Design, synthesis and evaluation of dialkyl 4-(benzo[d][1,3]dioxol-6-yl)-1, 4-dihydro-2,6-dimethyl-1-substituted pyridine-3,5-dicarboxylates as potential anticonvulsants and their molecular properties prediction

Article abstract of DOI:10.1016/j.ejmech.2013.06.006

The present study is on the development of dialkyl 4-(benzo[d][1,3]dioxol- 6-yl)-1,4-dihydro-2,6-dimethyl-1-substituted pyridine-3,5-dicarboxylate derivatives as isosteric analogues of isradipine and nifedipine, by the replacement of benzofurazanyl and 2-

Full text of DOI:10.1016/j.ejmech.2013.06.006

Accepted Manuscript
Design, Synthesis and evaluation of dialkyl 4-(benzo[d][1,3]dioxol-6-yl)-1,4-
dihydro-2,6-dimethyl-1-(4-substituted)pyridine-3,5-dicarboxylates as potential
anticonvulsants and their molecular properties prediction
G. Prasanthi, K.V.S.R.G. Prasad, K. Bharathi
PII:
S0223-5234(13)00374-7
10.1016/j.ejmech.2013.06.006
EJMECH 6234
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 December 2012
Revised Date: 25 May 2013
Accepted Date: 4 June 2013
Please cite this article as: G. Prasanthi, K.V.S.R.G. Prasad, K. Bharathi, Design, Synthesis and
evaluation of dialkyl 4-(benzo[d][1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethyl-1-(4-substituted)pyridine-3,5-
dicarboxylates as potential anticonvulsants and their molecular properties prediction, European Journal
of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.06.006.
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*Graphical Abstract (for review)
Click here to download Graphical Abstract (for review): Graphical abstract.pdf
ACCEPTED MANUSCRIPT
Graphical Abstract:
O
O
H3CH2COOC
COOCH2CH3
H3C
N
H
CH3
compound 8f
A novel series of 4-aryl-1,4-dihydropyridines (8a-8o) is designed and synthesized,
which displayed good anticonvulsant and antioxidant activities. Compound 8f exhibited good
anticonvulsant activity comparable to diazepam and also passed the Lipinski’s rule-of-five.

*Highlights (for review)
ACCEPTED MANUSCRIPT
Research Highlights:
 A series of 4-aryl-1,4-dihydropyridines were synthesized by Hantzsch reaction.
 All the compounds were evaluated for anticonvulsant and antioxidant activities.
 Compounds 8a, 8f and 8k showed good anticonvulsant activity.
 These compounds also obeyed the Lipinski’s rule-of-five.
 Selected compounds were also evaluated for antinociceptive activity.

*Revised Manuscript
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ACCEPTED MANUSCRIPT
Design, Synthesis and evaluation of dialkyl 4-(benzo[d][1,3]dioxol-6-yl)-1,4-
dihydro-2,6-dimethyl-1-(4-substituted)pyridine-3,5-dicarboxylates
as
potential anticonvulsants and their molecular properties prediction†.
G. Prasanthi, K.V.S.R.G. Prasad, K. Bharathi*.
Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam,
Tirupati- 517502, India.
†Part of the work was presented at Indo-US Symposium on “Frontiers in Medicinal
Chemistry and Drug Discovery” held in April (21-23) 2011at JSS University, Mysore, India.
*Corresponding author. Tel.: +919490922956, +919959008935
E-mail addresses: bharathikoganti@yahoo.co.in, prasanthi.gummalla@yahoo.com.

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Abstract
The present study is on the development of dialkyl 4-(benzo[d][1,3]dioxol-6-yl)-1,4-
dihydro-2,6-dimethyl-1-(4-substituted)pyridine-3,5-dicarboxylate derivatives as isosteric
analogues of isradipine and nifedipine, by the replacement of benzofurazanyl and 2-
nitrophenyl groups respectively with benzo[d][1,3]dioxo-6-yl group, as potential
anticonvulsants. Fifteen new derivatives (8a-8o) were synthesized and tested for
anticonvulsant activity using maximal electroshock and subcutaneous pentylenetetrazole
induced seizure methods. Compound 8f possessing free NH group in 1, 4-dihydropyridine
ring, diethyl ester functionality at the positions 3 and 5 showed significant anticonvulsant and
antioxidant activities. This was also supported by molecular properties prediction data.
Selected compounds were evaluated for antinociceptive activity in capsaicin induced
nociception assay at 10mg/kg body weight, but displayed no significant activity at the tested
dose.
Key words: benzofurazanyl; benzo[d][1,3]dioxo-6-yl; 1,4-dihydropyridine; anticonvulsant
activity; antinociceptive activity.

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1. Introduction
4-Aryl-1,4-dihydropyridines (4-aryl-1,4-DHPs) form a major class of drugs used as
potent calcium-channel blockers and were reported to be effective against majority of
convulsive procedures including electro and pentylenetetrazole convulsions. The recent
investigations on 1,4-dihydropyridines proved that they can modulate TRPV1 (transient
receptor potential vanilloid1) channels in a positive fashion and thus represent a lead
molecule for future pain therapeutics [1,2]. Seizures are the symptoms of epilepsy, which is a
common neurological disorder affecting about 1% of the world population. Despite all the
new antiepileptic drugs (AEDs) introduced in the past few decades, about 30% of the patients
with epilepsy are still not seizure free [3]. Therefore, there is a significant need to develop
new AEDs that are more potent and with fewer side effects.
Nifedipine and amlodipine are the prototype drugs of the class 4-aryl-1,4-DHPs
and have been subjected to several modifications (Fig 1). Their analogue, isradipine has
received particular attention as antihypertensive agent, calcium channel blocker and was also
reported to possess anticonvulsant activities [4-6]. Isradipine (4-(4-benzofurazanyl-1,4-
dihydro-2,6-dimethyl-3,5-pyridindicarboxylate, methyl 1-methylethyl) was evaluated for
anticonvulsant activity by maximal electroshock method (MES) in mice and reported to be
active at 5mg/kg dose [7,8]. Pharmacological and clinical studies have documented the
pathophysiological similarities in epilepsy and neuropathic pain models. Thus, antiepileptic
agents have good potential to manage neuropathic pain [9].
Based on these reports, we have recently begun a study on benzo[d][1,3] dioxole
derivatives as isosteric analogues of isradipine molecule, where benzofurazanyl moiety of
isradipine was replaced by benzo[d][1,3]dioxol-6-yl moiety. In this paper, we report the
molecular properties prediction, synthesis, characterization and evaluation for anticonvulsant,
antinociceptive and antioxidant activities of dialkyl4-(benzo[d][1,3]dioxol-6-yl)-1,4-dihydro-
2,6-dimethyl-1-(4-substituted)pyridine-3,5-dicarboxylate derivatives, 8a-8o in which the
benzo[d][1,3]dioxolyl moiety was attached to the fourth position of 1,4-dihydropyridine ring.
2. Results
2.1. Chemistry
Synthesis of benzo[d][1,3]dioxole derivatives of 1,4-dihydropyridine was carried out
via a one-pot multicomponent reaction (scheme 1). The condensation of commercially

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available piperonal, alkylacetoacetates and different substituted anilines in methanol provided
the compounds 8a-8j in good yields [10]. In the synthesis of asymmetrical 1,4-
dihydropyridine derivatives (8k-8o), the above method was modified to avoid the formation
of symmetrical 1,4-dihydropyridines. The reaction was carried out by condensation of
piperonal with ethylacetoacetate, followed by addition of methylacetoacetate and substituted
anilines and ammonia to obtain asymmetrical 1,4-DHPs as explained in scheme 2 [11,12].
The structures of compounds 8a-8o were confirmed by spectral data and elemental analysis.
2.2. Pharmacological evaluation.
The preclinical discovery and development of the new drug candidates for the
treatment of epilepsy are based mainly on the use of predictive animal models. In the present
study there are two in vivo screens used those include subcutaneous pentylenetetrazole
(scPTZ) seizures and maximal electroshock seizures.
The profile of anticonvulsant activity of compounds 8a-8o was evaluated by maximal
electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) tests after the
administration of test compounds (p.o) and reference standard drug (phenytoin, p.o;
diazepam, i.p) to male Wistar albino rats at the dose of 50 mg/kg body weight [13,14].
Seizure inducing pentylenetetrazole (80mg/kg, s.c) injection or maximal electroshock
(150mAmp for 0.2 sec) was applied 1h after the administration of the drug candidates. In
scPTZ induced method, the seizure response was observed for a maximum period of 300sec.
Compound 8f blocked the seizure activity at the maximum latency time used in the study.
Compounds 8a, 8b, 8f-8m and 8o showed significant anti-scPTZ activity (p<0.0001 vs.
control). Of these compounds, 8f, 8g and 8h exhibited good activity comparable to diazepam
(5mg/kg, i.p) as reference standard (Table 1). Other compounds, 8a, 8b, 8i-8k, 8m and 8o
showed moderate activity. Compounds 8d, 8e and 8n were devoid of anti-scPTZ activity. The
protection against PTZ induced mortality was also studied. Compound 8f exhibited 100%
protection and compounds, 8g, 8h and 8j showed moderate protection against mortality
(66%). Other compounds did not exhibit significant protection.
In the MES model, all the test compounds showed significant decrease in the duration
of limb extension and protection against electro convulsions except 8d and 8e (p>0.05 vs.
control) (Table 1). Among these derivatives, 8f and 8h exhibited good anti-MES protection
and other derivatives showed moderate protection. However, the activities of compounds 8a-
8o are less than that of phenytoin (30mg/kg, po).

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Of these derivatives, compounds 8f-8k and 8m showed good seizure inhibition
activity in scPTZ induced seizure method and are comparable to nifedipine at 30mg/kg,
whereas in MES induced method compounds 8f and 8h are comparable to nifedipine and
isradipine hind limb extension periods. Compound 8f was further subjected to dose-
dependent study at three doses (25, 50 and 100 mg/kg body weight) by PTZ induced and
MES induced seizure models (Table 2). The compound showed 41.6, 65.6 and 72.0
percentage protection respectively against electroconvulsive seizures. In PTZ model,
compound 8f showed equal latency periods at 50mg and 100mg/kg doses, but no protection
against mortality at 100mg/kg dose.
All the compounds were evaluated for in vitro antioxidant activity by DPPH free
radical scavenging assay. IC50 values were calculated and are given in table 3. Among these,
compounds 8f, 8i and 8n showed moderate radical scavenging activity (IC50 values ranging
from 363-386µg/ml).
The pathophysiological similarity between epilepsy and neuropathic pain has
prompted us to extend the study to capsaicin-induced nociception assay, to evaluate the
ability of the compounds in reducing neuropathic pain. Perusal of the results showed that
compounds 8a, 8b, 8f and 8o did not present any significant activity at the dose of 10mg/kg
(Table 4).
2.3. Calculation of drug-likeness properties
Drug-likeness can be considered as a delicate balance among the molecular properties
of a compound that influences its pharmacodynamics, pharmacokinetics and ultimately
ADME (absorption, distribution, metabolism and excretion) in human body like a drug [15].
These parameters allow ascertaining oral absorption, or membrane permeability that occurs
when evaluated molecules obey Lipinski’s rule-of-five [16]. Other parameters that included
are number of rotatable bonds, molecular volume, topological polar surface area, percentage
absorption and in vitro plasma protein binding.
The above mentioned parameters were calculated for 8a-8o and the results were
presented in table 5. It was observed that compounds 8a, 8f and 8k have optimum logP (<5),
lower in vitro plasma protein binding (<60%) and no violations from Lipinski’s rule-of-five.
3. Discussion

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The title compounds, 8a-8o were synthesized based on the lead molecule, isradipine.
The present work is related to the isosteric replacement of benzofurazanyl group in isradipine
with benzo[d][1,3]dioxo-6-yl moiety. Isradipine was reported to cause significant
anticonvulsant activity in mice using maximal electroshock model in a dose-dependent
manner at 1, 2.5 and 5.0 mg/kg [7,8].
Symmetric 1,4-DHP derivatives (8a-8j) were synthesized by following the Hantzsch
synthesis and asymmetric derivatives (8k-8o) by the modified Hantzsch synthesis. The
mechanism involved condensation of the aldehyde, β-ketoester (5, 6) and amines (7a-e)
followed by cyclisation and simultaneous elimination of three moles of water [17]. The
synthesis of symmetric 1,4-DHP derivatives (8a-8j) involves knoevenagel condensation of
piperonal and one molecule of alkylacetoacetate, followed by Michael addition of
aminoketone arising from the reaction of second molecule of alkylacetoacetate and amine.
Asymmetric 1,4-DHP derivatives were synthesized by slight modification of the reaction,
where piperonal reacts with ethylacetoacetate to produce knoevenagel product, which further
reacts with the methylacetoacetate and substituted amines to produce the compounds 8k-8o
[18].
The IR spectra of the title compounds showed a broad band at 3400-3300 cm-1
assignable to secondary amine group, a strong band at 1710-1680 cm-1 due to unsaturated
ester group on 1, 4-DHP basic nucleus at the positions 3 and 5. A band at 1450-1350cm-1
indicated symmetric and asymmetric stretching of nitro functional group and a band at 1050-
1
1020cm-1 showed C-N stretching. The HNMR spectrum of the compounds supported the
structures of 8a-8o. These compounds showed multiplets in the region of 6.4 to 8.1 ppm due
to aryl protons and a singlet at 5.80-5.95 ppm representing methylene protons of the
benzo[d][1,3]dioxole ring. The mass spectra of the compounds (8a-8o) showed the respective
molecular ion peaks and the data of elemental analysis of the compounds are within the limits
of ±0.4% of theoretical value.
In an attempt to study of the structure-activity relationships, the compounds are
designed by introducing a phenyl or substituted phenyl at position 1, benzo[d][1,3]dioxole
ring at position 4 and by varying the size of alkyl groups at positions 3 and 5 of 1,4-
dihydropyridine. It was previously reported that anticonvulsant activities of 1,4-DHPs are
strongly influenced by the nature of substitution at positions 1 and 4. The presence of phenyl
ring at fourth position (compounds 3& 4) increased the anticonvulsant activity when

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compared to 1,4-dihydropyridine derivatives with no substitution at position 4 (compounds
1&2; Fig 2) [19]. In our study, introduction of benzo[d][1,3]dioxolyl moiety in place of
phenyl group retained the activity indicating the importance of presence of 4-aryl
substitution. It was found that the anticonvulsant activity profile of the 4-
benzo[d][1,3]dioxole-1,4-dihydropyridines is significantly influenced by the nature of
substitution on the nitrogen atom at position 1. Compounds possessing no substitution at
position 1 and with free NH group available are found to be active (8a, 8f and 8k).
Compound 8f showed marked anticonvulsant activity in both the models and maximum
protection against mortality. Similar results were also reported with N-methylation of 4-
substituted-1,4-DHPs which caused a large decrease in anticonvulsant activity [20]. It is
probable that the presence of free NH might augment binding to the target via hydrogen
bonding.
Introduction of a phenyl group at position one resulted in decrease in the activity. The
presence of a nitro group on the phenyl ring further influenced the activity. Compounds with
2-nitrophenyl substitution are more potent than those containing nitro groups at third and
fourth positions of the phenyl ring. With an increase in size of the alkyl group of the ester
functionality, the anticonvulsant activity is also increased. The presence of ethyl group of 3,5-
diester functionality as in 8f-8j resulted in active analogs. It was supported by the literature
studies that bulkiness of ester functionality seems to be important for potency [20]. It is
probable that the presence of larger alkyl groups in the ester could have better interaction
with calcium channels and gain more potency. It was reported that existence of hindrance by
substituted groups prevent hydrolysis of esteric groups on 1,4-dihydropyridine ring [21]. The
presence of benzodioxolyl moiety at position 4 and 2,6-dimethyl groups on dihydropyridine
ring can induce steric hindrance on ester groups at third and fifth positions of
dihydropyridine. Hence, it is likely that the physiologically active compound in the seizure
studies is indeed the test compound, instead of a metabolite. Compound 8f was further
evaluated at 25mg and 100mg as it exhibited promising seizure inhibition and protection
against mortality at 50mg/kg dose. The activity was found to be dose dependent in both
scPTZ and MES models. The isosteric replacement of 2-nitrophenyl and benzofurazanyl
substitutions of nifedipine and isradipine respectively with benzodioxolyl group at position 4
of dihydropyridine ring retained the activity.
Perusal of the radical-scavenging ability data showed that the compounds exhibited
only moderate scavenging of DPPH free radical (Table 3). There was a good agreement

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between anticonvulsant activity and radical scavenging activity for compound 8f, thus
suggesting that anticonvulsant activity of compound 8f might involve antioxidant
mechanisms.
The study was extended to evaluate selected compounds for antinociceptive activity
by using capsaicin induced flinching assay in Swiss albino mice. Literature studies showed
that capsaicin, resiniferatoxin, N-oleoyldopamine etc act on vanilloid receptors (TRPV1)
producing nocicepton [22]. Compounds 8a, 8b, 8f and 8o which exhibited good
anticonvulsant activity were evaluated for capsaicin induced flinching model at 10mg/kg
body weight. None of the compounds displayed significant antinociceptive activity at the
tested dose. As these compounds were not evaluated at other higher doses, further studies are
required to test the antinociceptive profile of the compounds.
Calculation of drug-likeness properties
Drug-likeness of the compounds 8a-8o were estimated from predicted ADME values
and Molinspiration software and found to score well. In particular, optimum lipophilicity
(<5) and presence of hydrogen bonding donor make the molecules 8a, 8f and 8k likely to
have good drug-likeness and absorption. Number of rotatable bonds is important to know the
conformational changes, flexibility and for binding with receptors or channels [23].
Compounds 8a-8o were found to possess rotatable bonds 5-9 (<10), indicating high
conformational flexibility. It was observed from the data that the title compounds exhibited
%ABS ranging from 67.5 to 83.3%, while standard isradipine and nifedipine displayed 73.2
and 70.9 %ABS respectively.
Lipinski’s rule-of-five is widely used filter for drug-like properties and states that
most molecules with good membrane permeability have logP≤5, MW≤500, HBD≤5 and
HBA≤10. Of these compounds, 8a, 8f and 8k obeyed the rule and other compounds were
found to violate in one parameter i.e. partition co-efficient. Furthermore, compounds which
violated the rule are also deficient in hydrogen bonding donors, whereas compounds 8a, 8f
and 8k were found to have one HBD, which may also be a necessary requirement to exhibit
the activity.
The importance of presence of free NH group of 1,4-dihydropyridine is also
supported by the estimation of in vitro plasma protein binding using preADME studies. In
vitro plasma protein binding (%) for the compounds 8a-8o was given in table 5. Compounds

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containing free NH group i.e. 8a, 8f and 8k showed less than 90% in vitro plasma protein
binding thus suggesting weak plasma protein binding of these compounds [24].
4. Conclusion
The present study revealed that compound 8f showed promising anticonvulsant
activity comparable to phenytoin. The presence of free NH group of 1,4-DHP, 4-aryl
substitution and the size of the alkyl group of the ester functionality at positions 3 and 5 are
probably the desirable features for good anticonvulsant and antioxidant activities. Further, the
molecular properties prediction data supports that compounds 8a, 8f and 8k might involve
hydrogen bonding interaction with target site, displayed good in silico absorption and low
plasma protein binding, thus making them as potential drug candidates for antiepileptic
therapy.
5. Materials and methods
5.1 General
Aldehyde 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 diluted pellets on a Jasco
1
FTIR (FTIR-4100) Spectrophotometer. H NMR spectra were carried out on Jeol-400 MHz
NMR Spectrophotometer (JNM-400) using TMS as internal reference. Chemical shifts (δ
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. Mass spectral data was obtained on LCMS (schimadzu) APCI modelLC-2010 EV.
Elemental analyses were performed on Perkin Elmer 2400 C, H and N elemental analyser.
5.1.1. General method for the synthesis of dialkyl 4-(benzo (d)[1,3]dioxol-6-yl)-1,4-dihydro-
2,6-dimethyl-1-(substituted)pyridine-3,5-dicarboxylate (8a-8j). (Scheme-1)
To a solution of piperonal (0.01mol, 1.5g), methylacetoacetate (5) (0.03mol, 3.2ml) or
ethylacetoacetate (6) (0.03mol, 3.8ml) in methanol (20ml) was treated with ammonia solution
or substituted anilines (7a-e) (0.02mol) and refluxed for 8-36 hours. The completion of the
reaction was monitored by TLC using n-hexane: acetone (9:1). After the completion of the

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reaction, the mixture was cooled and evaporated to separate the solid. The crude compound
was purified by double recrystallization with methanol. The yield obtained was 52-60%.
5.1.1.1. Dimethyl 4-(benzo[d] [1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxyl ate (8a). Piperonal, methylacetoacetate and ammonia were refluxed in methanol
for 8h and obtained as pale yellow crystals, yield 52%, mp 175-178°C. Rf : 0.315; IR (KBr,
νmax, cm-1): 3391(N-H Str), 3070-3020(Ar C-H Str), 2294 (alk C-H Str), 1702(C=O
1
Str), 1086 (aliphatic C-N Str), 771(N-H Wag). H NMR (400MHz, CDCl3) δ (ppm):
1.7 (s, 6H, 2(CH3) at C-2 & C-6), 3.7 (s, 6H, 2 (CH3) at C-3 & C-5), 4.45 (s, 1H, C-4), 5.05
(br s, 1H, NH), 5.9 (s, 2H, O-CH2-O), 6.4 (s, 1H, Ar-H), 6.50-6.65 (d, 2H, J=7.48Hz, Ar-H).
APCI-MS: m/z = 344.9 (M)+, 345.9 (M+H)+. Anal. Calc. for C18H19NO6: C, 62.60; H, 5.55;
N, 4.06. Found: C, 62.35; H, 5.41; N, 4.01.
5.1.1.2. Dimethyl 4-(benzo[d] [1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethyl-1-phenyl pyridine -
3,5-dicarboxylate (8b). Piperonal, methylacetoacetate and aniline were refluxed in methanol
for 12h and obtained as pale yellow crystals, yield 52%. m.p 185-186°C. Rf : 0.31. IR (KBr,
νmax, cm-1): 3070(Ar C-H Str), 2950(Alk C-H Str), 1702 & 1691(C=O Str, α, β unsaturated
1
ester), 1036(Aliphatic C-N Str). H NMR (400MHz, CDCl3) δ (ppm): 1.7 (s, 6H, 2(CH3) at
C-2 & C-6) 3.7 (s, 6H, 2(CH3) at C-3 & C-5), 4.45 (s, 1H, CH at C-4), 5.9 (s, 2H, O -CH2-
O), 6.42 (d, 1H, J=8.0Hz, Ar -H), 6.5 (d, 1H, J=8.0Hz, Ar-H), 6.6 (t, 1H, J =7.6Hz, Ar-H),
6.7(d, 2H, J=7.44Hz, Ar-H), 7.01 (t, 2H, J=7.44Hz, Ar-H). APCI-MS: m/z = 421 (M)+, 422
(M+H)+. Anal. Calc. for C24H23NO6: C, 68.40; H, 5.50; N, 3.32. Found: C, 68.35; H, 5.45; N,
3.26
5.1.1.3.
Dimethyl4-(benzo[d][1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethyl-1-(2-nitrophenyl)
pyridine-3,5-dicarboxylate (8c). Piperonal, methylacetoacetate and 2-nitroaniline were
refluxed in methanol for 18h and obtained as Orange colour crystals with 56% yield. m.p.
235-237°C. Rf :0.72; IR (KBr) νmax, cm-1: 3024 (Ar C-H Str), 2925(Alk C-H Str),1702
(C=O Str, α, β unsaturated ester ), 1482 (N-O asym str),1326(N-O sym str).1H NMR
(400MHz, CDCl3) δ (ppm): 1.75 (s, 6H, CH3 at C-2 & C-6), 3.75 (s, 6H, CH3 at C-3& C-5),
4.45 (s, 1H, CH at C-4), 5.95 (s, 2H, O-CH2-O), 6.35 (s, 1H, Ar-H), 6.40-6.55 (d, 2H, J=8.0
Hz, Ar-H), 6.8 (d, 1H, J=7.3Hz, Ar-H), 7.2 (t, 1H, J=7.3Hz, Ar-H), 7.4 (t, 1H, J=7.3Hz, Ar-
H), 7.5-7.6(d, 1H, J=7.3Hz, Ar-H). APCI-MS: m/z = 465.9 (M)+, 466.9 (M+H)+. Anal. Calc.
for C24H22N2O8: C, 61.80; H, 4.75; N, 6.01. Found. C, 61.75; H, 4.71; N, 5.97

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5.1.1.4. Dimethyl 4-(benzo[d][1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethyl-1-(3-nitrophenyl)
pyridine-3,5-dicarboxylate (8d). Piperonal, methylacetoacetate and 3-nitroaniline were
refluxed in methanol for 24h and obtained as yellowish orange crystals with 57% yield. m.p.
251-253°C. Rf : 0.33; IR (KBr, νmax, cm-1) : 3070 (Ar C-H Str), 2994 (alk C-H Str), 1702
(C=C Str, α, β unsaturated ester ), 1482 (N-O asym.str), 1341(N-O sym str).1H NMR
(400MHz, CDCl3) δ (ppm): 1.75 (s, 6H, CH3 at C-2 & C-6), 3.75 (s, 6H, CH3 at C-3& C-5),
4.45 (s, 1H, CH at C-4), 5.95 (s, 2H, O-CH2-O), 6.35 (s, 1H, Ar-H), 6.49-6.55 (d, 2H, J=8.0
Hz, Ar-H), 6.8 (d, 1H, J=7.5Hz, Ar-H), 7.2 (t, 1H, J=7.5Hz, Ar-H), 7.4 (s, 1H, J=7.5Hz, Ar-
H), 7.55-(d, 1H, J=7.5Hz, Ar-H). APCI-MS: m/z = 466.9 (M+H)+. Anal. Calc. for
C24H22N2O8: C, 61.80; H, 4.75; N, 6.01. Found C, 61.76; H, 4.69; N, 5.95;
5.1.1.5. Dimethyl 4-(benzo[d][1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethyl-1-(4-nitrophenyl)
pyridine -3,5-dicarboxylate (8e). Piperonal, methylacetoacetate and 4-nitroaniline were
refluxed in methanol for 18h and obtained as dark yellow crystals with 55% yield. m.p. 265-
268°C. Rf : 0.305. IR (KBr, νmax, cm-1): 3101 (Ar C-H Str), 2893 (alk C-H Str), 1705
(C=O Str, α, β unsaturated), 1489 (N-O asym. str), 1349 (N-O sym. str).1H
NMR(400MHz, CDCl3) δ(ppm): 1.73 (s, 6H, CH3 at C-2 & C-6) , 3.75 (s, 6H, CH3 at C-3
& C-5), 4.5 (s, 1H, CH at C-4), 5.9(s, 2H, -O-(CH2)-O), 6.37 (s, 1H, Ar-H), 6.45-6.65 (d,
2H, J=6.7Hz, Ar-H), 6.8-7.9 (m, 4H, Ar-H). APCI-MS: m/z = 465.9 (M)+. Anal. Calc. for
C24H22N2O8: C, 61.80; H, 4.75; N, 6.01. Found C, 61.74; H, 4.71; N, 5.97;
5.1.1.6.
Diethyl
4-(benzo[d][1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethyl
pyridine-3,5-
dicarboxylate (8f). Piperonal, ethylacetoacetate and ammonia were refluxed in methanol for
12h and obtained as fine yellow crystals with 56% yield. m.p. 198-201°C. Rf :0.3 IR (KBr,
νmax, cm-1): 3401 (N-H Str), 3070 (Ar C-H Str), 2925(Alk C-H Str), 1691 (C=O
Str, α, β unsaturated), 800(N-H Wag).1H NMR (400MHz, CDCl3) δ (ppm): 1.3 (t, 6H,,
J=6Hz, CH3 at C-3 & C-5) ,1.71(s, 6H, CH3 gp at C-2 & C-6), 4.2 (q, 4H, J=6Hz, CH2 at C-3
& C-5), 4.44 (s, 1H, CH at C-4), 5.08 (br s, 1H, NH), 5.9 (s, 2H, O-CH2-O), 6.4 (s, 1H, Ar-
H), 6.48-6.65 (d, 2H, J=7.8Hz, Ar-H). APCI-MS: m/z = 372.9 (M)+, 373.9 (M+H)+. Anal.
Calc. for C20H23NO6: C, 64.33; H, 6.21; N, 3.75. Found: C, 64.08; H, 6.09; N, 3.85.
5.1.1.7. Diethyl 4-(benzo[d][1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethyl-1-phenylpyridine-3,5-
dicarboxylate (8g). Piperonal, ethylacetoacetate and aniline were refluxed in methanol for
16h and obtained as pale yellow crystals with 53% yield. m.p. 215-216°C. Rf :0.317. IR (KBr,
νmax, cm-1): 3070(Ar C-H Str), 2950(Alk C-H Str), 1702 & 1691(C=O Str, α, β unsaturated

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1
ester), 1036(Aliphatic C-N Str). H NMR(400MHz, CDCl3) δ (ppm):1.33 (t, 6H, CH3 at C-3
& C-5), 1.72 (s, 6H, CH3 at C-2 & C-6), 4.2 (q, 4H, J=5.8Hz, CH2 gp at C-3 & C-5), 4.65 (s,
1H, CH at C-4), 5.75 (s, 2H, -O-CH2-O-), 6.37(s, 1H, Ar-H), 6.40-6.55 (d, 2H, J=7.1Hz, Ar-
H),7.0-6.6 (m, 5H, Ar-H). APCI-MS: m/z = 449 (M)+. Anal. Calc. for C26H27NO6: C, 69.47;
H, 6.05; N, 3.12. Found: C, 69.48; H, 6.01; N, 3.25.
5.1.1.8. Diethyl 4-(benzo[d][1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethyl-1-(2-nitrophenyl)
pyridine-3,5-dicarboxylate (8h). Piperonal, ethylacetoacetate and 2-nitroaniline were refluxed
in methanol for 28h and obtained as light orange crystals with 58% yield. m.p. 258-259°C.
Rf :0.68. IR (KBr, νmax, cm-1): 3024 (Ar C-H Str), 2925(Alk C-H Str),1702 (C=O Str,
α, β unsaturated ester ), 1482 (N-O Asym str),1326(N-O sym str),1203(C-phenolic
gp), 1149(C-O-C Ester).1H NMR(400MHz, CDCl3) δ (ppm): 1.28 (t, 6H, J=4Hz CH3 at
C-3 & C-5),1.7 (s, 6H, CH3 at C-2 & C-6), 4.15 (q, 4H, J=4Hz, CH2 gp at C-3 & C-5 ),4.45
(s, 1H, CH at C-4), 5.9 (s, 2H, O-CH2-O-), 6.37 (s, 1H, Ar-H), 6.48-6.65 (d, 2H, J=6.9Hz,
Ar-H),7.95-6.8 (m, 4H, Ar-H). APCI-MS: m/z = 494.9 (M+H)+. Anal. Calc. for C26H26N2O8:
C, 63.15; H, 5.30; N, 5.67. Found: C, 63.08; H, 5.36; N, 5.85.
5.1.1.9. Diethyl 4-(benzo[d][1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethyl-1-(3-nitrophenyl)
pyridine-3,5-dicarboxylate (8i). Piperonal, ethylacetoacetate and 3-nitroaniline were refluxed
in methanol for 36h and obtained as pale orange crystals with 53% yield. m.p. 273-275°C. Rf
:0.35. IR (KBr νmax, cm-1): 3070 (Ar C-H Str), 2994 (alk C-H Str), 1702 (C=C Str, α, β
1
unsaturated ester ), 1482 (N-O asym.str), 1341(N-O sym str). H NMR (400MHz, CDCl3) δ
(ppm): 1.2 (t, 6H, J=4.4Hz, CH3 at C-3 & C-5),1.7(s, 6H, CH3 at C-2 & C-6), 4.15(q, 4H,
J=4.4Hz CH2 at C-3 & C-5),4.45 (s, 1H, CH at C-4 ), 5.95 (s, 2H, O-CH2-O-), 6.38 (s, 1H,
Ar-H), 6.7-6.45 (d, 2H, J=7.2Hz Ar -H),7.4 (s, 1H, Ar-H),7.6-6.68 (m, 3H, Ar-H). APCI-
MS: m/z = 494.9 (M+H)+. Anal. Calc. for C26H26N2O8: C, 63.15; H, 5.30; N, 5.67. Found: C,
63.21; H, 5.16; N, 5.55.
5.1.1.10. Diethyl 4-(benzo[d][1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethyl-1-(4-nitrophenyl)
pyridine -3,5-dicarboxylate (8j). Piperonal, ethylacetoacetate and 4-nitroaniline were refluxed
in methanol for 30h and obtained as yellow fine crystals with 52% yield. m.p. 287-289°C. Rf
: 0.325. IR (KBr, νmax, cm-1): 3101 (Ar C-H Str), 2893 (Alk C-H Str), 1705 (C=C,
1
C=O Str, α, β unsaturated), 1489 (N-O Asym. str), 1349 (N-O sym. str). H NMR
(400MHz, CDCl3) δ (ppm): 1.29 (t, 6H, J=6Hz CH3 at C-3 & C-5), 1.71 (s, 6H, CH3 at C-2 &
C-6), 4.17 (q, 4H, J=6Hz, CH2 at C-3 & C-5),4.43 (s, 1H, CH at C-H), 5.93 (s, 2H, -O-CH2-

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O-), 6.37 (s, 1H, Ar-H), 6.48-6.62 (d, 2H, J=7.8Hz, Ar-H), 6.8-7.9 (m, 4H, Ar-H). APCI-MS:
m/z = 493.9 (M)+. Anal. Calc. for C26H26N2O8: C, 63.15; H, 5.30; N, 5.67. Found: C, 63.25;
H, 5.18; N, 5.65.
5.1.2. General method for the synthesis of 3-ethyl, 5-methyl 4-(benzo[d][1,3]dioxol-6-
yl)-1,4-dihydro-2,6-dimethyl-1-(substituted)pyridine-3,5-dicarboxylate (8k-8o). (Scheme-2)
The synthesis of asymmetrical 1,4-DHPs (8k- 8o) were synthesized by the modified
Hantzsch method to avoid the formation of symmetrical1,4-DHPs.
To an ice-cooled solution of 10mmol (1.5g) of piperonal in 30ml of isopropanol,
10mmol (1.1ml) of the ethylacetoacetate were added and refluxed for five hours. Then six
mmol (0.8ml) of methylacetoacetate was added and the mixture was refluxed for 8 hours.
Without separation 2mmol of amine was added and the mixture was refluxed for 4-6 hours.
After cooling, the solvent was evaporated and the separated solid was purified by
recrystallization. (Yield: 30-35 %)
5.1.2.1. 3-ethyl, 5-methyl 4-(benzo[d][1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate (8k). Fine yellow crystals with 57% yield. m.p. 186-189°C. Rf: 0.3. IR (KBr,
νmax, cm-1): 3401 (N-H Str), 3070 (Ar C-H Str), 2925(Alk C-H Str), 1691 (C=O
1
Str, α,β-unsaturated), 800(N-H Wag). H NMR (400MHz, CDCl3) δ(ppm): 1.23 (t, 3H,
J=6Hz, CH3 at C-3), 1.7 (s, 6H, CH3 at C-2 & C-6), 3.75 (s, 3H, CH3 at C-5), 4.15 (q, 2H,
J=6Hz, CH2 at C-3), 4.45 (s, 1H, CH at C-4), 5.1(br s, 1H, NH), 5.75 (s, 2H, O-CH2-O), 6.4
(s, 1H, Ar-H), 6.62-6.5(d, 2H, J=7.8Hz, Ar-H). APCI-MS: m/z = 358.9 (M)+, 359.9 (M+H)+.
Anal. Calc. for C19H21NO6: C, 63.50; H, 5.89; N, 3.9. Found: C, 63.15; H, 5.91; N, 3.95.
5.1.2.2. 3-ethyl,5-methyl 4-(benzo[d][1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethyl-1-phenyl-
pyridine-3,5-dicarboxylate (8l). Pale yellow colour fine crystals with 52% yield. m.p. 196-
197°C. Rf : 0.319. IR (KBr, νmax, cm-1): 3070 (Ar C-H Str), 2950 (alk C-H Str), 1702 &
1
1691(C=O Str, α, β unsaturated ester), 1036(Aliphatic C-N Str). H NMR (400MHz, CDCl3)
δ (ppm): 1.2-1.3 (t, 3H, CH3 gp at C-3), 1.7 (s, 6H, CH3 at C-2 & C-6), 3.75 (s, 3H, CH3 at C-
5), 4.14 (q, 2H, J=8Hz CH2 gp at C-3), 4.45 (s, 1H, CH at C-4), 5.7 (s, 2H, O-CH2-O-), 6.36
(s, 1H, Ar-H), 6.4-6.5 (d, 2H, J=8Hz, Ar-H), 6.55-7.0 (m, 5H, Ar -H). APCI-MS: m/z = 435.1
(M)+, 436.1 (M+H)+. Anal. Calc. for C25H25NO6: C, 68.95; H, 5.79; N, 3.22. Found: C, 69.48;
H, 5.71; N, 3.25.

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5.1.2.3. 3-ethyl, 5-methyl 4-(benzo[d][1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethyl-1-(2-nitro
phenyl)pyridine-3,5-dicarboxylate (8m). Orange fine crystals with 55% yield. m.p. 246-
247°C. Rf: 0.63; IR (KBr, νmax, cm-1): 3024 (Ar C-H Str), 2925(Alk C-H Str),1702
1
(C=O Str, α, β unsaturated ester ), 1482 (N-O Asym str),1326(N-O sym str). H
NMR(400MHz, CDCl3) δ (ppm): 1.3 (t, 3H, J=4Hz CH3 at C-3), 1.75 (s, 6H, CH3 at C-2 &
C-6), 3.72 (s, 3H, CH3 at C-5), 4.17 (q, 2H, J=4Hz, CH2 at C-3 ), 4.45 (s, 1H, CH at C-4),
5.95 (s, 2H, -O-CH2-O-), 6.37 (s, 1H, Ar-H), 6.45-6.75 (d, 2H, J=8.5Hz, Ar-H), 8.0-6.75 (m,
4H, J=7.8Hz, Ar-H). APCI-MS: m/z = 479.9 (M)+, 480.9 (M+H)+. Anal. Calc. for
C25H24N2O8: C, 62.49; H, 5.03; N, 5.83. Found: C, 62.09; H, 5.01; N, 5.75.
5.1.2.4. 3-ethyl, 5-methyl 4-(benzo[d][1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethyl-1-(3-nitro
phenyl) pyridine-3,5-dicarboxylate (8n). Yellow crystals with 59% yield. m.p. 252-254°C.
Rf: 0.4; IR (KBr, νmax, cm-1) : 3070(Ar C-H Str), 2950(Alk C-H Str), 1702 & 1691(C=O Str,
1
α, β unsaturated ester), 1036(Aliphatic C-N Str). H NMR (400MHz, CHCl3) δ(ppm): 1.23
(t, 3H, J=4.2Hz, CH3 gp at C-3), 1.7 (s, 6H, CH3 gp at C-2 & C-6), 3.7 (s, 3H, CH3 at C-5 ),
4.2 (q, 2H, J= 4.2Hz, CH2 gp at C-3), 4.45 (s, 1H, CH at C-4), 5.9 (s, 2H, -O-CH2-O-), 6.35
(s, 1H, Ar- H), 6.45-6.65 (d, 2H, J=7.2Hz, Ar-H), 6.7-7.6 (m, 4H, Ar-H). APCI-MS: m/z =
480.1 (M+H)+. Anal. Calc. for C25H24N2O8: C, 62.49; H, 5.03; N, 5.83. Found: C, 62.15; H,
5.04; N, 5.05.
5.1.2.5. 3-ethyl, 5-methyl 4-(benzo[d][1,3]dioxol-6-yl)-1,4-dihydro-2,6-dimethyl-1-(4-nitro
phenyl)pyridine-3,5-dicarboxylate (8o). Pale yellow crystals with 57% yield. m.p. 267-
269°C. Rf :0.31; IR (KBr, νmax, cm-1): 3101 (Ar C-H Str), 2893 (Alk C-H Str), 1705
1
(C=C, C=O Str, α, β unsaturated), 1489 (N-O Asym. str), 1349 (N-O sym. str). H
NMR (400MHz, CDCl3) δ (ppm): 1.2 (t, 3H, J=4.4Hz, CH3 at C-3), 1.68 (s, 6H, CH3 at C-
2&C-6), 3.75 (s, 3H, CH3 at C-5), 4.2 (q, 2H, J=4.4Hz ,CH2 at C-3), 4.47 (s, 1H, CH at C-4),
5.9 (s, 2H, -O-CH2-O-), 6.37 (s, 1H, Ar-H ), 6.45-6.7 (d, 2H, J=8.1Hz, Ar-H), 6.75-7.98 (m,
4H, J=7.6Hz, Ar-H). APCI-MS: m/z = 481.9 (M+H)+. Anal. Calc. for C25H24N2O8: C, 62.49;
H, 5.03; N, 5.83. Found: C, 62.59; H, 5.10; N, 5.85.
5.2. Pharmacology
5.2.1. Experimental animals
Male Swiss albino mice (18-22g) and male Wistar rats (150-200g) were used as
experimental animals. They were obtained from King Institute of preventive medicine,
Guindy, Chennai-32. The animals were acclimatized at least a week under standard

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husbandary conditions, room temperature of 24±1°C, relative humidity 45-55% and a 12: 12
h light/dark cycle. The animals had free access to rodent pellet diet (Pranav Agro Industry,
Bangalore) and water under strict hygienic conditions. All animal experiment protocols were
approved by the Institutional Animal Ethical Committee (IAEC) of Annamacharya college
of Pharmacy, Rajampet, India (1220/a/08/CPCSEA/ANCP/06).
5.2.2. Acute toxicity studies
The study was conducted as per OECD-425 guidelines for testing of chemicals acute
oral toxicity [25]. The test was used to fix the safe dose for the compounds 8a-8o. Swiss
albino mice were divided into six groups each containing 10 animals and repeated for all the
drugs. Drugs were administered by oral route in different concentrations (2000, 1000, 500,
250, 100 and 50mg/kg body weight). The animals were observed for their death over a
period of 7days. The LD50 values were calculated by median lethal dose calculations and
dose were fixed as 50mg/kg body weight.
5.2.3. Evaluation of Anticonvulsant Activity
5.2.3.1. The Maximal Electric Shock test (MES)
The anticonvulsant property of the test compounds in this model was assessed by its
ability to protect against maximal electric shock induced convulsions. Male Wistar albino rats
were divided into 17 groups of six rats each. Group 1 was the control group received vehicle
(0.5% sodium carboxymethylcellulose); Group 2 received phenytoin (30 mg/kg, oral), Group
3 -17 received each of the test compounds 8a-8o respectively (50 mg/kg, oral), which were
prepared by suspending in 0.5% sodium carboxymethylcellulose. One hour after the
administration of vehicle, phenytoin or test compounds, maximal electric shock of 150mA
current for 0.2 sec was applied through corneal electrodes to induce convulsions using an
electroconvulsiometer (INCO, Ambala, India) and duration of hind limb tonic extension was
noted. Abolition or reduction in the duration of tonic extension was considered as the index
for anticonvulsant activity [26].
5.2.3.2. The Subcutaneous Pentylenetetrazole Seizure test (Sc PTZ)
This method utilizes a dose of pentylenetetrazole (PTZ), 80mg/kg subcutaneously in
rats that produces clonic seizures. The rats were divided into 17 groups of six rats each.
Group 1 animals were kept as control and were received vehicle; Group 2 received diazepam
(5 mg/kg, intraperitoneally), Group 3 -17 received the test compounds 8a-8o respectively

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(50mg/kg, oral), which were prepared by suspending in 0.5% sodium
carboxymethylcellulose. One hour after administration of vehicle, diazepam or test
compounds 8a-8o, PTZ (80mg/kg) was injected subcutaneously. The time of onset of clonic
convulsions and the protection against mortality were observed [27,28]. The maximum
latency time used in the study was fixed as 300sec. Further, compound 8f was subjected to
the dose-dependent activity in both PTZ and MES models at 25, 50 and 100 mg/kg doses.
5.2.4. Evaluation of antioxidant activity
5.2.4.1. Assay for scavenging of DPPH free radicals
The ability to scavenge 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) stable free radical
was determined by using DPPH method. In this method, 1 ml of test compound (10, 50, 100,
250, 500 µg/ml) in ethanol was added to 3.9 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 test compounds was calculated [29].
5.3. Antinociceptive activity
Capsaicin-induced nociceptive assay
Male Swiss mice (18-22g) were used for the method. Following the adaptation to the
experimental conditions, 20µl of capsaicin (1nmol/paw) was injected intraplantarly in to the
right hind paw, and the total number of flinches 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 8a, 8b, 8f and 8o using oral gavages (50mg/kg in a dose
volume of 0.5% sodium carboxymethylcellulose) 1 h prior to capsaicin injection [30].
5.4. Statistical Analysis
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.
5.5. Calculation of drug-likeness and ADME properties
The molecular properties like TPSA, cLogP, number of rotatable bonds and violations of
Lipinski’s rule-of-five were calculated using Molinspiration online property calculator tool kit

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[31]. Topological polar surface area was used to calculate the percentage of absorption
(%ABS) according to the equation: %ABS = 109 – [0.345× TPSA] [23]. In vitro plasma
protein binding values were obtained from ADME calculator [32].
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Table(s)
ACCEPTED MANUSCRIPT
Table 1: Anticonvulsant activity of dialkyl 4-[benzo [d][1,3]-dioxol)-6-yl]-1, 4-dihydro-2,
6-dimethyl-1-substitutedpyridine-3,5-dicarboxylates (8a-8o) at 50mg/kg body weight.
compounds
scPTZ a
Protection
MES a
against Limb extension (s) % protection
mean±SEM
Latency period (s)
mean±SEM
200±33***
200±12***
174±20**
138±13ns
mortality (%)
0
15±1**
12±1***
14±1**
18±1ns
27
8a
0
41.2
33.3
11.1
07.9
59.0
46.0
60.3
49.2
46.0
28.5
41.2
44.5
28.5
31.7
-------
69.8
66.6
-----
-----
8b
0
8c
0
8d
93±7ns
0
19±2ns
8e
>300***
100
66.6
66.6
33.3
66.6
33.3
0
8±1***
11±1***
8±1***
10±1***
11±1***
15±1**
12±1***
11±1***
15±**
8f
275±9***
293±4***
250±17***
237±28***
233±21***
221±25***
251±16***
136±14ns
8g
8h
8i
8j
8k
8l
33.3
0
8m
8n
225±10***
262±13***
--------
33.3
33.3
---------
33.3
-----
-----
14±1**
--------
8o
diazepam
phenytoin
nifedipine
control
Ascorbic
acid
6±1***
7±1***
21±1
215±3***
83±4
-----
------
a The test compounds were administered 1h before the injection of PTZ (80 mg/kg, sc), MES (150 m.A, 0.2 sec), control: 0.5% sodium
carboxymethylcellulose. Reference stanadard: diazepam (5mg/kg, ip) and phenytoin (30mg/kg, oral). Latency time was observed for 300sec
in scPTZ. Values are expressed as mean±SEM, n=6. One way analysis of variance (ANOVA) followed by Dunnett’s method.*** p<0.0001
vs control, ** p<0.05 vs control, ns p>0.05 vs control.
#Isradipine was reported to show limb extension period 9±1 (mean±SEM) [8].

ACCEPTED MANUSCRIPT
Table 2: Anticonvulsant activity of diethyl 4-[benzo [d][1,3]-dioxol)-6-yl]-1, 4-dihydro-
2, 6-dimethyl-1-substitutedpyridine-3,5-dicarboxylate (8f) at 25 and 100mg/kg body
weight.
compound
dose
scPTZ induced model b
Latency period
MES method b
mg/kg
%
Protection Limb extension %
Mean±SEM(s) against mortality Mean±SEM(s) protection
Control
83±3.8
262±13***
-----
194±3***
>300***
>300***
---
25±1
---
Diazepam
5
66.6
-----
100
100
0
------
----
Phenytoin
30
25
50
100
6±1***
14±1***
8±1***
7±1***
74.6
41.6
65.6
72.0
8f
8f
8f
b The test compounds were administered 1h before application of MES (150 m.A, 0.2 sec)/ the injection of PTZ (80mg/kg, sc), control:
0.5% sodium carboxymethylcellulose. Reference standard: diazepam (5mg/kg, ip) / phenytoin (30mg/kg, oral). Values are expressed as
mean ± SEM, n=6. One way analysis of variance (ANOVA) followed by Dunnett’s method.
*** p < 0.0001 vs control.

ACCEPTED MANUSCRIPT
Table 3: Antioxidant activity of dialkyl 4-[benzo[d][1,3]-dioxol)-6-yl]-1, 4-dihydro-2, 6-
dimethyl-1-substitutedpyridine-3,5-dicarboxylates (8a-8o) in DPPH free radical
scavenging activity
O
O
R1OOC
COOR
CH3
N
CH3
Ar
compound
R
R1
Ar
IC50(µg/ml)c
604
CH3
CH3
CH3
CH3
CH3
CH3
CH3 C6H5
CH3 C6H4-2-NO2 2354
CH3 C6H4-3-NO2 521
CH3 C6H4-4-NO2 1289
H
8a
8b
8c
8d
542
8e
C2H5 C2H5
C2H5 C2H5 C6H5
H
375
443
8f
8g
C2H5 C2H5 C6H4-2-NO2 874
C2H5 C2H5 C6H4-3-NO2 363
C2H5 C2H5 C6H4-4-NO2 597
8h
8i
8j
CH3
CH3
CH3
CH3
CH3
---
C2H5
C2H5 C6H5
C2H5 C6H4-2-NO2 933
C2H5 C6H4-3-NO2 386
C2H5 C6H4-4-NO2 854
H
467
740
8k
8l
8m
8n
8o
----
----
32
Ascorbic acid
c Reduction of DPPH free radical by the test compounds at various concentrations was expressed as IC value, which was estimated in
50
ethanol solution, absorbance was measured at 517 nm.

ACCEPTED MANUSCRIPT
Table: 4: Antinociceptive activity of dialkyl 4-[benzo[d][1,3]-dioxol)-6-yl]-1, 4-dihydro-
2, 6-dimethyl-1-substitutedpyridine-3,5-dicarboxylates (8a, 8b, 8f and 8o) in capsaicin
induced flinching assay at 10mg/kg
Compound
Number of flinches
(Mean ± SEM)
12±2
% of activity
control
8a
------
21
0
9.5±1ns
12±1ns
11±1ns
11±1ns
8b
8f
8
8o
4
The test compounds were administered 1h before the injection of capsaicin (20µl, 1nmol/paw), control: 0.5% sodium
carboxymethylcellulose. Values are expressed as mean ± SEM, n=6. One way analysis of variance (ANOVA) followed by Dunnett’s
method.

ACCEPTED MANUSCRIPT
Table 5: Structural and pharmacokinetic properties of the 1,4-dihydropyridine
derivatives 8a-8o.
compoun %ABS M.W cLogP nrotb HBA HBD volume MR
d
TPSA Viola- iPPB
tions
≤1
0
Rule
8a
8b
8c
8d
8e
8f
-----
80.3
83.3
67.5
67.5
67.5
80.3
83.3
67.5
67.5
67.5
80.3
83.3
67.5
67.5
67.5
<500 ≤5
---
5
6
7
7
7
7
8
9
9
9
6
7
8
8
8
5
6
<10 <5
-----
40-130 ----
83.10
----
345.3 3.524
421.4 5.466
466.4 5.377
466.4 5.401
466.4 5.425
373.4 4.276
449.5 6.218
494.5 6.129
494.5 6.153
494.5 6.177
359.3 3.90
435.4 5.842
480.4 5.753
480.4 5.777
480.4 5.801
343.3 3.571
346.3 3.072
7
1
0
0
0
0
1
0
0
0
0
1
0
0
0
0
1
1
303.37 87.87
61.0
91.1
93.4
93.4
92.3
69.5
91.1
92.4
92.2
91.4
65.1
91.1
92.8
92.4
91.8
72.8
41.4
7
375.16 113.65 74.31
398.49 119.80 120.1
398.49 119.80 120.1
398.49 119.80 120.1
1
10
10
10
7
1
1
1
336.97 97.10
83.1
0
8g
8h
8i
7
408.76 122.89 74.3
432.10 129.03 120.1
432.10 129.03 120.1
432.10 129.03 120.1
1
10
10
10
7
1
1
8j
1
8k
8l
320.17 92.49
83.1
0
7
391.96 118.27 74.3
1
8m
8n
8o
10
10
10
8
415.30 124.4
415.30 124.4
415.30 124.4
296.69 87.11
302.78 87.89
120.1
120.1
120.1
103.5
110.4
1
1
1
isradipine 73.2
nifedipine 70.9
0
8
0
cLogP- calculated partition co-efficient, nrotb- number of rotatable bond, HBA-number of hydrogen bond acceptors, HBD-number of
hydrogen bond acceptors, MR-molar refractivity, TPSA-molecular polar surface area, violations- number of violations from Lipinski rule-
of-five, iPPB- in vitro plasma protein binding (%), %ABS- percentage absorption.

Figure(s)
ACCEPTED MANUSCRIPT
Figure 1. Lead structures for target compounds 8a-8n.
N
N
O
NO2
Cl
H3COOC
COOCH3
H3COOC
COOCH2CH3
(H3C)2HCOOC
COOCH3
H3C
N
H
CH3
H3C
N
H
CH2OCH2CH2NH2
H3C
N
H
CH3
Nifedipine
Amlodipine
Isradipine
Figure 2. Structures of 1,4-dihydropyridine derived pharmacologically active drug
candidates
H3COOC
COOCH3
H3CH2COOC
COOCH2CH3
H3C
N
H
CH3
H3C
N
H
CH3
(1)
(2)
H3CH2COOC
COOCH2CH3
H3COOC
COOCH3
H3C
N
H
CH3
H3C
N
H
CH3
(4)
(3)

ACCEPTED MANUSCRIPT
Scheme 1. Synthetic protocol of the symmetric dialkyl 4-{(benzo (d)[1,3]dioxo)-6-yl}-1,4-
dihydro-2,6-dimethyl-1-(substituted)pyridine-3,5-dicarboxylates (8a-8j)
O
O
O
COOR
O
CH2
C
Ar-NH2 (7a-e)
+
H3C
O
reflux
8-24 hrs
ROOC
CH3
COOR
CH3
(5)R=CH3
(6)R=C2H5
CHO
N
Ar
(8a-j)
R
Ar
8a
8b
8c
8d
8e
8f
CH3
H
CH3
C6H5
CH3
C6H4-2-NO2
C6H4-3-NO2
C6H4-4-NO2
H
CH3
CH3
C2H5
C2H5
C2H5
C2H5
C2H5
8g
8h
8i
C6H5
C6H4-2-NO2
C6H4-3-NO2
C6H4-4-NO2
8j