Crocetti L et al. (2024), 3,8-Disubstituted Pyrazolo[1,5-a]quinazoline as...

XB-ART-61016

Int J Mol Sci 2024 Oct 09;2519:. doi: 10.3390/ijms251910840.

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3,8-Disubstituted Pyrazolo[1,5-a]quinazoline as GABAA Receptor Modulators: Synthesis, Electrophysiological Assays, and Molecular Modelling Studies.

Crocetti L , Guerrini G , Melani F , Mascia MP , Giovannoni MP .

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As a continuation of our study in the field of GABAA receptor modulators, we report the design and synthesis of new pyrazolo[1,5-a]quinazoline (PQ) bearing at the 8-position an oxygen or nitrogen function. All the final compounds and some intermediates, showing the three different forms of the pyrazolo[1,5-a]quinazoline scaffold (5-oxo-4,5-dihydro, -4,5-dihydro, and heteroaromatic form), have been screened with an electrophysiological technique on recombinant GABAAR (α1β2γ2-GABAAR), expressed in Xenopus laevis oocytes, by evaluating the variation in produced chlorine current, and permitting us to identify some interesting compounds (6d, 8a, 8b, and 14) on which further functional assays were performed. Molecular modelling studies (docking, minimization of complex ligand-receptor, and MD model) and a statistical analysis by a Hierarchical Cluster Analysis (HCA) have collocated these ligands in the class corresponding to their pharmacological profile. The HCA results are coherent with the model we recently published (Proximity Frequencies), identifying the residues γThr142 and αHis102 as discriminant for the agonist and antagonist profile.

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Genes referenced: gabarap

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	Scheme 1. Reagents and conditions: (a) DMF, sodium acetate, reflux temperature, 2–5 h; (b) HCl for obtaining 3a, reflux temperature; H3PO4 for obtaining 3c–e at 150 °C.
	Scheme 2. Reagents and conditions: (a) LiAlH4/THF abs., 50–60 °C; (b) toluene, Pd/C 10%, reflux temperature; (c) glacial AcOH/HBr 33% (1:1), reflux temperature; (d) H3PO4, 130 °C.
	Scheme 3. Reagents and conditions: (a) DMF abs., K2CO3, RX, 50–60 °C, 3–6 h; (b) DCM, NIS, 40 °C, 1 h.
	Scheme 4. Reagents and conditions: (a) POCl3/PCl5, reflux temperature, 5 h; (b) EtOH/DCM (1:2), NaBH4, room temperature, 2 h; (c) toluene, Pd/C 10%, reflux temperature, 10 h; (d) HCl 6M/AcOH (1:3), 100 °C, 20 h; (e) SOCl2, reflux temperature, 1 h; (f) DCM, suitable alcohol, 50 °C; 2–5 h.
	Scheme 5. Reagents and conditions: (a) HCl, reflux temperature, 12 h; (b) DCM, NIS, 40–50 °C, 3 h.
	Scheme 6. Reagents and conditions: (a) MeOH, HCOONH4, Pd/C 10%, room temperature, 4 h; (b) toluene, Pd/C 10%, reflux temperature; (c) NaOH 10% solution, 100 °C, and 8 h, then HCl; (d) (i) SOCl2, reflux temperature, 1 h, and (ii) DCM, suitable alcohol, 50 °C, 2–5 h.
	Scheme 7. Reagents and conditions: (a) benzyl bromide, K2CO3 anhydrous, DMF anhydrous, 80 °C, 2 h.
	Figure 1. Concentration–response curves of compounds on GABA-induced Cl− currents in Xenopus laevis oocytes expressing recombinant α1β2γ2L-GABAA receptors. Data are expressed as the percentage modulation of the response induced by GABA at EC5-10 values and are the mean ± SEM of values obtained from two to nine oocytes.
	Figure 2. Compound 6d and 8b antagonized the potentiation of GABA-induced Cl− currents by lorazepam in Xenopus laevis oocytes expressing recombinant α1β2γ2L-GABAA receptors. Data are expressed as the percentage modulation of the response induced by GABA at EC5-10 values and are the mean ± SEM of values obtained from four oocytes. * p < 0.05. ** p < 0.01.
	Figure 3. Compound 8a and 14 antagonized by flumazenil in the potentiation of GABA-induced Cl− currents by lorazepam in Xenopus laevis oocytes expressing recombinant α1β2γ2L-GABAA receptors. Data are expressed as the percentage modulation of the response induced by GABA at EC5-10 values and are the mean ± SEM of values obtained from four oocytes. * p < 0.05.
	Figure 4. Compound 6d reduced the potentiation of GABA-induced Cl− currents by CGS 9895 in Xenopus oocytes expressing recombinant α1β2 GABAA receptors. Data are expressed as the percentage potentiation of the response induced by GABA at the EC5-10 value and are the mean + S.E.M of values obtained from 3 to 6 oocytes. ** p < 0.01.
	Figure 5. HCA for ligand (conformations)–amino acid (αHis102, γThr142, αSer205) hydrogen bond lengths (method of WARD.D2 and Euclidean distance).
	Figure 6. Hydrogen bonds among the agonist 8a (blue), the antagonist 8b (red), and γThr142, αHis102, and αSer205 in the binding site.
	Figure 7. Hydrogen bonds among the agonist 14 (blue), the antagonist 6d (red), and γThr142, αHis102, and αSer205 in the binding site.