Schiff Bases of Isatin and Adamantane-1-Carbohydrazide: Synthesis, Characterization and Anticonvulsant Activity

using pentylenetetrazole- (PTZ-) induced seizure model using phenobarbitone as a positive control. Our ﬁndings showed that compounds 18–23 provided signiﬁcant protection against PTZ-induced seizure, and maximum activities were associated with compound 23. Moreover, all investigated compounds increased the latency of induced convulsion and reduced the duration of epilepsy with compound 23 being the best. Interestingly, most of the synthesized molecules showed reduction in neurological symptoms and severity of the seizure. Molecular docking studies suggest GABA-A receptor as a potential target, and in silico ADME screening revealed that the pharmaceutical properties of compound 23 are within the speciﬁed limit. Thus, compound 23 was identiﬁed as a promising candidate that warrants further drug discovery processes.


Introduction
Epilepsy is one of the neurological disorders that involves abnormal electrical activity in the central nervous system, which results in spontaneous and recurrent seizures with or without loss of consciousness [1]. It is a group of diverse syndromes affecting more than 50 million people of different ages worldwide, and it poses a substantial economic burden in terms of health care services and lost productivity at work [2]. Furthermore, the presence and magnitude of related psychiatric conditions, such as anxiety and depressive disorders, make seizures worse, and can impair the quality of life in several domains [2]. Currently used antiepileptic drugs, such as phenytoin, phenobarbitone, and carbamazepine, are associated with serious side effects, and in several cases, they failed to manage seizures adequately. Moreover, studies have disclosed that around 30% of patients are resistant to the conventional antiepileptic therapy [3][4][5]. In this context, there is an urgent need to develop more efficacious antiepileptic drugs with reduced potential to induce side effects. Such new antiepileptic drugs should be explored based on original ideas to open new avenues for adequate control of this devastating disease [6][7][8]. Researchers now follow three main modalities in attempts to develop new antiepileptic drugs: (I) chemical optimization of currently existing drugs, (II) discovery of new therapeutic entities against novel potential therapeutic targets, and (III) conventional drug screening (nontarget-driven) approach [8].
Several isatin-based derivatives ( Figure 1) have been reported to possess anticonvulsant properties [13]. Some of these were reported for their enhanced antiepileptic activity through a significant elevation of GABA levels in the brain [14].
Adamantane (5) (Figure 2) and its derivatives were revealed in the literature to possess broad-spectrum pharmacological activities [15]. By virtue of its hydrophobic nature, adamantane was extensively adopted in drug discovery as a modifier of the pharmaceutical and pharmacokinetic properties of several drug classes [16][17][18]. To this end, incorporation of adamantane moiety has been shown to enhance CNS penetration of the modified structures, and consequently, this has been considered of value in promoting CNS drug targeting [15]. In this regard, adamantane derivatives (6)(7)(8) (Figure 2) have been documented to display excellent anticonvulsant activity via different modes of action including the blockade of the NMDA-activated channel [19][20][21][22][23]. Despite the long clinical success of conventional antiepileptic drugs, it has been established that the concept of "one-drug-one-target-one-disease" is unsatisfactory in treating this heterogeneous disease. Furthermore, newgeneration antiepileptic drugs have shown low efficacy in adequate controlling of seizures [24]. us, there is an urgent need to deliver new potent antiepileptic drugs.
Recently, literature has grown up around the theme of molecular hybridization as an efficient drug design strategy that affords ligands to be able to interact with multiple targets of interest [25]. is strategy involves the fusion of two pharmacophoric moieties in a single structural framework [26].
Considering all these factors into account, the study presented here aims to contribute to the growing area of antiepileptic drug discovery by exploring new molecular architecture ( Figure 3) designed through hybridization of two pharmacophoric moieties, namely, isatin and adamantane.
e resultant hybrid molecules are expected to possess an enhanced CNS penetration, together with their ability to modulate multiple targets involved in the etiology of epilepsy. Different substitution patterns with different electronic and/or hydrophobic environments were adopted to get an insight into their possible SAR.
All the synthesized compounds were purified by recrystallization from the appropriate solvent, and their chemical structures were verified by recording their IR, 1 H, and 13 C NMR.
Ester (10) and hydrazide (11) showed strong absorptions at 1730 and 1613 cm −1 , respectively, indicating the presence of carbonyl functionality in each case. Furthermore, two distinct absorption bands become visible at 3328 and 3280 cm −1 indicating NH 2 stretching in 11. 1-Hdroxymethylisatins 15 and 16 showed broad bands ranging from 3313 to 3446 cm −1 corresponding to the free OH group. N-Alkyl isatin 17 displayed absorption bands at 3091 cm −1 and 2972 cm −1 corresponding to aromatic C-H stretching and aliphatic C-H stretching, respectively. e IR spectra of Schiff bases 18-23 were apparently distinct from their respective intermediates. To this end, the appearance of a sharp band in the range of 1670-1690 cm −1 denotes stretching of hydrazide carbonyl (CONHN) in all Schiff bases. In addition, bands around 1700 cm −1 correspond to amide carbonyl (CONH) functionality in compounds 18-20. e 1 H NMR spectra of the synthesized compounds are in consistence with the proposed chemical structures. For Schiff bases 18-23, the spectra displayed the adamantane-1-carbohydrazide and the indole sets of protons in a manner consistent with their designed structures. e 15 aliphatic protons in the adamantane moiety are exhibited as multiplets in a range of δ 1.59-2.13 ppm [30]. 1 H NMR spectra of 18 and 21 displayed the four aromatic protons of the indole ring ranging from δ 6.45 to 8.22 ppm, which are consistent with the reported data [31,32]. In 5-substituted isatin derivatives, 19,20,22, and 23, the three aromatic hydrogens are relatively shifted downfield due to 5-substitution by the electron-withdrawing group. Compounds 18-20 showed a broad singlet peak at δ 9.02-11.44 ppm integrated to one proton assigned for the deshielded ArNHCO proton of the indole ring. However, this peak is not displayed for N-alkylated compounds (21)(22)(23). 1-Hydroxy methylated products 21 and 22 displayed a broad singlet peak at δ 8.70-8.89 ppm integrated to one proton assigned to hydroxyl group hydrogen. Moreover, a singlet signal at δ 3.58-3.94 ppm integrated for 2H is ascribed to ArNCH 2 . 1 H NMR spectrum of N-benzyl derivative 23 exhibited five aromatic hydrogens attributed to the benzyl group protons at δ 7-7.5 ppm beside the aromatic protons of the indole ring.
As a general pattern, the 13 C NMR spectra in all compounds are in consistence with the chemical structures and with the previously reported data [32][33][34].
e most observable trend in 13 C NMR spectra is related to the number of peaks displayed at the aromatic region that are always equal to the sum of the equivalent aromatic carbons for each compound. e 13 C NMR spectra exhibit characteristic signals resonating around δ 160. 28 (18)(19)(20)(21)(22)(23) along with the positive control, phenobarbital, at two dose levels (30 and 100 mg/kg). On the contrary, the negative control group received only 5% methylcellulose. Contrary to what has been reported [35], phenobarbital at the two tested doses, 30 and 100 mg/kg, showed 25% and 100% protection against PTZ-induced seizure, respectively ( Figure 4). Compound 18 at 100 mg/kg displayed a protection rate of 58% (Figure 4), while at the lower dose, it was inactive. Insertion of the bromo group at C5 of the indole ring rendered compound 19 inactive at the two tested doses ( Figure 4). On the contrary, chloro substitution at the same position as in compound 20 led to significant protection (40%) at the low dose, while no activity was observed for this compound at the higher dose ( Figure 4). Hydroxymethylation of position 1 for Schiff bases 18 and 19 afforded compounds 21 and 22, respectively. ese compounds demonstrated significant seizure protection rates at the low dose (>40%), while their bioactivity reduced at the higher doses. ese results propose that manipulation of position 1 by polar functionality improves the anticonvulsant activity. Based on this, we tried to introduce the nonpolar motif through N-benzylation of the most potent compound 22 to furnish the more lipophilic compound 23. Being superior to the standard drug phenobarbitone in terms of seizure protection rate, compound 23 emerged as the most potent as it displayed 67% seizure protection at the low dose of 30 mg/kg and 58% at the higher dose of 100 mg/kg as shown in Figure 4.
Correlation between lipophilicity and anticonvulsant activity was reported [36][37][38]. For anticonvulsant agents to be effective, they have to attain adequate concentration in the brain through the efficient crossing of BBB. All compounds investigated here were found to have CLogP values (Table 1) above 2, which is an important feature for effective CNS penetrability [38]. It is worthnoting that the most  Journal of Chemistry efficacious compound 23 has a CLogP value of 6.952, which exceeds that of the other compounds. In this regard, the high potency for compound 23 could be partially explained based on its higher lipophilicity, which directly influences the CNS delivery.

Effect of Synthesized Compounds on Latency and
Duration of PTZ-Induced Seizure. e time in seconds of the onset of the seizure, expressed as the latency of convulsion, and the duration of the PTZ-induced seizure were obtained for the six synthesized compounds and phenobarbital at 30 and 100 mg/kg doses. As illustrated in Table 2, compounds 18-23 increased the latency of convulsion with compound 23 exhibiting a significant delay on the onset of PTZ-induced seizures (1263 ± 501.2 sec) compared to the positive control (467.5 ± 56.41 sec) at 30 mg/kg dose. ese results are consistent with those disclosed in the literature which described the influence of isatin-inspired compounds on latency time of convulsions [14]. Furthermore, except compound 20 at the low dose, all the synthesized Schiff bases were found effective in shortening the epileptic duration (Table 2) with significant effect observed for compounds 18 and 21-23. Compounds 18,22, and 23 at low doses (30 mg/kg) were more effective than the standard drug at the same dose, pointing to their seizure protection ability at relatively lower doses. It is worthwhile to mention that these compounds displayed better protection at lower doses (30 mg/kg) rather than the higher doses (100 mg/ kg) as shown in Table 2.

Effect of the Investigated Compounds on Neurological Signs of PTZ-Induced Seizure.
e neurological signs and severity of the PTZ-induced seizure were observed for 1 hour after the administration of compounds 18-23 along with phenobarbitone. As shown in Table 3, all candidate compounds demonstrated a reduction in PTZ-induced neurological signs of seizures. e most potent compound 23 was proved to be better than the standard drug in minimizing these signs at the dose (30 mg/kg).

Neurotoxicity of the Investigated Compounds.
Neurotoxicity of compounds 18-23, along with the standard drug, was assessed for 1 hour after the induction of the seizure. To this end, compounds 18-23, together with phenobarbitone, exhibited CNS depressive effects such as somnolence and sleep after 1 hour of seizure induction, as depicted in Table 4. While compounds 19 and 21 revealed signs of CNS toxicity such as lack of exploratory behavior and catalepsy, the most potent candidate 23 at 30 mg/kg displayed a similar pattern of neurological toxicity to the standard drug at the same tested dose.

Docking on GABA A Receptor (3IP9)
. GABA is the majorCNS inhibitory neurotransmitter involved in the pathophysiology of epilepsy [39]. erapeutic agents acting through the modulation of the GABA A receptor have been reported to inhibit seizures in both experimental animals and humans [40]. It has been reported that PTZ mediates its convulsive action through direct inhibition of the GABAergic process in the CNS [41]. Since the synthesized compounds antagonized the convulsion induced by PTZ, it probably may exert its anticonvulsant activities through activation of GABAergic transmission in similar mechanisms of barbiturates such as phenobarbital [42]. Molecular docking is a powerful tool for investigating the potential action mode of biodynamically active molecules. erefore molecular docking analysis has been carried out to assess the potential molecular interaction patterns of compounds 18-23 with the GABA A receptor. e docking energies (kcal/mol) of the synthesized compounds against phenobarbitone are summarized in Table 1. As shown in Table 1, compounds 18-23 exhibited lower docking energies ranging from −7.6 to −8.6 kcal/mol, which are relatively far less than the standard drug phenobarbitone (−5.903 kcal/mol). is could be partially explained by the formation of more stable complexes as a consequence of better positioning of these compounds in the active center of the receptor. While the relatively less active compound 22 displayed the lowest binding energy (−8.662 kcal/ mol), the most potent compound 23 showed a binding energy of −7.875 kcal/mol. is could be rationalized by the relatively high hydrophobicity of compound 23 (CLogP = 6.952), which facilitates the penetration of 23 into the brain in high concentration levels. e 3D interaction of the GABA A receptor with phenobarbital and compound 23 is shown in Figure 5, indicating hydrophobic interaction of the isatin moiety, which is further augmented by deep longitudinal insertion of the benzyl group in the hydrophobic pocket. Such an interaction has been documented in the literature for potent anticonvulsant compounds proposing the necessity of aryl/alkyl hydrophobic group as key structural elements [43]. us, the superior activity of 23 appears to be due to the unique existence of the benzyl group which aid to (i) Hydrophobic interaction with the binding site; (ii) Increase CNS penetrability.

Drug-Likeness Study.
Prediction of ADMET properties of the titled compounds such as partition coefficient (CLogP), topological polar surface area (TPSA), and number of rotatable bonds (Nrotb), in addition to violations of Lipinski's and Veber's parameters, was performed using SwissADME server. As shown in Table 1   Data analyzed by one-way ANOVA followed by Dunnett's test (n � 4); * P < 0.05, * * P < 0.01, and * * * P < 0.001 when compared with the control group.  Journal of Chemistry Veber's parameters. us, these compounds have positive drug-likeness such as good oral bioavailability and favorable CNS penetrability and hence could be envisioned as promising and potential leads for further investigations.

General.
e starting adamantane-1-carboxylic acid, isatins, and alkyl halides were commercially available from Sigma-Aldrich, USA. All solvents and reagents were of analytical grade and were used without further purification. Precoated silica gel TLC plates were used to monitor reaction progress; a UV lamp (254 nm) was used to visualize the spots. e purity of synthesized compounds was checked by TLC using ethyl acetate and n-hexane (1 : 1) as the mobile phase. Melting points were determined on Electrothermal Karl kolb (scientific technical supplier, Germany) and were uncorrected. Infrared (IR) spectra (Supplementary S1) were recorded as KBr disk using Shimadzu IR apparatus, University of Khartoum, Sudan, and data are given in ῡ (cm −1 ). NMR spectra (Supplementary S2) were determined utilizing either DMSO-d 6 or CDCl 3 as the solvent and recorded on Bruker Avance 111 NMR Spectrophotometer operating at 400 MHz for 1 H and at 100 MHz for 13 C NMR at the research center, College of Pharmacy, Cairo University, Egypt.

Anticonvulsant Activity
Experimental Animals. Wistar albino rats of both sexes, weighing about 70-130 g, were housed under standard environmental conditions of temperature (12 hours light/dark cycle; 24 ± 2°C; 30-70% humidity) and had ad libitum access to purified water and food. All animals fasted overnight prior to the experiment with free access to water. Animals were adapted to the laboratory condition for a week before initiating the experiments. Prior to starting the experiment, all experimental animal protocols were approved by the Institutional Animal Ethical Committee (IAEC) of Faculty of Pharmacy, Omdurman Islamic University, constituted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
Chemicals. Standard convulsive agents, PTZ was obtained from Sigma-Aldrich (St. Louis, MO, USA), and phenobarbital was obtained from Shanghai Pharmaceutical Company (Khartoum, Sudan).

Experimental Protocol.
is investigation was performed following the Antiepileptic Drug Development Program Protocol (ADDP) [44]. Six synthetic compounds (18)(19)(20)(21)(22)(23) were investigated for anticonvulsant activity in albino rats, which were divided into fifteen groups (N � 4). All groups were injected subcutaneously in a loose fold of skin on the back of the neck with PTZ (70 mg/kg) as a chemical epileptic inducer. In the first group, rats were orally administered methylcellulose 5% as the vehicle control (negative control), and in the second and third groups, rats were orally (p. o) administered phenobarbital 30 and 100 mg/kg, respectively, as reference anticonvulsant drugs for comparison. e remaining groups were orally (p. o) administered synthetic compounds each in different doses (30 and 100 mg/kg) one hour before s. c PTZ 70 mg/kg. Animals were placed in isolation cages to minimize stress and then were observed for 30 min, 1, 4, and 24 hours for signs of neurological deficits and anticonvulsant activities.
Measuring of Anticonvulsant Activities. Protection rate % against PTZ-induced seizure was measured by the following equation: protection rate % � N C -N T /N C × 100, where N C is the number of seizure attacks during 1 hour after seizure induction in the control group and N T is the number of seizure attacks during 1 hour after seizure induction in the treated group. Furthermore, convulsion latency was measured as the onset time (seconds) of convulsion after seizure induction. Moreover, the duration of convulsion (seconds) was calculated by following the formula, time after completion of convulsion − the onset time of convulsion.
Statistical Analysis. Data were expressed as protection %, seizure duration, and latency time in seconds ± SEM and analyzed by two-tailed Student's test to determine the significance of the difference between the control group and rats treated with the test compounds. e difference in results was considered significant when the P value <0.05. All statistical calculations were carried out using Graph-Pad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) [45].

Computational Studies
Molecular Docking. e 3D structure of the GABA A receptor was downloaded from the protein data bank (PDB ID: 3IP9). e energy of this target was minimized via Cresset Flare software [46]. e chemical structures of compounds were drawn via ChemDraw software, and the 3D structure was generated in mol2 with Open Babel software [47]. e default settings of Cresset Flare software were used for the docking process [46].
Drug-Likeness Studies. e prediction of ADMET properties, the number of rotatable bonds, topological polar surface area (TPSA), and the number of hydrogen bond donors (HBD) and acceptors (HBA) for all synthesized compounds were estimated using SwissADME (available at http://www. swissadme.ch.com).

Conclusion
In the present work, six adamantane-isatin conjugates (18)(19)(20)(21)(22)(23) were synthesized with the aim of investigating their anticonvulsant activity. Compound 23 was the most efficacious and provided significant protection against PTZinduced seizure with minimal neurotoxicity. Molecular docking studies suggested GABA A receptor as a potential target, and in silico ADME screening revealed that the pharmaceutical properties of compound 23 are within the specified limit.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request. Journal of Chemistry 9