Discovery of New Antiproliferative Imidazopyrazole Acylhydrazones Able To Interact with Microtubule Systems

Abstract

Even though immunotherapy has radically changed the search for anticancer therapies, there are still many different pathways that are open to intervention with traditional small molecules. To expand our investigation in the anticancer field, we report here a new series of compounds in which our previous pyrazole and imidazopyrazole scaffolds are linked to a differently decorated phenyl ring through an acylhydrazone linker. Preliminary tests on the library were performed at the National Cancer Institute (USA) against the full NCI 60 cell panel. The best compounds among the imidazopyrazole series were then tested by immunofluorescence staining for their inhibition of cell proliferation, apoptosis induction, and their effect on the cell cycle and on microtubules. Two compounds, in particular 4‐benzyloxy‐3‐methoxybenzyliden imidazopyrazole‐7‐carbohydrazide showed good growth inhibition, with IC50 values in the low‐micromolar range, and induced apoptosis. Both compounds altered the cell‐cycle phases with the appearance of polyploid cells. Immunofluorescence analysis evidenced microtubules alterations; tubulin polymerization assays and docking studies suggested the tubulin system to be the possible, although not exclusive, target of the new acylhydrazone series reported here.

Insights into the Pharmacological Activity of the Imidazo−Pyrazole Scaffold

“…[26] Therefore, platelets could be a simple, economic and suitable cellular model based on a causal relationship between inflammation and tumorigenesis. [2,27] As previous imidazoÀ pyrazoles 2 blocked ROS production in platelet and to confirm anti-inflammatory activity of imidazoÀ pyrazole core already reported, [16,17] the inhibition of platelet aggregation and ROS production on human platelets were evaluated for the most active compounds 3 a, 3 e, 4 c, 5 g and 5 h. As reported in Table 4, compounds 3 a and 4 c showed the lower IC 50 values for platelet aggregation and ROS production inhibition, suggesting that the shift of the acylhydrazonic substituent from position 7 to position 6 (derivatives 5 g,h) or the insertion of bulky substituents on the catechol ring (derivative 3 e) have a detrimental effect on this activity. This specific trend agrees with previous results regarding compounds 2, where more embedded 2 a and 2 b (more similar to 3 e, 5 g and 5 h) resulted less active as ROS production inhibitors than 2 c, characterized by a smaller substituent on para position of catechol moiety.…”

“…As previous compounds 2 (particularly 2 a and 2 b) interfere with tubulin system, [16] the ability of new imidazoÀ pyrazoles 3 e and 5 h (close analogues of 2 a and 2 b) to bind the colchicine binding site in the polymeric complex tubulin α/tubulin β/ stathmin4 (PDB ID: 6XER) [31] was evaluated by docking simulations (AutoDock 4.2). [32] Both enantiomers of the two selected ligands were considered, whereas the stereochemistry of the hydrazone CH=N group was kept fixed in the E conformation, as assessed by NMR study.…”

“…[11,12] More recently, other imidazoÀ pyrazoles (compounds 2, Figure 1), sharing with a number of new anticancer drugs a differently decorated acylhydrazone moiety, [13][14][15] evidenced interesting antiproliferative and antioxidant properties. [16,17] Interestingly, compounds with bulky substituents at para position of catechol ring (derivatives 2 a and 2 b, Figure 2) evidenced a remarkable anticancer activity through the interaction with different intracellular systems, including tubulin, [16] whereas derivatives characterized by a smaller substituent (e. g., 2 c, Figure 2) inhibited reactive oxygen species (ROS) production. [17] To further extend the structure-activity relationships (SARs) of derivatives 2 and to identify novel imidazoÀ pyrazoles endowed with antiproliferative/anti-inflammatory properties, we designed and synthesized a new library (twenty compounds) of imidazoÀ pyrazole compounds 3-5 ( Figure 2, Table 1).…”

“…Moreover, some derivatives proved to reduced angiogenesis process in VEGF‐stimulated human umbilical vein endothelial cells (HUVEC) and in neuroblastoma cell lines [11,12] . More recently, other imidazo−pyrazoles (compounds 2 , Figure 1), sharing with a number of new anticancer drugs a differently decorated acylhydrazone moiety, [13–15] evidenced interesting antiproliferative and antioxidant properties [16,17] . Interestingly, compounds with bulky substituents at para position of catechol ring (derivatives 2 a and 2 b , Figure 2) evidenced a remarkable anticancer activity through the interaction with different intracellular systems, including tubulin, [16] whereas derivatives characterized by a smaller substituent (e. g., 2 c , Figure 2) inhibited reactive oxygen species (ROS) production [17] …”

“…Then, for the most promising molecules, we assessed cytotoxicity, ROS production inhibition and in silico pharmacokinetics and drug‐likeness properties. Finally, as previous imidazo−pyrazoles 2 a , b resulted active as tubulin polymerization inhibitors, [16] molecular docking and molecular dynamic simulations on tubulin system were carried out for the most potent antiproliferative compounds.…”