Of the acidic hydroxyazoles involved hydroxypyrazole had the

Of the acidic hydroxyazoles involved, hydroxypyrazole had the weakest acidic profile (pKa in the range of 6–7), although highly deprotonated at physiological pH. During hit optimization, the two pyrazole ring positions available for substitution provide an opportunity for a better exploration of the chemical space, which allows for accessing additional binding areas of the target protein [23]. For these reasons, and in order to identify new hits besides 1, a series of pyrazoles that were already present in our library [24,25], were initially assayed for PfDHODH activity. This screening identified 7a (Fig. 3), as the best PfDHODH inhibitor in the μM range. Pyrazole 7a is characterized by the presence of a bulky substitution at position 5 and a carboxylic Thiorphan synthesis function at position 4. With the aim of more extensive study of its SAR, in the following the 7a structure was extensively investigated by modulating four different positions (series 2, Fig. 3). The synthetic strategies used for obtaining the two designed series of compounds are presented here and fully discussed together with the compounds’ biological profile in enzymatic and cell-based assays. The X-ray crystallography structures of the ligand - PfDHODH complexes of the most representative compounds were also determined in order to identify experimentally the binding poses.
Result and discussion
Conclusions We herein describe the synthesis of two series of new PfDHODH inhibitors based on the hydroxyazole scaffold. In the first series of compounds, the 2-hydroxy-3,5-dichlorophenyl moiety of 1 [21], was replaced with three different hydroxyazole systems (3 as best compound). The second series includes a modulation of a 3-hydroxypyrazole-4-carboxylic acid, a scaffold that had never before been explored in the field of PfDHODH inhibition. This led to the identification of 7e, which selectively inhibited PfDHODH in vitro and achieved a single digit μM IC50 value. 7e was also active on the parasite (40.7 μM). The crystal structures of the complexes between the most interesting compounds in the two series (compounds 3 and 7e), and PfDHODH led to the identification of their binding modes, which is essential for the subsequent application of a hit-to-lead process necessary to improve their activity and drug-like properties. These studies are under development and will be the subjects of forthcoming publications.
Experimental section
Notes
PDB ID codes The atomic coordinates and structure factors of PfDHODH in complex with compounds 3 (PDB id: 6I55) and 7e (PDB id: 6I4B) have been deposited in the RCSB Protein Data Bank.
Acknowledgements This research was supported by funding from the University of Turin, Ricerca Locale 2016 and 2017 (Grant numbers LOLM_RILO_17_01, LOLM_RILO_18_01, BOSD_RILO_17_01 and BOSD_RILO_18_01) and from Ministero degli Affari Esteri e della Cooperazione Internazionale (Grant number PGR00978). FTMC and CHA labs are supported by CNPq and FAPESP grants and they are CNPq research fellows. GCC and TAT received FAPESP (n° 2015/20774-6) and CAPES fellowships, respectively. The authors would like to thank Davide Bonanni for the helpful discussions on the compound binding mode and for preparing the final crystallographic figures, Livio Stevanato for maintaining the NMR instrumentation and Dale James Matthew Lawson for proofreading the final manuscript.
Dihydroorotate dehydrogenase (DHODH) is an enzyme essential to pyrimidine de novo biosynthesis. It catalyzes oxidative conversion of dihydroorotate to orotate using the co-factors flavin mononucleotide (FMN) and ubiquinone (CoQ) in the redox process. Since blocking pyrimidine biosynthesis has an antiproliferative effect on rapidly dividing cells, inhibitors of human DHODH (DHODH) have been pursued and developed for the treatment of cancer, and immunological disorders, such as rheumatoid arthritis and multiple sclerosis., , The DHODH enzymes of parasitic pathogens such as (DHODH) and are also attractive targets for the development of new therapeutics to combat malaria and sleeping sickness., ,