FR2778181A1 - NEW VEGETABLE OIL COMPOUNDS FOR USE IN THE PHARMACEUTICAL AND COSMETIC INDUSTRY, PARTICULARLY FOR THE INHIBITION OF CELL GROWTH - Google Patents

Abstract

The invention concerns a compound of formula (I) wherein: R' is H or a carboxylic acid acyl radical (a); R is a C13-C21 hydrocarbon chain capable of comprising 1 to 4 ethenoid or acetylene unsaturations.

Description

NEW VEGETABLE HUTLE COMPOUNDS FOR USE

  IN THE PHARPIMACEUTICAL INDUSTRY AND COST iETIOQUE, NOTA MMVENT

  FOR THE NI-IIBITION OF CELL GROWTH

  The present invention relates to new compounds which can be used more particularly in the pharmaceutical and cosmetic industry and which in particular have an action of inhibiting cell growth which is useful, in particular in

  the treatment of inflammation and cancer.

  More particularly, it has been possible to demonstrate in certain vegetable oils, in particular in avocado oil, the presence of compounds which exhibit

  marked properties of inhibition of cell proliferation.

  The present invention relates to compounds as described above of formula:

OH O

  R 'a.O r R in which: R' is H or the acyl radical of a carboxylic acid Ri -C - OH; il O R is a hydrocarbon chain comprising from 13 to 21 carbon atoms, preferably 15 or 17 carbon atoms, and which may contain from I to 4 unsaturations

  ethylenic and / or acetylenic.

  Among these compounds, it is more particularly necessary to mention the compounds in which the radical R is chosen from | 9 lç ú 00f r- 0o 9 [SIúI Zrl OH C \ l 0 le O HO LV Zd IZ L tl IL \ V≥X w 1 L Id 91 S1 ú1SI Zl9 1Z LI bl 11

91I Tú1 ZI

L S

61 u_ = 9V Id

G61 U IS

  X V = tld The references corresponding to the compounds of the fraction P l and to the compound of the fraction P2, the additional indices are those which appear in the

present description.

  These compounds preferably being chosen from those for which R ′ is H or a radical R 1 - C o R 1 is an alkyl radical from C 1 to C 6, preferably the radical o methyl. These compounds can be used alone or in combination, in particular in the oily fractions containing them, to produce pharmaceutical compositions, as well as cosmetic compositions, in general these compositions will preferably be used topically, but it is possible to

  provide for systemic applications, especially oral.

  The compounds according to the invention can also, in certain cases, be

  used as a food supplement.

  The compositions according to the present invention are more particularly useful for the production of medicaments intended to inhibit proliferation. cellular and, taking into account the analyzes which have been made, they will be more particularly usable as agent intervening in the inflammatory process and also in the treatment of cancers and non-cancerous pathologies linked to cell proliferation, such as the treatment of certain types of adenoma like prostatic adenoma. The compounds according to the present invention can also be

  usable as anti-viral agent.

  These compounds can be used either in pure form, for example in synthetic form, but also in the form of a mixture or even in the form of an oily fraction or of a concentrate still contained in the original oil, in particular

avocado oil.

  This is why the present invention also relates to fractions.

  oily rich in at least one of the compounds mentioned above.

  The compounds according to the present invention can be obtained by different routes, either synthetic or hemi-synthetic, but are preferably

obtained by extraction.

  More particularly, the compounds according to the present invention are obtained by a process in which they are extracted from a vegetable oil by any suitable process, in particular by extraction, for example using a solvent, by

  pressing, chromatography, and / or molecular distillation.

  Vegetable oil or oily concentrate can be obtained by any

  known process, in particular by extraction using a solvent and / or by pressure.

  The extraction using a solvent can be carried out by any suitable solvent, alcohol such as EtOH, MeOH, BuOH, alkane such as hexane, chlorinated solvent, CH2Cl2 or CHCl3,

  ether or ester such as AcOEt or their mixtures.

  Generally, the treatment conditions are below 80 ° C. for 8 hours, that is to say that temperatures below 80 ° C. are preferably used for less than 8 hours. It should be understood that by conditions "below 80 ° C. for 8 hours" is meant to designate conditions in which the cyclization does not take place or little, for example less than 10% of cyclized product. This does not mean that the temperature is necessarily lower than C, in fact, it is possible to use temperatures of 100 C or more but only a few minutes or tens of minutes, on the contrary temperatures of C or less over more than 8 hours, 24 hours for example, can lead to cyclization. Preferably, the different fractions are treated so that there remains water, of the order of a few percent, in fact when the product is dehydrated, the cyclization begins. The compounds according to the invention are preferably extracted at

from avocado oil.

  In a preferred mode of preparation of the compounds according to the present invention, the avocados are cut into strips a few millimeters thick and dried at a temperature below 85 ° C., preferably in a thin layer at 70-80 ° C.

  the residual humidity after drying is close to 5%.

  The dried avocados are crushed and the oil is extracted by pressure, filtered and dried at 70 ° C under 0.5 mm of mercury or under equivalent conditions. The dried oil is fractionated by molecular distillation at 240 C under 103 mm of mercury in order to obtain a distillate which represents from 5 to 10% of the oil. This extract is enriched to -40% in compounds according to the present invention. It is possible to vary the distillation conditions, provided that a fraction which represents 5 to

% of the original oil.

  It is also possible to obtain the compounds according to the present invention by extraction using a cold solvent, that is to say at a temperature of the order of 15 to 25 ° C. for example. By choosing the solvents, one can obtain concentrates very rich in compounds directly from the fruit pulp but one can also use several successive extractions. In order to isolate the compounds, a preparative liquid chromatography is carried out on the silica column from the above fractions, with the phase being

  mobile, for example, hexane-isopropanol (90:10).

  For most applications, the compounds can be used in

  mixing without isolation.

  The tests which have been carried out on the compounds according to the present invention, of which certain results will be found in the examples, show that some of the compounds according to the present invention inhibit cell proliferation, for example human dermal fibroblasts, at concentrations of 100 gg / ml in the experiments carried out but that some of them inhibit nearly 50% of the proliferation of the same fibroblasts at much lower concentrations, of the order of 10 pg / ml. On the other hand, the same compounds have no effect on the biosynthesis of type 1 collagen, which constitutes the main matrix macromolecule produced by fibroblasts, it seems, taking into account the analysis of all the results, that the products in question or the fractions that contain them do not change the

  biosynthesis of the extracellular matrix.

  Tests carried out on CCL39 cell show similar results on the following three tests: * cell proliferation induced by KGF * cell proliferation induced by b-FGF * cell proliferation induced by E-GF The compositions of the invention can be used both for curative as preventive, as main therapy or as adjunct therapy. The compounds according to the present invention and the fractions which contain them can

  also be used as a nutritional supplement.

  The compounds according to the present invention can also be used as precursors of furan derivatives. In fact, by heating at a temperature above 80 ° C. for an extended time, in particular more than 8 hours, in particular 24 hours, or at temperatures above 100 ° C. for a few hours, of these compounds or of the fractions which contain them, the corresponding furan derivatives are obtained, which are also useful in industry

pharmaceutical and cosmetic.

  The remarks made previously for the 80 C / 8 hours couple in the preparation of the products according to the invention is also valid in the case of cyclization. In this case, it is a couple of temperature and heating time which

  provides more than 10% cyclization, preferably more than 50%.

  The invention therefore also relates to processes in which, during one of the process steps, before extraction or after, the precursors or a fraction which contains them is heated under conditions above 80 ° C. for 8 hours,

  preferably it is heated for a few hours at 100 ° C. or more.

  The invention also relates to the fractions thus obtained, in particular the oily fraction containing furan derivatives. The furan derivatives of avocado oil have been described in particular in Farines, M. et al., 1995, J. of Arn. Oil Chem.

Soc. 72, 473.

  Other characteristics and advantages of the present invention will appear

  on reading the examples below.

EXAMPLE 1

  Methods for extracting, separating and isolating the compounds g from fresh pulp are ground for 2 minutes in a mixer in the presence of 200 ml of EtOH. The solution, filtered through Buchner and concentrated in vacuo to 1/4 of the volume of

  is called concentrate A. The pellet is taken up in 150 ml EtOAcMeOH (30:70).

  After filtration, washing with 50 ml MeOH and concentration under vacuum at 1/4 of the starting volume, concentrate B is obtained. Each of the concentrates A and B is extracted with 2 times ml CH2Cl2. The CH2Cl2 solutions are dried with Na2SO4, then evaporated. Two extracts A1 and BI are obtained. These two extracts are combined, taken up in 2 volumes of hexane and purified on bleaching earth "nevergreen". After evaporation, the residue, dissolved in a minimum of CHC13, is placed at the top of a column of silica of the brand "Amicon" 20-45.gm (20 x 300 mm) and eluted with 500 ml CHCI3 then 250 mi CHCI3- MeOH (98: 2). Fractions are collected in 60 ml volumes. Fractions 4 and 5 contain product 1 (P1), fractions 8, 9 and 10 contain product 2 (P2). These products were separated by preparative HIPLC on a Partisil OD S2 column. Product 1

  gives 4 main peaks, product 2 gives 1 main peak.

  Product identification The chemical structure of the molecules corresponding to the main peaks thus isolated was determined by infrared spectrophotometry, RMIN of the proton and GC / SM in chemical ionization (CH4). Analysis of the infrared spectra of all the compounds studied highlights the presence of a vibration of OH valence around 3400 cm'l due to a hydroxyl function and the existence of a large methylene group -CH2- between 2840 and 2940 cm '. The compounds P1 have a vibration of valence C = O at 1740 cm 'specific for the esters and C-O at 1240 cml specific for the short chain acetate type acid esters. P1A4, P1A7 and P2A7 have a C = O vibration at 1715 cm 'from acyclic ketones. PlA6 and PiA9 have a vibration C = O at 1670 cm 'strongly displaced by an unsaturation in cc. P1A7, P2A7 and PlA9 have a HC = ethylenic vibration at 3020 cm ". P1A4 has a spectrum characteristic of a saturated product. The GC / SM analyzes carried out on the trimethylsilylated derivatives of the alcohol functions made it possible to determine the molecular masses and the formulas crude of each compound. The molecular mass of the products Pl corresponds in all cases to a crude formula R-C9H1704Si, that is to say if one disregards the trimethylsilyl group, to alcohols R-C6H904. molecular of the product P2 corresponds to a crude formula C27H5403Si2, which corresponds by ignoring the trimethylsilyl groups in alcohol C2lH3803 The RMvIN spectra of the proton systematically present for all the Pl compounds a singlet (3 protons) at around 2 ppm representing the acetate methyl group, a doublet doubled at around 3.95 and 4 ppm for each of the two protons in 1, a solid around 4.20, attributable to the proton in 2 coupled with the two hydrogen atoms in I and the two hydrogen atoms in 3. A doublet at around 2.50 ppm for PIA4 and PIA7 or 2.65 ppm for PlA6 and PlA9 (effect of the double bond in 5-6) is attributable to the set of two protons at 3. A triplet towards 2.35 (2 protons) is attributable to the two hydrogen atoms at 5, coupled with the protons at 6, for the compounds PIA4 and PiA7. This signal from the methylene group is not observed for PIA6 and PIA9 which

  present; on the other hand, an ethylenic proton in 5 to 6 ppm in the form of a doublet.

  The compounds PIA7 and P1A9, with two unsaturations on the aliphatic chain, exhibit a chemical shift of the ethylenic protons at 12, 13 and 15, 16

  (4 H, m) between 5.1 and 5.3 ppm.

EXAMPLE 2

  Furanic compounds a) Method of extraction of total lipids 300 to 500 mg of fresh avocado pulp frozen with liquid nitrogen and crushed or the weight of dried pulp obtained from approximately 400 mg of fresh pulp, are ground mortar, stirred with 10 ml of MeOH-CHCI3 (1: 1) then centrifuged. The pellet is taken up in the same volume of solvent. The whole is stirred and then centrifuged. The organic phases are combined, evaporated under a stream of nitrogen at room temperature. The residue is extracted twice with CHCl3 and dried over Na2SO4. The solution is filtered on a Gelman ACRO LC 13 filter (0.45 μm), washed with 1.5 ml of CHCl 3 and

  evaporated under a stream of nitrogen at room temperature.

  From 6 different avocados, 2 pulp fragments were taken. One has

  directly subjected to the previous extraction, a fresh pulp oil is obtained.

  The other was dried in an oven for 24 hours at 80 C before being subjected to extraction.

former.

  In dry pulp oil, there are few compounds according to the invention, the compounds are almost entirely cyclized in furanic form, from 50 to 170 mg per 100 g of fresh starting pulp, while pulp oil fresh is rich in compounds according to the invention and contains few fiuranic derivatives, from 0 to 10 mg for

100 g of fresh pulp.

  b) Extraction method according to progressive polarity gradient 9 g of fresh crushed pulp are extracted with 4 times 15 ml of n-hexane. The cell pellet is taken up in 4 times 15 ml of CHCl 3, 4 times 15 m! ethyl ether then with 3 times 15 ml of the following solvents: EtOAc, n-BuOH, H2O. The 6 phases

  organics are collected and evaporated.

  After evaporation of the solvents under reduced pressure, each of the extracts was placed in an oven at 80 ° C. for 24 hours and then purified for the qualitative analysis of the furan derivatives. It revealed the presence of furan compounds in the

  "ethyl acetate" and "butanolic" phases.

  30. A new extraction on fresh pulp with ethyl acetate, concentrated under a stream of nitrogen, was analyzed by 2-dimensional thin layer chromatography. The first migration of the extract was carried out in ethyl ether revealing two different spots of Rf. Then the thin layer was placed in an oven, 24 hours at 80 C, before being subjected to the second migration in heptane, a system for developing furan compounds. The revelation then showed that the two previous spots had given rise to furan compounds during the passage of the thin layer in an oven. The two spots corresponding to the compounds

  according to the invention are transformed under the effect of heat treatment.

EXAMPLE 3

  Method for preparing a fraction rich in compounds according to the invention The avocados are cut into strips a few millimeters thick and dried in thin layers at 70-80 C; the residual moisture after drying is close to

5%.

  The dry avocados are ground. The oil is extracted by pressure, filtered and

  dried at 70 ° C. under 0.5 mm of mercury.

  The dried oil is fractionated by molecular distillation at 240 C under

'3 mm of mercury.

  The distillate, 5 to 10% of the oil, is an enriched extract (30 to 40%) in

  compounds according to the present invention.

  Preparative liquid chromatography on a silica column, with the hexane-isopropanol mobile phase (90:10) allows to isolate a purified mixture

  products according to the invention (1.5 to 5 g).

EXAMPLE 4

  Measurement of the activity of inhibiting cell proliferation of the compounds according to the invention In what follows, preparation D corresponds to the unsaponifiables of avocado, preparation E corresponds to the fraction H. that is to say the fraction in which the majority of the compounds according to the present invention have been cyclized in furanic form, preparation J corresponds to the mixture of Pl type products, as

  in preparation K it corresponds to product P2.

  Cell culture The cells used are human fibroblasts from the foreskin of young children. They were obtained by the explants technique and cultivated in DMEN (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal calf serum (SVF). The cultures are kept in an oven at 5% CO2, 37 C, and the

  medium is changed every three days.

  Study of cell proliferation Cell proliferation was measured by incorporation of tritiated thymidine. The fibroblasts were seeded in 24 wells of 2 cm 2 dishes at a density of 20,000 cells / well in DMEN + 10% FCS (1 ml). 24 hours after seeding, the cultures were incubated with the avocado preparations D, EJ and K (dissolved beforehand in absolute ethanol), at concentrations of 0.5, 1, 10 and 100 gg / ml (depending on experience). Parallel control cultures were incubated in the absence or presence of ethanol (at the dilutions used in the series treated with the solutions of avocado extracts). 20 hours later, [3H] -thymidine was added to the

  cultures in a small volume at a rate of 1 pCi / ml. An identical volume of [3H] -

  thymidine was added to control cultures that received no treatment. In

  all the experiments, each test was carried out on 4 identical wells.

  At the end of the incubation, the medium was eliminated and the cell layer was rinsed twice with PBS in order to eliminate the free radioactivity. Then, the cells received I ml of cold 5% trichloroacetic acid (TCA). After 30 minutes at 4 ° C., the TCA was removed and the cell mats washed three times with 5% TCA. 500% l of 0.1 N sodium hydroxide were added in order to dissolve the proteins and the multi-well plates placed at 50 C for 1 hour. After homogenization by pipetting, 400 Ptl of the lysate

  were counted in liquid scintillation.

  Results Four experiments were carried out and the results of three of them are presented in Table 1 where the incorporation of thymidine was expressed, therefore cell proliferation as a percentage of each respective control. Substances D and E inhibit the proliferation of fibroblasts human dermals for concentrations of 100 pg / ml, the other concentrations used do not modify this parameter, Substances J and K at concentrations of 10 gg / ml inhibit at least% the proliferation of dermal fibroblasts, and this effect is d '' the more marked as one increases the concentration of substance, until total proliferation is halted

(100 pig / ml).

  30. All of these results show that substances D and E are more moderate inhibitors of the proliferation of dermal fibroblasts than substances J and K. Additional tests carried out on the biosynthesis of collagen show that the compositions according to the present invention have no clear action on

collagen synthesis.

he

Claims (22)

1) Compound of formula: OH O   R / 0e R in which: R 'is H or the acyl radical of a carboxylic acid Ri - C - OH; el O   R is a hydrocarbon chain of 13 to 21 carbon atoms and which may contain   1 to 4 ethylenic or acetylenic unsaturations.   2) Compound according to claim 1, characterized in that the radical R   is a hydrocarbon chain having 15 or 17 carbon atoms.   3) Compound according to claim 2, characterized in that the radical R is chosen from: PIA4R = 1 19 PIA6 'R = 7 19 1213 1516 it 14 17 21 6 1213 1516 7 11 14 17 21   Mention may also be made of the compound P2 A7 OH O HO 1213 1516 3,521 21 OC OC   4) Compound according to one of claims I to 3, characterized in that the   radical R 'is H or a radical Rl -C o R1 is a C1 to C6 alkyl radical. o If   5) Compound according to one of claims I to 4, characterized in that the radical R 'is H or CH3 -C wl o   6) Process for the preparation of a compound according to one of claims I to   5, characterized in that it is extracted from a vegetable oil.   7) Method according to claim 6, characterized in that the compound is   extracted from avocado oil.   8) Method according to one of claims 6 or 7, characterized in that the   compound is extracted from an oil or an oily concentrate obtained by   extraction of fruit pulp using a solvent or by pressure.   9) Method according to claim 8, characterized in that the extraction solvent is an alkane, an alcohol, a chlorinated solvent, an ether, an ester or their mixtures. ) Method according to claim 9, characterized in that the solvent is   chosen from: hexane, MeOH, EtOH, BuOH, CHCI3. CHCI2 and AcOEt.   11) Method according to one of claims 6 to 10, characterized in that it is   conducted under conditions below 80 C for 8 hours.   12) Method according to one of claims 6 to 10, characterized in that   the extraction comprises at least one step of molecular distillation of the vegetable oil.   13) Method according to one of claims 6 to 12, characterized in that the   compound comes from an oily concentrate by extraction using a hexane phase isopropanol.   14) Method according to one of claims 6 to 13, characterized in that   the oil is extracted from dried avocado at a temperature below 80 C.   15) Oily fraction rich in compound according to one of claims I to   4, characterized in that it is obtained by implementing the method according to one of claims 6 to 14.   16) Furan derivative obtained by heating a compound according to one of   claims I to 5 or a fraction containing it according to claim 15, characterized   in that said compound or said fraction is heated to a temperature above  C, in particular greater than 100 C.   17) Fraction rich in furan derivative according to claim 16, characterized in that it is obtained by heating an oily fraction according to the   claim 15 at a temperature above 80 C.   18) Fraction according to one of claims 16 or 17, characterized in that   the furan derivatives are obtained by heating, under conditions greater than 80 C for 8 hours, avocados.   19) Pharmaceutical composition, characterized in that it comprises   as active ingredient a compound according to one of claims I to 5 or a fraction   according to one of claims 15 to 18.   ) Cosmetic composition, characterized in that it comprises by way of   active ingredient a compound according to one of claims I to 5 or a fraction according to one of claims 15 to 18.   21) Composition according to one of claims 19 or 20, characterized in that   that it is intended for topical application.   22) Use of a compound according to one of claims I to 5 or a   fraction according to claims 15 for the production of medicaments intended for   inhibit cell proliferation.   23) Use according to claim 22, characterized in that the   drug is intended to treat the inflammatory process.   24) Use according to claim 23, characterized in that the   drug is intended for the treatment of cancer.   ) Use of a compound according to one of claims I to 5 for the   production of antiviral drugs.