Design, Synthesis and Biochemical Evaluation of Novel Ethanoanthracenes and Related Compounds to Target Burkitt's Lymphoma

Abstract

Lymphomas (cancers of the lymphatic system) account for 12% of malignant diseases worldwide. Burkitt's lymphoma (BL) is a rare form of non-Hodgkin's lymphoma in which the cancer starts in the immune B-cells. We report the synthesis and preliminary studies on the antiproliferative activity of a library of 9,10-dihydro-9,10-ethanoanthracene based compounds structurally related to the antidepressant drug maprotiline against BL cell lines MUTU-1 and DG-75. Structural modifications were achieved by Diels-Alder reaction of the core 9-(2-nitrovinyl)anthracene with number of dienophiles including maleic anhydride, maleimides, acrylonitrile and benzyne. The antiproliferative activity of these compounds was evaluated in BL cell lines EBV^- MUTU-1 and EBV⁺ DG-75 (chemoresistant). The most potent compounds 13j, 15, 16a, 16b, 16c, 16d and 19a displayed IC₅₀ values in the range 0.17-0.38 μM against the BL cell line EBV^- MUTU-1 and IC₅₀ values in the range 0.45-0.78 μM against the chemoresistant BL cell line EBV⁺ DG-75. Compounds 15, 16b and 16c demonstrated potent ROS dependent apoptotic effects on the BL cell lines which were superior to the control drug taxol and showed minimal cytotoxicity to peripheral blood mononuclear cells (PBMCs). The results suggest that this class of compounds merits further investigation as antiproliferative agents for BL.

Keywords: 9,10-dihydro-9,10-ethanoanthracene; Burkitt’s lymphoma; DG-75; MUTU-1; anthracene; apoptosis; maprotiline; nitrostyrene.

Figure 1

Chemical structures of compounds with reported activity against Burkitt’s lymphoma: compounds 1–7, maprotiline 8, ethanoanthracene 9 and nitrostyrene lead compounds 10a–c with target ethanoanthracene structure.

Scheme 1

Synthesis of maleimides 11a–s and nitrovinylanthracenes 12a–f and nitroethylanthracene 12g. Reagents and conditions: (a) RNH₂, diethyl ether, reflux, 1 h. (b) NaOAc, Ac₂O, 120 ^oC, 30 min. (15–70%); (c) Piperidine acetate, excess nitroalkane (CH₃NO₂, CH₃CH₂NO₂ or CH₃CH₂CH₂NO₂), 90 ^oC, N₂, 1.5 h. (50–99%); (d) NaBH₄, (CH₃)₂CHOH, CH₂Cl₂, RT, 24 h, 85%.

Scheme 2

Synthesis of ethanoanthracenes 13a–n (Series I, Table 1), 14a–c (Series II, Table 1), 16a–n (Series IIIA, Table 2), 17a–n, 18 (Series IIIB, Table 3): Reagents and conditions (a) Toluene, 90 °C, 48 h.

Scheme 3

Synthesis of ethanoanthracene dimer 15 (Series 1, Table 1): Reagents and conditions: (a) Toluene, 90 °C, 48 h., (10%).

Scheme 4

Synthesis of ethanoanthracenes 19a–f (Series IV, Table 6). Reagents and conditions: (a) R¹R²C=CHR³, (NCC=CH₂, CH₃CH₂OCOC=CH₂, CH₃OCOC=CH₂, CH₃OCO(CN)C=CH₂), Toluene, 90 °C, 48 h; (b) H₂SO₄, R-OH, (R = CH₃, CH₂CH₃), reflux, 6 h.

Figure 2

X-ray crystallographic molecular structures of 16c, 16j, 17l, 17n, 19a and 20a with atomic displacement shown at 50% probability. Only major disordered moiety shown for 17l. Non-hydrogen atoms labelled where possible.

Scheme 5

Synthesis of ethanoanthracenes 20a–g (Series V, Table 7), 21a–k (Series VI, Table 8) and 23a–k (Series VII, Table 9). Reagents and conditions: (a) Anthranilic acid, CCl₃CO₂H, isoamyl nitrite, 0 °C then toluene, 90 °C, 1 h; (b) NaBH₄, (CH₃)₂CHOH, CH₂Cl₂; (c) Toluene, 90 °C, 48 h.

Figure 3

Overlay of 9,10-dihydroethanoanthracene compounds 13j, 16a, 16b and 19a (yellow) with maprotiline 8 (cyan). The atoms are coloured by element type: oxygen = red, nitrogen = blue, chlorine = green.

Figure 4

Ethanoanthracene nitrostyrene compounds 16a–d, 13j, 19a, 15 induce apoptosis in Burkitt’s lymphoma MUTU-1 cell line. Induction of apoptosis in Burkitt’s lymphoma MUTU-1 cell line following treatment for 24 h with selected ethanoanthracene nitrostyrene compounds (0.2–10 μM) or a comparative control (taxol) in MUTU-1 cell line is determined using Annexin V and propidium iodide staining/FACS. (A) Taxol, 12, 16a; (B) 16b, 16c, 16d; (C) 13j, 19a, 15. Ethanol was used as the vehicle. Values represent the mean of three independent experiments.

Figure 4

Ethanoanthracene nitrostyrene compounds 16a–d, 13j, 19a, 15 induce apoptosis in Burkitt’s lymphoma MUTU-1 cell line. Induction of apoptosis in Burkitt’s lymphoma MUTU-1 cell line following treatment for 24 h with selected ethanoanthracene nitrostyrene compounds (0.2–10 μM) or a comparative control (taxol) in MUTU-1 cell line is determined using Annexin V and propidium iodide staining/FACS. (A) Taxol, 12, 16a; (B) 16b, 16c, 16d; (C) 13j, 19a, 15. Ethanol was used as the vehicle. Values represent the mean of three independent experiments.

Figure 5

Ethanoanthracene nitrostyrene compounds 16a–d, 13j, 19a, 15 induce apoptosis in Burkitt’s lymphoma DG-75 cell line. Induction of apoptosis in Burkitt’s lymphoma DG-75 cell line following treatment for 48 h with selected ethanoanthracene nitrostyrene compounds (0.2–10 μM) or a comparative control (taxol) in DG-75 cell line is determined by Annexin V and propidium iodide staining/FACS: (A) Taxol, 12, 16a; (B) 16b, 16c, 16d; (C) 13j, 19a, 15. Ethanol was used as the vehicle. Values represent the mean of three independent experiments.

Figure 5

Ethanoanthracene nitrostyrene compounds 16a–d, 13j, 19a, 15 induce apoptosis in Burkitt’s lymphoma DG-75 cell line. Induction of apoptosis in Burkitt’s lymphoma DG-75 cell line following treatment for 48 h with selected ethanoanthracene nitrostyrene compounds (0.2–10 μM) or a comparative control (taxol) in DG-75 cell line is determined by Annexin V and propidium iodide staining/FACS: (A) Taxol, 12, 16a; (B) 16b, 16c, 16d; (C) 13j, 19a, 15. Ethanol was used as the vehicle. Values represent the mean of three independent experiments.

Figure 6

Lactate dehydrogenase (LDH) assay of ethanoanthracenes 15, 16b and 16c. Cells were treated with ethanoanthracene compounds 15, 16b and 16c (1 μM and 10 μM) for 24 or 48 h. Induction of necrotic cell death was determined by measuring LDH release in MUTU-I (A,C) and DG-75 cell lines (B,D), with control lysis solution (100% necrosis). Values are shown the mean of three independent experiments.

Figure 7

In Vitro antiproliferative effect of compounds 15, 16b and 16c on (A) PBMCs (24 h), (B) MUTU-1 cell line (24 h) and (C) DG-75 cell line (48 h) at 1 μM and 0.5 μM.

Figure 8

Effect of antioxidant pre-treatment on viability of DG-75 cells treated with compounds 15, 16b and 16c.

Figure 9

Summary of SAR for 9,10-dihydro-9,10-ethanoanthracenes.