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

Abstract

As a continuation of our study in the field of GABA_A 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 GABA_AR (α1β2γ2-GABA_AR), 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.

1. Introduction

The GABA_A receptor (GABA_AR) belongs to the LGIC (Ligand-Gated Ion Channel) family arranged in a pentameric fashion. When the neurotransmitter GABA (γ-aminobutyric acid) interacts with the orthosteric binding sites in the receptor, the opening of the channel allows the influx of the chloride ions, driving the inhibitory neurotransmission and inducing postsynaptic hyperpolarization. Five subunits belonging to different families (α, β, γ, ρ, δ, ε, π, θ) concur to form the pentamer with various isoforms (α1-6, β1-3, γ1-3, ρ1-3, δ, ε, π, θ) and by convention, each subunit has a principal (+, plus) and a complementary side (−, minus). The most representative and prevalent pentamer contains two alpha, two beta, and one gamma subunit, and shows different and specific binding sites for GABA and benzodiazepines. The neurotransmitter GABA binds the two orthosteric binding sites to the interface between the α−/β+ subunits in the extracellular domain (ECD), thus suggesting that the two GABA-binding sites may not be functionally equivalent, with one having a threefold higher affinity for GABA than the other [1].

The ‘classic benzodiazepines’ (such as diazepam) bind the canonical site at the interface of the α+/γ− subunits in the ECD, even if three low-affinity binding sites located in the β/α and γ/β interface transmembrane domain (TMD) [2,3] and one between the α+/β− interface (the so-called low-affinity diazepam binding site) [2,4] and defined as the “pyrazoloquinolinone binding site” [5] have been evidenced and confirmed by the cryogenic electron microscopy (cryo-EM). The most significant GABA_A receptors present in the SNC and binding the ‘classic benzodiazepines’ are α1β2γ2-GABA_ARs, the most numerous population of receptors, followed by α2β2γ2- and α3β2γ2- and α5β2γ2-GABA_ARs. The α4β2γ2- and α6β2γ2-GABA_AR do not bind the classic benzodiazepines since these alpha isoforms show an arginine101 (R101) instead of a histidine101 (H101), a fundamental residue involved in the agonist (diazepam, alprazolam) or antagonist (flumazenil) binding. On the other hand, these last two types of receptors (α4β2γ2- and α6β2γ2-GABA_AR) could be involved in the potential therapeutic action of ‘pyrazoloquinolinones’ on neuropsychiatric disorders, as reported since the first decade of the 2000s [6,7,8,9,10,11].

Continuing our research on pyrazoloquinazolines (PQs) as ‘benzodiazepine receptor ligands’ [12], we report here the synthesis of new pyrazolo[1,5-a]quinazoline derivatives bearing at the 8-position an oxygen or nitrogen function, never inserted before, as well as other different groups/atoms to evaluate the ability of these heteroatoms to interact, through their lone pair, with receptor protein. 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 GABA_AR (α1β2γ2-GABA_AR), expressed in Xenopus laevis oocytes, by evaluating the variation in produced chlorine current, and where possible, a pharmacological profile has been defined (agonist/antagonist); then, molecular modelling studies (docking, minimization of complex ligand–receptor, and MD model) and a statistical analysis have been performed to further validate the model we recently published [13] by comparing the biological results and the prediction data.

2. Results and Discussion

2.1. Chemistry

The synthesis of the 5-oxo-4,5-dihydropyrazolo[1,5-a]quinazoline and of the corresponding 4,5-dehydro derivatives is depicted in Scheme 1, Scheme 2, Scheme 3, Scheme 4, Scheme 5, Scheme 6 and Scheme 7.

The cyclization reaction of the commercial 2-hydrazinyl benzoic acids (1a–e), variously substituted at position 4 or 5 (1a = 4-NO₂, 1b = 5-NO₂, 1c = 4-Cl, 1d = 4-Br, 1e = 4-I) with ethyl 2-cyano-3-ethoxyacrilate in DMF, gave the corresponding ethyl 5-oxo-4,5-dihydropyrazolo[1,5-a]quinazoline carboxylate substituted at the 8-position (2a, [14] 2c [12], 2d–e), or 7-position (2b). The next decarboxylation at position 3 was made in conc. HCl for compound 2a and H₃PO₄ for compounds 2c–e, affording compounds 3a, already obtained by Zhang with a different synthetic route [15], and 3c–e (Scheme 1).

The previously synthesized 8-methoxypyrazolo[1,5-a]quinazolin-5(4H)-one 3f [16] was chosen as the starting product for obtaining compound 5, Scheme 2. Following the already reported method [16], 3f was aromatized by treatment with LiAlH₄ in anhydrous THF and then with Pd/C 10% to achieve 4a [16], which, in turn, was subjected to demethylation with AcOH/HBr 33%, giving the final product 5 in low yield and in a very long time. Thus, to improve this reaction, we first transformed by demethylation with H₃PO₄ compound 3f in 8-hydroxypyrazolo[1,5-a]quinazolin-5(4H)-one at 3g and then, the reduction of the lactam function with LiAlH₄ in anhydrous THF gave the desired compound 5 directly, without the need to use Pd/C in toluene.

Scheme 3 depicts the 8-O-alkylation of the 8-hydroxypyrazolo[1,5-a]quinazoline 5, in the standard conditions (DMF/K₂CO₃/RX) to obtain compounds 4b–g; the further iodination with NIS/DCM at position 3 of 4b–g and 4a [16] gave the final 3-iodopyrazolo[1,5-a]quinazoline 8-alkyl/8-alkyl(hetero)aryl 6a–g.

The ethyl 8-nitro and the 7-nitro-5-oxo-4,5-dihydropyrazolo[1,5-a]quinazoline carboxylates 2a and 2b, respectively, were subjected to aromatization (Scheme 4) through a synthetic way already exploited in our laboratory. The transformation of 2a and 2b in the corresponding 5-chloro derivative with POCl₃/PCl₅ (7a and 7b [14]) and the subsequent C-Cl bond reduction with NaBH₄/DCM/EtOH gave the corresponding 4,5-dihydropyrazolo[1,5-a]quinazoline 8a and 8b in a good yield. The further treatment with Pd/C toluene at reflux permitted the aromatization of the pyrimidine ring of the scaffold, easily affording the ethyl 8/7-nitropyrazolo[1,5-a]quinazoline-3-carboxylate 9a and 9b. The next acid hydrolysis (HCl 6M/AcOH, 1:3) of the ethyl ester function of 9a,b gave the corresponding 3-carboxylic acid 10a,b, which underwent the standard esterification reaction (SOCl₂, DCM/suitable alcohol) to obtain the final 3-(hetero)arylalkyloxy carbonyl derivatives (11a–d).

The ethyl 8-nitropyrazolo[1,5-a]quinazoline-3-carboxylate 9a was decarboxylated in conc. HCl at reflux temperature, and the easily obtained 3-H derivate 12 was transformed into the corresponding 3-iodo derivative 13 using NIS/DCM, Scheme 5.

Finally, Scheme 6 depicts the synthetic route to obtain the esters 17a,b, bearing the amino group at position 8. The starting product 9a was treated with ammonium formate in methanol and Pd/C, affording the ethyl 8-amino-4,5-dihydropyrazolo [1,5-a]quinazoline-3-carboxylate 14, which, by treatment with toluene/Pd/C at reflux, was converted into compound 15; the latter was subjected to hydrolysis in an alkaline medium, and after the standard work-up, the 3-carboxylic acid 16 was recovered. Treatment with thionyl chloride and then with the suitable alcohol in DCM permitted us to obtain the 3-(2-methoxybenzyloxy)carbonyl- and the 3-(2-thienylmethoxy)carbonyl- derivatives 17a,b. Compound 15 was also alkylated in standard conditions with benzyl bromide, obtaining N-benzyl (18) and N, N-dibenzyl derivatives (19), respectively, as shown in Scheme 7.

2.2. Biological Evaluation

As anticipated in the Introduction, the compounds were screened through electrophysiological techniques on recombinant α1β2γ2L-GABA_A receptors expressed in Xenopus laevis oocytes. The effects of compounds tested at 1–100 µM on the modulation of GABAA receptor function were assessed, and products for which no change in the chlorine current was recorded were also tested in the presence of the agonist lorazepam to determine whether they did not effectively bind the receptor or they exhibited an antagonist profile. Following this preliminary screening, we observed that all the 5-oxo-4,5-dihydro derivatives (compounds 2a–e, 3a, and 3c–g), as well as the aromatic pyrazolo[1,5-a]quinazolines lacking the substituent at position 3 (4a–g, 5, and 12), do not bind the GABA_A receptor, suggesting that on the one hand the dihydro form is not appropriate for the target, and on the other hand that the substituent at position 3 is needed for the activity. Also, the 8-amino PQs 18 and 19, N-benzyl- and N, N-dibenzyl derivatives, respectively, do not bind the GABA_A receptor, indicating that position 8 does not permit the presence of bulky substituents.

Instead, the remaining final compounds substituted at positions 3 and 7/8 show a certain ability to bind the GABA_A receptor. The most interesting electrophysiological results were obtained for compounds 6d, 8a, 8b, and 14 (Figure 1). Among the 3-iodo derivatives, compound 6d, which does not modulate the chlorine current, was evaluated for its ability to antagonize the full agonist lorazepam (1 µM) and its antagonist profile is evident in Figure 2. All other compounds bearing an alkoxy (6a–c) or a benzyloxy group (6e–f) and the 8-nitro 13 generally modulate the GABAA current in a discontinued manner, providing nonsignificant results (see Figure S1 panel A, Supporting Information, which depicts the profile of all compounds). Moving to the 3-ester derivatives bearing a nitro or an amino group at the 7/8-position of the pyrazolo[1,5-a]quinazoline scaffold (8a,b, 9a,b, 11a–d, 14, 15, 17a,b (Figure S1 panel B, Supporting Information)), some interesting results emerge from electrophysiological assays for the two isomers’ ester derivatives 8a and 8b (8-nitro and 7-nitro derivatives, respectively). Compound 8a enhances the chlorine current in the recombinant α1β2γ2L-GABA_AR, reaching +57% at 100 µM; on the contrary, the 7-nitro isomer 8b is an α1β2γ2L-GABA_AR antagonist, Figure 1. Among the 8-amino derivatives (14, 15, 17a,b, Figure S1 panel C, Supporting Information), the ethyl 8-amino-4,5-dihydropyrazolo[1,5-a]quinazoline-3-carboxylate 14 emerges, enhancing the chlorine current until +85% at 100 µM (Figure 1). Finally, the aromatic analogue of 14 (compound 15) as well as the 3-aryl(hetero)alkyl ester derivatives 17a,b are able to modulate the chlorine current only weakly or not at all.

The functional assay for 6d, 8a, 8b, and 14 (Figure 2 and Figure 3) confirms the antagonist profile at the α1β2γ2L-GABA_AR for 6d and 8b, since these two compounds reduce the potentiation of the lorazepam-induced chlorine current (Figure 2). The agonists’ profile at the α1β2γ2L-GABA_AR of 8a and 14 was then confirmed since their induced chlorine currents were reduced to 6% and to about 20%, respectively, by the antagonist flumazenil (Figure 3).

As is well known, in addition to the high-affinity benzodiazepine binding site, the pentamer αβγ-GABA_AR also shows a benzodiazepine low-affinity site, located in the extracellular domain at the α+/β− interface [2,4,17]. The pyrazoloquinolinone CGS 9895 is a null modulator (antagonist) at the high-affinity benzodiazepine binding site, but it is also able to act as a positive allosteric modulator [7]. According to several reports [8,11], it is considered that drugs acting at the “non-canonical” α+/β− low-affinity binding site might display potential clinical relevance; for example, they could be beneficial for long-term epilepsy treatment since they could interact with a broader variety of GABA_A receptor subtypes such as δ, ε, and π subunit-containing GABA_AR. Therefore, we decided to further investigate compound 6d in order to highlight a possible interaction with this ‘non-canonical’ Bz site. The data obtained with GABA_A receptors devoid of the γ subunit (α1β2) (Figure 4) indicate that, indeed, compound 6d (10 µM) reduces in a statistically significant manner the potentiation of the GABA_A receptor induced by CGS 9895 (3 µM) (about −85%), suggesting that it binds the α+/β− low-affinity site too and might display potential clinical relevance.

2.3. Molecular Modelling Studies

To explain the different behaviour of the new ligands, the distances of the hydrogen bonds between the ligands and the amino acids of the active site were measured. The calculation was performed on 20 compounds, 6a–g, 8a,b, 9a,b, 11a–d, 14, 15, and 17a,b.

The hydrogen bond lengths were measured on the structures, completely relaxed (i.e., at a minimum potential energy), of the ligand–receptor complex. The position of the ligand in the ligand–receptor complex was identified with the automatic docking programme AUTODOCK [18]. From AUTODOCK, it is possible to obtain more than one probable position (i.e., complex conformation) depending on the molecular flexibility. Thus, when this occurred, all the probable conformations were completely relaxed before measuring the hydrogen bond lengths. Lengths (H-acceptor atom) expressed in Angstroms (Å) measured in the various relaxed conformations of the ligand–receptor complexes are reported in

Table 1. Hydrogen bond lengths with Amino acids involved in the ligand–receptor interaction.

	phe100 a	his102 a	thr142 a	lys156 a	ser159 a	ser205 a	thr207 a	tyr210 a
6a		2.17				2.55
6b			2.36			2.08
6c		2.58	2.83			2.16
6d						2.08
6e_1						1.97
6e_2			2.28			2.62
6e_3
6f_1		2.61	2.26			2.4	1.91
6f_2			2.05			2.16	1.98
6f_3			2.85
6f_4		2				1.83
6g						2.18
8a	2.61	1.98	1.88			2.04
8b	2.58	1.97				2.19
9a	2.81	1.92	1.89			1.91
9b_1	2.59	2.03	1.92				1.92
9b_2	2.56	2.75	2.65			1.92		2.83
11a				2.56		1.96
11b_1		1.98	1.96			1.84
11b_2		2.31		2.49		2.82	2.59	2.49
11c_1				2.02		1.93		2.08
11c_2						1.97
11d_1		1.98	1.87				2.96	2.63
11d_2				1.94		1.96
13	2.71	1.92				2.81
14		2.46	1.89		1.92
15		2.33	2.06		2.14	1.93
17a_1					1.79	2.74
17a_2	2.71	1.87				2.12
17a_3	2.54	2.12	2.07			2.52	2.16
17b_1			1.93			1.86	2.87
17b_2					1.91	2.44	1.81
m b	2.63	2.19	2.17	2.25	1.94	2.16	2.28	2.51
sd c	0.10	0.28	0.34	0.32	0.15	0.29	0.46	0.32

^a Hydrogen bond length, expressed in Angstrom (Å), calculated on all conformations of complex ligand-binding site. ^b Average of hydrogen bond length for each residue. ^c Standard deviation.

(as can be seen for compounds 6e, 6f, 9b, 11b, 11c, 11d, 17a, and 17b, the measurements were performed on multiple conformations).

The residues involved in hydrogen bond interaction with most compounds are αHis102, γThr142, and αSer205. The relatively “short” mean (m) hydrogen bond length for each residue, 2.19Å, 2.17Å, and 2.16Å, respectively, indicates strong bonds, while the relatively large standard deviation (sd) (0.28, 0.34, and 0.29, respectively) suggests a significant difference in bond strength between the ligands under consideration.

Using the hydrogen bond lengths between ligands and the amino acids αHis102, γThr142, and αSer205 as variables for a Hierarchical Cluster Analysis (HCA) [19], it was possible to classify the ligands into two clusters (Figure 5).

HCA was performed using, as a similarity parameter, the Euclidean distance calculated in the three-dimensional space generated by the three variables (hydrogen bond lengths). WARD.D2 was chosen as the aggregation method.

The use of other or a greater number of variables (lengths) did not lead to an improvement in the classification; indeed, in some cases, classification errors occurred.

Biological results discussed above demonstrate that compounds 8a and 14 exhibit an agonist profile, while compounds 8b and 6d act as antagonists. Supposing that a similar interacting force with amino acids in the binding site drove the binding of an agonist or antagonist, we could speculate that Cluster 1 (where 8a and 14 are) gathered ligands whose conformations interact with the binding site as agonists and Cluster 2 (where 8b and 6d are) gathered ligands whose conformations bind the site as the antagonist. Thus, the HCA classification indicates a correspondence with a probable profile. Moreover, it is to be noted that for the ligands for which Autodock generates more conformations, the collocation could be non-univocal.

In

Table 2. Hierarchical Cluster Analysis (HCA) for the new compounds.

Compound	HCA a	A b	B b
13	AN	1	1
14	AG	1	1
15	AG	0	0
11a	AN	1	1
11b_1	AG	0	1
11b_2	AN	1
11c_1	AN	1	1
11c_2	AN	1
11d_1	AG
11d_2	AN
17a_1	AN	1	1
17a_2	AN	1
17a_3	AG	0
17b_1	AG	0	1
17b_2	AN	1
6a	AN	1	1
6b	AG	0	0
6c	AG	0	0
6d	AN	1	1
6e_1	AN	1	1
6e_2	AG	0
6e_3	AN	1
6f_1	AG
6f_2	AG
6f_3	AN
6f_4	AN
6g	AN
8a	AG	1	1
8b	AN	1	1
9a	AG	1	1
9b_1	AG	0	0
9b_2	AG	0
Accuracy %		64	76

^a Activity predicted by HCA. ^b Correspondence with biological results.

, we report the HCA classification (agonist/antagonist (AG/AN), agreeing to Cluster 1 or 2) for the new 20 compounds and the correspondence with the biological results is reported in columns A and B. The value 1 in the A column indicates that the HCA collocation is in accordance with the biological test, while the same value in column B indicates that, at least, one conformation agrees with biological results; otherwise, the value 0 indicates no accordance. If columns A and B are empty, the biological results are unclear. The accuracy of the HCA ranges from about 60 to 75%, considering that not all poses of each compound are correctly located by the statistical method. From the HCA results, it could be speculated that the hydrogen bond lengths among αHis102, γThr142, αSer205, and ligands permit the discrimination of agonists from antagonists. Agonists show a stronger hydrogen bond interaction (shorter bond) with γThr142 than antagonists; the same thing happens with αHis102, while with αSer205, the bond length between agonists and antagonists is similar, and thus not relevant.

For the sake of clarity, the images of the minimized ligand–receptor complexes of the most interesting ligands (6a, 8a, 8b, and 14) are reported below. The minimized ligand–receptor complexes of the two isomers 8a and 8b (8-nitro- and 7-nitro-4,5-dihydropyrazolo[1,5-a]quinazolin-3-carboxylate, respectively), to which the electrophysiological assays assigned an agonist (8a) and antagonist profile (8b), gave interesting information that allowed us to explain their different profiles. Figure 6 reports the complex ligand–receptor of the two isomers 8a (blue) and 8b (red), where the hydrogen bonds with γThr142, αHis102, and αSer205 are clearly evident.

In particular, compound 8a (collocated in agonist class) engages with γThr142 and αHis102 strong hydrogen bonds (length 1.88 Å and 1.98 Å, respectively) through the oxygen carbonyl group of the ethyl ester moiety at the 3-position and the oxygen of the 8-nitro group. Compound 8b (collocated in antagonist class) engages only a strong hydrogen bond with αHis102 (length 1.97 Å) through the 7-nitro group oxygen atoms; the carbonyl oxygen of the ester function does not engage the hydrogen bond with γThr142 (length 3.32 Å) probably because the whole molecule is shifted in the site since the driving force of the accommodation is the hydrogen bond engaged by the 7-nitro group with αHis102.

Also, the agonist compound 14 (blue in Figure 7) in the minimized complex shows strong hydrogen bonds with γThr142 (length 1.89 Å) and weak hydrogen bonds (length 2.4 Å) with αHis102, while the antagonist 6d (red in Figure 7) forms a very weak hydrogen bond with γThr142 (length 2.74 Å) and no hydrogen bond with αHis102. The Proximity Frequency (PF) values calculated by the molecular dynamics simulation (60 ns) [13] (see Supporting Information) for compounds 8a and 8b have highlighted that the agonist 8a shows a Proximity Frequency with αVal203-γThr142 higher than the antagonist 8b. These results are coherent with PFs’ model in which the agonist compounds were simultaneously close to the αVal203 and γThr142 amino acids (with a frequency of 37% compared to the frequency of 16% found by the antagonist compounds) while the antagonist compounds were simultaneously close to the αHis102, αTyr160, and γTyr58 amino acids (with a frequency of 35% against a frequency of 13% for agonist compounds).

3. Materials and Methods

3.1. Chemistry

All melting points were determined on a Büchi apparatus (New Castle, DE, USA) and are uncorrected. Extracts were dried over Na₂SO₄, and the solvents were removed under reduced pressure. Merck F-254 commercial plates (Merck, Durham, NC, USA) were used for analytical TLC to follow the course of reactions. Silica gel 60 (Merck 70-230 mesh, Merck, Durham, NC, USA) was used for column chromatography. ¹H-NMR, and ¹³C-NMR, spectra were recorded on an Avance 400 instrument (Bruker Biospin Version 002 with SGU, Bruker Inc., Billerica, MA, USA). Chemical shifts (δ) are in parts per million (ppm) approximated by the nearest 0.01 ppm, using the solvent as an internal standard. Coupling constants (J) are in Hz; they were calculated by Top Spin 3.1 and approximated by 0.1 Hz. Data are reported as follows: the chemical shift, multiplicity (exch, exchange; br, broad; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; or a combination of those, e.g., dd), integral, assignments, and coupling constant. All new compounds had a purity >95%; microanalyses indicated by symbols of the elements were performed on a Perkin-Elmer 260 elemental analyzer for C, H, and N, and they were within ±0.4% of the theoretical values.

General procedure for the synthesis of compounds 2d–e: The starting hydrazine (1d-e) (1.0 mmol) was reacted with ethyl 2-cyano-3-ethoxyacrylate (1.0 mmol) in abs. DMF (3 mL), and sodium acetate (1.1 mmol) was added. The reaction was maintained at reflux temperature until the starting material disappeared in TLC (eluent toluene/ethyl acetate/acetic acid, 8:2:1, v/v/v). After the addition of ice and water to the final solution, the formed precipitate was filtered and washed with water, ethanol, and diethyl ether. The recrystallization with ethanol gave a pure product.

Ethyl 8-bromo-5-oxo-4,5-dihydropyrazole[1,5-a]quinazolin-3-carboxylate, 2d

From hydrazine 1d; yield = 61%; mp = 227–228 °C. ¹H-NMR (400 MHz, CDCl₃) δ 1.39 (t, 3H, CH₂CH₃, J = 7.2 Hz), 4.38 (q, 2H, CH₂CH₃, J = 7.2 Hz), 7.62 (dd, 1H, H7, J = 8.4 Hz, J = 1.6 Hz), 8.05 (s, 1H, H2), 8.17 (d, 1H, H6, J = 8.4 Hz), 8.37 (d, 1H, H9, J = 1.2 Hz), 9.57 (bs, 1H, NH, exch.). IR cm⁻¹ 3172, 1703, 1681. Anal. C₁₃H₁₀N₃O₃Br (C, H, N).

Ethyl 8-iodo-5-oxo-4,5-dihydropyrazole[1,5-a]quinazolin-3-carboxylate, 2e

From hydrazine 1e; yield = 58%; mp = 207–209 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 1.27 (t, 3H, CH₂CH₃, J = 6.8 Hz), 4.27 (q, 2H, CH₂CH₃, J = 6.8 Hz), 7.91–7.85 (m, 2H, H7, and H6), 8.16 (s, 1H, H2), 8.38 (s, 1H, H9), 11.65 (bs, 1H, NH, exch.). IR cm⁻¹ 3154, 1702, 1680. Anal. C₁₃H₁₀N₃O₃I (C, H, N).

General procedure for the synthesis of compounds 3a, and 3c–e: To 0.6 mmol of the starting ethyl ester, 2a was decarboxylated by using conc. HCl (30 mL) at reflux temperature, while 2c-e were treated with polyphosphoric acid (2.6 mmol) and maintained at 150 °C. When the starting material disappeared, evaluating by TLC (eluent toluene/ethyl acetate/methanol, 8:2:1.5, v/v/v), the reaction (suspension or solution) was added to ice/water, and if a precipitate formed, this was worked up as usual; otherwise, the solution was extracted with ethyl acetate; the final solid was purified by recrystallization by a suitable solvent.

8-Nitropyrazolo[1,5-a]quinazolin-5(4H)-one, 3a [15]

By treatment of 2a with conc. HCl; from water. Yield = 65%; mp = >300 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 5.99 (d, 1H, H3, J = 2.0 Hz), 7.84 (d, 1H, H2, J = 1.6 Hz), 8.30 (m, 2H, H6 and H7), 8.61 (d, 1H, H9, J = 1.2 Hz), 11.93 (bs, 1H, NH, exch.). Anal. C₁₀H₆N₄O₃ (C, H, N).

8-Chloroxypyrazolo[1,5-a]quinazolin-5(4H)-one, 3c

By treatment of 2c with polyphosphoric acid; from ethanol. Yield = 70%; mp = 279–281 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 5.88 (d, 1H, H3, J = 1.6 Hz), 7.49 (dd, 1H H7, J = 8.8 Hz, J = 2 Hz), 7.78 (d, 1H, H2, J = 1.6 Hz), 8.00 (d, 1H, H9, J = 1.2 Hz), 8.09 (d, 1H, H6, J = 8.8), 12.25 (bs, 1H, NH, exch.). IR cm⁻¹ 3134, 1681. Anal. C₁₀H₆N₃OCl (C, H, N).

8-Bromoxypyrazolo[1,5-a]quinazolin-5(4H)-one, 3d

By treatment of 2d with polyphosphoric acid; from ethanol. Yield = 72%; mp = 268–270 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 5.90 (d, 1H, H3, J = 1.6 Hz), 7.64 (dd, 1H H7, J = 8.8 Hz, J = 2 Hz), 7.78 (d, 1H, H2, J = 1.6 Hz), 8.01 (d, 1H, H6, J = 8.8), 8.16 (d, 1H, H9, J = 1.2 Hz), 12.25 (bs, 1H, NH, exch.). Anal. C₁₀H₆N₃OBr (C, H, N).

8-Iodoxypyrazolo[1,5-a]quinazolin-5(4H)-one, 3e

By treatment of 2e with polyphosphoric acid; from ethanol. Yield = 71%; mp = >300 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 5.88 (d, 1H, H3, J = 2.0 Hz), 7.78 (m, 3H; H2, H6, and H7), 8.36 (d, 1H, H9, J = 1.2 Hz), 12.21 (bs, 1H, NH, exch.). ESI-MS calcd. for C₁₀H₆N₃OI, 311.07; found: m/z 328.10 [M + H]⁺. Anal. C₁₀H₆N₃OI (C, H, N).

8-Hydroxypyrazolo[1,5-a]quinazolin-5(4H)-one, 3g: In a sealed tube, compound 4a [16] (0.6 mmol) was treated with phosphoric acid at 120 °C; after monitoring by TLC (eluent toluene/ethyl acetate/methanol, 8:2:1.5, v/v/v), the final suspension was treated with water and the precipitate, filtered, was purified by ethanol. Yield = 65%; mp = >300 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 5.82 (d, 1H, H3, J = 1.6 Hz), 6.84 (dd, 1H H7, J = 8.4 Hz, J = 2 Hz), 7.35 (d, 1H, H9, J = 2.4 Hz), 7.72 (d, 1H, H2, J = 1.6 Hz), 7.93 (d, 1H, H6, J = 8.4), 10.88 (bs, 1H, OH, exch.), 11.90 (bs, 1H, NH, exch.). Anal. C₁₀H₇N₃O₂ (C, H, N).

8-Hydroxypyrazolo[1,5-a]quinazoline, 5

From 3g, 8-hydroxypyrazolo[1,5-a]quinazolin-5(4H)-one, pathway a in Scheme 2: Compound 3g (0.35 mmol) was suspended in THF abs (15 mL) heated at 50–60 °C and a LiAlH₄ pellet (1:1.5) was added and maintained at refluxed temperature for 8 h. Monitoring the reaction by TLC (toluene/ethyl acetate/methanol, 8:2:1.5, v/v/v), it was possible to evidence the formation of the 4,5-dihydro derivative, which immediately converts into the 4,5-dehydro derivative. At the end of the reaction, after cooling to room temperature, the careful addition of ice/water caused gas evolution, and then the final solution was extracted with ethyl acetate. After the normal work-up, the residue was recovered by water. Yield 65%.

From 4a, 8-methoxypyrazolo[1,5-a]quinazoline [16], pathway c in Scheme 2: Compound 4a (0.35 mmol) was suspended in a mixture of AcOH/HBr 33% (20 mL, 1/1) and was refluxed for 72 h, monitoring the reaction by TLC (toluene/ethyl acetate/methanol, 8:2:1.5, v/v/v). The final solution was added to water, neutralized, extracted with ethyl acetate, and worked up as usual. The residue was recovered with water; yield 35%.

The final product 5 was purified by a chromatography column (TLC eluent), mp = 268–270 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 6.70 (d, 1H, H3, J = 1.6 Hz), 7.03 (dd, 1H H7, J = 8.4 Hz, J = 2 Hz), 7.60 (d, 1H, H9, J = 2.4 Hz), 7.99 (d, 1H, H6, J = 8.4), 8.09 (d, 1H, H2, J = 1.6 Hz), 8.83 (s, 1H, H5), 11.14 (bs, 1H, OH, exch.). Anal. C₁₀H₇N₃O (C, H, N).

General procedure for the synthesis of compounds 4b–g: To 0.6 mmol of 5 in DMF abs (1.5 mL), we added anhydrous K₂CO₃ (1.2 mmol) and stirred for ten minutes, and then added the suitable alkylant (0.66 mmol, 1:1.1), maintaining the reaction at 80 °C. When the starting material disappeared (3–6 h), evaluated by TLC (eluent toluene/ethyl acetate/methanol, 8:2:1.5, v/v/v), ice/water were added and the precipitate was filtered, washed with water, and purified by recrystallization.

8-(c-Propylmethoxy)pyrazolo[1,5-a]quinazoline, 4b

Using c-propylbromide; yield 55%; mp = 103–105 °C, ethanol. ¹H-NMR (400 MHz, CDCl₃) δ 0.40 (m, 2H, CH₂ c-prop); 0.70 (m, 2H, CH₂ c-prop); 1.35 (m, 1H, CH₂ c-prop), 4.04 (d, 2H CH₂, J = 7.2 Hz), 6.73 (d, 1H, H3, J = 1.6 Hz), 7.12 (dd, 1H H7, J = 8.4 Hz, J = 2.0 Hz), 7.77 (d, 1H, H9, J = 2.4 Hz), 7.82 (d, 1H, H6, J = 8.4), 8.07 (d, 1H, H2, J = 1.6 Hz), 8.76 (s, 1H, H5). Anal. C₁₄H₁₃N₃O (C, H, N).

8-(2-Propynyloxy)pyrazolo[1,5-a]quinazoline, 4c

Using propargyl bromide; yield 71%; mp = 151–153 °C, ethanol. ¹H-NMR (400 MHz, CDCl₃) δ 2.61 (t, 1H, HC≡, J = 2.4 Hz), 4.93 (d, 2H CH₂, J = 2.4 Hz), 6.78 (d, 1H, H3, J = 1.6 Hz), 7.15 (dd, 1H H7, J = 8.4 Hz, J = 2.0 Hz), 7.87 (d, 1H, H6, J = 8.4), 7.95 (d, 1H, H9, J = 2.4 Hz), 8.10 (d, 1H, H2, J = 1.6 Hz), 8.79 (s, 1H, H5). Anal. C₁₃H₉N₃O (C, H, N).

8-(Benzyloxy)pyrazolo[1,5-a]quinazoline, 4d

Using benzyl bromide; yield 66%; mp = 145–149 °C, ethanol. ¹H-NMR (400 MHz, CDCl₃) δ 5.32 (s, 2H CH₂), 6.86 (d, 1H, H3, J = 2.0 Hz), 7.21 (dd, 1H H7, J = 8.4 Hz, J = 2.0 Hz), 7.38 (m, 1H, Ph), 7.43 (m, 2H, Ph), 7.49 (d, 2H, Ph, J = 7.2 Hz), 7.90 (d, 1H, H6, J = 8.4), 7.99 (d, 1H, H9, J = 2.4 Hz), 8.13 (d, 1H, H2, J = 1.6 Hz), 8.85 (s, 1H, H5). Anal. C₁₇H₁₃N₃O (C, H, N).

8-(2-Methylbenzyloxy)pyrazolo[1,5-a]quinazoline, 4e

Using 2-methylbenzyl chloride; yield 66%; mp = 145–149 °C, i-propanol. ¹H-NMR (400 MHz, CDCl₃) δ 2.41 (s, 3H, CH₃), 5.27 (s, 2H CH₂), 6.78 (d, 1H, H3, J = 2.0 Hz), 7.16 (dd, 1H H7, J = 8.4 Hz, J = 2.0 Hz), 7.29 (m, 3H, Ph), 7.45 (d, 2H, Ph, J = 7.2 Hz), 7.85 (d, 1H, H6, J = 8.4), 7.99 (d, 1H, H9, J = 2.4 Hz), 8.10 (d, 1H, H2, J = 1.6 Hz), 8.79 (s, 1H, H5). Anal. C₁₈H₁₅N₃O (C, H, N).

8-(2-Methoxybenzyloxy)pyrazolo[1,5-a]quinazoline, 4f

Using 2-methoxybenzyl chloride; yield 73%; mp = 116–118 °C, i-propanol. ¹H-NMR (400 MHz, CDCl₃) δ 3.85 (s, 3H, OCH₃), 5.33 (s, 2H CH₂), 6.75 (d, 1H, H3, J = 2.0 Hz), 6.87 (d, 1H, Ph, J = 8.8 Hz), 6.92 (m, 2H, Ph), 7.17 (dd, 1H H7, J = 8.4 Hz, J = 2.0 Hz), 7.48 (d, 2H, Ph, J = 7.2 Hz), 7.83 (d, 1H, H6, J = 8.4), 7.99 (d, 1H, H9, J = 2.4 Hz), 8.08 (d, 1H, H2, J = 1.6 Hz), 8.76 (s, 1H, H5). Anal. C₁₈H₁₅N₃O₂ (C, H, N).

8-(Pyridin-4-ylmethoxy)pyrazolo[1,5-a]quinazoline, 4g

Using 4-bromomethylpyridine hydrobromide; yield 80%; mp = 158–162 °C, i-propanol. ¹H-NMR (400 MHz, CDCl₃) δ 5.28 (s, 2H CH₂), 6.77 (d, 1H, H3, J = 2.0 Hz), 7.20 (dd, 1H H7, J = 8.4 Hz, J = 2.0 Hz), 7.50 (d, 2H, Py, J = 5.6 Hz), 7.89 (d, 1H, H9, J = 1.6 Hz), 7.91 (d, 1H, H6, J = 8.8), 8.08 (d, 1H, H2, J = 1.6 Hz), 8.67 (d, 2H, Py, J = 6.0 Hz), 8.79 (s, 1H, H5). Anal. C₁₆H₁₂N₄O (C, H, N).

General procedure for the synthesis of compounds 6a–g: To 0.5 mmol of starting material (4a–g) in DCM (5 mL), NIS (1.3 mmol) was added and stirred at 80 °C. When the starting material disappeared (1 h), evaluated by TLC (eluent toluene/ethyl acetate/methanol, 8:2:1.5, v/v/v), the solution was diluted with DCM (10 mL), and the organic layer was washed with a 5% NaOH solution; then, after the usual work-up, the residue was recovered by an ethanol/water solution and purified by recrystallization.

3-Iodo-8-methoxypyrazolo[1,5-a]quinazoline, 6a [16]

3-Iodo-8-(c-propylmethoxy)pyrazolo[1,5-a]quinazoline, 6b

Yield 55%; mp = 165–167 °C, ethanol. ¹H-NMR (400 MHz, CDCl₃) δ 0.41 (m, 2H, CH₂ c-prop); 0.70 (m, 2H, CH₂ c-prop); 1.35 (m, 1H, CH₂ c-prop), 4.04 (d, 2H CH₂, J = 7.2 Hz), 7.14 (dd, 1H H7, J = 8.4 Hz, J = 2.0 Hz), 7.74 (d, 1H, H9, J = 2.4 Hz), 7.85 (d, 1H, H6, J = 8.4), 8.09 (s, 1H, H2), 8.82 (s, 1H, H5). Anal. C₁₄H₁₂N₃OI (C, H, N).

3-Iodo-8-(2-propynyloxy)pyrazolo[1,5-a]quinazoline, 6c

Yield 78%; mp = 179–181 °C, ethanol. ¹H-NMR (400 MHz, CDCl₃) δ 2.61 (t, 1H, HC≡, J = 2.4 Hz), 4.92 (d, 2H CH₂, J = 2.4 Hz), 7.18 (dd, 1H H7, J = 8.4 Hz, J = 2.0 Hz), 7.90 (d, 1H, H6, J = 8.4), 7.92 (d, 1H, H9, J = 2.4 Hz), 8.18 (s, 1H, H2), 8.84 (s, 1H, H5). ¹³C-NMR (400 MHz, CDCl₃) δ 29.70, 56.50, 97.28, 99.37, 116.12, 130.28, 142.97, 150.87. Anal. C₁₃H₈N₃OI (C, H, N).

3-Iodo-8-(benzyloxy)pyrazolo[1,5-a]quinazoline, 6d

Yield 70%; mp = 176–178 °C, ethanol. ¹H-NMR (400 MHz, CDCl₃) δ 5.29 (s, 2H CH₂), 7.20 (dd, 1H H7, J = 8.4 Hz, J = 2.0 Hz), 7.39 (m, 1H, Ph), 7.43 (m, 2H, Ph), 7.48 (d, 2H, Ph, J = 7.2 Hz), 7.87 (d, 1H, H6, J = 8.4), 7.92 (d, 1H, H9, J = 2.4 Hz), 8.10 (s, 1H, H2), 8.84 (s, 1H, H5). ¹³C-NMR (400 MHz, CDCl₃) δ 70.93, 87.05, 96.62, 113.22, 116.94, 127.75, 128.57, 128.81, 130.43, 135.36, 138.18, 142.81, 151.75, 163.95. Anal. C₁₇H₁₂N₃OI (C, H, N).

3-Iodo-8-(2-methylbenzyloxy)pyrazolo[1,5-a]quinazoline, 6e

Yield 70%; mp = 209–211 °C, i-propanol. ¹H-NMR (400 MHz, CDCl₃) δ 2.41 (s, 3H, CH₃), 5.26 (s, 2H CH₂), 7.18 (dd, 1H H7, J = 8.4 Hz, J = 2.0 Hz), 7.26 (m, 3H, Ph), 7.44 (d, 2H, Ph, J = 7.2 Hz), 7.88 (d, 1H, H6, J = 8.4), 7.94 (d, 1H, H9, J = 2.4 Hz), 8.12 (s, 1H, H2), 8.84 (s, 1H, H5). ¹³C-NMR (400 MHz, CDCl₃) δ 18.99, 52.59, 69.58, 96.44, 113.30, 116.98, 126.24, 128.95, 129.04, 130.46, 130.66, 133.29, 137.04, 147.06, 152.13, 164.10. Anal. C₁₈H₁₄N₃OI (C, H, N).

3-Iodo-8-(2-methoxybenzyloxy)pyrazolo[1,5-a]quinazoline, 6f

Yield 90%; mp = 154–156 °C, i-propanol. ¹H-NMR (400 MHz, CDCl₃) δ 3.90 (s, 3H, OCH₃), 5.33 (s, 2H CH₂), 6.94 (d, 1H, Ph, J = 8.8 Hz), 6.99 (t, 1H, Ph, J = 7.6 Hz), 7.20 (dd, 1H H7, J = 8.4 Hz, J = 2.0 Hz), 7.33 (t, 1H, Ph, J = 7.2 Hz), 7.48 (d, 1H, Ph, J = 7.6 Hz), 7.86 (d, 1H, H6, J = 8.4), 7.97 (d, 1H, H9, J = 2.4 Hz), 8.10 (s, 1H, H2), 8.83 (s, 1H, H5). Anal. C₁₈H₁₄N₃O₂I (C, H, N).

3-Iodo-8-(pyridin-4-ylmethoxy)pyrazolo[1,5-a]quinazoline, 6g

Yield 68%; mp = 190–192 °C, i-propanol. ¹H-NMR (400 MHz, CDCl₃) δ 5.34 (s, 2H CH₂), 7.22 (dd, 1H H7, J = 8.4 Hz, J = 2.0 Hz), 7.48 (d, 2H, Py, J = 5.6 Hz), 7.90 (d, 1H, H9, J = 1.6 Hz), 7.92 (d, 1H, H6, J = 8.8), 8.10 (s, 1H, H2), 8.68 (d, 2H, Py J = 6.0 Hz), 8.85 (s, 1H, H5). Anal. C₁₆H₁₁N₄OI (C, H, N).

General procedure for the synthesis of compounds 7a,b: To 0.5 mmol of starting material (4, 5), POCl₃ (3 mL) and PCl₅ (0.5 mmol) were added and stirred at reflux temperature for 5 h. When the starting material disappeared, evaluated by TLC (eluent toluene/ethyl acetate/methanol, 8:2:1.5, v/v/v), the solution was evaporated under vacuum and the addition of ice/water gave a precipitate, which was filtered and used as such for the next step.

Ethyl 8-nitro-5-chloropyrazolo[1,5-a]quinazoline-3-carboxylate, 7a

Yield 85%; mp = 199–202 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 1.32 (t, 3H CH₃, J = 6.8 Hz), 4.33 (q, 2H, CH₂, J = 7.2 Hz), 8.47 (dd, 1H H7, J = 7.2 Hz, J = 2.0 Hz), 8.58 (d, 1H, H6, J = 8.8), 8.69 (s, 1H, H2), 9.01 (d, 1H, H9, J = 2.0 Hz). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.77, 60.69, 106.16, 117.60, 117.93, 124.13, 130.85, 139.53, 143.85, 145.47, 146.86, 154.07, 161.43. Anal. C₁₃H₉N₄O₄Cl (C, H, N).

Ethyl 7-nitro-5-chloropyrazolo[1,5-a]quinazoline-3-carboxylate, 7b

Yield 85%; mp = 199–202 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 1.33 (t, 3H CH₃, J = 6.8 Hz), 4.33 (q, 2H, CH₂, J = 7.2 Hz), 8.62 (d, 1H, H9, J = 8.4), 8.69 (s, 1H, H2), 8.85 (dd, 1H H8, J = 8.0 Hz, J = 2.0 Hz), 8.96 (d, 1H, H6, J = 2.0 Hz). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.77, 60.69, 106.16, 117.60, 117.93, 124.13, 130.85, 139.53, 143.85, 145.47, 146.86, 154.07, 161.43. Anal. C₁₃H₉N₄O₄Cl (C, H, N).

General procedure for the synthesis of compounds 8a,b: A solution of 0.6 mmol of starting material (7a,b) in ethanol/DCM (15 mL/25 mL) was added to sodium borohydride in a large excess (6.0 mmol) and the reaction was maintained at room temperature under stirring for 2 h. The final suspension was evaporated to dryness and the residue was recovered with water, filtered, and washed with water.

Ethyl 8-nitro-4,5-dihydropyrazolo[1,5-a]quinazoline-3-carboxylate, 8a

Yield 83%; mp = 222–224 °C, ethanol. ¹H-NMR (400 MHz, DMSO-d₆) δ 1.24 (t, 3H CH₃, J = 6.8 Hz), 4.18 (q, 2H, CH₂, J = 7.2 Hz), 4.65 (s, 2H, CH₂), 7.31 (s, 1H NH, exch.), 7.58 (d, 1H, H6, J = 8.8), 7.76 (s, 1H, H2), 8.00 (dd, 1H H7, J = 7.2 Hz, J = 2.0 Hz), 8.14 (d, 1H, H9, J = 2.0 Hz). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.93, 42.74, 59.62, 94.74, 108.37, 120.59, 128.20, 128.80, 134.92, 143.13, 148.73, 148.75, 162.78. IR, cm⁻¹ (nujiol) 3373, 1674, 1527, 1350. Anal. C₁₃H₁₂N₄O₄ (C, H, N).

Ethyl 7-nitro-4,5-dihydropyrazolo[1,5-a]quinazoline-3-carboxylate, 8b

Yield 78%; mp = 239–241 °C, i-propanol. ¹H-NMR (400 MHz, DMSO-d₆) δ 1.24 (t, 3H CH₃, J = 6.8 Hz), 4.18 (q, 2H, CH₂, J = 7.2 Hz), 4.65 (s, 2H, CH₂), 7.39 (s, 1H NH, exch.), 7.69 (d, 1H, H9, J = 2.0 Hz), 7.79 (s, 1H, H2), 8.22 (m, 2H, H6, and H8). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.93, 42.70, 59.63, 93.00, 114.69, 122.05, 123.24, 125.08, 139.08, 143.93, 144.77, 149.34. IR, cm⁻¹ (nujiol) 3373, 1674, 1527, 1350. Anal. C₁₃H₁₂N₄O₄ (C, H, N).

General procedure for the synthesis of compounds 9a,b: To 0.5 mmol of starting material (8a,b) in toluene (50 mL), we added Pd/C 10% and the reaction was maintained at refluxed temperature for 10 h, monitoring the reaction by TLC (eluent toluene/ethyl acetate/methanol, 8:2:1.5, v/v/v). Then, the suspension was filtered and the solution was evaporated under vacuum; the precipitate was recovered with i-propanol.

Ethyl 8-nitropyrazolo[1,5-a]quinazoline-3-carboxylate, 9a

Yield 56%; mp = 228–230 °C, ethanol. ¹H-NMR (400 MHz, DMSO-d₆) δ 1.32 (t, 3H CH₃, J = 6.8 Hz), 4.33 (q, 2H, CH₂, J = 7.2 Hz), 8.47 (dd, 1H H7, J = 7.2 Hz, J = 2.0 Hz), 8.57 (d, 1H, H6, J = 8.8), 8.69 (s, 1H, H2), 9.03 (d, 1H, H9, J = 2.0 Hz), 9.52 (s, 1H, H5). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.85, 60.50, 110.71, 121.04, 132.30, 146.46, 156.06. Anal. C₁₃H₁₀N₄O₄ (C, H, N).

Ethyl 7-nitropyrazolo[1,5-a]quinazoline-3-carboxylate, 9b

Yield 82%; mp = 239–241 °C, ethanol. ¹H-NMR (400 MHz, DMSO-d₆) δ 1.32 (t, 3H CH₃, J = 6.8 Hz), 4.32 (q, 2H, CH₂, J = 7.2 Hz), 8.57 (d, 1H, H9, J = 8.4), 8.68 (s, 1H, H2), 8.79 (dd, 1H H8, J = 8.0 Hz, J = 2.0 Hz), 9.28 (d, 1H, H6, J = 2.0 Hz), 9.54 (s, 1H, H5). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.84, 60.51, 117.05, 126.38, 129.92. Anal. C₁₃H₁₀N₄O₄ (C, H, N).

General procedure for the synthesis of compounds 10a,b: To a mixture of HCl conc. and AcOH (1:3, 4 mL/12 mL), 0.5 mmol of starting material (30–31) was added and maintained at reflux temperature for 20 h. The final suspension of acid was filtered and used as such for the next step.

8-Nitropyrazolo[1,5-a]quinazoline-3-carboxylic acid, 10a

Yield 35%; mp > 300 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 8.45 (d, 1H, H6, J = 8.8), 8.56 (d, 1H H7, J = 7.2 Hz), 8.62 (s, 1H, H2), 8.99 (s, 1H, H9), 9.48 (s, 1H, H5), 12.69 (bs, 1H, OH, exch.). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.85, 60.50, 110.71, 121.04, 132.30, 146.46, 156.06. Anal. C₁₁H₆N₄O₄ (C, H, N).

7-Nitropyrazolo[1,5-a]quinazoline-3-carboxylic acid, 10b

Yield 30%; mp = >300 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 8.58–8.78 (m, 3H, H2, H8, and H9), 9.26 (d, 1H, H6, J = 2.0 Hz), 9.52 (s, 1H, H5), 12.7 (bs, 1H OH, exch.). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.84, 60.51, 117.05, 126.38, 129.92. Anal. C₁₁H₆N₄O₄ (C, H, N).

General procedure for the synthesis of compounds 11a–d: Starting carboxylic acid (10a,b, 0.5 mmol) was treated with SOCl₂ and maintained at reflux temperature for 1 h; after the evaporation of the excess reagent, DCM (10 mL) and the suitable alcohol (0.7 mmol) were added. The reaction was monitored by TLC (eluent toluene/ethyl acetate/acetic acid, 8:2:1, v/v/v) and at the end, the solvent evaporated, and the residue was recovered by ethanol and recrystallized by ethanol.

2-Methoxybenzyl 8-nitropyrazolo[1,5-a]quinazoline-3-carboxylate, 11a

From 10a and 2-methoxybenzyl alcohol; yield 25%; mp = 165–166 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 3.81 (s, 3H, OCH₃), 5.35 (s, 2H, CH₂), 8.45 (d, 1H, H6, J = 8.8), 8.56 (d, 1H H7, J = 7.2 Hz), 8.73 (s, 1H, H2), 8.02 (s, 1H, H9), 9.55 (s, 1H, H5). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.85, 60.50, 110.71, 121.04, 132.30, 146.46, 156.06. Anal. C₁₉H₁₄N₄O₅ (C, H, N).

Thien-2-ylmethyl 8-nitropyrazolo[1,5-a]quinazoline-3-carboxylate, 11b

From 10a and 2-thienylmethanol; yield 25%; mp = 176–178 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 5.54 (s, 2H, CH₂), 7.03 (m, 1H, H4 thiophene), 7.26 (m, 1H, H3 thiophene), 7.54 (d, 1H, H5 thiophene, J = 4.4 Hz), 8.48 (d, 1H, H6, J = 8.8), 8.57 (d, 1H H7, J = 7.2 Hz), 8.70 (s, 1H, H2), 9.01 (s, 1H, H9), 9.53 (s, 1H, H5). ¹³C-NMR (400 MHz, DMSO-d₆) δ 60.44, 110.67, 121.06, 122.17, 127.31, 128.04, 129.13, 132.38, 135.56, 138.68, 146.14, 156.02, 161.64. Anal. C₁₆H₁₀N₄O₄S (C, H, N).

2-Methoxybenzyl 7-nitropyrazolo[1,5-a]quinazoline-3-carboxylate, 11c

From 10b and 2-methoxybenzyl alcohol; yield 30%; mp = 176–178 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 3.72 (s, 3H, OCH₃), 4.51 (s, 2H, CH₂), 6.89 (d, 1H, Ph, J = 6.0 Hz), 7.16 (m, 3H, Ph), 7.52 (d, 1H, H9, J = 8.4), 7.68 (s, 1H, H2), 8.25 (d, 1H, H8, J = 8.0 Hz), 8.43 (s, 1H, H6), 9.13 (s, 1H, H5). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.85, 60.50, 110.71, 121.04, 132.30, 146.46, 156.06. Anal. C₁₉H₁₄N₄O₅ (C, H, N).

Thien-2-ylmethyl 7-nitropyrazolo[1,5-a]quinazoline-3-carboxylate, 11d

From 10b and 2-thienylmethanol; yield 20%; mp = 201–204 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 4.64 (s, 2H, CH₂), 6.92 (m, 1H, H4 thiophene), 6.98 (m, 1H, H3 thiophene), 7.48 (d, 1H, H5 thiophene, J = 4.4 Hz), 7.55 (d, 1H, H9, J = 8.4 Hz), 7.69 (s, 1H, H2), 8.19 (d, 1H H8, J = 8.8 Hz), 8.43 (d, 1H, H6, J = 1.2 Hz), 9.19 (s, 1H, H5). ¹³C-NMR (400 MHz, DMSO-d₆) δ 60.44, 110.67, 121.06, 122.17, 127.31, 128.04, 129.13, 132.38, 135.56, 138.68, 146.14, 156.02, 161.64. ESI-MS calcd. for C₁₆H₁₀N₄O₄S, 354.04; found: m/z 354.04 [M + H]⁺. Anal. C₁₆H₁₀N₄O₄S (C, H, N).

8-Nitropyrazolo[1,5-a]quinazoline, 12: Ethyl ester 9a (100 mg, 0.35 mmol) was suspended in HCl conc (20 mL) and maintained at reflux temperature until the starting material disappeared, evaluated by TLC (eluent toluene/ethyl acetate/methanol, 8:2:1.5, v/v/v). The acid phase was extracted with ethyl acetate and washed twice with water. The evaporation to dryness gave a white residue, which was recovered by water, filtered under suction, and recrystallized by ethanol. Yield 30%; mp = 160–162 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 7.00 (d, 1H, H3, J = 2.0 Hz), 8.34 (d, 1H, H2, J = 2.0 Hz), 8.53 (d, 1H, H6, J = 8.8 Hz), 8.74 (dd, 1H, H7, J = 8.2 Hz, J = 2.4 Hz), 9.21 (d, 1H, H9, J = 2.0 Hz), 9.28 (s, 1H, H5). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.85, 60.50, 110.71, 121.04, 132.30, 146.46, 156.06. Anal. C₁₀H₆N₄O₂ (C, H, N).

3-Iodo-8-nitropyrazolo[1,5-a]quinazoline, 13: To a solution of compound 12 (100 mg, 0.47 mmol) in methylene chloride (10 mL), we added NIS (1:1.1) and maintained it at 40–50 °C until the starting material disappeared in TLC (eluent toluene/ethyl acetate, 8:2, v/v). The final yellow solution was washed with water made alkaline with NaOH and, after the usual work-up, evaporated to dryness. The residue was recovered by ethanol and recrystallized by the same solvent. Yield 30%; mp = 160–162 °C. ¹H-NMR (400 MHz, CDCl₃) δ 7.24 (s, 1H, H2), 8.19 (d, 1H, H6, J = 8.8 Hz), 8.36 (d, 1H, H7, J = 8.2 Hz), 9.00 (s, 1H, H9), 9.29 (s, 1H, H5). ¹³C-NMR (400 MHz, DMSO-d₆) δ 109.58, 120.48, 122.15, 132.18, 135.86, 148.32, 150.65, 153.41. Anal. C₁₀H₅N₄O₂I (C, H, N).

Ethyl 8-amino-4,5-dihydropyrazolo[1,5-a]quinazoline-3-carboxylate, 14: A solution of the starting product (9a, 0.7 mmol, 200 mg) in methanol (20 mL), ammonium formate (0.21 mmol, 132 mg), and a catalytic amount of Pd/C 10% was maintained at room temperature, under stirring, for 4 h. The final suspension, monitored by TLC (eluent toluene/ethyl acetate/methanol, 8:2:1.5, v/v/v), was filtered and evaporated to dryness and the final residue was recovered with water, filtered, and separated by a chromatography column (eluent toluene/ethyl acetate/methanol, 8:2:1.5, v/v/v) as the faster band running. Yield 45%; mp = 145–148 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 1.23 (t, 3H CH₃, J = 6.8 Hz), 4.16 (q, 2H, CH₂, J = 7.2 Hz), 4.32 (s, 2H, CH₂); 5.27 (bs, 2H, NH₂, exch.); 6.33 (dd, 1H, H7, J = 8.0 Hz, J = 2.0 Hz), 6.91 (d, 1H, H9, J = 2.0 Hz), 6.87 (d, 1H, H6, J = 8.0 Hz), 6.92 (bs, 1H, NH, exch.), 7.61 (s, 1H, H2). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.81, 60.16, 93.57, 103.34, 110.61, 115.91, 131.48, 138.12, 145.28, 145.59, 154.04, 156.04, 162.83. IR nujiol cm⁻¹: 3360, 3196, 1713,1587, 1263, 1111. Anal. C₁₃H₁₂N₄O₂ (C, H, N).

Ethyl 8-aminopyrazolo[1,5-a]quinazoline-3-carboxylate, 15: Starting material 14 (1.0 mmol) was refluxed with toluene and Pd/C 10% for 5 h, and when the final product was completely formed (evaluated by TLC; eluent toluene/ethyl acetate/methanol, 8:2:1.5, v/v/v), the final suspension was filtered and the solution evaporated to dryness, giving the desired compounds. Yield 55%; mp = 158–162 °C, ethanol. ¹H-NMR (400 MHz, DMSO-d₆) δ 1.29 (t, 3H CH₃, J = 6.8 Hz), 4.26 (q, 2H, CH₂, J = 7.2 Hz), 6.91 (m, 3H, H7, and NH₂, exch.), 7.30 (s, 1H, H9), 7.84 (d, 1H, H6, J = 8.8 Hz), 8.42 (s, 1H, H2), 8.86 (s, 1H, H5). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.81, 60.16, 93.57, 103.34, 110.61, 115.91, 131.48, 138.12, 145.28, 145.59, 154.04, 156.04, 162.83. IR nujiol cm⁻¹: 3360, 3196, 1713, 1587, 1263, 1111. Anal. C₁₃H₁₂N₄O₂ (C, H, N).

8-Aminopyrazolo[1,5-a]quinazoline-3-carboxylic acid, 16: The starting product 15 was suspended in a NaOH 10% solution and maintained at 150 °C for 8 h. When the reaction ended, evaluated by TLC (eluent toluene/ethyl acetate/methanol, 8:2:1.5, v/v/v), the solution was treated with ice/water and then acidified by acetic acid until pH 5–6. The precipitate was filtered and washed with ethanol and diethyl ether. Yield 97%; mp > 300 °C. ¹H-NMR (400 MHz, DMSO-d₆) δ 6.71 (m, 3H, NH₂, exch., H7), 7.26 (s, 1H, H9), 7.77 (d, 1H, H6, J = 8.0 Hz), 8.11 (s, 1H, H2), 8.65 (s, 1H, H5). Anal. C₁₁H₈N₄O₂ (C, H, N).

General procedure for the synthesis of compounds 17a,b: The carboxylic acid 16 (0.5 mmol) was treated with SOCl₂ and maintained at reflux temperature for 1 h; after the evaporation of the excess reagent, DCM (10 mL) and the suitable alcohol (0.7 mmol) were added. The reaction was monitored by TLC (eluent toluene/ethyl acetate/acetic acid, 8:2:1, v/v/v) and at the end, the solvent evaporated, and the residue was treated with an alkaline aqueous solution and the precipitate was filtered and recrystallized by ethanol.

2-Methoxybenzyl 8-aminopyrazolo[1,5-a]quinazoline-3-carboxylate, 17a

From 16 and 2-methoxybenzyl alcohol; yield 20%; mp = 182–185 °C. ¹H-NMR (400 MHz, CD₃CN-d₃) δ 3.85 (s, 3H, OCH₃), 5.37 (s, 2H, CH₂), 6.96–7.03 (m, 3H, Ph), 7.33 (m, 1H, H7), 7.44 (bs, 2H, NH₂, exch.), 7.51 (m, 2H, H9 and Ph), 7.84 (d, 1H, H-6, J = 8.8), 8.43 (s, 1H, H2), 8.86 (s, 1H, H5). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.85, 60.50, 110.71, 121.04, 132.30, 146.46, 156.06. Anal. C₁₉H₁₆N₄O₃ (C, H, N).

Thien-2-ylmethyl 8-aminopyrazolo[1,5-a]quinazoline-3-carboxylate, 17b

From 16 and 2-thienylmethanol; yield 30%; mp = 178–181 °C. ¹H-NMR (400 MHz, CD₃CN-d₃) δ 5.49 (s, 2H, CH₂), 6.93 (m, 3H, H7 and H3, H4 thiophene), 7.21 (m, 1H, H5 thiophene), 7.40 (m, 3H, H9, NH₂, exch.), 7.80 (d, 1H, H6, J = 8.4), 8.39 (s, 1H, H2), 8.81 (s, 1H, H5). ¹³C-NMR (400 MHz, DMSO-d₆) δ 59.91, 93.67, 102.79, 110.68, 116.00, 127.25, 127.81, 128.82, 131.32, 138.16, 139.18, 145.37, 145.92, 154.07, 156.28, 162.20. Anal. C₁₆H₁₂N₄O₂S (C, H, N).

General procedure for the synthesis of compounds 18 and 19: Compound 15 (0.2 mmol) was suspended in anhydrous DMF (2 mL), and anhydrous K₂CO₃ (0.4 mmol) was added, maintaining the suspension under stirring at room temperature for 30 min. After adding benzyl bromide (0.2 mmol), the reaction was maintained at 80 °C until the starting material disappeared. The final solution was treated with water and then extracted with DCM (10 mL × 3vv), and the organic layer was worked up as usual. The residue was purified by a chromatography column (eluent toluene/ethyl acetate/acetic acid, 8:2:1 v/v/v), obtaining the 8-N-benzylamino (18) and the 8-dibenzylamino derivative (19).

Ethyl 8-benzylaminopyrazolo[1,5-a]quinazoline-3-carboxylate, 18

The second band running, yield 25%, mp = 175–176° C. ¹H-NMR (400 MHz, DMSO-d₆) δ 1.28 (t, 3H, CH₃, J = 6.8 Hz); 4.25 (q, 2H, CH₂, J = 7.2 Hz); 4.50 (d, 2H, CH₂, J = 6.0 Hz); 7.02 (d, 1H, H7, J = 8.8 Hz); 7.13 (s, 1H, H9); 7.21 (t, 1H benzyl, J = 6.0 Hz); 7.29–7.35 (m, 4H, benzyl); 7.86 (d, 1H, H6, J = 8.0 Hz), 8.39 (s, 1H, H2), 8.85 (s, 1H, H5). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.82, 46.15, 60.13 110.75, 127.48, 127.63, 129.08, 130.94, 130.63, 145.30, 153.92, 154.85, 162.75. Anal. C₂₀H₁₈N₄O₂ (C, H, N).

Ethyl 8-dibenzylaminopyrazolo[1,5-a]quinazoline-3-carboxylate, 19

The first band running, yield 25%, mp = 168–170° C. ¹H-NMR (400 MHz, CDCl₃) δ 1.41 (t, 3H, CH₃, J = 6.8 Hz); 4.25 (q, 2H, CH₂, J = 7.2 Hz); 4.85 (s, 2H, CH₂, J = 6.0 Hz); 7.02 (dd, 1H, H7, J = 2.8, Hz J = 8.8 Hz); 7.23–7.34 (m, 5H, benzyl); 7.66 (s, 1H, H9); 7.74(d, 1H, H6, J = 8.0 Hz), 8.41 (s, 1H, H2), 8.86 (s, 1H, H5). ¹³C-NMR (400 MHz, DMSO-d₆) δ 14.82, 46.15, 60.13 110.75, 127.48, 127.63, 129.08, 130.94, 130.63, 145.30, 153.92, 154.85, 162.75. Anal. C₂₇H₂₄N₄O₂ (C, H, N).

3.2. Molecular Docking Studies

3.2.1. Molecular Docking

All the 3D structures of the molecules were designed (“DS ViewerPro 6.0 Accelrys Software Inc.”, San Diego, CA, USA). The structure of the binding site was obtained from the Human α1β2γ2-GABAA receptor subtype in complex with GABA and flumazenil, conformation B (PDB ID 6D6T) [2], considering all the amino acids within a distance of about 2 nm from the structure of the flumazenil. The ligands were placed at the binding site through AUTODOCK 4.2 [18]. We collected the minimum number of ligand-binding site complex conformations to cover at least 90% of the poses found by AUTODOCK.

3.2.2. Complex Potential Energy Minimization

For the potential energy minimization of the complexes’ ligand–enzyme, we used the GROMACS v5.1 programme and conducted it in vacuum [20].

The partial atomic charge of the ligand structures was calculated with CHIMERA using the AM1-BCC method, and the topology was created with ACPYPE based on the routine Antechamber. For the potential energy calculation, the AMBER99sb force field parameters were applied. A conjugate gradient algorithm for energy minimization, with the tolerance of 10.0 kJ mol⁻¹ nm⁻¹ (the minimization is converged when the maximum force is smaller than this value), was used.

3.2.3. Molecular Dynamics Simulation

A 60 ns MD simulation was performed for all complexes using the GROMACS v5.1 programme and conducted in vacuum [20]. The partial atomic charge of the ligand structures was calculated with CHIMERA [21] using the AM1-BCC method, and the topology was created with ACPYPE [22] based on the routine Antechamber [23]. The OPLS-AA/L all-atom force field [24] parameters were applied to all the structures. To remove bad contacts, the energy minimization was performed using the steepest descent algorithm until convergence was achieved or for 50,000 maximum steps. The next equilibration of the system was conducted in two phases:

(1)

A canonical NVT ensemble, a 100 ps position restraint of molecules at 300 K, was carried out using a temperature coupling thermostat (velocity rescaling with a stochastic term) to ensure the proper stabilization of the temperature [25].

(2)

An isothermal isobaric NPT ensemble, a 100 ps position restraint of molecules at 300 K and 1 bar, was carried out without using barostat pressure coupling to stabilize the system. These were then followed by a 60 ns MD run at 300 K with position restraints for all protein atoms. The Lincs algorithm [26] was used for bond constraints to maintain rigid bond lengths.

The initial velocity was randomly assigned, taken from Maxwell–Boltzman distribution at 300 K, and computed with a time step of 2 fs, and the coordinates were recorded every 0.6 ns for an MD simulation of 60 ns. The conformations collected during the simulated trajectory were 100 in total. The ‘Proximity Frequencies’ (PFs) [13] with which the 100 conformations of each binding site ligand complex intercept two or more amino acids during the dynamics simulation have been calculated. The ‘Proximity Frequency’ (PF) is the frequency with which the ligand was, during the molecular dynamics simulation, at a distance of less than 0.25 nm from an amino acid of the binding site and also, simultaneously, from 2 and 3 amino acids of the binding site.

3.2.4. Hierarchical Cluster Analysis (HCA)

A Hierarchical Cluster Analysis was performed by R [19] using the Euclidean distance and ward D2 minimum variance method.

3.3. Biological Experiment

3.3.1. Expression of Human Receptor Subunits

A mixture of pCDM8-based vectors for the a1, b2, or g2L subunits of human GABAA receptors (total of 1.5 ng of DNA, comprising equal amounts of α, β, and γ subunit vectors), or an equal amount of α and β receptors for the expression of α1β2 receptors, was injected into the animal pole of X. laevis oocytes as described [27] with the use of a microdispenser (Drummond Scientific, Broomwall, PA, USA). The injected oocytes were maintained at 13 °C in sterile modified Barth’s solution [MBS: 88 mM NaCl, 1 mM KCl, 10 mM HEPES-NaOH (pH 7.5), 0.82 mM MgSO₄, 2.4 mM NaHCO₃, 0.91 mM CaCl₂, 0.33 mM Ca(NO₃)₂] supplemented with streptomycin (10 mg/L), penicillin (10,000 U/L), gentamicin (50 mg/L), theophylline (90 mg/L), and pyruvate (220 mg/L).

3.3.2. Electrophysiology

Electrophysiological measurements were performed in oocytes 2 to 4 days after DNA injection. Oocytes were placed in a rectangular chamber (volume~100 µL) and perfused at a rate of 1.7 mL/min with MBS at room temperature with the use of a roller pump (Cole-Parmer, Chicago, IL, USA) and 18-gauge polyethylene tubing (Clay Adams, Parsippany, NJ, USA). Oocytes were impaled at the animal pole with two glass electrodes (0.5 to 10 MΩ) filled with 3 M KCl and were clamped at −70 mV with the use of an oocyte clamp (model OC725C; Warner Instruments, Hamden, CT). Currents were measured and analyzed with the pClamp 9.2 software (Molecular Devices, Union City, CA, USA). GABA (Sigma, St. Louis, MO, USA) was dissolved in MBS and applied to the oocytes for 30 s. Oocytes were perfused with test drugs for 30 s either in the absence of the agonists or in their presence at the EC5-10 (the concentration of an agonist that induces a peak current equal to 5 to 10% of the maximal current elicited by the maximal concentration of the agonist). Compounds were first dissolved in DMSO at a concentration of 10 mM and then diluted in MBS to the final concentrations. In each experiment, control responses were determined before and 10/15 min after the application of the drug.

3.3.3. Statistics

A statistical analysis was performed on normalized data using the Kruskal–Wallis test followed by Dunn’s post hoc test or the Mann–Whitney test using Graph Pad Prism 7 (Graph Pad Software, Inc., San Diego, CA, USA).