FR2689397A1 - Use of di:tert-butyl-4-hydroxy-benzylidenyl-indoline-2-one - as an antioxidant, prostanoid synthesis inhibitor and platelet anti aggregant to treat atheroma, arteriosclerosis inflammatory diseases, etc. - Google Patents

Abstract

Use of 3-(3,5-ditert-butyl-4- hydroxybenzylidenyl) indoline-2-one derivs. and their base addn. salts (when R1 = H) in medicines, is claimed. In (I) R1 = H or CH3. 3-(3,5-ditert-butyl- 4-hydroxybenzylidenyl) indoline-2-one is pref. claimed. USE - (I) are antioxidants and protect cellular lipids and low density lipoproteins (LDL) from peroxidn. They have a strong inhibiting effect on the biosynthesis of prostanoids. They have a platelet antiaggregant effect. Their pharmacological activity is better than that of probucol. Use of (I) and compsns. contg. (I) in the treatment and prevention of ailments due to or linked to peroxidn. phenomena, to disorders of the synthesis of prostanoids, and/or to problems of platelet aggregation, in the treatment of central or peripheral ischaemic disorders, inflammatory diseases, pain, metabolic diseases, atheroma, arteriosclerosis, respiratory diseases, asthma, emphysema, disorders of immunological origin, lupus erythematosus, allergic reactions, certain cancers, cerebral or peripheral ageing and in the prevention and treatment of damage due to surgical trauma or the reperfusion of organs, is claimed, partic. atheroma, arteriosclerosis and inflammatory diseases. Daily dosage is 1-200mg, in one or two doses. Claimed dosage is 1-50mg. (I) appear to be nontoxic; no problems were observed at 1g/kg (p.o. mouse).

Description

 The invention relates to the use of derivatives of 3- (3,5-ditertbutyl-4-hydroxybenzylidenyl) indoline-2-one and their addition salts with a pharmaceutically acceptable base, for obtaining medicaments.

dans laquelle R1 représente un atome d'hydrogène ou un radical méthyle, et de leurs sels d'addition, lorsque R1 représente un atome d'hydrogène, avec une base pharmaceutiquement acceptable, pour l'obtention de médicaments.More particularly, the present invention relates to the use of the compounds of formula (I)

in which R1 represents a hydrogen atom or a methyl radical, and their addition salts, when R1 represents a hydrogen atom, with a pharmaceutically acceptable base, for obtaining medicaments.

 Among the pharmaceutically acceptable bases which can be used to salify the compounds used according to the invention, there may be mentioned, by way of examples and without limitation, sodium hydroxide, potassium hydroxide, triethylamine, diethylamine, ethanolamine , arginine, lysine, and diethanolamine.

The compounds of formula (I) have been described in the publication of
ZHUNGIETU et al. (Khim. Geterotsikl. Soedin., 1973, (1): pages 40-44) as reaction products of indoles and hydroxyindole with certain aldehydes but no pharmacological activity has been mentioned.

 The Applicant has discovered that the compounds of formula (I) surprisingly have very interesting pharmacological properties, and that they are therefore useful for obtaining medicaments.

 Indeed, the compounds used according to the present invention have very important antioxidant properties. Pharmacological studies have in particular shown that the compounds used according to the present invention are endowed with remarkable protective activities in the context of peroxidation processes of cellular lipids and low density lipoproteins (LDL).

 Furthermore, the compounds used according to the present invention have the particularity of having a powerful inhibitory effect on the biosynthesis of prostanoids. They also have significant anti-platelet aggregation power.

The pharmacological activities of the compounds used according to the invention are in particular much higher than those of probucol, a commercial compound of the prior art.

We can therefore expect compounds used according to the invention, which have both inhibitory properties of lipid peroxidation, biosynthesis of prostanoids, and platelet aggregation, a particularly new and beneficial action in conditions where a peroxidation of membrane lipids, dysregulation of prostanoid synthesis and / or platelet aggregation disorders.

 The compounds of formula (I) can thus be used for obtaining medicaments useful in the treatment or the prevention of affections due or related to phenomena of peroxidation, to dysregulations of the synthesis of prostanoids, and / or to disorders platelet aggregation, in the treatment of central or peripheral ischemic disorders, inflammatory diseases, pain, metabolic diseases, atheroma, arteriosclerosis, respiratory diseases, asthma, emphysema, immunological diseases, lupus erythematosus, allergic reactions, certain cancers, cerebral or peripheral aging, and in the prevention and treatment of damage due to surgical trauma and reperfusion of organs.

 The present invention also relates to pharmaceutical compositions containing one of the derivatives of formula I, or one of its addition salts with a pharmaceutically acceptable base, alone or in combination with one or more inert non-toxic excipients.

 Among the pharmaceutical compositions according to the invention, mention may be made, by way of examples and without limitation, of those which are suitable for oral, parenteral, nasal, rectal, perlingual, ocular or pulmonary administration and in particular injectable preparations, aerosols, eye or nasal drops, simple, film-coated or coated tablets, capsules, capsules, suppositories, creams, ointments, and dermal gels.

 The useful dosage varies according to the age and weight of the patient, the route of administration, the nature of the condition and any associated treatments and ranges from 1 mg to 200 mg per 24 hours in 1 to 2 doses.

The examples which follow illustrate the invention and do not limit it in any way.

Example A: STUDY OF THE ANTIPEROXIDANT ACTIVITY OF THE COMPOUNDS OF FORMULA
(I) USED ACCORDING TO THE INVENTION
The action of the compounds used according to the invention, capable of trapping OH radicals, has been studied on the one hand, on the spontaneous peroxidation of lipids and on the other hand, on the peroxidation induced by the Fe2 + - ascorbate system (10 WM - 250 pM), and this on homogenates of rat brains.

 During the measurement of the spontaneous lipid peroxidation, the homogenates of the brain of rats are placed in the presence or in the absence of the test compounds for 60 minutes at 37 ° C. The reaction is stopped at OOC and the assay of malondialdehyde is carried out at using thiobarbituric acid Lipid peroxidation is determined by the substances reacting with thiobarbituric acid expressed in nanomoles of malondialdehyde.

 During the measurement of the induced lipid peroxidation, the methodology is identical to that previously described except for the addition to the homogenate of the radical-inducing system: Fe2 + ascorbate. The reference substances are probucol and vitamin E.

 The concentrations of the tested compounds inhibiting the peroxidation of the substrate by 50% are calculated.

 It appeared that the compounds of formula (I) used according to the invention have a particularly intense anti-oxidant activity since, they allow an increase of more than 100% of the anti-oxidant activity compared to probucol and of more than 300% compared Vitamin E, which is the natural antioxidant in the human body. This remarkable result occurs whether the peroxidation is spontaneous or induced by a chemical system.

Example B: STUDY OF THE PROTECTIVE POWER OF THE LDL OXIDATION OF
COMPOUNDS OF FORMULA (I) USED IN ACCORDANCE WITH THE INVENTION
The capacity of the compounds used according to the invention to decrease the proportions of oxidized LDL was measured as follows
A 24 hour incubation is carried out bringing together native LDLs, a Cu2 + system generating free radicals and the compounds to be tested.

The results are obtained after analysis of the medium by a high performance chromatographic technique: the FPLC (Fast Protein Liquid
Chromatography). The protective power of the compound tested is determined after comparison of the chromatogram obtained with that of the reference positive control: probucol.

It is clear that the compounds used according to the invention have a very high protective power and significantly superior to that of probucol.

Example C: STUDY OF THE INHIBITORY ACTIVITY OF THE COMPOUNDS OF FORMULA (I)
USED ACCORDING TO THE INVENTION ON PROSTANOID SYNTHESIS 1) STUDY OF THE INHIBITORY ACTIVITY ON PROSTANOID SYNTHESIS
FROM CYCLOOXYGENASE
The aim of this study is to measure the inhibitory activity of the molecules used according to the invention on the secretion of prostaglandin
E2 (PGE2), one of the main prostanoids produced by the cyclooxygenase of human granulocytes stimulated by the calcium ionophore A23187.

PROTOCOL
- Isolation of human granulocytes:
Human venous blood from healthy donors who have not taken medication for 2 weeks is collected in polypropylene tubes containing 1 volume of anticoagulant (2.73% citric acid, 4.48% sodium citrate, glucose 2%) for 10 volumes of blood.

 Within one hour of collection, 6% dextran is added to the blood (0.3 cm3 / cm3 of blood). After an incubation of 30 min at 37 C, the plasma rich in white blood cells is centrifuged at the speed of 100 g for 5 min at 40 C.

 The pellet is resuspended in 3 cm3 of 0.83% NH4Cl (to lyse the contaminating red blood cells) and centrifuged at a speed of 100 g for 5 min at 40 C.

 The pellet rich in mono- and polynucleated white blood cells is recovered in 5 cm3 of phosphate buffer (pH 7.4) of the following composition (in mM): 137: NaCl; 2.68: KCl; 8.1: Na2HPO4; 1.47: KH2PO4; 0.9: CaCl2 0.5: MgCl2 and deposited on 3 cm3 of a type 400 ficoll solution at a density of 1.077.

 After centrifugation at a speed of 420 g for 30 min at 40 C, the pellet rich in granulocytes is resuspended in 5 cm3 of phosphate buffer and centrifuged at a speed of 100 g, 5 min at 4 C. Finally, the granulocytes are counted and the density is adjusted to 3 × 10 6 cells / cm 3 of phosphate buffer.

- Stimulation of granulocytes by calcium lionophore A23187
The cells (3 × 10 6 cells / cm 3) are preincubated at 370 ° C. for 15 min in the absence or in the presence of the products to be tested at the desired concentration. Then the cells are stimulated for 15 min at 370 C with the
A23187 at 5 x 10-6 M (stock solution at 10-2 M in DMSO). The basal rate is measured from cells receiving neither test products nor
A23187.

 The reaction is stopped in ice and the supernatant is recovered after centrifugation at a speed of 250 g for 5 min at 40 C.

- PGE2 dosage:
The quantity of PGE2 produced is measured by a radioimmunological test (RIA). A calibration range is carried out under the same conditions with common concentrations of PGE2.

- Results:
The compounds of formula (I) used according to the invention surprisingly show an inhibitory activity on the synthesis of prostanoids derived from cyclooxygenase much greater than that of probucol.

 For example, at a concentration of 10-5 M, the compounds of formula (I) used according to the invention allow an inhibition of the production of PGE2 by 88% whereas the probucol only gives an inhibition of the order 40%.

2) STUDY OF THE INHIBITING ACTIVITY ON THE SYNTHESIS OF PROSTANOIDS
FROM LIPOXYGENASE
The inhibitory activity on the synthesis of prostanoids of the compounds of formula (I) used according to the invention is measured on human polynuclear cells washed, in the presence or in the absence of the compound to be tested, after activation of these cells by calcium (ionophore calcium A 23187).

 The production of the main prostanoid, derived from lipoxygenase, produced by human polynuclear cells: leukotriene B4 (LTB4) is measured by a radioimmunological test.

 The compounds of formula (I) used according to the invention therefore surprisingly show an activity which inhibits the synthesis of prostanoids derived from lipoxygenase much greater than that of probucol.

 By way of example, at a concentration of 10-5 M, the compounds of formula (I) used according to the invention allow an inhibition of the production of LTB4 greater than 95% whereas the probucol only gives an inhibition of the around 40%.

CONCLUSION
Studies I and 2 of Example C show that the compounds used according to the invention have an intense inhibitory activity on the synthesis of prostanoids.

Example D: STUDY OF THE ANTI-INFLAMMATORY ACTIVIST OF THE COMPOUNDS USED
ACCORDING TO THE INVENTION
PRINCIPLE
An experimental inflammation, of an immunological nature, is produced by injecting an anti-rat serum into the plantar pad of the rat's paw. The inflammatory reaction results in edema of the paw.

 The aim of this study is to investigate the anti-inflammatory effect of the compounds used according to the invention in the model of edema of immunological origin in the rat.

The parameters measured are:
- the volume of the edema,
- the generation of prostaglandins E2 (PGE2),
PROTOCOL
The animals used in this study are male Wistar rats weighing 350 to 400 g.

- Animals
Localized edema is produced at time tO of the study by an intraplantar injection of 0.1 cm 3 of rabbit anti-rat serum.

 Negative control animals receive an intraplant injection of 0.1 cm3 of physiological fluid.

- Treatment of animals
The products are suspended in gum arabic.

 One hour before the induction of edema, the animals receive the products, administered orally, by esophageal probe at a dose of between 3 and 30 mg / kg.

 Positive control animals as well as negative controls receive gum arabic by esophageal probe.

- Measurement of the volume of the edema:
Edema is characterized by an increase in the volume of the leg, determined using a water plethysmometer.

 An initial measurement of the volume of the paw is carried out before any treatment.

 Another measurement is carried out 2 hours after the induction of the edema.

- Measurement of PGE2:
The animals are sacrificed just after the plethysmometric measurement.

 The inflamed region is removed and placed in a pH 3.0 solution containing 33% CH3CN.

 After homogenization, the PGE2 are extracted on an ethyl C2 column (Amersham) and eluted with methyl ester formic acid.

 After evaporation under nitrogen, the PGE2 are dissolved in 200 μl of phosphate buffer.

 PGE2 concentrations are measured by radioimmunoassay.

RESULTS
It appears that the compounds used according to the invention, from a dose of 3 mg / kg, allow a very significant volume inhibition of the edema caused and also lead to a significant reduction in the production of prostaglandin E2, which is one of the mediators major inflammation.

Example E: STUDY OF ACUTE TOXICITY
The acute toxicity was assessed after oral administration to groups of 3 mice (20 + 2 grams) of increasing doses (0.1 - 0.25 - 0.50 - 0.75 - lg / kg) of compounds of formula ( I). The animals were observed at regular intervals during the first day and daily for the 2 weeks following the treatment.

 It appears that the compounds of formula (I) are completely non-toxic. No death is observed after administration of a dose of 1g.kg-1. There are no symptoms after administration of this dose.

Example F: PHARMACEUTICAL COMPOSITION: TABLETS
5 mg tablets of 3- (3,5-ditert-butyl-4-hydroxybenzylidenyl) indoline-2-one
Preparation formula for 1000 tablets: 3- (3,5-ditert-butyl-4-hydroxybenzylidenyl) indoline-2-one. . . 5 g
Wheat starch. . . . . . . . . . . . . . . . . . . . . . . . . 60g
Lactose. . . . . . . . . . . . . . . . . . . . . . . . . . . 45g
Magnesium stearate. . . . . . . . . . . . . . . . . . . . 0.5 g
Silica. . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2g
Hydroxypropylcellulose. . . . . . . . . . . . . . . . . . . . 0.5 g

Claims (10)

 in which R1 represents a hydrogen atom or a methyl radical, and their addition salts, when R1 represents a hydrogen atom, with a pharmaceutically acceptable base, for obtaining medicaments.

 CLAIMS 1) Use of the compounds of formula (I) 2) Use according to claim 1 of 3- (3,5-ditert-butyl-4 hydroxybenzylidenyl) indoline-2-one and its addition salts with a pharmaceutically acceptable base, for obtaining medicaments. 3) Use according to claim 1 of the compounds of formula (I) for obtaining medicaments useful in the treatment or prevention of conditions due or linked to peroxidation phenomena, to dysregulations of prostanoid synthesis, and / or platelet aggregation disorders, in the treatment of central or peripheral ischemic disorders, inflammatory diseases, pain, metabolic diseases, atheroma, arteriosclerosis, respiratory diseases, asthma, emphysema, immunological diseases, lupus erythematosus, allergic reactions, certain cancers, cerebral or peripheral aging, and in the prevention and treatment of damage due to surgical trauma and reperfusion of organs . 4) Use according to claim 1 of the compounds of formula (I) and their addition salts with a pharmaceutically acceptable base for obtaining medicaments in the treatment of atheroma. 5) Use according to claim 1 of the compounds of formula (I) and their addition salts with a pharmaceutically acceptable base for obtaining medicaments in the treatment of arteriosclerosis. 6) Use according to claim 1 of the compounds of formula (I) and their addition salts with a pharmaceutically acceptable base for obtaining medicaments in the treatment of inflammatory diseases. 7) Use according to claim 1 of 3- (3,5-ditert-butyl-4 hydroxybenzylidenyl) indoline-2-one and its addition salts with a pharmaceutically acceptable base for obtaining medicaments in the treatment of the atheroma. 8) Pharmaceutical composition containing as active ingredient at least one compound of formula (I) according to claim 1 or one of its salts with a pharmaceutically acceptable base in combination with one or more pharmaceutically acceptable excipients or vehicles. 9) Pharmaceutical composition according to claim 8 containing the aetif principle at a dose of 1 to 50 mg. 10) Use of a pharmaceutical composition according to claim 8 in the treatment or prevention of conditions due to or related to peroxidation phenomena, to dysregulations of prostanode synthesis, and / or to platelet aggregation disorders, in the treatment of central or peripheral ischemic disorders, inflammatory diseases, pain, metabolic diseases, atheroma, arteriosclerosis, respiratory diseases, asthma, emphysema, diseases of immunological origin, lupus erythematosus, allergic reactions, certain cancers, cerebral or peripheral aging, and in the prevention and treatment of damage due to surgical trauma and reperfusion of organs.