Synthesis and Evaluation of Anticonvulsant Activity of Some Quinazoline Analogues

Aim: A new series of Quinazoline 4(3H)-one derivative were prepared by reacting quinazoline 4(3H)-one hydrazide with substituted aromatic aldehydes. Quinazoline is used as a potent pharmacological agent with various biological activities such as antimicrobial, antiviral, antitumor, convulsion, anxiety, anti-inﬂammatory, and analgesic. In this background, we have synthesized a series of Quinazoline 4(3H)-one derivatives (4a-4f) and screened for their anticonvulsant activity.  
Methods: In this work, Schiff bases were prepared by treating quinazoline 4(3H)-one hydrazide with aromatic aldehydes. Six compounds (4a-4f) were screened for anticonvulsant activity by Isoniazid (INH) and Pentylenetetrazole (PTZ) induced convulsions in mice. 
Results: All the compounds were given satisfactory reaction yields that representing the efficiency of the employed synthetic route. In INH induced convulsion model, delayed the onset of convulsion significantly 4a, 4b, 4d, 4e, 4f when compared to an induction control group. Whereas delayed onset of convulsion was non-significant for 4c. In PTZ induced convulsion model, delayed the onset of convulsion significantly 4a, 4d, 4e, 4f when compared to induction control group. Whereas delayed onset of convulsion was non-significant for 4b and 4c. 
Conclusion: This indicates the anticonvulsant activity to these derivatives which might be due to potentiating GABA activity in the CNS. This anticonvulsant activity was due to presence of electron-donating group like OH, NH2, OCH3 and electron-withdrawing group like CF3 at 2nd and 4th position of aromatic ring attached to hydrazide.


INTRODUCTION
Epilepsy is the most common neurological disorder that causes unprovoked, recurrent seizures and affects all ages of people. About 65 million people are suffering from epilepsy in worldwide. It arises somewhat more in males than in females. Epilepsy is a non-curable disorder, but the disorder can be managed with antiepileptic drugs that will be prescribed on severity of symptoms, health and how well ketogenic diet Brain surgery respond to therapy as well as various strategies like vagus nerve stimulator [1]. Antiepileptic agents are neither curative nor preventive and are active exclusively as a means of controlling the symptoms of epilepsy. They can directly acts on ion channels or indirectly impact on synthesis, metabolism, or function of neurotransmitters or receptors that control channel opening and closing. Antiepileptic drugs are exhibited their action by various mechanism such as gamma aminobutyric acid (GABA) enhancers, sodium channel blockers, glutamate blockers, calcium channel inhibitors. These drugs are effectively reducing the seizures, whereas their therapeutic efficacy is overcome by some unwanted side effects such as drowsiness, megaloblastic anemia, ataxia, gastrointestinal disturbance, etc. [2]. Despite the broad and growing array of antiepileptic drugs available for treatment, approximately 30% of epileptic patients have inadequate seizure control and a further 25% suffer from significant adverse effects. Thus there is an ongoing need to develop more antiepileptic drugs that are effective and endowed with improved safety profiles.
Nitrogen containing heterocycles are amongst the most advantaged molecular scaffolds of pharmaceuticals, in which quinazoline is significant landmark that is present in a total of 9 U.S. FDA approved pharmaceuticals [3]. Quinazoline derivatives are the most active organic compounds that have a varied biological activities. They are widely utilized in pharmacy and agrochemicals, such as, fluquinconazole fungicide which is used for the control of agriculture diseases [4]. Numerous articles have been reported about the biological activity of quinazoline derivatives, including their antibactericidal, and anti-tumor activities [5,6]. 4(3H)-quinazolinone has a skeleton consisting of two fused rings in which two nitrogen atoms are attached to a carbon and a carbonyl group linked to one of the nitrogen [7]. This skeleton is found in the structure of some natural products and a number of drugs that were used to treat certain diseases [8,9]; therefore, their synthesis was specially targeted by chemists and pharmacists [10]. Literature survey reveals that natural quinazolinones and their synthetic analogues possess a variety of pharmacological activities including central nervous system (CNS) related disorders such as convulsion, anxiety [11][12][13][14][15], anti-inflammatory, analgesic [16][17][18][19], antiviral [20], antitumor [21][22][23] and antimicrobial [24].

Chemistry
All of the compounds were characterized by IR, 1H, and 13C NMR spectra recorded with Bruker WM-300 in deuterated DMSO at 400 and 100 MHz, respectively using tetra methyl silane (TMS) as the internal standard. All chemical shifts are described on the δ scale. Thinlayer chromatography (TLC) was carried out using Merck silica gel 60 F-254 plates (layer thickness: 0.25 mm) and a UV lamp and all solvents were distilled before use.

Synthesis
A mixture of 2-propyl-4H-3, 1-benzoxazinone, and ammonium acetate is irradiated in a MW bath reactor at 100 W and 120°C for 2-5 min. The resulting crude is filtered off, washed with cold water and recrystallized from petrol ether to give compound 1.
To a solution of quinazolinone, 1 in DMF K 2 CO 3 was added. The reaction mixture is stirred at 60°C for 30 min, then KI is added and the formed mixture is stirred for another 15 min. Solution of respective alkylating agents in DMF is dropped slowly into the mixture. After 4 h at 60 °C, the mixture is allowed to cool down and then is poured into ice-cold water. The solid that formed is scrubbed with water, filtered and crystallized using the suitable solvent to give the respective compounds 2.

Fig. 1. Synthesis of Quinazoline 4(3H)-one derivatives
A suspension of the ester of 2 and hydrazine hydrate in absolute ethanol was refluxed for 3 h. The reaction mixture was concentrated and then cooled down to RT. The formed solid was filtered and recrystallized from EtOH to give acetohydrazide 3.
An equimolar mixture of acetohydrazide 3 and aromatic aldehyde in 30 mL absolute EtOH was refluxed for 3 h. The reaction mixture was left overnight and the separated solid was filtered off and recrystallized from EtOH to give compound 4a-f.

Isoniazid (INH) induced convulsions in mice
In the present study, 48 mice (

Pentylenetetrazole (PTZ) induced convulsions in mice
In PTZ induced convulsion model, 48 mice (18-30g) were divided into eight groups. Test compounds 4a, 4b, 4c, 4d, 4e and 4f 100 mg/kg i.p. were suspended in CMC solution. Diazepam 10 mg/kg i.p. used as a standard. After 60 minutes of dose administration, pentylenetetrazole (80 mg/kg) was administered to all mice to induce convulsions. Immediately after PTZ injection, each mice was kept in a separate cage and observed for convulsions. The onset of convulsions and percentage protection were recorded in each group and compared against a control group. All results were statistically analyzed by one way ANOVA followed by Turkey-Kramer multiple comparison test [26].

Physicochemical Characterization of Quinazoline 4(3H)-One Derivatives
The table below shows the physicochemical characterization of the compounds estimated in this study (Table 1). All the compounds were given satisfactory reaction yields and properly separated from the reaction mixture, that representing the efficiency of the employed synthetic route.
The Characterization of synthesized derivatives were carried out by using Infrared spectroscopy (IR), Proton NMR (H1 NMR) and Carbon NMR (C13 NMR) for structure elucidation.
Quinazoline 4(3H)-one derivatives showed anticonvulsant effect in Isoniazid and PTZ induced convulsions in mice.

CONCLUSION
Convulsions being an important symptom of epilepsy precipitates either due to enhanced activity of excitatory neurotransmitters like Glutamate or depletion of inhibitory neurotransmitters like GABA in the CNS. Current pharmacotherapy for convulsions reported limited therapeutic outcomes and are associated with many deleterious side effects [27,28]. This indicates the need to develop new drugs for the treatment of convulsions with improved therapeutic outcome. Hence in the present study, preclinical trials for different derivatives of Quinozoline compounds were carried out for anticonvulsant activity in chemical (Isoniazid and PTZ) induced convulsions in mice.
INH was reported to inhibit GABA synthesis and glutamic acid decarboxylase by inhibiting pyridoxine phosphokinase and thereby depleting GABA concentration in the CNS [27,28,29] and precipitate the convulsions [30]. In present study derivatives 4a, 4b, 4c, 4d, 4e, 4f at 100 mg/kg,i.p, significantly delayed the mean onset of convulsion when compared to induction control group. This indicates the anticonvulsant activity to these derivatives which might be due to antagonizing the INH effect on GABA synthesis. PTZ induced convulsions are a well-established animal model for absence seizure. PTZ is GABA antagonists that leads to CNS stimulation which produces convulsion in mice [30,31]. Quinazoline derivatives 4a, 4d, 4e, 4f prolonged mean onset of convulsions significantly when compared to induction control group. This indicates the anticonvulsant activity to these derivatives which might be due to potentiating GABA activity in the CNS. This anticonvulsant activity was due to presence of electron donating group like OH, NH2, OCH3 and electron withdrawing group like CF3 at 2 nd and 4 th position of aromatic ring attached to hydrazide.
On the basis of result, it can be concluded that Quinazoline 4(3H)-one derivatives exhibited anticonvulsant activity. All synthetic compounds of Quinazoline 4(3H)-one derivatives showed

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
The present study did not involve Patients.

ETHICAL APPROVAL
This project was approved by the Animal Ethics Committee from Dr. D. Y. Patil College of Pharmacy, Akurdi, Pune (DYPCOP/IAEC/ 2020/04).