WO2006089881A1 - Use of phenanthrene derivatives as anti-inflammatory agents, synthesis method and intermediate products - Google Patents

Abstract

The invention concerns the use of phenanthrene derivatives of formula (I), wherein R1 and R2 represent a hydroxyl or methoxy radical; R3 represents a hydrogen atom or a hydroxyl radical optionally protected by a suitable protecting group; R4 represents a hydrogen atom or a methoxy radical, for making a medicine for inhibiting inflammatory reactions. The invention also concerns a method for dermo-cosmetic skin treatment which consists in applying on the skin a composition comprising at least one phenanthrene derivative of formula (I). Finally, the invention concerns a method for synthesis of said phenanthrenes of formula (I) as well as the intermediate products.

Description

 Use of phenanthrene derivatives as anti-inflammatory agents, synthesis process and intermediate products

The present invention relates to the use of phenanthrene derivatives as anti-inflammatory agents. The present invention also relates to a process for the synthesis of these phenanthrene derivatives as well as the intermediate products synthesized during this process.

The inflammatory reaction is a host defense response to external aggression. This reaction may in particular be mediated by compounds belonging to the class of prostaglandins and leucitrienes. In this case, the central molecule of the inflammatory reaction is arachidonic acid, the inflammatory reaction and all of its metabolisms being called "prostaglandin cascade". Arachidonic acid is practically not present in the free state in the cellular cytoplasm, it is essentially present at the cellular level in the form of an ester at the level of membrane phospholipids. Under the action of an enzyme, phospholipase A ₂ , arachidonic acid is released and metabolized in two ways: the lipo-oxygenase pathway that leads to the synthesis of leukitrienes and the cyclooxygenase pathway that triggers synthesis. prostanoids.

The treatment of the inflammatory component of cutaneous pathologies by the topical route generally involves the use of local corticoids, the side effects of which are well known. Nonsteroidal anti-inflammatory agents have little or no activity when administered topically. The search for topically active non-steroidal anti-inflammatory agents remains a major focus of research in pharmacology, dermatology and dermo-cosmetics.

For the purposes of the present invention, the term "phenanthrene derivatives" means compounds of the general formula

qui peuvent éventuellement être substitués en position 2, 3, 4, 5, 6, 7, 8 et 9 par des radicaux choisis dans le groupe constitué par OH, OCH₃, OC₂H₅, CH₃ et C₂H₅. Lorsque deux substituants sont portés par des atomes de carbone adjacents, ils peuvent éventuellement former ensemble un hétérocycle -0-CH₂-O-. Les principaux dérivés phénanthrèniques connus ont été isolés à partir d'extraits de plantes appartenant à la famille des Orchidaceae. Ainsi divers dérivés phénanthrèniques ont été isolés, principalement à partir de différentes Orchidaceae (P. L. Majumder and R. C. Sen, Pendulin, a polyoxygenated phenanthrene derivative from the Orchidaceae Cymbidum pendulum, Phytochemistrγ, Vol. 30, pp. 2432-2434, 1991 ; P. Tuchinda et al, Phenanthrenes of Eulophia nuda, Phytochemistry, Vol. 27, no 10, pp. 3267-3271, 1988 ; Shimuzu et al, anti-inflammatory constituents of topically applied crude drugs III. Constituents ant anti-inflammatory effect of Paraguayan crude drug « Tamanda cuna », Chem. Pharm. Bull., 36(1 1)447-4452, 1988).

which may optionally be substituted in the 2, 3, 4, 5, 6, 7, 8 and 9 position with radicals selected from the group consisting of OH, OCH ₃ , OC ₂ H ₅ , CH ₃ and C ₂ H ₅ . When two substituents are carried by adjacent carbon atoms, they may optionally together form a heterocycle -O-CH ₂ -O-. The main known phenanthrene derivatives have been isolated from plant extracts belonging to the Orchidaceae family. Thus, various phenanthrene derivatives have been isolated, mainly from different Orchidaceae (PL Majumder and RC Sen, Pendulin, a polyoxygenated phenanthrene derivative from the Orchidaceae Cymbidum pendulum, Phytochemistry, Vol 30, pp. 2432-2434, 1991, P. Tuchinda et al., Phenanthrenes of Eulophia nuda, Phytochemistry, Vol 27, No. 10, pp. 3267-3271, 1988; Shimuzu et al, anti-inflammatory constituents of topically-applied crude drugs. III Constituents ant anti-inflammatory effect of Paraguayan crude drug "Tamanda cuna", Chem Pharm Bull., 36 (11) 447-4452, 1988).

Phenanthrene derivatives, isolated from extracts of Orchidaceae, are described as having activity on the gastrointestinal system, antipyretic activity, antioxidant activity and spasmolytic activity.

Tamier, the main plant of the Dioscoreaceae family, is a fairly common plant in European undergrowth. She wears a number of vernacular names such as "beaten women's herb", "fire root", "wild vine", ... The tamer is a climbing plant, perennial by a rhizome, the stem is voluble, herbaceous, hail, streaked, green or reddish. The tamier can reach 3 m in height, the rhizome is voluminous and fleshy, gray with whitish section, covered with a kind of cork. The leaves are entire, petiolate, cordiform at the base and terminally pointed at the apex, with webbed venation. The insertion of the leaves on the stem is alternating. The flowers are grouped into axillary inflorescences. The male flowers and female flowers are not borne by the same foot: the Tamier is a dioecious species. The fruit is a small globose berry, fleshy and shiny, initially green then becoming red when ripe. Maturation takes place in August and in November.

Tamier is known to have many traditional medicinal properties, including diuretic and purgative properties, anti-inflammatory properties, but also analgesic. Reisch et al. were the first to demonstrate the presence of phenanthrene derivatives in the rhizome of Tamier (Reisch and al., structure and biocheraistry of natural phenanthrene derivatives, Herba Hung, 1970, 9 (2), 43-48; Reisch et al., Chemistry of Natural Substances. Nitrogen-free phenenthrene derivatives from Tamus communis rhizomes, Tetrahedron Lett, 1969, 2, 67-68). Phenanthrene derivatives have been described as growth regulators and dormancy inducers.

Confosarin, which has been isolated from Orchidaceae Bulbophyllum reptans, FAa confusa from Bulbophyllum gymnopus, and Catasetum barbatum, is known to exhibit anti-inflammatory action (Shimuzu et al., Anti-inflammatory constituents of topically applied crude drugs III Anti-inflammatory constituents of Paraguayan crude drug Tamanda cuna, Chem Pharm Bull, 36 (11) 447-4452, 1988). It answers the following formula:

In general, phenanthrene derivatives, in particular confusarin, exhibit genotoxic activity which limits their use as a drug. There is also a need for products, natural or obtained by organic synthesis, which have anti-inflammatory activity but are not very genotoxic.

Surprisingly, the inventors have discovered that certain phenanthrene derivatives exhibit an anti-inflammatory activity, while being little genotoxic. These derivatives can be isolated from common Tamier extracts or can be obtained by organic synthesis.

The present invention relates to the use of phenanthrene derivatives of general formula (I)

(I) in which R1, R2 and R3 represent, independently of one another, a hydroxyl radical or a methoxy radical (-OMe), R4 represents a hydrogen atom or a hydroxyl radical optionally protected by a suitable protecting group, with the exception confusarin, for the manufacture of a medicament for inhibiting inflammatory reactions. Advantageously, R1 represents the methoxy radical and R2 or

R3 represents the hydroxyl radical, still more preferably R1 represents the methoxy radical, R2 represents the hydroxyl radical and R3 represents the methoxy radical.

The phenanthrene derivatives according to the invention advantageously punctuate the inhibition of inflammatory reactions which involve an involvement of the arachidonic cascade. The phenanthrene derivatives according to the invention are not very genotoxic, they can therefore effectively be used, in humans or animals, as anti-inflammatory agents.

For the purposes of the present invention, the term "appropriate protective group of the hydroxyl function" means any substituent capable of protecting the hydroxyl radical from any undesirable reaction that may take place. As an example of a suitable protecting group for the hydroxyl function, there may be mentioned in particular a methyl ether, for example methoxymethyl or benzyloxymethyl, a benzyl, a trityl, a substituted ethyl ether, for example 2,2,2- tricholoethyl, a silylated ether

(SiMe ₃ , ShBuMe ₂ , SiC ₆ H ₅ Me ₂ ), an ether prepared by reaction between the hydroxyl radical and a carboxylic acid (acetate, benzoate) or a tetrahydropyran.

The phenanthrene derivatives of formula (I) can be isolated from Tamier extracts, in particular from Tamus communis. They are advantageously isolated from extracts of rhizomes of Tamier, including rhizomes of Tamus communis. The phenanthrene derivatives of formula (I) can also be obtained by organic synthesis. The inventors have also discovered that confusarin can be isolated from Tamier extracts, especially Tamus communis, advantageously from extracts of rhizomes, or be obtained by organic synthesis.

The medicament according to the invention is advantageously intended for the treatment and / or the prevention of inflammatory pathologies, in particular joint pathologies, such as rheumatoid arthritis, cutaneous and mucosal disorders of the intestinal tract, such as Crohn's disease, of the circulatory system, connective tissue and central nervous system. The medicament according to the invention is more particularly intended for the treatment and / or prevention of inflammatory skin diseases such as atopic eczema, contact dermatitis, inflammatory dermatoses, irritative dermatitis, acne, psoriasis, scleroderma, pityriasis versicolor, skin aging, photo-immuno suppression, vitiligo.

The medicament according to the invention comprises between 0.001% and 50% by weight, advantageously 0.001% and 30% by weight, of phenanthrene derivatives of formula (1) and a pharmaceutically acceptable excipient.

The drug may also contain the usual adjuvants in the pharmaceutical field.

According to the present invention, the medicament is formulated to be administered topically, orally, subcutaneously, injectably, rectally and genitally. The drug is advantageously formulated to be administered topically

When the medicament is formulated to be administered topically, it may be in the form of a solution or lotion for external use, a cream, ointment, milk, lotion, gel, Ointment, serum, paste, foam, aerosol, patch or stick.

The modes of administration, the dosages and the optimal dosage forms of the compounds and compositions according to the invention can be determined according to the criteria generally taken into account in the establishment of a pharmaceutical treatment adapted to a patient such as, for example, age. or the body weight of the patient, the severity of his general condition, the tolerance to treatment, the side effects noted, the type of skin.

The subject of the present invention is also a method for dermo-cosmetic treatment of skins, in particular oily or dry skins, exposed or not to ultraviolet radiation, characterized in that it consists in applying to the skin a composition comprising at least one derivative phenanthrene of formula (I).

The dermo-cosmetic treatment method advantageously allows the treatment of selected skins among sensitive, irritated, intolerant, allergic-prone skin, aged, presenting a cutaneous barrier disorder, presenting cutaneous redness, or presenting a non-pathological immunological disorder or imbalance related to intrinsic, extrinsic or hormonal aging.

The composition used in the dermo-cosmetic treatment method according to the invention advantageously comprises between 0.001 and 50% by weight of phenanthrene derivatives of formula (I) and a cosmetically acceptable excipient. The composition may also contain the usual adjuvants in the cosmetic field.

According to the present invention, the dermo-cosmetic composition is formulated to be administered topically, orally, subcutaneously, injectably, rectally and genitally. The dermo-cosmetic composition is advantageously formulated to be administered topically.

When the dermo-cosmetic composition is formulated to be administered topically, it may be in the form of a solution or lotion for external use, a cream, ointment, milk, lotion, gel, ointment, serum, paste, mousse, aerosol, patch or stick. The modes of administration, the dosages and the optimal dosage forms of the compositions according to the invention can be determined according to the criteria generally taken into account in the establishment of a cosmetic treatment, preferably a dermatological treatment, adapted to a patient such as, for example the age or body weight of the patient, the severity of his general condition, the tolerance to treatment, the observed side effects, the type of skin.

The present invention also relates to the particular phenanthrene derivatives of formulas (VI) and (VII):

Ces dérivés (VI) et (VII) ont été isolés à partir d'extraits de rhizomes de Tamier, notamment de Tamus communis. Ils peuvent également être obtenus par synthèse organique.

These derivatives (VI) and (VII) were isolated from extracts of Tamier rhizomes, including Tamus communis. They can also be obtained by organic synthesis.

The present invention also relates to a process for the organic synthesis of a compound of formula (I), including confusarin, by deprotection of a compound of formula (II)

dans laquelle R'i, R'₂ et R'₃ représentent, indépendamment les uns des autres, une fonction hydroxyle protégée par un groupement protecteur approprié ou un radical méthoxy, et R'₄ représente un atome d'hydrogène ou une fonction hydroxyle protégée par un groupement protecteur approprié.

in which R'i, R ' ₂ and R' ₃ represent, independently of each other, a hydroxyl function protected by an appropriate protective group or a methoxy radical, and R ' ₄ represents a hydrogen atom or a protected hydroxyl function by an appropriate protective group.

The appropriate protective group of the hydroxyl function is as defined above. According to an advantageous variant of the invention, the hydroxyl function is protected by a benzyl group.

According to an advantageous variant of the invention, the radical R ' ₁ represents the methoxy radical and a radical R' ₂ or R ' ₃ represents a hydroxyl function protected by a suitable protective group. Even more advantageously, R ' ₁ represents the methoxy radical, R' ₂ represents a hydroxyl function protected by a suitable protective group and R ' ₃ represents the methoxy radical.

The conditions of the deprotection step will depend on the protective group of the hydroxyl function employed. These conditions are well known to those skilled in the art. For example, if the hydroxyl function (s) is (are) protected by a benzyl group, the deprotection step may be a hydogenolysis step in the presence of palladium.

According to an advantageous variant of the invention, the compound of formula (I) obtained following the deprotection step is further purified by any method known to those skilled in the art. The compound of formula (II) is advantageously obtained by radical cyclization of a stilbene of formula (III)

dans laquelle R'i, R'₂ R'₃ et R'₄ ont la même signification que pour la formule (II). Selon une variante avantageuse de l'invention, l'étape de cyclisation du stilbène de formule (III) est une cyclisation radicalaire. Cette cyclisation radicalaire est avantageusement réalisée en présence d'hydrure de tributylétain et d'AIBN (azobisisobutyronitrile). Le rendement molaire de cette étape de cyclisation est avantageusement supérieur à 40%.

in which R'i, R ' ₂ R' ₃ and R ' ₄ have the same meaning as for formula (II). According to an advantageous variant of the invention, the cyclization step of the stilbene of formula (III) is a radical cyclization. This radical cyclization is advantageously carried out in the presence of tributyltin hydride and AIBN (azobisisobutyronitrile). The molar yield of this cyclization step is advantageously greater than 40%.

The compound of formula (III) is obtained by Wittig reaction between a compound of formula (IV)

et un composé de formule (V)

and a compound of formula (V)

Phosphonium bromide is generated in situ from bromide (IV) and triphenylphosphine at a temperature of about ^{0 °} C for about one hour in DMF. After addition of a solution of aldehyde (V) in DMF and MeOLi (previously prepared from MeLi in MeOH) and stirring of the reaction mixture for about 16 hours at about 90 ⁰ C, the stilbénique derivative (III) is obtained with a molar yield greater than 50%, optionally in the form of a mixture of isomers. The present invention finally relates, as intermediates, to the derivatives of formula (III)

dans laquelle R'j, R'₂, R'₃ et R'₄ ont la même signification que pour la formule (II). Selon une variante avantageuse de l'invention, le radical R'i représente le radical méthoxy et un radical R'₂ ou R'₃ représente une fonction hydroxyle protégée par un groupement protecteur approprié. D'une manière encore plus avantageuse, le radical R'i représente le radical méthoxy, R'₂ représente une fonction hydroxyle protégée par un groupement protecteur approprié et R'₃ représente le radical méthoxy.

in which R'j, R ' ₂ , R' ₃ and R ' ₄ have the same meaning as for formula (II). According to an advantageous variant of the invention, the radical R ' ₁ represents the methoxy radical and a radical R' ₂ or R ' ₃ represents a hydroxyl function protected by a suitable protective group. Even more advantageously, the radical R ' ₁ represents the methoxy radical, R' ₂ represents a hydroxyl function protected by a suitable protective group and R ' ₃ represents the methoxy radical.

In the following examples, which are given by way of illustration and which do not therefore delimit the scope of the present invention, the following abbreviations are used to designate the phenanthrene derivatives tested:

SR34: 3,7-dihydroxy-2,4,8-trimethoxyphenanthrene SR46: 3,7-dibenzyloxy-2,4,8-trimethoxyphenanthrene

SR69: 4,7-dihydroxy-2,3,8-trimethoxydihydrophenanthrene SR82: 4,7-dibenzyloxy-2,3,8-trimethoxyphenanthrene SR216: 2,7-dihydroxy-3,4,8-trimethoxyphenanthrene (confusarin) SR237: 3-hydroxy-2,4,8-trimethoxyphenanthrene SR223: 3-hydroxy-7-benzyloxy-2,4,8-trimethoxyphenanthrene

A34: 4,7-dihydroxy-2,4-dimethoxyphenanthrene In these examples, the following nomenclature is used:

The letters A, B and C correspond to the name of each of the cycles: we will speak for example of the cycle C. The numbers correspond to the generally accepted numbering of carbon atoms of phenanthrene derivatives (there are other nomenclatures in which the carbon atoms are numbered in another way).

Thus the SR34 derivative corresponds to the phenanthrene derivative of formula (I) substituted with a hydroxyl radical on carbon atoms numbered 3 and 7 and by a methoxy radical on carbon atoms numbered 2, 4 and 8.

Fractions from the Tamier rhizome extract are obtained as follows. The plant extract is made on 250 g of Tamier rhizome, Tamus communis, previously ground, supplemented with ethyl acetate. The whole is heated to reflux with stirring. The mixture is then filtered on Bϋchner. The filtrate recovered is evaporated to dryness on a rotary evaporator to recover 2.5 g of dry extract.

In the examples which follow, the extract thus recovered is called "total extract of Tamier" or "total extract of Tamus communis". This extract contains confusarin.

From the extract are obtained fractions on an open silica column followed by elution with solvents of increasing polarity:

CHCVhexane 30/70: 16 fractions that are not recovered; CHCl 3 / hexane 70/30: 9 fractions, the first 2 are pooled (fraction A) and the other 7 are pooled (fraction B);

Pure CHCI ₃ : 12 fractions, the first 2 are pooled (fraction C), the 4th and 5th fractions are pooled (fraction D).

Fraction D is refracted on a silica column and eluted with pure CHCl ₃ , thus obtaining crystals of A34. These fractions include the following phenanthrene derivatives:

3,7-dihydroxy-2,4,8-trimethoxyphenanthrene; 7-hydroxy-2,3,4-trimethoxyphenanthrene; 2,7-dihydroxy-3,4,8-trimethoxy-phenanthrene; 8-hydroxy-2,3,4,7-tétraméthoxyphénanthrène.

The following examples illustrate the invention and are not limiting. Example 1: Anti-inflammatory Activity of Phenanthrene Derivatives (Tracer PG6KFlα)

In this example, we evaluated the activity of two SR 34 and SR 46 phenanthrene compared to that of the total extract of Tamus communis and one of these fractions on PG6KF1 production induced in the keratinocyte by a cascade stimulant. arachidonic acid, calcium ionophore A23187. Principle: measurement of the production of prostaglandin PGόKFlα produced by the human keratinocyte after stimulation with the calcium ionophore A23187 (cylcooxygenase pathway) by means of an Elisa test.

The keratinocyte is the most represented cell at the level of the epidermis. In response to numerous extracellular factors present in its environment, it releases various biologically active mediators, including prostaglandins and leukotrienes, which play an important role in the initiation and modulation of skin inflammatory reactions and which are also involved in the regulation of of the immune response. The keratinocyte is a good model of study in cutaneous pharmacology, which makes it possible to determine, in vitro, the capacities of various molecules to modulate the production of these mediators resulting from the metabolism of arachidonic acid.

In this example, we were particularly interested in a prostaglandin, PGόKFlα which is one of the major metabolites produced by the stimulated keratinocyte, and representative of the modulation of the production of arachidonic acid metabolites from the cyclo pathway. -oxygénase. Materials and methods

- Cell line: HaCaT human keratinocytes - Culture plates 6 wells: 35 mm in diameter

> κ Reagents:

- RPMI culture medium (Roswell Park Memorial Institute, Moore Middle, GIBCO supplier)

- Phosphate buffer PBS pH 7.4 (phosphate buffer saline: saline solution at physiological pH, supplier GIBCO)

- Trypsin EDTA (supplier GIBCO)

- Fetal calf serum (SVF, supplier GIBCO) - EIA system (Enzyme Immuno Assay, supplier EUROMEDEX) -Ionophore calcium A23187 (supplier SIGMA)

> κ Weighted products:

Phenanthrenes SR 34 and SR 46 (15 mg solubilized respectively in 300 μl of ethanol and 300 μl of ethyl acetate).

- Total extract of Tamus communis and fraction B (10 mg solubilized in 200 μl of DMSO)

> κ Protocol:

The keratinocyte suspension in RPMI 10% FCS is distributed in 6-well plates (1x10 ⁶ cells / well), and incubated for 16 hours at 37 ^° C. in a 5% CO ₂ atmosphere. The keratinocytes are then rinsed with PBS to remove the non-adherent cells and then exposed to the test products included in RPMI without

SVF (which could interfere in the dosage).

SR 34 and SR 46 are solubilized respectively at a concentration of 50 mg / ml of ethanol and ethyl acetate and are then taken up in RPMI without FCS. The concentrations tested in culture are 1 μg / ml, 3 μg / ml and 10 μg / ml for SR 34 and 1 μg / ml, 3 μg / ml, 10 μg / ml, 20 μg / ml and 40 μg / ml for SR 46. these non-cytotoxic concentrations, the final concentration of ethanol is less than 0.025% and that of ethyl acetate less than 0.1%. The total extract of Tamus communis and a fraction (fraction B) are solubilized at a concentration of 50 mg / ml of DMSO and are then taken up in RPMI without FCS. The concentration tested in culture is 30 μg / ml. The different concentrations were chosen after a cytotoxicity evaluation test and are not cytotoxic.

For each lot, 3 wells are made. The cells are preincubated for 60 minutes with the test products and then a stimulating agent of the arachidonic acid cascade, the calcium ionophore, is added for 5 hours: the calcium ionophore A23187 is used at a concentration of 2.5 μM. After these 5 hours of culture, the culture media of each of the wells are recovered, centrifuged at 3000 rpm and stored at -80 ^° C. The production of prostaglandin 6KF 1a for each of the tests is measured by

Elisa kit (supplier EUROMEDEX). Principle of the Elisa test: immunoenzymatic assays allow the determination of antigens and antibodies through the use of a marker, which is a molecule whose detection makes it possible to identify these elements. In the Elisa method, these markers are enzymes. The principle is based on a competition between the antigen that is PGôFKlα contained in the samples (analyte), and the antigen conjugated to an enzyme (PGόFKlα, conjugated to the enzyme). This competition is carried out for a limited number of antibodies previously fixed to a microplate on which the tests are carried out. The antibody is in default with respect to the total amount of antigen (analyte and conjugated antigen). When the reaction reached equilibrium, the antibodies bound analyte and conjugated antigen in the same proportion as they are in the initial reaction mixture. Non-fixed substances are removed by washing. The analyte is by definition in variable quantity: the more it is abundant, the less the antibodies fix the conjugated antigen and vice versa. The addition of a chromogenic substrate will allow the assay of the antigens: indeed, in contact with the conjugate, a color reaction is performed and the optical density of each sample can then be measured spectrophotometer. The more the reaction is stained, the more antibody-conjugated antigens there are and the less the analyzed sample contains PGOFK _J ; : in this case, the tested product limits the synthesis of PGόFKjα and therefore has an anti-inflammatory power. Conversely, the less the reaction is stained, the lower the amount of antibody-bound conjugated antigens, and the more the sample analyzed contains PG6FKiα: the test product does not inhibit the synthesis of PGόFKjo; and does not have a good anti-inflammatory power. Results For comparison, all the results are given in Table 1. They are expressed in picograms of prostaglandin 6KFIa. for 1x10 keratinocytes. The statistical analysis of the results is carried out by an analysis of variance and by the Dunnett test (*** significant at the PO ₅ OOl threshold).

Table 1 Conclusion

The Phenanthrenes SR 34 and SR 46, the total extract of Tamus communis and its fraction B were evaluated, in terms of anti-inflammatory activity, on the release of prostaglandin 6KF1a by the keratinocytes. Each of the products has been tested at different concentrations.

The significant production of PG6KFlα induced 1Α23187 (1270pg / l X l O ⁶ cells) is released widely (+ 380%) of the basal production of control (334pg / l X lO ⁶ cells). => The phenanthrene SR 34 shows a significant anti-inflammatory activity for the three concentrations evaluated with a percentage inhibition of the release of PGόKFlα a ranging from 25% to 90%, this effect is dose-dependent.

=> The phenanthrene SR 46 has no anti-inflammatory activity in terms of inhibition of PG6KFlα release and this whatever the concentrations used. This result makes it possible to suggest that at least one hydroxyl group carried by one of two axillary aromatic rings (A and C) is necessary for the anti-inflammatory activity.

=> The total extract of Tamus communis has a significant anti-inflammatory activity at the concentration of 30μg / ml with a percentage of PG6KF1 release inhibition of 76%.

=> The fraction B of Tamus communis has a significant anti-inflammatory activity at the concentration of 30 μg / ml with a percentage inhibition of PGόKFlα release of 92%.

Example 2 Anti-Inflammatory Activity of Phenanthrene Derivatives (Tracer PGόKFlα)

In this example, we evaluated the activity of three SR 34, SR 69 and SR 82 phenanthrene on the production of PG6KF1 <x induced in the keratinocyte by a stimulator of the arachidonic acid cascade, the calcium ionophore A23187. The procedure is the same as in Example 1, only the tested products change: Phenanthrenes SR 34, SR 69 and SR 82 (10 mg solubilized respectively in 50 μl of ethanol for SR 34 and 50 μl of ethyl acetate for the other two).

The SR34, SR69 and SR82 are solubilized at a concentration of 200 mg / ml of ethanol for SR34 and of ethyl acetate for the other two and are then taken up in RPMI without FCS. The concentrations tested are 1 μg / ml, 3 μg / ml and 10 μg / ml. At these non-cytotoxic concentrations, the final concentration of ethanol is less than 0.025% and that of ethyl acetate less than 0.1%. Results For comparison, all the results are given in Table 2. They are expressed in picograms of prostaglandin 6KF la for 1x10 ⁶ keratinocytes. The statistical analysis of the results is carried out by an analysis of variance and by Dunnett's test.

*** significant at threshold P <0.001).

Table 2

Conclusion

The significant production of PG6KFl0f induced by 1Α23187 fl020pg / lx JO ⁶ cells) largely detaches (+ 525%) from the basal production of the control (194 μg / l × 10 ⁶ cells).

=> Phenanthrenes SR 34 and SR 69 show a significant anti-inflammatory activity, for the three concentrations evaluated, on the release of PG6KFlα: Table 3

Tableau 3

Table 3

The activity of SR 34 is perfectly reproducible from one experiment to another.

=> The phenanthrene SR 82 does not exhibit anti-inflammatory activity in terms of inhibition of PGόKFlα release. and that whatever the concentrations used. This result makes it possible to suggest that at least one hydroxyl group carried by one of two axillary aromatic rings (A and C) is required for anti-inflammatory activity

Example 3: Anti-inflammatory activity of phenanthrene derivatives (Tracer PGόKFlα)

In this example, we evaluated the activity of seven phenanthrene SR34, SR46, SR69, SR82, SR216, SR223 and SR237 on the production of PGόKFlα induced in the keratinocyte by a stimulant of the arachidonic acid cascade, the calcium ionophore A23187. The procedure is the same as in Examples 1 and 2. The results obtained for the phenanthrenes tested are given in Table 4:

Table 4

* "-" means that the molecule tested has no effect on the production of PGόKFlα Quantity of product tested: 3μg / mL

Products whose hydroxyl functions are protected by benzyl groups (coded as SR42 and SR82) do not show any anti-inflammatory activity, suggesting that at least one hydroxyl group carried by one of the two axillary aromatic rings (A and C) is necessary for anti-inflammatory activity.

The product SR216, which is confusarin, also has an anti-inflammatory activity. However, this activity is less important than SR34 or SR237.

EXAMPLE 4 Anti-inflammatory Activity of Phenanthrene Derivatives (PGόKFlα Tracer)

In this example, we evaluated the activity of extracts obtained from the rhizome of Tamus communis on the production of PGόKFlα induced in the keratinocyte by a stimulant of the arachidonic acid cascade, the ionophore calcium A23187 (2.5 μM). The procedure is as in Examples 1 to 3. The results obtained for the fractions tested are given in Table 5 below:

Table 5

The three fractions and A34 exhibit significant anti-inflammatory activity at a concentration of 1 μg / ml.

Example 5 Study of the Genotoxic Activity by the Micronucleus Test on L5178Y Mouse Lymphoma Cells on the SR237 Product and on the Total Tamier Extract

The in vitro Micronucleus assay is a test that can detect mitotic delay, apoptosis, chromosomal breaks, loss of one or more chromosomes, and non-disjunction simultaneously.

Micronuclei consist of fragments of chromosome (s) or a

(or more) whole chromosome (s) that did not migrate to one of the poles at anaphase. They result either from chromosomal breaks or from an alteration of the structure and / or functioning of a part of the mitotic apparatus, since whole chromosomes may not migrate towards the poles.

Principle: L5178Y cells in the exponential growth phase are treated in 96-well microplates with different doses of the product to be studied without metabolic activation. After the periods of treatment and recovery, the cells are collected, fixed, spread on slides and stained. The number of micronuclei per 1000 mononuclear cells is determined by microscopic reading (500 x immersion). A product resulting in a statistically significant increase in the number of micronuclei compared to the negative control with a dose / effect relationship is genotoxically concluded in this test.

L5178Y cells are cells of a mouse lymphoroma. This is a continuous transformed line. Method:

Cell culture: an ampoule stored at -18O ⁰ C containing about 3 to

4.10 ⁶ cells is thawed. L5178Y cells are resuspended in 40 ml Fischer's medium containing 10% horseradish serum (FMI 0) to remove the freezing liquid containing 10% DMSO. After centrifugation for 5 minutes at 900 rpm the supernatant is removed and replaced with 40 ml of FM10. The cells are resuspended by suction-discharge using a pipette of

10 ml for single use then transferred to a culture flask of 80 cm ² . The vial is screwed slightly and incubated in an oven at 5% CO ₂ at 37 ^° C. for 72 hours. A cell density of the order of 7 to 9.10 cells / ml is then obtained.

The genotoxicity tests are performed in duplicate: 24-hour treatment without metabolic activation, followed by a recovery time of 20 hours. Product preparation

The maximum dose is set at 2000 μg / ml in final concentration. Product soluble in an aqueous solvent: a stock solution (MS) at 40 mg / ml is prepared then the daughter solution (SF): 0.1 ml of SM + 0.9 ml of FM 10 = 4 mg / ml Product soluble in DMSO: a stock solution (MS) at 200 mg / ml is prepared then the daughter solution (SF): 0.025 ml of MS + 1.225 ml of FM 10 = 4 mg / ml.

Successive dilutions are carried out in microplates (3 plates are used):

1. Distribution 200 μl of the daughter solution in the first well of 2 successive rows,

2. Distribution of 100 μl of FM10 in all other wells,

3. Collection of 100 μl of the first well using a multichannel pipette and dilutions of wells by suction-discharge,

4. 100 μl rejection of the last well. Cell addition: L5178Y cells are counted at the Mallassez hematimeter. The cell suspension is prepared in FM 10: 4. 10 6 cells / ml. 100 μl of the appropriate suspension are distributed by the multichannel pipette into the corresponding plates. At the end of the treatment period, the plates are centrifuged for 5 minutes at

900 rpm. The treatment medium is removed by inversion. Two consecutive washes are performed by resuspension of the cells by adding 200 μl of FM10 to all the wells. After the last wash, 200 μl of FM10 are again added, the cells resuspended and the plates are reincubated for 20 hours. Harvest

After centrifugation, the medium is removed and replaced with the washing medium (medium FM1 O + 0.1% of pluronic acid), itself, after centrifugation, removed and replaced by the hypotonic medium (medium FMO / 2 + 0 , 1% pluronic acid). After centrifugation, the supernatant is removed and then a fixer (mixture 3 volumes of absolute ethanol + 1 volume of acetic acid) is added. Then, after a waiting time, the cells are spread on numbered slides. Coloring

After drying for at least 24 hours in ambient air, the slides are stained for 10 minutes in a solution of 2% Giemsa in water. They are then rinsed with water. Reading

The doses used for the determination of the frequency of micronucleated cells, in the case of a toxic product, must induce a relative toxicity compared to the solvent control around 50% for the maximum concentration, between 50 and 25% for the intermediate dose. and less than 25% for the low dose. The relative toxicity is calculated for each dose by the ratio [Average Absorbance of the Dose] / [Average Absorbance of the Control].

The criteria for determining a micronucleus are: • Intensity of staining of the micronucleus <with that of the main nucleus,

• Diameter less than 1/3 of the area of the main core,

• Clearly surrounded by a nuclear membrane, • Non-refractive, not attached to the main nucleus by a nucleopiasmic bridge,

* No contact with the main nucleus and located inside the cytoplasm.

The reading is carried out on 1000 mononucleated cells having a well preserved cytoplasm. To exclude apoptosis or DNA fragmentation phenomena, mononuclear cells containing more than 5 micronuclei are not counted. Results interpretation

For each of the doses tested, a statistical comparison of the results is performed using the CHl 2 test.

A product is classified as genotoxic to L5178Y mouse lymphoma cells in culture if it induces a statistically significant increase in the number of micronuclei compared to the control, if this increase involves at least one doubling compared to the control. and if there is a dose-effect relationship.

Results

Culture and treatment

Cells: Mouse Lymphoma L5178Y in TO Culture

Duration of treatment: 24 hours followed by 20 hours of recovery

* Tamier communis extract features: dark brown viscous extract

Micronucleus test: doses studied: 60-30-15 μg / ml solvent: DMSO positive control: Mitomycin C: 0.025 μg / ml

The results of the study of genotoxic activity by the micronucleus microscopy test on L5178Y mouse lymphoma cells, during the test without metabolic activation for a 24-hour treatment followed by 20 hours of recovery, are given in the following table 6.

* Chi-square test table 6

Conclusion Tamier Communis extract caused a statistically significant increase in the number of micronuclei in L5178Y mouse lymphoma cells after the 24-hour treatment in the absence of metabolic activation followed by a recovery period at the maximum analysable concentration of 60. μg / ml with 21 micronucleated cells for 2000 mononuclear cells vs 5 for the solvent control. Under these conditions, the Tamier Communis extract therefore exhibits significant genotoxic activity with respect to L5178Y mouse lymphoma cells after treatment in the absence of metabolic activation followed by a period of 20 hours of recovery, during an in vitro micronucleus test screening test carried out in micromethod and without repetition. * SR237 Features: Low density white powder

Micronucleus test: doses studied: 31, 25-15,63-7,81 μg / ml solvent: DMSO positive control: Mitomycin C: 0,025 μg / ml The results of the study of the genotoxic activity by the micronucleus test in micromethode on L5178Y mouse lymphoma cells, during the test without metabolic activation for a 24-hour treatment followed by 20 hours of recovery, are given in the following Table 7.

* chi-square test table 7

In the following Table 8, the results correspond to data obtained from 13 independent tests during the study of genotoxic activity by the in vitro micronucleus test on L5178Y mouse lymphoma cells (micromethode), without metabolic activation for a treatment of 24 hours followed by 20 hours of recovery.

Table 8 Conclusion SR237 did not cause any statistically significant increase in the number of micronuclei on L5178Y mouse lymphoma cells after a 24 hour treatment in the absence of metabolic activation followed by a recovery time of 20 hours. Under these conditions, SR237 therefore does not exhibit genotoxic activity with respect to L5178Y mouse lymphoma cells in the absence of metabolic activation, during a screening test of the in vitro micronucleus test carried out in vitro. micromethode and without repetition.

In the examples which follow, the percentages are expressed in moles, unless otherwise indicated.

Example 6 Synthesis of SR34 or 3,7-dihydroxy-2,4,8-trimethoxyphenanthrene (RN-118169-17-8) of Formula

3,7-dihydroxy-2,4,8-trimethoxyphenanthrene is a product that has been isolated from Orchidaceae Bulbophyllum reptans, Cymbidium pendulum, Glottis livida (Lindley) Schltr. (Orchidaceae), Thumia alba and Eulophia muda. This phenanthrene was synthesized by radical cyclization of a brominated stilbene derivative, followed by hydrogenolysis of the benzyl groups. The stilbene derivative has, in turn, been prepared by Wittig reaction between a benzaldehyde and a benzyl bromide.

1) Synthesis of benzaldehyde of formula:

This derivative was easily prepared in two stages from commercial syringaldehyde by bromination (86%) followed by benzylation (89%), giving an overall yield of 77%.

2) Synthesis of benzyl bromide of formula

Le bromure est synthétisé en 4 étapes à partir du 2,3-dihydroxybenzaldéhyde, commercial, avec un bon rendement global de 65% : i) O-méthylation régiosélective du 2,3-dihydroxybenzaldéhyde ; ii) benzylation du groupement hydroxy restant ; iii) réduction de l'aldéhyde par NaBH_{4 ;} et iv) bromation de l'alcool primaire

The bromide is synthesized in 4 steps from commercial 2,3-dihydroxybenzaldehyde, with a good overall yield of 65%: i) Regioselective O-methylation of 2,3-dihydroxybenzaldehyde; ii) benzylation of the remaining hydroxy group; iii) reduction of the aldehyde by NaBH _4; and iv) bromination of primary alcohol

3) Synthesis of the Stilbene Derivative of Formula

The stilbene derivative (Z) is prepared by Wittig reaction between phosphonium derived from benzyl bromide and benzaldehyde.

The phosphonium bromide is generated in situ from benzyl bromide and triphenylphosphine at ^{0 °} C for one hour in DMF. After adding a solution of benzaldehyde in DMF and then MeOLi (previously prepared from MeLi in MeOH) and stirring the reaction mixture for 16 hours at 90 ^° C., the stilbénique derivative is obtained with a yield varying from 67 to 76 ^° C. %, in the form of a mixture of isomers (of = 60-80%). Under these conditions, the Wittig reaction is known to selectively give stilbene Z.

4) Synthesis of the dibenzyl derivative SR46 of formula:

Le dérivé dibenzylé SR46 est obtenu par cyclisation radicalaire intramoléculaire du dérivé stilbénique (Z)

The dibenzyl derivative SR46 is obtained by intramolecular radical ring closure of the stilbene derivative (Z)

The majority isomer (Z) was reacted under free radical conditions in the presence of tributyltin hydride and AlBN in degassed toluene and kept at reflux for about 6 hours. This cyclization reaction led us to dibenzyloxyphenanthrene with a correct and reproducible yield (59-61%) in relatively concentrated media (0.08M). However, the derivative SR46 was never able to be obtained pure but always in mixture with traces of derivative of AIBN

(probably [Me ₂ (CN) C] ₂ , δ (Me) 1.5ppm), this despite successive purifications.

The hydrogenolysis of the benzyl groups was thus carried out on the SR46 / dimer derivative mixture to conduct, after 24 hours at 20 bar of H _2, with phenanthrenediol SR34 in good yields (68-74%). The SR34 derivative can be obtained pharmacologically pure after purification on a silica column and trituration in petroleum ether. A high hydrogen pressure must be maintained over a long period because of the difficult hydrogenolysis of the second benzyl group; TLC monitoring clearly shows the very rapid formation of a mono-benzyl derivative and the disappearance of the latter with a much slower kinetics.

A double benzylation reaction then makes it possible to obtain benzyloxyphenanthrene SR46 with 80% yield and a degree of purity satisfactory for pharmacological tests.

Example 7 Synthesis of 2,7-dihydroxy-3,4,8-trimethoxyphenanthrene (SR216) or conrusarin (RN108909-02-0) of formula

Confusarin or 2,7-dihydroxy-3,4,8-trimethoxyphenanthrene is a product that until now has been isolated from Orchiacae Bulbophyllum reptans, Eia confusa from Bulbophyllum gymnopus, and Catasetum barbatum.

This phenanthrene was synthesized according to a strategy identical to that used for SR34, that is to say by radical cyclization of a brominated stilbene derivative followed by hydrogenolysis of the benzyl groups. The stilbene derivative was, in turn, prepared by Wittig reaction between a benzaldehyde and a benzyl bromide whose synthesis was described in the previous example.

à partir de l'acide gallique.The main difficulty of this new synthesis lay in the preparation of α-bromo benzaldehyde. In fact, the dissymmetry of this compound made it necessary to implement and develop regioselective O-methylation and aromatic bromination reactions. 1) synthesis of Pα-bromo benzaldehyde of formula;

from gallic acid.

A first step of acetylation of methyl gallate in the presence of NEt ₃ leads to methyl 3,4,5-triacetoxybenzoate with 94% yield. The use of NEt ₃ in place of pyridine allows a significant increase in yield and simplifies the treatment. When the triacetoxylated derivative is heated at 50 ^° C. in DMF in the presence of K ₂ CO ₃ and MeI, a regioselective alkylation reaction takes place to yield methyl 3,5-diacetoxy-4-methoxybenzoate with 75% of performance.

The basic hydrolysis of diacetate (K ₂ CO ₃ , MeOH-H ₂ O, TA, 19h) gives the dihydroxy ester of formula

avec un excellent rendement. Ce benzoate de méthyle doit ensuite être réduit pour obtenir le benzaldéhyde correspondant.

with excellent performance. This methyl benzoate must then be reduced to obtain the corresponding benzaldehyde.

At this stage of the synthesis, the dihydroxy benzaldehyde must be monobrominated then the OH groups must be differentiated by two different alkylations (methylation and benzylation) to finally obtain the desired compound.

Due to the activation of the aromatic ring by the two hydroxy functions, obtaining a monobrominated derivative was the main difficulty of the bromination reaction. Indeed, despite the implementation of a single equivalent of NBS and the careful monitoring of the reaction, the monobrominated derivative has always been obtained (from 42 to 53%) mixed with the dibromo derivative (from 36 to 20%). . These two products could nevertheless be easily separated on a chromatographic column. 2) Synthesis of the Stilbene Derivative of Formula

Le dérivé stilbénique est obtenu par réaction de Wittig selon un mode opératoire identique à celui utilisé pour SR 34 (exemple 2) à partir de l'α-bromo benzaldéhyde et du bromure benzylique.

The stilbene derivative is obtained by Wittig reaction according to a procedure identical to that used for SR 34 (Example 2) from α-bromo benzaldehyde and benzyl bromide.

In this case, the reaction is stereospecific since only the Z isomer was isolated and no trace of the E isomer could be detected.

3) Synthesis of the dibenzyl derivative SR82 of formula:

The radical cyclization was then carried out to yield phenanthrene SR82 in reproducible yields and equivalent to those obtained for the synthesis of SR 34. However, in this case, the mixing problem with AIBN derivatives was not encountered and the SR82 derivative could be obtained pharmacologically pure by simple purification on a chromatographic column. After hydrogenolysis of the benzyl groups, confusarin is obtained with 85% yield.

Example 8 Synthesis of 3-hydroxy-7-benzyloxy-2,4,8-trimethoxyphenanthrene (SR223) of Formula

Phenanthrene SR223 is the mono benzylated analogue at position 7 of SR 34. Like the latter, the stilbene synthesis methodology has been retained. It was then enough for us to differentiate the protective groups of the two hydroxy functions carried respectively by the precursors of the ring A (benzaldehyde) and of the ring C (benzyl bromide) of the final phenanthrene. Due to the speed of preparation of this compound, our choice was to protect the OH of α-bromo benzaldehyde in the form of a /? Αra-methoxybenzyl ether (OPMB). As for benzyl bromide, it remains unchanged. Indeed, unlike the benzyl group, the PMB group can be removed by acid hydrolysis. After bromination of the syringaldehyde (86%), the phenol is reacted in the presence of β-methoxybenzyl chloride, NaI and K ₂ CO ₃ at reflux of acetone to reach Pα-bromo benzaldehyde with an excellent overall yield (84%). %).

Stilbene is obtained by Wittig reaction between benzyl bromide and α-bromo benzaldehyde with 70% yield as a mixture of ZIE-85/15 isomers. The (Z) isomer is then placed under free radical ringing conditions to reach the di-protected phenanthrene which is finally acid hydrolyzed under reflux of acetic acid to yield the monohydroxylated derivative SR223. The overall yield is 13%.

Example 9: Synthesis of 3-hydroxy-2,4,8-trimethoxyphenanthrene (SR237) of Formula

Phenanthrene SR 237 does not have a hydroxy function on ring C. Therefore, its synthesis involves the preparation of a new benzyl or β-methoxyl bromide of formula:

This gold / β-methoxyl benzyl bromide was prepared in 3 steps from salicylaldehyde with 86% overall yield. Nevertheless, the benzyl alcohol precursor bromide and o-methoxybenzaldehyde are commercial The phenanthrene SR237 is finally obtained according to the reaction sequence of the previous examples (Wittig / radical cyclization / hydrogenolysis) with an overall yield of 22% from this benzyl bromide ort / 20-methoxylated.

The Wittig reaction mainly leads to the cis isomer but with a lower diastereoisomeric excess (50% instead of 70%).

Claims

 claims Use of phenanthrene derivatives of formula (I)

in which R 1, R 2 and R 3 represent a hydroxyl radical or a methoxy radical, R 4 represents a hydrogen atom or a hydroxyl radical optionally protected by a suitable protecting group, with the exception of confusarin, for the manufacture of a medicament intended to inhibit inflammatory reactions. 2. Use according to claim 1, characterized in that R1 represents the methoxy radical and R2 or R3 represents the hydroxyl radical. 3. Use according to claim 1 or 2, characterized in that R1 represents the methoxy radical, R2 represents the hydroxyl radical and R3 represents the methoxy radical. 4. Use according to any one of claims 1 to 3, characterized in that the medicament is intended for the treatment and / or prevention of inflammatory diseases, including joint pathologies, such as rheumatoid arthritis, cutaneous-mucous lesions intestinal tract, such as Crohn's disease, circulatory system, connective tissue and central nervous system. 5. Use according to any one of claims 1 to 4, characterized in that the medicament is for the treatment and / or prevention of inflammatory skin diseases such as atopic eczema, contact dermatitis, inflammatory dermatoses , irritative dermatitis, acne, psoriasis, scleroderma, pityriasis versicolor, skin aging, photo-immuno suppression, vitiligo. 6. Use according to any one of claims 1 to 5, characterized in that the drug comprises between 0.001 and 50% by weight of phenanthrene derivatives of formula (I). 7. Use according to any one of claims 1 to 6, characterized in that the drug is topically applied. 8. A method of cosmetic treatment of skins, especially oily or dry skin, exposed or not to ultraviolet radiation, characterized in that it consists in applying to the skin a composition comprising at least one phenanthrene derivative of formula (I). 9. Cosmetic treatment method according to claim 8, characterized in that the composition comprises between 0.001 and 50% by weight of phenanthrene derivatives of formula (I). 10. Phenanthrene derivatives of formulas (VI) and (VII):

(VII)