Microwave-Assisted Facile Synthesis and anticonvulsant evaluation of Novel N-( 3-chloro-2-oxo-4-substituted phenyl azetidin-1-yl )-2-( 1 , 3-dioxoisoindolin-2-yl ) acetamides

Herewith, we report the design and synthesis of a series of N-(3-chloro-2oxo-4-substituted phenyl azetidin-1-yl)-2-(1,3-dioxoisoindolin-2yl)acetamide 7(a-l) derivatives, obtained by condensation of Schiff’s base and chloroacetyl chloride in dimethyl formamide as solvent and few drops of triethyl amine as a catalyst under microwave irradiation for about 3-4 min (700 W) at 800C based on four component pharmacophoric model to get structural prerequisite indispensable for anticonvulsant activity. The synthesized derivatives were investigated for CNS depressant, maximal electroshock seizure (MES), subcutaneous pentylenetetrazole (sc-PTZ) induced seizure and neurotoxicity screening. Most of the compounds were found to be potent in MES model. The anticonvulsant screening data shows that 65% of the compounds were found to be active against MES model when compared to 35% sc-PTZ model.


Introduction:
Epilepsy is a chronic disorder affecting 50 million people worldwide [1][2][3].It is characterized by recurrent seizures due to abnormal excessive and synchronous neuronal activity in the brain.
Epilepsy, being one of the most common and serious neurological disorder is characterized by recurrent seizures which results from a temporary electrical disturbance of the brain due to an imbalance between excitatory and inhibitory neurotransmitters.About one third of the patients do not respond well to current multiple drug therapy [4,5].
A global campaign against epilepsy conducted by World Health Organization (WHO) in partnership with International Bureau for Epilepsy (IBE) and International League against Epilepsy (ILAE) suggested that around 1% of world population at any time is afflicted with this neurological disorder [6,7].Phenytoin, carbamazepine, lamotrigine, sulfamate and topiramate are recent antiepileptic drugs which have been clinically effective against different types of seizures.
Moreover, these drugs cause various side effects such as drowsiness, gastrointestinal disturbance, hepato-toxicity, and megaloblastic anemia.
In the present work, our objective was to design and synthesize new compounds having dioxoindolin moiety coupled with azetidinone nucleus via amide linkage, as a pharmacophore, with the hope to get compounds with enhanced anticonvulsant activity.Thus, novel, N-(3-chloro-2-oxo-4substituted phenyl azetidin-1-yl)-2-(1,3-dioxoisoindolin-2-yl) acetamide 7(a-l) derivatives were synthesized as antiepileptic drugs that shows similar mode of action on neuronal sodium channels as phenytoin [25].All the synthesized titled compounds comprised of the essential pharmacophoric elements that are necessary for good anticonvulsant activity as suggested by Unverferth et al. [26], which are indicated by rectangles in Fig. 1.The essential structural features which could be responsible for an interaction with the active site were a hydrophobic unit (R), an electron donor (D) group, and a hydrogen donor/acceptor (HBD) unit [27].The title compounds were synthesized by microwave method as it gives less pollution, shorter reaction time, increased rate of reaction, more yield, cleaner and greener eco-friendly synthetic protocol.

Chemistry
The synthetic protocol employed for the synthesis of N-(3-chloro-2-oxo-4-substituted phenyl azetidin-1-yl)-2-(1,3-dioxoisoindolin-2-yl)acetamide 7(a-l) derivatives is presented in Scheme 1. Nsubstituted benzylidene/methylene-2-(1,3-dioxo isoindolin-2-yl) acetohydrazides 6(a-l) was obtained as per procedure [28] which upon reaction with chloro acetyl chloride in DMF and using few drops of triethyl amine as a catalyst under microwave irradiation for about 3-4 min (700 W), gives final derivatives7(a-l).The purity of the synthesized compounds was checked by TLC and melting points were determined in open capillary tubes on a Buchi 530 melting point apparatus and are uncorrected.The assignments of the structures were based on elemental and spectral data.The physical data of the synthesized compounds is presented in Table 1.The proposed structures of final compounds were confirmed by the data obtained from IR, NMR, Mass and elemental analysis.All the synthesized compounds were evaluated for their anticonvulsant activity by MES and sc-PTZ model and have shown best protection against MES test (Table 2).In MES test, the anticonvulsant activity of the newly synthesized compounds was carried out at 0.5 and 4 h at the dose of 100 mg/kg.The compounds 7a, 7d, and 7e have shown best protection at both time intervals.In MES test, the compounds 7b, 7c, 7g, and 7h showed protection at 0.5 h, while compounds 7f, 7i and 7j

Neurotoxicity screening
In neurotoxicity screening, the compounds 7a, 7c, 7d, 7e, 7g, 7i and 7j were found to be nontoxic at a dose of 100 mg/kg while compounds 7b, 7f, 7h and 7k were found to be toxic at the same dose after 4 h (Table 2).

Behavioural activity
From the behavioural activity of synthesized compounds using actophotometer, the compounds 7a, 7c and 7e showed no behavioural despair effect when compared to diazepam at 0.5 h.The compounds 7a, 7b, 7e, and 7h showed no behavioural despair effect when compared to diazepam at 4 h (Table 3).All the other compounds were found to decrease behavioural activity of the animals at 100 mg/kg compared to diazepam.Each value represents the mean SEM significantly different from the control at p<0.05; ns denotes not significant at p<0.05 (Student's t-test); locomotor activity score was measured for 10 min.b) The compound was tested at dose level of 4 mg/kg (i.p.).

Experimental Chemistry
All reagents and solvents were used as obtained from the supplier or recrystallized/redistilled unless otherwise noted.Synthetic microwave oven Milestone micro synth system was used for synthesis of final title compounds.The progress of the reaction was monitored by TLC, silica gel-G (Merck) coated aluminium plates, visualized by iodine vapor.Infrared (IR) and nuclear magnetic resonance ( 1 H NMR and 13 C NMR) spectra of the synthesized compounds were recorded on JASCO FTIR (PS 4000) using KBr pellets and Bruker Avance II (400MHz) instruments, respectively.Chemical shifts are reported in parts per million (ppm), using TMS as an internal standard.Elemental analyses (C, H, and N) were undertaken with a Shimadzu FLASHEA112 analyzer and all analyses were consistent with theoretical values (within ±0.5%) unless indicated.The mass spectra were recorded on a Waters MicroMass ZQ 2000 spectrometer.

General
The solid product formed was filtered, dried and recrystallized from ethanol.The structures of the final compounds of the series 7(a-l) were confirmed by the spectral data and elemental analysis as given below [29].

Table 2 : Anticonvulsant and neurotoxicity screening of the synthesized compounds 7(a-l).
Dose 100 mg/kg of the compound was administered and the protection and neurotoxicity were measured after 0.5 and 4 h.The figures indicate the minimal dose required to cause protection or neurotoxicity in 50% or more of the animals.The dash ( -) indicates the absence of anticonvulsant activity or neurotoxicity.(X) denotes not tested.