EXTRACTION AND SPECTROSCOPIC AUTHENTICATION OF THYMOQUINONE FROM NIGELLA SATIVA AND THE SYNTHESIS, CHARACTERISATION AND IN VITRO HIV-1 REVERSE TRANSCRIPTASE INHIBITION OF PYRIDINONE ANALOGUES

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) remain central to HIV-1 therapy, but their durability is limited by resistance mutations in the reverse transcriptase (RT) non-nucleoside binding pocket. Thymoquinone (TQ), the principal quinone of Nigella sativa, has a broad pharmacological profile but no established anti-HIV pharmacophore. We report the experimental arm of an integrated programme that uses TQ as a seed for RT-directed pyridinone leads. TQ was isolated from cold-pressed N. sativa seed oil by silica-gel column chromatography and recrystallisation, and authenticated by proton and carbon NMR and high-resolution mass spectrometry (HRMS): crude oil yield 32% w/w of seed, TQ content 1.1% w/w, isolated TQ 0.9% w/w of oil at 96% purity, melting point 42–44 °C; HRMS confirmed C₁₀H₁₂O₂. Three leads were prepared and characterised: a virtual-screening pentacenedione (Ligand 1), a rationally designed pyrazolopyridinyl pyridin-2(1H)-one (Ligand 2, TQ-PZP-03) and a 3D-QSAR-benchmark pyridinone (Ligand 3, VC1). Recasting the quinone as a pyridin-2(1H)-one removed the reactive Michael-acceptor motif and installed a lactam N–H hydrogen-bond donor. In a colorimetric HIV-1 RT inhibition assay benchmarked against efavirenz (EC₅₀ 4.1 µg mL⁻¹), TQ and Ligand 3 were the most active test compounds (EC₅₀ 11.7 and 19.4 µg mL⁻¹; rated high), while Ligands 1 and 2 were weaker (105 and 67.1 µg mL⁻¹). The results establish TQ and the adamantyl-pyridinone as tractable, chemically validated scaffolds for optimisation as broad-spectrum RT inhibitors. Thaizolidine, Biological activities, etc.