Novel pyrazolo[3,4-d] pyrimidine derivatives bearing carbon-aryl(heteryl)idene moieties were synthesized via a condensation reaction of 5-aminopyrazoles and cyclic lactams. The preparation of the target compounds employed bioisosterism, where a pyrazole ring was a major replacement. Fifteen target compounds were investigated for their antiproliferative activity on five human cancer cell lines; derivative (E)− 1-methyl-9-(3,4,5-trimethoxybenzylidene)− 6,7,8,9-tetrahydropyrazolo[3,4-d]pyrido[1,2-a]pyrimidin-4(1H)-one (10k) showed the highest activity (IC₅₀ value (0.03–1.6 μM), on selected cell lines. Results of an in vivo experiment on an HT-29 xenograft nude mouse model also confirmed that 10k inhibited tumor growth. The proposed anticancer mechanism of 10k in HT-29 and HCT-116 cells was that 10k caused G2/M phase arrest in cancer cells and decreased the mitochondrial membrane potential (Δψmt). Additional studies were conducted on HUVEC, where 10k significantly inhibited HUVEC cell migration, adhesion, and tube formation activity. Molecular modeling studies revealed that 10k forms hydrogen bonds with cys-919 of vascular endothelial growth factor receptor 2 (VEGFR-2) and inhibit VEGFR-2 kinase activity. Moreover, tubulin polymerization assay results showed that 10k formed hydrogen bonds with Asn-101 and Gln-11 of tubulin. Furthermore, it could change the aberration of microtubule arrangements in HUVEC and inhibit tubulin polymerization. These results indicate that the main anticancer activity of 10k may be mediated by anti-vascular effects and inhibition of tubulin polymerization in pre-clinical trials.