CDK2 x Tubulin

A novel chrysin-ferrocene Schiff base (CFSB) was synthesised as a potential anticancer agent. CFSB demonstrated high cytotoxicity against cancer cells with HepG2 (liver) being the most susceptible (IC₅₀ = 3.11 µM). The compound was less toxic towards normal MRC5 cells and exhibited ∼5-fold selectivity towards most cancer cells. CFSB caused G1-phase arrest, induced caspase-dependent apoptosis by increasing Bax/Bcl2 ratio and reduced metastasis by decreasing MMP9 in HepG2. Furthermore, CFSB was inactive against CDK2, EGFR, TrkA and VEGFR, but it strongly inhibited topoisomerase II (IC₅₀ = 20 µM) with potency comparable to etoposide (IC₅₀ = 15 µM), while weak inhibition was observed against tubulin (IC₅₀ = 76 µM). DFT calculations revealed that CFSB had desirable reactivity, while docking indicated high binding affinity with topoisomerase II. Molecular dynamics and MM-GBSA analyses showed that CFSB-topoisomerase II complex was stable with favourable binding energies, while in silico ADMET studies showed drug-like properties for CFSB.

A series of new pyrazolo[1,5-a]pyrimidine derivatives was designed and synthesized as dual CDK2/tubulin polymerization inhibitors. MTT cytotoxicity assay was conducted against five cancer cell lines and one normal cell line. Compounds 6h, and 6q displayed the highest antiproliferative activity, with average IC₅₀ values of 7.01 and 17.37 μM, respectively, against the tested cancer cell lines. MDA-MB-231 triple-negative breast cancer (TNBC) cells were the most sensitive cell lines to 6q and 6h with IC₅₀ values of 6.44 and 12.75 μM, respectively. Cell cycle analysis of MDA-MB-231 cells post-treatment with 6h and 6q revealed remarkable arrest at S/G2-M transition. Both compounds increased the total apoptotic population in MDA-MB-231 apoptosis detection flowcytometry assay. Evaluation of apoptotic markers through ELISA assays of intrinsic pathway markers; P53, Bax, Bcl-2, cytochrome-c, caspase-9, extrinsic pathway markers; caspase-8 and the executioner caspase-3 showed activation of both intrinsic and extrinsic pathways in apoptosis induction. Both compounds were able to inhibit cyclin-dependent kinase-2 CDK2/Cyclin A in enzyme assay with IC₅₀ values of 3.41 ± 0.15 and 6.37 ± 0.41 μM for 6q and 6h, respectively. Tubulin polymerization kinetic assay disclosed that compound 6h was able to inhibit tubulin polymerization by 70.9 % a more potent effect compared to 45.7 % for colchicine indicating CDK2/Tubulin dual Inhibition properties. The effect of 6h was further confirmed by its effect on reducing detectable β-tubulin levels by 43 %. Density functional theory (DFT) computational studies were performed to explain the electronic arrangement, energy gap and reactivity descriptors of compounds 6h and 6q. Molecular docking manifested strong interaction of both 6h and 6q with catalytic residues of CDK2 binding pocket with binding scores of -7.0582 and -6.3965 kcal/mol, respectively. Molecular docking of 6h at the tubulin colchicine binding site CBS disclosed strong multiple interactions with both α and β tubulin residues with a binding score of -7.61 kcal/mol. ADME prediction study predicted favorable pharmacokinetic properties. Both 6h and 6q provided the basis for the development of a more potent series of pyrazolo[1,5-a] pyrimidine-based CDK2/tubulin dual inhibitors.

In this study, we synthesized a new-generation library of colchicine derivatives via cycloaddition of colchicine utilizing position C-8 and C-12 diene system regioselectivity with aryne precursor to generate a small, focused library of derivatives. We assessed their anticancer activity against various cancer cell lines like MCF-7, MDA-MB-231, MDA-MB-453, and PC-3. Normal human embryonic kidney cell line HEK-293 was used to determine the toxicity. Among these derivatives, silicon-tethered compound B-4a demonstrated the highest potency against breast cancer cells. Subsequent mechanistic studies revealed that B-4a effectively modulates cell cycle regulatory kinases (CDK-2 and CDK-4) and their associated cyclins (cyclin-B1, cyclin-D1), inducing apoptosis. Additionally, B-4a displayed a noteworthy impact on tubulin polymerization, compared to positive control flavopiridol hydrochloride in a dose-dependent manner, and significantly disrupted the vimentin cytoskeleton, contributing to G1 arrest in breast cancer cells. Moreover, B-4a exhibited substantial anti-metastatic properties by inhibiting breast cancer cell migration and invasion. These effects are attributed to the down-regulation of major epithelial to mesenchymal transition (EMT) factors, including vimentin and Twist-1, and the upregulation of the epithelial marker E-cadherin in an apoptosis-dependent manner.