Design, synthesis, and in vivo antiepileptic evaluation of novel quinazolinone-phthalimide derivatives

Abstract

Epilepsy impacts millions of individuals globally, with around one-third of patients not responding to existing antiepileptic medications. As a result, in the current study, quinazolinone-phthalimide derivatives were designed, synthesized, and characterized by ¹H NMR, ¹³C NMR, FTIR, and Mass spectrometry. To assess antiepileptic activity, two different animal seizure models pentylenetetrazole and lithium-pilocarpine-induced seizure models were employed. The results indicated that the efficacy of compounds 8j and 8k, in terms of latency and seizure count, was comparable to that of diazepam, with no observed mortality, suggesting a favorable safety profile. Also, antagonistic activity on the GABA_A receptor was confirmed in an animal model in the presence of flumazenil. Docking studies also showed that both compounds interact with key residues in the benzodiazepine-binding pocket and penetrate deeper into the GABA_A receptor binding site. Molecular dynamics confirmed the stability of complexes. Thus, quinazolinone-phthalimide derivatives emerge as promising candidates for further development and investigation as antiepileptic agents.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-43166-8.

Introduction

Epilepsy is a prevalent neurological disorder causing recurrent seizures that can result in cognitive impairment, brain injuries, and other neurological deficits, and if left untreated, may lead to cognitive dysfunction, brain damage, and other neurological deficits. It affects people of all ages and has significant social, economic, and psychological impacts. Epilepsy-related premature death risk is three times higher than general population, with the highest mortality rate in low- and middle-income countries^1,2.

Commonly used antiepileptic drugs (AED), including phenobarbital, phenytoin, and carbamazepine, are less effective and have poorly tolerated side effects^3,4. Despite significant progress in therapy in recent years, management of seizures remains unsatisfactory, with almost 30% of patients experiencing uncontrolled seizures⁵. This necessitates the introduce new anticonvulsant medications that are more effective and have fewer side effects^6–8.

The mechanism of epileptic seizures lies in the dysfunctional balance of excitatory and inhibitory neurotransmission in the central nervous system, giving rise to abnormal neuronal activity and excessive discharge. AEDs currently available can be divided into four main classes based on the molecular mechanism of action including voltage-dependent Na⁺ and/or Ca²⁺ channel modulators, GABA-mediated inhibition enhancers or other GABA system modulators, ionotropic glutamate receptor-mediated inhibitors of synaptic excitement; and synaptic neurotransmitter release modulators⁹.

At present, quinazolines have attracted significant attention as potential candidates for the development of novel AEDs, such as anticonvulsant agents. These agents protect against seizures by targeting GABA receptors and upregulating GABAergic signaling. In particular, quinazolin-4(3 H)-one derivatives have demonstrated anticonvulsant activity via GABAergic glutamatergic and ion-gated voltage^10–12. Methaqualone (A, Fig. 1) and its analogs are good examples of anticonvulsant agents affecting GABA_A receptors. As another example, compound B provided 100% protection in the psychomotor seizure mouse test and exhibited strong binding to the GABA_A receptor¹³. In the following, ligand-based design has generated promising candidates such as compound C, which exhibited significant anticonvulsant activity with ED₅₀= 21.2 mg/kg in the MES test¹⁴. Thioquinazolin-4(3 H)-one derivatives are also synthesized and were found to possess anticonvulsant activity. In particular, compound D, which displayed 100% protection in the seizure animal model, is likely to exert its action via interaction with the GABA_A receptor, making this compound a robust anticonvulsant candidate¹⁵.

Fig. 1.

The general structures of newly designed compounds alongside previously reported antiseizure agents.

Phthalimide ring has demonstrated significant anticonvulsant activity, and hence several potent anticonvulsants have been developed based on this nucleus. Compound E demonstrated strong effects in the PTZ-induced seizure model, increasing the clonic seizure threshold and displaying potency against tonic seizures, comparable to phenytoin. The meta-nitro derivative was more effective than its ortho and para counterparts¹⁶. Another study on the isoindoline-1,3-dione backbone showed high potency of the 4-methoxyphenyl derivative (compound F) in animal models of seizures¹⁷. Compound G, featuring a phthalimide pharmacophore linked to an aliphatic chain, showed significant activity in both the ipPIC and scSTY models, likely through GABA mediation, and exhibited lower neurotoxicity than amide derivatives¹⁸. In PTZ- and MES-induced seizure models, compound H provided dose-dependent protection, with the best protection being observed at 100 mg/kg. In vivo, screening validated its activity against generalized tonic-clonic and absence seizures. Motor-improvement tests showed no evidence of neurotoxicity, and mechanistic studies showed that the isoindole moiety was responsible for its anticonvulsant activity¹⁹.

Encouraged by the promising pharmaceutical profiles of 1,3-isoindolinedione and quinazolin-4(3 H)-one as potent anticonvulsant entities, new anticonvulsant compounds were designed and synthesized. The anticonvulsant potential of these compounds was evaluated using the pentylenetetrazole (PTZ)-induced seizure model in mice and the lithium-pilocarpine-induced status epilepticus model in rats, aiming to explore their efficacy and therapeutic potential in epilepsy management.

Results and discussions

Chemistry

The synthetic pathway is illustrated in Scheme 1. Briefly, 2-(3-bromopropyl)isoindoline-1,3-dione (compound 3) was synthesized by dissolving isoindoline-1,3-dione (compound 1) in dimethylformamide (DMF), followed by the addition of 1,3-dibromopropane (compound 2). The reaction mixture was stirred at room temperature for 24 h, then extracted with dichloromethane. The crude product was purified via column chromatography (yield: 55%). In the next step, following a green synthesis approach, amine derivatives (compound 5a-n) were dissolved in water, and isatoic anhydride (compound 4) was gradually added. After 3 h, the precipitate (compound 6) was filtered (yield: 90%). In the subsequent ring-closing step, carbon disulfide was added dropwise to the ethanolic solution of 6 at 0 °C in 1:1 molar ratio. The reaction mixture was then refluxed for 12 to 15 h to obtain compound 7a-n (yield: 83%).

Scheme 1.

Procedure for synthesis of 8a-n.

Finally, a nucleophilic substitution reaction was carried out by adding compound 7a-n and potassium carbonate in DMF followed by the addition of compound 3 to the mixture and the resultant was stirred at 80 °C overnight to synthesize 8a-n as the final products.

In vivo pentylenetetrazol-induced seizure model (GABAergic evaluation)

PTZ is a GABAA receptor antagonist that inhibits GABAergic neurotransmission and is, therefore, the predominant inhibitory system in the human brain to induce seizures. This model is particularly useful for evaluating the efficacy of drugs targeting the GABAergic system²⁰. Male NMRI mice were divided into 17 equal groups, with n = 5. To induce the seizure model, PTZ was administered as a single intraperitoneal (IP) injection at a dose of 70 mg/kg. The weight of the 8–10-week-old mice used in the study ranged from 25 to 30 g. The groups were classified as follows: NC group represents the negative control, DZP refers to diazepam (2 mg/kg) known as the positive control, TLD represents thalidomide (10 mg/kg), known as another positive control, and 8a–n corresponds to the treated group with a dose of 10 mg/kg.

The latency

Compounds 8a–n at the dose of 10 mg/kg were screened for antiepileptic activity in the PTZ-induced seizure model for 1 h postinjection of the 70 mg/Kg PTZ. The latency time (time to first seizure) results are visualized in Fig. 2, with statistical analysis indicating significant differences between treatment groups. SAR analysis revealed that compounds 8j with 6.79 min (R: ethylphenyl) and 8k with 6.95 min (R: cyclohexyl) exhibited no significant differences in latency times compared to diazepam with 6.98 min (2 mg/kg), indicating potent anticonvulsant effects. Compounds 8a (3-chlorophenyl), 8b (benzyl), 8d (cyclopentyl), 8f (2,3-dimethylphenyl), 8 g (4-methylphenyl), and 8n (cyclopropyl) displayed activity similar to thalidomide (10 mg/kg) with no significant differences, prolonging seizure onset. In contrast, compounds 8c (phenyl), 8e (2-chloro-4-methylphenyl), 8 h (3-methylphenyl), 8i (3-methoxyphenyl), 8 L (4-methoxyphenyl), and 8 m (butyl) did not significantly differ from the negative control group, indicating weaker effects on seizure latency.

Fig. 2.

The in vivo evaluation of the synthesized compounds at the onset of the first seizure in the PTZ seizure model. Tukey test which was used for comparison in which * was representative for p < 0.05, ** was representative for p < 0.01 and *** was representative for p < 0.001.

The results indicate that electron-donating substituents 8 g, 8 h, 8i, and 8 L decrease the strength of anticonvulsant activity. Electron-donating groups increase the electron density of the aromatic ring, leading to weaker π–π stacking interactions with essential aromatic residues in the GABA_A receptor binding pocket. The methoxy group alters the receptor interactions required for deep receptor-cavity binding. Cyclohexyl in 8k creates favorable conditions that enhance blood-brain barrier (BBB) permeability, as described in Sect.  2.6, thereby increasing drug efficacy. The same trend, to a lesser extent, was observed in 8n, which has cyclopropyl substituents with improved BBB permeation. The linker extension leads to improved time latency results, which are demonstrated in ethylphenyl 8j and benzyl 8b.

Number of seizures

As shown in Fig. 3, there was no significant difference in the number of seizures between diazepam (2 mg/kg) and thalidomide (10 mg/kg), indicating their comparable anticonvulsant effects after 60 min. Most synthesized compounds also showed no significant differences compared to these reference drugs, confirming their high potency in reducing seizure frequency. However, exceptions were observed with 8e (2-chloro-4-methylphenyl) and 8 m (cyclopentyl), which did not reduce seizure frequency significantly compared to the control group. The SAR analysis showed that compounds 8j and 8k had the lowest seizure incidence, making them the most effective treatments for seizure control. This could be due to good BBB permeability and better GABA_A receptor binding capacity. However, there is no significant difference between 8a, 8b, 8 h, 8i, and 8 L, with moderate to good potency. The low effectiveness of 8 m was attributed to linear butyl substitution.

Fig. 3.

The number of seizures in the PTZ-induced seizure model. Tukey test which was used for comparison in which * was representative for p < 0.05, ** was representative for p < 0.01, *** was representative for p < 0.001 and **** was representative for p < 0.0001.

Number of mortality

Mortality was evaluated in the PTZ-induced seizure model (n = 5 animals per group). The negative control group showed 80% mortality (Fig. 4). In contrast, both thalidomide and diazepam exhibited 0% mortality at the tested doses. Among the synthesized derivatives, compounds 8k (cyclohexyl), 8j (ethylphenyl), and 8 g (4-methylphenyl) also showed 0% mortality at 10 mg/kg, suggesting a favorable acute safety profile under the experimental conditions. The corresponding contingency data (alive/dead distribution) and repetation are provided in Table S1 in the Supporting Information.

Fig. 4.

Mortality rate in the PTZ-induced seizure model (n = 5 per group). Mortality (%) is presented for each group.

Evaluation of GABA-A receptor involvement in the anticonvulsant effects of compounds 8j and 8k using flumazenil

To further investigate whether GABA_A receptors mediate their anticonvulsant effects, a co-administration test was performed using the most potent synthesized compounds (8j and 8k) with the selective GABA_A receptor antagonist flumazenil (2 mg/kg). However, if compounds 8j and 8k act as GABA_A receptor modulators to exert their anticonvulsant effects, flumazenil should reverse or attenuate their actions. As expected, flumazenil significantly reduced latency in diazepam-treated mice (p = 0.001), validating the model (Fig. 5). Both 8j and 8k significantly increased seizure latency (p < 0.05) compared to the control group. However, their response to flumazenil did not differ significantly from that of diazepam, suggesting that their anticonvulsant effects may involve GABA_A receptor modulation.

Fig. 5.

Effect of flumazenil on antiseizure activity of 8j and 8k.

In vivo lithium-pilocarpine seizure model (cholinergic mechanism & status epilepticus)

After the PTZ test, a lithium-pilocarpine model was used to extend the evaluation of seizure latency . This model generates seizures by increasing excitatory cholinergic activity, mediated by M1 receptor stimulation, in the hippocampus, striatum, and cortex. Pilocarpine, an agonist of muscarinic receptors, causes excessive excitation of neurons and induction of seizures. Lithium pretreatment lowers the required pilocarpine dose and amplifies seizure susceptibility by activating T cells and increasing IL-1 levels, which increase BBB permeability.

Compounds 8j and 8k were selected for evaluation in the lithium–pilocarpine model based on their higher anticonvulsant activity in the PTZ-induced seizure assay. In addition, compound 8e was included because it showed the lowest effectiveness in reducing seizure latency and seizure frequency in the PTZ model. This method allowed testing whether 8e would maintain the same activity pattern in a seizure model using cholinergic stimulation.

The results were consistent with previous findings, showing that compounds 8j and 8k, the most potent analogs, significantly improved latency time compared to the negative control group (p < 0.05). Additionally, there was no significant difference between these compounds and diazepam or thalidomide, used as positive controls. In contrast, compound 8e showed no significant difference from the negative control group (Fig. 6).

Fig. 6.

The in vivo evaluation of the synthesized compounds on latency in the lithium-pilocarpine seizure model.

Molecular docking simulation

Molecular docking studies were performed to gain insight into the binding mode of the synthesized compounds within the benzodiazepine binding pocket of the GABA_A receptor. Table 1 presents the calculated binding energies (kcal/mol) of all synthesized analogs. The reference compounds methaqualone (− 6.86 kcal/mol), diazepam (− 5.79 kcal/mol), and thalidomide (− 5.57 kcal/mol) exhibited moderate binding affinities within the receptor cavity. The analysis revealed that compounds 8j and 8k showed stronger binding interactions with the benzodiazepine binding site, as evidenced by lower binding energies (-7.60 and − 7.44 kcal/mol, respectively). The values indicate better interaction of these analogs than that of the reference drugs. Also, the potency of derivatives in most cases shows a direct relationship with their binding energies.

Table 1.

Binding energies of synthesized analogs within the binding site of the GABA_A receptor.

Entry	Binding energy	Entry	Binding energy
8a	-6.95	8 h	-5.80
8b	-6.32	8i	-6.50
8c	-5.36	8j	-7.60
8d	-5.78	8k	-7.44
8e	-6.08	8 L	-5.92
8f	-5.53	8 m	-5.17
8 g	-5.79	8n	-5.91

The superimposed conformations of these compounds are presented in Fig. 7, demonstrating a similar orientation within the α1/γ2 interface of the receptor, which corresponds to the classical benzodiazepine binding site.

Fig. 7.

Superimposition of the structures of thalidomide (red), diazepam (purple), 8j (yellow), and 8k (green) in the α1/γ2 subunit of the GABA_A receptor.

The 2D interaction pattern of the mentioned compound is shown in Fig. 8. Importantly, interaction patterns of 8j and 8k are almost consistent with those of diazepam, methaqualone and thalidomide. In particular, positive control compounds interacted with amino acids Tyr58 and Phe77 (γ-chain) and with Tyr210, Phe100, His102, and Ser205 (α-chain), all involved in binding and crucial for receptor binding and activation. As these interactions are observed in 8j and 8k, these compounds may act through a mechanism similar to that of these well-established compounds.

Fig. 8.

Interactions of methaqualone (A), diazepam (B), thalidomide (C), 8j (D), and 8k (E) with the GABA_A receptor.

Moreover, the deeper fitting of 8j and 8k into the benzodiazepine pocket, with additional interactions with Asn60 and Thr142 in the γ-subunit, may imply a more potent or specific modulation of the GABA_A receptor, which could contribute to their observed anticonvulsant effects. Overall, the docking results provide strong support for the hypothesis that 8j and 8k interact with the GABA_A receptor and have potential as therapeutic agents for seizure disorder.

Molecular dynamics simulations

A molecular dynamics study of the most active compounds (8j and 8k) was performed, comparing them with the GABAA receptor. Structural studies using cryo-electron microscopy have revealed the receptor pentameric composition, typically composed of multiple subunits, including α, β, and γ. Each subunit contains approximately 400 to 500 residues, making the receptor complex and functionally sophisticated. In this study, the entire receptor, including all subunits, was subjected to molecular dynamics simulations²¹.

As exhibited in Fig. 9, the GABA_A receptor complex system showed a gradual increase in RMSD, reaching a peak of 6 Å at 10 ns, then decreasing and stabilizing with fluctuations around 4 Å, followed by another increase to 6 Å. Given the large structure of this complex, such behavior is reasonable.

Fig. 9.

RMSD of GABA_A (blue), GABA_A-8j (orange) and GABA_A- 8k (green).

Next, the behavior of 8k was assessed. This system demonstrated an increase to about 5 Å, exhibiting structural rearrangements, followed by sharp fluctuations up to 15 ns. After this, the RMSD plateaued at approximately 2 Å from 25 ns onward, indicating stable binding. In contrast, the 8j showed sharp fluctuations from 0 to 27 ns, followed by stabilization after 30 ns, with an RMSD of about 2 Å. These results confirm that the two derivatives interact effectively with the receptor complex benzodiazepine binding sites, suggesting their potential antiseizure activity.

The root mean square fluctuation (RMSF) of the 8j was also assessed and showed a RMSF value less than 3 Å, indicating stable binding (Fig. 10a). The 8k showed no deviation beyond 3 Å, confirming the stability of the ligand Fig. 10b.

Fig. 10.

The RMSF of 8j (a) and 8k (b) during MD simulation.

Pharmacokinetic profile of 8a-n

The pharmacokinetic properties of these derivatives were evaluated using in silico models to determine their potential as therapeutic agents (Table 2). All compounds pass the lipophilic role of 5, exhibiting their drug-like properties. The in silico Caco-2 permeability values are expressed as log Papp (cm/s), and compounds with predicted Papp > 0.90 cm/s were considered to exhibit high intestinal permeability. The MDCK permeability values are reported as Papp (cm/s), and values > 10 × 10^− 6 cm/s are considered high permeability. These results confirmed that all compounds exhibit suitable intestinal absorption and membrane penetration, supporting their potential for effective oral administration. Analysis of BBB penetration is reported as logBB (the logarithmic ratio of brain to plasma drug concentration). Compounds with logBB values above 0.3 are considered capable of readily crossing the BBB. The ability of these derivatives to cross the BBB more effectively, may enhance their suitability for targeting neurological disorders. Moreover, current results indicated that none of the compounds could be considered significant P-glycoprotein (P-gp) substrates, resulting in a decrease in drug efflux and resistance. Their favorable pharmacokinetic profiles, particularly the balance between BBB penetration and P-gp-mediated efflux, make them a promising class of agents for CNS disorders in general and as potential antiepileptic agents in particular.

Table 2.

The pharmacokinetic characteristics of 8a-n.

Compound	Lipinski rule	Caco-2 permeability (log cm/s)	MDCK Permeability (log cm/s)	Pgp -substrate	BBB penetration (log BB)
8a	+	-4.755	1.7e-05	0.006	0.634
8b	+	-4.693	2.1e-05	0.089	0.47
8c	+	-4.734	2.2e-05	0.01	0.646
8d	+	-4.693	2.3e-05	0.115	0.934
8e	+	-4.786	1.9e-05	0.001	0.743
8f	+	-4.777	2.2e-05	0.001	0.679
8 g	+	-4.772	2e-05	0.007	0.61
8 h	+	-4.793	2.1e-05	0.005	0.657
8i	+	-4.784	2.4e-05	0.005	0.515
8j	+	-4.770	1.9e-05	0.138	0.527
8k	+	-4.710	2.2e-05	0.094	0.925
8 L	+	-4.725	2.4e-05	0.006	0.469
8 m	+	-4.621	2.1e-05	0.02	0.744
8n	+	-4.678	2.4e-05	0.16	0.93

Caco-2 permeability is expressed as log Papp (cm/s); MDCK permeability is expressed as Papp (cm/s); BBB penetration is expressed as logBB (logarithmic ratio of brain to plasma drug concentration). Compounds with logBB > 0.3 are considered to cross the BBB readily.

Conclusion

In the current study, a new series of quinazolinone-phthalimide derivatives were designed and evaluated as possible anticonvulsant agents. Notably, both compounds 8j (ethylphenyl) and 8k (cyclohexyl) exhibited significant anticonvulsant activity in the PTZ-induced seizure model, with latency times of 6.79 min and 6.95 min, respectively, similar to that of diazepam (6.98 min). These compounds markedly delayed seizure onset, indicative of increased GABAergic efficacy, which is essential for seizure suppression. Significantly, 8j and 8k displayed the fewest seizures, comparable with diazepam and thalidomide, further corroborating their potency in reducing seizure frequency. A good safety profile was demonstrated by the 8j and 8k with no mortality up to 10 mg/kg. Moreover, the involvement of GABA_A receptors in the anticonvulsant effects of 8j and 8k was confirmed by their response to flumazenil. Molecular docking studies further confirmed the interaction of 8j and 8k with the GABA_A receptor with binding energies of -7.60 and − 7.44 kcal/mol, respectively. The molecular dynamics simulation also confirmed the stability of the complexes. In the lithium-pilocarpine seizure model, 8j and 8k significantly increased the latency time like diazepam, which further supported their use as anticonvulsant compounds. The findings of this study indicate that 8j and 8k can be lead candidates for targeting GABA_A receptors as anticonvulsant agents.

Method and materials

Chemicals and Instrumentation

All chemicals and solvents except for Active pharmaceutical ingredients including Thalidomide (Tofigh Darou, Tehran, Iran), diazepam (Behvazan company, Tehran, Iran), pilocarpine HCl (Sina Daru Pharmaceutical Company, Tehran, Iran), flumazenil (Darupakhsh, Tehran, Iran) and scopolamine butyl bromide (Hyoscine N-butylbromide, Chemi Darou Company, Tehran, Iran) were in synthetic grade and purchased from Merck (Germany) and Sigma (U.S.A) and used without further purifications.

Characterization of synthetic compounds were investigated by spectroscopic methods. Proton nuclear magnetic resonance (¹H NMR) spectra were measured on a Bruker FT-500 spectrometer (Bruker, Darmstadt, Germany) at 500 MHz using tetramethyl silane (TMS) as the internal standard in pure deuterated solvent, DMSO-d₆. Chemical shifts were quoted on δ scale in parts per million (ppm). Mass spectra of the obtained products were evaluated by a HP (Agilent Technologies) 5937 mass selective detector (USA).

General procedure for synthesis of 2-(3-bromopropyl)isoindoline-1,3-dione (compound 3)

Based on Qingmin Wang et al. study, to obtain compound 3, a solution of 1,3-dibromopropane was added dropwise in 1.5 to 1 molar ratio to the prepared solution of potassium phthalimide dissolved in dimethyl formamide. The reaction mixture was stirred for 24 h at room temperature. Upon completion of the reaction as determined by TLC, the reaction mixture was quenched with water and extracted with dichloromethane. The dichloromethane extract was washed with brine and dried by magnesium sulfate and concentrated under vacuum. Further purification with column chromatography (petroleum ether and ethyl acetate) to obtain white powder in 55% yield²². To improve synthesis yield, optimization strategies such as using alternative polar aprotic solvents, increasing the reaction temperature, and applying phase-transfer catalysis can be considered.

General procedure for synthesis 2-thioxo-2,3-dihydroquinazolin-4(1 H)-one derivatives (compound 7a-n)

As depicted in scheme 1 different 2-amino-N-aryl/alkyl benzamide derivatives were prepared by reaction of (1:1) isotoic anhydride and different amines in water. The reaction last for 3 h and completion of reaction was checked by TLC. The subsequent precipitation was obtained by filtration and washed with water and dried. The obtained white/yellowish powder (6a-n) was used for next step without further purification²³. To obtain 8a-n, carbon disulfide was added dropwise to ethanolic solution of 6a-n at 0 °C in 1:1 molar ratio. The reaction mixture was then refluxed for 12 to 15 h. Then water and 1:1 HCl were added to the reaction mixture, and the precipitate was washed many times with water to obtain a pale white precipitate in 65–85% yield²⁴.

General procedure for synthesis of 2-(3-((3-alky/aryl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)propyl)isoindoline-1,3-dione derivatives (compound 8a-n)

The equimolar solution of compound 7a-n and potassium carbonate in DMF was prepared and stirred for 10–15 min. Subsequently, the 1.3 molar ratio of compound 3 was added to the mixture and the resultant was stirred at 80 °C overnight. Upon completion of the reaction, monitored by TLC, the mixture was poured into ice-water, and the solid product was filtered, washed with water, and dried to yield compounds 8a-n in 75–82% yield²⁵.

2-(3-((3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)thio)propyl)isoindoline-1,3-dione (8a)

FT-IR (KBr): 3060 (CH aromatic), 2922 (CH aliphatic), 1703 (C = O), 1682 (C = C aromatic).¹H NMR (400 MHz, DMSO) δ 8.03 (d, J = 7.9 Hz, 1H, H₅,_{Quinazolinone}), 7.85 (s, 4 H, H_{Ar, Isoindolinedione}), 7.70 (t, J = 7.7 Hz, 1H, H_3Cl−Ar), 7.67–7.52 (m, 3 H, H_{Ar, Quinazolinone}), 7.42 (t, J = 6.7 Hz, 2 H, H_3Cl−Ar), 7.19 (d, J = 8.1 Hz, 1H, H_3Cl−Ar), 3.68 (t, J = 6.4 Hz, 2 H, CH₂-N), 3.16 (t, J = 7.9 Hz, 2 H, CH₂-S), 2.01 (dd, J = 13.1, 6.2 Hz, 2 H, CH₂). ¹³C NMR (101 MHz, DMSO) δ 168.44 (2 C=O_{Isoindolinedione}), 161.10 (C=O_{Quinazolinone}), 156.97, 147.52, 137.74, 135.22, 134.88 (2 C), 133.86, 132.08 (2 C), 131.42, 130.36, 130.00, 128.87, 126.95, 126.33, 126.19, 123.48 (2 C), 119.93, 37.02 (C-N), 29.54, 28.32 (C-S).MS (EI) m/z calculated for C₂₅H₁₈ClN₃O₃S [M]⁺ = 475.9; found = 475.9.

2-(3-((3-benzyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)propyl)isoindoline-1,3-dione (8b)

FT-IR (KBr): 3050 (CH aromatic), 2934 (CH aliphatic), 1717 (C = O), 1661 (C = C aromatic). ¹H NMR (400 MHz, DMSO-d₆) δ 8.08 (d, J = 7.8 Hz, 1H, H₅,_{Quinazolinone}), 7.86 (m, J = 4.5 Hz, 4 H, H_{Ar, Isoindolinedione}), 7.71 (t, J = 7.6 Hz, 1H, H_Ar), 7.44 (t, J = 7.5 Hz, 1H, H_Ar), 7.36–7.18 (m, 6 H, H_Ar), 5.28 (s, 2 H, CH_2Benzyl), 3.71 (t, J = 6.5 Hz, 2 H, CH₂-N), 3.25 (t, J = 7.3 Hz, 2 H, CH₂-S), 2.04 (p, J = 6.8 Hz, 2 H, CH₂). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.47 (2 C=O_{Isoindolinedione}), 161.32 (C=O_{Quinazolinone}), 157.07, 147.21, 136.10, 135.19, 134.87 (2 C), 132.15 (2 C), 129.02 (2 C), 127.80 (2 C), 127.08, 127.02, 126.44, 126.18, 123.51(2 C), 119.12, 47.19 (C_Benzyl), 36.97 (C-N), 29.32, 28.29 (C-S). MS (EI) m/z calculated for C₂₆H₂₁N₃O₃S [M]⁺ = 455.5; found = 454.9.

2-(3-((4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio)propyl)isoindoline-1,3-dione (8c)

FT-IR (KBr): 3219 (CH aromatic), 2817 (CH aliphatic), 1760 (C = O), 1621 (C = C aromatic). ¹H NMR (400 MHz, DMSO) δ 8.02 (d, J = 7.8 Hz, 1H, H₅,_{Quinazolinone}), 7.84 (s, 4 H, H_{Ar, Isoindolinedione}), 7.69 (t, J = 7.7 Hz, 1H, H_Ar), 7.53 (d, J = 5.9 Hz, 3 H, H_Ar), 7.46–7.36 (m, 3 H, H_Ar), 7.19 (d, J = 8.1 Hz, 1H, H_Ar), 3.66 (t, J = 6.4 Hz, 2 H, CH₂-N), 3.13 (t, J = 7.3 Hz, 2 H, CH₂-S), 2.00 (p, J = 6.8 Hz, 2 H, CH₂). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.42 (2 C=O_{Isoindolinedione}), 161.16 (C=O_{Quinazolinone}), 157.53, 147.57, 136.35, 135.12, 134.85 (2 C), 132.08 (2 C), 130.19, 129.87 (2 C), 129.81(2 C), 126.95, 126.24, 126.17, 123.47 (2 C), 119.92, 37.03 (C-N), 29.52, 28.22 (C-S). MS (EI) m/z calculated for C₂₅H₁₉N₃O₃S [M]⁺ = 441.5; found = 441.

2-(3-((3-cyclopentyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)propyl)isoindoline-1,3-dione (8d)

FT-IR (KBr): 3081 (CH aromatic), 2950 (CH aliphatic), 1705 (C = O), 1662 (C = C aromatic). ¹H NMR (400 MHz, DMSO) δ 8.00 (d, J = 7.8 Hz, 1H, H₅,_{Quinazolinone}), 7.86 (q, J = 4.9 Hz, 4 H, H_{Ar, Isoindolinedione}), 7.66 (t, J = 7.5 Hz, 1H, H_{Ar, Quinazolinone}), 7.39 (t, J = 7.5 Hz, 1H, H_{Ar, Quinazolinone}), 7.19 (d, J = 8.1 Hz, 1H, H_{Ar, Quinazolinone}), 4.77 (p, J = 8.3 Hz, 1H, CH_Cyclopentyl), 3.75 (t, J = 6.5 Hz, 2 H, CH₂-N), 3.27 (t, J = 7.2 Hz, 2 H, CH₂-S), 2.16 (dt, J = 12.7, 6.9 Hz, 2 H, CH_2Cyclopentyl), 2.08 (p, J = 6.7 Hz, 2 H, CH₂), 1.95 (m, 2 H, CH_2Cyclopentyl), 1.88–1.79 (m, 2 H, CH_2Cyclopentyl), 1.57 (m, 2 H, CH_2Cyclopentyl). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.49 (2 C=O_{Isoindolinedione}), 160.92 (C=O_{Quinazolinone}), 157.07, 146.57, 134.87 (2 C), 134.81, 132.15 (2 C), 126.57, 126.22, 125.84, 123.51 (2 C), 120.15, 59.81 (C_Cyclopentyl), 37.08 (C-N), 29.86, 28.59 (2C_Cyclopentyl), 28.07 (C-S), 26.03 (2C_Cyclopentyl). MS (EI) m/z calculated for C24H23N3O3S [M]⁺ = 433.5; found = 434.3.

2-(3-((3-(2-chloro-4-methylphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)thio)propyl) isoindoline-1,3-dione (8e)

FT-IR (KBr): 3074 (CH aromatic), 2919 (CH aliphatic), 1709 (C = O), 1690 (C = C aromatic). ¹H NMR (400 MHz, DMSO) δ 8.02 (d, J = 7.8 Hz, 1H, H₅,_{Quinazolinone}), 7.84 (s, 4 H, H_{Ar, Isoindolinedione}), 7.69 (t, J = 7.7 Hz, 1H, H_Ar), 7.60 (s, 1H, H_Ar), 7.50 (d, J = 8.1 Hz, 1H, H_Ar), 7.41 (t, J = 7.6 Hz, 1H, H_Ar), 7.31 (d, J = 7.9 Hz, 1H, H_Ar), 7.18 (d, J = 8.1 Hz, 1H, H_Ar), 3.67 (t, J = 6.4 Hz, 2 H, CH₂-N), 3.17–3.11 (m, 2 H, CH₂-S), 2.41 (s, 3 H, CH₃), 2.01 (p, J = 7.1 Hz, 2 H, CH₂). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.43 (2 C=O_{Isoindolinedione}), 161.14 (C=O_{Quinazolinone}), 157.23, 147.52, 137.88, 135.22, 135.17, 134.85 (2 C), 133.94, 132.22, 132.07 (2 C), 130.13, 128.67, 126.95, 126.28, 126.18, 123.47 (2 C), 119.91, 37.02 (C-N), 29.54, 28.28 (C-S), 19.95 (C-Ph). MS (EI) m/z calculated for C₂₆H₂₀ClN₃O₃S [M]⁺ = 489.1; found = 489.1.

2-(3-((3-(2,3-dimethylphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)thio)propyl)isoindoline-1,3-dione (8f)

FT-IR (KBr): 3031 (CH aromatic), 2930 (CH aliphatic), 1718 (C = O), 1607 (C = C aromatic). ¹H NMR (400 MHz, DMSO-d₆) δ 8.05 (d, J = 7.2 Hz, 1H, H₅,_{Quinazolinone}), 7.84 (s, 4 H, H_{Ar, Isoindolinedione}), 7.70 (d, J = 7.4 Hz, 1H, H_Ar), 7.41 (d, J = 7.5 Hz, 1H, H_Ar), 7.32 (d, J = 7.5 Hz, 1H, H_Ar), 7.23 (d, J = 8.2 Hz, 2 H, H_Ar), 7.14 (d, J = 7.7 Hz, 1H, H_Ar), 3.65 (t, J = 13.0 Hz, 2 H, CH₂-N), 3.20 (dd, J = 14.7, 7.0 Hz, 1H, CH-S), 3.09 (dd, J = 14.5, 6.9 Hz, 1H, CH-S), 2.31 (s, 3 H, CH₃), 2.03–1.97 (m, 2 H, CH₂), 1.90 (s, 3 H, CH₃). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.39 (2 C=O_{Isoindolinedione}), 160.73 (C=O_{Quinazolinone}), 157.61, 147.70, 138.44, 135.23, 135.17, 134.83 (2 C), 132.08 (2 C), 131.73, 127.35, 127.03, 126.93, 126.32, 126.25, 123.47, 123.40 (2 C), 119.66, 37.01 (C-N), 29.23, 28.29 (C-S), 20.34 (C-Ph), 13.91 (C-Ph); MS (EI) m/z calculated for C₂₇H₂₃N₃O₃S [M]⁺ = 469.6; found = 469.3.

2-(3-((4-oxo-3-(p-tolyl)-3,4-dihydroquinazolin-2-yl)thio)propyl)isoindoline-1,3-dione (8 g)

FT-IR (KBr): 3055 (CH aromatic), 2928 (CH aliphatic), 1707 (C = O), 1676 (C = C aromatic). ¹H NMR (400 MHz, DMSO-d₆) δ 8.01 (d, J = 7.9 Hz, 1H, H₅,_{Quinazolinone}), 7.84 (s, 4 H, H_{Ar, Isoindolinedione}), 7.68 (t, J = 7.7 Hz, 1H, H_Ar), 7.40 (t, J = 7.6 Hz, 1H, H_Ar), 7.31 (d, J = 8.1 Hz, 2 H, H_Ar), 7.25 (d, J = 8.1 Hz, 2 H, H_Ar), 7.19 (d, J = 8.1 Hz, 1H, H_Ar), 3.66 (t, J = 6.5 Hz, 2 H, CH₂-N), 3.12 (t, J = 7.2 Hz, 2 H, CH₂-S), 2.39 (s, 3 H, CH₃), 2.00 (p, J = 6.7 Hz, 2 H, CH₂). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.41(2 C=O_{Isoindolinedione}), 161.19 (C=O_{Quinazolinone}), 157.79, 147.56, 139.82, 135.06, 134.83 (2 C), 133.71, 132.07 (2 C), 130.37 (2 C), 129.49 (2 C), 126.94, 126.18, 126.16, 123.46 (2 C), 119.89, 37.03 (C-N), 29.54, 28.17 (C-S), 21.33 (C-Ph). MS (EI) m/z calculated for C₂₆H₂₁N₃O₃S [M]⁺ = 455.5; found = 455.2.

2-(3-((4-oxo-3-(m-tolyl)-3,4-dihydroquinazolin-2-yl)thio)propyl)isoindoline-1,3-dione (8 h)

FT-IR (KBr): 3059 (CH aromatic), 2926 (CH aliphatic), 1704 (C = O), 1681 (C = C aromatic).

¹H NMR (400 MHz, DMSO-d₆) δ 8.02 (d, J = 7.8 Hz, 1H, H₅,_{Quinazolinone}), 7.84 (s, 4 H, H_{Ar, Isoindolinedione}), 7.68 (t, J = 7.7 Hz, 1H, H_Ar), 7.41 (t, J = 7.5 Hz, 2 H, H_Ar), 7.33 (d, J = 7.8 Hz, 1H, H_Ar), 7.23–7.14 (m, 3 H, H_Ar), 3.66 (t, J = 6.5 Hz, 2 H, CH₂-N), 3.12 (h, J = 6.6 Hz, 2 H, CH₂-S), 2.36 (s, 3 H, CH₃), 2.00 (p, J = 7.1 Hz, 2 H, CH₂). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.41 (2 C=O_{Isoindolinedione}), 161.14 (C=O_{Quinazolinone}), 157.57, 147.55, 139.44, 136.24, 135.10, 134.84 (2 C), 132.07 (2 C), 130.85, 130.05, 129.65, 126.92, 126.77, 126.22, 126.17, 123.46 (2 C), 119.89, 37.04 (C-N), 29.48, 28.26 (C-S), 21.25 (C-Ph). MS (EI) m/z calculated for C₂₆H₂₁N₃O₃S [M]⁺ = 455.5; found = 455.1.

2-(3-((3-(3-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)thio)propyl)isoindoline-1,3-dione (8i)

FT-IR (KBr): 3039 (CH aromatic), 2925 (CH aliphatic), 1708 (C = O), 1674 (C = C aromatic). ¹H NMR (400 MHz, DMSO-d₆) δ 8.03 (d, J = 7.9 Hz, 1H, H₅,_{Quinazolinone}), 7.85 (s, 4 H, H_{Ar, Isoindolinedione}), 7.69 (t, J = 7.7 Hz, 1H, H_Ar), 7.43 (m, 2 H, H_Ar), 7.19 (d, J = 8.1 Hz, 1H, H_Ar), 7.14–7.03 (m, 2 H, H_Ar), 6.96 (d, J = 7.7 Hz, 1H, H_Ar), 3.78 (s, 3 H, O-CH₃), 3.68 (t, J = 6.5 Hz, 2 H, CH₂-N), 3.14 (t, J = 7.4 Hz, 2 H, CH₂-S), 2.01 (p, J = 6.8 Hz, 2 H, CH₂). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.45 (2 C=O_{Isoindolinedione}), 161.07 (C=O_{Quinazolinone}), 160.36, 157.53, 147.55, 137.41, 135.12, 134.87 (2 C), 132.09 (2 C), 130.55, 126.95, 126.24, 126.15, 123.48 (2 C), 121.84, 119.94, 115.78, 115.55, 55.90 (C-O), 37.02 (C-N), 29.44, 28.26 (C-S). MS (EI) m/z calculated for C₂₆H₂₁N₃O₄S [M]⁺ = 471.5; found = 471.1.

2-(3-((4-oxo-3-phenethyl-3,4-dihydroquinazolin-2-yl)thio)propyl)isoindoline-1,3-dione (8j)

FT-IR (KBr): 3027 (CH aromatic), 2940 (CH aliphatic), 1715 (C = O), 1667 (C = C aromatic). ¹H NMR (400 MHz, DMSO-d₆) δ 8.05 (d, J = 7.8 Hz, 1H, H₅,_{Quinazolinone}), 7.85 (m, 4 H, H_{Ar, Isoindolinedione}), 7.68 (t, J = 7.6 Hz, 1H, H_Ar), 7.42 (t, J = 7.5 Hz, 1H, H_Ar), 7.36–7.30 (m, 2 H, H_Ar), 7.27–7.22 (m, 3 H, H_Ar), 7.20 (d, J = 8.1 Hz, 1H, H_Ar), 4.22–4.13 (m, 2 H, CH_{2Ethylbenzene}-N), 3.76 (t, J = 6.5 Hz, 2 H, CH₂-N), 3.31 (t, J = 7.2 Hz, 2 H, CH₂-S), 2.99–2.91 (m, 2 H, CH₂-Ph), 2.08 (p, J = 6.8 Hz, 2 H, CH₂). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.51 (2 C=O_{Isoindolinedione}), 160.86 (C=O_{Quinazolinone}), 156.44, 147.12, 138.21, 135.00, 134.87 (2 C), 132.14, 129.09 (2 C), 129.08 (2 C), 127.14, 126.81, 126.32, 126.13, 123.51 (2 C), 119.19, 45.77 (N-C_Ethylbenzene), 37.01 (C-N), 33.79 (C-Ph), 29.19, 28.26 (C-S). MS (EI) m/z calculated for C₂₇H₂₃N₃O₃S [M]⁺ = 469.6; found = 469.3.

2-(3-((3-cyclohexyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)propyl)isoindoline-1,3-dione (8k)

FT-IR (KBr): 3091 (CH aromatic), 2922 (CH aliphatic), 1712 (C = O), 1678 (C = C aromatic). ¹H NMR (400 MHz, DMSO) δ 7.98 (d, J = 7.9 Hz, 1H, H₅,_{Quinazolinone}), 7.86 (m, 4 H, H_{Ar, Isoindolinedione}), 7.64 (t, J = 7.7 Hz, 1H, H_Ar), 7.38 (t, J = 7.5 Hz, 1H, H_Ar), 7.16 (d, J = 8.1 Hz, 1H, H_Ar), 4.07 (m, 1H, CH_Cyclohexyl), 3.75 (t, J = 6.4 Hz, 2 H, CH₂-N), 3.26 (t, J = 7.2 Hz, 2 H, CH₂-S), 2.08 (p, J = 6.8 Hz, 2 H, CH₂), 1.79 (d, J = 12.2 Hz, 2 H, CH_2Cyclohexyl), 1.69–1.56 (m, 4 H CH_2Cyclohexyl), 1.20 (dq, J = 24.6, 12.0 Hz, 4 H, CH_2Cyclohexyl). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.47 (2 C=O_{Isoindolinedione}), 161.46 (C=O_{Quinazolinone}), 156.62, 146.54, 134.87 (2 C), 134.80, 132.14 (2 C), 126.59, 126.19, 125.78, 123.49 (2 C), 120.29, 61.13 (N-C_Cyclohexyl), 37.12 (C-N), 29.89, 28.46 (C-S), 27.96 (2C_Cyclohexyl), 26.30 (2C_Cyclohexyl), 25.25 (C_Cyclohexyl). MS (EI) m/z calculated for C₂₅H₂₅N₃O₃S [M]⁺ = 447.6; found = 448.3.

2-(3-((3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)thio)propyl)isoindoline-1,3-dione (8 L)

FT-IR (KBr): 3080 (CH aromatic), 2935 (CH aliphatic), 1715 (C = O), 1550 (C = C aromatic).

¹H NMR (400 MHz, DMSO-d₆) δ 8.03 (d, J = 7.8 Hz, 1H, H₅,_{Quinazolinone}), 7.86 (s, 4 H, H_{Ar, Isoindolinedione}), 7.69 (t, J = 7.6 Hz, 1H, H_Ar), 7.42 (t, J = 7.5 Hz, 1H, H_Ar), 7.29 (d, J = 8.8 Hz, 2 H, H_Ar), 7.20 (d, J = 8.1 Hz, 1H, H_Ar), 7.04 (d, J = 8.8 Hz, 2 H, H_Ar), 3.83 (s, 3 H, O-CH₃), 3.68 (t, J = 6.5 Hz, 2 H, CH₂-N), 3.12 (t, J = 7.3 Hz, 2 H, CH₂-S), 2.00 (p, J = 6.8 Hz, 2 H, CH₂). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.46 (2 C=O_{Isoindolinedione}), 161.36 (C=O_{Quinazolinone}), 160.37, 158.24, 147.60, 135.08, 134.88 (2 C), 132.11 (2 C), 130.98 (2 C), 128.73, 126.98, 126.20, 126.16, 123.50 (2 C), 119.93, 114.99 (2 C), 55.90 (C-O), 37.06 (C-N), 29.54, 28.18 (C-S). MS (EI) m/z calculated for C₂₆H₂₁N₃O₄S [M]⁺ = 471.5; found = 470.9.

2-(3-((3-butyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)propyl)isoindoline-1,3-dione (8 m)

FT-IR (KBr): 3100 (CH aromatic), 2931 (CH aliphatic), 1716 (C = O), 1587 (C = C aromatic). ¹H NMR (400 MHz, DMSO-d₆) δ 8.02 (d, J = 7.9 Hz, 1H, H₅,_{Quinazolinone}), 7.86 (m, 4 H, H_{Ar, Isoindolinedione}), 7.66 (t, J = 7.7 Hz, 1H, H_Ar), 7.39 (t, J = 7.6 Hz, 1H, H_Ar), 7.17 (d, J = 8.1 Hz, 1H, H_Ar), 3.97 (t, J = 7.8 Hz, 2 H, N-CH_2Butyl), 3.74 (t, J = 6.5 Hz, 2 H, CH₂-N), 3.28 (t, J = 7.2 Hz, 2 H, CH₂-S), 2.08 (p, J = 6.9 Hz, 2 H, CH₂), 1.61 (p, J = 7.6 Hz, 2 H, CH_2Butyl), 1.32 (h, J = 7.4 Hz, 2 H, CH_2Butyl), 0.90 (t, J = 7.3 Hz, 3 H, CH_3Butyl). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.47 (2 C=O_{Isoindolinedione}), 160.89 (C=O_{Quinazolinone}), 156.54, 147.11, 134.87, 134.86 (2 C), 132.14 (2 C), 126.79, 126.21, 126.06, 123.49 (2 C), 119.15, 44.05 (C_Butyl-N), 37.01 (C-N), 29.92 (C_Butyl), 29.15, 28.23 (C-S), 20.06 (C_Butyl), 13.99 (C_Butyl). MS (EI) m/z calculated for C₂₃H₂₃N₃O₃S [M]⁺ = 421.5; found = 421.3.

2-(3-((3-cyclopropyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)propyl)isoindoline-1,3-dione (8n)

FT-IR (KBr): 3050 (CH aromatic), 2925 (CH aliphatic), 1705 (C = O), 1586 (C = C aromatic). ¹H NMR (400 MHz, DMSO-d₆) δ 7.96 (d, J = 7.9 Hz, 1H, H₅,_{Quinazolinone}), 7.92–7.70 (m, 4 H, H_{Ar, Isoindolinedione}), 7.60 (t, J = 7.7 Hz, 1H, H_Ar), 7.34 (t, J = 7.5 Hz, 1H, H_Ar), 7.10 (d, J = 8.1 Hz, 1H, H_Ar), 3.73 (t, J = 6.5 Hz, 2 H, CH₂-N), 3.20 (t, J = 7.3 Hz, 2 H, CH₂-S), 2.78 (tt, J = 7.3, 4.1 Hz, 1H, CH_Cyclopropane), 2.06 (p, J = 6.9 Hz, 2 H), 1.13 (q, J = 6.7 Hz, 2 H, CH_{2Cyclopropane}), 0.88 (p, J = 5.3 Hz, 2 H, CH_{2Cyclopropane}). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.44 (2 C=O_{Isoindolinedione}), 161.91 (C=O_{Quinazolinone}), 159.44, 146.88, 134.83 (2 C), 134.60, 132.11 (2 C), 126.63, 125.90, 125.81, 123.46 (2 C), 119.77, 37.11 (C-N), 29.11, 28.27 (C-S), 26.75 (1 C), 11.17 (2 C).MS (EI) m/z calculated for C₂₃H₂₃N₃O₃S [M]⁺ = 405.4; found = 405.1.

Animal study

All methods were performed in accordance with the relevant guidelines and regulations. In this regard, the animals were kept in a controlled environment with a temperature of 25 ± 2 °C and a 12/12-hour light/dark cycle. They had free access to food and water, except during the experimental procedures when they were removed from their cages. The study was approved by the Animal Ethics Committee of Tehran University of Medical Sciences, Tehran, I.R. Iran, under ethical approval No. IR.TUMS.AEC.1400.004 complying with ARRIVE guideline. Animals were euthanized according to the AVMA guideline through anesthetic condition by administering pentobarbital 100 mg/kg intravenously²⁶.

Animal model of seizure using PTZ

PTZ is a GABA _A receptor antagonist that induces seizures by inhibiting this receptor and affecting the reticular formation, diencephalic regions, and caudal hypothalamus. The procedure first involves IP injection of synthesized derivatives (10 mg/kg) then ward after 30 min PTZ at a dose of 70 mg/kg was IP injected and all mice were followed up for 1 h. The 5% DMSO (vehicle) as negative control, thalidomide (10 mg/kg), diazepam (2 mg/kg) as positive controls were also screened. Male NMRI mice were divided into 17 groups of 5 animals each for the PTZ model. The test was conducted via intraperitoneal (IP) injection. Additionally, ten synthesized compounds were administered intraperitoneally at a dose of 10 mg/kg, equivalent to thalidomide, to groups of five mice each. After the injections, each mouse was placed alone in cages with metal mesh, which allowed them to move freely and prevented the behaviors of one mouse from affecting the seizures of another. Flumazenil (2 mg/kg, IP), was also administered to reveal the action mechanism.

Animal model of seizure using lithium-pilocarpine

To further assess the potency of the two most active compounds (8j and 8k) and the least active compound (8e), lithium-pilocarpine model was performed. A total of six groups were created from male Wistar rats. Lithium chloride (127 mg/kg) was administered IP to each rat. Nineteen hours after hyoscine (1 mg/kg, IP) was administered to minimize the peripheral cholinergic side effects of pilocarpine hydrochloride (HCl). It was also followed by concurrent administration of the synthesized compounds (8j, 8k, and 8e at 10 mg/kg, IP), diazepam (10 mg/kg, IP) and thalidomide (10 mg/kg, IP), as a positive control, and 5% DMSO as a negative control. After 30 min, pilocarpine hydrochloride was administered to induce seizure. Latency to seizure onset in the lithium-pilocarpine model was recorded for each rat.

In silico studies

Molecular docking

The X-ray crystallographic structure of human full-length α1β3γ2L GABA_A receptor in complex with diazepam was analyzed during the current study are available in the Worldwide ProteinData Bank with the PDB ID of 6HUP repository (https://www.rcsb.org/structure/6HUP).

To prepare ligands, the Marvin Sketch applet (Marvin package, ChemAxon Company) was primarily used for drawing 2D structures of compound 8a-n which were then converted to 3D structures minimized in energy via MM2 algorithm in PDB file format by the mean of Chem3D ultra version 8.0. AutoDock 4.2 software was utilized. All input files were prepared by AutoDockTools 1.5.7 (ADT), Autogrid 4.2, and Autodock 4.2 to perform molecular docking studies of the synthesized compound on the benzodiazepine site of the GABA_A receptor. The non-polar hydrogens were merged and the addition of Kollman charges was done for receptor preparation. The Grid box was 60 Å in XYZ. The Lamarckian genetic algorithm was used for conformational search and the rest of the parameters were set as default²⁷. The Discovery Studio visualizer was utilized for visualizing 2D and 3D ligand-receptor interactions²⁸.

Molecular dynamics simulation

Docked GABA_A complexes with the most active compounds (8j and 8k) were used for molecular dynamics simulation, using the PDB code 6HUP and diazepam as the binding site, as per the Schrödinger package. The complexes were solvated with explicit SPC water molecules and positioned at the center of an orthorhombic box under periodic boundary conditions. Counterions from a 0.15 M NaCl solution were added to neutralize the system, mimicking real cellular ionic concentrations. The MD protocol consisted of three steps: minimization, pre-production, and production MD simulations.

Pharmacokinetic properties

The pharmacokinetic characteristics of synthesized compound 8a-n were determined in silico using ADMET Lab 2.0 online platform^29,30.

Statistical analysis

The statistical analysis was performed using GraphPad Prism 10.1.2. To compare latency between groups, a one-way analysis of variance (ANOVA) was conducted, followed by the Tukey posthoc test.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Fatemeh Moradkhani synthesized compounds, conducted biological and in silico studies, and wrote the initial draft. Mehdi Asadi supervised the chemistry part of the study. Ahmad Reza Dehpour supervised animal study. Mohsen Amini supervised synthesis and compound identification. Massoud Amanlou is responsible for project administration, financial support for the project, supervising the modeling section, and writing, reviewing, and editing the final version of the manuscript. All authors read and approved the final version of the article.

Funding

The authors would like to acknowledge the support of the Tehran University of Medical Sciences.

Data availability

The datasets generated and/or analyzed during the current study are available in the Worldwide ProteinData Bank with the PDB ID of 6HUP repository ( [https://www.rcsb.org/structure/6HUP](https:/www.rcsb.org/structure/2Y9X) ).

Declarations

Competing interests

The authors declare no competing interests.