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We here in describe the molecular hybridization approach, to synthesize novel ring constrained analogues cinnamide derivatives 5a-5l as potential anticonvulsant, antinociceptive and antioxidant activities. The designing of compounds involved in combining the structural features i.e., morpholine, piperidinyl, piperazine and arylpiperazines with cinnamoyl pharmacophoric group. [A series of ring constrained analogues cinnamide derivatives was synthesized as potential anticonvulsant, antinociceptive and antioxidant activities by combining the key pharmacophoric group. The piperazine moiety and other analogues, also the certain features of SB366791 were hybridized/ clubbed to obtain the title compounds.] Of these, compound 5e with 4-flourophenyl substitution on piperazine ring exhibited highest activity in capsaicin induced model and anticonvulsant models. Further, all the derivatives studied for molecular and preadmet properties.
Cinnamides have a long history of human use possessing variety of biological properties, such as antineoplastic, antidepressant, antimycobacterial, anticonvulsant and antinociceptive activities, etc [1-6]. A series of N-aryl trans cinnamides were reported as novel, potent and selective competitive inhibitor of human and rat TRPV1 and thus display antinociceptive activity (AMG9810, SB-366791) [7-9]. The derivatives of m-(trifloromethyl) cinnamide and some (E) and (Z) N-alkyl-alpha, beta-dimethyl cinnamides were also reported to possess anticonvulsant activity [10-12]. The past decades have demonstrated many attempts to identify the structural features of compounds crucial for anticonvulsant and antinociceptive activities. As a result it was proved that one of the important core fragments possessing nitrogen containing heterocyclic system attached to the other aromatic system via a carbonyl or methylenyl group. In view of the above investigations, the present study is focussed on synthesizing different nitrogen containing heterocyclic systems like piperidine, morpholine, piperazine and N-arylpiperazines attached to the styryl carbonyl moiety antinociceptive and anticonvulsant activities. [13, 14].
Epilepsy and neuropathic pain are disorders characterised by either inappropriate spontaneous neuronal activity or excessive neuronal activity in response to physiological stimuli. These disorders are currently managed by drugs that are capable of dampening neuronal excitability . Drugs commonly used for epilepsy therapy include carbamazepine, Phenytoin, ethosuximide, lamotrigine, gabapentin, etc . Of these, gabapentin and carbamazepine are approved for the treatment of neuropathic pain and lamotrigine has demonstrated efficacy for neuropathic pain in clinical trials . Pharmacological and clinical studies have documented the pathophysiological similarities in epilepsy and neuropathic pain models. Thus, antiepileptic agents may have good potential to manage neuropathic pain . Due to the shared pathomechanism of many central and peripheral nervous system disorders, several antiepileptic and antinociceptive drugs may have efficacy to reduce the oxidative stress/ antioxidant therapy.
Based on the importance of the cinnamides, piperazine and N-arylpiperazine pharmacophoric moieties for antiepileptic and antinociceptive activities, it is planned to combine these potential scaffolds in to a novel hybrid molecule. We have designed and synthesized a novel series of cinnamide derivatives containing morpholinyl, piperidinyl, piperazinyl and N-arylpiperazinyl moieties as ring constrained analogues possessing promising biological activities.
Results and discussion
Synthesis of 1-(4-substitutedpiperazin-1-yl)prop-2-ene-1-one derivatives was carried out by simple and feasible procedures (scheme 1). Here, cinnamicacid was treated with thionylchloride, a versatile and highly reactive chlorinating agent, which produced cinnamoyl chloride by the nucleophilic substitution of chlorine. This further reacted with secondary amines like piperidine, morpholine, piperazine and substituted piperazines by dehydrohalogenation reaction affording the title compounds 3a-3l as depicted in scheme 1. The structures of the compounds were confirmed by IR, 1HNMR, mass and elemental analysis. The IR spectra of the compounds showed a broad band in the range of 1655-1640cm-1 assignable to the C=O stretching in amide functional group and a band at 1640-1610 cm-1 indicating the C=C stretching in trans cinnamoyl group. The mass spectra of the compounds (5a-5l) showed the molecular ion peaks and the elemental analysis for the compounds are within the limits of ±0.4% of theoretical value.
The 1H NMR spectrum of the compounds supported the structures of 3a-3l. These compounds showed a doublet in the region of 6.35-7.60 ppm due to alkenyl protons in cinnmoyl moiety (J=12.0-15.8 Hz), a broad doublet or a triplet in the region of 2.1-2.8 ppm and 3.2-3.9 ppm indicated the protons of the heterocyclics like piperidinyl, morpholinyl and piperazinyl substitutions. A multiplet in the region of 6.5- 7.5 ppm indicated the aromatic protons present in the phenyl ring.
Preclinical discovery and development of new drug candidates for the evaluation of both antiepileptic and antinociceptive activities are based on the use of animal models. The profile of antinociceptive activity of the compounds 5a-5l was evaluated in capsaicin-induced and formalin induced nociceptive methods after the oral administration of the compounds to the male Swiss albino mices at the dose of 10 mg/kg and observations were made after 1h. The results were shown in tables 2&3, fig 3, 4 and 5. All the compounds 5a-5h, 5j and 5k exhibited the significant activity in capsaicin induced nociception except 5i and 5l. Compound 5e with 4-florophenyl substitution exhibited highest activity (73.5%), which may be due to the presence of electron withdrawing p-floro substitution present on phenyl piperazinyl moiety. whereas, compounds 5f and 5d possessing 4-chlorophenyl and phenyl substitutions at the 4th position of piperazine also showed promising activity (64.7 and 61.7%). The isosteric replacement of N-phenyl ring in 5d with pyridin-2-yl and pyrimidin-2-yl as in 5h and 5i resulted in a drastic decrease in activity. Compounds 5j, 5k and 5l are structurally related to 5d, 5f and 5h respectively with decreased antinociceptive activity. This may be due to the presence of methylenedioxy group present on 3rd and 4th positions of phenyl ring of cinnamoyl moiety.
In formalin induced nociception method, all compounds 5a-5l exhibited significant activity. Isosteric replacement of morpholinyl moiety in 5b with piperazine as in 5c increased the activity, which may be due to the interaction of free NH with target site. Presence of pheny ring at the 4th position of piperazine as in 5d decreased the activity, whereas 4-florophenyl as in 5e and 5k, and 4-chlorophenyl as in 5f exhibited good antinociceptive activity. The compounds are not comparable to tramadol in exhibiting the protection against formalin induced nociception.
The second phase of formalin induced nociception method indicates anti-inflammatory response. Here, compounds 5b, 5c, 5e and 5k exhibited significant activity. Out of these, compounds possessing morpholine ring showed good anti-inflammatory activity. Compound 5c possessing free piperazine ring also showed good activity, followed by 4-fluorophenylpiperazinyl substitution. Other compounds did not exhibit significant anti-inflammatory response.
The antiepileptic profile of the compounds 5a-5l was evaluated by two methods, subcutaneous pentylenetetrazole induced and maximal electroshock induced methods. All the compounds exhibited significant protection except 5c and 5g against pentylenetetrazole induced seizures. Compounds possessing p-florophenyl substitution on piperazine ring as in 5f exhibited good latency periods and comparable to the standard drug, diazepam. Comparison of the activity of the bioisosteric analogues as in 5a, 5b and 5c showed increased latency periods in the order of increased lipophilicity (5c<5b<5a). Compounds 5b, 5d and 5e exhibited maximum protection against mortality and are comparable to standard drug, diazepam.
In case of maximal electroshock induced method, all the compounds 5a-5l showed significant protection. Compounds possessing pyrimidin-2-yl substitution as in 5h displayed good protection against electroshock induced seizures (100%) and is more potent than the standard drug, phenytoin. Compounds with phenyl, 4-florophenyl and morpholinyl substitutions as in 5d, 5e and 5b respectively showed less limb extension periods and thus pronounced seizure inhibitory activity and are comparable to the standard drug, Phenytoin.
All the synthesized compounds were also evaluated for in vitro antioxidant activity by three methods. Compound 5i exhibited good scavenging activity (IC50=7.4 µg) against DPPH free radicals and is more potent than the standard ascorbic acid (IC50=10.6 µg), which may be due to the N-pyrimidin-2-yl substitution. Table 6 shows the ability of the test compounds to scavenge nitric oxide in the IC50 ranges from 6.9-40.2 µg. Among these, compound possessing pyrimidin-2-yl substitution as in 5i exhibited highest activity (IC50=6.9 µg) and comparable to ascorbic acid (IC50=7.1 µg). The compound having 4-nitrophenyl substitution (5g) showed the highest activity (IC50=76 µg) in super oxide scavenging assay, more active than ascorbic acid (IC50=163.5 µg).
Drug-likeness is a qualitative concept, estimated from the molecular properties that affect their absorption, distribution, metabolism and excretion (ADME) of a compound by using Molinspiration software for the compounds under study [--]. Some basic molecular descriptors such as partition coefficient (log P), molecular weight (MW), or hydrogen bond acceptors and donors in a molecule indicate membrane permeability and bioavailability [--]. Number of rotatable bonds explains the conformational changes and flexibility of molecules and for the binding to the receptors. It is revealed that number of rotatable bonds should be â‰¤10 to pass the oral bioavailability [--]. Compounds 5a-5l possesses 2-4 rotatable bonds and therefore, exhibits the optimum conformational flexibility.
Molecular polar surface area (TPSA) is a very useful parameter to predict the transport properties of drugs like intestinal absorption and blood-brain barrier crossing. TPSA and volume are inversely proportional to the percentage of absorption (%ABS) and calculated using the equation: %ABS= 109 ± 0.345 Ã- TPSA, [--]. It was observed that all the title compounds exhibited a great %absorption ranging from 85 to 100% (table 5). The Lipinski's rule of five states that most molecules with good membrane permeability have logP â‰¤ 5, molecular weight â‰¤ 500, number of hydrogen bond acceptors â‰¤ 10 and number of hydrogen bond donors â‰¤ 5 and widely used as a filter for drug-likeness. Furthermore, none of the compounds violated Lipinski's parameters, making them potentially promising agents for antiepileptic and antinociceptive therapy.
The absorption and distribution parameters like oral bioavailability, in vitro plasma protein binding and blood-brain barrier penetration were calculated from preADME predictor, a molecular descriptor tool [--]. Oral drug absorption can be predicted using in vitro models like human intestinal absorption (%HIA), Caco2 cell (PCaco2) and MDCK cell (PMDCK) permeabilities (table 6). As per the criterion compounds of the series (5a-5l) showed %HIA ranging from 96-100%, optimum Caco2 cell permeability (4-70 nm/sec) and compounds 5a-5e and 5l displayed optimum MDCK cell permeability (25-500 nm/sec). Compounds 5a-5f and 5l showed optimum penetration into CNS via the blood-brain barrier.
The present study revealed that compound 5e showed promising anticonvulsant and antinociceptive activities. The presence of 4-florophenyl substitution on piperazine ring may be responsible //desirable feature to show the anticonvulsant and antinociceptive effect. Molecular properties prediction data support that, the compounds existence as drug-likeness.
Aldehydes and esters were procured from Sigma-Aldrich and Merck chemicals. All other chemicals are of AR grade. Purity of the samples was monitored by TLC analysis using Precoated aluminium plates (Merck), coated with Silica Gel (Kieselgel 60) with F254 indicator. Melting points were determined in open capillaries using Analab melting point apparatus and were uncorrected. IR spectra were recorded as KBr pellets on a Jasco FTIR (FTIR-4100) Spectrophotometer. 1H NMR spectra were carried out on Jeol-400 MHz NMR Spectrophotometer (JNM-400) using TMS as internal reference. Chemical shifts (Î´ values are given in parts per million (ppm) using CDCl3 as solvent coupling constants (J) in Hz. Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet.
Accurate masses were obtained on LCMS (schimadzu) APCI modelLC-2010 EV. Elemental analyses were performed on Perkin Elmer 2400 C, H, N elemental analyzer.
General method for the synthesis of compounds 5a-5l
To 0.01 mol of compound 3 or 4 dissolved in acetone, 0.01mol of different secondary amines and 2-3 drops of triethylamine were added at room temperature and continuously stirred for 10 -12h to obtain the compounds 5a-5l. Completion of the reaction was monitored by TLC plates.
synthesis of methylenedioxycinnamic acid (2)
A mixture of piperonal (0.165 mol), malonic acid (0.36 mol), dry pyridine (75ml) and piperidine (1.5 ml) is refluxed for one hour (a rapid evolution of carbondioxide takes place). The cooled reaction mixture was poured into water containing enough hydrochloric acid to dissolve pyridine. The precipitated acid was filtered and washed with water to obtain compound 2 (88% yield).
synthesis of cinnamoyl chloride (3) or methylenedioxycinnamoyl chloride (4).
0.01mol of cinnamicacid (1, 1.66g) or compound (2, 2.1g) dissolved in dichloromethane, 0.01mol of thionylchloride was added at room temperature and allowed for stirring for 5 h. After the completion of reaction, excess of thionylchloride was removed by distillation to obtain the compound 3 or 4. These compounds were used without further purification for next step.
PHYSICAL AND SPECTRAL DATA OF THE COMPOUNDS
synthesis of the (E)-3-phenyl-1-(piperidin-1-yl) prop-2-en-1-one // (E)-3-phenyl-1-(piperidin-1-yl) acrolein (5a): 1.66g of cinnamoylchloride (3, 0.01mol) was dissolved in acetone, 1ml of (0.01 mol) piperidine and 2-3 drops of triethylamine was added at room temperature with continuous stirring for 10 -12h to obtain the compound 5a as white colour powder with 58% yield. M.p. 143°C. Î»max 285 nm, Rf: 0.225. IR (KBr) Î½max, cm-1: 3095, 3080 & 3060 (Ar C-H str), 3029 (trans =CH str), 1652 (amide C=O str), 1631 (trans C=C str). 1H NMR (400MHz, CHCl3) Î´ (ppm): 2.45-2.55 (dt, 6H, J=4.7 Hz, 3CH2 of piperidine), 3.4 (t, 4H, J=5.2 Hz 2CH2 of piperidine), 6.35-6.45 (d, 1H, J=15.6 Hz, -CH=CH- of cinnamoyl group), 7.35-7.45 (d, 1H, J=15.67Hz, -CH=CH- of cinnamoyl group). APCI-MS: m/z = 215.9 (M)+, 217.9 (M+2H)+.
synthesis of the (E)-1-morpholino-3-phenylprop-2-en-1-one // (E)-1-morpholino-3-phenyl-acrplein (5b): 1.66g of compound 3 was dissolved in acetone, 1ml of (0.01mol) morpholine and proceeded as in 5a obtain the compound 5b as white crystalline powder, yield 57%. m.p 148°C. Î»max 275 nm. Rf : 0.28, IR (KBr) Î½max, cm-1: 3075 & 3056 (Ar C-H str), 3012 (trans =CH str), 2987 (C-H str), 1652 (amide C=O str), 1631 (trans C=C str). 1H NMR (400MHz, CHCl3) Î´ (ppm): 2.45-2.55 (dt, 6H, J=5.2Hz, 2CH2 of morpholine), 3.4 (t, 4H, J=5.2 Hz 2CH2 of morpholine), 6.35-6.45 (d, 1H, J=14.8 Hz, -CH=CH- of cinnamoyl group), 7.35-7.45 (d, 1H, J=14.8 Hz, -CH=CH- of cinnamoyl group). APCI-MS: m/z = 216.9 (M)+, 218.9 (M+2H)+.
synthesis of the (E)-3-phenyl-1-(piperazin-1-yl) prop-2-en-1-one // (E)-3-phenyl-1-(piperazinyl) acrolein (5c): 1.86g of (0.01mol) N-boc-piperazine and proceeded as in 5a. The obtained compound was subjected for the deprotection and produced the compound 5c as white crystalline powder, yield 62%. m.p 180°C. Î»max 282nm. Rf: 0.235. IR (KBr) Î½max, cm-1: 3095 & 3060 (Ar C-H str), 3029 (trans =CH str), 1638 (amide C=O str), 1599 (Ar C-C str). 1H NMR(400MHz, CHCl3) Î´ (ppm): 2.30-2.45 (br d, 4H, J=4.2 Hz, 2CH2 of Piperazine, 3.4-3.6 (br t, 4H, J=4.2Hz, 2CH2 of Piperazine), 6.0 ( br s, 1H, NH of piperazine), 6.5 (d, 1H, J=12.5 Hz, -CH=CH-C6H5), 7.12-7.35 (m, 5H, Ar-H), 7.6 (d, 1H, J=12.5 Hz, -CH=CH-C6H5). APCI-MS: m/z = 216.9 (M)+, 217.9 (M+H)+.
synthesis of the (E)-3-phenyl-1-(4-phenylpiperazin-1-yl) prop-2-en-1-one (5d): 1.62g of (0.01mol) N-phenylpiperazine and proceeded as in 5a to obtain the compound 5d as shiny white crystalline flakes, yield 78%. M.p. 146°C. Î»max 276.3 nm. Rf : 0.29. IR (KBr) Î½max, cm-1: 3098 & 3063 (Ar C-H str), 3027 (trans =CH str), 1638 (amide, C=O str). 1H NMR (400MHz, CHCl3) Î´(ppm): 3.28-3.36 (br d, 8H, 4CH2 of piperazine), 6.79 (d, 1H, J=15.8 Hz, -CH=CH-C6H5), 6.89-7.49 (m, 10H, Ar-H), 7.51-7.55 (d, 1H, J=15.8 Hz, -CH=CH-C6H5). APCI-MS: m/z = 292.9 (M)+.
synthesis of the (E)-1-(4-(4-fluorophenyl) piperazin-1-yl)-3-phenylprop-2-en-1-one (5e): 1.82g of (0.01mol) N-(4-florophenyl)-piperazine and proceeded as in 5a to obtain the compound 5e. It was obtained as white silky powder, yield 72%. M.p. 142°C. Î»max 325 nm, Rf : 0.215, IR (KBr) Î½max, cm-1: 3097 & 3070 (Ar C-H str), 3024 (trans =C-H str), 2994,2950 & 2893 (alkyl CH2 C-H str), 1641(amide C=O str). 1H NMR (400MHz, CHCl3) Î´ (ppm): 3.28-3.40 (br d, 8H, 4CH2 of piperazine), 6.35-6.45 (dd, 1H, J=13.8HZ, -CH=CH-C6H5), 6.5-7.65 (m, 9H, J=7.29Hz, J=8.1Hz Ar-H), 7.3-7.4 (dd, 1H, J=13.8HZ, -CH=CH-C6H5). APCI-MS: m/z = 310.9 (M)+, 292.1 (C19H19N2O)+.
synthesis of the (E)-1-(4-(4-chlorophenyl) piperazin-1-yl)-3-phenylprop-2-en-1-one (5f): 1.96g of (0.01mol) N-(4-chlorophenyl)-piperazine and proceeded as in 5a to obtain the compound 5f. It was obtained as white silky crystals, yield 71%.. M.p. 155°C, Î»max 277 nm, Rf: 0.25. IR (KBr) Î½max, cm-1: 3091 & 3057 (Ar C-H str), 3024 (trans =C-H str), 2984 & 2943 (alkyl CH2 C-H str), 1641(amide C=O str). 1H NMR (400MHz, CHCl3) Î´ (ppm): 3.28-3.40 (br d, 8H, 4CH2 of piperazine), 6.25-6.35 (dd, 1H, J=12.2 Hz, -CH=CH-C6H5), 6.45-7.45 (m, 9H, Ar-H), 7.35-7.45 (dd, 1H, J=12.2Hz, -CH=CH-C6H5). APCI-MS: m/z = 326.9 (M+H)+.
synthesis of the (E)-1-(4-(4-nitrophenyl) piperazin-1-yl)-3-phenylprop-2-en-1-one (5g): 2.07g of (0.01mol) N-(4-nitrophenyl)-piperazine was used to obtain the compound 5g and appeared as pale yellow colour, yield 67%. M.p. 175°C, Î»max 407 nm, Rf : 0.25. IR (KBr) Î½max, cm-1: 3086 (Ar C-H str), 3020 (trans =C-H str), 2989 & 2933 (alkyl CH2 C-H str), 1645 (amide C=O str). 1H NMR (400MHz, CHCl3) Î´ (ppm): 3.25-3.40 (br d, 8H, 4CH2 of piperazine), 6.20-6.35 (dd, 1H, J=12.5 Hz, -CH=CH-C6H5), 6.45-7.45 (m, 9H, Ar-H), 7.35-7.45 (dd, 1H, J=12.5Hz, -CH=CH-C6H5). APCI-MS: m/z = 337.1 (M)+.
synthesis of the (E)-3-phenyl-1-(4-(pyridin-2-yl) piperazin-1-yl) prop-2-en-1-one (5h): 1.63g of (0.01mol) N-(pyridin-2-yl)-piperazine was used to obtain the compound 5h appaered as white crystals, yield 64%. M.p. 125°C. Î»max 290 nm, Rf : 0.275. IR (KBr) Î½max, cm-1: 3094 & 3076(Ar C-H str), 1625 (amide C=O str). 1H NMR (400MHz, CHCl3) Î´ (ppm): 2.15-2.25 (t, 4H, J=4.2 Hz, 2CH2 of piperazine), 3.8-3.9 (t, 4H, J=4.2 Hz, 2CH2 of piperazine), 6.35-6.45 (d, 1H, J=14.5 Hz, -CH=CH-C6H5), 6.9-8.1 (m, 9H, J=7.35Hz, J=7.9 Hz, Ar-H ), 7.6-7.7 (d, 1H, J=14.5 Hz, -CH=CH-C6H5). APCI-MS: m/z = 292.9 (M)+, 214.9 (M+2H)+.
synthesis of the (E)-3-phenyl-1-(4-(pyrimidin-2-yl) piperazin-1-yl) prop-2-en-1-one (5i): 1.64g of (0.01mol) N-(pyrimidin-2-yl)-piperazine used in the 5a procedure to obtain the compound 5i as dull White colour crystals, yield 68%. M.p. 140°C. Î»max 401 nm, Rf : 0.28. IR (KBr) Î½max, cm-1: 3079 & 3072 (Ar C-H str), 2926 (C-H str, CH2 ), 1625 (amide C=O str). 1H NMR (400MHz, CHCl3) Î´ (ppm): 2.15-2.25 (t, 4H, J=4.5 Hz, 2CH2 of piperazine), 3.8-3.9 (t, 4H, J=4.5 Hz, 2CH2 of piperazine), 6.35-6.45 (d, 1H, J=14.2 Hz, -CH=CH-C6H5), 6.8-8.1 (m, 9H, Ar-H ), 7.6-7.7 (d, 1H, J=14.2 Hz, -CH=CH-C6H5). APCI-MS: m/z = 293.9 (M)+.
synthesis of the (E)-3-(benzo[d] [1,3]dioxol-6-yl)-1-(4-phenyl)piperazin-1-yl)prop-2-en-1-one (5j): 2.1g of (0.01mol) compound 4 was dissolved in acetone, 1.62g of (0.01mol) N-(phenyl)-piperazine and 2-3 drops of triethylamine was added at room temperature with continuous stirring for 10 -12h to obtain the compound 5j and appeared as white powder with 66% yield. M.p. 252°C. Î»max 322 nm, Rf : 0.235. IR (KBr) Î½max, cm-1: 3098 & 3075 (Ar C-H str), 1638 (amide C=O str). 1H NMR (400MHz, CHCl3) Î´(ppm): 3.1-3.3 (br d, 4H, J=5.7Hz, 2CH2 of piperazine), 3.75-3.90 (br d, 4H, J=5.7Hz, 2CH2 of piperazine), 5.85 (s, 2H, CH2 of -OCH2-O), 6.35-6.45 (d, 1H, J=15.8Hz, CH gp of -CH=CH-C6H5), 7.55-7.65 (d, 1H, J=15.8Hz, CH gp of -CH=CH-C6H5), 6.85-7.45(m, 8H, Ar-H). APCI-MS: m/z = 335.9 (M)+, 215.0 (C13H15N2O)+.
synthesis of the (E)-3-(benzo[d] [1, 3]dioxol-6-yl)-1-(4-(4-fluorophenyl)piperazin-1-yl)prop-2-en-1-one (5k): 1.82g of (0.01mol) N-(4-florophenyl)-piperazine used and proceeded as in 5j to obtain the compound 5k as white powder with 60% yield. M.p. 250°C. Î»max 315 nm, Rf: 0.24. IR (KBr) Î½max, cm-1: 3058 (Ar C-H str), 2964 (C-H str in CH2), 1638 (amide C=O str). 1H NMR(400MHz, CHCl3) Î´(ppm): 3.1-3.2 (t, 4H, J=4.2Hz, 2CH2 of piperazine), 3.65-3.80 (br d, 4H, J=4.9Hz, 2CH2 of piperazine) 5.9 (s, 2H, CH2 of -OCH2-O), 6.65-6.75 (dd, 1H, J=15.3Hz, CH of -CH=CH-C6H5), 7.55-7.70 (d, 1H, J=15.3Hz, CH of -CH=CH-C6H5), 7.05-7.45 (m, 7H, Ar-H). APCI-MS: m/z = 354.9 (M+H)+.
synthesis of the (E)-3-(benzo[d][1,3]dioxol-6-yl)-1-(4-(pyridin-2-yl)piperazin-1-yl)prop-2-en-1-one (5l): 1.63g of (0.01mol) N-(pyridin-2-yl)-piperazine used in 5j procedure to obtain the compound 5l. It was obtained as white powder with 60% yield. M.p. 249°C. Î»max 317 nm, Rf :0.255. IR (KBr) Î½max, cm-1: 3064 (Ar C-H str), 2973 (C-H str, CH2), 1625 (amide C=O str). 1H NMR (400MHz, CHCl3) Î´ (ppm): 2.15-2.25 (t, 4H, J=4.2 Hz, 2CH2 of piperazine), 3.8-3.9 (t, 4H, J=4.2 Hz, 2CH2 of piperazine), 6.35-6.45 (d, 1H, J=14.5 Hz, -CH=CH-C6H5), 6.9-8.1 (m, 9H, J=7.35Hz, J=7.9 Hz, Ar-H ), 7.6-7.7 (d, 1H, J=14.5 Hz, -CH=CH-C6H5). APCI-MS: m/z = 336.9 (M)+.
Male Swiss Albino mices (18-22g) and Male wistar rats (150-200g) were used as experimental animals and were obtained from King Institute Of Preventive Medicine, Guindy, Chennai-32. The animals were maintained in well-ventilated room by maintaining the temperature 23±2°C with natural 12±1 h day-night cycle in the polypropylene cages. They were fed ad libitum with balanced rodent pellet diet and water throughout the experimental period. The animals were sheltered for one week and prior to the experiment they were acclimatized to laboratory temperature. The protocol was approved by Institutional Animal Ethics Committee constituted for the purpose as per CPSCEA guidelines (1220/a/08/CPCSEA/ANCP/06).
Acute toxicity study:
The study was conducted as per OECD-425 guide lines for testing of chemicals acute oral toxicity  and used to fix the safe dose for the compounds 5a-5l. Swiss albino mice were divided into 14 groups each containing 5 animals. Drugs were administered by oral route in different concentrations (2000, 1000, 500, 250, 100, 50, 20 and 10mg/kg body weight). The animals were observed for their death over a period of 7days. The LD50 values were calculated by up and down method and dose was fixed as 10mg/kg body weight.
Evaluation of Anticonvulsant Activity
The Subcutaneous Pentylenetetrazole Seizure test (Sc PTZ)
This method utilizes a dose of Pentylenetetrazole (PTZ) 80mg/kg in rats that produces clonic seizures. The Male wistar rats were divided into 14 groups of six rats each. Group 1 was the control group received vehicle; Group 2 received 5 mg/kg body weight of Diazepam, Group 3 -14 received the 10mg/kg body weight of compounds 5a-5l respectively, which were prepared by suspended in 0.5% sodiumcarboxymethylcellulose. All the drugs were administered 1h prior to the PTZ administration and the latency period of the seizures and the mortality were observed [---].
The Maximal Electric Shock test (MES)
The anticonvulsant property of the drug in this model was assessed by its ability to protect against Maximal Electric Shock induced convulsions. Male Wistar albino rats were divided into 14 groups of six rats each. Group 1 was the control group received vehicle; Group 2 received 30 mg/kg body weight of Phenytoin, Group 3 -14 received the test compounds 5a-5l respectively. Maximal Electric Shock of 150mA current for 0.2 sec was applied through corneal electrodes to induce convulsions in the control, standard and test compounds treated animals [---].
Evaluation of antinociceptive activity:
Method for Capsaicin-induced nociception:
Male Swiss mice (18-22g) were used for the method and followed the adaptation to the experimental conditions. 20µl of capsaicin (1nmol/paw) was injected subplantarly in to the right hind paw, and the total number of flinchings of the injected paw was measured individually for 5 min and used as a measurement of nociception. The animals were treated with control and test compounds 5a-5l using oral gavages (10mg/kg) 1 h prior to capsaicin injection [---].
Method for formalin-induced nociception:
The mice were divided in to fourteen groups each containing six animals. Group 1 was the control group received vehicle; Group 2 received tramadol injection (5mg/ kg, s.c.), Group 3 -14 received the test compounds 5a-5l (10mg/kg, p.o) respectively, 1h prior to the formalin injection. Animals were injected intraplantarly with 20 Î¼l of 2.5% formalin solution (0.92% of formaldehyde, made up in saline solution 137 mM NaCl) . Mice were immediately placed in a glass cylinder 20 cm in diameter and observed from 0 to 30 min following formalin injection. The amount of time spent licking the injected paw was timed with a chronometer and was considered as indicative of nociception. The first phase of the nociceptive response normally peaked 5 min after the formalin injection and the second phase 15 to 30 min after the formalin injection, representing the neurogenic and inflammatory nociceptive responses, respectively.
Evaluation of antioxidant activity:
DPPH radical scavenging assay:
The ability to scavenge 2, 2-diphenyl-1-Picryl-Hydrazyl (DPPH) radical was determined by using DPPH method is as follows. 1 ml of test compound (5a-5i) (10, 50, 100, 250, 500 µg/ml) in ethanol and the reaction mixture was added to 4 ml of 0.004% methanol solution of DPPH and incubated in a dark place for 30 min. The absorbance of the samples was read at 517 nm. Ascorbic acid was used as reference standard. Percentage inhibition of DPPH free radical by the sample was calculated [---].
Measurement of nitric oxide activity:
The procedure is based on the principle that, sodiumnitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent. Scavengers of nitric oxide compete with oxygen, leading to reduced production of nitrite ions. For the experiment, sodiumnitroprusside (10 mM), in phosphate-buffered saline, was mixed with different concentrations of the test compounds 5a-5i, dissolved in water and incubated at room temperature for 15 min. After the incubation period, 0.5 ml of Griess reagent was added. The absorbance of the chromophore formed was read at 570 nm.
The results of anticonvulsant and antinociceptive activities were expressed as Mean ± SEM. The statistical significance of the differences between the groups was analyzed by one-way analysis of variance (ANOVA) followed by the Dunnett's multiple comparison test.
Calculation of molecular and ADME properties
The molecular properties like TPSA, miLogP, number of rotatable bonds and violations of Lipinski's Rule-of-Five were calculated using Molinspiration online property calculator tool kit . Topological polar surface area was used to calculate the percentage of Absorption (%ABS) according to the equation: %ABS = 109 - [0.345Ã- TPSA] .In vitro plasma protein binding values were obtained from ADME calculator.