In this work, 2′-alkoxymethyl substituted klavuzon derivatives were prepared starting from 2-methyl-1-naphthoic acid in eight steps. Anticancer potencies of the synthesized compounds were evaluated by performing MTT cell viability test over cancerous and healthy pancreatic cell lines, along with CRM1 inhibitory properties in HeLa cells by immunostaining and Topo I inhibition properties by supercoiled DNA relaxation assay. Their cytotoxic activities were also presented in hepatocellular carcinoma cells (HuH-7) derived 3D spheroids. Among the tested klavuzon derivatives, isobutoxymethyl substituted klavuzon showed the highest selectivity of cytotoxic activity against pancreatic cancer cell line. They showed potent Topo I inhibition while their CRM1 inhibitory properties somehow diminished compared to 4′-alkylsubstituted klavuzons. The most cytotoxic 2′-methoxymethyl derivative inhibited the growth of the spheroids derived from HuH-7 cell lines and PI staining exhibited time and concentration dependent cell death in 3D spheroids.Copyright © 2020 Elsevier Inc. All rights reserved.