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. 2018 Dec;33(1):1225-1238.
doi: 10.1080/14756366.2018.1493473.

Design, synthesis, in vitro antiproliferative activity and apoptosis-inducing studies of 1-(3',4',5'-trimethoxyphenyl)-3-(2'-alkoxycarbonylindolyl)-2-propen-1-one derivatives obtained by a molecular hybridisation approach

Delia Preti  1 , Romeo Romagnoli  1 , Riccardo Rondanin  1 , Barbara Cacciari  1 , Ernest Hamel  2 , Jan Balzarini  3 , Sandra Liekens  3 , Dominique Schols  3 , Francisco Estévez-Sarmiento  4 , José Quintana  4 , Francisco Estévez  4
Affiliations

Design, synthesis, in vitro antiproliferative activity and apoptosis-inducing studies of 1-(3',4',5'-trimethoxyphenyl)-3-(2'-alkoxycarbonylindolyl)-2-propen-1-one derivatives obtained by a molecular hybridisation approach

Delia Preti et al. J Enzyme Inhib Med Chem. 2018 Dec.

Abstract

Inhibition of microtubule function using tubulin targeting agents has received growing attention in the last several decades. The indole scaffold has been recognized as an important scaffold in the design of novel compounds acting as antimitotic agents. Indole-based chalcones, in which one of the aryl rings was replaced by an indole, have been explored in the last few years for their anticancer potential in different cancer cell lines. Eighteen novel (3',4',5'-trimethoxyphenyl)-indolyl-propenone derivatives with general structure 9 were synthesized and evaluated for their antiproliferative activity against a panel of four different human cancer cell lines. The highest IC50 values were obtained against the human promyelocytic leukemia HL-60 cell line. This series of chalcone derivatives was characterized by the presence of a 2-alkoxycarbonyl indole ring as the second aryl system attached at the carbonyl of the 3-position of the 1-(3',4',5'-trimethoxyphenyl)-2-propen-1-one framework. The structure-activity relationship (SAR) of the indole-based chalcone derivatives was investigated by varying the position of the methoxy group, by the introduction of different substituents (hydrogen, methyl, ethyl or benzyl) at the N-1 position and by the activity differences between methoxycarbonyl and ethoxycarbonyl moieties at the 2-position of the indole nucleus. The antiproliferative activity data of the novel synthesized compounds revealed that generally N-substituted indole analogues exhibited considerably reduced potency as compared with their parent N-unsubstituted counterparts, demonstrating that the presence of a hydrogen on the indole nitrogen plays a decisive role in increasing antiproliferative activity. The results also revealed that the position of the methoxy group on the indole ring is a critical determinant of biological activity. Among the synthesized derivatives, compound 9e, containing the 2-methoxycarbonyl-6-methoxy-N-1H-indole moiety exhibited the highest antiproliferative activity, with IC50 values of 0.37, 0.16 and 0.17 μM against HeLa, HT29 and MCF-7 cancer cell lines, respectively, and with considerably lower activity against HL-60 cells (IC50: 18 μM). This derivative also displayed cytotoxic properties (IC50 values ∼1 μM) in the human myeloid leukemia U-937 cell line overexpressing human Bcl-2 (U-937/Bcl-2) via cell cycle progression arrest at the G2-M phase and induction of apoptosis. The results obtained also demonstrated that the antiproliferative activity of this molecule is related to inhibition of tubulin polymerisation. The presence of a methoxy group at the C5- or C6-position of the indole nucleus, as well as the absence of substituents at the N-1-indole position, contributed to the optimal activity of the indole-propenone-3',4',5'-trimethoxyphenyl scaffold.

Keywords: Microtubule; apoptosis; indole derivatives; structure–activity relationship; tumour cell growth.

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Figures

Figure 1.
Figure 1.
Structure of chalcone (1) reported by Ducki et al., combretastatin A-4 (CA-4, 2), indole-based chalcone derivatives 3–4 and general structure (5) of methyl/ethyl 3-[(3′,4′,5′-trimethoxyphenyl)thio/carbonyl]-5-substituted-1H-indole-2-carboxylates reported by De Martino et al.
Figure 2.
Figure 2.
Bioisosteric replacement of the sulphur or carbonyl group (X) of compound 5 with the 2-propen-1-one system (drawn in red) of chalcone 1 furnished a new class of indole-based chalcone conjugates of type A.
Figure 3.
Figure 3.
Tumour cells were incubated with vehicle (control), 10 µM (U-937) or 1 μM (U-937/Bcl-2) of the specified compound for 72 h and images were obtained with an inverted phase-contrast microscope.
Figure 4.
Figure 4.
Differential effects of compounds on cell viability of normal PBMCs versus U-937 and U-937/Bcl-2 cells. Human leukaemia, and quiescent and phytohemagglutinin-activated PBMCs [PBMC(+PHA)] from healthy human donors were cultured in the presence of 10 μM of each compound for 24 h. Values represent mean ± SE of three independent experiments each performed in triplicate. *p < 0.05, significantly different from the corresponding control.
Figure 5.
Figure 5.
(A) Photomicrographs of representative fields of cells stained with Hoechst 33258 to evaluate nuclear chromatin condensation (i.e. apoptosis) after treatment with 10 μM (U-937) or 1 μM (U-937/Bcl-2) of compounds 9d, 9e and 9f for 72 h. (B) U-937 and U-937/Bcl-2 cells were incubated with 10 or 1 μM, respectively, of the specified compound for 72 h, subjected to flow cytometric analysis using propidium iodide labelling, and the percentage of hypodiploid cells was determined by flow cytometry. Values represent means ± SE from three different experiments performed in triplicate. *p < 0.05, significantly different from control. (C) Representative histograms of flow cytometry after propidium iodide staining of U-937/Bcl-2 cells incubated in the absence or in the presence of 1 μM of the specified compound for 72 h.
Figure 6.
Figure 6.
Effects of JNK/SAPK inhibitor on cell cycle distribution in U-937 and U-937/Bcl-2. Cells were pre-treated with SP600125 (10 μM) for 1 h and then treated with the indicated compounds (10 μM in U-937 or 1 μM in U-937/Bcl-2) for 24 h and subjected to flow cytometry using propidium iodide staining. A representative histogram is shown. Values indicate the percentage of cells in sub-G1 (left) and G2-M phase (right) of the cell cycle.
Scheme 1.
Scheme 1.
Reagents: (a) MeOH or EtOH, conc. H2SO4, reflux; (b) POCl3, DMF, 0 °C to rt then Na2CO3; (c) R3-halide, NaH, DMF, rt, 2 h; (d) 3′,4′,5′-trimethoxyacetophenone, piperidine (cat.), MeOH or EtOH, reflux, 24 h.
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Grants and funding

DP is supported by the fund “Finanziamento delle attività base di ricerca 2017” (FFABR) of the University of Ferrara.
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