Molecular docking study and anticonvulsant activity of synthesized 4-((4,6-dimethyl-6H-1,3-thiazin-2-yl)phenylsulfonyl)urea/thiourea derivatives

2.2.1 General procedure for the synthesis of 4,6-dimethyl-2-phenyl-6H-1,3-thiazine (1)

Compound 1 was synthesized according to the reported procedures (Thakur et al., 2015).

2.2.2 General procedure for synthesis of 4-(4,6-dimethyl-6H-1,3-thiazin-2-yl)benzene-1-sulfonyl chloride (2)

The chlorosulfonation of synthesized compound (1) was carried out using chlorosulfonic acid solution. Two separate equimolar (0.06 mol) solutions of compound 1 and chlorosulfonic acid were prepared in 1,4-dioxane (60 ml) with stirring at room temperature. Both the solutions were mixed together to produce a homogenous exothermic mixture by stirring at room temperature for half an hour. The resulting solution was added to the crushed ice for precipitation of compound 2. The precipitated solid was filtered and recrystallized from ethanol. Yield 74% and m.p. 240–244 °C.

IR Vmax (KBr, in cm⁻¹): 1509–52 (Ar-C⚌C—), 1193 (C—N), 2360 (C—S—C), 1380 (C—CH₃), 1359 and 1177 (C—SO₂), 3058 (Ar—C—H); ¹H NMR (300 MHz, DMSO-d₆, δ): 5.60 (1H, d, C–H of thiazine), 3.46 (1H, m, CH-S of thiazine), 1.53 (3H, d, CH₃ of thiazine) 1.72 (3H, s, CH₃ of thiazine), 7.94–8.14 (4H, m, Ar-SO₂); FAB-MS (m/z): 301 [M⁺].

2.2.3 General procedure for synthesis of 1-(4-substitutedphenyl)-3-(4-(4,6-dimethyl-6H-1,3-thiazin-2-yl)phenylsulfonyl)urea/thiourea (3a–l) (Samanta et al., 2004)

Substituted phenylurea/phenylthiourea (Robert and Smith, 1948) (0.15 mol) was dissolved in alkaline sodium hydroxide solution (2 N) in a flask. To the resultant solution, chlorosulfonyl derivative (compound 2) was added slowly with constant stirring and maintained at 70 °C in water bath. In order to keep the reaction mixture alkaline further sodium hydroxide solution was added and allowed to cool to the room temperature. The solution was filtered and acidified with hydrochloric acid for complete precipitation.

2.3.1 Maximal electroshock seizure (MES) model

For the evaluation of anticonvulsant activity, observations carried after 0.5 h and 4 h of dose administration. MES was induced by 150 mA alternate current of 60 Hz for 0.2 s by means of ear clip electrodes. Prior placing the electrodes, a drop of 0.9% saline solution was poured into each ear. As a result of maximal seizure, initially tonic flexion and then tonic extension of hind limb occured. Abolition of hind limb extension shows the protection from seizure attack (Lotarski et al., 2014).

2.3.2 Pentylenetetrazole (PTZ) induced seizure test

The 0.9% sodium chloride solution was used to dissolve Pentylenetetrazole and administered subcutaneously at a dose of 85 mg/kg body weight. Severity of convulsion was noted in the control group. PTZ induces clonic seizures lasting for 3-5 s of forelimbs and/or hindlimbs. Animals were administered different doses (30, 100 and 300 mg/kg, i.p.) of test compounds and evaluated after 0.5, 4 h followed by PTZ administration. Animals were kept separately to reduce stress and observed for a period of 30 min for abolition of seizure threshold.

2.3.3 Neurotoxicity screening

Minimal motor impairment in rats was measured using standardized rotarod test. The rats were trained to balance on knurled plastic rod (1 inch diameter, 25 cm height), rotating at 6 rpm. The rats retained on the rotating rod for 1 min were selected for the study. Trained animals were treated with test compounds at doses of 100 and 300 mg/kg i.p. The neurotoxicity was defined as inability of rats to stay on rod for at least 1 min in each of four trials.

2.4.1 Chemistry

The route of synthesis given in scheme 1 was very handy for preparation of derivatives (3a–l) with high yield. 4,6-dimethyl-2-phenyl-6H-1,3-thiazine (1) was prepared according to the procedure reported in literature (Thakur et al., 2015). Addition of sulfonyl chloride group at the para position of aromatic ring in compound 1 was carried out by preparing two separate solution of chlorosulfonic acid and compound 1 in 1,4-dioxane. The solutions were mixed together at room temperature which produces 4-(4,6-dimethyl-6H-1,3-thiazin-2-yl)benzene-1-sulfonyl chloride (2). In the final step of synthesis, the compound 2 was stirred with different alkaline solution of phenylurea/thiourea in 2 N sodium hydroxide solution at 70 °C to form a homogenous mix. After cooling, the mix was completely neutralized with concentrated HCl to get precipitate of 1-(4-substitutedphenyl)-3-(4-(4,6-dimethyl-6H-1,3-thiazin-2-yl)phenylsulfonyl)urea/thiourea (3a–l) (Table 1).

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Scheme 1

Reagents and reaction conditions: (i) Chlorosulfonic acid, 1,4 dioxane, stirring at r.t., (ii) NaOH solution (2 N), conc. HCl, stirring at 70 °C.

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The structure of different synthesized compounds was elucidated by IR, ¹H NMR, mass spectra and elemental analysis. The absence of singlet peak around δ 6.0 ppm and δ 4.0 ppm in the NMR spectra of compound 2 shows the absence of —NH (of urea) moiety. Also absence of IR peak around 3400 cm⁻¹ confirmed the nonexistence of urea moiety. There were two IR peaks around 1177 and 1359 cm⁻¹, which assure the addition of –SO₂Cl group in compound 2. Further the structure of target molecule (3a–l) was elucidated by the presence of characteristic singlet peak around δ 6.0 ppm for —NH of urea and δ 4.0 ppm, δ 2.0 ppm for thiourea along with the IR peak around 3400 cm⁻¹. There was also a doublet peak at δ 5.2 ppm which characterizes the C–H of thiazine ring. Additional spectrum at δ 1.5 ppm and δ 1.7 ppm helps to elucidate the presence of methyl groups in thiazine ring. The multiplates around δ 7.8–8.1 ppm and δ 6.4–7.2 ppm conclude the presence of two aromatic rings in the structure. IR bands around 1680 for C⚌O and 3032 for Ar—C—H support the NMR data for the confirmation of the structure. Molecular weight and purity of compounds were supported by mass spectral data.

Two different models viz. subcutaneous pentylenetetrazole (scPTZ) and maximal electroshock seizure (MES) were used for the estimation of anticonvulsant activity. Three dose levels 30, 100 and 300 mg/kg by intraperitoneal (i.p.) route were used for study. All the synthesized derivatives except 3d and 3k are active in both the anticonvulsant models (MES and scPTZ). Out of all these, the compounds 3a, 3b and 3c are found as the most active derivatives devoid of neurotoxicity at any dose. Compounds 3g and 3h are also active but show neurotoxicity at higher dose. In scPTZ model, the compounds were positively active within half an hour but not so after 4 h, which suggest that the drug is fast acting but having short duration in this type of seizure. (Table 2).

Out of 3a, 3b and 3c, the compound 3b is the most active derivative containing electronegative chloride group showing anticonvulsant effect at 100 mg/kg body weight. Another electronegative derivative 3b containing hydroxyl group manifests the promising anticonvulsant effect at the same dose level except at 300 mg/kg body weight after 4 hrs in scPTZ model. The unsubstituted aromatic ring as in compound 3a is also a remarkable candidate for protection against MES induced convulsion after compounds 3b and 3c. The electron donating group (—CH₃) (3g and 3l) is less active as compare to the presence of electronegative atom in the same position. On the other hand an attempt for bioisosteric replacement of oxygen (sulfonylurea) with sulfur atom (sulfonylthiourea) is not convincing to get an efficient molecule, as it has less anticonvulsant activity with minor neurotoxicity (3g–l).

These results suggest that the synthesized derivatives are efficiently bound and inhibit voltage gated sodium channel to protect the spread of neuronal action potential by impairing the prolonged depolarization of neuronal membrane which helps to reduce the excitatory motor activity.

2.4.2 Docking study using open voltage gated sodium channel model

For comparing the effective binding of test compounds which have been docked to the active site, where the following amino acid residues were found in the protein i.e. 147 ARG, 151 ASN, 155 ASN, 161 ILE, 15 MET, 165 MET, 154 PRO, 162 PRO, 157 TRP, 218 LEU, 216 LYS, 222 PHE, 215 SER. All the designed derivatives were docked (Argus Lab) in the active site of open voltage gated sodium channel (Fig. 1).

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Figure 1

(a) Best possible pore of compound 3a (boll and stick structure) showing hydrogen bond (red color line) and bond distance, (b) best possible pore of compound 3b, (c) best possible pore of compound 3c, (d) Possible interaction of amino acids with phenytoin.

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The phenytoin is the standard ligand bearing the less docking score (−12.54) than the most active test compound. The oxygen atom of sulfonyl (—SO₂) and keto moiety of urea were taking part in the formation of hydrogen bond with channel protein. The amino acid residues 147 ARG, 154 PRO, 151 ASN and 150 MET formed a hydrophobic domain around the distal aromatic ring of test compounds. Out of all these the sulfonylurea derivatives, compounds 3a, 3b and 3c were showing the highest docking score without any toxic profile.

Compound 3a has highest docking score (−13.18) among the three, but the drug likeness (4.61) and drug score (0.64) were lower than the other two (3b and 3c). Compound 3a formed four hydrogen bonds, one with —NH group of indole ring in 157 TRP (2.28 Å), two with oxygen (–SO₂ and >C⚌O) of —NH₂ group of 155 ASN (2.73 Å and 2.99 Å) and the last one with 215 SER (2.93 Å) amino acid residues on the active site. Compound 3b having docking score −13.05 was also a suitable candidate for synthesis as it is having the highest drug likeness (6.92) with drug score (0.48) while the most favorable compound is 3c with good docking score (−13.10) as well as drug likeness (6.37) and highest drug score (0.66). Compound 3c is a hydroxyl derivative which helps to form an additional hydrogen bond with the oxygen atom of 151 ASN even in the hydrophobic domain. Out of the all sulfonylthiourea derivatives (3g–l), compound 3h was shown a considerable docking score (−13.08) with low drug likeness (4.80) and drug score (0.30), but due to toxic profile (minor mutagenic) cannot be suited for synthesis. The nitro derivatives 3d and 3k showed negative drug likeness and very low drug score with poor docking score made these unsuitable for synthesis (Table 3).