Synthesis of fluorinated tubastatin A derivatives with bi-, tri-, and tetracyclic cap groups: molecular docking with HDAC6 and evaluation of in vitro antitumor activity†

Herein, we report the synthesis of 16 tubastatin A derivatives with fluorinated bi-, tri-, and tetracyclic cap groups. Most derivatives show strong in vitro antitumor activity, achieving micromolar or sub-micromolar efficacy. The most promising compound, 4-(6-bromo-3,3-difluoro-1,2,3,4-tetrahydro-9H-carbozol-9-yl)methyl)-N-hydroxybenzamide (14f), demonstrated potent anti-proliferative effects against human nasopharyngeal carcinoma cells (SUNE1) and human breast cancer cells (MDA-MB-231), with IC₅₀ values of 0.51 μM and 0.52 μM, respectively. Notably, compound 4-((8-fluoroindeno[2,1-b]indol-5(6H)-yl)-N-hydroxybenzamide (13c) exhibited significant anti-proliferative activity against pancreatic cancer cells (SW1990), with an IC₅₀ of 2.06 μM and low cytotoxicity to normal cells. Overall, variations in the cap group from bi- to tri-, then to tetracyclic, and the introduction of fluorinated groups enhance the antitumor activity of these derivatives. Among them, difluoromethyl-modified tricyclic derivatives show a broad spectrum in vitro antitumor effect. Molecular docking studies indicate that these derivatives bind to Histone Deacetylase 6 (HDAC6) at low binding energies, ranging from −6.54 to −9.84 kcal mol⁻¹, through metal complexation, hydrogen bonding, π–π stacking, and π–cation interactions, which correlates with their good antitumor activity. Compound 4-((2-fluoro-5,6-dihydro-7H-benzo[c]carbazol-7-yl)methyl)-N-hydroxybenzamide (13a) with the lowest binding energy of −9.84 kcal mol⁻¹ exhibited the best in vitro antitumor activity against MCF-7, with IC₅₀ of 1.98 μM.