A new class of anti-proliferative activity and apoptotic inducer with molecular docking studies for a novel of 1,3-dithiolo[4,5- b]quinoxaline derivatives hybrid with a sulfonamide moiety

Abstract

A new series of 6-(pyrrolidin-1-ylsulfonyl)-[1,3]dithiolo[4,5-b]quinoxaline-2-ylidines 10a-f, 12, 14, 16, and 18 were designed, synthesized, and evaluated for their in vitro anticancer activity. The structures of the novel compounds were systematically characterized by ¹H NMR, ¹³C NMR, and elemental analysis. The synthesized derivatives were evaluated for their in vitro antiproliferative activity against three human cancer cell lines (HepG-2, HCT-116, and MCF-7) with more sensitivity to MCF-7. Moreover, three derivatives 10c, 10f, and 12 were the most promising candidates with sub-micromole values. These derivatives were further evaluated against MDA-MB-231, and the results displayed significant IC₅₀ values ranging from 2.26 ± 0.1 to 10.46 ± 0.8 μM and showed low cellular cytotoxicity against WI-38. Surprisingly, the most active derivative 12 revealed sensitivity towards the breast cell lines MCF-7 (IC₅₀ = 3.82 ± 0.2 μM) and MDA-MB-231 (IC₅₀ = 2.26 ± 0.1 μM) compared with doxorubicin (IC₅₀ = 4.17 ± 0.2 and 3.18 ± 0.1 M). Cell cycle analysis showed that compound 12 arrests and inhibits the growth of MCF-7 cells in the S phase with values of 48.16% compared with the untreated control 29.79% and exhibited a significantly higher apoptotic effect in MCF-7 with a value of 42.08% compared to control cell at 1.84%. Furthermore, compound 12 decreased Bcl-2 protein 0.368-fold and activation on pro-apoptotic genes Bax and P53 by 3.97 and 4.97 folds, respectively, in MCF-7 cells. Compound 12 exhibited higher inhibitory activity to EGFR^Wt, EGFR^L858R, and VEGFR-2 with IC₅₀ values (0.19 ± 0.009, 0.026 ± 0.001, and 0.42 ± 0.021 μM) compared with erlotinib (IC₅₀ = 0.037 ± 0.002 and 0.026 ± 0.001 μM) and sorafenib (IC₅₀ = 0.035 ± 0.002 μM). Finally, in silico ADMET prediction presented that 1,3-dithiolo[4,5-b]quinoxaline derivative 12 obeys the Lipinski rule of five and the Veber rule with no PAINs alarms and moderately soluble properties. Additionally, toxicity prediction revealed that compound 12 demonstrated inactivity to hepatotoxic carcinogenicity, immunotoxicity, mutagenicity, and cytotoxicity. Moreover, molecular docking studies showed good binding affinity with lower binding energy inside the active site of Bcl-2 (PDB: 4AQ3), EGFR (PDB: 1M17), and VEGFR (PDB: 4ASD).

Fig. 1. Rational study involved the most bioactive cores as quinazoline (quinoxaline analog), pyrrolidine, and sulfonamide moiety, and our newly designed 1,3-dithiolo[4,5-b]quinoxaline derivatives.

Scheme 1. Synthetic pathways of the 2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonyl chloride 3, 3-chloro-6-(pyrrolidin-1-ylsulfonyl)quinoxalin-2(1H)-one 6, and 2,3-dichloro-6-(pyrrolidin-1-ylsulfonyl)quinoxaline 7.

Scheme 2. Synthetic pathways of the target 6-(pyrrolidin-1-ylsulfonyl)-[1,3]dithiolo[4,5-b]quinoxaline-2-ylidines (10a–f).

Scheme 3. Illustration on the resection mechanism of bi-nucleophile 1,3-dithiolo with unsymmetrical from bi-electrophile and 2,3-dichloro derivative 7.

Scheme 4. Synthesis of novel [1,3]dithiolo[4,5-b]quinoxalines 12, 14, 16, and 18 linked to cyclohexyl, pyrazolyl, and pyrimidinyl moieties.

Fig. 2. Cell cycle distribution% assessment using FACS analysis (a) untreated cell; (b) compound 12 treated with MCF-7 at (IC₅₀ = 3.82 μM).

Fig. 3. The graph shows the apoptosis-inducing effects of the most active derivative 12 in MCF-7 when treated at its IC₅₀ for 24 h (a) untreated cell; (b) compound 10/MFC-7.

Fig. 4. Inhibitory percentage of 1,3-dithiolo[4,5-b]quinoxaline derivative 12 and positive controls (doxorubicin, erlotinib, and sorafenib) against EGFR and VEGFR-2 enzymes.

Fig. 5. Bioavailability radar chart generated by Swiss-ADME for (A) the most active compound 12, (B) doxorubicin, (C) erlotinib, and (D) sorafenib.

Fig. 6. Represented the 2D and 3D binding modes of (A) co-crystallized ligand and (B) compound 12 inside Bcl-2 binding pocket (PDB: 4AQ3).

Fig. 7. Represented the 2D and 3D binding modes of (A) co-crystallized ligand and (B) compound 12 inside the EGFR binding pocket (PDB: 1M17).

Fig. 8. Represented the 2D and 3D binding modes of (A) co-crystallized ligand and (B) compound 12 inside the VEGFR-2 binding pocket (PDB: 4ASD).