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Indole alkaloids as leads for developing effective anticancer drugs targeting multidrug resistant cancer cells
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Multidrug resistance (MDR) is the major obstacle for cancer chemotherapy. MDR is a multifactorial phenomenon that can result from several mechanisms, including an increased drug efflux, due to overexpression of ABC drug transporter proteins (P-gp, MRP1 and ABCG2), which transport anticancer drugs out of the cells, or a failure to undergo apoptosis. The development of ABC transporter inhibitors is a promising approach for overcoming MDR. The discovery of collateral sensitivity agents (CS) and the development of effective apoptosis inducers have also been considered realistic strategies. Thus, the main goal of this work was to find out new effective anticancer indole alkaloids from African plants (Apocynaceae and Rubiaceae families), targeting MDR cancer cells. The phytochemical study of the alkaloid fraction of the methanol extract of Tabernaemontana elegans yielded three novel (4-6) and five known (7-11) bisindole alkaloids of the vobasinyl-iboga type and three monoterpene indole alkaloids of the corinanthe type (1-3). Moreover, aiming at generating a small library of monoterpene indole alkaloids, the chemical derivatization of the two epimeric monoterpene indole alkaloids dregamine (1) and tabernaemontanine (2),isolated in large amount from this species, allowed the preparation of 47 derivatives. In this way, the chemical transformation of the ketone group at C-3 of both compounds (1 and 2) afforded several aromatic and aliphatic imines (1.1-1.8 and 2.1-2.6) and azines (1.16-1.27 and 2.9-2.20), a thioketone (1.9), alcohols (1.10 and 2.7), and esters (1.11 and 2.8). The N-acyl/alkylated compounds 1.12 1.15 were also obtained. From the alkaloid fraction of the methanol extract of Voacanga thouarsii, one new monoterpene indole alkaloid (16) of the iboga type and four known (12-15), along with one indole alkaloid of the sarpagine type (17) were isolated. Moreover, a new phenol, 3,4,5-trimethoxyphenol-1-O-β-D-glucopyranosyl-4′,6′-O-(E)-dicoumaroyl ester (19), and several known phenolic compounds (18, 20-22), one steroid (23) and one triterpene (24) were also obtained. The phytochemical study of Psychotria capensis yielded two norisoprenoids (28-29) and three phenolic compounds (25-27). The structures of the compounds were established from their physical and spectroscopic data (IR, MS, 1D and 2D NMR -COSY, HMBC, HMQC and NOESY-experiments). The indole alkaloids 4-8, 9, 1.1-1.5 and 2.1-2.5 were evaluated for their ability as apoptosis inducers in HCT116, and SW620 colon and HepG2 liver cancer cells. The cytotoxicity of the compounds was evaluated in the three cell lines by the MTS and lactate dehydrogenase assays. The apoptosis induction studies included Guava ViaCount flow cytometry assays, nuclear morphology evaluation by Hoechst staining, and caspase-3/7 activity assays. To explore the association of compounds-induced growth inhibition with regulation of cell cycle progression, the cellular DNA content was determined by flow cytometry, analysing the percentage of cells in different phases of the cell cycle. Moreover, in order to determine the molecular pathways by which compounds exerted their pro-apoptotic effect, key apoptosis proteins were evaluated by immunoblot analysis, using total protein extracts from HCT116, SW620 or HepG2 cells exposed to compounds. The most significant results were obtained for some hydrazones (1.4, 2.2 and 2.4) and several bisindole alkaloids (4-7 and 9). Structure-activity relationships for compounds 1, 2 and their derivatives (1.1-1.5 e 2.1-2.5) showed that the replacement of the carbonyl group of the parent compounds 1 and 2 by a hydrazone moiety, bearing a bromo-pyridine (1.4 and 2.4) or a phenyl group (2.2) seems to be responsible for their activity, which might also depend on the stereochemistry of the tetrahedral stereocenter at C-20. The induced inhibition of proliferation of HCT116 cells by bisindole alkaloids 4 and 5 was associated with G1 phase arrest, while bisindole alkaloids 6 and 9 induced G2/M cell cycle arrest. In addition, immunoblot analysis showed that exposure to compound 7 reduced the expression of anti-apoptotic proteins in the tree cell lines. The monoterpene indole alkaloids 1, 2 and their derivatives 1.1-1.15, 2.1-2.8 were also evaluated for their effects on the reversion of MDR in cancer cells mediated by P-glycoprotein using a human ABCB1-transfected mouse T-lymphoma cell model. SAR analysis showed that different substituents at C-3 and at the indole nitrogen led to different ABCB1 modulatory effects. A remarkable enhancement in MDR reversal activity was found for the imine derivatives 1.4, 1.7, 2.3 and 2.4, sharing a new aromatic moiety. Some of these indole alkaloids (1.2-1.4, 1.7-1.9, 1.12, 1.13 e 2.2-2.4) were assayed for their antiproliferative effects in combination with doxorubicin; the results showed that the tested compounds synergistically enhanced the effect of the antitumor drug. The anti-MDR reversal activity of azine derivatives (1.16-1.27 and 2.9-2.20) was evaluated using as models transfected cancer cells NHI-3T3 overexpressing P-gp, and a transfected HEK293 cell line overexpressing either MRP1 or ABCG2. In both cancer cell lines overexpressing P-gp or MRP1, a considerable MDR reversing activity was observed for compounds with an aromatic azine moiety. The strongest activity as P-gp inhibitors was found for the epimeric azines 1.16 and 2.9, which were found to be selective for this transporter. Instead, compounds 1.22 and 2.15 inhibited selectively MRP1 drug-efflux. No compound was able to inhibit ABCG2. Azine derivatives (1.16-1.27 and 2.9-2.20) were also evaluated as collateral sensitivity agents. Compounds 2.12, with an aromatic azine moiety at C-3, and compounds 1.24, 1.25, 2.17 and 2.18, sharing a new aliphatic -C=N-N=C-R at this carbon, were considered CS agents killing selectively BHK-21-MRP1 overexpressing cells. Among the compounds with selective cytotoxic activity against cancer cells overexpressing MRP1, compounds 1.23-1.25 were able to increase the depletion of glutathione by MRP1, thus triggering cell death through apoptosis. In conclusion, several indole alkaloids of both natural origin or obtained by derivatization are promising potential lead structures as MDR reversers.
Descrição
Tese de doutoramento, Química Farmacêutica e Terapêutica, Universidade de Lisboa, Faculdade de Farmácia, 2017
Palavras-chave
Teses de doutoramento - 2017