Síntesis y evaluación biológica frente a Óxido Nítrico Sintasa de nuevas pirazolinas y derivados de urea y tiourea N, N'-disustituidos

Resumo

INTRODUCTION Nitric oxide (NO) is an inorganic free radical and an important biomessenger that regulates several physiological functions in the nervous, immune, and cardiovascular systems.[1] NO is synthesized from the enzyme catalysis of L-arginine to L-citrulline in several cell types by a family of nitric oxide synthase (NOS) isoenzymes with consumption of molecular oxygen. Native NOS is a homodimeric enzyme. Each monomer consists of an N-terminal oxygenase domain with a catalytic heme active site and a cofactor tetrahydrobiopterin (BH4) binding site, and a C-terminal electron-donating reductase domain binding flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide (NADPH).[2,3] The linker between the two functional domains is a calmodulin (CaM) binding motif that enables electron flow from the oxygenase domain to the reductase domain.[4] In mammalians, three isoforms of NOS have been identified: neuronal (nNOS), endothelial (eNOS) and inducible NOS (iNOS).[5] nNOS and eNOS are constitutive and regulated by intracellular Ca/CaM. They continually produce low levels of NO used for nerve function and blood regulation, respectively. While, iNOS produces large toxic bursts of NO to fight pathogens, and is not Ca-dependent.[6,7] To exert appropriate functions, NO synthesis by the three isozymes is under tight regulation. Thus, overproduction of NO by nNOS has been associated with neurodegenerative disorders such as Alzheimer's, Parkinson's or Huntington's diseases,[8-10] and the inducible isoform seems to be responsible for the massive NO production in pathologies such as arthritis, colitis, tissue damage, cancer or several inflammatory states.[11-13] The NO underproduction by eNOS has been associated with hypertension and atherosclerosis.[14,15] Accordingly, inhibition of nNOS or iNOS, but not of eNOS, could provide an effective therapeutic approach. Previously, our research group has described a series of nNOS inhibitors with a kynurenine,[16] kynurenamine,[17] or 4,5-dihydro-1H-pyrazole structure.[18] AIMS The aim of this PhD dissertation is the development of potential NOS inhibitors. The design, synthesis and biological evaluation against the iNOS and nNOS isoforms of new pyrazolines and N,N¿-disubstituted urea and thiourea derivatives is proposed. The specific aims are the following: 1. Synthesis of the proposed compounds with their corresponding methodologies. 2. Unequivocally characterization of the synthesized molecules using one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry techniques (HRMS) as well as elemental analysis. 3. In vitro biological activity evaluation against nNOS and iNOS of all final products and against eNOS of the most active ones. 4. Assessment of the qualitative and quantitative structure-activity relationships (QSAR) of the three families of compounds. 5. Conducting molecular modeling studies to design and propose a binding mode for these new compounds to i/nNOS. RESULTS A. Pyrazoline derivatives This family of compounds has been designed basing on the results of previous pyrazolines published by our group.[18] In these compounds, the activation and deactivation of the aromatic ring was increased, through R1 substituents, keeping the cyclopropyl and phenyl groups in R2, as well as changing the size and flexibility of the phenyl moiety.[19] The in vitro biological results demonstrate that the increase of R2 size substituent improves the iNOS, as well as the nNOS inhibitory activity. Nevertheless, regarding the R1 substituent in the aromatic ring, electron-withdrawing groups enhance iNOS inhibition, whereas electron-donating substituents get better the nNOS inhibition. This fact is confirmed by docking studies which show the better orientation of 3h (R1 = 4,5-Cl2, R2 = CH2CH2Ph) in iNOS and 3r (R1 = 2,3,4-(OMe)3, R2 = CH2CH2Ph) in nNOS. This last derivative is the most active nNOS inhibitor of all the tested compounds (IC50 = 400 µM), with a good nNOS/iNOS selectivity. B. Kynurenamine-urea and thiourea derivatives These products were designed from the previously published kynurenamines, carrying a urea or thiourea substituted group, isosteric to the final guanidine moiety of L-arginine, the natural substrate of NOS.[20,21] In general, all compounds show, in vitro, better inhibition against iNOS than nNOS, being the chlorinated series the most active compounds. In addition, the urea residue seems to play an important role in compounds selectivity. Thioureas similarly inhibited both isoenzymes, while ureas selectively inhibited iNOS. The urea 5n (R1 = Cl, R2 = Et, X = O) was the most active iNOS inhibitor with IC50 = 100. This was confirmed by docking and MD simulations studies, which showed the more favorable orientation of 5n in iNOS establishing good interactions with the enzyme. Also, this compound did not inhibit eNOS, demonstrating the selectivity necessary to avoid the adverse effect of hypertension. C. 3-Hydroxypropyl urea and thiourea derivatives These compounds have been designed by the reduction of the carbonyl group of the above mentioned kynurenamine-urea and thiourea derivatives. The synthetic route performed to this new family could also be applied to the last family allowing us to obtain the molecules 5 with less synthetic steps (from 8 to 5) and duplicating the global yield. Moreover, using the MW, we could shorten the urea and thiourea formation time from 18h to 20 min with good yield ranging between 60 and 90%. Regarding the biological results, this family mostly inhibits better the neuronal NOS isoform than the inducible one. Furthermore, thioureas exhibit higher inhibition than ureas for both isoforms. Among all the tested compounds, 4g (R1 = OMe, R2 = Me, X = S) shows the best nNOS (IC50 = 130 µM) and iNOS (IC50 = 180 µM) inhibition values without inhibiting eNOS. Such compound could be useful to fight pathologies where both i/nNOS are implicated such as neurodegenerative diseases. REFERENCES 1. Moncada, S. R. M. J.; Palmer, R. M. L.; Higgs, E. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 1991, 43, 109-142. 2. Griffith, O. W.; Stuehr, D. J. Nitric Oxide Synthases: Properties and Catalytic Mechanism. Annu. Rew. Physiol. 1995, 57, 707-736. 3. Roman, L. J.; Martásek, P.; Masters, B. S. S. Intrinsic and extrinsic modulation of nitric oxide synthase activity. Chem. Rev. 2002, 102, 1179-1190. 4. Knowles, R. G.; Moncada, S. Nitric oxide synthases in mammals. Biochem. J. 1994, 298(Pt 2), 249-258. 5. Zhou, L.; Zhu, D-Y. Neuronal nitric oxide synthase: structure, subcellular localization, regulation, and clinical implications. Nitric Oxide-Biol. Chem. 2009, 20, 223-230. 6. Groves, J. T.; Wang, C. C. Nitric oxide synthase: models and mechanisms. Curr. Opin. Chem. Biol. 2000, 4, 687-695. 7. Aktan, F. iNOS-mediated nitric oxide production and its regulation. Life sci. 2004, 75, 639-653. 8. Wilcock, D. M.; Lewis, M. R.; Van Nostrand, W. E.; Davis, J.; Previti, M. L.; Gharkholonarehe, N.; Vitek, M. P.; Colton, C. A. Progression of amyloid pathology to Alzheimer's disease pathology in an amyloid precursor protein transgenic mouse model by removal of nitric oxide synthase 2. J. Neurosci. 2008, 28, 1537-1545. 9. Calabrese, V.; Mancuso, C.; Calvani, M.; Rizzarelli, E.; Butterfield, D. A.; Stella, A. M. G. Nitric oxide in the central nervous system: neuroprotection versus neurotoxicity. Nat. Rev. Neurosci. 2007, 8, 766-775. 10. Deckel, A. W. Nitric oxide and nitric oxide synthase in Huntington's disease. J. Neurosci. Res. 2001, 64, 99-107. 11. Kröncke, K. D.; Fehsel, K.; Kolb-Bachofen, V. Inducible nitric oxide synthase in human diseases. Clin. Exp. Pharmacol. Physiol. 1998, 113, 147-156. 12. Lechner, M.; Lirk, P.; Rieder, J. Inducible nitric oxide synthase (iNOS) in tumor biology: the two sides of the same coin. Seminars in cáncer biology 2005, Vol. 15, No. 4, 277-289. Academic Press. 13. Zamora, R.; Vodovotz, Y.; Billiar, T. R. Inducible nitric oxide synthase and inflammatory diseases. Mol. Med. 2000, 6, 347. 14. Taddei, S.; Virdis, A.; Ghiadoni, L.; Sudano, I.; Salvetti, A. Endothelial dysfunction in hypertension. J. Cardiovasc. Pharmacol. 2001, 38, S11-S14. 15. Napoli, C.; de Nigris, F.; Williams-Ignarro, S.; Pignalosa, O.; Sica, V.; Ignarro, L. J. Nitric oxide and atherosclerosis: an update. Nitric oxide 2006, 15, 265-279. 16. Camacho, E.; León, J.; Carrión, A.; Entrena, A.; Escames, G.; Khaldy, H.; Acuña-Castroviejo, D.; Gallo, M. A.; Espinosa, A. Inhibition of nNOS activity in rat brain by synthetic kynurenines: Structure-activity dependence. J. Med. Chem. 2002, 45, 263-274. 17. Entrena, A.; Camacho, M. E.; Carrión, M. D.; López-Cara, L. C.; Velasco, G.; León, J.; Escames, G.; Castroviejo-Acuña, D.; Tapias, V.; Gallo, M. A.; Vivó, A.; Espinosa, A. Kynurenamines as neural nitric oxide synthase inhibitors. J. Med. Chem. 2005, 48, 8174-8181. 18. Camacho, M. E.; León, J.; Entrena, A.; Velasco, G.; Carrión, M. D.; Escames, G.; Vivó, A.; Acuña-Castroviejo, D.; Gallo, M. A.; Espinosa, A. 4, 5-Dihydro-1 H-pyrazole Derivatives with Inhibitory nNOS Activity in Rat Brain: Synthesis and Structure-Activity Relationships. J. Med. Chem. 2004, 47, 5641-5650. 19. Carrión, M. D.; Chayah, M.; Entrena, A.; López, A.; Gallo, M. A.; Acuña-Castroviejo, D.; Camacho, M. E. Synthesis and biological evaluation of 4,5-dihydro-1H-pyrazole derivatives as potential nNOS/iNOS selective inhibitors. Part 2: Influence of diverse substituents in both the phenyl moiety and the acyl group. Bioorg. Med. Chem. 2013, 21, 4132-4142. 20. Chayah, M.; Carrión, M. D.; Gallo, M. A.; Jiménez, R.; Duarte, J.; Camacho, M. E. Development of Urea and Thiourea Kynurenamine Derivatives: Synthesis, Molecular Modeling, and Biological Evaluation as Nitric Oxide Synthase Inhibitors. ChemMedChem 2015, 10, 874-882. 21. Chayah, M.; Carrión, M. D.; Gallo, M. A.; Choquesillo-Lazarte D.; Camacho, M. E. NMR assignments and structural characterization of new thiourea and urea kynurenamine derivatives nitric oxide synthase inhibitors. Magn. Reson. Chem. 2015, DOI: 10.1002/mrc.4295.