A new series of pregnenolone derivatives was synthesized by parallel synthesis. Their anticancer activity against cancer cell lines was evaluated and SAR established. Display omitted ► The synthesis of a series of pregnenolone derivatives 2– 107 has been described which were evaluated for their cytotoxicity against HepG2 and MDA-MB-231 cell lines. ► Among all derivatives, the heterocyclic enone 8 (IC 50 = 0.74 μM/mL against HepG2), its pyrazoline derivative 48 (IC 50 = 0.91 μM/mL against MDA-MB-230 cancer cell lines) were identified as the most active compounds. ► All ( O-carboxymethyl) oxime, hydazones and pyrazoles derivatives of pregnenolone were found to be inactive against both the cancer cell lines. Pregnenolone ( 1) was used as a template to develop new anticancer compounds. Ring-D modification of 1 resulted in the synthesis of benzylidenes 2– 17, pyrazolines 18– 76, pyrazoles 85– 91, hydrazones 77– 84, and oximes 92– 107 derivatives. The structure of compound 107 was also deduced through single crystal X-ray diffraction studies. The inclusion of furanyl and pyridyl rings to pregnenolone skeleton increases the cytotoxicity of all compounds significantly. Among benzylidene derivatives, only heterocyclic enone 8 (IC 50 = 0.74 μM/mL against HepG2), and 17 (IC 50 = 4.49 μM/mL against HepG2, IC 50 = 5.01 μM/mL against MDA-MB-230 cancer cell line) exhibited a significant activity. The cytotoxicity data of pyrazoline derivatives 18– 76 revealed that only furanyl bearing pyrazolines 40, 42– 44, 48, and 49 exhibited significant activities. While all ( O-carboxymethyl) oximes, hydazones, and pyrazoles derivatives of pregnenolone did not show any significant activity against both the cell lines. Thus the furanyl bearing enone 8 (IC 50 = 0.74 μM/mL against HepG2), and its pyrazoline derivative 48 (IC 50 = 0.91 μM/mL against MDA-MB-230 cancer cell lines) were identified as the most active compounds in all derivatives of pregnenolone.