HYDROSOLUBLE HYDROXYBISPHOSPHONIC DERIVATIVES OF DOXORUBICIN

Abstract

The invention relates to hydrosoluble hydroxybisphosphonic derivatives of doxorubicin of formula (I) and the pharmaceutically acceptable salts thereof. The invention also relates to the use of said compounds as a drug, especially in the treatment of a bone tumour. The invention further relates to the pharmaceutical compositions comprising such compounds, the methods for the synthesis thereof and synthesis intermediates.

##STR00001##

Claims

1. A compound of the following general formula (I): ##STR00023## or a pharmaceutically acceptable salt thereof, wherein X represents a bivalent group selected from: ##STR00024## with n and m each independently representing an integer between 1 and 6, ##STR00025## with p and q each independently representing an integer between 1 and 6, and ##STR00026## with t representing an integer between 1 and 6, these groups being bound to the imine function of the compound of formula (I) via their terminal nitrogen or oxygen atom and to the hydroxybisphosphonic acid function of the compound of formula (I) via their terminal carbon atom.

2. The compound according to claim 1, wherein n, m, p, q and t each independently represent an integer between 1 and 4.

3. The compound according to claim 1, which is selected from the following compounds: ##STR00027## and pharmaceutically acceptable salts thereof.

4. (canceled)

5. (canceled)

6. (canceled)

7. A pharmaceutical composition comprising at least one compound according to claim 1 and at least one pharmaceutically acceptable carrier.

8. (canceled)

9. (canceled)

10. (canceled)

11. A method for preparing a compound according to claim 1 comprising the following steps: (a) reaction of doxorubicin or a salt thereof with a compound of the following formula (A): ##STR00028## or a salt thereof, wherein X is as defined in claim 1, to give a compound of formula (I) according to claim 1, and (b) optionally, salification of the compound of formula (I) obtained in the preceding step (a) to give a pharmaceutically acceptable salt thereof.

12. A method for preparing a compound according to claim 1 for which X represents a group (1) comprising the following steps: (i) coupling reaction of a compound of the following formula (B): ##STR00029## or a salt thereof, with a compound of the following formula (C): ##STR00030## or a salt thereof, wherein n and m are as defined in claim 1, and doxorubicin or a salt thereof, the coupling reaction being carried out in one step in the presence of the compound of formula (B) or a salt thereof, the compound of formula (C) or a salt thereof, and doxorubicin or a salt thereof or in two steps by first coupling the compound of formula (B) or a salt thereof with doxorubicin or a salt thereof before carrying out the coupling with the compound of formula (C) or a salt thereof, or by first coupling the compound of formula (B) or a salt thereof with the compound of formula (C) or a salt thereof before carrying out the coupling with doxorubicin or a salt thereof, to give a compound of formula (I) according to claim 1 for which X represents a group (1), and (ii) optionally, salification of the compound of formula (I) obtained in the preceding step (i) to give a pharmaceutically acceptable salt thereof.

13. A compound of the following formula (A) or (B): ##STR00031## wherein X represents a bivalent group selected from: ##STR00032## with n and m each independently representing an integer between 1 and 6, ##STR00033## with p and q each independently representing an integer between 1 and 6, and ##STR00034## with t representing an integer between 1 and 6, these groups being bound to the imine function of the compound of formula (I) via their terminal nitrogen or oxygen atom and to the hydroxybisphosphonic acid function of the compound of formula (I) via their terminal carbon atom, or a salt thereof.

14. The compound according to claim 13, which is selected from the following compounds: ##STR00035## and salts thereof.

15. A method for treating a bone tumor comprising administering to a person in need thereof an effective amount of a compound according to claim 1.

16. The method according to claim 15, wherein the bone tumor is selected from primary bone tumors; bone metastases; and multiple myeloma.

17. The method according to claim 16, wherein the primary bone tumor is osteosarcoma, chondrosarcoma, giant cell tumor or Ewing's sarcoma.

18. A method for treating a bone tumor comprising administering to a person in need thereof an effective amount of a pharmaceutical composition according to claim 7.

19. The method according to claim 18, wherein the bone tumor is selected from primary bone tumors; bone metastases; and multiple myeloma.

20. The method according to claim 19, wherein the primary bone tumor is osteosarcoma, chondrosarcoma, giant cell tumor or Ewing's sarcoma.

Description

FIGURES

[0064] FIG. 1 represents the change over time of the mean tumor volume (mm.sup.3) of an HOS tumor developed in mice in the control (CT), compound (II), compound (III), doxorubicin, vector 5, and doxorubicin+vector 5 groups.

[0065] FIG. 2 represents the radiographies at Day 27 of the tumorous paws of the 10 mice of each control (CT), compound (II) and compound (III) group.

EXAMPLES

[0066] Abbreviations used:

[0067] DMSO: Dimethylsulfoxide

[0068] DPBS: Dulbecco's Phosphate-Buffered Saline

[0069] EDTA: Ethylenediaminetetraacetic acid

[0070] ES: Electrospray

[0071] HPLC: High-performance liquid chromatography

[0072] MS: Mass spectrometer

[0073] yld: Yield

[0074] NMR: Nuclear magnetic resonance

[0075] sat: Saturated

[0076] RT: Room temperature

[0077] TFA: Trifluoroacetic acid

[0078] THF: Tetrahydrofuran

[0079] TRIS: 2-Amino-2-(hydroxymethyl)propane-1,3-diol

[0080] 1. Synthesis of Compounds According to the Invention

[0081] Compound (II):

##STR00013##

[0082] Step 1:

##STR00014##

[0083] Compound 1 (3 g, 11.7 mmol) is solubilized in DMSO (15 ml) at 80° C. then is added to a solution of compound 2 (5 g, 47.2 mmol) in DMSO (30 ml) at 80° C. The transparent solution obtained is stirred 3 h at RT. The reaction mixture is diluted with a mixture of methanol (120 ml) and water (120 ml). To the transparent solution obtained, methanol (600 ml) is added with stirring followed by diethyl ether (150 ml) and stirring is continued for 5 min at RT. The precipitate obtained is filtered and washed with methanol, then solubilized completely in 150 ml of water at RT for 15 min. To the solution obtained, methanol (600 ml) is added, followed by diethyl ether (150 ml) and stirring is continued for 5 min at RT. The precipitate obtained is filtered, washed with methanol followed by diethyl ether, then dried under vacuum at RT. Compound 4 (3.2 g, yld=58%) is obtained. .sup.1H NMR spectrum (D.sub.2O, 400 MHz) δ, ppm: 3.87; (2H, s), 3.70; (2H, s), 3.24; (2H, s).

[0084] Step 2:

##STR00015##

[0085] The synthesis of compound 5 is described in the application WO 2012/130911. Under argon, a solution of compound 4 (1060 mg, 2.26 mmol) in 24 ml of a THF/H.sub.2O/TFA (20:20:1) mixture is added all at once to a solution of compounds 3 (918 mg, 1.58 mmol) and 5 (1028 mg, 2.26 mmol) in 24 ml of the THF/H.sub.2O/TFA (20:20:1) mixture. The dark red solution obtained is stirred at RT for 16 h protected from light. An Et.sub.2O/MeOH (5:1) mixture (360 ml) is then added to the solution while maintaining stirring and the red precipitate suspension obtained is stirred for 5 min at RT. The precipitate is filtered and washed with Et.sub.2O then dried under vacuum. The red powder obtained is solubilized in 10 ml of NaHCO.sub.3sat:H.sub.2O (1:1) mixture and the solution obtained is introduced into a C18 column then eluted with a gradient of [600 ml of 3% MeOH/H.sub.2O+2 ml of 20% NH.sub.3/H.sub.2O] to [600 ml of 50% MeOH/H.sub.2O+2 ml of 20% NH.sub.3/H.sub.2O]. The final product leaves starting from about 450 ml of eluent in about 150 ml of eluent. The fractions containing the final product are evaporated under vacuum to about 10 ml of solution remaining which is then diluted with MeOH (40 ml) followed by Et.sub.2O (200 ml). The mixture obtained is stirred until a red precipitate forms, which is stirred for 5 min at RT, then filtered, washed with Et.sub.2O, and dried under vacuum at RT. Compound (II) (526 mg, yld=23%) is obtained.

[0086] .sup.1H NMR spectrum (1% TFA-D/DMSO-D6, 400 MHz) δ, ppm: 14.07; (1H, s), 13.36; (1H, s), 11.97; (1H, s), 11.24; (1H, s), 10.53; (1H, s), 10.48; (1H, s), 10.30; (1H, s), 9.80; (1H, s), 8.05; (1H, s), 7.95; (2H, s), 7.84; (2H, m), 7.68; (1H, dd), 7.46; (1H, s), 7.40; (1H, d), 7.33; (1H, t), 7.02; (1H, d), 5.30; (1H, s), 4.99; (1H, s), 4.61; (1H, t), 4.5-3.7; (12H, m), 3.6-2.85; (14H, m), 3.01; (1H, d), 2.81; (3H, s), 2.27; (4H, m), 2.13; (2H, m), 1.85; (1H, t), 1.65; (1H, d), 1.18; (3H, d). .sup.31P NMR spectrum (1% TFA-D/DMSO-D6, 162 MHz) 8, ppm: 18.23. Mass spectrum (ES-) (m/z): [M-H].sup.−1386. Purity by HPLC analysis (C18, H.sub.2O/EDTA/NH.sub.3-MeCN): 97%.

[0087] Compound (III):

##STR00016##

[0088] Step 1:

##STR00017##

[0089] With stirring, compound 5 (1 g, 2.2 mmol) is added to a solution of compound 2 (1 g, 9.4 mmol) in water (40 ml) at 95° C. To the transparent solution obtained, TFA (0.8 ml) is added and the mixture is stirred 30 min at the same temperature. The mixture is then placed at RT and NaBH.sub.3CN (0.6 g, 9.5 mmol) is added, then stirring is continued for 24 h at RT. The reaction mixture is introduced on a C18 column which is eluted with a gradient of [600 ml of 3% MeOH/H.sub.2O] to [600 ml of 50% MeOH/H.sub.2O]. The excess compound 2 is eluted after about 150 ml of eluent and the final product 7 is eluted after about 400 ml of eluent in a volume of about 200 ml of eluent. The fractions are evaporated under vacuum to about 10 ml of solution remaining which is then diluted by stirring with MeOH (200 ml) followed by Et.sub.2O (300 ml). The precipitate obtained is filtered, washed with Et.sub.2O and dried under vacuum at RT. Compound 7 (780 mg, yld=71%) is obtained.

[0090] .sup.1H NMR spectrum (D20, 400 MHz) δ, ppm: 7.26; (1H, t), 6.97; (2H, m), 6.89; (1H, dd), 4.10; (2H, t), 3.89; (2H, s), 3.60-3.10; (4H, s), 2.82; (3H, s), 2.31; (2H, m), 2.19; (2H, m). .sup.31P NMR spectrum (D.sub.2O, 162 MHz) δ, ppm: 16.87. Mass spectrum (ES-) (m/z): [M-H].sup.−500.

[0091] Step 2:

##STR00018##

[0092] The mixture of compounds 7 (250 mg, 0.5 mmol) and 3 (290 mg, 0.5 mmol) is solubilized in 6 ml of a THF/H.sub.2O (1:1) mixture at 80° C., stirred at this temperature for 3 min, then evaporated to dryness under vacuum. The dried mixture obtained is left under vacuum for 5 min at 80° C. and the stoppered round-bottom flask is rapidly cooled to RT with cold water. The solid obtained is solubilized with stirring at 30-40° C. in a mixture of NaHCO.sub.3sat (4 ml), water (4 ml) and THF (8 ml). The very dark transparent solution obtained is diluted with water (50 ml) and introduced on a C18 column. The column is eluted with a gradient of 600 ml of 3% MeOH/H.sub.2O to 600 ml of MeOH. Compound (III) is eluted after about 400 ml of eluent in a volume of about 200 ml of eluent. The fractions are evaporated under vacuum to about 10 ml of solution remaining which is diluted by stirring with MeOH (100 ml) followed by Et.sub.2O (300 ml). The red precipitate formed is filtered, washed with Et.sub.2O and dried under vacuum at RT. Compound (III) (160 mg, yld=32%) is obtained.

[0093] .sup.1H NMR spectrum (D.sub.2O/THF-D8=3/1, 400 MHz) δ, ppm: 8.03; (2H, m), 7.81; (1H, d), 7.40; (1H, t), 7.21; (1H, s), 7.07; (1H, d), 5.40; (1H, s), 5.17; (1H, t), 4.42-3.99; (11H, m), 3.80-3.35; (6H, m), 3.14-3.95; (4H, m), 2.73-2.35; (7H, m), 2.22; (1H, t), 2.02; (1H, m), 1.47; (1H, m), 1.44; (3H, d). .sup.31P NMR spectrum (D.sub.2O/THF-D8=3/1, 162 MHz) δ, ppm: 14.62. Mass spectrum (ES-) (m/z): [M-H].sup.−1025. Purity by HPLC analysis (C18, H.sub.2O/EDTA/NH.sub.3-MeCN): 98%.

[0094] Compound (IV):

##STR00019##

[0095] Step 1:

##STR00020##

[0096] To a transparent solution of commercial product 8 (300 mg, 1.57 mmol) in THF (2 ml), triethylamine (218 μl, 1.57 mmol) is added and the homogeneous mixture is cooled to 0° C. At this temperature, the solution of product 10 (190 mg, 1.57 mmol) in THF (600 μl) is gradually added and the reaction mixture is stirred 15 min at 0° C.

[0097] The precipitate is rapidly filtered in order to collect the transparent solution under argon. To this solution, compound 11 (1.4 g, 4.71 mmol) is added and the transparent mixture obtained is stirred for 1 h at RT. Methanol (3 ml) is added and after 10 min of stirring the mixture is concentrated under vacuum to dryness at 36° C. To the residue obtained, TFA (2 ml) is added and the white precipitate formed is stirred at RT for 1 h, then the volatiles are evaporated to dryness at 36° C. The oil obtained is treated with methanol followed by ether and the white precipitate formed is filtered, washed with ether and dried. Compound 13 is obtained (205 mg, yld=55%).

[0098] .sup.1H NMR spectrum (D.sub.2O, 400 MHz) 8, ppm: 4.49; (2H, t). .sup.31P NMR spectrum (D.sub.2O, 162 MHz) δ, ppm: 14.18.

[0099] Step 2:

##STR00021##

[0100] Under argon, a solution of compounds 3 (270 mg, 0.46 mmol) and 13 (100 mg, 0.42 mmol) in 3 ml of a THF/H.sub.2O/TFA (20:20:1) mixture is stirred at RT for 16 h protected from light. 20 ml of an NaHCO.sub.3sat:H.sub.2O (1:1) mixture is added and the solution obtained is introduced into a C18 column then eluted with a gradient of [600 ml of 3% MeOH/H.sub.2O+2 ml of 20% NH.sub.3/H.sub.2O] to [600 ml of 50% MeOH/H.sub.2O+2 ml of 20% NH.sub.3/H.sub.2O]. The final product leaves starting from about 210 ml of eluent in about 150 ml of eluent. The fractions containing the final product are evaporated under vacuum to about 5 ml of solution remaining which is then diluted with methanol (40 ml) then with diethyl ether (200 ml). The mixture obtained is stirred until a red precipitate forms, which is stirred for 5 min at RT, then filtered, washed with diethyl ether, and dried under vacuum at RT. Compound (IV) (160 mg, yld=47%) is obtained.

[0101] .sup.1H NMR spectrum (D.sub.2O, 400 MHz) δ, ppm: 7.66; (1H, t), 7.38; (2H, m), 5.48; (1H, s), 4.84; (1H, s), 4.62; (2H, m), 4.40; (2H, s), 4.29; (1H, m), 3.90; (3H, s), 3.87; (1H, s), 3.73; (1H, t), 2.90; (1H, d), 2.57; (1H, d), 2.35; (1H, d), 2.15-1.92; (3H, m), 1.33; (3H, d).

[0102] .sup.31P NMR spectrum (D.sub.2O, 162 MHz) δ, ppm: 15.19; (1P, d), 15.08; (1P, d). Mass spectrum (ES+) (m/z): [M-H].sup.−807. Purity by HPLC analysis (C18, H.sub.2O/EDTA/NH.sub.3-MeCN): 99%.

[0103] 2. Solubility of Compounds (II), (III) and (IV) Compared with Products (a) and (b).

[0104] The application WO 2012/130911 describes the derivative (b) below derived from coupling between a modified doxorubicin moiety and an HBP-type vector. This compound (b) is insoluble in water and poorly soluble in water supplemented with TRIS, which makes such a compound difficult to use.

[0105] By using the same HBP vector as in the application WO 2012/130911, the product (a) derived from the coupling of doxorubicin with this vector 5 is also insoluble in water, in water with organic additives (Tween®, PEG, glycerin, glucose, TRIS) or with inorganic additives (sodium carbonate or bicarbonate), and in organic solvents (ethanol or dimethylsulfoxide or mixtures thereof with water), which makes this product difficult to use.

##STR00022##

[0106] The solubility of products (II), (III) and (IV) according to the invention is greatly improved in relation to the solubility of molecules (a) and (b) (see table below), which facilitates the use of such compounds as medicinal products.

TABLE-US-00001 (II) (III) (IV) (a) (b) DPBS or around around around insoluble insoluble water 255 mM 160 mM 315 mM 2 mg/ml not tested insoluble around solution of 1 mM TRIS in water

[0107] 3. Biological Tests

[0108] Materials and Methods:

[0109] The antitumor activity of compounds (II) and (III) according to the invention was evaluated in an orthotopic, xenogeneic murine model of osteolytic osteosarcoma induced by paratibial injection of the human tumor line HOS (human osteosarcoma, ATCC) in immunosuppressed mice. The activity of the molecules according to the invention was compared with that of the two main subunits comprising them (doxorubicin and vector 5) alone or in combination. The treatments, on 10 animals per group, began the day following tumor induction and were administered intraperitoneally 1 to 2 times per week for 5 weeks. Tumor volumes were measured twice per week using a slide caliper and radiographic evaluation was carried out once per week. Tumor volume, expressed in mm.sup.3, is calculated according to the following formula: V=(L×l.sup.2)/2 (L and l, expressed in mm, correspond to the large length and the small length of the tumor, respectively).

[0110] The following treatment groups were thus studied, each on 10 mice:

TABLE-US-00002 Treatment administered intraperitoneally. (Injection volume: Groups 10 mL/kg) 1 Control (solvent: DPBS) 2×/wk 2 Compound (II) 25 μmol/kg-2×/wk (2.5 mM in DPBS) 3 Compound (III) 25 μmol/kg-2×/wk (2.5 mM in DPBS) 4 Doxorubicin 3.7 μmol/kg-1×/wk (0.37 mM in 0.9% NaCl.sub.aq) 5 Vector 5 25 μmol/kg-2×/wk (2.5 mM in DPBS) 6 Doxorubicin 3.7 μmol/kg-1×/wk (0.37 mM in 0.9% NaCl.sub.aq) Vector 5 25 μmol/kg-2×/wk (2.5 mM in DPBS)

[0111] Results:

[0112] Toxicity.

[0113] Administered at a dose of 12 mg/kg (20 μmol/kg), doxorubicin is responsible for very early weight loss in the animals (weight loss >10% requiring euthanasia of the animals after 2 weeks of treatment) and for cardiotoxicity. Compounds (II), (III) and (IV) used at a molar equivalent dose do not induce these signs of toxicity. With the aim of treating the tumor more effectively, the bifunctional molecules can even be used at higher doses than the anticancer agents alone (knowing that the typical doxorubicin treatment dose is 2 mg/kg (3.7 μmol/kg)). A study for determining the maximum usable dose thus showed similar weight loss in the mice when compounds (II), (III) and (IV) were used intravenously or intraperitoneally at a dose 13 times higher than doxorubicin alone.

[0114] Antitumor Properties.

[0115] (see FIGS. 1 and 2) Compounds (II) and (III) significantly reduce the size of extraosseous tumors measured in relation to the control group and to the groups treated with vector 5 and/or doxorubicin.

[0116] Comparison of the tumor volumes of the various groups is possible up to Day 27, however. After this date, the animals of the control, doxorubicin and vector 5 groups had to be euthanized because of excessively large tumor volumes. Moreover, at Day 32, all the animals of the doxorubicin +vector 5 group have a tumor greater than 1500 mm.sup.3 in volume whereas only one mouse from the compound (II) group exceeds this threshold.

[0117] Prevention of Bone Lesions Associated with Tumor Development.

[0118] Compounds (II) and (III) reduce metaphyseal fractures, alteration of cortical integrity, and new formation of ectopic bone compared with the control, doxorubicin, vector 5, and doxorubicin +vector 5 groups. At Day 27, whereas 90% of the control group animals have fractures of the tibial metaphysis, the seat of tumor development, these fractures are not detected in any of the mice treated with compounds (II) and (III). The lesions observed in the doxorubicin group are equivalent to those of the control group (significant osteolysis on the cortex surface and metaphyseal fractures). Vector 5 (vector 5, and doxorubicin+vector 5 groups) partly reduces the metaphyseal fractures, but unfortunately large osteolytic lesions were observed on the cortical surfaces. On the other hand, the compounds according to the invention significantly prevent the osteolysis associated with tumor development in this osteosarcoma model.