FR2893618A1 - New quaternary onium metal complexes useful for treating cancer - Google Patents

Abstract

Quaternary onium metal complexes (I) are new. Quaternary onium metal complexes of formula [(R)xM1>Xm]2+>[M2>Zn.2M3>Yp.M3>Wq]2-> (I) are new. R = quaternary phosphonium, ammonium, isothiuronium or sulfonium; M1> = a metal with an oxidation number of 4; M2> = a metal with an oxidation number of 2 or 3; M3> = a metal with an oxidation number of 3; X, Y, Z, W = halide ions; m = 0 or 2 or fractions of which the sum is 2; x = 2 or 4; n, p, q = 3 or 4 or fractions of which the sum is 3 or 4. ACTIVITY : Cytostatic. Bis(triphenylphosphine)germane tris(tetrachlorocerate) tetrachlorovanadate had IC50 of 0.0041 mu M against U25l brain tumor cells, 0.0078 mu M against HeLa uterine cancer cells and 0.0085 mu M against Hepg 2 liver cancer cells. MECHANISM OF ACTION : None given.

Description

The present invention relates to new metal complexes

quaternary onium which have interesting pharmacological properties, in particular for the treatment of cancer. The present invention also relates to a process for obtaining metal complexes of quaternary onium as well as to the pharmaceutical compositions containing them. The vast majority of cancer patients undergo chemotherapy. The judicious choice of the appropriate anti-tumor agent or mixture of appropriate anti-tumor agents is an ongoing challenge in the face of the evolution of the tumor response to treatment. In recent years, mitochondrial medicine has developed from the discovery that cancerous diseases are associated with dysfunction of mitochondria. The first generation of compounds capable of targeting mitochondria was developed by Madar et al. [(1999): Enhanced uptake of (11C TPMP) in canine brain tumor: PET studies. J. Nuc. Med. 40, 1180-5]. These are phosphonium derivatives labeled with fluorine F-18. Preclinical studies on tumor cells and models have shown that labeled phosphonium derivatives can detect solid tumors and differentiate their malignancy from inflammation. These derivatives accumulate intensely in the neoplasm but very little in inflammation [Madar I et al (2001): Enhanced accumulation of [3H] triphenyl methyl phosphonium (TPMP) in breast tumors in vivo: comparison with MIBI. JNM 42, 278P; Madar I et al (2002 Feb.): Preferential accumulation of (3H) tetraphenyl phosphonium in non small cell lung carcinoma in mice: comparison 25 with 99mTc-MIBI. J. Nucl. Med. 43 (2), 234-8]. It has also been demonstrated by Guo et al. [(2001): Allelic losses in Ora Testdirected biopsies of patients with prior upper aerodigestive tract malignancy. Clin. Cancer Res. 7, 1963-8] that mitochondrial probes detect genetically modified cells long before traditional histology can identify a cellular abnormality. Using positron emission tomography (PET), the phosphonium derivatives mentioned above made it possible to measure the tumor response to chemotherapy. These phosphonium derivatives are excellent tools for developing apoptotic anti-tumor agents because they allow their mechanism of action to be tested and efficacy to be evaluated directly in patients. On the other hand, lipophilic cationic tetraphenylphosphonium salts are novel anticancer agents potentially useful in the treatment of neoplasia. Tetraphenylphosphonium chloride (TPP) is the basic compound of the series of tetraphenylphosphonium salts mentioned above, which especially includes (triphenylphosphonium methyl) benzaldehyde chloride (drug A) and [4-hydrazinocarboxy-1- chloride. butyl] tris- (4-dimethylaminophenyl) phosphonium (drug B). These compounds selectively inhibit in vitro the growth of cell lines of different carcinomas (breast, colon, pancreas, kidney and hypopharynx) compared to that of unaltered cell lines (monkey or rat kidney epithelial cells) [Rideout, D et al (1989): Phosphonium salts exhibiting selective anticarcinoma activity in vitro. Anti-Cancer Drug Design 4, 265-280; Rideout, D et al (1994): Mechanism of inhibition of FaDu hypopharyngeal carcinoma cell growth by tetraphenylphosphonium chloride. Int. J. Cancer 57, 247-253]. It appears that these drugs accumulate intracellularly depending on the mitochondrial potential of the cell membrane. The highly negative plasma membrane potentials characteristic of neoplastic cells are believed to be responsible for the accumulation and toxicity of phosphonium salts in malignant cells [Lampidis TJ et al (1985): Selective killing of carcinoma cells in vitro by lipophilic- cationic compounds: a cellular basis. Biomed. Pharmacother. 39, 220-226].

Mitochondrial toxicity is also believed to be responsible for the cytotoxicity of these compounds. Inhibition of mitochondrial respiration has been demonstrated with TPP [Rideout, D et al (1989): Phosphonium salts exhibiting selective anticarcinoma activity in vitro. Anti-Cancer Drug Design 4, 265-280].

The other phosphonium derivatives apparently have a similar mechanism of action because the sensitivity profiles determined with tumor cells are very similar.

In order for chemotherapy treatment to be effective, drugs must be available which bind more selectively to neoplastic cells than to normal cells or healthy cells and which exhibit high cytotoxicity for malignant cells at doses which do not induce side effects that are too important for the body. It is known that metal ions, such as gallium, palladium, ruthenium, rhodium, cerium, lanthanium and vanadium ions, are effective anti-tumor agents. To this end, mention may be made, for example, of patent EP 0 059 148 which describes the use of gallium chloride as an anticancer agent; EP 0 599 881, which describes gallium complexes useful as anticancer agents. The following articles can also be cited: - Bode AM, Dong Z. 2002. The paradox of arsenic: molecular mechanisms of cell transformation and chemotherapeutic effects. Crit. Rev. Oncol. Hematol. 42 (1): 5-15 24. - Collery P, Keppler B, Madsoulet C, Desoize B. 2002. Gallium in cancer treatment. Crit. Rev. Oncol. Hematol. 42 (3): 283-296. - Desoize B, Madoulet C. 2002. Particular aspects of platinum compounds used at present in cancer treatment. Crit. Rev. Oncol. Hematol. 42 (3): 317-325. 20 - Evangelou AM. 2002. Vanadium in cancer treatment. Crit. Rev. Oncol. Hematol. 42 (3): 249-265. - Katsaros N, Anagnostopoulou A. 2002. Rhodium ans its compounds as potential agents in cancer treatment. Crit. Rev. Oncol. Hematol. 42 (3): 297-308. - Melendez E. 2002. Titanium complexes in cancer treatment. Crit. Rev. Oncol. Hematol. 42 (3): 309-315. - Richardson DR. 2002. Iron chelators as therapeutic agents for the treatment of cancer. Crit. Rev. Oncol. Hematol. 42 (3): 267-281. - Tiekink ERT. 2002. Gold derivatives for the treatment of cancer. Crit. Rev. Oncol. Hematol. 42 (3): 225-248. Metal complexes have now been found which can combine several metal ions into a single molecule. These complexes are quaternary onium metal complexes which have very interesting pharmacological properties. In particular, they are very selective and exhibit an anti-tumor activity which is similar, or even superior, to the corresponding metal ions. The quaternary onium metal complexes according to the invention correspond to the general formula: [(R) L M 'Xm] 2+ [M "Z". 2M ,,, YP M ,,, Wq] z (I) in which: - R is a quaternary onium chosen from phosphoniums, quaternary ammoniums, isothiouronium or sulfonium; - M 'represents at least one metal having an oxidation degree of 4; - M "represents at least one metal having an oxidation degree of 2 or 3; - M" 'represents at least one metal having an oxidation degree of 3; - X, Y, Z and W, which are identical or different, are halide ions, preferably chloride ions; - m is equal to 0, or else is an integer equal to 2 or more fractions whose sum is equal to 2; - Legal equal to 2 or 4; - n, p, q are integers equal to 3 or 4 or fractions whose sum is 3 or 4. Preferably, M 'is chosen from the following metals: silicon, titanium, germanium, zirconium, vanadium, tin, hafnium. M "when it has an oxidation degree of 2, can be a metal from groups IIA, VIII, IB and IIB of the Mendeleeff periodic table; when it has an oxidation degree of 3, it can be chosen from the metals of groups IIIB, VIII and IIIA of the Mendeleeff periodic table. Preferably, M "is chosen from Fe, Co, Sn, Pd and Pt. M" 'has an oxidation degree of 3 and can be chosen from the metals of groups IIIB, VIII and IIIA of the Mendeleeff periodic table. Examples of metals suitable for M "'are Ce, V, Cr, Fe, Co, Gr, Ce and V. Preferably, R is chosen from the: - quaternary phosphonium of formula ùP + (R1) 3 in which R1 is a phenyl or a dimethylamino group; - quaternary ammonium of formula (R2) 4N + _ in which R2 is a C1-C6 alkyl group, in particular methyl, butyl, pentyl or hexyl; - sulfonium of formula (R3) 3S + _ in which R3 is a C1-C4 alkyl, or an alkylsulfonium of formula: [CH5_CH3] R, in which R is a hydrocarbon residue of a C6-C24 fatty acid; - isothiouronium of formula S + ûC (NR4) 2 in which R4 is hydrogen or a C1-C4 alkyl or of formula:

+ ~~ N H RùSùC. NH2, wherein R is a hydrocarbon residue of a C 6 -C 24 fatty acid, preferably R is octyl, lauryl or dodecyl.

Among the compounds of formula (I) above, particularly preferred are those in which R is a triphenyl phosphonium of formula deP + (C6H5) 3 •

Among these compounds, those in which M ′ is germanium, M ″ is

vanadium and M "'is cerium are particularly preferred.

These complexes, which are capable of complexing metal ions, are preferably cationic surfactants which possess an HLB (equilibrium

15 hydrophilic / lipophilic) of at least 7, determined by the log of the octanol-water partition coefficient, i.e. the distribution coefficient of the compound tested between the oily phase and the aqueous phase.

The quaternary onium metal complexes according to the invention are pharmaceutically acceptable compounds, the toxicity of which can be controlled by adjustment of the dose and the schedule of administration.

The complexes according to the invention can be obtained by the process which consists of:

1) reacting an onium precursor with a compound of formula M'Xm in a water / ethanol mixture, at room temperature.

2) reacting the resulting halide under reflux with a compound of the formula M "Zn and a compound of the formula M" 'YP and optionally with a compound of the formula M "' Wq in an alcohol / water mixture.

According to an implementation variant of the process, an evaporation of the water / ethanol mixture is carried out at the end of step 1).

The onium precursor to be used in the process of the invention is chosen from phosphines of formula HP (RI) 3, amines of formula (R2) 3NH; sulfur derivatives capable of giving the above sulfonium ions as well as isothioureas of formula HS formuleC (NR4) 2 'In general, the complex obtained is crystallized in the same alcohol / water mixture, which is preferably a methanol / water mixture . In the complexes according to the invention, the metals M ′, M ″ and M ″ ″ are chosen from metals known for their pharmaceutical properties, in particular their anti-tumor properties. Preferably, the metals M ′, M ″ and M ″ ″ are chosen to from the following metals: germanium, gallium, palladium, ruthenium, rhodium, cerium, lanthanium or vanadium. The compounds of the invention are "metallosurfactants" which are capable of forming micelles in water when the alkyl groups are hydrocarbon residues of fatty acids, in particular dodecyl groups. The invention also relates to pharmaceutical compositions which contain as active principle a complex according to the invention in combination with a pharmaceutically acceptable vehicle suitable for oral or parenteral administration. The compounds of the invention can be administered in varying doses depending on the condition of the patient and the tumor to be treated; these doses can be, for example, from 10 to 60 mg per kg and per day. The present invention will now be described in more detail by the following illustrative examples.

EXAMPLE 1: Synthesis of bis (triphenylphosphine) germane t is (tetrachlorocerate) (tetrachlorovanadate). 0.125 g (0.6 mmol) of germanium chloride (GeCl4) was added slowly and with stirring to 0.65 g (0.23 mmol) of triphenylphosphine in 20 ml of acetone. After 30 minutes of stirring, a mixture of 0.6 mmol of 30 VCl3 and 0.18 mmol of CeCl3.7H2O, dissolved in 2 ml of a methanol / water mixture (1: 1 v / v) was added. . The resulting mixture was refluxed for one hour, then concentrated and finally cooled slowly to give crystals which were filtered and washed with methanol / ether (1:10 v / v). The crystals obtained were dried and 1.2 g of bis (triphenylphosphonium) germane tris (tetrachlorocerate) (tetrachiorovanadate) of formula: ++ V CI4 CeCl4-CeCl3 CecI3 Ge were obtained. . The physicochemical characteristics of the product thus obtained are as follows: 1,637.77 - molecular weight - IR spectrum 687.66 cm-1 (GeûP) - mass spectroscopy 51 (42), 77 (39), 183 (26%) , 277 (100%) - H-1 NMR 7.22-7.69 (m, 30 H, Ar-H) - ultraviolet adsorbance in 255 nM a solution of DMF - elemental analysis:. Calculated C 26.4; H 1.97; CI 34.64; P 3.78; Ce 25.67; V 3.11. found C 26.1; H 2.23; Cl 34.44; P 3.20; Ce 25.89; V 3.07 EXAMPLE 2: Synthesis of bis (triphenylphosphine) dichlorogermane bis (trichlorocerate) (tetrachlorovanadate). 0.6 mmol of germanium chloride was added slowly and with stirring to 1.2 mmol of triphenylphosphine in 20 ml of acetone. After evaporation of the solvent, the bis (triphenylphosphonium) dichlorogermane thus obtained was dissolved in a methanol / water mixture (1: 1 v / v).

To this dissolved compound was added a mixture of VCl3 (0.0006 mol) and CeCl3 (0.0018 mol) dissolved in a methanol / water mixture. The mixture was heated at reflux for one hour, concentrated and cooled slowly to give a precipitate which was filtered off and washed with ethanol / ether (1:10 v / v). The product was dried and there was obtained 0.91 g of bis (triphenylphosphonium) dichlorogerman, tetrachlorocerate, tetrachlorovanadate, bis (trichlorocerate) corresponding to the formula: ++ V Cl 4_ CeC14 CeCI3 This3 PHARMACOLOGICAL TESTS * Inhibitory effects The cell line cancerous Ehrlich ascites (CAE cells) used in the present study, was provided by the National Cancer Institute in Cairo (Egypt) and maintained in female mice of the Swiss albino type, by intraperitoneal transplantation of 2 weekly , 5 x 106 tumor cells per mouse. CAE cells were obtained by needle aspiration under aseptic conditions. The cells were tested for their viability and the dead cells were demonstrated by staining according to the technique of Lazurus et al. Cancer (1996) Chemother.Res. 50: 543. Ascites fluid was diluted in sterile physiological saline such that 0.1 ml of fluid contained 2.5 x 106 cells counted microscopically using a haemocytometer. Compound AB9 was tested in vitro by mixing in a series of 25 test tubes of 0.1 ml of tumor cells, 0.8 ml of RPMI 1640 medium (medium containing 1% I-glutamine, 10% serum of fetal calf and 1% streptomycin) and 0.1 ml of test compound at different concentrations (corresponding to 10, 20, 25, 50 and 95 g / ml). The test tubes were incubated at 37 ° C for two hours and then centrifuged. The supernatant was separated from the cells by aspiration and the cells were stained with trypan blue. The percentage of non-viable cells (CNV) was calculated by the following equation: number of non-viable cells% of CNV = / 100 total cells _ 10 An increase in the percentage of CNV depending on the dose of test compound was obtained. . There was thus obtained 10, 40, 80, 100% of CNV at respective doses of 10, 20, 25, 50, 95 gg / ml. Compound AB9 was also tested on other cell lines and determined for 50% inhibitory concentrations (50% IC). The cell lines used are lines 0251; Hela and Hepg 2. Monolayers of cells were incubated with compound AB9 for 48 hours at 37 ° C. under an atmosphere charged with 5% CO2 according to the protocol described by Skehan P., Storeng R. et al. (1990), New colorimetric cytotoxicity assay for anticancer drug testing. J. Nat1. Cancer Institute 82: 1107-1112. The following results were obtained: • U251 cell line (brain tumor cell line): IC50 = 0.0041 mol / I • Hela cell line (uterine cancer cell line): IC50 = 0, 0078 'mol / I 25 • Hepg 2 cell line (liver cancer cell line): IC50 = 0.0085! Mol / I * In vivo tests on solid tumors or Ehrlich ascites. Female Swiss albino mice (20-25 g) were used in the following tests. They were placed in special cages with free access to 30 food rations and water. • Determination of the 50% lethal dose (LD 50). To determine the non-toxic dose, the doses which cause acute toxicity were identified by proceeding as follows: - female mice of the Swiss albino type were treated with different concentrations of compound AB9 ranging from 15 to 120 mg / kg of body weight dissolved in 0.1 ml of CMC and sterile physiological serum in the appropriate amount. These concentrations were injected intraperitoneally into 6 mice for each concentration.

The doses responsible for acute toxicity were determined by analysis of probabilities and the non-toxic dose of 25 mg / kg body weight was chosen for the in vivo study below. The probability analysis according to the method of Hardison et al. "Nine functions for probability distribution" SUGI supplemental library users' guide (1983); Sas institute Inc .; Cary, NC., Was used to calculate the 50% lethal dose (LD 50) in mice and the recorded mortality was detected 40 days after treatment. • Study of survival. A group of 20 mice which were not inoculated with the tumor cells received the same concentration of CMC and physiological saline. They served as 20 control mice and enabled the survival rate to be calculated. Eighty mice were inoculated with 2.5.106 tumor cells in 0.1 ml sterile physiological saline intramuscularly in the right front paw or intraperitoneally and left for 10 days until the diameter of the tumors was reached. reaches about 10 mm3. These mice were then divided into four groups: Group I: group of mice bearing solid tumors which were injected with CMC and physiological saline; Group II: group of mice bearing solid tumors which have been injected with compound AB9 in solution in CMC and physiological saline; Group III: group of mice bearing ascites tumors injected with CMC and physiological saline; Group IV: group of mice bearing ascites tumors which have been injected with compound AB9 dissolved in CMC and physiological saline.

We compared the survival of the different groups of mice and we observed: - an increase in the percentage of surviving mice in the mice treated with compound AB9 compared to the control mice: 30% of the mice died on the 21st day in the control group whereas no mice died in the group of mice bearing solid tumors and treated with compound AB9. The difference is highly significant (p <0.0007) according to Student's t test. It was observed that 100% of the mice were alive on day 24 in mice treated with compound AB9 while only 70% were alive in control mice (90% on day 15; 80% on day 18 and 70%. the 21st day). The results of the various tests above are collated in the appended FIG. 1 which gives the percentage of survival (ordinates) as a function of the days of treatment). It was found that there was no difference between the treated mice and the control mice for the ascites form of the tumor. As a result, the compound according to the invention is not suitable for tumors having rapid hematological growth. On the other hand, the high efficiency observed with solid tumors is very promising for the compounds according to the invention. 20 • Study of tumor volume. In this study, we also used mice of the Swiss albino type weighing 25-30 g, kept at room temperature (24 2 C) with alternating night / day cycles of 12 hours and fed with a standard laboratory diet and water ad libitum.

Solid tumors were produced by intramuscular inoculation with 0.2 ml of CAE cells, containing 2.5 x 106 viable CAE cells in the right thigh of the lower limb of each female Swiss albino mouse. Mice which had a palpable tumor 9 days after inoculation were used for this study.

The change in tumor volume (VT) was calculated on days 9, 13, 14 and 20 after inoculation by the following formula: Tumor volume (mm3) = 0.52 AB2 in which: - A is the minor axis of the tumor; - B is the major axis of the tumor. Mice with a palpable tumor were treated by local injection (im) into the intramuscular tumor at a dose of 25 mg / kg body weight, 0.9% saline solution of compound AB9 or compound AB'9. every other day, for a total of 7 injections over 13 days. At the start of the experiment, the mice were divided into four groups of 10 mice each: Group I: group of mice not treated (controls) with CAE cells; Group II: group of mice inoculated with CAE cells receiving no other treatment; Group III: group of mice inoculated with CAE cells and treated with compound AB9 as indicated above; Group IV: group of mice inoculated with CAE cells and treated with compound AB'9 as indicated above. The results are shown in FIG. 2 which is a graph giving the tumor volume (mm3) as a function of the duration of the treatment. The values reported on this graph are the mean standard deviation values of 10 mice / group. Differences from controls were considered significant at p <0.05 (*) or at p <0.001 (***).

Claims (7)

1. Metal complexes of quaternary onium corresponding to the formula: [(R) LM 'Xm] 2+ [M "Zn. 2M ,,, Yp. M ,,, Wq] z (I) in which: - R is a quaternary onium chosen from phosphoniums, quaternary ammoniums, isothiouronium or sulfonium; - M 'represents at least one metal having an oxidation degree of 4; - M "represents at least one metal having an oxidation degree of 2 or 3; - M "'represents at least one metal having an oxidation degree of 3; - X, Y, Z and W which are identical or different, are halide ions, preferably chloride ions; - m is equal to 0, or else is an integer equal to 2 or more fractions whose sum is equal to 2; -Lis equal to 2 or 4; - n, p, q are integers equal to 3 or 4 or fractions whose sum is 3 or 4. 2. Complexes according to claim 1, characterized in that M 'is Ge. 3. Complexes according to claim 1, characterized in that M "is Va. 4. Complexes according to claim 1, characterized in that M "'is Ce. 5. Complexes according to any one of claims 1 to 4, characterized in that it is bis (triphenylphosphine) germane tris (tetrachlorocerate) (tetrachlorovanadate). 6. Complexes according to any one of claims 1 to 4, characterized in that it is bis (t iphenylphosphine) dichlorogermane bis (trichlorocerate) tetrachloro (vanadate) tetrachlorocerate. 7. Pharmaceutical composition containing as active principle a complex according to any one of claims 1 to 6 in combination with a pharmaceutically acceptable vehicle.