, a radical of mono - or bicycloalcoyl optionally substituted by one or more alkyl radicals comprising from 1 to 7 carbon atoms, one of the carbon atoms of the radical of mono - or bicycloalcoyl optionally being asymmetric, a phenyl radical, a radical halophenyl, nitrophenyl radical or a phenyl radical substituted by one or more alkyl radicals having 1 to 7 Da are carbon atoms, trifluoromethyl or by radicals or radical-COOY wherein Y is a hydrogen atom or an alkyl radical having from 1 to 5 carbon atoms.

The compounds of the invention are inhibitors of lipoxygenase and cyclogenase. They may be used in therapy. human for their non-steroidal antiinflammatory, antithrombotic, anti-allergic, antiischemic and antianaphylactic.

The invention relates, also, a process for the preparation of these catechol derivatives, said process comprising reacting, at reflux, in an anhydrous alkanol, the s - (dihydroxy 3.4 phenyl) isothiourea of formula II, below, on a tosylate of formula III, below, wherein R is defined as above.

The reaction is, preferably, performed by refluxing the starting materials in the anhydrous methanol 48 to 72 hours during, under an inert atmosphere. The s - (dihydroxy 3.4 phenyl) isothiourea II is, preferably, used in the form of its hydrochloride. The group represented by R in formula tosylate III can be racemic or optically active.

The s - (dihydroxy 3.4 phenyl) isothiourea II can be prepared by a method derived from that described by J DANEKE, Û. JAENKE, B PANKOW and H.W. WANZLICK, Tetrahedron Letters and 1970, 15, 1271. The tosylate III can be prepared by a method derived from that described by A. STRITWIESER dy A.C., wais areas dy, J 92. Chem., 1962, 27, 290.

The invention relates, finally, therapeutic compositions that use as an active ingredient one of the compounds defined above.

The invention will be, incidentally, more preferably comprised by describing the examples that follow.

Example 1

(methyl-2 thio-butyl) -4 cathecol (R-CH2 -=^- CH2 (CH2^) - OH,,- HC.,)

In a one liter reactor equipped with a magnetic stirrer and placed under nitrogen flow, is dissolved 22 grams (0.1 moles) of s - (dihydroxy 3.4 phenyl) isothiourea in 50 ml of anhydrous methanol. The solution is heated and added a solution of sodium methanolate containing 9.2 g sodium (4 equivalents) for 150 ml of anhydrous methanol. Is poured 24.2 grams (0.1 moles) of tosylate methyl 2 in solution in 50 ml of butyl methanol, the temperature being carried in the vicinity of the boiling point of the methanol.

The reaction mixture is maintained at reflux for 48 hours and then the solvent is evaporated under reduced pressure (2000 Pascal). The residue is taken up by demineralized water and acidified by hydrochloric acid to 20%. After extraction with ethyl ether, the organic phase is washed with water and then dried over anhydrous sodium sulfate. After removal of ethyl ether, a red oil is obtained which is purified by chromatography on silica gel using a mixture of hexane/ethyl acetyl (95/5 by volume) as eluent. The yield is 54%. The identity and the structure of this compound has been confirmed by spectroscopy and elemental microanalysis by PMR-with the following results.

62.08 7.69 15.18 14.99% Calculated 62.24 7.60 15.06 15.10% found

Example 2

(+) - (methyl 2 butyl Ske) -4 catechol

(≈ - R-OH₂~HC (HC^) - OH_:HM~_: )

By doing the in the example 1 but using the tosylate £- methyl-2 butyl tosylate instead of methyl 2 butyl, the above compound was obtained in a yield of 54%. It is an oil of. yellow color;

[V] Q.³ =+ 22.52, c=0.6, chloroform. The identity and the structure of this compound has been confirmed by spectroscopy and elemental microanalysis PMR-with the following results.

62.21 7.67 15.22 15.01% Calculated 62.24 7.60 15.06 15.10% found

Example 3

by menthyl thiourea 4 catechol (the R=a menthyl)

By doing the in the example 1 but using the tosylate of menthyl instead of the tosylate methyl 2 butyl, the above compound A was obtained with a yield of 8%..c'is a solid that melts at 124" C. (Kofler). The identity and the structure of this compound has been confirmed by spectroscopy and elemental microanalysis PMR-with the following results.

% Calculated 68.53 8.63 11.41 11.43% found 68.48 8.74 ' 11.23 11.28

Example 4

(+) - thiourea 4 by menthyl catechol (≈ R-L by menthyl)

By performing•as in the example 1 but using the tosylate GBP - a menthyl instead of the tosylate methyl 2 butyl* the above compound was achieved with a efficiency 10 t-. I. a solid melts at 119 * 0 (Kofler). : ^ ** D * 3p t-O|3.98, C. = 5, ethanol.•' L. 'identity and the structure of this compound have been confirmed spectroscopically MRT and microanalysis elemental with the following results.

' C. :■% calculated 68.53 8.63 11.41 11.43

67.99 8.37 11.22 12.05% Found

Example 5

(methyl-1 heptvle alkylthio) -4 catechol (where R=- CH2 (CH2.₃) - (HC^)₅zçh₃ 1

In operating as in example 1 but using the tosylate methyl 1 to heptane instead of the tosylate béthyle-a 2. butyl, the above compound was obtained in a yield of 26 %. It is an oil. The identity and the structure this compound was confirmed spectroscopically MRT and microanalysis elemental with the following results.

H 0 SEC.

. 65.90 8.65 12.74 12.50% Calculated 66.10 8.70 12.58 12.60% found

Example 6

(+) -(methyl-1 thio-heptyl) -4 catechol

(R-CH2 - - (HC₃) - (hC₂ )₅ - hC₃ )

The EH by proceeding as in the example 1 but using the tosylate ' GBP - méthyla L of heptane instead of the tosylate ^ - methyl butyl, the above compound was obtained in a yield 6.5 the c %.^O is an oil._D +=7, c=0.7, chloroform. The identity and structured of this compound has been confirmed by spectroscopy and elemental microanalysis SME with the following results. V.

H-O, O

66.10 8.70 12.58 ' 12.60 65.97 8.68 12.83 12.71

Example 7

phenyl thiourea r4 catechol (where R=phenyl)

By performing ' as in I^O example 1 but by using the tosylate phenyl instead of the tosylate salt. , of methylating 2 butyl, the above compound was obtained in a yield of 17%. It is a solid that melts at 164 "C. (Kofler)" the identity and its structure have. been confirmed, by, spectroscopy and elemental microanalysis MRT, -, with lèsrésultats following. ... , -

By doing the in the example 1 but using ' tosylate cyclohexyl instead of the tosylate methyl 2 butyl, the above compound was obtained in a yield of 31%. It is a solid that melts at 148 °c (Kofler). The identity and the structure of this compound has been confirmed by spectroscopy and elemental microanalysis MRT.... with. the ^ following results.

% Calculated

% Found

C - H O-O

64.25 7.20 14.26 14.29% Found 64.19 7.31 14.23 14.27% calculated

Example 9

(dimethyl-3.5) phenyl thiourea 4 catechol

(Where R=(dimethyl-3_F. 5) phenyl)

By doing the in the example 1 but using the tosylate of dimethyl-3.5 phenyl instead of the tosylate methyl 2 butyl, the above compound was obtained in a yield of 23%. It is an oil. The identity and the structure of this compound has been confirmed by spectroscopy and elemental microanalysis PMR-with the following results.

% calculated

% Found

O-H

S

68.26 5.73 12.99 13.02 68.20 3.93 12.91 12.96

Example 10

(di trifluoromethyl-2.6) phenyl thiourea 4 catechol

(■the R=(di trifluoromethyl-2.6) phenyl)

By doing the in the example 1 but by using the tosylate (di trifluoromethyl-2.6) phenyl instead of the tosylate methyl 2 butyl, the above compound was obtained in a yield of 13%. It is a solid that melts at 110" C. (Kofler). The identity and the structure of this compound has been confirmed by spectroscopy and elemental microanalysis PMR-with the following results.

H 0

F

% Calculated

% Found

47.46 2.28 9.03

47.36 2.55 8.98

9.05 32.18 8.98 32.13 Q6417 PHARMACOLOGY

The lipoxygenases (the LOS) convert arachidonic acid (AA cells) in hydroxy derivatives and leukotrienes. These products are potent pharmacological agents are expected to play important roles in inflammatory disorders or hypersensitivity. The LOS more AA and mainly act on the

the leukotrienes lo and duct 5

the lo and 12 leads to the hydroxyaspartic acid 12 (12 HPETE) peroxyeicosatetraenoic and other hydroxy compounds ≈ 12,

the conduit 15 HPETE lo and 15 and at d¹ other hydroxysteroid compounds 15,

and, in quantities substantially lower, the LO and lo and 11 8..

1) Search "in-vitro" 7 potential inhibitory activity of compounds on the lipoxygenase of the soybean

The method employed was that Corey E.J. and Al (j pieces. Bitter. Mal. Share, 104, 1750 - 1752? 1982). In a final volume of 1.8 ml, 0.2 m were mixed aerated with a borax buffer 500 units of soybean lipoxygenase. At the time of testing the inhibitors, added in 0.6 ml of liquid for that final concentrations range from 10 m to 10 m and then in a preincubation during 10 minutes to -8 room temperature. The reaction is started by the addition of arachidonic acid has 10 m is then method to..... , -4 incubating at room temperature for 90 minutes and is determined by measuring 15 by-HPETE to 236 nm absorption. For each experiment, was used as a control lipoxygenase boiled to account for any possible uptake of the compounds to the wavelength of the experiment. The percentage of enzyme activity was calculated for each concentration and the amount of substance required for 50% inhibition of enzyme activity was calculated by linear regression on a set of data points corresponding to the logarithm of the percentage molar concentration of inhibition.

- 11 -

The V ^ çvf■.

2) Study 1VII. thereinibitthe Ithe potential "in-vitro" of 1' aniothe n sub âroxide (0..).

The method employed was idanticue than LED chiefly, RF, J 9jor>and kk.e. Arfors (Res. Microvascular. 22

239 - 254; 1981), it is to say the iéduction cytochrome

3 + 3 +

the c (Cyt c-) was performed in a system comprising

. 3 + - 5

0, 9 - ii mm in hypoxanthine, 5.10 m of Cyt c in a bicarbonate buffer ph - 7.35 (0, 132 Μ NaCI, 4.7 lo and "³M KCI, 2, 1)~³ The m CaCl₂ , 1.2 10"³ W MgS0₄ , 0, 018 Μ NaHC0₃ ). The^: reaction is started by the addition of xanthine oxidase to the concentration of 9.07 Û / milliliter. The increase in absorption at 550 nm was measured at 37' C in a cell spectrophotometric thermostatic, every minute, during 4 min.

Each compound was added before experienced xanthine oxidase. The activity unit was defined as a change of 0,001 unit/min. The percentage of enzyme activity was calculated for each concentration of each compound experienced and the quantity of substance required to inhibit 50% of the enzyme (IC₅₀ ) was calculated by linear regression on a set of data points corresponding to the logarithm of the percentage molar concentration of inhibition. The corresponding values are reported in the following table.

3) Experiment " in-vitro¹¹ compounds on metabolism of arachidonic cascade human platelet microsomes

This experiment was run in enzymatic glassware silicone according to the method of P. an HO, e. Walters and hr, by Sullivan (prostaglandin 12, 951; 1976). The reaction mixture containing 50 mm tris-HCl buffer, at pH ≈ 7.9, 5 mm of tryptophan-2, 2 m of methemoglobin, 0.2 mg of powder microsomes, more the compound to be tested, in a total volume of 0.2 ml, incubated at 37 °c during was 5 minutes before the addition of 10 1 μ of 20 μΚ -arachidonic acid C.¹⁴ (0.08 juCI). After 5 other minute incubation, the reaction is then stopped by addition of 10 1 μ the m citric acid.

The reaction mixture was then extracted four times with 0.5 ml of anhydrous ethyl ether and then dried over sodium sulfate. The residue was then resuspended in about 40 1 μ ether and then chromatographed on silica gel plates. The eluent used was ethyl ether/methanol/acetic acid (90/1/2). The Rf values were measured relative to arachidonic acid. The chromatography in thin slabs were then exposed on ultra-film for about 24 hours KPL. The identification of the different staining was performed according to standard methods (in PGA₂ , BGP₂ , THE PGE₂ , TFP_2c , PXB₂ , arachidonic acid) in the same solvent system.

Quantitative results were obtained by measuring density of the developed film using a transmission densitometer (E.C. 910) combined with an integrator Hewlett-Packard 3390a. Standard as positive for the specific inhibition of thromboxane synthetase and cyclooxygenase were used 1 'imidazole and 1' indomethacin.

The results are reported in the following table.

Inhibiting cyclooxygenase

The activity of the substances of the cyclooxygenase is quantified by the two spots corresponding to the PGE₂ and DPP DSS ^ (PGEs ratio₂/ DPP DSS₂ ).

4) Inhibition test¹¹ vitro " prostaglandin synthetase inhibitors on the microsomes of seminal vesicle water hammer

This experiment was run according to the methods of Baumann and ai (Naunyn-to-Schmiedeberg ' e Briefs. Apotex. 307, 73;

1979) and Takeguchi, c and ai (Pharmacol. 10, 2372, 1971 *). Tagged molecules by enzymatic determination has been performed in glassware silicone. The reaction mixture, containing 50 mm tris-HCl buffer, ph=8.3, in the presence of MMI of reduced glutathione (GSH are), of 0.55 mm hydroquinone, and 50 of the test compound μ receptors powder microsomes of seminal vesicle pressure surges in a total volume of 0.2 ml, was incubated for

5 minutes at 37 ^ ℮ prior to addition of 10 1 μ acid

-6

m-labeled arachidonic 10 14 (0.08/ICF). After 30 minute incubation, shaking other, the reaction was stopped by addition of 10 1 μ the m citric acid.

The reaction mixture was then extracted four times with 0.5 ml of anhydrous ethyl ether and then dried over sodium sulfate. The residue has then been resuspended in about 40 μ 1 ether followed by chromatography on silica gel plates. The eluent was L!/ methanol/ethyl ether (45/1/2) acetic acid. The Rf values were measured relative to arachidonic acid. The chromatography in thin plates have then been exposed on ultrathin film for about 20 hours KPL. The identification of spots was performed according to standard methods (PGEs PGEs ^₂ , PGG'S_the , TFP_2a , THE PGA ^ IN PGA₂ , BGP₁ , BGP₂ ) in the same solvent system. Quantitative results were obtained by densitometry and are reported in the following table.

5) Experiment " in-vitro¹¹ compounds as inhibitors potential xanthine oxidase

The activity of xanthine oxidase was determined by the method of Horn. Kalckar (j pieces. Toxicol. Mal. 167, 429 - 443, 1947) which measures, by ' spectrophotometric pathway, the formation of uric acid.

In a cuvette spectrophotometric, there is xanthine oxidase such that the final concentration is 0.01 unit/ml. of, phosphate buffer 0, 05 Μ, ph=7.4, or the inhibitor. The reaction is started by the addition of xanthine to the final concentration of 5.10 ^ Μ. The release of uric acid was followed in all 30 seconds over 295 nm 2 min (linear phase). A unit of activity was defined as the change of 0,001 unit/min.. the percentage of enzyme activity was calculated for each concentration of the tested compounds as well as the amount of substance required to inhibit 50% of the enzyme (IC₅₀ ), linear regression on a set of data points corresponding to the logarithm of the percentage molar concentration of inhibition.

The results are reported in the following table.

6) Lipoxygenase inhibitory human leukocytes

a) inhibition of 5 - lipoxygenase and 12 - polymorphonuclear leukocytes

1. Incubation of 15 x 10⁶ human leukocytes/ml.

2 + 2 +..

with 2 mm of CA, 0.5 îuH Mg in the presence of inhibitors to 37³ C. for 20 min.

2. Stimulation with 1 μ receptors said ionophore (a23l87)/ml for 4 min,

3. Interruption of incubation by adding a volume of methanol.

4. Chromatographic analysis by RP-HPLC, . c18 column, 5 / α.

5. Measuring the height of the peaks and comparison with the internal standard (PGB is₂ ).

20 08417

b) inhibition of 5 -, - 12 - and 15 - lipoxygenases polymorphonuclear leukocytes

. Experimental protocol

1. Incubating 11 x 10⁶ human leukocytes/ml with the inhibitors during 20 min (2 mm CA and ' + 2. 0.5 mm mgs^{+ 2} ) to 37 °c.

2. Stimulation by .10 the IQ VBE1 D.¹ arachidonic acid and 1 q-μ ionophore (Α 23187)/π ΐ 1 during 4 min.

3. Interruption of incubation by adding D.¹ a volume of methanol.

4. RP-HPLC is chromatographic analysis.

5. Measuring the height of the peaks and comparison with the internal standard.