Ant-ligands molecules and biological applications

Abstract

ANT-ligands having a substituted nitrogeneous heterocycle A wherein A is a substituted pyrimidinone of formula I ##STR00001##
wherein R1 is (CH2)n-COOH; (CH2)n-COOR; (CH2)n-CONHR; (CH2)n-CON(R, R); (CH2)n-OH; (CH2)n-OR; (CH2)n-OAr; (CH2)n-C(R,R)(CH2)n-OH, R and R, in the above radicals, being identical or different and representing H or a C1-C12 alkyl or cycloalkyl radical; and Ar is a phenyl or Het., Het. representing an heterocyclic radical with one or several hetero atoms selected between N, S and O, said phenyl or heterocycle being optionally substituted by one or several atoms, groups or radicals selected from halogen atoms such as Cl, Br, I, or halogenated groups such as CCl3 or CF3; one or several OH, OR, COOH or COOR groups; a phenyl; a linear or branched C1-C12 alkyl radical; NHCOR; or CN; said groups occupying the same or different positions on the phenyl or heterocyclic radical; a linear or branched C1-C12 alkyl radical; a linear or branched C2-C12 alkylene radical; (CH2)n-C3-C6 cycloalkyl radical; (CH2)n Ar or (CH2)n-Het.; (CH2)n-NHCOR; (CH2)n-NH2; (CH2)n-N(R,R); (CH2)n-NHCOOH; (CH2)n-NHCOOR; NH(CH2)n-COOH; NH(CH2)n-COOR; R2 is (CH2)n-Ar, Ar being such as above defined and being optionally substituted such as above defined; a linear or branched C1-C12 alkyl or C2-C12 alkenyl radical, (CH2)n-OH; (CH2)n-OR; (CH2)n-CO-Het; (CH2)n-NHCOR; (CH2)n-NH2; (CH2)n-N(R,R); (CH2)n-COOH; (CH2)n-COOR; a linear or branched C1-C12 alkyl radical; (CH2)n-C(R)CHC(R)CH2; R3 forms a phenyl or an heterocyclic condensed group with the two adjacent carbons of the pyrazinone residue, said condensed group being optionally substituted such as above defined for Ar and Het.; and/or condensed to a cyclohexyl or oxanyl group, in turn optionally substituted such as above defined for Ar; n is 0 or an integer from 1 to 5; or A is a substituted pyrimidine of formula II ##STR00002##
wherein R4 is a CONHAr radical, optionally substituted such as above defined; R5 forms a phenyl or heterocyclic group condensed to the two adjacent carbon groups of the pyrazine residue, said phenyl or heterocyclic group being optionally substituted such as above defined, and
Ar being such as above defined with respect to formula I
or A is a substituted pyridine group of formula III ##STR00003##
wherein,
Ar and R2 are as above defined with respect to formula I.

Claims

1. A compound of Formula (I) ##STR00021## Wherein R1 is (CH.sub.2).sub.nCOOH; (CH.sub.2).sub.nC3-C6 cycloalkyl radical; (CH.sub.2).sub.n-Het.; (CH.sub.2).sub.nNHCOR; (CH.sub.2).sub.nNH.sub.2; (CH.sub.2).sub.nNHCOOH; R in the above radicals, representing H or a C1-C12 alkyl or cycloalkyl radical Het. representing an heterocyclic radical with one or several hetero atoms selected between N, S and O, said heterocycle being optionally substituted by one or several atoms, groups or radicals selected from Cl, I, F or CF.sub.3; one or several OH, OR.sub.10, COOH or COOR.sub.10 groups; a linear or branched C1-C12 alkyl radical; NHCOR.sub.10; or CN; said groups occupying the same or different positions on the heterocyclic radical; R, in the above radicals, representing H or a C1-C12 alkyl or cycloalkyl radical; wherein R.sub.10 in the above radicals represents H or a C.sub.1-C.sub.12 alkyl or cycloalkyl radical; R2 is (CH.sub.2).sub.nAr, Ar is a phenyl, said phenyl being optionally substituted by one or several atoms, groups or radicals selected from Cl, I, F or CF.sub.3; one or several OH, OR, COOH or COOR groups; said groups occupying the same or different positions on the phenyl radical; (CH.sub.2).sub.nCOOH R3 forms a thienyl group with the two adjacent carbons of the pyrimidinone residue, said thienyl being optionally substituted as above defined for Ar; and n is 0 or an integer from 1 to 5; wherein said compounds of Formula (I) are selective for ANT protein.

2. A compound of claim 1, wherein R1 is selected from the group consisting of (CH.sub.2).sub.nCOOH; (CH.sub.2).sub.nNH.sub.2; (CH.sub.2).sub.nNHCOR; or (CH.sub.2).sub.n Het., wherein Het. is a pyridyl radical.

3. A compound of claim 1, wherein R2 is a (CH.sub.2).sub.n-phenyl group, said phenyl group being substituted by at least one of Cl, I, F, CF.sub.3, or OR, wherein R represents H or a C1-C12 alkyl or cycloalkyl radical.

4. The compound of claim 1, wherein said compounds are administered to a subject at 0.1 mg/kg to 100 mg/kg body weight.

5. The compound of claim 4, wherein said compounds are administered daily or weekly.

6. A pharmaceutical composition comprising a therapeutically effective amount of at least one compound of claim 1, and pharmaceutically acceptable inert carrier.

7. The pharmaceutical composition of claim 6, further comprising a therapeutic agent selected from the group consisting of chemotherapeutics, apoptosis modulators, antimicrobial, antiviral, antifungal or anti-inflammatory agents.

8. A pharmaceutical composition comprising at least one compound of claim 1, wherein said composition induces cell death.

9. A method of targeting an ADP/ATP translocator ANT, wherein said method comprises administering an effective amount of an ANT ligand to the target, wherein the ANT ligand comprises a compound of claim 1.

10. The method of claim 9, which is performed in a subject in need thereof.

11. The method of claim 9, which is performed in vitro.

12. The method of claim 9, 10 or 11, wherein said ANT ligand comprises at least one of: ##STR00022## ##STR00023## ##STR00024##

13. A method for inducing cell death by targeting an ADP/ATP translocator ANT in cellula, comprising administering an effective amount of at-least one compound of claim 1.

14. The method of claim 9, wherein said targeting of said ADP/ATP translocator by said compound is a cancer therapy.

15. The method of claim 9 wherein said subject is mammalian.

16. The method of claim 15, wherein said subject is human.

Description

(1) Other characteristics and advantages are given in the following examples which refer to FIGS. 1 to 8, wherein:

(2) FIG. 1: Illustrates in silico molecular docking to find new ANT-ligands: (A) Structure of the carboxyatractyloside (CAT)-bovine ANT1 complex (adapted from (26)) (B) Prediction by computer analysis of the carboxyatractyloside (CAT) localization in the human ANT2 binding.

(3) FIG. 2: The ANT-ligand is pro-apoptotic on HT-29 and BxPC3 tumor cell lines (A) Chemical structure of compound 1 (B) Multiparametric analysis (chromatin condensation, mitochondrial transmembrane potential (Lm) loss, plasma membrane permeabilization, phosphatidylserine exposure) of cellular effects of the ANT-ligand on BxPC3 cell line after 48 h treatment. (C) Effects of compound 1 on ADP/ATP translocator activity of ANT measured on isolated mitochondria from mice liver or HT-29 tumor cell line (IC50 is given in M based on ANT assays) and on viability of HT-29 and BxPC3 tumor cell lines (LD50 is given in M based on MTT assays at 48 h).

(4) FIG. 3: Compound 1 induced-cell death is caspase-dependent

(5) Compound 1 induces classical hallmarks of apoptosis: mitochondrial potential (Lm) loss (Dioc6), phosphatidylserine exposure (Annexin-V+), plasma membrane permeabilization (PI+) and chromatin condensation as shown by multiparametric analysis. Apoptosis induced by the ANT-ligand is inhibited by pan-caspase inhibitors (z-VAD-fmk, Q-VD-OPH) but not by the cathepsin B inhibitor (Z-FA-fmk).

(6) FIG. 4: Compound 1 induces low toxicity on normal fibroblast Wi-38 A) Expression pattern of ANT isoforms in HT-29, BxPC3 and Wi-38 (normal lung fibroblasts) cell lines after RT-PCR reaction on total RNA (B) Multiparametric analysis (mitochondrial transmembrane potential, plasma membrane permeabilization, phosphatidylserine exposure) of cellular effects of the ANT-ligand on Wi-38 cell line after 48 h and 72 h treatments.

(7) FIG. 5: Target validation of compound 1 using ANT-deficient yeasts (A) Quantitative estimation of yeasts viability at 48 h after 2 h incubation with compound 1 (B) Illustration of WT (W303) and JL1-3 (LANT1, 2 and 3) yeast strains growth on plates at 48 h after 2 h incubation with compound 1.

(8) FIG. 6: Optimization by structure-Activity Relationship studies

(9) The table shows the effects of optimized compounds on HT-29, BxPC3, MiaPaca, Wi38 cell viability (LD50 in M); on ANT activity in mice liver and HT-29 tumor cell line mitochondria (IC50 in M); on swelling (DS50 in M) and Lm parameters (DP50 in M) in mice liver mitochondria (Mitotrust platform); and on viability of wild-type (W303) and ANT-deficient (JL1-3) yeasts strains (ED50 in M).

(10) FIG. 7: Chemical structures of optimized compounds in structural family 1.

(11) FIG. 8: Effects of compounds (family 2 & 3) on cell lines and isolated mitochondria.

(12) The table shows the effects of compounds on HT-29, BxPC3, MiaPaca, Wi-38 cell viability (LD50 in M); on ANT activity in mice and HT-29 tumor cell line mitochondria (EC50 in M); on swelling (DS50 in M) and Lm parameters (DP50 in M) in mice liver mitochondria.

(13) ADP/ATP Translocase Activity Assay

(14) The ANT activity assay is an indirect measure of ATP translocation from isolated mitochondria in exchange of ADP followed by NADPH formation in the medium. This assay is using a complex system of ATP detection constituted of enzymes (hexokinase, glucose-6phosphate-dehydrogenase), a substrate (glucose) and a co-substrate (NADP+) allowing NADP+ reduction in NADPH. The method is adapted from (10), with modifications: reactions in microplates, no pre-loading of mitochondria with ATP, detection of NADP+ reduction by fluorescence (Spectrofluorimeter Infinite M200, Tcan), incorporation of AP5A (P1P5diadenosine-5-pentaphosphate) to inhibit the adenylate kinase-dependent ATP synthesis (IC50: dose inducing 50% of carboxyatractyloside inhibition activity).

(15) Viability Assay and Characterisation of Cell Death

(16) MTT assay was used to evaluate the viability of a large range of human cell lines in presence of small molecules. Dose-response experiments allow us to determine a lethal-dose 50 (LD50; dose killing 50% of the cellular population) for each compound on a particular cell type after a 48-hour incubation. This viability assay is used as a first screening assay to identify cell-permeant molecules able to induce cell death (cytotoxic) or growth delay (cytostatic) among the 956 molecules of the ANT-ligands library. We have chosen to select molecules having an LD50 below 50 M on HT-29 (colon adenocarcinoma) or BxPC3 (pancreatic adenocarcinoma) cell lines. These molecules come into the ANT activity screening assays and the efficient ANT-inhibitors (IC50 below 50 M) are investigated for their mechanisms of cell death induction. Indeed, the characterisation of cell death consists in a multiparametric analysis of treated-cells by flow cytometry (FacsCalibur, Becton Dickinson) where can be measured (1) the loss of mitochondrial trans-membrane potential (Lm; DIOC6 labelling), (2) the plasma membrane permeabilization (Propidium Iodide labeling) and (3) the phosphatidylserines exposure (Annexin-V-fitc labelling).

(17) Scheme of Synthesis of Compounds 1-6 and 8-16

(18) ##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020##

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