Synthesis and Biological Evaluation of Novel Benzothiazole Derivatives as Potential Anticonvulsant Agents

New benztriazoles with a mercapto-triazole and other heterocycle substituents were synthesized and evaluated for their anticonvulsant activity and neurotoxicity by using the maximal electroshock (MES), subcutaneous pentylenetetrazole (scPTZ), and rotarod neurotoxicity (TOX) tests. Among the compounds studied, compound 2-((1H-1,2,4-triazol-3-yl)thio)-N-(6-((3-fluorobenzyl)oxy)benzo[d]thiazol-2-yl)acetamide (5i) and 2-((1H-1,2,4-triazol-3-yl)thio)-N-(6-((4-fluorobenzyl)oxy)benzo[d] thiazol-2-yl)acetmide (5j) were the most potent, with an ED50 value of 50.8 mg/kg and 54.8 mg/kg in the MES test and 76.0 mg/kg and 52.8 mg/kg in the scPTZ seizures test, respectively. They also showed lower neurotoxicity and, therefore a higher protective index. In particular, compound 5j showed high protective index (PI) values of 8.96 in the MES test and 9.30 in the scPTZ test, which were better than those of the standard drugs used as positive controls in this study.


Introduction
February ninth, 2015 was the first International Epilepsy Day, and it helped more people raise awareness and understanding about epilepsy. Such efforts are urgently needed, because with more than 50 million people presenting with epilepsy worldwide, epilepsy is the most common, chronic, serious neurological disease. In 2013, 119,000 deaths worldwide were attributable to epilepsy. Currently, 40% of patients in high-income countries and more than 70% of patients in developing countries do not get the treatment they need, because of the high expense or low availability of the appropriate drugs [1][2][3]. Therefore, there is a pressing need to develop more effective antiepileptic drugs (AEDs) endowed with an improved safety profile.

Pharmacology and Structure-Activity Relationship
The anticonvulsant activity evaluation of compounds 2-((1H-1,2,4-triazol-3-yl) thio)-N-(6alkoxybenzo[d]thiazol-2-yl)acetamide (5a-m) were determined using the MES test, which is a mechanism-independent animal seizure model that enables the identification of compounds preventing seizure spread [26]. It should be noted that the MES model remains the most useful tool for the identification of new anticonvulsants, despite significant advances in epilepsy research in the past several years [27]. The MES seizure model was used for preliminary (phase I) screening of compounds 5a-m. They were administered to mice intraperitoneally (i.p.) at the fixed dose of 100 mg/kg and the anticonvulsant protection was observed at two post-treatment times: 0.5 and 4 h. The method applied here allowed the determination of the number of animals (in a group consisting of three mice) protected against electrically-induced seizures as well as the estimation of the time course of anticonvulsant activity, including quick-acting (0.5 h) or long-acting properties (4 h). The results are presented in Table 1. The preliminary pharmacological screening revealed that five compounds (5b, 5c, 5g, 5i, and 5j) showed 100% anticonvulsant protection in the 0.5 h period and some of them (5c, 5i, and 5j) still had a little activity in the 4 h period. One compound, 5h, showed 67% anticonvulsant protection in the 0.5 h period, but no activity in the 4 h period. None of the compounds presented neurotoxicity at the dose of 100 mg/kg. Based on the above preliminary data, six active compounds were screened at the dose of 30 mg/kg in mice (i.p.) at the two post-treatment times (0.5 h and 4 h). As shown in Table 2, only two compounds, 5i and 5j, showed about 33% anticonvulsant protection activity in the 0.5 h period but no activity in the 4 h period.
The following structure-activity relationships (SAR) were obtained, while analyzing the preliminary screening of the synthesized compounds. Among the six alkyl chain-substituted derivatives, 5b and 5c showed better activities, and 5c still presented some activities in the 4 h period. However, with the increase in length, the activities of the compounds did not increase. Compound 5g, substituted with a benzyl group at the 6-position of the benzothiazole core, showed moderate activity at 100 mg/kg. Thus, the F, Cl, and CF3 groups were subsequently added onto the benzyloxy group of 5g at different positions, yielding compounds 5h-m. Substituent position on the phenyl ring also influenced anticonvulsant activity in the 6-fluorobenzyl derivatives as m-F = p-F > o-F. However, the 6-chlorobenzyl derivatives showed no activity at the dose of 100 mg/kg. Compound 5m, substituted with a trifluoromethyl at the 3-position of the benzyl group, also showed no activity, regardless of the period (0.5 h or 4 h) at the dose of 100 mg/kg. Based on the above-mentioned results, six compounds

Pharmacology and Structure-Activity Relationship
The anticonvulsant activity evaluation of compounds 2-((1H-1,2,4-triazol-3-yl) thio)-N -(6-alkoxybenzo[d]thiazol-2-yl)acetamide (5a-m) were determined using the MES test, which is a mechanism-independent animal seizure model that enables the identification of compounds preventing seizure spread [26]. It should be noted that the MES model remains the most useful tool for the identification of new anticonvulsants, despite significant advances in epilepsy research in the past several years [27]. The MES seizure model was used for preliminary (phase I) screening of compounds 5a-m. They were administered to mice intraperitoneally (i.p.) at the fixed dose of 100 mg/kg and the anticonvulsant protection was observed at two post-treatment times: 0.5 and 4 h. The method applied here allowed the determination of the number of animals (in a group consisting of three mice) protected against electrically-induced seizures as well as the estimation of the time course of anticonvulsant activity, including quick-acting (0.5 h) or long-acting properties (4 h). The results are presented in Table 1. The preliminary pharmacological screening revealed that five compounds (5b, 5c, 5g, 5i, and 5j) showed 100% anticonvulsant protection in the 0.5 h period and some of them (5c, 5i, and 5j) still had a little activity in the 4 h period. One compound, 5h, showed 67% anticonvulsant protection in the 0.5 h period, but no activity in the 4 h period. None of the compounds presented neurotoxicity at the dose of 100 mg/kg. Based on the above preliminary data, six active compounds were screened at the dose of 30 mg/kg in mice (i.p.) at the two post-treatment times (0.5 h and 4 h). As shown in Table 2, only two compounds, 5i and 5j, showed about 33% anticonvulsant protection activity in the 0.5 h period but no activity in the 4 h period.
The following structure-activity relationships (SAR) were obtained, while analyzing the preliminary screening of the synthesized compounds. Among the six alkyl chain-substituted derivatives, 5b and 5c showed better activities, and 5c still presented some activities in the 4 h period. However, with the increase in length, the activities of the compounds did not increase. Compound 5g, substituted with a benzyl group at the 6-position of the benzothiazole core, showed moderate activity at 100 mg/kg. Thus, the F, Cl, and CF 3 groups were subsequently added onto the benzyloxy group of 5g at different positions, yielding compounds 5h-m. Substituent position on the phenyl ring also influenced anticonvulsant activity in the 6-fluorobenzyl derivatives as m-F = p-F > o-F. However, the 6-chlorobenzyl derivatives showed no activity at the dose of 100 mg/kg. Compound 5m, substituted with a trifluoromethyl at the 3-position of the benzyl group, also showed no activity, regardless of the period (0.5 h or 4 h) at the dose of 100 mg/kg. Based on the above-mentioned results, six compounds (5b, 5c, 5g, 5h, 5i, and 5j) were selected from all the compounds for the next step and were tested at a dose of 30 mg/kg. As shown in Table 2, at the dose of 30 mg/kg, only m-F and p-F substituted compounds presented some anticonvulsant activities in the 0.5 h period. (5b, 5c, 5g, 5h, 5i, and 5j) were selected from all the compounds for the next step and were tested at a dose of 30 mg/kg. As shown in Table 2, at the dose of 30 mg/kg, only m-F and p-F substituted compounds presented some anticonvulsant activities in the 0.5 h period.
a Maximal electroshock (MES): doses of 100 mg/kg were administrated intraperitoneally in mice. The animals were examined at two times: 0.5 h and 4 h after administration; b n1/n2: the animals protected/the animals tested.

Comp. R MES (30 mg/kg) a 0.5 h 4 h 5b
n-C3H7 According to the bioisosterism, the mercapto-triazole ring of compounds 5i and 5j was replaced with other heterocycles, such as imidazole, triazole, tetrazole, and 3-amino-1,2,4-triazole. Compounds 6, 7, 8, and 9 were designed and synthesized. Their anticonvulsant activities were evaluated at the dose of 100 mg/kg, and screening results are shown in Table 3. The compounds, substituted with other heterocycles, hardly showed any anticonvulsant activities in the 0.5 h or 4 h period. Thus, we concluded that when the mercapto-triazole ring in compounds 5a-m was replaced by other heterocycles (i.e., imidazole, triazole, tetrazole, and 3-amino-1,2,4-triazole), the resultant compounds, 6a-b, 7a-b, 8a-b, and 9a-b, hardly presented any activities compared with the compounds containing the mercapto-triazole ring.    According to the bioisosterism, the mercapto-triazole ring of compounds 5i and 5j was replaced with other heterocycles, such as imidazole, triazole, tetrazole, and 3-amino-1,2,4-triazole. Compounds 6, 7, 8, and 9 were designed and synthesized. Their anticonvulsant activities were evaluated at the dose of 100 mg/kg, and screening results are shown in Table 3. The compounds, substituted with other heterocycles, hardly showed any anticonvulsant activities in the 0.5 h or 4 h period. Thus, we concluded that when the mercapto-triazole ring in compounds 5a-m was replaced by other heterocycles (i.e., imidazole, triazole, tetrazole, and 3-amino-1,2,4-triazole), the resultant compounds, 6a-b, 7a-b, 8a-b, and 9a-b, hardly presented any activities compared with the compounds containing the mercapto-triazole ring.
On the basis of the preliminary screening results, compounds 5i and 5j were subjected to the next phase of trials regarding the quantification of their anticonvulsant activity (indicated by ED50 MES) in mice.
The MES and subcutaneous pentylenetetrazole (scPTZ) seizure models represent the two most widely used animal seizure models in the search for new AEDs. The scPTZ test employs chemicallyinduced myoclonic seizures and allows the identification of agents raising the seizure threshold. This test is related to human generalized absence seizures [28]. Thus, the quantitative analysis of compounds 5i and 5j, the most potent compounds in the MES test, was studied in the scPTZ test to obtain ED50 PTZ. The quantitative neurotoxicity data (indicated by TD50) of compounds 5i and 5j was obtained from the rotarod test.
The results of the quantitative tests are reported in Table 4, along with the data from carbamazepine and phenytoin as positive drug control groups. The quantitative i.p. data in mice confirmed the safe and potent anticonvulsant activity of 5i and 5j. As shown in Table 4, both compounds showed a weaker anticonvulsant activity than the control drug carbamazepine (the ED50 MES was 11.8 mg/kg) in the MES seizure model. However, they showed a stronger anticonvulsant activity than valproic acid (the ED50 MES was 216.9 mg/kg) and better activities in the scPTZ test than all the drugs used as positive controls in this study. Especially, compound 5j showed higher safety with lower neurotoxicity than compound 5i, resulting in higher PI values (8.96 in the MES test and 9.30 in the scPTZ test).
On the basis of the preliminary screening results, compounds 5i and 5j were subjected to the next phase of trials regarding the quantification of their anticonvulsant activity (indicated by ED 50 MES) in mice. The MES and subcutaneous pentylenetetrazole (scPTZ) seizure models represent the two most widely used animal seizure models in the search for new AEDs. The scPTZ test employs chemically-induced myoclonic seizures and allows the identification of agents raising the seizure threshold. This test is related to human generalized absence seizures [28]. Thus, the quantitative analysis of compounds 5i and 5j, the most potent compounds in the MES test, was studied in the scPTZ test to obtain ED 50 PTZ. The quantitative neurotoxicity data (indicated by TD 50 ) of compounds 5i and 5j was obtained from the rotarod test.
The results of the quantitative tests are reported in Table 4, along with the data from carbamazepine and phenytoin as positive drug control groups. The quantitative i.p. data in mice confirmed the safe and potent anticonvulsant activity of 5i and 5j. As shown in Table 4, both compounds showed a weaker anticonvulsant activity than the control drug carbamazepine (the ED 50 MES was 11.8 mg/kg) in the MES seizure model. However, they showed a stronger anticonvulsant activity than valproic acid (the ED 50 MES was 216.9 mg/kg) and better activities in the scPTZ test than all the drugs used as positive controls in this study. Especially, compound 5j showed higher safety with lower neurotoxicity than compound 5i, resulting in higher PI values (8.96 in the MES test and 9.30 in the scPTZ test).

General Information
Melting points were determined in open capillary tubes and were uncorrected. IR spectra were recorded (in KBr) on IR Prestige-21. 1 H-NMR and 13 C-NMR spectra were measured on an AV-300 (Bruker, Switzerland), and all chemical shifts were given in ppm relative to tetramethylsilane. Mass spectra were measured on an AXIMA CFR Plus MALDI-TOF (Shimadzu, Japan). The chemicals were purchased from Aldrich Chemical Corporation.

Synthesis of 6-Hydroxy-2-aminobenzothiazole (2)
A mixture of 6-methoxy-2,3-dihydrobenzo[d]thiazol-2-amine (1) (10 g, 55.56 mmol) and 40 mL of hydrobromic acid (48% water solution) was refluxed for 20 h. The mixture was allowed to cool to room temperature and neutralized with NaOH solution to pH 7-8. The precipitate was filtered and washed with water. The filtrate was stirred with 100 mL hot water for 0.5 h and the remaining precipitate was filtered to yield a brown solid, compound 2.

General Procedure for the Synthesis of 6-Alkoxy-2-aminobenzothiazoles (3a-m)
A mixture of compound 2 (2 g, 12 mmol), potassium carbonate (2 g, 14.4 mmol), appropriate alkyl bromide or benzyl chloride derivatives (1.32 mmol), and a catalytic amount of benzyltriethylamine chloride (TEBA) in 50 mL acetone was heated under reflux for 18-24 h. After removing the solvent under reduced pressure, 80 mL of hot water was poured into the flask and the mixture was stirred for 0.5 h to eliminate the excess of potassium carbonate. The remaining precipitate was filtered to yield a russet solid (3a-m), which was used without further purification.

General Procedure for the Synthesis of 2-Chloro-N-(6-alkoxybenzo[d]thiazol-2-yl)acetamide (4a-m)
6-Alkoxy-2-aminobenzothiazoles (3a-m) (20 mmol) was dissolved in 30 mL acetone and 30 mmol of chloroacetyl chloride were added under cold conditions. The reaction was stirred for 9-10 h at room temperature. Water was added to the reaction mixture after the solvent (acetone) was removed under pressure-reducing conditions. The mixture was stirred for 0.5 h and filtered to yield a crude product. It was then purified by silica gel column chromatography with dichloromethane to yield a white solid 4a-m.
The yield, melting point, analytical data and spectral data of each compound are given below.             A solution of 2-chloro-N-(6-alkoxybenzo[d]thiazol-2-yl)acetamide (5 mmol), imidazole (6 mmol), and sodium hydroxide (6 mmol) in N,N-dimethylformamide (10 mL) was stirred at room temperature for 12 h. Half of the solvent was then evaporated and the solution was poured in 50 mL water and then extracted with ethyl acetate (30 mLˆ3). The ethyl acetate layer was dried over anhydrous MgSO 4 . Evaporation of the solvent yielded a crude product, which was recrystallized from dichloromethane to yield a white solid.  5.08 (s, 2H, imidazole -CH 2 -). 13 13  A mixture of 2-chloro-N-(6-alkoxybenzo[d]thiazol-2-yl)acetamide (5 mmol), 1H-tetrazole (6 mmol), and sodium hydroxide (6 mmol) in N,N-dimethylformamide (10 mL) was stirred at room temperature for 10 h. The solution was then poured in 50 mL of water and extracted with ethyl acetate (30 mLˆ3). The ethyl acetate layer was dried over anhydrous MgSO 4 . Evaporation of the solvent provided a crude product, which was recrystallized from dichloromethane to obtain a white solid.

Pharmacology
All compounds were evaluated for anticonvulsant activities with KunMing mice in the 18-22 g weight range purchased from the Laboratory of Animal Research, College of Pharmacy, Yanbian University. The animals were maintained on a 12 h light/dark cycle and allowed free access to food and water, except during the time they were removed from their cages for testing. The experimental substances were dissolved in dimethylsulfoxide (DMSO) and administered intraperitoneally (i.p.) in a volume of 0.1 mL/20 g body weight. The test method with reference to the Antiepileptic Drug Development (ADD) program [29,30].

MES Screening Test
Seizures were elicited with a 60-Hz alternating current of 50 mA intensity applied via corneal electrodes for 0.2 s. Protection against the spread of MES-induced seizures was defined as the abolition of the hind leg, and tonic maximal extension component of the seizure. The MES test was performed at 30 min after compound administration.

ScPTZ Seizures Screening Test
At 0.5 h after the administration of the test compound, 100 mg/kg PTZ (which 100% of the animals showed clonic seizure) dissolved in saline was administered subcutaneously. The animals were placed in individual cages and observed for 0.5 h. The median effective doses (ED 50 ), which showed 50% protection, were recorded.

Neurotoxicity Screening Test
The neurotoxicity of the compounds was measured in mice using the rotarod test. Mice were tested on a knurled plastic rod (diameter 3.2 cm) rotating at 6 rpm for 1 min, at 30 min after compound administration. Neurotoxicity was measured by the inability of the animal to maintain equilibrium on the rod for at least 1 minute in each of the trials.