4-OXO-N-(4-HYDROXYPHENYL)RETINAMIDE DERIVATIVES AS THERAPEUTIC AGENTS FOR THE TREATMENT OF CANCER

Abstract

The present invention relates to a compound having formula (I) below or a pharmaceutically acceptable salt thereof:

##STR00001##

wherein:

X is —COOH or NH.sub.2;

R is a straight or branched C.sub.1-C.sub.10 alkylene chain; and

R.sub.1 is H, straight or branched C.sub.1-C.sub.10 alkyl, aryl, or R.sub.2CO— wherein R.sub.2 is straight or branched C.sub.1-C.sub.10 alkyl, for use as antitumoral agents.

Claims

1. A compound of the following formula (I) or a pharmaceutically acceptable salt thereof: ##STR00007## wherein: X is —COOH or NH.sub.2; R is a straight or branched C.sub.1-C.sub.10 alkylene chain; and R.sub.1 is H, straight or branched C.sub.1-C.sub.10 alkyl, aryl, or R.sub.2CO— wherein R.sub.2 is straight or branched C.sub.1-C.sub.10 alkyl.

2. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 wherein X is —COOH.

3. The salt of a compound of formula (I) according to claim 2 with an alkali or alkaline-earth metal, an organic amine or an amino acid.

4. The salt of a compound of formula (I) according to claim 3 with sodium, potassium, lithium, calcium or magnesium.

5. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 wherein X is NH.sub.2.

6. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 wherein R is a straight or branched C.sub.1-C.sub.6 alkylene chain.

7. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 wherein R.sub.1 is H.

8. A compound of formula (I) according to claim 1 selected from the group consisting of: sodium 2-[3-[(1E,3E,5E,7E)-9-(4-hydroxyanilino) -3,7-dimethyl-9-oxo-nona-1,3,5,7-tetraenyl]-2,4,4-trimethyl-cyclohex-2-en-1-ylidene]amino]oxyacetate (compound 1a); sodium 2-[3-[(1E,3E,5E,7E)-9-(4-hydroxyanilino)-3,7-dimethyl-9-oxo-nona-1,3,5,7-tetraenyl]-2,4,4-trimethyl-cyclohex-2-en-1-ylidene]amino]oxybutyrate (compound 1b); sodium 2-[3-[(1E,3E,5E,7E)-9-(4-hydroxyanilino)-3,7-dimethyl-9-oxo-nona-1,3,5,7-tetraenyl]-2,4,4-trimethyl-cyclohex-2-en-1-ylidene]amino]oxyhexanoate (compound 1c).

9. A medicament comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1.

10. An antitumoral or chemopreventive agent comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1.

11. A method for treatment of solid or haematological, both metastatic and non-metastatic, tumors, comprising providing and applying an effective amount of the antitumoral or chemopreventive agent according to claim 10.

12. The method according to claim 11, wherein the treatment is that of breast cancer, ovarian cancer, prostate cancer, colorectal cancer, mesothelioma and other sarcomas, neuroblastoma, lymphoma, leukaemia and melanoma.

13. A combination of a compound of formula (I) according to claim 1 with one or more medicaments selected from the group consisting of antimitotic agents, compounds used in standard chemotherapy, natural or synthetic retinoids, epigenetic drugs, or (tumoral or non-tumoral) target-specific medicaments.

14. The combination according to claim 13 wherein the compound of formula (I) and the further medicaments are administered at the same time or sequentially, in any order.

15. A pharmaceutical composition comprising at least one compound of formula (I) according to claim 1 as active ingredient and a pharmaceutically acceptable carrier and/or eluent.

16. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 2 wherein R is a straight or branched C.sub.1-C.sub.6 alkylene chain.

17. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 3 wherein R is a straight or branched C.sub.1-C.sub.6 alkylene chain.

18. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 4 wherein R is a straight or branched C.sub.1-C.sub.6 alkylene chain.

19. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 5 wherein R is a straight or branched C.sub.1-C.sub.6 alkylene chain.

20. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 2 wherein R.sub.1 is H.

Description

EXAMPLES

Example 1

Preparation of Compound 1a

[0047] 17 mL of a solution of NH.sub.2OCH.sub.2COOH.1/2HCl (3a, 410 mg, 3.75 mmols) and CH.sub.3COONa (166 mg, 2.39 mmols) in 50% aqueous EtOH was added to a suspension of compound 2 (4-oxo-4-HPR, 700 mg, 1.73 mmols) in EtOH (11 mL). The reaction was kept under stirring at room temperature for 24 hours. The solvent was then removed at low pressure and the residue was diluted with cold H.sub.2O (20 mL), milled, and finally filtered under vacuum. 739 mg (1.54 mmols) of compound 4a (4-(carboxymethoxyimino)fenretinide) was obtained. Yield: 89%

[0048] Melting point: 121.8° C.

[0049] .sup.1H-NMR (CDCl.sub.3) δ: 7.43 (2H, d, J=8.2 Hz); 7.08 (1H, s); 6.99 (1H, dd, J=11.60; 14.6 Hz); 6.82 (2H, d, J=8.2 Hz); 6.44-6.16 (4H, m); 5.82 (1H, s); 5.32 (2H, s); 2.72 (2H, t, J=6.4 Hz); 2.44 (3H, s); 2.04 (3H, s); 1.91 (3H, s); 1.65 (2H, t, J=6.4); 1,12 (6H. s).

[0050] .sup.1H-NMR (DMSO-d.sub.6) δ: 9.78 (1H, s); 9.16 (1H, bs); 7.42 (2H, J=8.24 Hz); 7.05-6.89 (1H, m); 6.68 (2H, d, J=8.24); 6.47-6.24 (4H, m); 6.00 (1H, s); 4.57 (2H, s); 2.67-2.55 (2H, m); 2.33 (3H, s); 2.00 (3H, s); 1.82 (3H, s); 1.82 (3H, s); 1.63-1.49 (2H, m); 1.07 (6H, s).

[0051] 15.4 mL of an 0.1N solution of NaHCO.sub.3 was added to a suspension of 4a (739 mg, 1.54 mmols) in 10 mL of H.sub.2O The reaction was left under stirring for about 22 hours. The solvent was then removed at low pressure. 760 mg (1.52 mmols) of compound 1a was obtained. Yield: 99%

[0052] Melting point: 188.6° C. (with dec.)

[0053] .sup.1H-NMR (DMSO-d.sub.6) δ: 9.8 (1H, s); 9.38 (1H, bs); 7.42 (2H, d, J=8.2 Hz); 6.97 (1H, dd, J=14.3; 11.60 Hz); 6.68 (2H, d, J=8.2 Hz); 6.44-6.24 (4H, m); 6.01 (1H, s); 4.12 (2H, s); 2.61-2.53 (2H, m); 2.32 (3H, s); 2.00 (3H, s); 1.82 (3H, s); 1.58-1.47 (2H, m); 1.05 (6H, s).

Example 2

Preparation of Compound 1b

[0054] 0.5 mL of a solution of 3b (17 mg, 0.11 mmols) and CH.sub.3COONa (5 mg, 0.07 mmols) in 50% aqueous EtOH was added to a suspension of compound 2 (20 mg, 0.05 mmols) in EtOH (0.5 mL). The reaction was kept under stirring at room temperature for 24 hours. The solvent was removed at low pressure, the residue was taken up with ethyl acetate, and the solution was washed with H.sub.2O and dried on Na.sub.2SO.sub.4. The crude product was then purified by preparative chromatography in CH.sub.2Cl.sub.2: CH.sub.3OH 95: 5. 20 mg (0.04 mmols) of compound 4b was obtained.

[0055] (4-(carboxypropoxyimino)fenretinide) (yellow glass). Yield: 80%

[0056] .sup.1H-NMR (CDCl.sub.3) δ: 7.39 (2H, m); 7.12 (1H, s); 6.96 (1H, dd, J=10.6, 14.4); 6.78 (2H, m); 6.37-6.14 (4H, m); 5.80 (1H, s); 4.18 (2H, t, J=6.0) 2.60 (2H, t, J=6.6); 2.50 (2H, t, J=7.3); 2.41 (3H, s); 2.07-1.97 (2H, m); 2.02 (3H, s); 1.90 (3H, s); 1.89-1.76 (2H, m); 1.07 (6H, s).

[0057] 280 μL of an 0.1N solution of NaHCO.sub.3 was added to a suspension of 4b (18 mg, 0.035 mmols) in 0.5 mL of H.sub.2O. The reaction was left under stirring for about 20 hours. The solvent was then removed at low pressure. 15 mg (0.028 mmols) of compound 1b (4-(carboxypropoxyimino) fenretinide sodium salt) was obtained. Yield: 81%.

[0058] .sup.1H-NMR (DMSO-d.sub.6) δ: 9.80 (1H, s); 7.40 (2H, m); 6.95 (1H, dd, J=11.4, 14.4); 6-66 (2H, m); 6.42-6.24 (4H, m); 5.99 (1H, s); 3.97 (2H, t, J=6.5); 2.31 (3H, s); 1.98 (3H, s); 1.92-1.79 (2H, m); 1.83 (3H, s); 1.79-1.63 (2H, m); 1.51 (2H, t, J=7.2); 1.04 (6H, s).

[0059] Preparation of Compound 3b (4-aminooxybutyric acid hydrochloride): ethyl 4-(1,3-dioxo-1,3-dihydroisoindol-2-yloxy)butyrate.

[0060] N-hydroxyphthalimide (1.11 g, 6.64 mmols) and K.sub.2CO.sub.3 (1.84 g, 13.28 mmols) were added to a solution of ethyl 4-bromobutyrate (1 mL, 6.64 mmols) in anhydrous DMF (6.5 mL). The solution was left under stirring at room temperature overnight. When the solvent had been evaporated, the reaction mixture was taken up with ethyl acetate and washed with a saturated solution of NaCl. The organic phase was dried on Na.sub.2SO.sub.4 and the solvent was evaporated. The product was crystallised from isopropanol (1.67 g). Yield: 91%.

[0061] Melting point: 48.5° C.

[0062] .sup.1H-NMR (DMSO-d.sub.6) δ: 7.86 (4H, m); 4.16 (2H, t, J=6.4); 4.07 (2H, t, J=7.0); 2.53 (2H, t, J=7.3); 1.97-1.84 (2H, m); 1-19 (3H, t, J=7.0).

[0063] 4-(1,3-dioxo-1,3-dihydroisoindol-2-yloxy)butyric acid.

[0064] A solution of LiOH.H.sub.2O (0.8 g, 19.1 mmols) in ethanol (5.8 mL) and H.sub.2O (5.8 mL) was added to a solution of ethyl 4-(1,3-dioxo-1,3-dihydroisoindol-2-yloxy)-butyrate (1.65 g, 5.97 mmols) in THF (18 mL), and the solution was left to react at room temperature overnight. When the solvents had been evaporated, a 6N solution of HCl (6 mL) was added and the crude product was extracted with ethyl acetate. The combined organic phases were washed with a saturated solution of NaCl and dried with Na.sub.2SO.sub.4. The product was then crystallised from ethyl acetate. 1.1 g of product was obtained. Yield: 74%.

[0065] .sup.1H-NMR (DMSO-d.sub.6) δ: 11.25 (1H, bs); 7.83 (1H, d, J=7.0); 7.65-7.48 (2H, m); 7.39 (1H, d, J=7.3); 3.89 (2H, d, J=7.3); 2.4 (2H, t, J=7.3); 1.89-1.74 (2H, m).

[0066] 4-aminooxy-butyric acid hydrochloride. A suspension of 4-(1,3-dioxo-1,3-dihydro-isoindol-2-yloxyloxy)-butyric acid (400 mg, 1.61 mmols) in a solution of 3N HCl (2 mL) was heated to reflux for 2 hours. The phthalic acid was filtered, the filtrate was evaporated, and the product was crystallised from methanol. 166 mg of product 3b was obtained. Yield: 66%.

[0067] Melting point: 140° C.

[0068] .sup.1H-NMR (DMSO-d.sub.6) δ: 10.89 (3H, bs); 4.00 (2H, d, J=6.4); 2.31 (2H, t, J=7.3); 1.88-1.73 (2H, m).

[0069] Bibliography: Cyclic hydroxamates, especially multiply substituted [1,2]oxazinan-3-ones, Wolfe, Saul et al. Canadian Journal of Chemistry, 81(8), 937-960; 2003; Method for the synthesis of oxazinone amino acid derivatives, Wolfe, Saul et al. PCT Int. Appl., 2003018565, 6 Mar. 2003.

Example 3

Preparation of Compound 1c

[0070] 0.65 mL of a solution of 3c (27 mg, 0.15 mmols) and CH.sub.3COONa (7 mg, 0.1 mmols) in 50% aqueous EtOH was added to a suspension of compound 2 (30 mg, 0.07 mmols) in EtOH (0.56 mL). The reaction was placed under stirring at room temperature for 24 hours. The solvent was removed at low pressure, the residue was taken up with ethyl acetate, and the solution was washed with H.sub.2O and dried on Na.sub.2SO.sub.4. The crude product was then purified by preparative chromatography in CH.sub.2Cl.sub.2:CH.sub.3OH 95:5. 16 mg (0.03 mmols) of compound 4c (4-(carboxypentoxyimino) fenretinide) was obtained. Yield: 43%

[0071] .sup.1H-NMR (CDCl.sub.3) δ: 7.41 (2H, m); 7.14 (1H, s); 7.07-6.90 (1H, m); 6.80 (2H, m); 6.41-6.13 (4H, m); 5.82 (1H, s); 4.12 (2H, t, J=6.1); 2.62 (2H, t, J=6.4); 2.49-2.3 (2H, m); 2.43 (3H, s); 2.03 (3H, s); 1.92 (3H, s); 1.82-1.40 (8H, m); 1.09 (6H, s).

[0072] 280 μL of an 0.1N solution of NaHCO.sub.3 was added to a suspension of compound 4c (15 mg, 0.028 mmols) in 0.5 mL of H.sub.2O. The reaction was left under stirring for about 20 hours. The solvent was then removed at low pressure. 11 mg (0.02 mmols) of compound 1c was obtained. Yield: 71%.

[0073] .sup.1H-NMR (DMSO-d.sub.6) δ: 9.84 (1H, s); 7.40 (2H, m); 7.02-6.86 (1H, m); 6.66 (2H, m); 6.44-6.23 (4H, m); 6.00 (1H, s); 3.98 (2H, t, J=6.0); 2.31 (3H, s); 2.11-2.03 (2H, m); 1.98 (3H, s); 1.83 (3H,s); 1.83 (3H,s); 1.88-1.74 (2H, m); 1.64-1.34 (2H, m); 1.32-1.17 (2H, m)1.03 (6H, s).

[0074] Preparation of Compound 2c (6-aminooxy hexanoic acid):

[0075] 6-(1,3-dioxo-1,3-dihydro-isoindol-2-yloxy)-hexanoic acid. N-hydroxy-phthalimide (2.44 g, 15 mmols) and triethylamine (6.3 ml, 45 mmols) were added to a solution of 6-bromohexanoic acid (3 g, 15 mmols) in anhydrous DMF (30 mL). The solution was left under stirring at ambient temperature for 48 hours. The precipitate formed was filtered, and when the solvent had been evaporated the crude product was purified on Sephadex LH-20 with a 3:1:1 mixture of hexane:acetone: ethyl ether. 2.2 g of product was obtained. Yield: 53%.

[0076] .sup.1H-NMR (CDCl.sub.3) δ: 7.95-7.68 (4H, m); 4.23 (2H, t, J=6.4); 2.43 (2H, t, J=7.0); 1.93-1.68 (4H, m); 1.67-1.51 (2H, m).

[0077] 6-aminooxy-hexanoic acid. A suspension of 6-(1,3-dioxo-1,3-dihydro-isoindol-2-yloxy)-hexanoic acid (120 mg, 0.43 mmols) in a solution of 3N HCl (2 mL) was heated to reflux for 3 hours. The precipitate was removed by filtration, the water was evaporated, and the product was crystallised from methanol. 66 mg of product 3b was obtained. Yield: 84%.

[0078] .sup.1H-NMR (DMSO-d.sub.6) δ: 10.88 (3H, bs); 3.95 (2H, t, J=6.3); 2.18 (2H, t, J=7.3); 1.62-1.41 (4H, m); 1.36-1.20 (2H, m).

Example 4

Preparation of Compound 1d

[0079] A suspension of compound 2 (23 mg, 0.05 mmoli) in ethanol (500 μL) was treated with a solution of 2-aminoethoxyamine dihydrochloride (16 mg, 0.11 mmol) and anhydrous sodium acetate (6 mg, 0.07 mmol) in 50% aqueous ethanol (500 μL). The resulting mixture was stirred at room temperature for 24 h. The solvent was removed under reduced pressure and the crude was purified by preparative RP-18 chromatography in CH.sub.3OH/H.sub.2O9:1 to give 18 mg of compound 1d. Yield: 76%. mp 134° C.

[0080] .sup.1H-NMR (CH.sub.3OH-d.sub.4) δ: 7.37 (2H, d, J=8.5 Hz), 7.05 (1H, dd, J=11.3, 14.6), 6.74 (2H, d, J=8.5), 6.48-6.20 (4H, m), 5.99 (1H, s), 4.33-4.25 (2H, m), 3.28-3.22 (2H, m), 2.70 (2H, t, J=6.41 Hz), 2.37 (3H, s), 2.04 (s, 3H), 1.91 (3H, s), 1.62 (2H, t, J=6.4).

Example 5

Pharmacological Experiments

[0081] Experiments In Vitro

[0082] The antiproliferative activity of the various compounds 1a-c (in both monotherapy and combined treatment) on the different tumour cell lines was evaluated by sulphorhodamine B assay after 72 hours of treatment, and the dose able to inhibit 50% of growth (IC.sub.50) was calculated for the monotherapy (the results are summarized in Table 1 and Table 3), while the Kern Index (KI) was determined as the synergism/antagonism refractive index for the combined treatment (the results are depicted in FIG. 2).

[0083] As regards to the evaluation of the mechanism of action, ROS generation was determined after 5 hours of treatment with the use of the 5-(and -6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) probe, while the cell cycle was evaluated by staining with propidium iodide. Cytofluorimetric analysis (FACS) was conducted in both cases.

[0084] Experiments In Vivo

[0085] To evaluate the plasma levels of salt 1a, 4-oxo-4-HPR (120 mg/kg) and salt 1a (sodium 4-aminoxyacetate-4-HPR) (60 and 100 mg/kg) were administered i.p. to nude mice for 4 consecutive days (once a day), and the plasma levels were evaluated by HPLC 5 hours after the last administration (the results are summarized in Table 2).

[0086] As regards to mesothelioma, the mice were inoculated s.c. with STO cells, and treatment with the salt 1a began one day after tumor cell inoculation (doses: 30 and 60 mg/kg i.p.; 5 days/week for 4 weeks). The control mice were treated with the same solvent as used to dissolve the salt. The animals were examined twice a week to check their weight and any signs of toxicity. Tumour growth in the various groups (control and treated) was evaluated, and the differences were statistically analysed (the results are shown in FIG. 3a).

[0087] For the ovarian model, nude mice were inoculated i.p. with IGROV-1 cells, and treatment with the salt 1a began one day after tumor cell inoculation (doses: 30, 60 and 90 mg/kg i.p.; 5 days/week for 4 weeks). The control mice were treated with the same solvent as used to dissolve the salt. The animals were examined twice a week to check their weight and any signs of toxicity. The survival time of the various groups (control and treated) was evaluated, and the differences were statistically analysed. The experiments were conducted in duplicate, and comparable results were obtained (the results are shown in FIG. 3b).

[0088] As regards the breast cancer model, human breast cancer cells (MDA-MB-231) were inoculated into the mammary fat pad of NOD/SCID-gamma mice, and treatment with the salt 1a began one week after tumor cell inoculation (dose: 90 mg/kg; 4 days/week for 5 weeks). The animals were examined twice a week to check their weight and any signs of toxicity. Tumour growth in the various groups (control and treated) was evaluated, and the differences were statistically analysed. The experiments were conducted in duplicate, and comparable results were obtained (the results are shown in FIG. 3c).

TABLE-US-00001 TABLE 1 Antiproliferative activity of salt 1a in different tumour cell lines Tumour cell line IC.sub.50 (μM) Ovary A2780 2.78 IGROV-1 3.82 SKOV-3 5.96 Breast T47-D 3.95 MDA-MB-231 3.97 BT-474 3.69 Neuroblastoma SK-N-BE 1.55 SK-N-SH 1.9 Sk-N-5Y 1.3 Mesothelioma STO 1.48 MESO 1.28 Lymphoma JVM-2 1.11

TABLE-US-00002 TABLE 2 In vivo plasma levels Compound Dose Plasma level 4-oxo-4-HPR 120 mg/Kg 0.8 μM ± 0.4 Salt 1a  60 mg/Kg 60.3 μM ± 2.52 Salt 1a 100 mg/Kg 63.7 μM ± 2.08 4-oxo-4-HPR: 5% DMSO, 5% cremophor, H.sub.2O. Salt1a: 5% DMSO, H.sub.2O.

TABLE-US-00003 TABLE 3 Antiproliferative activity of salts 1b (LOM1098) and 1c (LOM1133) in different tumour cell lines IC.sub.50 (μM) Tumour cell line LOM1098 LOM1133 Ovary A2780 3.59 2.37 IGROV-1 3.54 2.19 Breast T47-D 4.20 3.00 MDA-MB-231 4.26 2.95