WO1990007520A1 - Oxysteryle phosphates, their synthesis and use as pharmaceutical agents - Google Patents

Abstract

Oxysteryle phosphates according to formula (I), where R1 is an oxysteryle radical; where R2 is H or a radical derived from: (a) a natural or synthetic nucleotide structure, (b) a non-nucleotide anticancer agent, (c) an immunodepressive or immunostimulant agent, (d) a component showing an affinity for specific receptors or sites; and where R3 is selected from: -O-H, and salts and pharmaceutically acceptable acide derivatives thereof. Application to the pharmaceutical industry.

Description

OXYSTERYL PHOSPHATES, SYNTHESIS AND USE COKKE PHARfriACEUTICAL AGENT

The present invention relates to oxysteryl phosphates, their method of synthesis and their use in particular as anti-cancer agents or immunosuppressants.

Recently, the pharmacological properties of the derivatives oxidized at various positions of the skeleton or the side chain of cholesterol or of its biogenetic precursors such as desmosterol and lanosterol have been discovered (cf. "Polyoxygenated sterols and triterpenes: chemical structures and biological activities" B. LUU in "Biological activities of oxysterols", Editions INSERM, Vol. 166, 1988, pages 23-40). These derivatives have a cytotoxic activity on cancer cells, this activity is selective since at determined concentrations, they are cytotoxic for cancer cells and without action on non-tumor cells. The mechanism of this action, one of the targets of which is the cell membrane, is original compared to that of known conventional anticancer drugs, the action of which is generally located at the level of cell division. For more explanations, see the article cited above and the article "Inhibitory effect of an oxygenated cholesterol on the induction and progression of DMBA mammary carcinoma in the rat" by O.H. Iversen, A. Kolberg, I. Smith-Kielland, A. Stabursvik, Wirchows, Arch. (Cell. Path.), 1986, 51, 313-320.

These compounds are also capable of inhibiting lymphocyte activation (blastogenesis and allogenic stimulation, in particular). This immunosuppression inhibits the appearance of IL-2 receptors on the surface of lymphocytes and the production of this growth factor. These oxysterols have an activity similar to that of immunodé¬presseurs commonly used in clinical like cyclosporine A and dexamethasone; but their mechanism of action is different: they intervene at the membrane level by affecting the activation of protein kinase C. However, these products have such a low solubility in the aqueous phase that it is difficult to use their property in vivo, especially in the treatment of cancerous tumors or in the prevention of organ transplant rejection. In order to improve the solubility of these products, attempts have been made to use detergents and ingredients but without any interesting results.

A second solution consisted in grafting hemisuccinate groups in order to increase the water solubility of these compounds. The corresponding alkaline salts made it possible to test the properties of said compounds in vivo (cf. "In vivo anti-tumor activity of water-soluble derivatives of 7-hydroxycholesterols" by S. Rong, C. Bergmann, B. Luu, JP Beck and G.Ourisson, CR Acad. Sci. 1985, 300, 89-94). These derivatives however have drawbacks: on the one hand their solubility in bi-distilled water remains relatively low (1 to 2%), while their solubility in physiological solute is even lower; on the other hand, their pH is not compatible with certain therapeutic treatments. Finally, their toxicity is high because the DL-50 is around 200 mg / kg. This is why one of the aims of the present invention is in particular to provide compounds derived from oxysterols which are significantly more soluble than hemisuccinates and which, if possible, exhibit improved therapeutic properties.

Another object of the present invention is to provide compounds of the above type allowing the association of the above oxysterols and of compounds having a complementary action in the field of immunomodulators and anti-cancer agents.

Another object of the present invention is to provide soluble compounds, derived from the above oxysterols, which are associated with molecules allowing the targeting of said oxysterols at specific sites.

These aims and others which will appear subsequently are achieved by means of oxysteryl phosphates of formula I:

0 _R _ | - 0— R. (D

3 I '

0 IR „ where R. is an oxysteryl radical, where R_ is H or a radical derived from: a) a natural or synthetic nucleotide structure, b) a non-nucleotide anticancer agent, c) an immunosuppressive or immunostimulatory agent, d) a component having an affinity for specific receptors or sites; where R, is chosen from: -OH or its especially alkaline salts, and derivatives of pharmaceutically acceptable acids.

The oxysterols, from which the oxysteryl radicals are derived, correspond to the family of compounds described in the article by B. LUU in "Biological activities of oxysterols, JP Beck, A. Crastes by Paulet Editeurs, Colloque INSERM, vol. 166, 1988, pages 23-40 Among these compounds, mention should be made of polyoxygenated ergosterol compounds, ganoderic acid derivatives, stigmastane derivatives, hippuristanol derivatives and pentacyclic triterpenes, especially maytenfolic acid. ,.

More specifically, the group R. oxysteryl has the following formula derived from cholesterol:

dans laquelle — indique une possible double liaison, v indique une liaison cis ou trans, dans laquelle R . ., Ri _?' -^~* _\ représentent indépendamment H ou CH,, dans laquelle R . , R „ et R^ représentent indépendamment H, =0, -OH, R. n'existant pas si la position 24-25 est occupée par une double liaison, à la con dition que l'un au moins des R^, R-, R, est une fonction oxygénée.

in which - indicates a possible double bond, v indicates a cis or trans bond, in which R. ., Ri _? '- ^~ * _\ independently represent H or CH ,, in which R. , R „and R ^ independently represent H, = 0, -OH, R. not existing if position 24-25 is occupied by a double bond, provided that at least one of R ^, R -, R, is an oxygen function.

The phosphate group is advantageously fixed in position 3. Among the preferred compounds, mention may be made of the compounds in which R. is 25 hydroxycholester-3 yl, 7 hydroxycholester-3 yl, 7.22 dihydroxycholester-3 yle, 22.25 dihydroxycholester-3 yle, 7.25 dihydroxycholester-3 yle.

The radical R_ may be a radical derived from a nucleotide structure, in particular derivatives of natural nucleotides with a purine or pyrimidine ring such as: - adenine

- thymine

- guanine

- uraciie

- Cytosine or synthetics, in particular halogen derivatives such as 5-fluorouracil, or else nucleotide derivatives known for their anti-cancer, anti-viral or immunomodulatory activity.

The radical R- can be in particular:

où R' est un groupement méthyle, éthyle, propyle, isopropyle, hydroxy, méthoxy, éthoxy ou fluor ; où R" est un groupement hydroxy, N, ou CN.

where R 'is a methyl, ethyl, propyl, isopropyl, hydroxy, methoxy, ethoxy or fluorine group; where R "is a hydroxy group, N, or CN.

The most conclusive tests are those where R 'is hydrogen, methyl or fluorine and / or R "is hydroxy.

The radical R _? can also be derived:

- a non-nucleotide anti-cancer agent,

- an immunostimulating peptide such as MDP and its derivatives,

- immunosuppressants of various classes such as corticosteroids, dexamethasone for example or peptides such as cyclosporine.

Finally, the radical R_ can be an element ensuring the targeting of the molecule on specific receptors or sites; thus it can be a simple or complex carbohydrate radical intended to target galactose receptors for example.

Agents such as antibodies or antigens can also be used to target complementary elements.

In some cases, the radical R_ may be grafted via a grafting element in particular: - to allow grafting of the molecules from which R- derives, which do not have OH functions, or

- to allow the molecules to be removed and limit the steric effects of bulky radicals.

The radical R- can be a simple ose, which makes it possible to significantly increase the solubility of the compound in a nonionic and non-toxic manner.

The radical R-, can also simply be hydrogen or a corresponding salt.

The radical R essentially has the aim of ensuring good solubilization of the phosphates according to the invention while respecting the pH constraints of the biological media to be treated; it is generally such that an anion or a pair of ions is formed once in the biological medium to be treated. The ionization state of R, and its associated ions depends on the desired pH.

The most interesting compounds will be described in more detail in the examples. The compounds according to the present invention can be synthesized according to the techniques described for the synthesis of oligodeoxyribonucleotides and phospholipids "Synthesis of oligodeoxyribonucleotide by a continuous flo, phosphotriester method on a Kieselguhr / polyamide support" by M.J. Gait, H.W.D. Matthes, M. Singh, B.5. Sproat et RC Titmas ", Nucleic Acid Res„, 1982, 10, 6243-6254, "Current methods of phosphorylation of biological molecules", LA Slatin, Synthesis, 1977 (November), 737-753, and "A simple and effective chemical phosphorylation procedure for biomoiecules ", W. Bann orth, A. Trzeciak, Helv. Chim. Acta, 1987, 10, 1-12. The use of bifunctional phosphorylating reagents in particular those described in the article" Preparation of ribonucieoside 3 ' -0-phosphoramidites and their application to the automated soiid phase synthesis of oligonucleotides "by N. Usman, RT Pon,. Ogilvie, Tetrahedron Lett., 1985, n ° 38, 4567-4570, has proved to be particularly advantageous.

The invention also relates to a process for the preparation of compound of formula I in which the compound of formula I is deprotected, certain reactive functions of which have been protected during synthesis.

Among the protective groups, mention should be made more particularly of the protective groups of the phosphite or phosphate group such as the CNCH-CH-- groups.

Similarly, in the case where the coupled compound is a nucleotide, it is necessary to eliminate the protective groups, in particular OH functions.

These protected compounds I can be obtained by phosphorylation, direct or indirect, simultaneous or successive, of the compounds R. -OH, and R - OH where the reactive functions of R. and R. ^* will have been protected, then possibly coupling of the phosphorylated compound with the non-phosphorylated compound. Advantageously, a method is used comprising the phosphorylation step which consists in reacting the compound R. OH where R. , same value as in formula I, with a phosphine of formula IV:

s s

R 0 - P. (IV)

10

where R 2 and R _g are similar or different amino diaicoyl groups in which the similar or different alkyl groups have from 1 to 7 carbon atoms and are preferably branched; where R. Q is a protective group such as CNCH-CH--.

The intermediate compound thus obtained of formula V:

allows a very large number of derivatives to be obtained by coupling said compound of formula V with a compound having a -OH function, from which any other reactive functions will have been protected, in the presence of tetrazole or of a compound capable of forming pairs ion with secondary amines followed by oxidation of the phosphite triester obtained.

Examples of such syntheses are detailed in the experimental part.

Another object of the present invention is to provide a pharmaceutical composition which contains as active principle at least one of the phosphates according to formula I. These phosphates can be used alone or as an active ingredient in combination with other active ingredients and with vectors commonly used in this type of therapy. These compositions are more particularly intended for the fight against cancer or for causing immunosuppression useful in particular in the prevention of transplant rejection.

The present invention further relates to a process for the preparation of anti-cancer and / or immunosuppressive pharmaceutical compositions containing compounds of formula I.

These compositions are produced by mixing, alone or in combination with other active ingredients, the compounds of formula I with the vectors commonly used in the matter and in particular the aqueous vectors, in particular the phosphates according to the invention have good solubility in physiological solutions this allows their use for the preparation of compositions to be administered by injection by intravenous or intraperitoneal route.

Finally, according to the present invention, the sterols have been made soluble by fixing a phosphate group of formula VI on a hydroxyl bond:

The following nonlimiting examples allow those skilled in the art to better understand the parameters for implementing the invention.

Example 1 Phosphorylation of Polyoxygenated Sterols

1 mmol of 7-triethylsilyoxycholesterol is co-evaporated with anhydrous acetonitrile (3 × 50 ml), then it is dissolved in 30 ml of CH 2 Cl-. 0.5 ml of bis- (diisopropylamine) -2-cyanoethyl phosphine and 70 mg of tetrazolide-diisopropylamine are then added thereto. We let them react to room temperature and under argon for two hours. Once the reaction is complete, rapid chromatography of the reaction mixture on silica gives the corresponding product with a yield of 98% (eluent: hexane / ethyl ether with 1% triethylamine). This phosphorylation is schematized by the following reaction:

Example 2 Coupling of the Nucleotide with the Phosphorylated Polyoxygenated Sterols

1 mmol of a previously obtained phosphorylated sterol and 1 mmol of a protected nucleotide are co-evaporated separately with anhydrous acetonitrile (3 × 50 ml). The sterol thus obtained is then dissolved in 5 ml of ethyl ether and added to 50 ml of an acetonitrile solution containing 1 mmol of nucleotide and 0.5 mmol of tetrazole. After 6 hours of reaction, the phosphite triester thus obtained is oxidized to the phosphate triester with m-chloroperbenzoic acid (2 hours). The product is purified by chromatography on silica (eluent: methanol / methylene chloride / triethylamine - Yield 60%). This coupling is schematized by the following reaction:

R '= H; F

Example 3: Deprotection of the Protective Groups of the Sterol and of the Nucleotide.

All the protective groups are deprotected by the action of 0.36% HCl in THF at 0 ° C for two hours. A reaction mixture is poured into 200 ml of dichloromethane and the organic phase is washed with an NaCl solution until pH 7. Chromatography on silica gives the deprotected product (eluent: methanol / methylene chloride mixture; yield 90%). This deprotection is schematized by the following reaction:

Example 4 Deprotection of the Protective Grouping of the Phosphate

The product obtained above is dissolved in methanot and a 25% ammonia solution is added in order to have a final concentration of 5% ammonia. After 2 hours of stirring, it is evaporated to dryness. The phosphate diester is obtained in the form of the sodium salt by passing the product over a Dowex 50 ion exchange column (Na form) with a yield of 100%. This deprotection is carried out by the following reaction:

2) Dowex-50 (Na ⁺ ) R - H; OH R '= H; F Example 5: Biological tests of the compounds according to the present invention.

To simplify the presentation of subsequent tests, the following notations have been chosen:

JB 69 (or 69): sodium salt of the ester of the monophosphoric acid of 7 P hydroxycholesterol and of 2-deoxyuridine,

JC 40 (or 40) s sodium salt of the ester of monophosphoric acid of 7 P, 25 dihydroxycholesterol and 2-deoxyuridine,

7 βOHC: 7> hydroxycholesterol (compound of the prior art),

7β, 25 OHC: 7 P *, 25 dihydroxycholesterol (compound of the prior art).

During preliminary tests, the phosphate of 7 β-hydroxychole-steryie and 5-fluorouracil gave in vivo results (Krebs II carcinoma, leukemia P388) superior to those obtained with the metal salts of bis-hemisuccinate of 7 ^ - hydroxycholesteryl. Cytotoxicity on lymphomas a) counting with Trypan blue

This technique has been used to measure the cytotoxic activity of 69 on murine and human lymphomas. After 48 hours of culture in a medium containing 2% of FCS (fetal calf serum), various murine lymphomas (RDM-4, EL-4, YAC) are completely lysed at concentrations of oxysterols (69 or 7βOHC) of 20 μM (table 1).

Table 1% inhibition on different lymphomas after 48 h of culture in 2 SVS Human lymphomas (Molt 4) are much more sensitive to 69 than to 7POHC (under the same culture conditions). 7β, 25 OHC is more active than 70OHC and 69 on murine lymphomas which can be explained by the activity probably conferred by hydroxyl in position 25. b) release of chromium 51

Different types of lymphomas were labeled with chromium 51, then subjected to the lysis of cholesterol derivatives in order to study the cytotoxity during the first 2 to 4 hours of culture. The lysis of lymphomas is demonstrated by the release of chromium 51 into the culture medium after 2 hours (or 4 hours of incubation), the results are collated in Table 2:

Table 2:% release of 51 C, r (after 2- h- incubation)

69 exerts an extremely rapid cytotoxic activity, while free 7pOHC not soluble in aqueous medium requires 24 to 48 hours of culture to exert a cytotoxic action. Compared to 69, even a very high dose of 7β, 25 OHC (50 μM) is not toxic on lymphomas during the first 2 to 4 hours of culture whereas concentrations 10 times lower (5 μM) are toxic on 24 hours of culture. 69, which is soluble in an aqueous medium (more than 30% in physiological saline) therefore has a kinetics of action faster than its non-phosphorylated correspon¬ dants.

From Table 2, it also appears that the cytotoxic action of 69 strongly depends on the concentration of SVF in the culture medium. (just like 7βOHC and 7β, 25 OHC). The addition of cholesterol in the culture medium makes it possible to partially reverse the cytotoxic action of the oxysterols: non-soluble or water-soluble derivatives.

The addition of BSA practically does not modify the cellular inhibition obtained with the insoluble oxysterols. On the other hand, the activity of the water-soluble derivatives is very strongly dependent on the concentration of BSA. 1% of BSA completely reverses the cytotoxic action of 69 to 40 μm. This difference in behavior between 7 ^ OHC and its water-soluble derivative could be explained by the presence of the phosphate group which would promote bonds to serum albumin. c) Incorporation of ³ H thymidine

In parallel, to the cytotoxicity tests determined by counting with Trypan blue and by salting out with chromium, experiments are carried out with the incorporation of ³ H Thymidine; another method to estimate cell proliferation. The results obtained are collated in Table 3 s

Table 3: Percentage of incorporation of ³ H Thymidine

EL-4 lymphomas in culture 48 h in a medium containing 2 or 10

% FCS (with the addition of ³ H Thymidine 6 hours before the end of the culture).

The incorporation of ³ H Thymidine reflects the cytotoxic activity of

69, but there is a very strong increase in the incorporation of ³ H

Thymidine at non-toxic doses of 69. This could be interpreted by a molecular effect of 69: blocking in S phase during cell proliferation. Cell cycle studies are used to assess the percentage of cells in each phase of the cycle. After addition of non-toxic doses of 69 to EL-4 cells cultured 3 to 6 days, an increase in the percentage of cells in Gl and S phases is observed, and a decrease in the percentage of cells in G2 phase. This effect is dose dependent. Example 6: In Vivo Tests

The antitumor activity of 69 was demonstrated with OFI mice to which the KREBS 2 tumor was transplanted. 100% remission is obtained with two successive injections i.p. 1.5 mg 69.2 hours after injection of the tumor. A similar effect was observed with mice carrying P388 leukemia (5 mg / kg in a single injection). EXAMPLE 7 Immunomodulation a) in vitro

In vitro 69 has a marked immunosuppressive activity, comparable to that of its non-phosphorylated correspondent, 7? OHC. C57 B6 mouse spleen lymphocytes were stimulated in vitro by a non-specific mitogen: concavanaline A. 69 is added at the start of stimulation and the culture is carried out for 42 hours followed by the addition of ³ H Thymidine then cultivation continues for another 6 hours. Immunosuppression is dependent on the concentration of fetal calf serum (SVF). The results are collated in Table 4.

Table 4: Percentage of inhibition of stimulation by concanavalin A in in vitro immunomodulation tests. b) Ex vivo

The purpose of the ex vivo tests is to detect activity of 69 in vivo in mice treated with it for several days. The activity of the product on animals is then demonstrated thanks to experiments carried out in vitro.

The LD 50 intraperitoneally of 69 is approximately 250 mg / kg. To avoid any phenomenon of acute toxicity, a treatment is carried out with a dose of 50 mg / kg distributed over two daily injections, this for 4 days. One day after the end of this treatment, the mice are sacrificed.

Peritoneal cells, spleen, thymus are removed mouse by mouse.

The effects of 69 are determined by the cell count in the presence of Trypan blue. The number of cells present in the spleens, the thymus, the peritoneal exudates are determined:. compared to controls, the cellularity of the spleen is unchanged,

. in the thymus, the cellularitis is clearly reduced compared to the controls, there remains only about 20% of cells, only the cells are affected during this treatment,. in the peritoneal cavity, the number of cells recovered is clearly higher in the treated mice. An increase in the number of macrophages and a migration of neutrophils into the abdominal cavity should be noted.

Responses to mitogens Response to stimulation with different mitogens (concavanaline

A, lipopolysaccharides) is increased for spienic cells of mice treated before 69. Although the cellularite in the thymus decreases by approximately 60%, a strong increase in stimulation is observed up to 300% of the thymocytes treated compared to the thymocytes of control mice. This phenomenon, similar to that observed in the case of corticosteroids, could be interpreted by a decrease in prethymic cells. Functional tests: cytotoxicity

. LAK: the activity of LAK (Lymphokine activated killer) is reduced in the treated mice,

. CTL: the activity of CTL (cytotoxic T lymphocyte) is reduced in the treated mice,

. IL-1: the IL-1 activity of peritoneal macrophages stimulated or not by LPS, is greatly reduced after treatment with interoperitoneal therapy with 69.

From previous examples, it has been demonstrated that 69 has excellent anti-tumor and immunosuppressive activity in vitro and in vivo thanks to its good solubility in aqueous medium (30% in physiological water). 69 is therefore a product capable of curing mice carrying the KREBS 2 tumor but also of modifying immune responses after a 4-day in vivo treatment. It is also worth highlighting the remarkable activity of 69 for the treatment of human lymphomas.

Tests carried out on the so-called "40" compound have confirmed the activity of this new family of water-soluble oxygenated phosphates. the cytotoxic activity of 40 is increased on murine lymphomas compared to 69; similarly, the immunosuppressive effect in vitro and ex vivo is accentuated with 40.

Claims

1. Oxysteryl phosphates of formula I

where R. - is an oxysteryl radical, where R-, is H or a radical derived from: a) a natural or synthetic nucleotide structure, b) a non-nucleotide anti-cancer agent, c) an immunosuppressive or immunostimulant agent, d) a component having an affinity for specific receptors or sites; where R, is chosen from: -OH or its pharmaceutically acceptable salts and derivatives of acids. 2. Phosphates according to claim 1, characterized in that the group R, has the following formula:

in which - - indicates a possible double bond, <v ~ - indicates a bond in cis or in trans, in which Ri, _» Ri _. R- _I - _J independently represent H or CH ,, in which R ^, R - and R _fi independently represent H, “O, -OH, R ^ not existing if the position 24-25 is occupied by a double bond , with the co dition that at least one of R., R „R, is an oxygen function. 3. Phosphates according to either of Claims 1 and 2, characterized in that the said oxysteryl group is 7 hydroxycholesteryl, the 25 hydroxycholesteryle, 7.25 dihydroxycholesteryle, 7.22 dihydroxycholeste 22.25 dihydroxycholesteryle, the phosphate being fixed in position 3. 4. Phosphates according to one of claims 1 to 3, characterized in that R_ is a nucleotide. 5. Phosphates according to claim 4, characterized in that the said nucleotide has the formula III:

where R 'is a methyl, ethyl, propyl, isopropyl, hydroxy, methoxy, ethoxy or fluorine group; where R "is a hydroxy group, N, or CN. 6. Phosphates according to claim 5, characterized in that R 'is fluorine, hydroxy or methyl and R "is hydroxy. 7. Phosphates according to claim 2, characterized in that they have the following formula;

in which R., R_ and R ^ independently represent H or OH, R 'represents H or F, X is a cation ensuring the neutrality of the compound. 8. Process for the preparation of compounds of formula I, characterized in that the compound of formula I is deprotected, certain reactive functions of which have been protected during synthesis. 9. Synthesis method according to claim 8, characterized in that the protected compounds I are obtained by phosphorylation, direct or indirect, simultaneous or successive, of the compounds R. -OH, and R ₂ -OH where the reactive functions of R.- and R. will have been protected. 10. Method according to claim 9 for the synthesis of phosphates according to one of claims 1 to 7, characterized in that it comprises the phosphorylation step which consists in reacting the compound R. OH where R, has the same value than in formula I, with a phosphine of formula IV: (IV) ^R 9 where Rg and _Rg are dialkyl amino groups the same or different which alcoyies similar or different groups have from 1 to 7 carbon atoms and are preferably branched; where R. _Q is a protective group; to obtain an intermediate compound of formula V: o - o - P - OR 10 \ (V) R, and in that the compound V is coupled with a compound R ..- OH in the presence of tetrazole, optionally reducing the phosphite triester thus obtained. 1 1. Pharmaceutical composition characterized in that it contains at least one compound according to one of claims 1 to 7. 12. Pharmaceutical composition according to claim 1 1, characterized in that it comprises vectors commonly used in the field of administration of anti-cancer and immunodé¬ pressers. 13. Composition according to one of claims 1 1 and 12 in the form of an injectable solution. 14. Process for rendering sterols water-soluble, characterized in that a phosphate group of formula VI is fixed on a hydroxy bond: R ₃ -P— (VI) 0R ₂