Bioorganic Chemistry 71 (2017) 135–145

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Bioorganic Chemistry

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Synthesis, molecular modeling studies and anticonvulsant activity ofcertain (1-(benzyl (aryl) amino) cyclohexyl) methyl esters

http://dx.doi.org/10.1016/j.bioorg.2017.01.0210045-2068/� 2017 Elsevier Inc. All rights reserved.

⇑ Corresponding author.E-mail address: mnaboulenein@yahoo.com (M.N. Aboul-Enein).

Walaa Hamada Abd-Allah a, Mona Elsayed Aboutabl b, Mohamed Nabil Aboul-Enein c,⇑,Aida Abdel Sattar El-Azzouny c

aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Misr University for Science & Technology, 6th of October City, EgyptbMedicinal and Pharmaceutical Chemistry Department, Pharmacology Group, Pharmaceutical and Drug Industries Research Division, National Research Centre (ID: 60014618), 33El Bohouth St., P.O. 12622, Dokki, Giza, EgyptcMedicinal and Pharmaceutical Chemistry Department, Medicinal Chemistry Group, Pharmaceutical and Drug Industries Research Division, National Research Centre (ID:60014618), 33 El Bohouth St., P.O. 12622, Dokki, Giza, Egypt

a r t i c l e i n f o

Article history:Received 4 November 2016Revised 22 December 2016Accepted 29 January 2017Available online 27 February 2017

Keywords:Anticonvulsants1-(Benzyl (aryl) amino) cyclohexyl) methylestersMolecular modelingEpilepsy

a b s t r a c t

A series of (1-(benzyl (aryl) amino) cyclohexyl) methyl esters 7a-n were prepared and screened for theiranticonvulsant profile. Screening of these esters 7a-n and their starting alcohols 6a and 6b revealed thatcompound 7kwas the most potent one in the scPTZ screening test with an ED50 value of 0.0056 mmol/kgbeing about 10- and 164-fold more potent than phenobarbital (ED50 = 0.056 mmol/kg) and ethosuximide(ED50 = 0.92 mmol/kg) as reference drugs, respectively. Meanwhile, in the MES test, compounds 7b and7k at doses 0.0821 mmol/kg and 0.0334 mmol/kg, exerted 66% and 50% protection of the tested mice,respectively, compared with diphenylhydantoin, which exerted 100% protection at dose 0.16 mmol/kg.In the neurotoxicity screen test, almost all esters 7a-n did not show any minimal motor impairment atthe maximum administrated dose. The anticonvulsant effectiveness of esters 7a-n was higher than theircorresponding alcohols 6a and 6b. Compounds 7b and 7k exhibited pronounced anticonvulsant activitydevoid of neurotoxicity in minimal motor impairment test and hepatotoxicity in the serum enzyme activ-ity assay. 3D pharmacophore model using Discovery Studio 2.5 programs showed high fit value. Theobtained experimental results of sc-PTZ activity of compounds 7a-n was consistent with the molecularmodeling study.

� 2017 Elsevier Inc. All rights reserved.

1. Introduction

Epilepsy is a common chronic neurological disorder character-ized by recurrent unprovoked seizures [1]. These seizures repre-sent transient signs and/or symptoms of abnormal excessive orsynchronous neuronal activity in the brain [2]. Despite the exis-tence of an array of novel antiepileptic drugs (AEDs), however theycontrol the seizures in only 50% of patients or decreases the inci-dence in only 75% of patients [3,4]. Moreover, they are accompa-nied with drastic untoward effects like ataxia, hepatotoxicity, andmegaloblastic anemia [5,6]. Accordingly, search for new and effi-cient antiepileptics with lower toxicity still a paramount demand.

It was previously disclosed that several geminally disubstitutedcyclohexane derivatives elicit anticonvulsant profile such as Gaba-pentin [7]. Also, Aboul-Enein et al. [8,9] have described and studiedthe synthesis and the anticonvulsant potential of N-benzyl-N-(1-

((pyrrolidin-1-yl) methyl) cyclohexyl) benzenamine (Ia), N-benzyl-N-(1-((piperidin-1-yl) methyl) cyclohexyl) benzenamine(Ib), N-(4-methoxybenzyl)-N-(1-((piperidin-1-yl) methyl) cyclo-hexyl) benzenamine (Ic), N-(4-chlorobenzyl)-N-(1((cyclohexylamino)methyl) cyclohexyl) benzenamine (IIa), N-(1-((cyclohexylamino)methyl)cyclohexyl)-N-(4-nitrobenzyl)benzenamine (IIb)and N-(1-((cyclohexylamino) methyl) cyclohexyl)-N-(3,4,5-trimethoxybenzyl) benzenamine (IIc), which were found to exertremarkable activity (Fig. 1).

As a contribution to this field and in continuation to our previ-ous work on the search for new candidates as anticonvulsants [10–18], herein is reported the synthesis of certain (1-(benzyl (aryl)amino) cyclohexyl) methyl ester derivatives 7a-n, where a designmodification of compounds I and II was considered in which theamino moiety [A] linked to the methylene cyclohexyl scaffold isreplaced by either aliphatic or aromatic ester side chain Fig. 1.Moreover the penultimate alcohols 6a and 6b were screened fortheir anticonvulsant effect for the purpose of comparison withtheir corresponding target esters 7a-n.

136 W.H. Abd-Allah et al. / Bioorganic Chemistry 71 (2017) 135–145

2. Results and discussion

2.1. Chemistry

Synthesis of the target compounds 7a-n was illustrated inScheme 1. Thus, cyclohexanone underwent Strecker synthesis con-ditions with the appropriate aniline and potassium cyanide in gla-cial acetic acid to give the respective nitrile 1a and 1b. The nitrilegroup was subjected to partial hydrolysis using sulfuric acid atambient temperature to yield the amide derivatives 2a and 2b.Subsequent complete hydrolysis of the amide group was success-fully achieved using concentrated HCl to furnish the correspondingacids 3a and 3b. The target compounds 7a-n as well as their inter-mediates 4a-b to 6a-b were obtained as portrayed in Scheme 1.Benzoylation of the secondary amine moiety in compounds 3aand 3b was achieved using benzoyl chloride and triethylamine togive compounds 4a and 4b followed by esterification yielded thecorresponding methyl esters 5a and 5b. Reduction of the latterusing LiAlH4 in dry THF gave the respective penultimate alcohols6a and 6b [8,9]. The target aliphatic esters 7a, 7b, 7h and 7i wereobtained by acylation of alcohols 6a and 6b with the appropriateanhydrides, while the aromatic esters 7c-g and 7j-n were achievedthrough benzoylation of 6a and 6b using the appropriate benzoylchlorides.

2.2. Anticonvulsant activity

Adopting predictable animal models has been crucial for thediscovery of new bioactive entities for the treatment of epilepsy.The Epilepsy section of the National Institute of Neurological Disor-ders and Stroke (NINIDS) has assigned a protocol adopted as a stan-

Fig. 1. Structural similarities of gabapentin, certain antico

dard procedure by the Antiepileptic Drug Development (ADD)program named ‘‘the gold standard screen” [19]. The adoptedscreening procedure includes three in vivo animal models: (i) sub-cutaneous pentylenetetrazole (scPTZ) (ii) maximal electric shock(MES), and (iii) neurotoxicity screening tests. The first scPTZ testdistinguishes compounds that elevate seizure threshold whileMES screen evaluates the ability to prevent seizure spread. In addi-tion, the neurotoxicity test identifies the minimal motorimpairment.

The initial anticonvulsant activity (Phase I screening) of the syn-thesized esters 7a–n and their alcohols 6a and 6b expressed as per-centage (%) protection as well as their neurotoxicity werepresented in Table 1 using phenobarbital, ethosuximide anddiphenylhydantoin as reference drugs. The reported data showedthat all screened compounds at dose level from 0.0334 to0.1333 mmol/kg were effective in scPTZ screen while several estersof series 7a-n were active in different ratios in the MES screen testat the same dose levels. Regarding the scPTZ screen, in the (1-(benzyl (phenyl) amino) cyclohexyl) methyl ester series (7a-g)where R1 = H, compounds 7a (R2 = CH3, ED50 = 0.0602 mmol/kg),7b (R2 = CH3CH2CH2CH2; ED50 = 0.0236 mmol/kg), 7d (R2 = 4-Cl-C6H4; ED50 = 0.0099 mmol/kg), 7e (R2 = 4-CH3-C6H4;ED50 = 0.0410 mmol/kg), 7f (R2 = 4-NO2-C6H4; ED50 = 0.0213 -mmol/kg) and 7g (R2 = 4-NH2-C6H4; ED50 = 0.0566 mmol/kg)exhibited 100% protection against scPTZ-induced seizures at doselevels of 0.045–0.1333 mmol/kg, while compound 7c (R2 = C6H5;ED50 = 0.0277 mmol/kg) showed 83.3% at dose level of0.0751 mmol/kg. The chloro-substituent 7d in the 4-position ofthe aromatic ring linked to the ester moiety was the most activecongener among 7a-g series. This compound exhibited 5.7-foldmore anticonvulsant potency than phenobarbital as well as 93-

nvulsants and the designed target compounds 7a-n.

Scheme 1. Adopted procedures for the synthesis of compounds 7a-n. Reagents and conditions: (i) conc. H2SO4, r.t, 48 h; (ii) HCl, r.t, 24 h; (iii) benzoyl chloride, TEA, reflux,12 h (iv) methanol, p-toluene sulfonic acid, reflux, 24 h; (v) LiAlH4, THF, 6 h, r.t then reflux, 3 h; (vi) appropriate anhydride, r.t, 24 h, or appropriate acid chloride, reflux, 12 h.

W.H. Abd-Allah et al. / Bioorganic Chemistry 71 (2017) 135–145 137

fold more potent than ethosuximide. The different congeners ofseries 7a-g were arranged in the following decreasing order:7d > 7f > 7b > 7c > 7e > 7g > 7a.

Furthermore, the (1-(benzyl (p-tolyl) amino) cyclohexyl)methyl ester series 7h-n where R1 = CH3, compounds 7h(R2 = CH3; ED50 = 0.0718 mmol/kg), 7i (R2 = CH3CH2CH2CH2;ED50 = 0.01117 mmol/kg), 7j (R2 = C6H5; ED50 = 0.0205 mmol/kg),7k (R2 = 4-Cl-C6H4; ED50 = 0.0056 mmol/kg), 7l (R2 = 4-CH3-C6H4;ED50 = 0.0195 mmol/kg) and 7m (R2 = 4-NO2-C6H4; ED50 = 0.0176 -mmol/kg) and 7n (R2 = 4-NH2-C6H4; ED50 = 0.0534 mmol/kg)exhibited antiseizure profile (100% protection) against scPTZ-induced seizures at dose levels of 0.0334–0.1280 mmol/kg. The4-chloro phenyl ester 7k displayed marked anticonvulsant action(ED50 = 0.0056 mmol/kg) in this screening test. It showed 100%anticonvulsant protection at dose 0.0334mmole/kg, and exhibitedanticonvulsant potency 10- and 164-fold more than phenobarbitaland ethosuximide, respectively. The different congeners of (1-(benzyl (p-tolyl) amino) cyclohexyl) methyl esters 7h-n seriesshowed a decrease in the anticonvulsant activity in the followingorder: 7k > 7i > 7m > 7l > 7j > 7n > 7h.

It could be revealed from the scPTZ screening results of bothseries 7a-g and 7h-n that the chloro (7k, 7d) and the nitro (7m,7f) substituents at the 4-position of the aromatic ring linked to

the ester side chain enhanced the anticonvulsant effect. In addi-tion, elongation of the aliphatic ester side chain (7i and 7b) poten-tiated the anticonvulsant effect compared with theircorresponding esters (7h and 7a), respectively. In addition, alco-hols 6a (R1 = H) and 6b (R1 = CH3) displayed similar anticonvulsantprotection (83.3%) against scPTZ-induced seizures at 0.1523 and0.1454 mmol/kg dose levels with ED50 = 0.0823 and0.0810 mmol/kg, respectively. Therefore, it could be concludedthat the introduction of the ester scaffold in 7a-n potentiated theanticonvulsant effect compared with their corresponding alcohols6a and 6b. All screened compounds against scPTZ-induced seizuresshowed anticonvulsant activity in the following decreasing order:7k > 7d > 7i > 7m > 7l > 7j > 7f > 7b > 7c > 7e > 7n > 7g > 7a > 7h >6a > 6b.

The dose which gave 100% anticonvulsant protection in thescPTZ screening has been selected for MES test. In the ester series7a-g, compound 7b (R2 = CH3CH2CH2CH2) at dose level of0.0821 mmol/kg, showed 66% protection of the tested mice after0.5 h post administration compared with the reference drugdiphenylhydantoin, which exerted 100% protection at dose0.1600 mmol/kg. Moreover, compounds 7f (R2 = 4-NO2-C6H4) and7a (R2 = CH3) at doses 0.0450 mmol/kg and 0.1333 mmol/kg,respectively showed similar anticonvulsant protection of 50%. On

Table 2Liver enzyme, albumin and total protein determination of the most active compounds 7b and 7 k.

Compound ALTa (U/L) ASTb (U/L) ALPc (U/L) Albumin (g/dL) Total protein (g/dL)

Control 22.33 ± 1.49 78.60 ± 10.79 12.50 ± 1.09 2.49 ± 0.17 7.90 ± 0.317b 21.2 ± 1.59 76.00 ± 7.74 11.33 ± 0.80 2.50 ± 0.23 8.31 ± 0.357k 20.00 ± 1.83 76.28 ± 7.15 10.63 ± 0.73 2.51 ± 0.27 8.46 ± 0.32

Data are represented as mean ± SEM and were analyzed by ANOVA followed by Student Newman Keul test n = 8.a Alanine amino transferase.b Aspartate amino transferase.c Alkaline phosphatease.

Table 1Anticonvulsant activity and neurotoxicity of compounds 7a-n and 6a-b.

Compd No. Dosea Max. %protection

Neuro-toxicityb ED50 mg/kg (confidence limits) ED50mmol/kg (confidence limits) Fit value

mg/kg mmol/kg scPTZ MES

7a 45 0.1333 100 50 0/6 20.32 (23.6–17.46) 0.0602 (0.070–0.0517) 2.717b 30 0.0821 100 66 0/6 8.617 (9.140–8.123) 0.0236 (0.0250–0.0222) 2.837c 30 0.0751 83 nd 0/6 11.06 (11.94–10.25) 0.0277 (0.0299–0.0257) 2.797d 20 0.0462 100 16 0/6 4.27 (5.090–3.582) 0.0099 (0.0118–0.0083) 2.987e 45 0.1088 100 16 1/6 16.82 (18.42–15.36) 0.041 (0.0449–0.0374) 2.827f 20 0.0450 100 50 0/6 9.462 (11.15–8.027) 0.0213 (0.0251–0.0181) 2.957g 45 0.1085 100 16 0/6 23.48 (28.56–19.31) 0.0566 (0.0688–0.0466) 2.657h 45 0.1280 100 33 0/6 25.22 (31.56–20.15) 0.0718 (0.0898–0.0574) 2.627i 20 0.0527 100 20* 0/6 4.428 (5.467–3.586) 0.0117 (0.0144–0.0095) 2.977j 45 0.1088 100 16 0/6 8.495 (9.787–7.374) 0.0205 (0.0236–0.0178) 2.797k 15 0.0334 100 50 0/6 2.505 (3.439–1.825) 0.0056 (0.0077–0.0041) 2.997l 45 0.1053 100 50 0/6 8.327 (10.69–6.489) 0.0195 (0.0250–0.0152) 2.817m 20 0.0437 100 16 0/6 8.063 (10.19–6.380) 0.0176 (0.0222–0.0139) 2.887n 45 0.1050 100 16 0/6 22.87 (17.36–30.13) 0.0534 (0.0704–0.0405) 2.536a 45 0.1523 83 nd 0/6 24.32 (29.00–20.40) 0.0823 (0.0982–0.0690) 2.716b 45 0.1454 83 nd 2/6c 25.08 (30.05–20.90) 0.0810 (0.0971–0.0676) 2.83

a The minimal dose which exhibited the maximum anticonvulsant activity.b Rotarod test: Number of animals exhibiting neurotoxicity (falling of the rotarod)/number of animals tested. *n = 5.Control group demonstrated 0/6 in the neurotoxicity

test and were devoid of anticonvulsant activity.Reference standards: Phenobarbital [ED50 = 13.2 (15.90–6.80) mg/kg or 0.056 (0.068–0.029) mmol/kg] at dose 30 mg/kg(0.1300 mmol/kg) displayed 100% max. protection in scPTZ test. Ethosuximide [ED50 = 130 (150–111) mg/kg or 0.92 (1.06–0.78) mmol/kg] at dose 150 mg/kg (1.06 mmol/kg)displayed 100% max. protection in scPTZ test. Diphenylhydantoin at dose 45 mg/kg (0.1600 mmol/kg) showed the 100% max. protection in MES test [17].

c This value of compound 6b is equivalent to 66.7% neuroprotection when compared to the reference standards Phenobarbital and Diphenylhydantoin at dose 100 mg/kgwhich is the minimum dose that displayed bioactivity in half or more of the number of mice [25–27].

138 W.H. Abd-Allah et al. / Bioorganic Chemistry 71 (2017) 135–145

the other hand compounds 7d (R2 = 4-Cl-C6H4), 7e (R2 = 4-CH3-C6H4) and 7g (R2 = 4-NH2-C6H4) exhibited the lowest anticonvul-sant effect (16%) observed in this series at dose range from0.0462 to 0.1088 mmol/kg. Meanwhile, in the ester series 7h-7n,comparable protection was observed for compounds 7k (R2 = 4-Cl-C6H4) and 7l (R2 = 4-CH3-C6H4) at dose levels of 0.0334 mmol/kg and 0.1053 mmol/kg, respectively in the tested mice (50%).Other congeners of this series 7h (R2 = CH3), 7i (R2 = CH3CH2CH2-CH2), 7m (R2 = 4-NO2-C6H4), 7n (R2 = 4-NH2-C6H4) and 7j(R2 = C6H5) displayed protection in less than half (33%-16%) ofthe tested animals at dose levels range from 0.0437 mmol/kg to0.1280 mmol/kg. It was worth to mention that, almost all the com-pounds in the neurotoxicity test did not show any minimal motorimpairment at the maximum administrated dose except ester 7eand alcohol 6b (Table 1). Regarding the relation between the anti-convulsant effect of the 4-methyl substituted anilino analogues7h-n and their unsubstituted congeners 7a-g, the data of bothscPTZ-induced seizures and MES tests revealed that, it has morethan a little anticonvulsant influence. It has been shown thatscPTZ-induced seizures could be prevented by drugs that reduceT-type Ca2+ currents such as ethosuximide and also by those whichenhance gamma amino butyric acid type A (GABAA) receptor-mediated inhibitory neurotransmission such as phenobarbital[20–24]. Interestingly, all the screened compounds 7a-n werefound to control the seizures induced by scPTZ, it could be con-cluded that these compounds possibly exhibit their anticonvulsantpotential through their effect on Ca2+ channels and/or GABAactivation.

Liver toxicity could be considered from the serious side effectsof the marketed antiepileptic drugs. The most active compounds7k and 7b were chosen to be assessed for serum enzyme activityassay. Elevated liver enzymes are considered as specific markersof liver dysfunction. Therefore, assessment of liver enzymes levelwas carried out. The data of the analysis of hepatozymes ALT,AST and ALP, as well as albumin and total protein were illustratedin Table 2. The serum levels of ALT, AST, ALP, albumin and totalprotein were determined and represented as mean ± SEM. Com-pounds 7k and 7b did not exhibit any significant change inenzymes level, albumin and total protein as compared to controlwhich indicated no signs of liver toxicity and ensure that thesecompounds were devoid of hepatotoxicity.

2.3. Molecular modeling study

This study was performed in order to gain further evidence forthe anticonvulsant activity of compounds 7a-n. The generation ofan anticonvulsant hypothesis was performed by running the com-mon feature pharmacophore model protocol in Discovery Studio2.5 software (Accelerys, Inc.: San Diego, CA.).

The fundamental structural features of antieplieptic drugs andseveral anticonvulsant compounds involve the prescence of hydro-gen bond acceptor (HBA), ring aromatic (RA) and hydrophobic moi-ety (HY) moiety. The training sets used to build the pharmacophoreand contain the essential structural features were illustrated inFig. 2 and the generated hypothetical model in Fig. 3. These fea-tures could be responsible for an interaction with the active site

III IV V ED50 = 0.49[31] ED50 = 0.15[28] ED50 = 0.071[17]

VII

VI VII VIII ED50 = 0.012[17] ED50 = 0.45 [32] ED50 = 0.019[17]

IX X ED50 = 0.25 [29] ED50 = 0.23[33]

XI XII Carbamazipine ED50 = 0.19 [32] ED50>0.42 [31]

Fig. 2. Representative examples of anticonvulsants (training sets). ED50 = 0.49[31] ED50 = 0.15[28] ED50 = 0.071[17]; ED50 = 0.012[17] ED50 = 0.45 [32] ED50 = 0.019[17];ED50 = 0.25 [29] ED50 = 0.23[33]; ED50 = 0.19 [32] ED50 > 0.42 [31].

W.H. Abd-Allah et al. / Bioorganic Chemistry 71 (2017) 135–145 139

with the specific receptor. Table 3 showed the distances and anglesbetween structural features HBA, RA and HY for the anticonvul-sants (training sets) containing the fundamental structural featuresof antiepileptic drugs [28–30].

2.3.1. Pharmacophore generation involves

1. Selection of the training set compounds (Fig. 2) which containthe essential pharmacophoric features as mentioned.

2. Conformational generation to ensure conformational flexibilityof ligands of training sets.

3. Alignment of the training sets and determining the essentialcommon features to generate the model.

4. 4-Application of forcefield CHARMm, then energyminimization.

5. Application of common feature pharmacophore in order toselect the features (HBA, RA, HY), then press the knob ‘‘run”to generate the hypothesis. The programme will give tenhypotheses and the hypothesis ranked number 7 was chosenas it involves the required features).

2.3.2. Validation of the generated pharmacophoreTen hypotheses were generated and the one ranked number 7

was chosen as the valid ideal hypothesis based on the following:(a) the hypothesis showed full mapping of all its features withoutany steric clashes together with high fit values with the trainingsets (compounds III-XI, e.g., Fig. 4), (b) retrospectively, the simu-lated fit values of test set compounds (7a-n) with the hereabovegiven hypothesis were more consistent with the experimentalresults than other hypotheses, (c) the database search study for

Table 3The constraint distances and angles between the features of the generated anticon-vulsant pharmacophore model.

Dimensions Features of the given anticonvulsantmodel

Constraint distances (Å) betweenfeatures

HBA-RA: 5.39HY-RA: 8.40HY-HBA: 6.43

Constraint angles between features HBA-RA vector: 46.06RA-HBA vector: 116.60HY-RA-HBA: 5.11HY-HBA vector: 140.04HY-RA vector: 119.47

Fig. 3. Pharmacophore model of anticonvulsants generated by discovery studio 2.5.Pharmacophoric features are coloured coded: green for hydrogen bond acceptor(HBA), orange for aromatic region and light blue for hydrophobic region (HY).

140 W.H. Abd-Allah et al. / Bioorganic Chemistry 71 (2017) 135–145

examining the affinity of such hypothesis with the molecularstructures of MiniMaybridge databases revealed that only 15 hitshave been reached from the databases (2000 compounds) [34].Such a low number of the recognized database molecules by suchpostulated hypothesis may add an advantage and selectivity to ourhypothesis, which involved three main features namely; hydrogenbonding acceptor (HBA), ring aromatic (RA) and one hydrophobicfeature (HY) (see Fig. 5).

Fig. 4. (A and B) Constraint distances and angles for pharmacophoric model of the traingreen, and orange and light blue represent hydrogen bonding acceptor (HBA), ring arom

3. Conclusion

The anticonvulsant activity of (1-(benzyl (aryl phenyl) amino)cyclohexyl) methyl esters 7a-n and their starting alcohols 6a and6bwas discussed. The target esters 7a-n exhibited 100% protectionagainst scPTZ-induced seizures at dose levels from 0.0334 mmol/kgto 0.1333 mmol/kg, compared with their corresponding startingalcohols 6a and 6b which demonstrated 83.3% protection at doses0.1523 and 0.1454 mmol/kg, respectively. The 4-chloro phenylderivative 7k displayed the highest anticonvulsant potential(ED50 = 0.0056 mmol/kg) in the scPTZ screening test. It showed100% protection against induced seizures at dose 0.0334mmole/kg. It exhibited 10- and 164-fold more potency than that of pheno-barbital and ethosuximide, respectively. Accordingly, the introduc-tion of the ester scaffolds in 7a-n potentiated the anticonvulsanteffect compared with their respective alcohols 6a and 6b andmightbe considered as prodrugs due to their increased lipophilicity.

In the MES test, compounds 7b and 7k at doses 0.0821 mmol/kgand 0.0334 mmol/kg, exerted 66% and 50% protection of the testedmice, respectively, compared with diphenylhydantoin, whichexerted100%protection at dose 0.16 mmol/kg. Other screened com-pounds exhibited from 50% to 16% protection at doses range from0.1333 mmol/kg to 0.0437 mmol/kg. Interestingly, compounds 7band 7k which provided the most protection in MES and scPTz testsrespectively were devoid of any liver toxicity. It is worth tomentionthat, in the neurotoxicity screen test almost all esters 7a-n did notshow any minimal motor impairment at the maximum adminis-trated dose. Conclusively, esters 7k and 7b could be considered aspromising anticonvulsant leads. Additionally, molecular simulationstudy including fitting to anticonvulsant 3D-pharmacophoremodelusing Discovery Studio 2.5 programs showed high-fit values. Thegenerated pharmacophore model can be used to the in vitro predic-tion of the anticonvulsant activity of new chemical candidates con-taining the previously mentioned features.

4. Experimental section

4.1. Chemistry

All melting points were determined using Electrothermal Capil-lary melting point apparatus. Infrared (IR) spectra were recordedas thin film (for oils) in NaCl discs or as KBr pellets (for solids) withJASCO FT/IR-6100 spectrometer and values are represented in cm�1.1HNMR(500 MHz)and 13CNMR(125 MHz) spectrawere carriedouton Jeol ECA 500 MHz spectrometer using TMS as internal standardand chemical shift values were recorded in ppm on d scale. The 1H

ing sets (III-XII), test sets (7a-n) and phenobarbital. The chemical features colouredatic (RA) and hydrophobic features (HY), respectively.

IIVV

k7d7

Phenobarbital, fit value = 2.45

Fig. 5. Mapping of anticonvulsants pharmacophore fragments of the leads V, VII, test set compounds 7d, 7 k and Phenobarbital, respectively.

W.H. Abd-Allah et al. / Bioorganic Chemistry 71 (2017) 135–145 141

NMR datawere represented as follows: chemical shifts, multiplicity(s. singlet, d. doublet, m. multiplet, br. broad), number of protons,and type of protons. The 13CNMRdatawere represented as chemicalshifts and type of carbons. Mass spectral data were obtained withelectron impact (EI) ionization technique at 70 eV from a FinniganMat SSQ-7000 Spectrometer. Elemental analyses were carried outin Microanalytical Units at National Research Centre and CairoUniversity and the result werewithin ±0.4% of the theoretical value.Purification was performed using column chromatography and sol-vent system petroleum ether (40–60): ethylacetate (1:1) for com-pounds 6a and 6b and chloroform: ethylacetate (9:1) for compounds 7a-n. Also, silica gel precoated aluminum cards with fluorescentindicator at 254 nm from Merck were used for TLC. Visualizationwas performed by illumination with UV light source (254 nm).

4.1.1. General procedure for synthesis of 1- (arylamino)cyclohexanecarbonitrile (1a and 1b)

A solution of 9.75 g (0.15 mol) of potassium cyanide in 25 ml ofwaterwas addeddropwise to a solutionof 14.7 g (0.15 mol) of cyclo-

hexanone and 0.15 mol of aniline or p-toluidine in 75 ml of glacialacetic acid. The mixture was stirred at room temperature for 24 h.The precipitated product was filtered, washed with water, driedand crystallized from petroleum ether (40–60 �C) to afford 1a or 1b.

4.1.1.1. 1-(phenylamino)cyclohexanecarbonitrile (1a). Yield: 75%;white solid, m.p. 74–76 �C [35].

4.1.1.2. 1-[(4-methylphenyl)amino]cyclohexanecarbonitrile (1b).Yield: 75%; yellowish white solid, m.p. 76–78 �C [36].

4.1.2. General procedure for synthesis of (arylamino)cyclohexanecarboxamides (2a and 2b)

The appropriate nitrile derivative 1a and 1b (0.125 mol) wasstirred in cold concentrated sulfuric acid (20 mL) at room temper-ature for 48 h. The reaction mixture was poured over crushed iceand rendered alkaline with ammonium hydroxide solution (25%).The precipitated amide was filtered off, washed with water, driedand recrystallized from ethanol to give 2a and 2b.

142 W.H. Abd-Allah et al. / Bioorganic Chemistry 71 (2017) 135–145

4.1.2.1. 1-(phenylamino)cyclohexanecarboxamide (2a). Yield: 85%;white solid, m.p. 148 �C [36].

4.1.2.2. 2-[(4-methylphenyl)amino]cyclohexanecarboxamide (2b).Yield: 85%; white solid, m.p. 154 �C [37].

4.1.3. General procedures synthesis of 1- (arylamino)cyclohexanecarboxylic acid (3a and 3b)

A mixture of 0.1 mol of the appropriate carboxamides 2a or 2bin 50 ml concentrated hydrochloric acid was refluxed for 12 hunder vigorous stirring. After cooling, the precipitated materialwas filtered, dissolved in aqueous NaOH (10%), then filtered fromany insoluble substances, neutralized with acetic acid to precipi-tate the corresponding acids, which was filtered, washed withwater, dried and crystallized from ethanol to give 3a and 3b.

4.1.3.1. 1-(phenylamino)cyclohexane-1-carboxylic acid (3a). Yield:60%; white solid, m.p. 141–2 �C [8,9].

4.1.3.2. 2-(p-tolylamino)cyclohexane-1-carboxylic acid (3b). Yield:85%; white solid, m.p. 172 �C [38].

4.1.4. General procedure for synthesis of 1- (arylamino)cyclohexanecarboxylic acid (4a and 4b)

To a solution of 0.05 mol of 1-(N-aralkylbenzamido) cyclohex-anecarboxylic acid (3a or 3b) and 15 g (0.15 mol) of triethylamine(TEA) in 50 ml of dry benzene was added dropwise under stirringand cooling 0.05 mol of benzoyl chloride. The reaction mixturewas refluxed for 12 h and the precipitated triethylaminehydrochloride was filtered off. The filtrate was dried (anhydrousNa2SO4) and evaporated under vacuum. The residual solid wascrystallized from ethanol to give solid crystals of the correspondingacids 4a and 4b.

4.1.4.1. 2-(1-(N-phenylbenzamido)cyclohexyl)acetic acid (4a). Whitesolid, m.p. 190–1 �C, yield 60% [8,9].

4.1.4.2. 2-(1-(N-(p-tolyl) benzamido)cyclohexyl)acetic acid (4b). Buffsolid, m.p. 110 �C, yield 90%; IR (KBr, cm�1): 3470 (OH). 1H NMR(CDCl3): 1.28–1.99 (m, 10H, 5 � CH2, cyclohexyl), 6.59 (d, 2H,J = 8.5, Har.), 7.40 (d, 2H, J = 8.5, Har.), 7.43–8.06 (m, 5H, Har.), 9.07

(OH); 13C NMR (CDCl3): 21.0 (CH3), 21.2, 25.1, 31.2 (3 � CH2, cyclo-hexyl), 61.2 (Cq), 120.6, 128.4, 129.5, 129.6, 129.8 (5CHar.), 130.1,133.1, 141.4 (3Car.), 171.6, 175.0 (2C@O); MS (EI) m/z (%): 237.17([M]+, 60); Anal. calcd. for C21H23NO3: C, 74.75; H, 6.87; N, 4.15.Found: C, 74.85; H, 6.89; N, 4.35.

4.1.5. General procedure for synthesis of methyl 1-(N-aryl benzamido)cyclohexane-1-carboxylate (5a and 5b)

A solution of 0.01 mol of the acid 4a or 4b, 1.28 g (0.04 mol) ofdry methanol and 2 mg (0.01 mmol) p-toluene sulphonic acid in30 ml of dry toluene was refluxed under stirring for 24 h. The sol-vent was evaporated under vacuum and the residue was dissolvedin dichloromethane (DCM). The organic phase was washed withaqueous Na2CO3 (10%) followed by water. The organic layer wasdried over anhydrous Na2SO4, evaporated under vacuum and theresidual solid was recrystallized from methanol to afford com-pounds 5a and 5b.

4.1.5.1. Methyl 1-(N-phenylbenzamido)cyclohexane-1-carboxylate(5a). White solid, m.p. 114-5 �C; yield 60%. [9].

4.1.5.2. Methyl 1-(N-(p-tolyl)benzamido)cyclohexane-1-carboxylate(5b). Brownish oil, yield 83%; IR (KBr, cm1): 3470 (OH). 1H NMR(CDCl3): 1.32–2.32 (m, 10H, 5 � CH2, cyclohexyl), 3.91 (s, 3H,

OCH3), 7.39–7.43 (m, 5H, Har.), 7.46 (d, 2H, J = 8.5, Har.), 7.52 (d,

2H, J = 8.5, Har.); 13C NMR (CDCl3): 21.3 (CH3), 25.0, 31.0, 31.2

(3 � CH2, cyclohexyl), 52.2 (OCH3), 61.2 (Cq), 120.3, 127.1, 128.4,128.8, 129.6 (5CHar.), 129.6, 129.8, 140.4 (3 Car.), 165.7, 167.2(2C@O). MS (EI) m/z (%): C22H25NO3, 351.18 ([M]+, 3); MS (EI) m/z (%): 251.45 ([M]+, 40); Anal. calcd. for C22H25NO3: C, 75.19; H,7.17; N, 3.99. Found: C, 75.28; H, 7.31; N, 4.11.

4.1.6. General procedures for synthesis of (1-(benzyl(aryl)amino)cyclohexyl)methanol (6a and 6b)

A solution of 0.01 mol of the ester 5a or 5b in dry THF wasadded to a slurry of 5.7 g (0.15 mol) LiAlH4 in dry THF at 0 �C understirring. The temperature of the reaction mixture was raised grad-ually to room temperature and left overnight then refluxed for 5 h.The complex was decomposed using saturated solution of Na2SO4

and ethylacetate, filtered, dried over anhydrous Na2SO4 and evap-orated to afford the corresponding alcohol 6a or 6b as viscous oilwhich was purified through column chromatograghy.

4.1.6.1. (1-(benzyl(phenyl)amino)cyclohexyl)methanol (6a). Yellowviscous oil; yield 80%. [9,39].

4.1.6.2. (1-(benzyl(p-tolyl)amino)cyclohexyl)methanol (6b). Yellow-ish viscous oil; yield: 85%; IR (KBr, cm�1): 3357 (OH) and disap-pearance of 2C@O at 1705 and 1639; 1H NMR (CDCl3) 1.30–2.01

(m, 10H, 5 � CH2, cyclohexyl), 2.22 (s, 3H, CH3), 3.63 (s, 2H, CH2-OH), 4.60 (s, 2H, CH2-C6H5), 6.56 (d, 2H, J = 5, Har.), 7.25 (d, 2H,J = 5, Har.), 7.32–7.63 (m, 5H, Har.); 13C NMR (CDCl3) d ppm 21.1

(CH3), 23.4, 25.1, 29.7 (3 � CH2, cyclohexyl), 62.4 (CH2-C6H5),64.5 (Cq), 67.1 (CH2-OH), 103.9, 112.8, 116.8, 120.0, 127.0 (5CHar.),129.8, 141.2, 141.4 (3 Car.); MS (EI) m/z (%): 309.45 ([M]+, 2). Anal.calcd. for C21H27NO: C, 81.52; H, 8.79; N, 4.53. Found: C, 81.52; H,8.77; N, 4.52.

4.1.7. General method for preparation of (1-(benzyl(aryl)amino)cyclohexyl)alkyl acetates (7a and 7h) and butyrates (7b and7i)

To a stirred solution of 2 mmol of alcohol 6a or 6b in 20 ml CH2-Cl2, 2 mmol (0.19 ml) of acetic anhydrid, 2 mmol (0.28 ml) of tri-ethylamine and 0.01 mol of dimethylaminopyridine were added.The reaction mixture was stirred at room temperature for 24 h,then H2O was added and stirred for 15 min. The organic layerwas evaporated, stirred with 1 NHCl (20 ml), extracted with ethy-lacetate, washed with water, dried and evaporated under vacuumto give viscous oils which were purified through columnchromatography.

4.1.7.1. (1-(benzyl(phenyl)amino)cyclohexyl)methyl acetate(7a). Yellowish oil, yield 85%; IR (KBr, cm�1): 3470 (OH), 1738(C@O); 1H NMR (CDCl3): 1.41–2.13 (m, 10H, 5 � CH2, cyclohexyl),

4.45 (s, 2H, CH2-O-CO), 5.10 (s, 2H, CH2-C6H5), 7.05–7.19 (m,

10H, Har.), 13C NMR (CDCl3): 22.1 (CH3-CO), 25.3, 26.0, 32.5

(3 � CH2, cyclohexyl), 52.8 (CH2-C6H5), 61.1 (Cq), 66.9 (CH2-O-CO), 103.5, 126.2, 127.8, 128.1, 128.4, 130.6 (6CHar.), 141.2, 147.4(2 � Car.), 171.2 (C@O); MS (EI) m/z (%), 337.46 ([M]+, 5), 264.28(100). Anal. calcd. for C22H27NO2: C, 78.30; H, 8.06; N, 4.15. Found:C, 78.21; H, 8.09; N, 4.21.

4.1.7.2. (1-(benzyl(phenyl)amino)cyclohexyl)methyl butyrate(7b). Colourless oil, yield 85%; IR (KBr, cm�1) 3470 (OH), 1738(C@O); 1H NMR (CDCl3): 1.41–2.13 (m, 10H, 5 � CH2, cyclohexyl),

4.42 (s, 2H, CH2-O-CO), 4.45 (s, 2H, CH2-C6H5), 7.05–7.19 (m,

10H, Har.); 13C NMR (CDCl3): 9.3 (CH2-CH2-CO) 22.1 (CH3-CH2-

CH2), 25.3, 26.0, 27.6 (3 x CH2, cyclohexyl), 32.7 (CH2-CO-O), 52.9

W.H. Abd-Allah et al. / Bioorganic Chemistry 71 (2017) 135–145 143

(CH2-C6H5), 63.9 (Cq), 67.4 (CH2-O-CO), 100.0, 103.9, 127.8, 128.1,128.3, 130.6 (6CHar.), 141.2, 147.4 (2 � Car.), 174.5 (C@O); MS (EI)m/z (%): 365.52 ([M]+, 0.03), 264.07 (100). Anal. calcd. forC24H31NO2: C, 78.86; H, 8.55; N, 3.83. Found: C, 78.77; H, 8.32;N, 3.79.

4.1.7.3. (1-(benzyl(p-tolyl)amino)cyclohexyl)methyl acetate(7h). Yellowish oil, yield 85%; IR (KBr, cm�1) 3470 (OH), 1738(C@O); 1H NMR (CDCl3): 1.06–1.99 (m, 10H, 5 � CH2, cyclohexyl),

2.02 (s, 3H, CH3-CO), 2.22 (s, 3H, CH3-C6H5), 4.02 (s, 2H, CH2-O-

CO), 5.03 (s, 2H, CH2-C6H5), 7.18–7.26 (m, 9H, Har.); 13C NMR

(CDCl3): 20.9 (CH3-CO), 21.0 (CH3-C6H5), 25.2, 25.5, 32.3

(3 � CH2, cyclohexyl), 52.0 (CH2-C6H5), 66.3 (Cq), 66.5 (CH2-O-CO), 103.8, 120.0, 127.8, 128.2, 128.5 (5CHar.), 129.8, 135.9, 140.0(3 Car.), 170.9 (C@O); MS (EI) m/z (%): 351.49 ([M]+, 8). Anal. calcd.for C23H29NO2: C, 78.59; H, 8.32; N, 3.99. Found: C, 78.66; H, 8.41;N, 3.88.

4.1.7.4. (1-(benzyl(p-tolyl)amino)cyclohexyl)methyl butyrate(7i). Brownish oil, yield 90%; IR (KBr, cm�1): 3470 (OH), 1734(C@O); 1H NMR (CDCl3): 1.41–2.13 (m, 10H, 5 � CH2, cyclohexyl),

4.42 (s, 2H, CH2-O-CO), 4.45 (s, 2H, CH2-C6H5), 7.05–7.19 (m,

10H, Har.); 13C NMR (CDCl3): 14.1 (CH3-CH2-CH2), 21.0 (CH3-CH2-

CH2), 25.3, 26.3, 27.4 (3 x CH2, cyclohexyl), 31.8 (CH2-CO-O), 51.0

(CH2-C6H5), 64.2 (Cq), 66.2 (CH2-O-CO), 120.4, 127.0, 127.5,128.2, 128.6 (5CHar.), 129.5, 130.1, 136.1 (3 Car.), 174.7 (C@O);MS (EI) m/z (%): 379.25 ([M]+, 5), 185.23(100). Anal. calcd. forC25H33NO2: C, 79.11; H, 8.76; N, 3.69. Found: C, 79.23; H, 8.65;N, 3.70.

4.1.8. General method for synthesis of (1-(benzyl(aralkyl)amino)cyclohexyl)methyl benzoates (7c-g, 7j-n)

To a solution of 0.05 mol of alcohols 6a or 6b and 15 g(0.15 mol) of triethylamine (TEA) in 50 ml of dry benzene wasadded dropwise under stirring and cooling 0.05 mol of appropriatebenzoyl chloride. The reaction mixture was refluxed for 12 h andthe precipitated triethylamine hydrochloride was filtered off. Thefiltrate was dried (anhydrous Na2SO4) and evaporated under vac-uum to give the corresponding compounds which were purifiedusing column chromatography.

4.1.8.1. (1-(benzyl(phenyl)amino)cyclohexyl)methyl benzoate (7c)[39]. Yellowish white solid, m.p. 90 �C, yield 90%; IR (KBr, cm�1)3470 (OH), 1716 (C@O); 1H NMR (CDCl3): 1.29–2.04 (m, 10H,

5 � CH2, cyclohexyl), 4.42 (s, 2H, CH2-O-CO), 5.36 (s, 2H, CH2-C6H5), 7.06–7.11 (m, 10H, Har.) 7.12–8.15 (m, 5H, Har.); 13C NMR

(CDCl3): 25.4, 25.5, 32.4 (3 x CH2, cyclohexyl), 51.6 (CH2-C6H5),

64.1 (Cq), 66.9 (CH2-O-CO), 100.0, 103.9, 128.1, 128.3, 128.4,128.9, 129.6, 130.2, 131.2 (9CHar.), 139.7, 141.2, 146.4 (2Car.),166.7 (C@O). MS (EI) m/z (%): 399.53 ([M]+, 3), 264.09 (100). Anal.calcd. for C27H29NO2: C, 81.17; H, 7.32; N, 3.51. Found: C, 81.30; H,7.31; N, 3.52.

4.1.8.2. (1-(benzyl(phenyl)amino)cyclohexyl)methyl 4-chlorobenzoate(7d). Yellowish white solid, m.p. 140 �C, yield 87%; IR (KBr, cm�1):3470 (OH), 1722 (C@O); 1H NMR (CDCl3): 1.31–2.14 (m, 10H,

5 � CH2, cyclohexyl), 4.45 (s, 2H, CH2-O-CO), 5.06 (s, 2H, CH2-C6H5), 7.07–7.14 (m, 10H, Har.), 7.35 (d, 2H, J = 5, Har.), 7.92 (d,

2H, J = 5, Har.); 13C NMR (CDCl3): 23.5, 26.0, 32.8 (3 � CH2, cyclo-

hexyl), 52.9 (CH2-C6H5), 63.9 (Cq), 66.9 (CH2-O-CO), 103.8, 127.7,127.8, 128.3, 128.7, 130.6, 131.0, (8CHar.), 139.4, 141.0, 141.4,147.4 (4Car.), 165.7 (C@O); MS (EI) m/z (%): 433.18 ([M]+, 1.12),

264.17 (100). Anal. calcd. for C27H28ClNO2: C, 74.73; H, 6.50, Cl,8.17, N, 3.23. Found: C, 74.64; H, 6.48; Cl, 8.20, N, 3.26.

4.1.8.3. (1-(benzyl(phenyl)amino)cyclohexyl)methyl 4-methylben-zoate (7e). Yellowish white solid, m.p. 86 �C, yield 90%; IR (KBr,cm�1): 3470 (OH), 1730 (C@O); 1H NMR (CDCl3): 1.36–2.38 (m,

10H, 5 � CH2, cyclohexyl), 2.40 (s, 3H, CH3), 4.54 (s, 2H, CH2-O-

CO), 5.13 (s, 2H, CH2-C6H5), 7.12–7.27 (m, 10H, Har.), 7.94 (d, 2H,J = 7.5, Har.), 8.03 (d, 2H, J = 7.5, Har.), 13C NMR (CDCl3): 21.7

(CH3), 21.9, 22.2, 32.9 (3 � CH2, cyclohexyl), 53.1 (CH2-C6H5),

59.1 (Cq), 64.4 (CH2-O-CO), 100.0, 103.7, 129.1, 129.7, 129.8,130.7, 130.8, 131.2 (8CHar.), 141.0, 143.8, 145.6 (2Car.), 162.6(C@O); MS (EI) m/z (%): 413.55 ([M]+, 1.93), 264.16 (100). Anal.calcd. for C28H31NO2: C, 81.32; H, 7.56, N, 3.39. Found: C, 81.45;H, 7.48; N, 3.37.

4.1.8.4. (1-(benzyl(phenyl)amino)cyclohexyl)methyl 4-nitrobenzoate(7f) [39]. Yellowish solid, m.p. 120 �C, yield 80%; IR (KBr, cm�1):3470 (OH), 1723 (C@O); 1H NMR (CDCl3): 1.31–2.01 (m, 10H,

5 � CH2, cyclohexyl), 3.71 (s, 2H, CH2-O-CO), 4.42 (s, 2H, CH2-C6H5), 7.03–7.11 (m, 4H, Har.), 8.16–8.22 (m, 10H, Har.); 13C NMR

(CDCl3): 22.7, 24.6, 25.3 (3 x CH2, cyclohexyl), 52.9 (CH2-C6H5),

65.3 (Cq), 68.8 (CH2-O-CO), 100.0, 103.5, 123.1, 123.5, 128.0,128.27, 130.7, 131.3 (8CHar.), 139.0, 139.2, 147.2, 150.7 (4Car.),164.7 (C@O); MS (EI)m/z (%): 444.53 ([M]+, 23), 264.00 (100). Anal.calcd. for C27H28N2O4: C, 72.95; H, 6.35, N, 6.30. Found: C, 72.97; H,6.33; N, 6.29.

4.1.8.5. (1-(benzyl(phenyl)amino)cyclohexyl)methyl 4-aminobenzoate(7g) [39]. Brown solid, m.p. 115 �C, yield 75%; IR (KBr, cm�1) 3470(OH), 1689 (C@O); 1H NMR (CDCl3): 1.29–1.97 (m, 10H, 5 � CH2,

cyclohexyl), 4.31 (s, 2H, CH2-O-CO), 4.61 (s, 2H, CH2-C6H5), 5.30(br.s, 2H, NH2), 6.61–6.62 (m, 5H, Har.), 6.64 (d, 2H, J = 5, Har.),7.17 (d, 2H, J = 5, Har.), 7.83–7.88 (m, 4H, Har.), 13C NMR (CDCl3):

20.2, 31.3, 33.3 (3 � CH2, cyclohexyl), 51.2 (CH2-C6H5), 60.2 (Cq),

67.2 (CH2-O-CO), 100.0, 106.2, 113.3, 121.2, 122.2, 129.2, 131.1,132.4 (8CHar.), 139.2, 140.1, 148.5, 150.2 (4Car.), 166.5 (C@O); MS(EI) m/z (%): 414.56 ([M]+, 15), 264.07 (100). Anal. calcd. forC27H30N2O2: C, 78.23; H, 7.29, N, 6.76. Found: C, 78.35; H, 7.32;N, 6.55.

4.1.8.6. (1-(benzyl(p-tolyl)amino)cyclohexyl)methyl benzoate(7j). Colourless oil, yield 86%; IR (KBr, cm�1): 3470 (OH), 1717(C@O); 1H NMR (CDCl3): 1.29–2.04 (m, 10H, 5 � CH2, cyclohexyl),

2.23 (s, 3H, CH3), 4.41 (s, 2H, CH2-O-CO), 5.40 (s, 2H, CH2-C6H5),6.97–7.43 (m, 5H, Har.), 7.44 (d, 2H, J = 5, Har.), 8.05 (d, 2H, J = 5,

Har.), 8.07–8.11 (m, 5H, Har.); 13C NMR (CDCl3): 23.5 (CH3), 25.4,

25.5, 32.3 (3 � CH2, cyclohexyl), 51.1 (CH2-C6H5), 64.9 (Cq), 67.1

(CH2-O-CO), 100.0, 103.5, 116.9, 119.9, 128.3, 128.4, 128.5, 128.6(8CHar.), 129.0, 129.6, 129.8, 130.2 (4Car.), 166.5 (C@O); MS (EI)m/z (%): 413.55 ([M]+, 2.17). Anal. calcd. for C28H31NO2: C, 81.32;H, 7.56, N, 3.39. Found: C, 81.43; H, 7.59; N, 3.35.

4.1.8.7. (1-(benzyl(p-tolyl)amino)cyclohexyl)methyl 4-chlorobenzoate(7k). Yellowish white solid, m.p. 132 �C, yield 87%; IR (KBr, cm�1):3470 (OH), 1719 (C@O); 1H NMR (CDCl3): 1.19–1.93 (m, 10H,

5 � CH2, cyclohexyl), 2.84 (s, 3H, CH3), 4.35 (s, 2H, CH2-O-CO),

5.27 (s, 2H, CH2-C6H5), 6.50 (d, 2H, J = 5, Har.), 7.24 (d, 2H, J = 5,Har.), 7.27–7.31 (m, 5H, Har.), 7.84 (d, 2H, J = 5, Har.), 7.87 (d, 2H,

J = 5, Har.); 13C NMR (CDCl3): 21.1 (CH3), 25.4, 32.3, 45.3 (3 x CH2,

cyclohexyl), 51.2 (CH2-C6H5), 63.9 (Cq), 66.9 (CH2-O-CO), 103.8,116.7, 119.8, 127.9, 128.2, 128.34, 128.6 (7CHar.), 128.6, 130.9,

144 W.H. Abd-Allah et al. / Bioorganic Chemistry 71 (2017) 135–145

131.1, 139.3, 139.4 (5Car.), 165.7 (C@O). MS (EI) m/z (%): 448 ([M]+,0.47). Anal. calcd. for C28H30ClNO2: C, 75.07; H, 6.75, Cl, 7.91, N,3.13 Found: C, 75.19; H, 6.68; Cl, 7.83; N, 3.14.

4.1.8.8. (1-(benzyl(p-tolyl)amino)cyclohexyl)methyl 4-methylben-zoate (7l). Yellowish white solid, m.p. 118 �C, yield 85%; IR (KBr,cm�1): 3470 (OH), 1718 (C@O); 1H NMR (CDCl3): 1.32–2.03 (m,

10H, 5 � CH2, cyclohexyl), 2.37, 2.41 (2 s, 6H, 2CH3), 4.36 (s, 2H,

CH2-O-CO), 5.33 (s, 2H, CH2-C6H5), 6.90–7.21 (m, 5H, Har.), 7.29(d, 2H, J = 5, Har.), 7.91–7.96 (m, 4H, Har.), 13C NMR (CDCl3):

21.22, 21.7 (2CH3), 25.5, 25.6, 32.3 (3 x CH2, cyclohexyl), 52.0

(CH2-C6H5), 64.1 (Cq), 64.4 (CH2-O-CO), 100.0, 103.9, 116.8,119.8, 128.1, 129.1, 129.1, 129.6 (8CHar.), 136.2, 143.27, 143.6,143.8 (4Car.), 166.7 (C@O); MS (EI) m/z (%): 427.59 ([M]+, 13). Anal.calcd. for C29H33NO2: C, 81.46; H, 7.78, N, 3.28. Found: C, 81.47; H,7.79; N, 3.29.

4.1.8.9. (1-(benzyl(p-tolyl)amino)cyclohexyl)methyl 4-nitrobenzoate(7m). Brownish oil, yield 82%; IR (KBr, cm�1) 3470 (OH), 1701(C@O); 1H NMR (CDCl3): 1.20–1.57 (m, 10H, 5 � CH2, cyclohexyl),

2.24 (s, 3H, CH3), 3.82 (s, 2H, CH2-O-CO), 5.28 (s, 2H, CH2-C6H5),6.99–7.25 (m, 4H, Har.), 7.85 (d, 2H, J = 5, Har.), 8.20 (d, 2H, J = 5,

Har.), 8.23–8.31 (m, 4H, Har.); 13C NMR (CDCl3): 21.1 (CH3), 22.7,

29.7, 31.9 (3 � CH2, cyclohexyl), 53.1 (CH2-C6H5), 60.2 (Cq), 66.2

(CH2-O-CO), 123.6, 123.8, 127.5, 127.7, 128.2, 128.7, 129.7 (7CHar.),131.2, 132.5, 137.0, 148.2, 150.4 (5Car.), 166.3 (C@O); MS (EI) m/z(%): 458.22 ([M]+, 5.06). Anal. calcd. for C28H30N2O4: C, 73.34; H,6.59, N, 6.11. Found: C, 73.36; H, 6.57; N, 6.12.

4.1.8.10. (1-(benzyl(phenyl)amino)cyclohexyl)methyl 4-aminoben-zoate (7n). Brownish oil, yield 85%; IR (KBr, cm�1) 3470 (OH),1689 (C@O); 1H NMR (CDCl3): 1.21–1.56 (m, 10H, 5 � CH2, cyclo-

hexyl), 2.23 (s, 3H, CH3), 4.21 (s, 2H, CH2-O-CO), 5.0 (s, 2H, CH2-C6H5), 5.30 (br.s, 2H, NH2), 6.99–7.25 (m, 8H, Har.); 13C NMR

(CDCl3): 21.1 (CH3), 22.6, 29.7, 33.8 (3 � CH2, cyclohexyl), 52.8

(CH2-C6H5), 58.5 (Cq), 66.2 (CH2-O-CO), 112.2, 114.1, 120.0,127.5, 127.8, 128.8, 129.1 (7CHar.), 130.3, 138.5, 142.1, 148.4,152.9 (5Car.), 165.7 (C@O); MS (EI) m/z (%): 426.29 ([M-2], 0.14).Anal. calcd. for C28H32N2O2: C, 78.47; H, 7.53, N, 6.54. Found: C,78.48; H, 7.54; N, 6.55.

4.2. Anticonvulsant activity

4.2.1. Materials4.2.1.1. Animals. The anticonvulsant activity of the compoundsunder investigation 7a–n and 6a-bwas tested on adult male albinomice weighing 20–25 g. Animals were obtained from the AnimalsHouse Colony of the National Research Centre, Cairo, Egypt. Ani-mals were housed in polypropylene cages under standardized con-ditions of light and temperature (room temperature 23 ± 2 �C,relative humidity 55 ± 5%, 12 h light/dark cycle). Animal wereallowed free access to water and standard rat chow. All experimen-tal procedures involving animals were performed according to theguidelines of the ethical committee of the National Research Cen-tre for experimental animal use. Animals were allowed to acclima-tize to laboratory conditions before the start of the experiment. Allthe tested compounds were suspended in 7% Tween 80 as avehicle.

4.2.1.2. Subcutaneous pentylenetetrazole (scPTZ)-induced seizurestest. ScPTZ test was performed to access the production of thresh-old or minimal (clonic) seizures. PTZ prepared as an aqueous solu-tion was administered subcutaneously in the loose fold of the skinon the back of the mice neck at a dose of 85 mg/kg [40] which is

known to produces clonic seizures that lasts for a period of at leastfive seconds in 97 per cent (CD97) of the animals tested. Six micewere used in both the control and the experimental groups. Thetest is carried out by injecting PTZ subcutaneously 30 min after i.p. injection of the test compound. The animals were observed for30 min after PTZ administration for the occurrence of seizures. Athreshold convulsion is defined as one episode of clonicconvulsions which persist for at least a 5 s. Failure to observe suchthreshold seizure indicates the ability of the test compound toabolish of PTZ on seizure threshold and is considered as protection[41] and NIH reference [42].

4.2.1.3. Maximal electroshock seizure test (MES test). MES is a modelused to identify generalized tonic-clonic seizures. Mice were i.p.injected with the test compound. 30 min later, an electric currentof fixed current intensity of 25 mA and 0.2 s stimulus durationwas produced via Rodent Shocker generator (constant-currentstimulator Type 221, Hugo Sachs Elektronik, Freiburg, Germany)and delivered via ear-clip electrode producing electroconvulsionsin mice. The ability of the test compound to abolish the tonic hindlimb extension (i.e. 120 degree outstretching of the mouse hindlimb to the plane of the body axis) indicates its capability to inhibitMES-induced seizure spread [43].

4.2.1.4. Neurotoxicity. Neurotoxicity is determined using rotarodtest which is well established test for the detection of minimalneurological deficit. Animals were trained to maintain equilibriumon a rotating 1-inch-diameter knurled plastic rod at a speed of10 rpm for at least 1 min in each of three trials using a rotaroddevice (UGO Basile, 47600, Varese, Italy). Only animals that fulfillthis criterion were included in the experiment. The selectedtrained animals were divided into control and treated groups.The animals in the treated groups were given the test compoundsat doses that exerted 100% protection in the scPTZ test via i.p. routewhile, the control group received the vehicle. Thirty minutes afterthe administration of the test compound or vehicle, the mice wereplaced again on the rotating rod and the neurotoxicity was indi-cated by the inability of the animal to maintain equilibrium onthe rod for at least 1 min [44].

4.2.1.5. Serum liver enzyme activity. Mice were divided into 3groups (n = 8). Group 1 served as control group and received thevehicle (7% aqueous suspension of tween 80) only; Group 2received compound 7b (8.6 mg/kg, i.p.) dissolved in vehicle. Group3 received compound 7k (2.5 mg/kg, i.p.) dissolved in vehicle. Thetreatments were carried out for a period of 7 days. Twenty-fourhours after the last administration of the compounds, the animalswere anaesthetized. The blood samples were collected by cardiacpuncture followed by centrifugation at 3000 rpm for 10 min forthe separation of sera. The serum samples obtained were usedfor the analyses of the liver enzymes AST, ALT and ALP, as wellas total protein and albumin were determined using commerciallyavailable kits (Biodiagnostic, Egypt).

4.3. Molecular modeling

The generation of pharmacophore modeling method has beenwidely used in lead discovery and optimization as a key tool ofcomputer aided drug design. A hypothesis was formulated usinggeneration common feature pharmacophore model protocol in Dis-covery studio 2.5. The lead compounds III-XII, Fig. 2 which werereported to have anticonvulsant activity were used to generatecommon feature pharmacophore anticonvulsants. A set of confor-mational models of each structure of the lead compounds was per-formed and used to generate the common feature hypotheses,where ten hypotheses were generated.

W.H. Abd-Allah et al. / Bioorganic Chemistry 71 (2017) 135–145 145

Conflict of interest

The authors have declared no conflict of interest.

Acknowledgements

The authors thank the National Research Centre, Dokki, Giza,Egypt, for the support of this research through project No.10010302 (2013–2016).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bioorg.2017.01.021.

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