Novel Regioselective Synthesis of 1,3,4,5-Tetrasubstituted Pyrazoles and Biochemical Valuation on F1FO-ATPase and Mitochondrial Permeability Transition Pore Formation

Vincenzo Algieri; Cristina Algieri; Paola Costanzo; Giulia Fiorani; Antonio Jiritano; Fabrizio Olivito; Matteo Antonio Tallarida; Fabiana Trombetti; Loredana Maiuolo; Antonio De Nino; Salvatore Nesci

doi:10.3390/pharmaceutics15020498

Novel Regioselective Synthesis of 1,3,4,5-Tetrasubstituted Pyrazoles and Biochemical Valuation on F₁F_O-ATPase and Mitochondrial Permeability Transition Pore Formation

Pharmaceutics. 2023 Feb 2;15(2):498. doi: 10.3390/pharmaceutics15020498.

Affiliations

¹ Department of Chemistry and Chemical Technologies, University of Calabria, Via P. Bucci, Cubo 12C, 87036 Rende, CS, Italy.
² Department of Veterinary Medical Sciences, Mitochondrial Biochemistry Lab, Via Tolara di Sopra, 50, 40064 Ozzano Emilia, BO, Italy.
³ Department Molecular Sciences and Nanosystems, University Ca' Foscari Venezia, Via Torino 155, 30172 Venezia Mestre, VE, Italy.

Abstract

An efficient, eco-compatible, and very cheap method for the construction of fully substituted pyrazoles (Pzs) via eliminative nitrilimine-alkene 1,3-dipolar cycloaddition (ENAC) reaction was developed in excellent yield and high regioselectivity. Enaminones and nitrilimines generated in situ were selected as dipolarophiles and dipoles, respectively. A deep screening of the employed base, solvent, and temperature was carried out to optimize reaction conditions. Recycling tests of ionic liquid were performed, furnishing efficient performance until six cycles. Finally, a plausible mechanism of cycloaddition was proposed. Then, the effect of three different structures of Pzs was evaluated on the F₁F_O-ATPase activity and mitochondrial permeability transition pore (mPTP) opening. The Pz derivatives' titration curves of 6a, 6h, and 6o on the F₁F_O-ATPase showed a reduced activity of 86%, 35%, and 31%, respectively. Enzyme inhibition analysis depicted an uncompetitive mechanism with the typical formation of the tertiary complex enzyme-substrate-inhibitor (ESI). The dissociation constant of the ESI complex (K_i') in the presence of the 6a had a lower order of magnitude than other Pzs. The pyrazole core might set the specific mechanism of inhibition with the F₁F_O-ATPase, whereas specific functional groups of Pzs might modulate the binding affinity. The mPTP opening decreased in Pz-treated mitochondria and the Pzs' inhibitory effect on the mPTP was concentration-dependent with 6a and 6o. Indeed, the mPTP was more efficiently blocked with 0.1 mM 6a than with 1 mM 6a. On the contrary, 1 mM 6o had stronger desensitization of mPTP formation than 0.1 mM 6o. The F₁F_O-ATPase is a target of Pzs blocking mPTP formation.

Keywords: 1,3-dipolar cycloaddition; F1FO-ATPase; hydrazonyl chlorides; mitochondrial permeability transition pore; pyrazoles.

Grants and funding

This work was financed by the University of Bologna, Italy, the RFO grant to SN, and an Alma Idea 2022 grant to SN. This research was also funded by the University of Calabria and Calabria Region (PAC CA-LABRIA 2014–2020-Asse Prioritario 12, Azione B 10.5.12 CUP: H28D19000040006). We thank the Italian Ministry of University and Research (MUR) for two doctoral grants.