WO2003031381A2 - Synthesis of (z)-3,5,4'-trimethoxystilbene, and use thereof - Google Patents

Abstract

The invention concerns the field of organic chemistry and more particularly that related to the synthesis of novel molecules capable of exhibiting biological properties, in particular therapeutic and more particularly properties for preventing or treating certain diseases such as in particular cancer-type diseases. The invention concerns a novel method for chemical synthesis of (Z)-3,5,4'-trimethoxystilbene (compound R3), the compound obtainable by said method, the use of said compound as medicine in particular as synthetic anticancer drug at low production cost, the corresponding medicine and various uses of said compound (R3).

Description

 Process for the synthesis of (Z) -3,5,4'-trimethoxystilbene, compound obtained and uses of said compound, in particular as a medicament, in particular as an anticancer agent

The present invention relates to the field of organic chemistry and more particularly that related to the synthesis of new molecules capable of exhibiting biological properties, in particular therapeutic and more particularly properties in the field of prevention or treatment of certain diseases such as in particular diseases of the “cancer” type.

It relates to a new process for the chemical synthesis of (Z) -3,5,4'-trimethoxystilbene (hereinafter referred to as compound R3), the compound R3 capable of being obtained by said process, various uses of the compound synthesized (in particular as a medicament), in particular as a synthetic anticancer agent at low cost price, a medicament having said compound R3 as an active principle as well as a cosmetic composition.

Compound R3 is a derivative of resveratrol, a natural polyphenol found in the plant world.

The anti-cancer effects of polyphenols in food have been the subject of numerous studies. These have led to the study of the effects of resveratrol (3,5,4'-trihydroxy-trα "s-stilbene or isomer (E)), a natural constituent of many plants, vines and grapes. Resveratrol which has fungicidal properties is produced by the vine to fight against the development of the fungus Botritis cinerea. Research has made it possible to show that this compound possesses, as well in vitro, on human cancer cells, as in vivo, in cancer mice, interesting anti-cancer properties (F. Raul et al. “Anti-proliferative effect of resveratrol, a natural component of grapes and wine, on human colony cancer cells ", Cancer Lett. 158. 85-91, 2000 and" Resveratrol inhibits intestinal tumorigenesis and modulâtes host-defense related gene expression ^' in an animal model of human familial adenomatous polyposis ”, Nutrition and Cancer, 39, 102-107, 2001). However, said natural compound is, in its “trans” form, unstable and rapidly transformed by the cell. Thus, the problem posed by the present invention is that of making available to the public a new anticancer molecule which is both effective and of low cost.

To this end, the present invention provides a new synthesis of a molecule derived from resveratrol, namely, (Z) -3,5,4'- trimethoxystilbene, of formula I:

hereinafter designated as compound R3, in fact corresponding to the tri-methylated derivative of resveratrol.

Compound R3 has been tested for its possible anticarcinogenic properties. Biological studies have unexpectedly and surprisingly demonstrated that the compound R3 exhibits great stability and a remarkable anticancer action which turns out to be much superior (of the order of 100 times greater) than that of resveratrol.

By studying the cellular and molecular mechanisms of its action, it has been demonstrated that it has biological properties, in particular anticancer properties, similar to or even superior to those described for Taxotere ^® (docetaxel, Aventis, France) used in anticancer chemotherapy and radiotherapy.

The subject of the present invention is a process for synthesizing the compound according to the invention, characterized in that it essentially consists in exposing a sample of (E) -3,5,4'-trimethoxystilbene isomer dissolved in a solvent suitable for ultraviolet light with a wavelength culminating between 200 nm and 400 nm, this for an exposure time of between one hour and two hours for a quantity of (E) isomer of 10 ^"2 mole and, the if necessary, evaporating said solvent in order to collect the desired isomer (Z) The present invention also relates to the compound R3 capable of being obtained by the aforementioned process, the use of said compound R3 according to the invention as a medicament , especially for the preparation a drug for the treatment or prevention of a disease involving abnormal cell proliferation or growth, such as cancer.

According to another use, the compound according to the invention is used for the preparation of a medicament intended to treat or prevent cystic fibrosis.

A subject of the present invention is also the use of the compound according to the invention as an agent for depolymerization of the microtubular network of living human or animal cells, as an agent for inhibiting the polyamine synthesis of living human or animal cancer cells, as an agent for blockage of the cell cycle, in particular of the cell cycle of living human or animal cancer cells, as an agent for the treatment or prevention of hyperproliferative pathologies, in particular inflammatory conditions, infectious and / or parasitic diseases such as malaria and as an agent photoprotective, in particular for protection against UV-A and / or UV-B rays.

The present invention further relates to a medicament characterized in that it comprises, as active principle, at least the compound R3 according to the invention.

Finally, the present invention also relates to a cosmetic composition with photoprotective property, characterized in that it comprises, as active principle, at least the compound according to the invention.

The uses, medicaments and cosmetic compositions according to the invention are the subject of claims 10 to 23.

The invention will be better understood from the description below, which relates to a preferred embodiment, given by way of nonlimiting example, and explained with reference to the appended schematic drawings, in which: FIG. 1 represents an example of a synthetic diagram of the process according to the invention, FIG. 2 represents the growth curve of CaCo- 2 cells as a function of the concentration of compound R3 added (FIG. 2a) as well as the growth curve of IEC-6 cells as a function of the concentration of compound R3 added (FIG. 2b), FIG. 3 illustrates the double labeling of CaCo-2 cells with Annexin-FITC (FIG. 3a) and propidium iodide (FIG. 3b) after treatment with the compound R3, FIG. 4 represents an analysis of a cycle cell by flow cytometry of cancer cells treated with the compound R3 (FIG. 4a), FIG. 4b represents the analysis of the cycle of FIG. 4a in the absence of treatment with the compound R3, FIG. 5 represents the activity of the ODC as a function of the duration of the treatment with the compound R3 (FIG. 5a) and the activity of the AdometDC (SAMDC) as a function of the duration of the treatment with the compound R3 (FIG. 5b), FIG. 6 represents snapshots of the microtubular network of CaCo-2 cells under the influence of the compound R3 and two known compounds, FIG. 7 represents a competition curve between R3 and ligands labeled with tritium (vinblastine, colchicine) for their fixation on tubulin, Figure 8 shows histograms showing t the percentage of cells marked either by annexin V (apoptotic cells) or having a hyperdiploid character, and FIG. 9 represents a histogram illustrating the "in vivo" effects of R3 on the development of tumors after subcutaneous implantation of human tumor cells in nu / nu athymic mice.

According to a first characteristic, the process for synthesizing the compound according to the invention is characterized in that it essentially consists in exposing a sample of isomer (E) -3.5.4 ′ ~ trimethoxystilbene 5 dissolved in a solvent suitable for ultraviolet light with a wavelength peaking between 200 nm and 400 nm, this for an exposure time of between one hour and two hours for a quantity of (E) isomer of 10 ^" mole and, if applicable if necessary, evaporating said solvent in order to collect the desired isomer (Z).

Advantageously, the method according to the invention is characterized in that the culminating wavelength of the ultraviolet light chosen is preferably 254 nm. According to another characteristic, the irradiation time is preferably one hour for 10 ^"2 mole of isomer (E). Advantageously, the solvent used is preferably ethanol.

According to a preferred embodiment, the isomer (E) is obtained by preparation of a 4-methoxybenzyl halide, transformation of said halide into phosphonate, in parallel, 3,5-dihydroxybenzoic acid is transformed into 3,5-dimethoxybenzoate of methyl 2, then into 3,5-dimethoxybenzyl alcohol 3, oxidation of said alcohol to the corresponding aldehyde and finally transformation of 3,5-dimethoxybenzaldehyde 4 into the (E) - 3,5,4'-trimethoxystilbene isomer 5 by reaction with said phosphonate . Advantageously, the 4-methoxybenzyl halide is preferably 4-methoxybenzyl bromide 1.

According to a particularly advantageous embodiment, 3,5-dimethoxybenzaldehyde 4 is obtained by adding, according to the Corey method, pyridinium dichromate to 3,5-dimethoxybenzyl alcohol 3.

The present invention also relates to the compound capable of being obtained by the process according to the invention, characterized in that it is (Z) -3,5,4'-trimethoxystilbene.

Hereinafter, a preferred, nonlimiting example of synthesis of the compound R3 according to the invention will be described in more detail.

A) Example of a preferred chemical synthesis process for (Z) - 3,5,4 '-trimethoxystilbene or compound R3:

In what follows, all the melting points were measured, without correction, using capillary tubes and a device type B-510 no. 533640 marketed by the company Bϋchi. The nuclear magnetic resonance spectra of * H and ¹³ C were recorded with a Bruker AC type spectrometer at 200 MHz. The mass spectra come from an AutoSpec E Micromass installation, operating at 70 eV of energy and equipped with a device for direct introduction of the sample to be analyzed.

The measurement results are listed in the following table:

3.5 - C H10O3 45 - 48 ° C 166.16 1H, CDCI ₃ , 200MHz: 9.88 (1H, s, CHO); dimethoxybenzaldehyde 6.98 (2H, d, J = 2.3 Hz, H _2j6 ); 6.68 (1H, t, J = 2.3 Hz, H ₄ ); 3.82 (6H, s, 2 x OMe)

13 C, CDCI ₃ , 200 MHz: 192 (CHO); 161.3 (C ₃ , C ₅ ); 138.5 (C,); 107.2 (C ₂ , C ₆ ); 55.7 (2 x OMe)

(E) -3, 5, 4 'C17H1 ₈ O ₃ 56-57 ° C 270.33 1H, CDCl _3, 200 MHz: 3.85 (9H, s, 3 x triméthoxystilbène OMe); 6.40 (1H, t, J = 2.5 Hz, H ₄ ); 6.68 (2H, d, J = 2.5 Hz, H _{2> 6} ); 6.93 (2H, d, J = 9 Hz, H _{3. , 5.} ); 7.00 (2H, d, J = 16.5 Hz, vinyl); 7.48 (2H, d, J = 9 Hz, H ₂ -, ₆

¹³ C, CDCI ₃ , 200 MHz: 161 (C ₃ ); 159.5 (C ₅ ); 159.3 (C,); 139.7 (d); 130 (C _r ); 128.7 (C _p ); 127.8 _{(C2. 6.);} 126.6 (Cα); 114.2 (C _3. ); 113.8 (C _5. ); 104.4 (C _2.6 ); 99.7 (C ₄ ); 55.4 (OCH ₃ x 3)

Mass. m / z = 270 (M *, 100%); 258.0; 239.0; 228.1; 197.1; 150.1; 137.0; 121.1; 109.1

Physico-chemical data relating to the compounds used.

The dimethylformamide used in the context of the synthesis was distilled over a 0.4 nm molecular sieve and under reduced pressure. The dichloromethane used for step 4 was dried over calcium hydride. The 4-methoxybenzylic alcohol, the 3,5-dihydroxybenzoic acid and the triethylphosphite used below are products which are commercially available and which can be used as they are.

1. Preparation of 4-methoxybenzyl bromide 1

A volume of 6 ml of 48% concentrated hydrobromic acid was added all at once to 7.5 g (54 mmol) of 4-methoxybenzyl alcohol. The mixture was then vigorously stirred for 30 minutes at room temperature. Then 100 ml of diethyl ether were added and the acid phase was separated. After a second extraction with ether, the organic phases were combined, which were washed with, first of all sodium hydrogen carbonate, then with salt water, and which were finally dried with water. using magnesium sulfate. The 4-methoxybenzyl bromide 1 obtained is not very stable and was therefore immediately used in the next step. It is in the form of a colorless oil and 10.54 g of it have been obtained, which corresponds to a yield of 97%.

2. Preparation of methyl 3-5-dimethoxybenzoate 2

A mixture consisting of 5 g of 3,5-dihydroxybenzoic acid (32.4 mmol), 25 g of anhydrous potassium carbonate, 20 ml of dimethyl sulphate and 100 ml of acetone is brought to reflux for

4 hours. After filtration of the potassium carbonate and evaporation of the acetone, the remaining mixture was diluted with 100 ml of water and 200 ml of ether. Before being dried over magnesium sulfate, the ethereal phase was washed successively, twice with concentrated ammonia, once with water, twice with dilute hydrochloric acid and finally with salt water. The solvent evaporated, a crude product is obtained which is purified on a silica chromatographic column 60 sold by the company Merck using a hexane: ethyl acetate mixture 4: 1 (volume ratio). Colorless mass needles are thus obtained

6.17 g. This also corresponds to a yield of 97%. 3. Preparation of 3,5-dimethoxybenzyl alcohol 3

5 g (25.4 mmol) of 3,5-dimethoxybenzoate are added to a suspension of the lithium aluminum hydride (1.02 g, or 26.8 mmol) in 75 ml of diethyl ether methyl, contained in 25 ml of diethyl ether at a rate allowing a gentle reflux. After a reflux of 2.5 hours, the mixture is cooled, diluted with 50 ml of a 1: 1 mixture (volume ratio) water-ether and finally with dilute hydrochloric acid. The ethereal extract washed with water is dried over magnesium sulfate. Colorless needles with a mass of 3.86 g are obtained, which corresponds to a yield of 90%.

4. Preparation of 3.5-dimethoxybenzaldehyde 4

To 3.5 g (20.8 mmol) of 3,5-dimethoxybenzyl alcohol 3 in 30 ml of dried dichloromethane, 11.75 g (31.2 mmol) of pyridinium dichromate prepared according to the Corey method are added.

This method consists in adding in 20 minutes 15.8 g of pyridine to 20 g of chromium trioxide diluted in 20 ml of distilled water, taking care that the temperature does not exceed 20 ° C. After 15 minutes of vigorous stirring, 80 ml of acetone are added.

The orange crystals of pyridinium dichromate (25.5 g) are filtered after 12 hours of growth at -20 ° C, which corresponds to a yield of 68%. After stirring for 12 hours at room temperature, the reaction mixture is diluted using 60 ml of diethyl ether, filtered through a column filled with silica 60 sold by the company Merck to give, after evaporation of the solvent, 3, 1 g of a solid in the form of colorless needles. The yield is 90%. 5. Preparation of the isomer (EV3.5.4'-trimethoxystilbene 5

A mixture consisting of 14.5 ml (84.6 mmol) of triethylphosphite and 10.54 g (52.4 mmol) of 4-methoxybenzyl bromide 1 is heated at 120 ° C. for 6 hours. , a gaseous evolution of ethyl bromide occurs. After cooling to 0 ° C., 75 ml of dimethylformamide, previously distilled, and 2.97 g (55.2 mmol) of sodium methylate are added to the mixture and the mixture is stirred for one hour. Then 5.8 g (35 mmol) of the previously synthesized 3,5-dimethoxybenzaldehyde 4 are introduced therein. The solution obtained is stirred for one hour at room temperature and then another hour at 100 ° C. After cooling, the reaction mixture is stirred overnight, seized with water and extracted with ether. The organic phases are combined, washed with salt water, checked with litmus paper and finally dried over magnesium sulfate. The crude product is purified by chromatography on silica 60 with a mixture of hexane and ethyl acetate (4: 1) as eluent (volume ratio). 7.85 g of a solid are obtained in the form of colorless needles. The yield is 83%.

6. Preparation of (ZV3.5.4'-trimethoxystilbene (compound R3

A sample consisting of a solution of 1 ml at 10 ^"2 mole per liter of (E) -3,5,4'-trimethoxystilbene 5, is exposed to a peak ultraviolet light at 254 nm. The duration of the irradiation is after evaporation under reduced pressure of the solvent, the desired stereoisomer Z (R3) is obtained quantitatively.

The cost of the chemicals necessary to obtain a gram of 98% pure R3 compound is currently around 8 euros (or around 52 FRF). The scheme for the synthesis of compound R3 is summarized in Figure 1.

B) Biological effects of compound R3 according to the invention:

1) Inhibition by compound R3 of the growth of cancerous and / or transformed cells In order to demonstrate the activity of the compound R3 on the growth of cancer cells in vitro, the following tests were carried out.

Human colonic cancer cells of the CaCo 2 line and murine transformed cells of the IEC-6 line were treated 24 h after sowing with increasing concentrations of R3, ranging from 0.1 μM to 0.6 μM.

Figure 2 shows the cell growth curve

CaCo-2 (FIG. 2a) as a function of the concentration of compound R3 added, while FIG. 2b shows the growth curve of IEC-6 cells as a function of the concentration of compound R3. Each point on these two curves represents the mean value (± SEM) calculated from 8 samples. The legend of Figure 2a also applies to Figure 2b.

The results observed show that the compound R3 exerts a strong inhibitory action (inhibition of 70%) on the growth of the cancerous colonic cells CaCo-2 for concentrations greater than 0.2 μM, that is to say an inhibition similar to that obtained with resveratrol but for concentrations 150 times lower.

A complete arrest of cell growth of CaCo-2 cells is observed for a concentration of 0.3 μM in compound R3. With regard to IEC-6 cells, the compound R3 is even more active, since total inhibition of cell growth is already obtained at a concentration of compound R3 of 0.2 μM.

Since the IEC-6 line has a significantly higher proliferative power than CaCo-2 type cells, these results show that the effects caused by treatment with the R3 molecule are directly linked to the proliferative state of the cells.

2. Characterization of the antiproliferative effect of the compound R3

This study allows us to characterize the mechanism by which R3 inhibits cancerous cell proliferation.

Three main types of inhibitions have been described:

- necrosis, or direct and non-specific cytotoxicity of cells,

- apoptosis or programmed death of the cell, the term “cell suicide” is also used, and

- a simple blockage of the cell cycle. a) Aspecific cytotoxicity (cell necrosis):

Measuring the activity of intracellular enzymes released into the culture medium is a good indicator of cell lysis. Indeed, destruction of the cell membrane results in the release of the cytoplasmic content in the supernatant, and this in proportion to the number of cells destroyed.

Lactate dehydrogenase (LDH) is a ubiquitous intracytoplasmic enzyme, and its appearance in the external environment is a prime indicator for evaluating the cytotoxicity of a product (Decker and Lohmann-Matthes 1988).

Unexpectedly and surprisingly, it has been found that treatment of the CaCo-2 cells with the compound R3, causes a complete disappearance of the LDH activity which can no longer be detected in the extracellular medium and this even for concentrations high in R3 allowing to conclude that the growth inhibition observed in CaCo-2 cells is not the result of an aspecific cytotoxicity of the compound R3.

b) apoptosis

The term apoptosis is used to describe a particular mode of cell degeneration which, ultimately, results in cell death. It is an endogenous cellular process put in place as a last resort when an external or internal signal activates a metabolic pathway creating a significant dysfunction of the cell. Apoptosis is characterized by condensation and degradation of chromatin, reduction in cell volume and membrane deformation. One of the biochemical markers of apoptosis is a characteristic breakdown of DNA. Indeed, a DNA fragmentation is observed which results from the activation of an endonuclease selectively cleaving the DNA at sites located near the nucleosomal units. Mono- and oligonucleosomal fragments are thus generated. The detection of these fragments by electrophoresis on agarose gel is one of the most commonly used methods to qualify apoptosis.

Using this technique it could be seen that the R3 molecule did not cause such a process. After separation, a fragmentation similar DNA was obtained in the treated cells compared to the control cells. No DNA degradation could be demonstrated, even for high concentrations of compound R3 added. Treatment with the R3 molecule therefore does not induce an apoptotic process in CaCo-2 cells.

Another biochemical marker that can characterize apoptosis is the translocation of phosphatidyl serine from the cytoplasmic surface of the membrane to the outside of the cell membrane. The in vitro detection of this phenomenon can be visualized from the interaction between annexin V (a protein specifically binding phosphatidyl serine) and phosphatidyl serine. Annexin V is labeled with a specific fluorochrome and detection is carried out by flow cytometry.

The cells were therefore treated with 0.3 and 1 μM of R3 for 24 hours, then the analysis of the link between annexin V and phosphatidyl serine was carried out by flow cytometry (cf. FIG. 3a of the figure). 3).

It can be seen in said FIG. 3 a, that the treatment with the R3 molecule of CaCo-2 cells does not induce any variation in the profile of the labeling with annexin. There is no increase in the percentage of annexin-binding cells. The R3 molecule therefore does not cause the translocation of phosphatidyl serine. Therefore, this compound once again does not have the characteristics of an apoptosis inducer.

During this experiment, labeling with propidium iodide (PI) was carried out in parallel. The results are visible in Figure 3b of Figure 3. PI is an intercalator of DNA which is exclusively included by permeabilized cells. It is therefore excluded by viable cells (first peak), while it will be incorporated into dead cells (everything that is found after the first peak).

In FIG. 3b presented previously, it can be seen that cells incorporate this intercalant (peak on the far right in FIG. 3b of FIG. 3), both in the controls (presence of a few dead cells) and in the cells having undergone a treatment with the compound R3. However, we can see that R3 induces significant cell death as shown by the decrease in the number of cells forming the first peak. Thus, it can be seen that the higher the dose of R3, the higher the number of dead cells. This led to study the possible effects of the compound R3 on the course of the cell cycle. The results of this study are given below. c) Study by flow cytometry of the blocking of the cell cycle by the compound R3

Flow cytometry is defined as the precise study of isolated cells entrained by a liquid flow. It allows the qualitative and quantitative characterization of cells by determining the distribution and variation of the number of cells in each phase of the cell cycle, namely the phases Gi (preparation for entry into phase S), S (synthesis), G ₂ (preparation for entering phase M) and M (mitosis). This characterization is based on the fact that the cell has, during its development, a variable DNA content. We will find 2n chromosomes in phase G], while for cells in phase G ₂ the number of chromosomes present will be 4n. The intermediate values are those corresponding to the DNA synthesis phase, therefore to phase S of the cycle. In a cytometer, the channeled cells comparable to particles, cut the light beam of a laser and the signals which they emit in return are analyzed. These signals are a function of the amount of DNA present in the cell.

The CaCo-2 cells are treated with 0.3 μM of R3 over a period of 64 hours (Figures 4a and 4b). The distribution of cells in the different phases of the cell cycle was analyzed for sampling times of 16, 24, 40, 48 and 64 hours. Each curve represents the proportion of cells in the phase of the cycle concerned. The legend of Figure 4a also applies to Figure 4b.

A blockage of cells in the G ₂ / M phase (DNA duplication) is observable from 16 hours after treatment and is maintained approximately until 40 hours. During this period, an accumulation of cells blocked in the G2 / M phase is observed. It seems that beyond 24 hours, the compound R3 no longer makes it possible to completely maintain this blocking, this therefore results in a slight inflection of the curve. This phenomenon is not due to a possible reversibility of the product, but is probably associated with a slightly low concentration and which does not make it possible to affect all the cells in a pronounced way. This hypothesis is confirmed by the fact that the cells are again blocked. when 48 hours of fresh medium containing the R3 molecule is added. This blockage is much more pronounced than that observed previously since 85% of the cells are now blocked at the level of the G2 / M phase of the cell cycle. This therefore shows that the effects generated by the R3 molecule on the cell cycle are potentiated during successive treatments and are not reversible. It is interesting to note that the effects on the cell cycle which are induced by the compound R3 are similar to those exerted by another synthetic anticancer used in clinical practice, namely Taxotere ^® already mentioned above.

3. Study of the effects of compound R3 on the synthesis of polyamines

Polyamines are ubiquitous compounds which can be considered as growth factors for the tumor cell.

The endogenous metabolism of polyamines is regulated by 2 limiting enzymes called ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC or SAMDC). ODC catalyzes the decarboxylation of ornithine to putrescine and AdoMetDC catalyzes the decarboxylation of S-adenosyl-methionine into S-adenosyl-methionine decarboxylated, donor of aminopropyl group during the synthesis of spermidine and spermine.

Figures 5a and 5b show the activities of the ODC and the

SAMDC, expressed in pmol / h / mg protein. Each column represents the mean values (± SEM) of the ODC and AdoMetDC activities calculated from 3 samples. The legend of Figure 5a also applies to Figure 5b.

The activity of ODC within tumor cells is generally exacerbated, making it a potential target in the search for new anticancer agents. With this in mind, the effects of the compound

R3 have been studied on the activity of ODC and AdoMetDC (Figures 5a and

5b of figure 5).

It can be seen that a treatment with the compound R3 significantly affects the activity of the ODC, and this from the first treatment, however the intensity of the inhibition decreases after 48 hours of treatment. The R3 compound brought into contact with the cells during the first treatment is not in sufficient concentration to cause an irreversible blocking of the enzyme. This is verified by the fact that a second treatment carried out at 48 hours leads to a strong inhibition of the ODC. These results are further supported by the fact that the activity of AdoMetDC which did not undergo any significant variation during the first treatment is strongly inhibited during a second treatment.

Thus the compound R3 strongly inhibits the synthesis of polyamines (putrescine, spermidine and spermine) by the cancer cell which contributes to explain the powerful antiproliferative effects of this molecule.

4. Effects of compound R3 on the cytoskeleton

It has previously been possible to demonstrate that the R3 molecule causes a blockage of the cell cycle in the G2 / M phase. However, it is known that the passage of this phase is essential in cell division. Indeed, it is at this period of the cycle that the cell will be able to split and for this there is the play of key elements of the cytoskeleton. The microtubular network represents one of these elements.

Consequently, the repercussions that the R3 molecule could have on this network were also analyzed. The action of this compound has been compared with other compounds having an important and recognized effect on the microtubular network, namely nocodazole which causes depolymerization of microtubules, and taxol which on the contrary inhibits this depolymerization. The CaCo-2 cells are treated for 2 hours with the compound R3 (5 μM, 10 μM and 20 μM), taxol (10 μM) and nocodazole (20 μM). The structure of the microtubular network is then revealed by immunohistochemical methods.

As illustrated by the corresponding photo (bottom right of Figure 6), taxol by its anti-depolymerizing action freezes the microtubular network while nocodazole (photo bottom and left of Figure 6) significant disorganization of this network. It has been found that the compound R3 causes the same type of alterations as those which are observable when the cells are treated with nocodazole (photographs from the top right and the two photographs from the middle of FIG. 6). Compound R3 therefore falls into the class of agents causing depolymerization of the microtubular network and which lead to cell death.

By studying the effects of compound R3 on the growth of various cancer lines, it has been possible to provide significant data relating to the antiproliferative power of this compound on human or animal cancer cells. Thus, it has been shown that the R3 molecule has a powerful antiproliferative power causing blockage of cancer cells at the G2 / M phase of the cell cycle. This blockage is the result of the direct effect of this molecule on the microtubular network, since it causes depolymerization of this network leading to cell death (mummification of cells). Other work has also made it possible to show that the compound R3 is in no way toxic towards a line of normal lymphocyte cells even at high concentration, which makes it possible to envisage a real selectivity of the compound R3 according to the invention with respect to for cells in the active growth phase. This fact is remarkable and reinforces the interest of said compound R3 in cancer therapy since other anticancer currently in clinical use, including Taxotere ^®, do not show such selectivity of action.

More recent additional studies have also made it possible to highlight the following other properties:

5. “In vitro” studies

a) Identification of the R3 binding site on tubulin

The inventors of the present application have established that the compound R3 interferes with the microtubular network by causing a depolymerization of the latter.

There are three possible binding sites for the poisons of the microtubular network at the level of the β subunit of tubulin: the domain of periwinkle alkaloids, the sites of taxoids and that of colchicine. However, it was unexpectedly and surprisingly found that the effects obtained during the treatment of Caco-2 cells with R3, are similar to those described for other poisons of microtubules such as colchicine and the alkaloids of the periwinkle of Madagascar. (Vincristine, Vinblastine). Colchicine is not currently used as an anti-cancer agent due to its excessive toxicity. Compound R3, because of its low toxicity, could therefore represent an alternative compound of choice if the latter binds to the same domain as that of colchicine. To determine whether or not R3 binds to the colchicine binding site, a competitive study was therefore undertaken.

To this end, a tubulin-rich cell extract from Caco-2 cells was brought into contact with labeled colchicine and increasing concentrations of R3. The results obtained show in Figure 7 (where each point represents the average value (± SEM) calculated from 3 samples) that R3 causes a “dose-dependent” displacement of colchicine up to 5 μM, and that at above this threshold, whatever the dose of R3 used, 14% of the labeled colchicine remains linked to tubulin. These results suggest that R3 would bind either to the site of colchicine itself, or to a separate site, which would influence the binding of colchicine to its site.

Colchicine having applications in the treatment of cystic fibrosis, it is proposed, by analogy, the use of compound R3 according to the invention for the preparation of a medicament intended to treat or prevent cystic fibrosis.

b) Induction of an original apoptotic process in lymphoid cell lines:

In order to better characterize the degenerative process which the R3 compound involves on cancer cells, the aforementioned inventors have also carried out a characterization of the antiproliferative effects of R3 on cell lines of lymphoid origins. These cells have the advantage of not adhering to their culture support and therefore do not therefore have to be subjected to the digestive action of trypsin to be analyzed. This helps to maintain perfect cellular integrity.

The NH32 lymphoid line was used, the p53 gene of which was completely inactivated by homologous double recombination, this in order to also see whether the antiproliferative effect of R3 was dependent on the presence or not of this tumor suppressor gene. The same series of experiments as that used during the characterization of the antiproliferative power of R3 on the Caco-2 cell line was undertaken.

The results obtained show that R3 causes a comparable “dose-dependent” inhibition of cell growth in these two lines. However, contrary to what had been obtained in the previous results, a process of cell death of the apoptotic type was highlighted.

The TK6 and NH32 cells are treated with increasing doses of the compound R3 (0.1 to 1 μM) and the analysis of the link between annexin V and phosphatidyl serine is carried out by flow cytometry, after 16, 24, 40 and 48 hours of treatment (Figure 8). Treatment of the cells with the R3 compound has no significant effect during the first 24 hours of treatment regardless of the dose of R3 used. After 40 hours of treatment, a very significant increase in the percentage of cells outsourcing phosphatidyl serine is then obtained from a concentration of R3 of the order of 0.3 μM. This increase is “dose-dependent” in the two lines studied. However, the NH32 line seems less affected than the TK6 line. In an attempt to explain this trend towards a decrease in the number of cells externalizing phosphatidyl serine in NH32 type cells, the effect of R3 on the DNA content of the cells was examined. The results demonstrate once again that the action of the compound R3 causes blocking of the cells at the level of the G2 / M phase of the cell cycle. Suφrenantly after treatment with R3, a high percentage of cells had a particular profile in NH32 cells, namely a DNA content higher than normal. Treatment with R3 therefore causes a phenomenon of hypoidoidy.

As illustrated in Figure 8, we can see that this phenomenon is very marked for NH32 cells while it is relatively moderate for TK6 cells. This phenomenon would explain the slight difference observed in the level of externalization of phosphatidyl serine and suggests that the degenerative process caused by R3 is dependent on p53. It is conceivable that cells exhibiting a wild p53 (TK6) status would rapidly enter apoptosis under the action of p53, while the entry of the cell into a hypoidoid phase would cause a delay in this process. These results show that R3 behaves as a pro-apoptotic agent on these cancer cell lines.

6. Effects of R3 on other human cancer cell lines:

As shown in Table 1 below, the compound R3 according to the invention shows a higher activity on the lines which have the fastest replication times (TK6, NH32, KB). The antiproliferative effects of R3 are manifested on all the cancer lines tested.

Table 1: Inhibition of the growth of tumor cells by R3

IC₅₀ = concentration (micromolaire) en R3 nécessaire pour inhiber de 50 % la croissance des cellules.

IC ₅₀ = concentration (micromolar) in R3 necessary to inhibit cell growth by 50%.

7. "In vivo" studies: Effects of R3 on tumor development in nu / nu athvmic mice.

Figure 9 illustrates the effects of R3 on tumor development after subcutaneous implantation of human tumor cells in nu / nu athymic mice. Three weekly injections (intraperitoneal route) were carried out on six subjects at a dose of 100 mg / kg, then the tumors were removed 25 days after implantation.

As can be seen, the compound R3 inhibits the development of the tumor by 50% and R3 has no apparent toxicity on the animal (identical weight evolution in the 2 groups of mice).

The present invention therefore also relates to the compound R3 according to the invention for use as a medicament.

It also relates to the use of said compound R3 according to the invention for the preparation of a medicament intended to treat or prevent a disease involving an abnormal cell proliferation or growth, such as cancer, in particular by causing cell death apoptotic in lymphoid cell lines.

It also relates to its use as an agent for depolymerization of the microtubular network of human or animal living cells, in particular by providing that the (Z) -3,5,4'- trimethoxystilbene is fixed at least partially on the site of colchicine .

It also relates to the use of said compound R3 according to the invention as an agent for inhibiting the polyamine synthesis of human or living animal cancer cells, as an agent for blocking the cell cycle, in particular of the cell cycle of human cancer cells or live animals, as an agent for the treatment or prevention of hyproliferative pathologies, in particular inflammatory conditions, infectious and / or parasitic diseases (malaria) and as a photoprotective agent, in particular for protection against UV-A and UV- rays B. In the case of diseases involving blockage of the cell cycle, the use is further characterized in that said blockage of the cell cycle takes place at the G2 / M phase.

Advantageously, the use of compound R3 according to the invention is further characterized in that said blocking of the cell cycle is irreversible.

The present invention further relates to a medicament characterized in that it comprises, as active principle, at least the compound R3 ((Z) -3,5,4'-trimethoxystilbene). Finally, it also relates to a cosmetic composition with photoprotective property, characterized in that it comprises, as active principle, at least the compound R3 ((Z) -3,5,4'-trimethoxystilbene).

Among the numerous and important advantages of the compound R3 according to the invention, there may be mentioned: a chemical synthesis of convenient access,

- access to numerous analogues (alkylated derivatives) by the same synthetic route,

- low manufacturing cost (8 euros / g),

- biological efficacy in the treatment of cancers and of any pathology linked to hypereiferative states, and

- low toxicity towards normal cells (especially immune cells).

Thanks to the compound R3 of the present invention, it also becomes possible to provide a method of treatment or prevention of a disease in a mammal, in particular diseases involving an abnormal cell proliferation or growth such as diseases of the "cancer" type. or hypeifroliferative pathologies, in particular inflammatory states, infectious and / or parasitic diseases or a disease such as cystic fibrosis, said treatment or prevention method comprising the administration (by any possible route) to said mammal of a therapeutically effective amount of at least one R3 compound of general formula I or of a pharmaceutically acceptable derivative (salt, ester, etc.) of said R3 compound according to the present invention.

Of course, the invention is not limited to the embodiment described. Modifications remain possible, in particular from the point of view of the constitution of the various elements or by substitution technical equivalents, without departing from the scope of protection of the invention.

Claims

1. Process for the synthesis of (Z) -3,5,4'-trimethoxystilbene, characterized in that it essentially consists in exposing a sample of (E) -3,5,4'-trimethoxystilbene (5) -isomer in solution in a solvent suitable for ultraviolet light with a wavelength between 200 nm and 400 nm, for an exposure time of between 1 hour and 2 hours for a quantity of (E) isomer of 10 ^" mole and, where appropriate, evaporating said solvent in order to collect the desired isomer (Z). 2. Method according to claim 1, characterized in that the culminating wavelength of the ultraviolet light chosen is preferably 254 nm. 3. Method according to claim 1 or 2, characterized in that the irradiation time is preferably one hour for 10 ^"2 mole of isomer (E). 4. Method according to any one of claims 1 to 3, characterized in that the solvent used is preferably ethanol. 5. Method according to any one of claims 1 to 4, characterized in that the isomer (E) used is obtained by preparation of a 4-methoxybenzyl halide, transformation of said halide into phosphonate, in parallel, acid 3 , 5-dihydroxybenzoic acid is transformed into 3,5 methyl dimethoxybenzoate (2), then into 3,5-dimethoxybenzylic alcohol (3), oxidation of said alcohol into corresponding aldehyde and finally transformation of 3,5-dimethoxybenzaldehyde (4) into isomer ( E) -3,5,4'-trimethoxystilbene (5) by reaction with said phosphonate. 6. Method according to claim 5, characterized in that the 4-methoxybenzyl halide is preferably 4-methoxybenz bromide (1). 7. Method according to claim 5 or 6, characterized in that 3,5-dimethoxybenzaldehyde (4) is obtained by adding, according to the Corey method, pyridinium dichromate to 3,5-dimethoxybenzyl alcohol (3) . 8. Compound capable of being obtained by the process according to any one of claims 1 to 7, characterized in that it is (Z) - 3, 5, 4 '-trimethoxy stilbene. 9. A compound according to claim 8 for use as a medicament. 10. Use of the compound according to claim 8 or 9 for the preparation of a medicament intended to treat or prevent a disease involving an abnormal cell proliferation or growth, such as cancer. 11. Use according to claim 10, characterized in that the (Z) -3,5,4'-trimethoxystilbene causes pro-apoptotic cell death in the lymphoid cell lines. 12. Use of the compound according to claim 8 or 9 for the preparation of a medicament having an action of depolymerization of the microtubular network of living human or animal cells. 13. Use of the compound according to claim 12, characterized in that the (Z) -3,5,4'-trimethoxystilbene binds to tubulin at least partially on the site of colchicine. 14. Use of the compound according to claim 8 or 9 for the preparation of a medicament having an action of inhibiting the polyamine synthesis of living human or animal cancer cells. 15. Use of the compound according to claim 8 or 9 for the preparation of a medicament having a blocking action of the cell cycle, in particular of the cell cycle of living human or animal cancer cells. 16. Use of the compound according to claim 15, characterized in that the blocking of the cell cycle takes place at the G2 / M phase. 17. Use of the compound according to claim 16, characterized in that said blocking of the cell cycle is irreversible. 18. Use of the compound according to claim 8 or 9 for the preparation of a medicament intended to treat or prevent hypereiferative pathologies, in particular inflammatory conditions, infectious and / or parasitic diseases such as malaria. 19. Use of the compound according to claim 8 or 9 for the preparation of a medicament intended to treat or prevent cystic fibrosis. 20. Use of the compound according to claim 8 or 9 as a photoprotective agent, in particular for protection against UV-A and / or UV-B rays. 21. Medicament, characterized in that it comprises, as active principle, at least one compound according to claim 8 or 9. 22. Cosmetic composition with photoprotective property, characterized in that it comprises, as active principle, at least one compound according to claim 8. 23. Composition according to claim 22, characterized in that the active compound is activated by light.