N-hydroxybenzamides as HDAC inhibitors for the treatment of parasitic diseases

Abstract

Compounds of formula ##STR00001##
wherein n is 0, 1 or 2; X is ##STR00002##
and Ar is a benzyl substituted at the meta position by a C.sub.1 to C.sub.8 alkoxy group, or a pharmaceutically acceptable salt thereof, for treating, preventing or inhibiting a parasitic disease, such as toxoplasmosis, in a subject are provided. Methods for preparing the compounds are also presented.

Claims

1. A pharmaceutical composition comprising: a compound of formula (I): ##STR00071## wherein: n is 0, 1 or 2, X is ##STR00072## Ar is a benzyl substituted at the meta position by a C.sub.1 to C.sub.8 alkoxy group or a pharmaceutically acceptable salt thereof; and at least one anti-parasitic compound, selected from the group consisting of: miltefosin, antimony based drugs, meglumine antimoniate, sodium stibogluconate, amphotericin B, benzimidazol, nifurtimox, paromomycin, pentamidin and its derivatives, arsenic derivatives, melarsoprol and difluoromethylornithin.

2. The pharmaceutical composition according to claim 1, wherein the benzyl is substituted at the meta position by a C.sub.1 to C.sub.4 alkoxy group.

3. The pharmaceutical composition according to claim 1, wherein in the compound of formula (I) the benzyl is substituted at the meta-position by a methoxy group.

4. The pharmaceutical composition according to claim 3, wherein in the compound of formula (I) either n is 0 and X is ##STR00073## said compound corresponding to formula (Ia1), ##STR00074## or wherein n is 1 and X is ##STR00075## said compound corresponding to formula (Ia2) ##STR00076##

5. A method for treating a parasitic disease in a mammalian subject, comprising applying to the subject an effective amount of the pharmaceutical composition of claim 1.

6. The method of claim 5, wherein the parasitic disease is caused by a protozoan parasite of the family of the Trypanosomatidae selected from the genus Trypanosoma or the genus Leishmania, or the parasite Toxoplasma gondii.

7. A method for treating toxoplasmosis in a mammalian subject, comprising applying to the subject an effective amount of the pharmaceutical composition.

8. The pharmaceutical composition according to claim 1 comprising a pharmaceutical acceptable excipient.

9. The method of claim 5, wherein the mammalian subject is a human, dog or cat.

10. The method of claim 5, wherein in the compound of formula (I) in the pharmaceutical composition Ar is a benzyl substituted at the meta position by a C.sub.1 to C.sub.4 alkoxy group.

11. The method of claim 10, wherein Ar is a benzyl substituted at the meta-position by a methoxy group.

Description

(1) The present invention is illustrated, but is not limited to, by the following figures and examples.

(2) Document US2003/013757 A1, discloses a compound of formula:

(3) ##STR00057##
hereafter named D16.

(4) FIG. 1 illustrates inhibitory activity of compounds (Ia1), (Ia2), (Ib1), (Ib2), (Ib3), (Ib4), (Ic1), (Ic2) and hydroxybenzamide (named also ST3 in the present invention) for T. gondii proliferation in human foreskin fibroblasts. X-axis corresponds to Log of concentration of compounds in M. Y-axis corresponds to parasite growth inhibition percent.

(5) FIG. 2 illustrates the capacity of the different compounds of 600 nM to inhibit HDAC activity from HELA nuclear extract and from HDAC-recombinant proteins (HDAC 1, HDAC 3, HDAC 6, HDAC 8).

(6) FIG. 3 illustrates the inhibitory effect of 125 nM of compound 363 on deacetylase activity measured in total protein extracts of T. gondii.

(7) FIG. 4 illustrates the lack of inhibitory effect of D16 on type I RH-YFP proliferation.

(8) FIG. 5 illustrates the lack of inhibitory effect of D16 on type II Prugniaud T. gondii proliferation.

(9) FIG. 6 illustrates the potent inhibitory effect of compound 363 on type II Prugniaud T. gondii proliferation.

EXAMPLES

(10) I. Synthesis of a Compound of Formula (I) or (I)

(11) A compound of formula (I) is synthesized according to following steps:

(12) ##STR00058##
General Procedure for 2a-2h:

(13) Amine derivative 1a or 1b (1 eq.) was dissolved in dry DCM (0.2M). Triethylamine (3 eq.) and the acid chloride or sulfonyl chloride (1 eq.) were added successively and the reaction mixture was heated at 40 C. After the reaction was complete (TLC control), the reaction mixture was concentrated. The residue was taken up with EtOAc and the organic phase was washed with a solution of 1M HCl, a saturated solution of NaHCO.sub.3 and brine, dried over Na.sub.2SO.sub.4, filtrated and concentrated to afford 2a-2h. The products were used such as in the next step.

(14) General Procedure for 3a-3h:

(15) Derivative 2a-2h (1 eq.) was dissolved in THF (3 ml). A solution of LiOH (3 eq.) in 3 ml of water was added and the reaction mixture was heated at 40 C. overnight. The reaction mixture was concentrated. The residue was taken up with 30 ml of water and the aqueous phase was washed with 20 ml of EtOAc, then the aqueous phase was acidified to pH 2 with a solution of 1M HCl. The aqueous phase was extracted with 3*20 ml of EtOAc. The organic phases were combined and dried over Na.sub.2SO.sub.4, filtrated and concentrated to afford 3a-3h. The products were used such as in the next step.

(16) General Procedure for Compound of Formula (I) or (I):

(17) Derivative of formula (I) (1 eq.) was dissolved in dry DMF (5 ml). Ethyl chloroformate (1.2 eq.) and N-methylmorpholine (1.3 eq.) were added successively at 0 C. After 10 mn, a solution of hydroxylamine (2 eq.) in MeOH (10 ml) was added and the reaction mixture was warm up to room temperature and let overnight. The reaction mixture was concentrated. The residue was taken up with EtOAc and the organic phase was washed with a saturated solution of NaHCO.sub.3 and brine, dried over Na.sub.2SO.sub.4, filtrated and concentrated. The crude products were purified by flash chromatography to afford a compound of formula (I).

(18) Compound of formula (Ib1): .sup.1H NMR (200 MHz, DMSO-d6) 11.17 (s, 1H), 9.19 (t, J=4.0 Hz, 1H), 9.00 (s, 1H), 7.70 (m, 4H), 7.56 (m, 1H), 7.39 (m, 3H), 4.51 (d, J=4.0 Hz, 2H). HPLC rt=3.356 min. (purity 100%). The calculated MS ESI+H.sup.+ is 289.28 and experimentally obtained is 289.10.

(19) Compound of formula (Ib2): .sup.1H NMR (200 MHz, DMSO-d6) 10.40 (s br, 1H), 7.75 (m, 6H), 7.58 (m, 1H), 7.45 (m, 1H). HPLC rt=3.553 min. (purity 98%). The calculated MS ESI+H.sup.+ is 275.25 and experimentally obtained is 275.10.

(20) Compound of formula (Ib3): .sup.1H NMR (200 MHz, DMSO-d6) 11.17 (s, 1H), 9.02 (s, 1H), 8.37 (s, 1H), 7.60 (m, 4H), 7.50 (m, 2H), 7.29 (d, J=5.6 Hz, 2H), 4.07 (s, 2H). HPLC rt=3.635 min. (purity 97%). The calculated MS ESI+H.sup.+ is 325.30 and experimentally obtained is 325.10.

(21) Compound of formula (Ib4): .sup.1H NMR (200 MHz, DMSO-d6) 11.07 (s, 1H), 10.75 (s, 1H), 8.95 (s, 1H), 7.55 (m, 5H), 7.48 (m, 1H), 7.15 (d, J=5.8 Hz, 2H). HPLC rt=3.531 min. (purity 100%). The calculated MS ESI+H.sup.+ is 311.31 and experimentally obtained is 311.10.

(22) Compound of formula (Ic1): .sup.1H NMR (200 MHz, DMSO-d6) 11.18 (s, 1H), 9.13 (t, J=4.0 Hz, 1H), 9.01 (s, 1H), 8.09 (s, 1H), 7.98 (d, J=Hz, 2H), 7.95 (d, J=Hz, 2H), 7.70 (d, J=5.8 Hz, 2H), 7.38 (d, J=5.8 Hz, 2H), 4.52 (d, J=4.0 Hz, 2H), 2.73 (s, 3H). HPLC rt=3.828 min. (purity 96%). The calculated MS ESI+H.sup.+ is 368.43 and experimentally obtained is 368.10.

(23) Compound of formula (Ic2): .sup.1H NMR (200 MHz, DMSO-d6) 11.13 (s, 1H), 10.46 (s, 1H), 8.95 (s, 1H), 8.14 (s, 1H), 8.10 (d, J=4.2 Hz, 2H), 8.03 (d, J=4.2 Hz, 2H), 7.87 (d, J=4.4 Hz, 2H), 7.76 (d, J=4.4 Hz, 2H), 2.75 (s, 3H). HPLC rt=3.984 min. (purity 100%). The calculated MS ESI+H.sup.+ is 354.40 and experimentally obtained is 354.10.

(24) Compound of formula (Ia2): .sup.1H NMR (200 MHz, DMSO-d6) 11.17 (s, 1H), 8.99 (s, 1H), 8.58 (t, J=4.0 Hz, 1H), 7.67 (d, J=5.4 Hz, 2H), 7.29 (d, J=5.4 Hz, 2H), 7.19 (t, J=5.8 Hz, 1H), 6.80 (m, 3H), 4.30 (d, J=4.0 Hz, 2H), 3.73 (s, 3H), 3.45 (s, 2H). HPLC rt=3.370 min. (purity 100%). The calculated MS ESI+H.sup.+ is 315.34 and experimentally obtained is 315.10.

(25) Compound of formula (Ia1): .sup.1H NMR (200 MHz, DMSO-d6) 11.09 (s, 1H), 10.35 (s, 1H), 8.93 (s, 1H), 7.70 (d, J=5.8 Hz, 2H), 7.64 (d, J=5.8 Hz, 2H), 7.22 (t, J=5.4 Hz, 1H), 6.89 (m, 2H), 6.82 (d, J=4.8 Hz, 1H), 3.74 (s, 3H), 3.63 (s, 2H). HPLC rt=3.618 min. (purity 99%). The calculated MS ESI+H.sup.+ is 301.32 and experimentally obtained is 301.10.

(26) II. Inhibition of Histone Deacetylase Activity Derived from HELA Nuclear Extract, of Some Recombinant Proteins and of T. gondii Total Protein Extract

(27) HDAC inhibitory activity was determined by a fluorimetric HDAC assay kit (Active-motif, Belgium), according to manufacturer's instructions. Briefly, 30 L of HeLa nuclear extract were mixed with 5 L of 10 compound to be tested and 10 L of assay buffer. Fluorogenic substrate (10 L) was added, reaction was allowed to proceed for 30 min at room temperature, then stopped by the addition of a developer containing Tichostatin A. Fluorescence was monitored after 30 min at excitation and emission wavelengths of 360 and 460 nm, respectively. HDAC inhibitory activity of the compounds of formula (Ia1), (Ia2), (Ib1), (Ib2), (Ib3), (Ib4), (Ic1), (Ic2) was assayed and compared to that of hydroxybenzamide, which is an anti-parasitic compound described in PCT/EP2011/004055, to suberoylanilide hydroxamic acid (SAHA) a known inhibitor of HDAC activity, and to D16. The results are exposed in Table 1.

(28) TABLE-US-00001 TABLE 1 HELA Nuclear extracts IC50 Compounds cLogP Structure in nM SAHA (suberoylanilide hydroxamic acid) ND 2100 +/ 200 ST3 (hydroxybenzamide) 0.855 0 7865 +/ 908 Compound (Ib1), named also 345 1.545 866 +/ 400 Compound (Ib2), named also 349 1.844 633 +/ 41 Compound (Ib3), named also 350 1.420 8611 +/ 1540 Compound (Ib4) named also 351 1.719 881 +/ 83 Compound (Ic1), named also 360 1.724 399 +/ 137 Compound (Ic2), named also 361 2.022 1825* Compound (Ia2), named also 362 1.438 518 +/ 146 Compound (Ia1), named also 363 1.829 85 +/ 35 Compound D16 ND 7735 +/ 1300 ND: not determined cLogP is the calculated LogP Hela nuclear extracts IC50 represents the inhibitory activity of a compound.

(29) The results of Table 1 show that the 9 compounds tested represent HDAC inhibitory activity against HeLa cell nuclear extract, which contains primarily HDACs 1,2,6,8. HDAC inhibitory activity of these compounds, which is from about 1 M for the less active compounds (ST3, compound 350 and compound 361) to 85 nM for the more active ones (compound 363), is in the activity range of well-known HDAC inhibitors and is higher than the inhibitory activity of SAHA that has been shown to be in the micromolar range of activity (1300 nM) (Jiao et al., Eur. J Med. Chem. 2009. 44:4470-4476), IC.sub.50 of 2000 nM in our experiment (Table 1). The inhibitory activity of compound D16 in HELA Nuclear extracts was the lowest as compared to other compounds in Table 1, with an IC50 value of 7735+/1300 nM.

(30) The data of FIG. 2 show that the compound 363 is the most potent inhibitor of the human HDAC activity. The IC.sub.50 of 363 is of 257+/50, 160+/52, 45+/15 and 863+/45 nM for recombinant HDAC1, HDAC3, HDAC6 and HDAC 8 respectively. It is a 3 to 100 fold more potent inhibitor for HDAC activity than SAHA (IC.sub.50 of 627+/39, 170+/14, 1573+/800, 230+/130 nM for HDAC1, HDAC 3, HDAC6 and HDAC8 respectively).

(31) The IC.sub.50 of D16 is of more than 10 microM for recombinant HDAC1 and of 198+/40 nM for recombinant HDAC6.

(32) As showed in FIG. 3, at 125 nM the compound 363 expresses a strong inhibitory effect on deacetylase activity measured in total protein extracts of T. gondii. It inhibits 44% of the T. gondii deacetylase activity whereas SAHA inhibit only 22% of this activity. This suggests that the compounds express broad inhibitory activity against both human and protozoan histone deacetylase activity.

(33) The compound (I) or (I) according to the invention have a strong and more specific inhibitory effect on Histone deacetylase activity, advantageously on Histone deacetylase 1 and 6 activity.

(34) III. Inhibition of T. gondii and Leishmania Proliferation

(35) The potential of aforementioned 9 compounds to inhibit the proliferation of T. gondii and Leishmania was also investigated.

(36) To assess the drug activity on toxoplasma proliferation, human foreskin fibroblasts (HFF) were infected with GFP expressing parasites for 30 mns (Striepen et al., Mol Biochem Parasitol. 1998, 92(2):325-38). Cells were then washed and drugs added at various concentrations ranging from 0.1 to 10 uM. After 24 h, cells were washed and fixed (PBS-formaldehyde 2%) and nucleus stained with Hoechst 33258 (2 g/ml). The number of infected cells i.e cells harbouring a parasitophorous vacuole and the number of parasites per vacuole were determined using an Olympus ScanR microscope (Olympus).

(37) Leishmanicidal activity was determined according to a previously published protocol that used Leishmania infantum luciferase expressing strain (MHOM/MA/ITMAP269) (Sereno D & Lemesre J L., Antimicrob Agents Chemother 1997, 41(5): 972-976).

(38) Antiparasitic activity of a compound is represented by IC.sub.50, which is calculated with Prism software (Prism4 for MacOSX Version 5.0b, December 2008). The cytoxicity of a compound against the host cells of L. infantum and T. gondii, such as THP-1 or HFF cells, was determined with a MTT assay, according to previously described protocol (Sereno et al., Antimicrob Agents Chemother. 2001, 45(4):1168-73). THP-1 is derived from the peripheral blood of a 1 year old human male with acute monocytic leukemia.

(39) Antiparasitic activity and cytotoxicity of these compounds are summarized in FIG. 1 and table 2.

(40) TABLE-US-00002 TABLE 2 L. infantum T. gondi Compound cLogP IC50 IC50 HFF LD.sub.50 IS THP-1 LD.sub.50 IS SAHA >100 ND ND ND >200 ND ST3 0.855 29.3 +/ 14.4 >50 >400 ND 271.0 +/_30.0 ND 345 1.545 27.2 +/ 2.5 5.0 +/ 1.0 58.3 +/ 1.2 11.6 1.7. +/ 0.5 0.3 349 1.844 >60.0 >50 >400 ND 264 +/ 8.0 ND 350 1.420 >60.0 40.0 +/10.0 300.0 +/ 50.0 7.5 >400 10 351 1.719 >60.0 50 116.5 +/ 35 2.3 76.5-17.9-5.8 1.53 360 1.724 >60.0 4.9 +/ 0.15 22.6 +/ 9.5 4.5 2.2 +/ 0.5 0.4 361 2.022 >60.0 >10 25.0 +/ 4.0 1.5 >400 25 362 1.438 >60.0 52.5 +/ 10.4 >400 >8 30.5 +/ 3.0 0.5 363 1.829 >60.0 0.35 +/ 0.05 105 +/ 10.5 300 3.6 +/ 0.5 10.3 Antiparasitic activity: IC50 in M. Cytotoxicity: DL50 in M. IS: Selectivity index: LD50 (HFF or THP-1)/IC50 T. gondii.

(41) Of the 8 compounds synthesized of the present invention, 3 compounds (compounds 345, 360, 363) inhibit the proliferation of the T. gondii at concentrations below 10 M. Interestingly, all these 3 compounds are also the better HDAC inhibitor among 9 tested compounds (IC50 of 1.7 M, 2.2 M and 3.6 M respectively). The compound 345 is the sole compound having both anti-leishmanial and anti-toxoplasma activity (Table 2). ST3 is another compound also presenting anti-leishmanial activity. Amongst the 9 compounds synthetized in Table 2 which have an IC50 value ranging from more than 60 M to less than 1 M, the compound 363 is the most selective for the intracellular proliferative stage of T. gondii. The difference between the selectivity index of 300 in HFF cells and 10 in THP-1 cells can be explained by the fact that THP-1 cell line is known to be susceptible to hydroxamate derivatives and therefore might not be ideal to evaluate the cytotoxicity of hydroxamate derivatives against host cells (Sung et al., Apoptosis. 2010, 15(10):1256-69). The HEPG2 cell line is considered as a model system for the studies of liver metabolism and toxicity of xenobiotics. An IC.sub.50 superior to 15 M for 363 and a calculated index of selectivity of 42, confirm the high selectivity of 363 for T. gondii tachyzotes.

(42) The present results demonstrate an inhibitory activity of the compound according to the invention on Toxoplasma or on Leishmania proliferation.

(43) IV. Inhibition of RH-YFP Type I or Type II Prugniaud Toxoplasma Proliferation

(44) To compare the activity of compounds according to the invention to that of prior art on toxoplasma proliferation, human foreskin fibroblasts (HFF) were plated in 96-wells plate (10 000 cells per well). Twenty-four hours later, cells were infected with 40 000 parasites, either RH-YFP type I or type II Prugniaud parasites or tachyzoites (kindly provided by B. Striepen, Athens) for one hour. Cells were then washed and drugs (compound D16 of formula

(45) ##STR00070##
(FIG. 4 and FIG. 5) or compound according to the invention (363) (FIG. 6) added at various concentrations ranging from 0.025 to 0.8 uM. After 24 h, cells were washed and fixed (PBS-formaldehyde 2%) and nucleus stained with Hoechst 33258 (2 g/ml). The number of infected cells i.e. cells harbouring a parasitophorous vacuole and the number of parasites per vacuole was determined using an Olympus ScanR microscope (Olympus).

(46) As shown in FIG. 4, D16 has no inhibitory activity on type I RH-YFP parasites proliferation.

(47) As shown in FIG. 5, D16 has no inhibitory activity on type II Prugniaud parasites proliferation.

(48) As shown in FIG. 6, compound according to the invention 363 has a potent inhibitory activity on type II Prugniaud parasites proliferation.

(49) The present results demonstrate an inhibitory activity of the compound according to the invention on Toxoplasma type I and type II parasites proliferation.

(50) The present results also demonstrated an inhibitory activity of the compound according to the invention on Babesia proliferation, in particular the IC50 calculated for compound 363 was about 1 microM.

(51) The compound according to the invention has an anti-Babesia activity.