Design, Synthesis and Pharmacological Evaluation of Sulfonamide Derivatives Screened Against Maximal Electroshock Seizure Test

A series of substituted 4-amino-benzene sulfonamides were designed, keeping in view the structural requirement of pharmacophore. Lipinski rule of five has also been calculated; failure to Lipinski rule was not observed. The synthesized compounds were evaluated for anticonvulsant. Docking was performed through AutoDock Vina. Molecules have been screened out through docking. Compounds were synthesized and characterized through IR, 1HNMR, 13C NMR, Mass and elemental analysis. The anticonvulsant activity of the synthesized compounds was assessed using the MES model. In-silico biological activity spectrum, toxicological studies, predicted oral rat LD50 and possible mechanism of action were investigated. The compound, 4-[2-(4-Acetyl-phenylamino)-ethyl]-benzenesulfonamide (K23) is found most active among the synthesized compounds. *Corresponding author: Ajeet, Drug Design Laboratory, School of Pharmaceutical Sciences, IFTM University, Moradabad, Uttar Pradesh, India, Tel: +91-9236817425; E-mail: ajeet_pharma111@rediffmail.com Received February 06, 2018; Accepted February 21, 2018; Published February 28, 2018 Citation: Ajeet, Kumar A, Mishra AK (2018) Design, Synthesis and Pharmacological Evaluation of Sulfonamide Derivatives Screened Against Maximal Electroshock Seizure Test. Mol Biol 7: 206. doi: 10.4172/2168-9547.1000206 Copyright: © 2018 Ajeet, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
Epilepsy is one of the serious disorders with no age, sex or racial boundaries.Epilepsy imposes a large economic burden on health care systems of countries.There is also a hidden burden associated with stigma and discrimination against the patient and even their family in the community, workplace, and home.In general, the patients with epilepsy suffer severe emotional distress, behavioral disorders and extreme social isolation [1].
Epilepsy is usually associated with recurrent seizures.This implies that epilepsy is an ancient disorder; indeed, in all civilizations it can be traced as far back as medical records exist.In fact, epilepsy is a disorder that can occur in all mammalian species, probably more frequently as brains have become more complex.Epilepsy is also remarkably uniformly distributed around the world [2].
Being inspired from the previous developments, there is always an enthusiasm to design of novel sulphonamide with many scaffolds and to evaluate their possible pharmacological properties as well as mechanism of action.These novel designs will support for future drug developments for multiple diseases in human health care.There is always a chance to get a potential compound on hit and trial basis for design and synthesis of novel compounds.Beyond this, at present multidisciplinary protocols are being suggested to screen the designed-only compounds through computational methods to get the possibility of pharmacological importance of compounds prior to their synthesis and in-vitro experiments.Sequential computational procedures include: (i) Collection of information about scaffolds, linkers and side-groups for possible novel compound; (ii) Structural designing of location specific derivative combinations with one or more scaffold; (iii) Virtualscreening of activity of designed compounds through structure-activity-relationship (SAR) models; (iv) Ligand-protein docking studies of designed compounds; (v) Toxicity estimation of designed compound and (vi) Evaluation of possible pharmacological properties designed compounds.The newly designed compounds, which follow the criteria considered in multiple computational studies, are preceded for synthesis, structural elucidation and further experimental evaluation of activities.
In this paper, we have designed and synthesized compounds as carbonic anhydrase inhibitors, keeping in view the structural requirement of pharmacophore.All the designed compounds were initially gone through in-silico screening like Lipinski rule, QSAR modeling & screening and docking studies.The screened compounds were then synthesized and gone through analytical studies like IR, NMR and Mass spectroscopy.After that all the synthesized compounds were treated for pharmacological screening.An In-silico biological activity spectrum, toxicological studies, predicted oral rat LD50, possible mechanism of action and SAR studies were also investigated.
Steps followed for completing the current research work is shown in following illustration Figure 1.

Designing of molecules
Identification of pharmacophoric groups was done from the structures of well-known anticonvulsants.The structures of well-known anticonvulsants and identified pharmacophore are presented in Table 1 [9,10].The essential structural features which could be responsible for an interaction with the active site were a hydrophobic (HP) unit, an electron donor (D) group, and a hydrogen donor/acceptor (HD/HA) unit.

Docking studies
Docking tool: Docking was performed with AutoDock Vina (PyRx-Python Prescription 0.8) docking software [11].It is virtual screening software for computational drug discovery that can be used to screen libraries of compounds against potential drug targets.It enables medicinal chemists to run virtual screening form any platform and helps users in every steps of this process from data preparation to job submission and analysis of the results [12].
Receptor: Lyase (Oxo-acid)-Human Carbonic Anhydrase-I.Experimental: All the chemicals and solvents, purchased from Merck (India), Spectrochem (India), Sigma-Aldrich (India), Hi-Media (India) and S.D. Fine (India) were used without further purification.Thin layer chromatographic (TLC) analysis of compounds was performed on silica gel G coated glass plates.The adsorbent silica gel G was coated to a thickness of about 0.3 mm on previously cleaned TLC plates of 20x5 cm 2 using conventional spreader.The plates were placed in hot air oven at 105°C for 30 min.The solutions of compounds were applied as a spot on the activated plate about 2 cm above from the lower edge.The mobile phases were selected according to the polarity of compounds.
Melting points were determined by using open capillary melting point apparatus and are reported uncorrected.The 1H-NMR spectra were recorded on Avance-III, Bruker, 400 MHz High Resolution NMR spectrometer and C13-NMR were recorded with Avance-III, Bruker, 100 MHz.Signals were described as singlet (s), doublet (d), triplet (t) and multiplet (m).FT-IR spectra (KBr) were recorded on a Perkin-Elmer Spectrometer BX-II spectrophotometer.The mass spectra were recorded on a Waters Micro-Mass ZQ 2000 mass spectrometer.

Synthetic scheme
Shown in following illustration Figure 2.

Mechanism of reaction
A mechanism of reaction of given schemes has been depicted in Figure 3.

Synthesis of substituted 4-amino-benzenesulfonamides
For the synthesis of an appropriate amide, the substituted substituted chlorobenzene (0.009 mol) of an individual acid dissolved in 20 ml. of dry acetone was added drop wise to a stirred solution of suitable aminosulfonamide (0.0092 mol) and pyridine (0.0091 mol) in 50 ml. of dry acetone.After addition, the reaction mixture was stirred for about 12 hour at room temperature and then the solvent was evaporated under reduced pressure.The residue was dissolved in 100 ml ethyl acetate and the organic phase washed three times with 20 ml of distilled water (3×20 ml).Then 10% HCl solution was added until pH 1 was reached, and the organic phase was separated from the aqueous phase and washed three times with brine.The aqueous solutions were combined and extracted with ethyl acetate (3×50 ml).The ethyl acetate extracts were combined and dried over MgSO 4 and product obtained was filtered and evaporated under reduced pressure [13].

Pharmacology Anticonvulsant activity
Animals: Rats of weight (125-160 g) were used to study the effect of test drug on MES induced seizures via "Electro convulsometer".Female animals were excluded because of the fact that estrus cycle influences the seizure threshold.The protocol for animal based experiments was approved by Institutional Animal Ethical Committee, IFTM University, Moradabad (Resolution no.2015/837ac/PhD/04) as per guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, India).Animals were housed in polypropylene cages with dust free rice husk as a bedding material under laboratory condition with controlled environment of temperature 25°C ± 2°C, humidity (60% ± 10%) and 12 h light/dark cycle as per CPCSEA guidelines.They were provided with balanced food and water, before subjecting them to experimentation, the animals were given a week's time to get acclimatized with the laboratory conditions.The animals were fasted overnight before the experiment.
The synthesized compounds were suspended in 0.5% methyl cellulose and the test compound was usually manipulated with a mortar pestle to help preparation of suspension.In the preliminary screening by MES, each compound was administered, at dose level of 100 mg/ kg (i.g.,) and anticonvulsant and neurotoxicity was assessed at 30 min and 2 h intervals after administration with different groups at each time interval having same species of animals.

The Maximal Electroshock Seizure (MES) model or Maximal Seizure Pattern Test
In the MES test, an electrical stimulus of 0.2 s in duration (150 mA in rat at 60 Hz) is delivered via trans-auricular electrodes.Rats  are tested at 30 minutes and 2 hours following doses of 100 mg/kg of test compound.Rats are tested at time intervals between 30 min.and 2h following a standard dose of 100 mg/kg (i.g.).Abolition of the hind limb tonic extensor component indicates the test compound's ability to inhibit MES-induced seizure spread [14][15][16].Control animals were also treated with the same MES procedure as of the test animals.Different behavioral phases under MES model including Flexion (legs in particular are bent), Extensor (arms and legs being flung outwards), Clonus (sustained rhythmical jerking) and Stupor (reduced level of consciousness) were also recorded for their duration of action (Sec) so that a reduction in duration of phases can be observed as an additional parameter for MES.

Minimal neurotoxicity
For minimal toxicity, 3 assessments i.e. rota rod test positional sense test, and gait and stance test were used.
Toxicity induced by a compound is detected in rats using the standard rota rod test.Untreated control rats, when placed on a 6 rpm.Rotation rod can maintain their equilibrium for a prolonged period of time.Neurological impairment can be demonstrated by the inability of a mouse to maintain equilibrium for one minute in each of three successive trials [17].
Rats are examined for behavioral toxicity by the positional sense test and a gait and stance test.In the positional sense test, one hind leg is gently lowered over the edge of a table, whereupon the rat, experiencing neurological deficit, will fail to lift its leg quickly back to a normal position.In the gait and stance test, neurotoxicity is indicated by a circular or zigzag gait, ataxia, abnormal spread of the legs, abnormal posture, tremor, hyperactivity, lack of exploratory behavior and stupor.Neurotoxicity tests have been carried with a dose of 100 mg/kg body weight at 30 min and at 4 hours from dose administration.

In-silico biological activity spectrum
The biological activity spectrum of a chemical compound is the set of different types of biological activity that reflect the results of the compound's interaction with various biological entities.Biological activity is defined qualitatively ("yes"/"none") suggesting that the biological activity spectrum represents the "intrinsic" property of a substance depending only on its structure and physical-chemical characteristics.
PASS (Prediction of Activity Spectra for Substances) [18] is a software product designed as a tool for evaluating the general biological potential of an organic drug like molecule.PASS provides simultaneous predictions of many types of biological activity based on the structure of organic compounds.Thus, we have used PASS to estimate the biological activity profiles for virtual molecules.
Probability "to be active" estimates the chance that the studied compound is belonging to the sub-class of active compounds (resembles the structures of molecules, which are the most typical in a sub-set of "actives" in PASS training set).

Predictive toxicology
Chemical safety is an important concern of 'The Organization for Economic Co-operation and Development' OECD (http://www.oecd.org/chemicalsafety/). OECD runs 'Quantitative Structure-Activity Relationships Project' under the flag of assessment of chemicals.United States Environment Protection Agency (EPA) leads U.S. engagement with the OECD's Environment Policy Committee (EPOC) and related subsidiary bodies.EPA pays attention towards safer chemical research.Toxicity Estimation Software Tool (TEST) is an in-silico predictive toxicology application, which is used by EPA for screening of new coming compound during chemical research.TEST software includes a set of QSAR models for screening of small molecules against various toxicity parameters as defined at its website: (https://www.epa.gov/chemical-research/toxicity-estimation-software-tool-test).In the present study, we have used TEST software inbuilt QSAR models for estimation of mutagenicity as well as Oral rat 50% lethal dose (LD50) of newly synthesized sulphonamides.Predictive toxicology studies are performed for approval of safe chemical research.

Mutagenicity
It's a capacity to induce mutation, as it said in genetics, a mutagen is a physical or chemical agent that changes the genetic material, usually DNA, of an organism and thus increases the frequency of mutations above the natural background level.

Irritants
Irritants are chemicals that cause reversible skin damage (unlike corrosion, which is irreversible).Clinical signs of irritation include the development of a rash, inflammation, swelling, scaling, and abnormal tissue growth in the affected area.

In-silico mutagenicity and skin irritancy studies
The Ames laboratory test was developed in the 1970's by Bruce Ames, Professor of Biochemistry at UC-Berkeley, as a fast and sensitive assay of the ability of a chemical compound or mixture to induce mutations in DNA.
Ames In-silico test was performed for finding probability of being mutagenic and skin irritant with the help of trial version of Discovery Studio 3.5 using TOPKAT® (Toxicity Prediction by Komputer Assisted Technology) [19] and freely available version of TEST 4.2.1 (Toxicity Estimation Software Tool) (U.S. EPA, 2016) using Consensus method.

Predicted oral rat LD50
Lethal dose (LD50) is the amount of an ingested chemical substance that kills 50 percent to test animal.It is expressed in mg/kg, or milligrams of substance per kilogram of body weight.Commonly it is known as lethal doses.
Predicted oral rat LD50 has been calculated with help of freely available version of TEST 4.2.1 (Toxicity Estimation Software Tool) (U.S. EPA, 2016) using Consensus method.

Designed molecules
A list of designed molecules has been shown in Table 2.

Lipinski rule of five
An initial descriptor calculation has also performed in order to observe designed compounds with its drug ability property (Lipinski Rule of Five) (Table 3).
Lipinski rule of five [20] helps in distinguishing between drug like and non-drug like molecules.It predicts high probability of success or failure due to drug likeness for molecules complying with 2 or more of the following rules: • Molecular mass less than 500 Dalton None of the designed molecules are found to be Lipinski failure.Hence, it implies that all the 10 designed compounds have the drug ability properties/drug likeness properties.
So, it may be concluded that all these designed molecules may proceed for further screening.

Docking
For performing docking, all receptors have been downloaded from NCBI website with PDB ID 1AZM (Lyase-Human Carbonic Anhydrase-I), all the designed ligands have been docked with protein (receptor) with AutoDock Vina (PyRx-Python Prescription 0.8) [11] software having its default settings.Docking study of different proteins were performed with the designed inhibitors is given in Table 4 and Figure 4; number of hydrogen bonds & binding pattern such as element, type of bond, atom number and residue at binding site were evaluated.
On docking analysis, designed compound K16 has been found to be strongly docked with 1AZM with 6 hydrogen bonds and binding affinity of: 7.4 Kcal/mol.On residue study Thr199, His96, His119 and His94 were found to be significant.On the account of ligand oxygen atom is significant in binding with donor bonds, whereas significant element in receptor is nitrogen.
On docking analysis, designed compound K20 has been found to be strongly docked with 1AZM with 6 hydrogen bonds and binding affinity of: 7.3 Kcal/mol.On residue study Thr199, His96, His119 and His94 were found to be significant.On the account of ligand oxygen atom is significant in binding with donor bonds, whereas significant element in receptor is nitrogen.
On docking analysis, designed compound K22 has been found to be strongly docked with 1AZM with 6 hydrogen bonds and binding affinity of: 7.1 Kcal/mol.On residue study Thr199, His96, His119 and His94 were found to be significant.On the account of ligand oxygen atom is significant in binding with donor bonds, whereas significant element in receptor is nitrogen.
On docking analysis, designed compound K23 has been found to be strongly docked with 1AZM with 7 hydrogen bonds and binding affinity of: 7.5 Kcal/mol.On residue study Thr199, His96, His119, Tyr204 and His94 were found to be significant.On the account of ligand oxygen atom is significant in binding with donor bonds, whereas significant element in receptor is nitrogen.
On docking analysis, designed compound K25 has been found to be strongly docked with 1AZM with 6 hydrogen bonds and binding affinity of: 7.4 Kcal/mol.On residue study Thr199, His96, His119 and His94 were found to be significant.On the account of ligand oxygen atom is significant in binding with donor bonds, whereas significant element in receptor is nitrogen.

Experimental Compound characterization
After synthesizing the designed compounds, percentage yield, retention factor (R f ), melting point and elemental analysis (CHNS analysis) were performed.
The findings of physical and elemental data of the compounds are reported in Table 5.

Spectral characterization of synthesized substituted 4-aminobenzenesulfonamides
Compound no.K16.4-[(2-Acetyl-phenylamino)-methyl]benzenesulfonamide (Figure 5).MES activity and neurotoxicity studies are given in Table 6 and their various phase activities has been given in Table 7 As far as performance in MES model concerns, compounds K16 and K23 were observed comparable to standard drug Acetazolamide.Rest synthesized compounds have shown moderate performance.

IR (KBr, cm
While observing different phases of MES model induced convulsions, a reduction in duration of extensor was observed as compared to control.Extensor period of compounds K16 and K23 was in between 3.05 sec and 2.92 sec.The best and minimum duration of extensor among compounds K16 and K23 was 2.92 sec (For compound K23).

In-silico biological activity spectrum
Probability to be active of designed screened compounds against carbonic anhydrase have been calculated with the help of PASS and shown in Table 8.

In-silico mutagenicity and skin irritancy studies
Ames In-silico test results have been shown in Table 9 The model used in Ames In-silico test matches the predicted value with the experimental values used in modeling of software, depending upon them if probability comes 1 then the designed molecules may act as mutagenic.

Predicted oral rat LD50
The newly synthesized molecules were passed through TEST software inbuilt QSAR model for estimation of 'Oral rat 50 percent lethal dose' (LD50) (https://www.epa.gov/chemical-research/toxicityestimation-software-tool-test).Predicted oral rat LD50 of screened compound is presented in Table 10, where it is converted to g/kg instead of mg/kg.

Pharmacology of Compounds Anticonvulsant activity and neurotoxicity of substituted-4amino-benzenesulfonamides
A maximal electroshock seizure test has been performed with an electrical stimulus of 0.2 s in duration (150 mA in rat at 60Hz) is delivered via trans-auricular electrodes.Animals were tested at 30 minutes and 2 h following doses of 100 mg/kg of test compound.Rats are tested at time intervals between 30 min.and 2h following a standard dose of 100 mg/kg.Abolition of the hind limb tonic extensor component indicates the test compound's ability to inhibit MES-induced seizure spread.It was observed that no animal has got neurotoxicity during a period of 30 min.to 4 hours.

Possible mechanism of action of synthesized sulfonamide derivatives
Literature shows that variety of isoforms of Carbonic anhydrase is responsible as anticonvulsant target [21].Sulfonamides are the most important class of Carbonic Anhydrase Inhibitors (CAIs) [4,22,23] The inhibition and activation of CAs are well-understood processes: most types of classical inhibitors bind to the metal center [21].CO 2 , bicarbonate, and protons are essential molecules/ions in many important physiologic processes in all life kingdoms (Bacteria, Archaea, and Eukarya), throughout the tree of life, and for this reason, relatively high amounts of CAs are present in different tissues/cell compartments of most investigated organisms [22,23].
In many organisms, CAs enzymes are involved in crucial physiological processes connected with respiration and transport of CO 2 /bicarbonate, pH and CO 2 homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions (e.g.gluconeogenesis, lipogenesis, and ureagenesis), bone resorption, calcification, tumorigenicity and many other physiologic or pathologic processes (thoroughly studied in vertebrates).In algae, plants, and some bacteria they play an important role in photosynthesis and other biosynthetic reactions [24].Many such enzymes from vertebrates, nematodes, fungi, protozoa and bacteria are well-known drug targets [22].

Carbonic anhydrase inhibition as anticonvulsant effect
One potential factor that may contribute to seizure is the modification of the environmental pH (alkalosis increases the neuronal excitability) [25].The pH buffering of the extracellular and intracellular spaces is mainly carried out by the CO 2 /HCO 3 buffer.The ratio of these two species is regulated by the activity of the carbonic anhydrase which catalyses the reversible conversion of CO 2 and H 2 O into H + and HCO 3 - [26].Acetazolamide, Topiramate and Zonisamide (Figure 10) are a well-known Carbonic anhydrase inhibitors as anticonvulsants.These drugs act by blocking sodium and calcium channels, which leads to the suppression of neuronal hypersynchronization.

Conclusion
We have described the designing, synthesis, computational studies and pharmacological evaluation of synthesized compounds as anticonvulsant agents.The designed compounds were successfully synthesized and well characterized by analytical and spectral data.Docking studies showed good interaction with Lyase (Oxo-acid)-Human Carbonic Anhydrase-I (1AZM).The synthesized compounds showed moderate to good anticonvulsant activity in MES model.The in-silico studies proved them to be with good drug-likeness properties.Few of them showed promising results, especially 4-[2-(4-Acetylphenylamino)-ethyl]-benzenesulfonamide (K23), however, further clinical studies need to be carried.

Figure 1 :
Figure 1: Complete steps followed during research work.

Figure 3 :
Figure 3: Common reaction mechanism of schemes employed.

••••
High lipophilicity (AlogP less than 5) Less than 5 hydrogen bond donors (nHBD) Less than 10 hydrogen bond acceptors (nHBA) Molar refractivity (MR) should be between 40-130 • This filter helps in early preclinical development and could help avoid costly late-stage preclinical and clinical failures.

Table 3 :
Descriptors of Lipinski rule of five.

Table 4 :
Docking results of all the compounds with receptor 1AZM.

Table 5 :
Physical and elemental data of all the synthesized compounds.

Table 7 :
Anticonvulsant effect on various phases of MES at 30 min from dose administration.