Introduction

Clinical depression is a common neurological disorder [1]. The symptom of feelings of helplessness, sluggish thought and cognitive dysfunction has been associated with depression [2]. Approximately, 21 % of the world population has depression [3, 4]. A number of MAO inhibitors, tricyclic antidepressants, selective 5-HT reuptake inhibitors, and specific 5-HT–NE reuptake inhibitors are currently used in the treatment of depression. However, their therapeutic outcome is satisfactory for only two-thirds of patients. Side effects of these drugs, such as somnipathy, anxiety, and sexual disorders, were observed in one-third of patients [5–7]. Therefore, it is imperative to discover new effective drugs for the treatment of depression.

Fig. 1
figure 1

Design of compounds 6a–x, 7, 8, 9, 10, 11, 12, 13, and 14

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Table 1 Antidepressant activities of the compounds 6a, and 7–14 in the FST
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In our previous study [8], we reported that 5-alkoxy-tetrazolo[1,5-a]quinazolines (I) had antidepressant activity. Among these compounds, 5-(hexyloxy)tetrazolo[1,5-a]quinazoline displayed a significant antidepressant activity and decreased the immobility times by 62.2 % at a dose of 100 mg/kg. Additionally, considerable accumulating evidence indicates that as a group, the quinazolinone derivatives possess extensive biological activities, such as antidepressant [9], anti-inflammatory [10], antimicrobial [11], anticonvulsant [12], antihelmintics [13], and anticancer [14].

Following these findings, we selected tetrazolo [1,5-a]quinazolin-5(4H)-one (II) as the parent nucleus instead of the tetrazolo[1,5-a] quinazoline (III) in compound I, and then substituted the phenyl group attached to the NH group of the quinazolin-4(3H)-one to obtain compound 6a (Fig. 1). Based on 6a, we also synthesized derivatives 7–14 using bioisosterism as well as other strategies. Then, this series of derivatives were evaluated for antidepressant with the FST. Based on the results of a preliminary screening (Table 1), the derivatives with tetrazolium substitution displayed the best antidepressant activity and decreased the immobility times by 63.90 % at a dose of 50 mg/kg. Hence, 6a was chosen as the parent scaffold, and a series of 4-(substituted-phenyl)tetrazolo[1,5-a]quinazolin-5(4H)-ones (6b–x) were designed and synthesized accordingly. All the synthesized compounds were evaluated for antidepressant activities using the FST, while the tail suspension test (TST) was used to verify the antidepressant activity of compound 6v. Finally, we determined whether the decrease in the immobility time was related to the changes in motor activity using the open-field test and measured the content of 5-HT, NE, and MAO in the brain tissue to elucidate the possible mechanism involved in the homeostasis of brain monoamine neurotransmitters.

Results and discussion

Chemistry

The process used for the preparation of all the compounds is reported in this section, and differently substituted phenylamines were used as starting material. All the compounds were synthesized using a previously reported method with slight modifications as shown in Scheme 1. First, the substituted aniline (a-x) was dissolved in dimethyl sulfoxide (DMSO) and reacted with carbon disulfide and aqueous sodium hydroxide to obtain the sodium salts of substituted phenylcarbamodithioates (1a–x), which were then reacted with dimethyl sulfate to yield methyl substituted phenylcarbamodithioates (2a–x). Next, methyl anthranilate and anhydrous potassium carbonate were added to the solutions of 2a–x in ethanol to obtain the corresponding methyl 2-[(substituted phenylcarbamothioyl)amino] benzoate (3a–x). These intermediates were then converted to compounds 4a–x using a hydrolysis-based process and then reacted with hydrazine hydrate in ethanol to yield 2-hydrazono-3-substituted phenyl-2,3-dihydroquinazolin-4(1H)-ones (5a–x). These compounds were treated with sodium nitrite and diluted hydrochloric acid at a temperature below \(5~^ {\circ }\hbox {C}\), and then stirred at room temperature to obtain compounds (6a–x) [14]. To obtain the target compounds 7–14, compound 5a was reacted with formic acid, acetic acid, urea, carbon disulfide, diethyl oxalate, ammonia, pyruvic acid, and acetylacetone, respectively [15–20].

Pharmacology

The antidepressant activities of the synthesized compounds were investigated in mice using the FST, which is widely used for the preclinical screening of putative antidepressant agents [21–23]. In the FST, mice were forced to swim under inescapable conditions, and consequently adopted an immobility behavior following an initial period of agitation and flurry. The analysis of the data of all the synthesized compounds (Table 1) revealed the following structure–activity relationships. The antidepressant activity of the derivatives with different heterocyclic substituents was in the following order tetrazolium substitution \(>\) methyl-1,2,4-triazole-3-carboxylate substitution \(>\) 4,6-dimethyl-1,2,4-triazin-5(4H)-one substitution. The position of the substituent group on the phenyl ring of the phenyl-substituted derivatives influences their activity markedly in the FST. The derivatives with different electron-donating groups (methyl and phenoxy) on the phenyl ring showed higher activity with the \(p\hbox {-CH}_{3}\) than the \(m\hbox {-CH}_{3}\) group, while the phenoxy substituents were inactive. Further, the activity of the derivatives with halogen substituents on the phenyl ring was in the order \(o\hbox {-F} > o,p\hbox {-Cl}_{2} > o\hbox {-Cl} > m\hbox {-Br} > p\hbox {-Cl}\). Replacing the hydrogen atom with the electron-withdrawing moieties \(p\hbox {-F}\), \(m\hbox {-F}\), and \(p\hbox {-Br}\) yielded compounds with low activity. The electron-donating groups appeared to be more effective in enhancing activity than the electron-withdrawing moieties were.

In Table 2, most of the compounds (except 6d, 6i, 6k, 6m–n, 6p, and 6s–t) showed potent antidepressant activities at a dose of 50 mg/kg intraperitoneally (i.p.). In particular, administration of compounds 6l, 6u, and 6v significantly reduced the periods of persistent immobility times compared to that observed in the control group \((p < 0.001)\). Compounds 6l, 6u, and 6v reduced the immobility times to 14.67, 16.50, and 11.83 s, respectively, compared to the immobility time for FXT (17.83 s). Further, they exhibited higher DID (%) values (78.53, 75.85, and 82.69 %) than that of FXT (73.91 %) and, therefore, had higher levels of antidepressant activity than FXT.

Scheme 1
scheme 1

Synthesis of compounds 6a–x, 7, 8, 9, 10, 11, 12, 13 and 14. a \(\hbox {NaNO}_{2}\), 10 % HCl, \(0{-}3~^{\circ }\hbox {C}\), 6 h, b HCOOH, reflux, 12 h, c \(\hbox {CH}_{3}\hbox {COOH}\), reflux, 12–16 h, d \(\hbox {NH}_{2}\hbox {CONH}_{2}\), \(200~^{\circ }\hbox {C}\), 1.5 h, e \(\hbox {CS}_{2}\), KOH, ethanol, reflux, 8–12 h; f Diethyl oxalate, reflux, 1 h, g \(\hbox {NH}_{3}\). \(\hbox {H}_{2}\hbox {O}\), ethanol, rt, 3 h, h CH\(_{3}\)COCOOH, ethanol, reflux, 10 h, i \((\hbox {CH}_{3}\hbox {CO})_{2}\hbox {CH}_{2}\), 1,4-dioxane, reflux, 3 h

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To verify the antidepressant activity of the compounds, the TST was conducted, because it is commonly used to predict the activities of antidepressant agents. In this study, as shown in Table 3, compound 6v showed a good activity profile compared to FXT at a dose of 50 mg/kg.

Table 2 Antidepressant activities of compounds 6b–6x in the FST
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Table 3 Antidepressant activities of compound 6v and FXT in the FST
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As shown in Table 4, we constructed a dose–response curve for compound 6v for behavioral despair using the FST results. Administration of compound 6v significantly decreased the immobility time at doses of 25 and 50 but not 10 mg/kg. Therefore, compound 6v exerted a dose-dependent antidepressant effect.

Table 4 Antidepressant activities of compound 6v and FXT in the TST
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The open-field test is a robust test frequently used to evaluate the autonomic effects of drugs and the general activities of animals [24]. The results of this test revealed that compound 6v did not have a significant effect on the motor activities (i.e., crossing, rearing, and grooming) in mice (Table 5), suggesting that the antidepressant activity of 6v observed in the FST did not result from central nervous system (CNS)-stimulant properties.

The mechanism of depression is very complicated and the pathogenesis still remains unclear. The classical ‘monoamine hypothesis’ is one of the theories on the etiology of depression, it is considered that symptoms of depression result from anomalies in monoaminergic neurotransmission, primarily serotonin and noradrenaline pathways, and disturbances in serotonergic and noradrenergic transmissions have been generally postulated to form neurochemical background of depression [25, 26]. The weakness for the function of the monoamine system is not necessarily to depression, but increasing the level of monoamine in brain can lead to antidepressant effect [27]. Furthermore, studies show that fluoxetine can increase the levels of miR-16 in the brain 5-HT neurons, thereby inhibit 5-HT transporter (SERT) expression, to raise the level of 5-HT in the synaptic cleft [28]. The determination result of brain NE, 5-HT, and MAO concentration is presented in Table 6. After single drug treatment and FST, NE and 5-HT levels of all testing sample groups were increased, NE and 5-HT levels were increased to 0.281 and 1.124 ng/mg at a dose of 25 mg/kg and to 0.401 ng/mg and 1.499 ng/mg at a dose of 50 mg/kg, respectively. Compared with the control, the NE and 5-HT concentrations of mice brain in the testing sample groups were significantly increased (p \(<\)0.01 and p \(<\) 0.05, respectively) at dose of 50 mg/kg. However, there was no significant difference at a dose of 25 mg/kg. The MAO level of all testing groups was reduced, but compared with the control, it showed no significant difference.

Table 5 Effect of compound 6v in the open-field test
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Conclusion

A series of 4-(substituted-phenyl)tetrazolo[1,5-a]quinazolin-5(4H)-ones derivatives were synthesized and evaluated for antidepressant effects. Most of them showed antidepressant activity in the FST. In particular, 4-(p-tolyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6v) showed significant antidepressant activity with a higher efficacy than that shown by FXT in the FST and TST at an i.p. dose of 50 mg/kg. Furthermore, based on the detected content of 5-HT, NE, and MAO in the brain tissue, we can deduce that its underlying mechanism may be related to the regulation of the homeostasis of brain monoamine neurotransmitter. Finally, other possible mechanisms still need to be elucidated in further research studies.

Table 6 Effect of FXT and 6v on brain NE, 5-HT, MAO level in mice
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Experimental procedures

Chemistry

Melting points of the compounds were determined using open capillary tubes and were uncorrected. Infrared (IR) spectra were measured in potassium bromide (KBr) using an IR Prestige-21 (PerkinElmer, Waltham, MA, USA). The proton \((^{1}\hbox {H})\) and carbon-13 \((^{13}\hbox {C})\) nuclear magnetic resonance (NMR) spectra were recorded at 300 and 75 MHz, respectively, on an AV-300 spectrometer (Bruker, Switzerland), and all chemical shifts were provided in ppm relative to the tetramethylsilane (TMS; s, d, t, and m = singlet, doublet, triplet, multiplet, respectively). High-resolution mass spectra were measured using a matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)/TOF mass spectrometer (Bruker Daltonik, Germany). All chemicals used were either analytical reagent (AR) grade or purified using standard techniques.

Synthesis of methyl phenylcarbamodithioate derivatives 2a–x [29]

A substituted aniline (0.03 mol) was dissolved in DMSO (20 mL), and 1.5 mL of an aqueous solution of sodium hydroxide (20 M) was added simultaneously with 2.74 g carbon disulfide (0.036 mol) under stirring at room temperature to obtain the intermediates (1a–x). Then, 2–4 h later, dimethyl sulfate (0.03 mol) was added dropwise under stirring while keeping the reaction system in ice-cold water for 5 h. Finally, the reaction mixture was poured into ice-cold water, and the resulting solids were filtered, washed, and re-crystallized from ethanol to obtain pure compounds 2a–x.

Synthesis of 3-phenyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one derivatives 4a–x [30]

A solution with one of the 2a–x compounds (0.02 mol), methyl anthranilate (0.02 mol), anhydrous potassium carbonate (200 mg), and ethanol (40 mL) was refluxed for 25–33 h under stirring. The reaction mixture was poured into cold water, and the crude product of the methyl 2-[(phenylcarbamothioyl)amino] benzoate derivatives (3a–x) was isolated from water by filtration. These derivatives were transferred to a different flask and then refluxed in 10 % alcoholic sodium hydroxide solution for 2 h. After cooling to room temperature under stirring, dilute hydrochloric acid was added to the mother liquor, until the precipitate completely dissolved out. The precipitate formed (4a–x) was filtered to yield a off-white solid, which was used without further purification.

Synthesis of 2-hydrazono-3-phenyl-2,3-dihydroquinazolin-4(1H)-one derivatives 5a–x [31]

3-Phenyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one derivatives (4a–x, 0.01 mol), hydrazine hydrate (60 %, 0.12 mol), and ethanol (25 mL) were mixed in round-bottom flask and refluxed for 4 h with stirring under nitrogen. The reaction mixture was then poured into cold water, a crude product (5a–x) was filtrated, and purified using silica gel column chromatography with dichloromethane:methanol \((\hbox {CH}_{2}\hbox {Cl}_{2}:\hbox {CH}_{3}\hbox {OH}; 80:1)\) as the eluent.

Synthesis of 4-phenyltetrazolo[1,5-a]quinazolin-5(4H)-one derivatives 6a–x [14]

One of the compounds 5a–x (0.01 mol) was dissolved in dilute hydrochloric acid (20 mL) in a round-bottom flask, and 10 % sodium nitrite \((\hbox {NaNO}_{2}\), 0.69 g, 0.01 mol) solution was added dropwise in an ice-bath to ensure that the system temperature did not exceed \(5~^{\circ }\hbox {C}\). The solution was stirred at room temperature and monitored using thin-layer chromatography (TLC). At the end of the reaction, the reaction mixture was extracted with \(\hbox {CH}_{2}\hbox {Cl}_{2}\, (3 \times 20\, \hbox {mL})\). The combined organic extracts were washed with water, dried over anhydrous \(\hbox {MgSO}_{4}\), and evaporated to dryness, and the resulting residue was purified using column chromatography with \(\hbox {CH}_{2}\hbox {Cl}_{2 }\) as the eluent to yield products 6a–x.

4-Phenyltetrazolo[1,5-a]quinazolin-5(4H)-one ( 6a)

White solid, Yield: 34 %, mp: \(187\hbox {--}189~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR}\) (\(\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 7.49–8.51(m, 9H, Ar–H). \(^{13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 158.7, 150.7, 136.1, 133.9, 132.8, 130.3, 130.0, 129.9, 128.9, 127.7, 116.7, 116.0. IR (\(\hbox {KBr}, \hbox {cm}^{-1})\): 1703 (C=O), 1618, 1549, 1273, 1130. ESI-HRMS calcd for \(\hbox {C}_{14}\hbox {H}_{11}\hbox {N}_{5}\hbox {O}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 264.0880; found: 264.0887.

4-(2-Methoxyphenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6b)

White solid, Yield: 36 %, mp: \(228\hbox {--}230~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR}\, (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 3.79 (s, 3H,–\(\hbox {CH}_{3}\)), 7.14–8.49 (m, 8H, Ar-H). \(^{ 13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 158.6, 154.8, 150.6, 135.9, 132.9, 131.8, 130.3, 129.4, 128.7, 122.4, 121.4, 116.7, 116.0, 112.6, 55.9. IR \((\hbox {KBr}, \hbox {cm}^{-1})\): 1689 (C=O), 1613, 1552, 1284, 1254, 1172, 1138. ESI-HRMS calcd for \(\hbox {C}_{15}\hbox {H}_{12}\hbox {N}_{5}\hbox {O}_{2}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 294.0986; found: 294.0986.

4-(4-Methoxyphenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6c)

White solid, Yield: 37 %, mp: \(235\hbox {--}237~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 3.90 (s, 3H,–\(\hbox {CH}_{3})\), 7.10–8.49 (m, 8H, Ar–H). \(^{ 13}\hbox {C-NMR} (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 160.5, 159.0, 150.9, 136.1, 132.7, 130.3, 128.9, 128.8, 126.3, 116.7, 116.0, 115.2, 55.6. IR (KBr, \(\hbox {cm}^{-1})\): 1688 (C=O), 1612, 1551, 1285, 1252, 1175, 1138. ESI-HRMS calcd for \(\hbox {C}_{15}\hbox {H}_{12}\hbox {N}_{5}\hbox {O}_{2}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 294.0986; found: 294.0988.

4-(4-Propoxyphenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6d)

Off-white solid, Yield: 35 %, mp: \(217\hbox {--}219~^ {\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 1.09 (t, 3H, J = 7.50 Hz, –\(\hbox {CH}_{3})\), 1.89 (m, 2H, –\(\hbox {CH}_{2}\)–), 4.02 (t, 2H, J =6.00Hz, \(\hbox {--OCH}_{2}\)–), 7.09–8.50 (m, 8H, Ar–H). \(^{13}\hbox {C-NMR}\) (\(\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 160.1, 159.0, 151.0, 136.0, 132.8, 130.3, 128.8, 128.7, 126.1, 116.7, 116.0, 115.7, 69.9, 22.5, 10.5. IR (KBr, \(\hbox {cm}^{-1})\): 1690 (C=O), 1616, 1552, 1283, 1251, 1175, 1136. ESI-HRMS calcd for \(\hbox {C}_{17}\hbox {H}_{16}\hbox {N}_{5}\hbox {O}_{2}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 322.1299; found: 322.1304.

4-(4-Butoxyphenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6e)

White solid, Yield: 40 %, mp: \(187\hbox {--}189~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 1.02 (t, 3H, J = 7.50 Hz, –\(\hbox {CH}_{3})\), 1.50–1.86 (m, 4H, –\(\hbox {CH}_{2}\)–), 4.06 (t, 2H, J = 6.00 Hz, \(\hbox {--OCH}_{2}\)–), 7.09–8.50 (m, 8H, Ar–H). \(^{13}\)C-NMR  (CDCl\(_{3}\), 75 MHz) \(\delta \): 160.1, 159.0, 153.0, 136.0, 132.8, 130.3, 128.8, 128.7, 126.1, 116.7, 116.0, 115.6, 68.1, 31.2, 19.3, 13.8. IR (KBr, \(\hbox {cm}^{-1})\): 1693 (C=O), 1620, 1553, 1279, 1250, 1175, 1134. ESI-HRMS calcd for \(\hbox {C}_{18}\hbox {H}_{18}\hbox {N}_{5}\hbox {O}_{2}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 336.1455; found: 336.1447.

4-(4-(Pentyloxy)phenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6f)

White solid, Yield: 37 %, mp: \(172\hbox {--}174~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 0.96 (t, 3H, J = 6.00 Hz, –\(\hbox {CH}_{3})\), 1.30–1.86 (m, 6H, –\(\hbox {CH}_{2}\)–), 4.04(t, 2H, J = 6.00 Hz, \(\hbox {--OCH}_{2}\)–), 7.08–8.49(m, 8H, Ar–H). \(^{ 13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 160.1, 159.0, 151.0, 136.0, 132.7, 130.3, 128.9, 128.7, 126.1, 116.7, 116.0, 115.6, 68.4, 28.9, 28.2, 22.4, 11.0. IR (KBr, \(\hbox {cm}^{-1})\): 1693 (C=O), 1620, 1553, 1278, 1250, 1176, 1134. ESI-HRMS calcd for C\(_{19}\hbox {H}_{20}\hbox {N}_{5}\)O\(_{2}^{+}\) ([M + H]\(^{+})\): 350.1612; found: 350.1614.

4-(4-(Hexyloxy)phenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6g)

White solid, Yield: 38 %, mp: \(166\hbox {--}168~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 0.97 (t, 3H, J = 6.00 Hz, -\(\hbox {CH}_{3})\), 1.37-1.87 (m, 8H, –CH\(_{2}\)–), 4.05 (t, 2H, J = 6.00 Hz, \(\hbox {--OCH}_{2}\)–), 7.09–8.50 (m, 8H, Ar–H). \(^{ 13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 160.1, 159.0, 151.0, 136.0, 132.8, 130.3, 128.8, 128.7, 126.1, 116.7, 116.0, 115.6, 68.4, 31.6, 29.1, 25.7, 22.6, 14.1. IR (KBr, \(\hbox {cm}^{-1})\): 1694 (C=O), 1620, 1553, 1277, 1250, 1179, 1134. MS m / z 364 (M+1). ESI-HRMS calcd for \(\hbox {C}_{20}\hbox {H}_{22}\hbox {N}_{5}\hbox {O}_{2}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 364.1678; found: 364.1761.

4-(4-(Octyloxy)phenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6h)

Yellow solid, Yield: 41 %, mp: \(174\hbox {--}176~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 0.92 (t, 3H, J = 6.00Hz, –\(\hbox {CH}_{3})\), 1.33–1.89 (m, 12H, –CH\(_{2}\)–), 4.05 (t, 2H, J = 7.50 Hz, \(\hbox {--OCH}_{2}\)–), 7.09–8.50 (m, 8H, Ar–H). \(^{13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 160.1, 159.0, 151.0, 136.0, 132.8, 130.3, 128.8, 128.7, 126.1, 116.7, 116.0, 115.7, 68.4, 31.8, 29.3, 29.3, 29.2, 26.0, 22.7, 14.1, IR (KBr, \(\hbox {cm}^{-1})\): 1695 (C=O), 1620, 1554, 1277, 1250, 1181, 1134. ESI-HRMS calcd for \(\hbox {C}_{22}\hbox {H}_{26}\hbox {N}_{5}\hbox {O}_{2}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 392.2081; found: 292.2080.

4-(4-(Decyloxy)phenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6i)

White solid, Yield: 35 %, mp: \(159\hbox {--}160~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 0.89 (t, 3H, J = 6.00Hz, \(\hbox {--CH}_{3})\), 1.29–1.87 (m, 16H, –CH\(_{2}\)–), 4.03 (t, 2H, J = 6.00 Hz, \(-\hbox {OCH}_{2}\)–), 7.07–8.48 (m, 8H, Ar–H). \(^{13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 160.1, 159.0, 151.0, 136.0, 132.8, 130.3, 128.8, 128.7, 126.1, 116.7, 116.0, 115.7, 68.4, 31.8, 29.3, 29.3, 29.2, 26.0, 22.7, 14.1, IR (KBr, cm\(^{-1}\)) : 1696 (C=O), 1620, 1553, 1275, 1250, 1182, 1134. ESI-HRMS calcd for \(\hbox {C}_{24}\hbox {H}_{30}\hbox {N}_{5}\hbox {O}_{2}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 420.2394; found: 420.2394.

4-(4-(Dodecyloxy)phenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6j)

White solid, Yield: 32 %, mp: \(152\hbox {--}154~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 0.89 (t, 3H, J = 7.50Hz, \(\hbox {--CH}_{3})\), 1.06–1.87 (m, 20H, –\(\hbox {CH}_{2}\)–), 4.03 (t, 2H, J = 6.00 Hz, \(-\hbox {OCH}_{2}\)–), 7.07–8.49 (m, 8H, Ar–H). \(^{13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 160.1, 159.0, 150.9, 136.0, 132.7, 130.3, 128.8, 128.7, 126.0, 116.7, 116.0, 115.6, 68.4, 32.0, 31.9, 29.7, 29.6, 29.6, 29.4, 29.2, 26.1, 26.0, 22.7, 14.1, IR (KBr, cm\(^{-1}\)): 1697 (C=O), 1620, 1553, 1274, 1250, 1183, 1134. ESI-HRMS calcd for \(\hbox {C}_{26}\hbox {H}_{34}\hbox {N}_{5}\hbox {O}_{2}+ ([\hbox {M} + \hbox {H}]^{+})\): 448.2707; found: 448.2707.

4-(4-(Tetradecyloxy)phenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6k)

White solid, Yield: 27 %, mp: \(146\hbox {--}148~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 0.88 (t, 3H, J = 6.00Hz, \(\hbox {--CH}_{3})\), 1.05–1.85 (m, 24H, –CH\(_{2}\)–), 4.03 (t, 2H, J = 6.00 Hz, \(-\hbox {OCH}_{2}\)–), 7.07–8.49 (m, 8H, Ar–H). \(^{13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 160.1, 159.0, 150.9, 136.0, 132.7, 130.3, 128.9, 128.7, 126.0, 116.7, 116.0, 115.6, 68.4, 31.9, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3, 29.3, 29.2, 29.2 26.0, 22.7, 14.1, IR (KBr, cm\(^{-1}\)): 1697 (C=O), 1620, 1553, 1274, 1250, 1183, 1134. ESI-HRMS calcd for \(\hbox {C}_{28}\hbox {H}_{38}\hbox {N}_{5}\hbox {O}_{2}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 476.3020; found: 474.3025.

4-(2-Fluorophenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6l)

White solid, Yield: 41 %, mp: \(184\hbox {--}186~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 7.35–8.51 (m, 8H, Ar–H). \(^{ 13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 159.5, 158.1, 156.1, 150.1, 136.3, 132.9, 132.3, 132.2, 130.4, 129.7, 129.0, 125.3, 125.3, 121.6, 121.4, 117.4,117.2, 116.3, 116.1. IR (KBr, \(\hbox {cm}^{-1})\): 1699 (C=O), 1622, 1558, 1283, 1136. ESI-HRMS calcd for \(\hbox {C}_{14}\hbox {H}_{\mathrm{9}}\hbox {FN}_{5}\hbox {O}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 282.0786; found: 282.0782.

4-(3-Fluorophenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6m)

White solid, Yield: 32  %, mp: \(200\hbox {--}202~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 7.25–8.50 (m, 8H, Ar–H). \(^{ 13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \):164.6, 161.3, 158.5, 150.4, 136.4, 134.9, 134.8, 132.7, 131.2, 131.1, 130.3, 129.0, 123.7, 123.6, 117.5, 117.2, 116.4, 116.1, 115.9, 115.6. IR (KBr, \(\hbox {cm}^{-1})\): 1699 (C=O), 1624, 1556, 1284, 1138. ESI-HRMS calcd for \(\hbox {C}_{14}\hbox {H}_{\mathrm{9}}\hbox {FN}_{5}\hbox {O}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 282.0786; found: 282.0793.

4-(4-Fluorophenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6n)

White solid, Yield: 52  %, mp: \(249\hbox {--}251~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 7.29–8.50 (m, 8H, Ar–H). \(^{ 13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 164.7, 161.4, 158.7, 150.6, 136.3, 132.8, 130.3, 129.8, 129.7, 129.0, 117.2, 116.9, 116.5, 126.0. IR (KBr, \(\hbox {cm}^{-1})\) : 1697 (C=O), 1621, 1555, 1282, 1133. ESI-HRMS calcd for \(\hbox {C}_{14}\hbox {H}_{\mathrm{9}}\hbox {FN}_{5}\hbox {O}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 282.0786; found: 282.0786.

4-(2-Chlorophenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6o)

White solid, Yield: 46  %, mp: \(181\hbox {--}182~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 7.52–8.51 (m, 8H, Ar–H). \(^{ 13}\hbox {C-NMR}(\hbox {CDCl}_{3}, 75\,\hbox {MHz})\) \(\delta \): 158.1, 150.0, 136.4, 132.9, 132.7, 131.8, 131.6, 131.0, 130.4, 130.1, 129.0, 128.5, 116.3, 116.1. IR (KBr, \(\hbox {cm}^{-1}\)): 1697 (C=O), 1620, 1553, 1281, 1132. ESI-HRMS calcd for \(\hbox {C}_{14}\hbox {H}_{9}\hbox {ClN}_{5}\hbox {O}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 298.0490; found: 298.0484.

4-(3-Chlorophenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6p)

White solid, Yield: 50 %, mp: \(208\hbox {--}210~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 7.41–8.50 (m, 8H, Ar–H). \(^{ 13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 158.5, 149.7, 136.4, 135.5, 134.7, 132.7, 130.9, 130.4, 130.3, 129.1, 128.3, 126.1, 116.4, 116.1. IR (KBr, \(\hbox {cm}^{-1})\): 1697 (C=O), 1621, 1554, 1283, 1134. ESI-HRMS calcd for \(\hbox {C}_{14}\hbox {H}_{9}\hbox {ClN}_{5}\hbox {O}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 298.0490; found: 298.0490.

4-(4-Chlorophenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6q)

White solid, Yield: 40 %, mp: \(278\hbox {--}280~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR}\) (\(\hbox {DMSO-{ d}}_{6,}\) 300 MHz) \(\delta \): 7.58–8.37(m, 8H, Ar–H). \(^{ 13}\hbox {C-NMR}\) (\(\hbox {DMSO-{ d}}_{6}\), 75 MHz) \(\delta \): 159.3, 151.4, 136.5, 134.6, 134.1, 133.1, 130.7, 130.0, 129.6, 129.1, 117.7, 115.9. IR (KBr, \(\hbox {cm}^{-1})\): 1696 (C=O), 1620, 1550, 1281, 1133. ESI-HRMS calcd for \(\hbox {C}_{14}\hbox {H}_{9}\hbox {ClN}_{5}\hbox {O}^{+} ([\hbox {M} + \hbox {H}]^{+})\): 298.0490; found: 289.0490.

4-(3-Bromophenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6r)

White solid, Yield: 33  % , mp: \(221\hbox {--}223~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 7.46–8.50 (m, 8H, Ar–H). \(^{ 13}\hbox {C-NMR}(\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 158.5, 148.8, 140.8, 136.4, 134.8, 133.3, 132.7, 131.1, 130.3, 129.1, 126.6, 123.1, 116.4, 116.1. IR (KBr, \(\hbox {cm}^{-1})\): 1699 (C=O), 1620, 1551, 1275, 1134. ESI-HRMS calcd for \(\hbox {C}_{14}\hbox {H}_{9}\hbox {BrN}_{5}\hbox {O}^{+} ([\hbox {M} + \hbox {H}]^{+})\): 341.9985; found: 341.9985.

4-(4-Bromophenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6s)

White solid, Yield: 38 %, mp: \(254\hbox {--}255~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR}\, (\hbox {DMSO-{ d}}_{6}\), 300 MHz) \(\delta \): 7.52–8.36 (m, 8H, Ar–H). \(^{ 13}\hbox {C-NMR}\, (\hbox {DMSO-{ d}}_{6}\), 75 MHz) \(\delta \): 159.3, 151.3, 136.5, 134.5, 133.1, 133.0, 131.0, 129.6, 129.1, 123.1, 117.7, 115.9. IR (KBr, \(\hbox {cm}^{-1})\): 1696 (C=O), 1620, 1550, 1281, 1136. ESI-HRMS calcd for \(\hbox {C}_{14}\hbox {H}_{9}\hbox {BrN}_{5}\hbox {O}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 341.9985; found: 341.9985.

4-(o-Tolyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6t)

White solid, Yield: 42 %, mp: \(158\hbox {--}160~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 2.19 (s, 3H, \(\hbox {Ar--CH}_{3})\), 7.33–8.52 (m, 8H, Ar–H). \(^{13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 158.4, 150.3, 136.2, 135.9, 133.0, 132.9, 131.8, 130.6, 130.3, 128.9, 128.1, 127.7, 116.5, 116.1, 17.7. IR (KBr, \(\hbox {cm}^{-1})\): 1695 (C=O), 1614, 1551, 1178, 1129. ESI-HRMS calcd for \(\hbox {C}_{15}\hbox {H}_{12}\hbox {N}_{5}\hbox {O}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 278.1036; found: 278.1031.

4-(m-Tolyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6u)

White solid, Yield: 46 %, mp: \(195\hbox {--}197~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 2.48 (s, 3H, Ar\(\hbox {--CH}_{3})\), 7.30–8.51 (m, 8H, Ar–H). \(^{ 13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 158.8, 151.6, 140.2, 136.1, 133.7, 132.8, 131.0, 130.3, 129.8, 128.9, 128.2, 124.6, 116.7, 116.0, 21.4. IR (KBr, \(\hbox {cm}^{-1})\): 1695 (C=O), 1616, 1552, 1178, 1128. ESI-HRMS calcd for \(\hbox {C}_{15}\hbox {H}_{12}\hbox {N}_{5}\hbox {O}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 278.1036; found: 278.1031.

4-(p-Tolyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6v)

White solid, Yield: 43 %, mp: \(239\hbox {--}240~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 2.49 (t, 3H, Ar\(\hbox {--CH}_{3})\), 7.36–8.51 (m, 8H, Ar–H). \(^{ 13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \):158.8, 150.8, 140.3, 136.1, 132.8, 131.2, 130.6, 130.3, 128.9, 127.4, 116.7, 116.0, 21.4. IR (KBr, \(\hbox {cm}^{-1})\): 1693 (C=O), 1613, 1550, 1179, 1130. ESI-HRMS calcd for \(\hbox {C}_{15}\hbox {H}_{12}\hbox {N}_{5}\hbox {O}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 278.1036; found: 278.0756.

4-(4-(Trifluoromethyl)phenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6w)

White solid, Yield: 31 %, mp: \(254\hbox {--}256~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 7.68–8.52 (m, 8H, Ar–H). \(^{ 13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 158.4, 150.2, 136.8, 136.5, 132.7, 130.4, 129.1, 128.4, 127.1, 127.0, 127.0, 116.3, 116.1. IR (KBr, \(\hbox {cm}^{-1})\): 1697 (C=O), 1614, 1553, 1281, 1130. ESI-HRMS calcd for \(\hbox {C}_{15}\hbox {H}_{9}\hbox {F}_{3}\hbox {N}_{5}\hbox {O}^{ +}\, ([\hbox {M} + \hbox {H}]^{+})\): 332.0754; found: 332.0756.

4-(2,4-Dichlorophenyl)tetrazolo[1,5-a]quinazolin-5(4H)-one (6x)

Pink solid, Yield: 35 %, mp: \(238\hbox {--}240~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 7.38–8.49 (m, 7H, Ar–H). \(^{13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 158.4, 158.3, 146.1, 136.5, 134.7, 134.0, 132.7, 131.5, 130.7, 130.4, 130.0, 129.1, 127.2, 116.1. IR (KBr, \(\hbox {cm}^{-1})\): 1707 (C=O), 1618, 1555, 1273, 1134. ESI-HRMS calcd for \(\hbox {C}_{14}\hbox {H}_{8}\hbox {Cl}_{2}\hbox {N}_{5}\hbox {O}^{ +}\, ([\hbox {M} + \hbox {H}]^{+})\): 332.0100; found: 332.0096.

Synthesis of 4-phenyl-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one (7)  [15]

A solution of compound 5a (0.01 mol) and formic acid (25 mL) was refluxed overnight under stirring and then poured into cold water. The target compound (7) was obtained by filtration, washing with water, drying, and purification using silica gel column chromatography with \(\hbox {CH}_{2}\hbox {Cl}_{2}:\hbox {CH}_{3}\hbox {OH}\) (100 : 1) as the eluent.

White solid, Yield: 30 %, mp: \(303\hbox {--}305~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 7.48–8.48 (m, 9H, Ar–H), 8.81(s, 1H, –N=CH–). \(^{13}\hbox {C-NMR}\, (\hbox {CDCl}_{3}\), 75 MHz) \(\delta \): 158.5, 148.9, 135.5, 134.2, 134.1, 132.5, 130.8, 129.8 129.6, 128.1, 127.5, 117.3, 114.7. IR (KBr, \(\hbox {cm}^{-1})\): 1688 (C=O), 1614, 1599, 1300, 1148. ESI-HRMS calcd for \(\hbox {C}_{15}\hbox {H}_{11}\hbox {N}_{4}\hbox {O}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 263.0927; found: 263.0934.

Synthesis of 1-methyl-4-phenyl[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one (8) [15]

Compound 5a (0.01 mol) was dissolved in acetic acid (25 mL), stirred and refluxed overnight, and then poured into ice water. The precipitated product was filtrated and purified by silica gel column chromatography with \(\hbox {CH}_{2}\hbox {Cl}_{2}:\hbox {CH}_{3}\hbox {OH}\) (100:1) to a pure product (8).

White solid, Yield: 31 %, mp: \(303\hbox {--}305~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 2.46 (s, 3H, \(\hbox {--CH}_{3})\), 7.34–8.47 (m, 9H, Ar–H). \(^{13}\hbox {C-NMR}\, (\hbox {CDCl}_{3, }\)75 MHz) \(\delta \): 158.6, 149.5, 145.8, 135.1, 134.4, 134.2, 131.0, 129.8, 129.5, 128.2, 126.9, 117.8, 115.0, 15.7. IR (KBr, \(\hbox {cm}^{-1})\): 1682 (C=O), 1610, 1597, 1317, 1180. ESI-HRMS calcd for \(\hbox {C}_{16}\hbox {H}_{13}\hbox {N}_{4}\hbox {O}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 277.1084; found: 277.1079.

Synthesis of 4-phenyl-[1,2,4]triazolo[4,3-a]quinazoline-1,5(2H,4H)-dione (9) [16]

A mixture of compound 5a (0.01 mol) and urea (4.8 g, 0.08 mol) was melted by heating it to \(200~^{\circ }\hbox {C}\). The reaction was monitored using TLC until completion, and then, the system was cooled and washed with enough hot water to remove the excess urea. The target compound 9 obtained was then filtered, dried, and subjected to silica gel column chromatography using \(\hbox {CH}_{2}\hbox {Cl}_{2}:\hbox {CH}_{3}\hbox {OH}\) (60 : 1) as the eluent.

White solid, Yield: 36 %, mp: \(>\)300 \(^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {DMSO-}d_{6}\), 300 MHz) \(\delta \): 7.47–8.70 (m, 9H, Ar–H), 11.83 (s, 1H, NH). \(^{13}\)C-NMR (DMSO-\({ d}_{6}\), 75 MHz) \(\delta \): 159.1, 152.7, 151.1, 142.1, 135.3, 135.3, 134.4, 129.6, 129.5, 129.4, 128.9, 126.0, 117.3, 114.9. IR (KBr, \(\hbox {cm}^{-1})\): 3177 (N–H), 1748 (C=O), 1709 (C=O), 1614, 1491, 1352, 1287, 1128, 1031. ESI-HRMS calcd for \(\hbox {C}_{15}\hbox {H}_{11}\hbox {N}_{4}\hbox {O}_{2}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 279.0877; found: 279.0874.

Synthesis of 4-phenyl-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one (10) [17]

Potassium hydroxide (0.56 g, 0.01 mol) was added to an ice-cold solution of 5a (0.01 mol) in absolute ethanol (15 mL) followed by carbon disulfide (1.52 g, 0.02 mol) added dropwise under stirring. The mixture was then diluted with absolute ethanol (10 mL), and the reaction was monitored using TLC until completion. Then, the solvent was removed to obtain a residue, which was purified using silica gel column chromatography using a mixture of \(\hbox {CH}_{2}\hbox {Cl}_{2}:\hbox {CH}_{3}\hbox {OH}\) (60 : 1) as the eluent to yield product 10 .

Blue-gray solid, Yield: 30 %, mp: \(187\hbox {--}189~^{\circ }\hbox {C}\). \(^{1}\)H-NMR (DMSO-\(d_{6}\), 300 MHz) \(\delta \): 7.45–10.39 (m, 9H, Ar–H), 13.85 (s, 1H, NH). \(^{13}\)C-NMR (DMSO-\(d_{6}\), 75 MHz) \(\delta \): 162.2, 158.8, 147.0, 136.0, 134.9, 129.7, 129.6, 129.2, 129.0, 127.4, 118.5, 116.1. IR (KBr, \(\hbox {cm}^{-1})\): 1703 (C=O), 1618, 1549, 1273, 1130. ESI-HRMS calcd for \(\hbox {C}_{15}\hbox {H}_{11}\hbox {N}_{4}\hbox {OS}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 295.0648; found: 295.0652.

Synthesis of ethyl 5-oxo-4-phenyl-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinazoline-1-carboxylate (11)  [18]

A mixture of compound 5a (0.02 mol) and diethyl oxalate (30 mL) was stirred and refluxed for 1 h. The white precipitate (pure compound 11) obtained after cooling was collected using suction filtration and purified by washing with ethanol.

White solid, Yield: 38 %, mp: \(187\hbox {--}189~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR}\, (\hbox {DMSO-{ d}}_{6}\), 300 MHz) \(\delta \): 1.40 (t, 3H, J = 7.50 Hz, –\(\hbox {CH}_{3})\), 4.54 (m, 2H, J = 7.00 Hz, –\(\hbox {CH}_{2}\)–), 7.53–8.42 (m, 9H, Ar–H). \(^{13}\hbox {C-NMR} (\hbox {DMSO-{ d}}_{6}\), 75 MHz) \(\delta \): 159.0, 158.8, 151.3, 141.3, 135.5, 135.2, 133.5, 129.8, 129.6, 129.4, 129.2, 128.1, 119.1, 118.6, 63.3, 14.4. IR (KBr, \(\hbox {cm}^{-1})\): 1724 (C=O), 1692 (C=O), 1611, 1541, 1481, 1273, 1192. ESI-HRMS calcd for \(\hbox {C}_{18}\hbox {H}_{15}\hbox {N}_{4}\hbox {O}_{3}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 335.1139; found: 335.1139.

Synthesis of 5-oxo-4-phenyl-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinazoline-1-carboxamide (12) [18]

Compound 11 (0.01 mol) was added to a mixture of methanol (10 mL) and ammonia (10 mL), and then stirred for 3 h at room temperature. Then, the precipitate was collected using filtration, washed with water, dried, and then purified using silica gel column chromatography with \(\hbox {CH}_{2}\hbox {Cl}_{2} :\hbox {CH}_{3}\hbox {OH}\) (40 : 1) as the eluent to obtain compound 12.

Off-white solid, Yield: 30 %, mp: \(266\hbox {--}268~^{\circ }\hbox {C}\). \(^{1}\)H-NMR (DMSO-\(d_{6}\), 300 MHz) \(\delta \): 7.53–8.62 (m, 9H, Ar–H), 8.24 (s, 1H, NH), 8.66 (s, 1H, NH). \(^{13}\)C-NMR (DMSO-\({ d}_{6}\), 75 MHz) \(\delta \): 160.4, 158.8, 150.8, 144.2, 135.7, 135.3, 133.8, 129.7, 129.5, 129.4, 129.2, 127.8, 118.9, 118.4. IR (KBr, \(\hbox {cm}^{-1})\): 3468 (N-H), 3349 (N-H), 1701 (C=O), 1678 (C=O), 1612, 1584, 1440, 1288. ESI-HRMS calcd for \(\hbox {C}_{18}\hbox {H}_{12}\hbox {N}_{5}\hbox {O}_{2}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 306.0986; found: 306.0991.

Synthesis of 2-methyl-5-phenyl-1H-[1,2,4]triazino[4,3-a]quinazoline-1,6(5H)-dione (13)  [19]

A mixture of 5a (0.02 mol) and pyruvic acid (0.03 mol) in absolute ethanol and glacial acetic acid (20 mL, v/v, 1:1) was refluxed for 10 h under stirring. To remove the solvent, glacial acetic acid (20 mL) was added, and reflux continued for 46 h. The crystals that formed after cooling were collected and purified using column chromatography to obtain compound 13.

Off-white solid, Yield: 28 %, mp: \(187\hbox {--}189~^{\circ }\hbox {C}\). \(^{1}\)H-NMR (DMSO-\(d_{6}\), 300 MHz) \(\delta \): 2.88 (s, 3H, –\(\hbox {CH}_{3})\), 7.48–8.30 (m, 9H, Ar–H). \(^{13}\hbox {C-NMR} (\hbox {DMSO-}d_{6}\), 75 MHz) \(\delta \): 158.9, 152.1, 149.6, 146.0, 135.7, 135.7, 134.5, 129.8, 129.7, 129.3, 129.2, 127.0, 118.0, 116.5, 15.4. IR (KBr, \(\hbox {cm}^{-1})\): 1712 (C=O), 1686 (C=O), 1612, 1597, 1568, 1487, 1439, 1315. ESI-HRMS calcd for \(\hbox {C}_{17}\hbox {H}_{13}\hbox {N}_{4}\hbox {O}_{2}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 305.1033; found: 305.1040.

Synthesis of 2-(3,5-dimethyl-1H-pyrazol-1-yl)-3-phenylquinazolin-4(3H)-one (14)  [20]

A mixture of hydrazine 5a (0.01 mol) and acetylacetone (0.012 mmol) in 1,4-dioxane (20 mL) was stirred and heated while refluxing for 3 h. The mixture was diluted in methylene chloride (10 mL) and repeatedly washed with water to remove the solvent. Finally, the organic layer was dried over anhydrous \(\hbox {MgSO}_{4}\), concentrated, and purified by silica gel column chromatography with \(\hbox {CH}_{2}\hbox {Cl}_{2}\) as the eluent to obtain compound 14.

Beige solid, Yield: 34 %, mp: \(178\hbox {--}179~^{\circ }\hbox {C}\). \(^{1}\hbox {H-NMR} (\hbox {CDCl}_{3}\), 300 MHz) \(\delta \): 2.00 (s, 3H, N=C–CH\(_{3})\), 2.31 (s, 3H, C=C–CH\(_{3})\), 5.71 (s, 1H, C=CH–C), 7.17–8.38 (m, 9H, Ar-H). \(^{13}\hbox {C-NMR}\, (\hbox {CDCl}_{3, }\)75 MHz) \(\delta \): 162.5, 150.6, 146.2, 144.1, 141.4, 135.5, 135.0, 128.6, 128.0, 127.9, 127.9, 127.6, 121.3, 114.9, 106.8, 13.2, 11.5. IR (KBr, \(\hbox {cm}^{-1})\): 1692 (C=O), 1611, 1595, 1287, 1273, 1153. ESI-HRMS calcd for \(\hbox {C}_{19}\hbox {H}_{17}\hbox {N}_{4}\hbox {O}^{+}\, ([\hbox {M} + \hbox {H}]^{+})\): 317.1397; found: 317.1397.

Pharmacology

Kunming mice (18–24 g) were housed collectively in polycarbonate cages and maintained on a standardized light–dark cycle with food and water ad libitum for one week before the experiments commenced. The mouse NE, 5-HT, and MAO enzyme-linked immunosorbent assay (ELISA) kits were purchased from the Beijing Pines Biotechnology Co., Ltd., China. The NE bitartrate injection (Wuhan Yuanda Pharmaceutical Co., Ltd., China), FXT (Cp, Shanghai Chinese and Western Pharmaceutical Co., Ltd., China), and test compounds were suspended in 0.3 % methyl cellulose and administered i.p. All doses of the drugs are expressed as mg/kg body weight.

Ethological tests in mice

FST

The FST is widely used for the evaluation of antidepressant activity. Kunming mice (22 \(\pm \) 2 g) were adopted for this study, and the methods used were previously described by Porsolt et al.  [21], and are available in the complete publication as referenced in this study.

TST

The TST was used to verify the antidepressant activity of the specific target compound and conducted following the previously reported methods of Steru et al.  [32, 33], with slight modifications.

Open-field test

The open-field test was used to evaluate the exploratory activity of the animals  [34]. The test was performed using the method described by Archer et al.  [35], with slight modifications.

Neurotoxicity (NT) screening [36, 37]

All test compounds were administered by i.p. injections. The animals were trained to stay on a 1-inch-diameter round rod rotating at 6 rpm for 1 min for the specified time. Each of the test mice was given three chances to maintain their balance on the rod for at least 1 min.

Biochemical tests in mice

Determination of NE, 5-HT, and MAO levels

Following treatment with the test compounds and FST, blood samples from the test animals were collected in the serum separator, allowed to clot for 0.5 h at \(37~^{\circ }\hbox {C}\), and then centrifuged for 10 min at \(3000 \times g\) at \(4~^{\circ }\hbox {C}\). Then, the serum was isolated and stored at \(-80~^{\circ }\hbox {C}\) for subsequent determination of NE, 5-HT, and MAO.

The determination of the concentrations of NE, 5-HT, and MAO was performed using the ELISA method. The optical density (OD) was read at 450 nm using a microplate reader within 15 min, and the NE, 5-HT, and MAO concentration was expressed in ng/mg or U/mg protein of the brain tissue.