Synthesis and biological evaluation of novel benzothiazole derivatives as potential anticonvulsant agents

Article abstract of DOI:10.3390/molecules21030164

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.

Full text of DOI:10.3390/molecules21030164

molecules
Article
Synthesis and Biological Evaluation of Novel
Benzothiazole Derivatives as Potential
Anticonvulsant Agents
Da-Chuan Liu, Hong-Jian Zhang, Chun-Mei Jin * and Zhe-Shan Quan *
Key Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain, Affiliated Ministry
of Education, College of Pharmacy, Yanbian University, Yanji 133002, China; 2013001048@ybu.edu.cn (D.-C.L.);
zhjzhishixiang@163.com (H.-J.Z.)
* Correspondence: cmjin@ybu.edu.cn (C.-M.J.); zsquan@ybu.edu.cn (Z.-S.Q.);
Tel./Fax: +86-433-243-6017 (C.-M.J.); +86-433-243-6020 (Z.-S.Q.)
Academic Editors: Shufeng Zhou and Wei-Zhu Zhong
Received: 6 January 2016 ; Accepted: 25 January 2016 ; Published: 29 February 2016
Abstract: 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.
Keywords: synthesis; benzothiazole; mercapto-triazole; anticonvulsant; maximal electroshock;
neurotoxicity; pentylenetetrazole
1. 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 [1–3]. Therefore, there is a pressing need to develop more effective antiepileptic drugs (AEDs)
endowed with an improved safety profile.
On the basis of a number of related materials, azoles and their derivatives have gained
much attention in recent years due to their potential biological applications linked to their
anticonvulsant [
Among them, heterocyclic compounds with the 3-mercapto-1,2,4-triazole substructure exhibit
a wide spectrum of biological activities [ 10]. Similarly, we also demonstrated that the
4], anti-inflammatory [5], anti-fungal [6], antiviral [7], and anticancer [8] activities.
9,
benzothiazole nucleus is a unique scaffold for further molecular exploration to synthesize novel
compounds. A literature survey revealed that benzothiazole analogs are associated with diverse
pharmacological effects [11–14], including anticonvulsant activity [15,16]. For this reason, and in
continuation to our efforts directed toward the synthesis of new heterocyclic compounds with
Molecules 2016, 21, 164; doi:10.3390/molecules21030164
www.mdpi.com/journal/molecules

Molecules 2016, 21, 164
2 of 13
anticonvulsant biological activities, in this study, we combined both biological components
(3-mercapto-1,2,4-triazole and benzothiazole) with an amide, which has anticonvulsant bioactivities
in some AEDs (such as carbamazepine and riluzole, also see references [17
2-((1H-1,2,4-triazol-3-yl)thio)-N-(6-alkoxybenzo[d]thiazol-2-yl)acetamide. To compare the compound
activities with those of other azoles, the mercapto-triazole in compound was replaced with other
heterocycles such as imidazole, triazole, tetrazole, and 3-amino-1,2,4-triazole, to obtain compounds
, and (Figure 1). Their anticonvulsant activities were evaluated using the maximal electroshock
,18]), to obtain a series of
5
6,
7
,
8
9
(MES) test and their neurotoxicity (TOX) was evaluated with the rotarod test in mice.
Figure 1. Benzothiazole and kinds of azoles combined with amide.
2. Results and Discussion
2.1. Chemistry
All the target compounds were synthesized according to Scheme 1. Compounds 3a–m were
prepared according to previous studies in our laboratory [19]. Compounds 3a
chloroacetyl chloride at room temperature in acetone to yield derivatives 4a
when derivatives 4a were allowed to react with different azoles, such as 1H-1,2,4-triazole-3-thiol,
imidazole, triazole, tetrazole, and 3-amino-1,2,4-triazole in refluxing dimethylformamide (DMF) in the
presence of NaOH [22 25], the 2-chlorine atom was substituted by these heterocycles, producing the
corresponding compounds: 2-((1H-1,2,4-triazol-3-yl)thio)-N-(6-alkoxybenzo[d]thiazol-2-yl)acetamide
5a ), N-(6-alkoxybenzo[d]thiazol-2-yl)-2-(1H-imidazol-1-yl)acetamide (6a ), N-(6-alkoxybenzo[d]
thiazol-2-yl)-2-(1H-1,2,4-triazol-1-yl)acetamide (7a–b), N-(6-alkoxybenzo[d]thiazol-2-yl)-2-(1H-tetrazol-1-yl)
acetamide (8a ), and 2-(3-amino-1H-1,2,4-triazol-1-yl)-N-(6-alkoxybenzo[d] thiazol-2-yl) acetamide
9a ). The structures of the targeted compounds were characterized using spectral methods, and all
spectral data corroborated the assumed structures.
–
m
were treated with
–
m [20,21]. Finally,
–m
–
(
–m
–b
–b
(
–b

Molecules 2016, 21, 164
3 of 13
Scheme 1. Synthetic route of target compounds. Reagents and conditions: (a) HBr (48 wt % in
H2O, 99.99% metals basis), reflux, 18 h; ( ) RBr/RPHCH2Cl, CH3COCH3, K2CO3, reflux, 18–24 h;
b
(c) ClCOCH2Cl, acetone, room temperature, 9–10 h; (d–h) DMF, NaOH, room temperature, 8–12 h.
2.2. 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 CF3 groups were subsequently added onto the
benzyloxy group of 5g at different positions, yielding compounds 5h
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
–m. Substituent position on the
,

Molecules 2016, 21, 164
4 of 13
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.
Table 1. Anticonvulsant activities screening (maximal electroshock test) and neurotoxicity screening in
mice at the dose of 100 mg/kg.
O
RO
S
N
N
NH
NH
S
N
5a-m
MES (100 mg/kg) a
Toxicity (100 mg/kg)
Comp.
R
0.5 h
4 h
0.5 h
4 h
1/3 b
3/3
3/3
0/3
1/3
1/3
3/3
2/3
3/3
3/3
0/3
0/3
0/3
5a
5b
5c
5d
5e
5f
5g
5h
5i
-CH3
n-C3H7
n-C4H9
n-C5H11
n-C6H13
0/3
0/3
1/3
0/3
0/3
0/3
0/3
0/3
1/3
1/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
n-C7H15
-CH2C6H5
-CH2C6H4 (o-F)
-CH2C6H4 (m-F)
-CH2C6H4 (p-F)
-CH2C6H4 (o-Cl)
-CH2C6H4 (m-Cl)
-CH2C6H4 (m-CF3)
5j
5k
5l
5m
a
Maximal electroshock (MES): doses of 100 mg/kg were administrated intraperitoneally in mice. The animals
b
were examined at two times: 0.5 h and 4 h after administration; n1/n2: the animals protected/the
animals tested.
Table 2. Anticonvulsant activities screening: MES test in mice at the dose of 30 mg/kg.
MES (30 mg/kg) a
Comp.
R
0.5 h
4 h
5b
5c
5g
5h
5i
n-C3H7
n-C4H9
-CH2C6H5
0/3
0/3
0/3
0/3
1/3
1/3
0/3
0/3
0/3
0/3
0/3
0/3
-CH2C6H4 (o-F)
-CH2C6H4 (m-F)
-CH2C6H4 (p-F)
5j
a
Maximal electroshock (MES): doses of 30 mg/kg were administrated intraperitoneally in mice.
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
heterocycles (i.e., imidazole, triazole, tetrazole, and 3-amino-1,2,4-triazole), the resultant compounds,
6a 7a 8a , and 9a , hardly presented any activities compared with the compounds containing
the mercapto-triazole ring.
–m was replaced by other
–
b,
–b
,
–
b
–b

Molecules 2016, 21, 164
5 of 13
Table 3. Anticonvulsant activities of compounds 6, 7, 8, and 9 in MES test.
MES (100 mg/kg)
Toxicity (100 mg/kg)
Comp.
R
0.5 h
4 h
0.5 h
4 h
6a
6b
7a
7b
8a
8b
9a
9b
-CH2C6H4 (m-F)
-CH2C6H4 (p-F)
-CH2C6H4 (m-F)
-CH2C6H4 (p-F)
-CH2C6H4 (m-F)
-CH2C6H4 (p-F)
-CH2C6H4 (m-F)
-CH2C6H4 (p-F)
0/3
0/3
0/3
0/3
0/3
0/3
1/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
1/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
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
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 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).
Table 4. Quantitative Pharmacological Parameters ED50, TD50, and PI Values in Mice.
PI d
a
c
ED50 scPTZ b (mg/kg)
Comp.
ED50 MES (mg/kg)
TD50 (mg/kg)
MES
scPTZ
5i
5j
CBZ
VPA
50.8 (37.0–69.8) e
54.8 (36.7–81.8)
11.8 (8.5–16.4)
76.0 (65.9–87.7)
52.8 (45.8–60.9)
>100
353.5 (309.3–404.1)
491.0 (429.5–561.2)
76.1 (55.8–103.7)
6.96
8.96
6.45
1.72
4.65
9.30
<0.76
1.56
216.9 (207.5–226.3)
239.4 (209.2–274.1)
372.9 (356.0–389.8)
a
ED50: median effective dose affording anticonvulsant protection in 50% of animals; b scPTZ: subcutaneous
c
pentylenetetrazole seizure test; TD50: median toxic dose eliciting minimal neurological toxicity in 50% of
animals; d PI: protective index (TD50/ED50); e 95% confidence intervals given in parentheses.

Molecules 2016, 21, 164
6 of 13
3. Experimental Procedures
3.1. General Information
Melting points were determined in open capillary tubes and were uncorrected. IR spectra were
1
recorded (in KBr) on IR Prestige-21. H-NMR and 13C-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.
3.2. Chemistry
3.2.1. 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.
3.2.2. 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.
3.2.3. 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.
3.2.4. General Procedure for the Synthesis of 2-((1H-1,2,4-Triazol-3-yl)thio)-N-(6-alkoxybenzo[d]
thiazol-2-yl)acetamide (5a–m)
A
mixture of 2-chloro-N-(6-alkoxybenzo[d]thiazol-2-yl)acetamide
(4a
–
m
)
(5 mmol),
1,2,4-triazol-3-thiol (6 mmol), and sodium hydroxide (6 mmol) in N,N-dimethylformamide
(10 mL) was stirred at room temperature for 12 h. After removing half of the solvent, 50 mL of
water was added to the reaction mixture, which was then extracted with ethyl acetate (30 mL
ˆ
3).
The combined layer of ethyl acetate was dried over anhydrous MgSO4. Evaporation of the solvent
provided a crude product, which was recrystallized from dichloromethane to yield a white solid.
The yield, melting point, analytical data and spectral data of each compound are given below.
2-((1H-1,2,4-Triazol-3-yl)thio)-N-(6-methoxybenzo[d]thiazol-2-yl)acetamide (5a). White solid in 52.5%. mp:
˝
208–209 C. 1H-NMR (300 MHz, DMSO)
δ
14.02 (s, 1H, triazole –NH–), 12.57 (s, 1H, –CO–NH–), 8.50
(s, 1H, triazole =CH–), 7.65 (d, J = 8.8 Hz, 1H, Ar–H), 7.58 (d, J = 2.5 Hz, 1H, Ar–H), 7.03 (dd, J = 8.8,
2.6 Hz, 1H, Ar–H), 4.20 (s, 2H, –S–CH2–), 3.80 (s, 3H, –OCH3). 13C-NMR (75 MHz, DMSO)
167.44,
δ

Molecules 2016, 21, 164
7 of 13
156.18, 155.77, 145.31, 144.89, 142.60, 132.79, 121.21, 114.97, 104.71, 55.61, 35.07. IR (KBr) cm´1: 1605.02,
1556.65 (C=N). Tof-MS: m/z [M + H]+ 322.26.
2-((1H-1,2,4-Triazol-3-yl)thio)-N-(6-propoxybenzo[d]thiazol-2-yl)acetamide (5b). White solid in 65.9%. mp:
169–170 ˝C. 1H-NMR (300 MHz, DMSO)
δ 14.09 (s, 1H, triazole –NH–), 12.50 (s, 1H, –CO–NH–), 8.51
(s, 1H, triazole =CH–), 7.64 (d, J = 8.8 Hz, 1H, Ar–H), 7.55 (s, 1H, Ar-H), 7.03 (dd, J = 8.8, 2.5 Hz, 1H,
Ar–H), 4.19 (s, 2H, –S–CH2–), 3.97 (t, J = 6.4 Hz, 2H, –OCH2–), 1.75 (dd, J = 13.9, 6.8 Hz, 2H, –CH2–),
0.99 (t, J = 7.3 Hz, 3H, –CH3). 13C-NMR (75 MHz, DMSO)
δ 167.41, 155.73, 155.57, 145.55, 145.23, 142.52,
132.77, 121.19, 115.33, 105.41, 69.54, 35.04, 22.09, 10.43. IR (KBr) cm´1: 1605.75, 1550.35 (C=N). Tof-MS:
m/z [M + H]+ 350.15.
2-((1H-1,2,4-Triazol-3-yl)thio)-N-(6-butoxybenzo[d]thiazol-2-yl)acetamide (5c). White solid in 40.6%. mp:
˝
172–173 C. 1H-NMR (300 MHz, DMSO) δ 14.11 (s, 1H, triazole –NH–), 12.45 (s, 1H, –CO–NH–), 8.58
(s, 1H, triazole =CH–), 7.63 (d, J = 8.8 Hz, 1H, Ar–H), 7.55 (s, 1H, Ar–H), 7.02 (dd, J = 8.8, 2.5 Hz, 1H,
Ar–H), 4.18 (s, 2H, –S–CH2–), 4.01 (t, J = 6.4 Hz, 2H, –OCH2–), 1.80–1.64 (m, 2H, –CH2–), 1.54–1.36
(m, 2H, –CH2–), 0.94 (t, J = 7.3 Hz, 3H, –CH3). 13C-NMR (75 MHz, DMSO)
δ 167.39, 155.73, 155.64,
145.41, 145.13, 142.57, 132.81, 121.18, 115.35, 105.50, 67.84, 35.12, 30.81, 18.77, 13.67. IR (KBr) cm´1
:
1605.78, 1556.09 (C=N). Tof-MS: m/z [M + H]+ 363.74.
2-((1H-1,2,4-Triazol-3-yl)thio)-N-(6-(pentyloxy)benzo[d]thiazol-2-yl)acetamide (5d). White solid in 37.2%.
mp: 107–108 ˝C. 1H-NMR (300 MHz, DMSO)
14.07 (s, 1H, triazole –NH–), 12.46 (s, 1H, –CO–NH–),
δ
8.51 (s, 1H, triazole =CH–), 7.63 (d, J = 8.8 Hz, 1H, Ar–H), 7.56 (s, 1H, Ar–H), 7.02 (dd, J = 8.8, 2.5 Hz,
1H, Ar–H), 4.19 (s, 2H, –S–CH2–), 4.00 (t, J = 6.5 Hz, 2H, –OCH2–), 1.84–1.64 (m, 2H, –CH2–), 1.39
(dd, J = 15.1, 10.5 Hz, 4H, –CH2–), 0.90 (t, J = 7.0 Hz, 3H, –CH3). 13C-NMR (75 MHz, DMSO)
δ 167.41,
155.72, 155.59, 145.60, 145.26, 142.51, 132.77, 121.18, 115.32, 105.38, 68.03, 35.05, 28.43, 27.75, 21.91, 13.92.
IR (KBr) cm´1: 1606.70, 1550.71 (C=N). Tof-MS: m/z [M + H]+ 378.24.
2-((1H-1,2,4-Triazol-3-yl)thio)-N-(6-(hexyloxy)benzo[d]thiazol-2-yl)acetamide (5e). White solid in 51.6%.
mp: 118–119 ˝C. 1H-NMR (300 MHz, DMSO)
δ 14.10 (s, 1H, triazole –NH–), 12.45 (s, 1H, –CO–NH–),
8.47 (s, 1H, triazole =CH–), 7.63 (d, J = 8.8 Hz, 1H, Ar–H), 7.55 (d, J = 2.4 Hz, 1H, Ar–H), 7.01 (dd,
J = 8.8, 2.5 Hz, 1H, Ar–H), 4.18 (s, 2H, –S–CH2–), 3.99 (t, J = 6.5 Hz, 2H, –OCH2–), 1.84–1.62 (m, 2H,
–CH2–), 1.51–1.11 (m, 6H, –CH2–), 0.88 (t, J = 6.9 Hz, 3H, –CH3). 13C-NMR (75 MHz, DMSO)
δ 167.41,
155.73, 155.60, 145.38, 145.16, 142.55, 132.78, 121.19, 115.33, 105.58, 68.92, 35.09, 30.50, 28.85, 28.65, 22.11,
13.66. IR (KBr) cm´1: 1606.09, 1552.62 (C=N). Tof-MS: m/z [M + H]+ 391.99.
2-((1H-1,2,4-Triazol-3-yl)thio)-N-(6-(heptyloxy)benzo[d]thiazol-2-yl)acetamide (5f). White solid in 67.7%.
mp: 147–148 ˝C. 1H-NMR (300 MHz, DMSO)
δ 14.11 (s, 1H, triazole –NH–), 12.45 (s, 1H, –CO–NH–),
8.57 (s, 1H, triazole =CH–), 7.63 (d, J = 9.1 Hz, 1H, Ar–H), 7.55 (s, 1H, Ar–H), 7.02 (dd, J = 8.8, 2.5 Hz,
1H, Ar–H), 4.18 (s, 2H, –S–CH2–), 3.99 (t, J = 6.5 Hz, 2H, –OCH2–), 1.83–1.65 (m, 2H, –CH2–), 1.49–1.19
(m, 8H, –CH2–), 0.88 (t, J = 7.0 Hz, 3H, –CH3). 13C-NMR (75 MHz, DMSO)
δ 167.40, 155.74, 155.65,
145.08, 145.00, 142.58, 132.83, 121.18, 115.35, 105.51, 68.16, 35.14, 31.25, 28.76, 28.45, 25.52, 22.05, 13.91.
IR (KBr) cm´1: 1606.15, 1550.45 (C=N). Tof-MS: m/z [M + H]+ 406.09.
2-((1H-1,2,4-Triazol-3-yl)thio)-N-(6-(benzyloxy)benzo[d]thiazol-2-yl)acetamide (5g). White solid in 49.9%.
mp: 181–182 ˝C. 1H-NMR (300 MHz, DMSO)
δ 14.12 (s, 1H, triazole –NH–), 12.49 (s, 1H, –CO–NH–),
8.58 (s, 1H, triazole =CH–), 7.75–7.64 (m, 2H, Ar–H), 7.48 (d, J = 6.8 Hz, 2H, Ar–H), 7.41 (dd, J = 11.3,
4.4 Hz, 2H, Ar–H), 7.34 (dd, J = 8.3, 5.7 Hz, 1H, Ar–H), 7.11 (dd, J = 8.8, 2.6 Hz, 1H, Ar–H), 5.15 (s, 2H,
–OCH2–), 4.19 (s, 2H, –S–CH2–). 13C-NMR (75 MHz, DMSO)
δ 167.96, 156.34, 155.65, 146.36, 145.36,
143.23, 140.45, 137.42, 133.15, 128.91, 128.31, 121.72, 116.01, 106.41, 70.24, 35.40. IR (KBr) cm´1: 1605.62,
1551.67 (C=N). Tof-MS: m/z [M + H]+ 398.26.

Molecules 2016, 21, 164
8 of 13
2-((1H-1,2,4-Triazol-3-yl)thio)-N-(6-((2-fluorobenzyl)oxy)benzo[d]thiazol-2-yl)acetamide (5h). White solid
in 58.3%. mp: 164–165 ˝C. 1H-NMR (300 MHz, DMSO)
14.10 (s, 1H, triazole –NH–), 12.51 (s, 1H,
δ
–CO–NH–), 8.56 (s, 1H, triazole =CH–), 7.76–7.70 (m, 1H), 7.69–7.64 (m, 1H), 7.59 (t, J = 7.1 Hz, 1H,
Ar–H), 7.49–7.38 (m, 1H, Ar–H), 7.32–7.21 (m, 2H, Ar–H), 7.12 (dd, J = 8.8, 2.5 Hz, 1H, Ar–H), 5.18
(s, 2H, –OCH2–), 4.20 (s, 2H, –S–CH2–). 13C-NMR (75 MHz, DMSO)
δ 167.96, 160.90 (244.5 Hz), 156.48,
155.49, 146.96, 145.36, 143.41, 133.18, 131.29, 131.24, 127.62, 125.02, 121.74, 115.98 (5.3 Hz) 115.73, 106.45,
64.64, 35.47. IR (KBr) cm´1: 1606.04, 1552.16 (C=N). Tof-MS: m/z [M + H]+ 415.52.
2-((1H-1,2,4-Triazol-3-yl)thio)-N-(6-((3-fluorobenzyl)oxy)benzo[d]thiazol-2-yl)acetamide (5i). White solid
in 41.6%. mp: 171–172 ˝C. 1H-NMR (300 MHz, DMSO)
δ 14.11 (s, 1H, triazole –NH–), 12.39 (s, 1H,
–CO–NH–), 8.52 (s, 1H, triazole =CH–), 7.75–7.60 (m, 2H, Ar–H), 7.52–7.39 (m, 1H, Ar–H), 7.36–7.24
(m, 2H, Ar–H), 7.22–7.06 (m, 2H, Ar–H), 5.18 (s, 2H, –OCH2–), 4.19 (s, 2H, –S–CH2–). 13C-NMR
(75 MHz, DMSO)
δ 167.82, 161.53 (243.75Hz), 156.39, 155.58, 146.32, 145.89, 143.62, 140.92, 133.28,
131.70, 123.25, 121.69, 115.82, 115.42 (21.5Hz), 115.24 (6.8 Hz), 106.61, 66.87, 35.52. IR (KBr) cm´1
:
1605.21, 1553.98 (C=N). Tof-MS: m/z [M + H]+ 415.68.
2-((1H-1,2,4-Triazol-3-yl)thio)-N-(6-((4-fluorobenzyl)oxy)benzo[d]thiazol-2-yl)acetamide (5j). White solid
in 43.2%. mp: 206–207 ˝C. 1H-NMR (300 MHz, DMSO)
14.09 (s, 1H, triazole –NH–), 12.43 (s, 1H,
δ
–CO–NH–), 8.46 (s, 1H, triazole =CH–), 7.77–7.61 (m, 2H, Ar–H), 7.58–7.42 (m, 2H, Ar–H), 7.33–7.17
(m, 2H, Ar–H), 7.15–7.04 (m, 1H), 5.13 (s, 2H, –OCH2–), 4.20 (s, 2H, –S–CH2–). 13C-NMR (75 MHz,
DMSO)
δ 167.62, 161.80 (242.3 Hz) 156.72, 156.15, 155.08, 146.10, 142.88, 133.19, 132.76, 130.08 (8.3 Hz),
121.24, 115.55, 115.26 (d, J = 21.4 Hz), 106.02, 69.13, 35.16. IR (KBr) cm´1: 1604.63, 1554.77 (C=N).
Tof-MS: m/z [M + H]+ 415.57.
2-((1H-1,2,4-Triazol-3-yl)thio)-N-(6-((2-chlorobenzyl)oxy)benzo[d]thiazol-2-yl)acetamide (5k). White solid
in 33.5%. mp: 198–199 ˝C. 1H-NMR (300 MHz, DMSO)
δ 14.11 (s, 1H, triazole –NH–), 12.51 (s, 1H,
–CO–NH–), 8.56 (s, 1H, triazole =CH–), 7.75–7.61 (m, 3H, Ar–H), 7.58–7.50 (m, 1H, Ar–H), 7.44–7.35
(m, 2H, Ar–H), 7.13 (dd, J = 8.8, 2.4 Hz, 1H, Ar–H), 5.20 (s, 2H, –OCH2–), 4.20 (s, 2H, –S–CH2–).
13C-NMR (75 MHz, DMSO)
δ 167.52, 156.13, 155.06, 146.21, 144.96, 143.05, 134.27, 132.82, 132.72, 130.18,
129.87, 129.39, 127.34, 121.32, 115.48, 106.10, 67.55, 35.10. IR (KBr) cm´1: 1604.77, 1558.48 (C=N).
Tof-MS: m/z [M + H]+ 431.54.
2-((1H-1,2,4-Triazol-3-yl)thio)-N-(6-((3-chlorobenzyl)oxy)benzo[d]thiazol-2-yl)acetamide (5l). White solid
in 54.3%. mp: 172–173 ˝C. 1H-NMR (300 MHz, DMSO)
δ 14.10 (s, 1H, triazole –NH–), 12.52 (m, 1H,
–CO–NH–), 8.55 (s, 1H, triazole =CH–), 7.74–7.62 (m, 2H, Ar–H), 7.55 (s, 1H, Ar–H), 7.50–7.34 (m, 3H,
Ar–H), 7.17–7.07 (m, 1H, Ar–H), 5.17 (s, 2H, –OCH2–), 4.20 (s, 2H, –S–CH2–). 13C-NMR (75 MHz,
DMSO)
δ 167.52, 156.04, 154.96, 145.53, 145.06, 142.96, 139.56, 133.17, 132.77, 130.33, 127.79, 127.40,
126.24, 121.30, 115.53, 106.09, 68.91, 35.10. IR (KBr) cm´1: 1605.28, 1557.79 (C=N). Tof-MS: m/z
[M + H]+ 431.49.
2-((1H-1,2,4-Triazol-3-yl)thio)-N-(6-((3-(trifluoromethyl)benzyl)oxy)benzo[d]thiazol-2-yl)acetamide
(5m).
White solid in 46.8%. mp: 182–183 ˝C. 1H-NMR (300 MHz, DMSO)
δ
14.15 (s, 1H, triazole –NH–),
12.54 (s, 1H, –CO–NH–), 8.53 (s, 1H, triazole =CH–), 7.90–7.77 (m, 2H, Ar–H), 7.75–7.57 (m, 4H, Ar–H),
7.21–7.09 (m, 1H, Ar–H), 5.26 (s, 2H, –OCH2–), 4.20 (s, 2H, –S–CH2–). 13C-NMR (75 MHz, DMSO)
δ
167.51, 156.09, 154.99, 146.22, 146.20, 145.86, 144.67, 143.05, 138.58, 132.80, 131.71, 129.56, 129.06,
124.32 (dd, J = 36.0, 3.3 Hz). 121.31, 115.55, 106.26, 69.08, 35.14. IR (KBr) cm´1: 1606.27, 1556.18 (C=N).
Tof-MS: m/z [M + H]+ 465.42.

Molecules 2016, 21, 164
9 of 13
3.2.5. General Procedure for the Synthesis of N-(6-Alkoxybenzo[d]thiazol-2-yl)-2-(1H-imidazol-1-yl)
acetamide (6a–b)
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 mL
Evaporation of the solvent yielded a crude product, which was recrystallized from dichloromethane to
yield a white solid.
ˆ
3). The ethyl acetate layer was dried over anhydrous MgSO4.
N-(6-((3-Fluo˝robenzyl)oxy)benzo[d]thiazol-2-yl)-2-(1H-imidazol-1-yl)acetamide (6a). White solid in 47.1%.
mp: 224–225 C. 1H-NMR (300 MHz, DMSO)
δ 12.55 (s, 1H, –CO–NH–), 7.72–7.62 (m, 3H, imidazole–H
and Ar–H), 7.45 (dd, J = 14.3, 7.4 Hz, 1H, Ar–H), 7.35–7.26 (m, 2H, Ar–H), 7.24–7.08 (m, 3H, imidazole–H
and Ar–H), 6.91 (s, 1H, imidazole–H), 5.17 (s, 2H, –OCH2–), 5.08 (s, 2H, imidazole –CH2–).13C-NMR
(75 MHz, DMSO)
δ 166.89, 162.56 (d, J = 243.5 Hz), 156.02, 155.63, 143.32, 140.38 (d, J = 7.5 Hz),
138.96, 133.19, 130.89 (d, J = 8.3 Hz), 128.58, 124.56, 121.87, 121.35, 116.08, 115.12 (d, J = 21.2 Hz),
114.95 (d, J = 22.5 Hz), 106.61, 69.46, 49.89. IR (KBr) cm´1: 1604.77, 1546.91 (C=N). Tof-MS: m/z
[M + H]+ 383.05.
N-(6-((4-Fluorobenzyl)oxy)benzo[d]thiazol-2-yl)-2-(1H-imidazol-1-yl)acetamide (6b). White solid in 56.0%.
˝
mp: 246–247 C. 1H-NMR (300 MHz, DMSO)
δ 12.52 (s, 1H, –CO–NH–), 7.73–7.63 (m, 3H, imidazole–H
and Ar–H), 7.60–7.46 (m, 2H, Ar–H), 7.27–7.19 (m, 2H, Ar–H), 7.14–7.08 (m, 1H, Ar–H), 7.04 (s, 1H,
imidazole–H), 6.92 (s, 1H, imidazole–H), 5.12 (s, 2H, –OCH2–), 5.09 (s, 2H, imidazole –CH2–). 13C-NMR
(75 MHz, DMSO)
δ 167.49, 162.26 (d, J = 243.7 Hz), 156.18, 155.61, 143.23, 138.88, 133.63, 133.18, 130.53
´1
(d, J = 8.3 Hz), 128.46, 121.77, 121.37, 116.09, 115.72 (d, J = 21.4 Hz), 106.51, 69.56, 48.99. IR (KBr) cm
:
1604.95, 1557.26 (C=N). Tof-MS: m/z [M + H]+ 383.00.
3.2.6. General Procedure for the Synthesis of N-(6-Alkoxybenzo[d]thiazol-2-yl)-2-(1H-1,2,4-triazol-1-yl)
acetamide (7a–b)
2-Chloro-N-(6-alkoxybenzo[d]thiazol-2-yl)acetamide (5 mmol) and sodium 1,2,4-triazole (6 mmol)
were dissolved in N,N-dimethylformamide (10 mL) and stirred for 10 h at room temperature. Half of the
solvent was then evaporated, the solution was poured in 50 mL water, and extracted with ethyl acetate
(30 mL
ˆ
3). The ethyl acetate layer was dried over anhydrous MgSO4. After removing the solvent
under reduced pressure, the crude product was obtained and recrystallized from dichloromethane to
yield a white solid.
N-(6-((3-Fluorobenzyl)oxy)benzo[d]thiazol-2-yl)-2-(1H-1,2,4-triazol-1-yl)acetamide (7a). White solid in
˝
55.1%. mp: 212–213 C. 1H-NMR (300 MHz, DMSO)
δ 12.73 (s, 1H, –CO–NH–), 8.59 (s, 1H, triazole–H),
8.03 (s, 1H, triazole–H), 7.77–7.64 (m, 2H, Ar–H), 7.52–7.40 (m, 1H, Ar–H), 7.37–7.27 (m, 2H, Ar–H),
7.23–7.10 (m, 2H, Ar–H), 5.33 (s, 2H, triazole –CH2–), 5.18 (s, 2H, –OCH2–). 13C-NMR (75 MHz, DMSO)
δ
166.31, 162.67 (d, J = 243.8 Hz), 156.09, 155.57, 152.04, 146.23, 142.99, 140.38 (d, J = 7.5 Hz), 133.21,
130.94 (d, J = 8.4 Hz), 124.07 (d, J = 2.6 Hz), 121.88, 116.15, 115.08 (d, J = 21.0 Hz), 114.77 (d, J = 21.8 Hz),
106.59, 69.42, 51.57. IR (KBr) cm´1: 1605.72, 1556.78 (C=N). Tof-MS: m/z [M + H]+ 383.65.
N-(6-((4-Fluorobenzyl)oxy)benzo[d]thiazol-2-yl)-2-(1H-1,2,4-triazol-1-yl)acetamide (7b). White solid in
˝
39.5%. mp: 227–228 C. 1H-NMR (300 MHz, DMSO)
δ 12.72 (s, 1H, –CO–NH–), 8.59 (s, 1H, triazole–H),
8.04 (s, 1H, triazole–H), 7.69 (dd, J = 5.6, 3.0 Hz, 2H, Ar–H), 7.53 (dd, J = 8.5, 5.7 Hz, 2H, Ar–H), 7.23
(t, J = 8.9 Hz, 2H, Ar–H), 7.12 (dd, J = 8.9, 2.5 Hz, 1H, Ar–H), 5.33 (s, 2H, triazole –CH2–), 5.13 (s, 2H,
–OCH2–). 13C-NMR (75 MHz, DMSO)
δ 166.26, 162.27(d, J = 242.25 Hz), 155.92, 155.70, 152.04, 146.22,
143.31, 133.65, 133.23, 130.54 (d, J = 8.3 Hz), 121.89, 115.72 (d, J = 21.4 Hz), 115.58, 106.52, 69.56, 51.56.
IR (KBr) cm´1: 1605.71, 1556.18 (C=N). Tof-MS: m/z [M + H]+ 383.57.

Molecules 2016, 21, 164
10 of 13
3.2.7. General Procedure for the Synthesis of N-(6-Alkoxybenzo[d]thiazol-2-yl)-2-(1H-tetrazol-1-yl)
acetamide (8a–b)
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 mL
The ethyl acetate layer was dried over anhydrous MgSO4. Evaporation of the solvent provided a crude
product, which was recrystallized from dichloromethane to obtain a white solid.
ˆ
3).
N-(6-((3-Fluorobenzyl)oxy)benzo[d]thiazol-2-yl)-2-(1H-tetrazol-1-yl)acetamide (8a). White solid in 48.3%.
mp: 205–206 ˝C. 1H-NMR (300 MHz, DMSO)
δ
12.86 (s, 1H, –CO–NH–), 9.45 (s, 1H, tetrazole–H),
7.76–7.65 (m, 2H, Ar–H), 7.49–7.40 (m, 1H, Ar–H), 7.32 (s, 1H, Ar–H), 7.30 (s, 1H, Ar–H), 7.21–7.11
(m, 2H, Ar–H), 5.67 (s, 2H, tetrazole–CH2–), 5.18 (s, 2H, –OCH2–). 13C-NMR (75 MHz, DMSO)
165.43,
162.66 (d, J = 243.6 Hz), 156.18, 155.60, 145.76, 143.13, 140.36 (d, J = 7.5 Hz), 133.18, 130.95 (d, J = 8.3 Hz),
δ
´1
124.10, 121.88, 116.19, 115.10 (d, J = 21.2 Hz), 114.78 (d, J = 22.1 Hz), 106.62, 69.43, 50.11. IR (KBr) cm
:
1606.36, 1556.98 (C=N). Tof-MS: m/z [M + H]+ 382.87.
N-(6-((4-Fluorobenzyl)oxy)benzo[d]thiazol-2-yl)-2-(1H-tetrazol-1-yl)acetamide (8b). White solid in 68.4%.
mp: 224–225 ˝C. 1H-NMR (300 MHz, DMSO)
12.86 (s, 1H, –CO–NH–), 9.46 (s, 1H, tetrazole–H),
7.69 (s, 2H, Ar–H), 7.59–7.47 (m, 2H, Ar–H), 7.23 (t, J = 8.6 Hz, 2H, Ar–H), 7.13 (dd, J = 9.0, 1.6 Hz,
1H, Ar–H), 5.68 (s, 2H, tetrazole–CH2–), 5.13 (s, 2H, –OCH2–). 13C-NMR (75 MHz, DMSO)
166.30,
δ
δ
162.26 (d, J = 243.5Hz), 156.09, 155.58, 145.76, 143.13, 133.66, 133.23, 130.60 (d, J = 8.3 Hz), 121.90,
116.09, 115.69 (d, J = 21.2 Hz), 106.60, 69.56, 50.95. IR (KBr) cm´1: 1606.01, 1555.99 (C=N). Tof-MS: m/z
[M + H]+ 382.99.
3.2.8. General Procedure for the Synthesis of 2-(3-Amino-1H-1,2,4-triazol-1-yl)-N-(6-alkoxybenzo[d]
thiazol-2-yl)acetamide (9a–b)
1H-1,2,4-triazol-3-amine (6 mmol), 2-chloro-N-(6-alkoxybenzo[d]thiazol-2-yl) acetamide (5 mmol),
and sodium hydroxide (6 mmol) in N,N-dimethylformamide (10 mL) were dissolved and stirred
at room temperature for 8 h. 50 mL of water was added to the reaction mixture, which was then
extracted with ethyl acetate (30 mL
MgSO4. After removing the solvent under reduced pressure, the crude product was obtained and then
recrystallized from dichloromethane to yield a white solid.
ˆ
3). The combined layer of ethyl acetate was dried over anhydrous
2-(3-Amino-1H-1,2,4-triazol-1-yl)-N-(6-((3-fluorobenzyl)oxy)benzo[d]thiazol-2-yl)acetamide (9a). White solid
˝
1
in 66.1%. mp: 235–236 C. H-NMR (300 MHz, DMSO)
δ 12.58 (s, 1H, –CO–NH–), 7.70 (s, 1H,
triazole–H), 7.69–7.65 (m, 1H, Ar–H), 7.50–7.41 (m, 1H, Ar–H), 7.37 (s, 1H, Ar–H), 7.34–7.26 (m, 2H,
Ar–H), 7.21–7.09 (m, 2H, Ar–H), 6.33 (s, 2H, triazole–NH2), 5.18 (s, 2H, –OCH2–), 4.93 (s, 2H, triazole
–CH2–). 13C-NMR (75 MHz, DMSO)
δ 166.72, 162.67 (d, J = 243.7 Hz), 156.25, 155.49, 146.61, 145.55,
143.32, 140.39 (d, J = 7.4 Hz), 133.19, 130.93 (d, J = 8.3 Hz), 124.06 (d, J = 2.6 Hz), 121.80, 116.07, 115.07
(d, J = 21.3 Hz), 114.76 (d, J = 21.9 Hz), 106.57, 69.42, 49.12. IR (KBr) cm´1: 1604.63, 1557.39 (C=N).
Tof-MS: m/z [M + H]+ 398.68.
2-(3-Amino-1H-1,2,4-triazol-1-yl)-N-(6-((4-fluorobenzyl)oxy)benzo[d]thiazol-2-yl)acetamide (9b). White solid
in 60.2%. mp: 249–250 ˝C. 1H-NMR (300 MHz, DMSO)
δ 12.51 (s, 1H, –CO–NH–), 7.72–7.64 (m, 2H,
tetrazole–H and Ar–H), 7.57–7.48 (m, 2H, Ar–H), 7.37 (s, 1H, Ar–H), 7.23 (t, J = 8.7 Hz, 2H, Ar–H),
7.11 (d, J = 9.0 Hz, 1H, Ar–H), 6.34 (s, 2H, triazole–NH2), 5.12 (s, 2H, –OCH2–), 4.93 (s, 2H, triazole
–CH2–). 13C-NMR (75 MHz, DMSO)
δ 167.26, 162.68 (d, J = 243.8 Hz), 156.25, 155.58, 146.81, 145.69,
143.23, 133.68, 133.20, 130.93 (d, J = 8.3 Hz), 121.80, 116.08, 115.70 (d, J = 21.3 Hz), 106.60, 69.55, 50.18.
IR (KBr) cm´1: 1605.88, 1556.08 (C=N). Tof-MS: m/z [M + H]+ 398.85.

Molecules 2016, 21, 164
11 of 13
3.3. 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].
3.3.1. 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.
3.3.2. 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 (ED50), which showed 50%
protection, were recorded.
3.3.3. 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.
4. Conclusions
In the present study, we described the synthesis and anticonvulsant activity evaluation of
2-((1H-1,2,4-triazol-3-yl)thio)-N-(6-alkoxybenzo[d]thiazol-2-yl) acetamide (5a
series of derivatives were also synthesized and evaluated for their anticonvulsant activity for SAR
studies, which included N-(6-alkoxybenzo[d]thiazol-2-yl)-2-(1H-imidazol-1-yl)acetamide (6a ),
N-(6-alkoxybenzo[d]thiazol-2-yl)-2-(1H-1,2,4-triazol-1-yl)acetamide (7a–b), N-(6-alkoxybenzo[d]thiazol2-yl)
-2-(1H-tetrazol-1-yl)acetamide (8a ), and 2-(3-amino-1H-1,2,4-triazol-1-yl)-N-(6-alkoxybenzo[d]thiazol
-2-yl)acetamide (9a ). All the compounds were tested for their anticonvulsant activity and
–m). In addition, several
–
b
–
b
–
b
neurotoxicity using the MES and rotarod tests at the dose of 100 mg/kg after i.p. Bioevaluation
demonstrated that the compounds possessing 1,2,4-triazole-3-thiol displayed the best anticonvulsant
activity and favorable PI. Among them, compounds 5i and 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 presented markedly decreased neurotoxicity and, therefore,
a higher protective index. Especially, compound 5j showed high PI values of 8.96 in the MES test and
9.30 in the scPTZ test, which were better than the standard drugs, carbamazepine and valproic acid,
used as positive controls in this study.
Acknowledgments: This work was supported by the National Natural Science Foundation of China (No. 81160382
and No. 81160409) and National Science and Technology Major Project of China (No. 2012ZX09103-101-044).
Author Contributions: Zhe-Shan Quan designed the compounds, planned synthetic pathways, Da-Chuan
Liu performed chemical synthesis, biological testing, analyzed the results of biological testing and wrote the
manuscript. Chun-Mei Jin and Hong-Jian Zhang performed part of chemical synthesis and biological testing.
All authors commented on the manuscript.

Molecules 2016, 21, 164
12 of 13
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
Moshé, S.L.; Perucca, E.; Ryvlin, P.; Tomson, T. Epilepsy: New advances. Lancet 2015, 385, 884–898. [CrossRef]
Van Nieuwenhuyse, B.; Raedt, R.; Sprengers, M.; Dauwe, I.; Gadeyne, S.; Carrette, E.; Delbeke, J.;
Wadman, W.J.; Boon, P.; Vonck, K. The systemic kainic acid rat model of temporal lobe epilepsy: Long-term
EEG monitoring. Brain Res. 2015, 1627, 1–11. [CrossRef] [PubMed]
3.
4.
The Lancet. A voice for people with epilepsy. Lancet 2015, 385, 482.
Deng, X.Q.; Song, M.X.; Zheng, Y.; Quan, Z.S. Design, synthesis and evaluation of the antidepressant and
anticonvulsant activities of triazole-containing quinolinones. Eur. J. Med. Chem. 2014, 73, 217–224. [CrossRef]
[PubMed]
5.
Sarigol, D.; Uzgoren-Baran, A.; Tel, B.C.; Somuncuoglu, E.I.; Kazkayasi, I.; Ozadali-Sari, K.; Unsal-Tan, O.;
Okay, G.; Ertan, M.; Tozkoparan, B. Novel thiazolo[3,2 b]-1,2,4-triazoles derived from naproxen with
´
analgesic/anti-inflammatory properties: Synthesis; biological evaluation and molecular modeling studies.
Bioorg. Med. Chem. 2015, 23, 2518–2528. [CrossRef] [PubMed]
6.
7.
Pandey, R.; Ahmad, Z.; Sharma, S.; Khuller, G.K. Nano-encapsulation of azole antifungals: Potential
applications to improve oral drug delivery. Int. J. Pharm. 2005, 301, 268–276. [CrossRef] [PubMed]
Barradas, J.S.; Errea, M.I.; D’Accorso, N.B.; Sepúlveda, C.S.; Talarico, L.B.; Damonte, E.B. Synthesis and
antiviral activity of azoles obtained from carbohydrates. Carbohydr. Res. 2008, 343, 2468–2474. [CrossRef]
[PubMed]
8.
9.
Tyagi, P.; Chandra, S.; Saraswat, B.S.; Yadav, D. Design, spectral characterization; thermal; DFT studies
and anticancer cell line activities of Co(II); Ni(II) and Cu(II) complexes of Schiff bases derived from
4-amino-5-(pyridin-4-yl) 4H-1;2;4-triazole-3-thiol. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2015
145, 155–164. [CrossRef] [PubMed]
,
Khanmohammadi, H.; Abnosi, M.H.; Hosseinzadeh, A. Synthesis, biological and computational study of
new Schiff base hydrazones bearing 3-(4-pyridine)-5-mercapto-1,2,4-triazole moiety. Spectrochim. Acta A Mol.
Biomol. Spectrosc. 2008, 71, 1474–1480. [CrossRef] [PubMed]
10. Hussein, W.M.; Vella, P.; Islam, N.U.; Ollis, D.L.; Schenk, G.; McGeary, R.P. 3-Mercapto-1,2,4-triazoles and
N-acylated thiosemicarbazides as metallo- -lactamase inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 380–386.
β
11. Agarwal, S.; Agarwal, D.K.; Gautam, N.; Agarwal, K.; Gautam, D.C. Synthesis and in vitro antimicrobial
evaluation of benzothiazole incorporated thiazolidin-4-ones derivatives. J. Korean Chem. Soc. 2014, 58, 33–38.
[CrossRef]
12. Singh, M.K.; Tilak, R.; Nath, G.; Awasthi, S.K.; Agarwal, A. Design, synthesis and antimicrobial activity of
novel benzothiazole analogs. Eur. J. Med. Chem. 2013, 63, 635–644. [CrossRef] [PubMed]
13. Stone, E.L.; Citossi, F.; Singh, R.; Kaur, B.; Gaskell, M.; Farmer, P.B.; Monks, A.; Hose, C.; Stevens, M.F.;
Leong, C.O.; et al. Antitumour benzothiazoles. Part 32: DNA adducts and double strand breaks correlate
with activity; synthesis of 5F203 hydrogels for local delivery. Bioorg. Med. Chem. 2015, 23, 6891–6899.
[CrossRef] [PubMed]
14. Ma, J.; Bao, G.; Wang, L.; Li, W.; Xu, B.; Du, B.; Lv, J.; Zhai, X.; Gong, P. Design, synthesis, biological evaluation
and preliminary mechanism study of novel benzothiazole derivatives bearing indole-based moiety as potent
antitumor agents. Eur. J. Med. Chem. 2015, 96, 173–186. [CrossRef] [PubMed]
15. Ugale, V.G.; Patel, H.M.; Wadodkar, S.G.; Bari, S.B.; Shirkhedkar, A.A.; Surana, S.J. Quinazolino-benzothiazoles:
Fused pharmacophores as anticonvulsant agents. Eur. J. Med. Chem. 2012, 53, 107–113. [CrossRef] [PubMed]
16. Siddiqui, N.; Rana, A.; Khan, S.A.; Bhat, M.A.; Haque, S.E. Synthesis of benzothiazole semicarbazones as
novel anticonvulsants—The role of hydrophobic domain. Bioorg. Med. Chem. Lett. 2007, 17, 4178–4182.
[CrossRef] [PubMed]
17. Hassan, M.Z.; Khan, S.A.; Amir, M. Design; synthesis and evaluation of N-(substituted
benzothiazol-2-yl)amides as anticonvulsant and neuroprotective. Eur. J. Med. Chem. 2012, 58, 206–213.
[CrossRef] [PubMed]
18. Tripathi, L.; Kumar, P.; Singh, R.; Stables, J.P. Design, synthesis and anticonvulsant evaluation of novel
N-(4-substituted phenyl)-2-[4-(substituted) benzylidene]-hydrazinecarbothio amides. Eur. J. Med. Chem.
2012, 47, 153–166. [CrossRef] [PubMed]

Molecules 2016, 21, 164
13 of 13
19. Liu, D.C.; Deng, X.Q.; Wang, S.B.; Quan, Z.S. Synthesis and anticonvulsant activity evaluation of
7-alkoxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-ones. Arch. Pharm. Chem. Life Sci. 2014, 347, 268–275.
[CrossRef] [PubMed]
20. Behbehani, H.; Ibrahim, H.M. 4-Thiazolidinones in heterocyclic synthesis: Synthesis of novel enaminones,
azolopyrimidines and 2-arylimino-5-arylidene-4-thiazolidinones. Molecules 2012, 17, 6362–6385. [CrossRef]
[PubMed]
21. Abdelazeem, A.H.; Gouda, A.M.; Omar, H.A.; Tolba, M.F. Design; synthesis and biological evaluation of
novel diphenylthiazole-based cyclooxygenase inhibitors as potential anticancer agents. Bioorg. Chem. 2014
57, 132–141. [CrossRef] [PubMed]
,
22. Zhang, Y.L.; Yang, K.W.; Zhou, Y.J.; La Curan, A.E.; Oelschlaeger, P.; Crowder, M.W. Diaryl-substituted
azolylthioacetamides: Inhibitor discovery of New Delhi metallo-β-lactamase-1 (NDM-1). ChemMedChem
2014, 9, 2445–2448. [CrossRef] [PubMed]
23. Yang, G.F.; Xu, L.; Lu, A.H. Synthesis and bioactivity of novel triazolo [1,5 a] pyrimidine derivatives.
´
Heteroatom Chem. 2001, 12, 491–496. [CrossRef]
24. Deb, P.K.; Kaur, R.; Chandrasekaran, B.; Bala, M.; Gill, D.; Kaki, V.R.; Akkinepalli, R.R.; Mailavaram, R.
Synthesis, anti-inflammatory evaluation, and docking studies of some new thiazole derivatives.
Med. Chem. Res. 2014, 23, 2780–2792. [CrossRef]
25. Papadopoulou, M.V.; Bloomer, W.D.; Rosenzweig, H.S.; Chatelain, E.; Kaiser, M.; Wilkinson, S.R.;
McKenzie, C.; Ioset, J.R. Novel 3-nitro-1H-1,2,4-triazole- based amides and sulfonamides as potential
antitrypanosomal agents. J. Med. Chem. 2012, 55, 5554–5565. [CrossRef] [PubMed]
26. Rogawski, M.A. Molecular targets versus models for new antiepileptic drug discovery. Epilepsy Res. 2006
68, 22–28. [CrossRef] [PubMed]
,
27. Castel-Branco, M.M.; Alves, G.L.; Figueiredo, I.V.; Falcão, A.C.; Caramona, M.M. The maximal electroshock
seizure (MES) model in the preclinical assessment of potential new antiepileptic drugs. Methods Find. Exp.
Clin. Pharmacol. 2009, 31, 101–106. [CrossRef] [PubMed]
28. Löscher, W. Critical review of current animal models of seizures and epilepsy used in the discovery and
development of new antiepileptic drugs. Seizure 2011, 20, 359–368. [CrossRef] [PubMed]
29. Krall, R.L.; Penry, J.K.; White, B.G.; Kupferberg, H.J.; Swinyard, E.A. Antiepileptic drug development: II.
Anticonvulsant drug screening. Epilepsia 1978, 19, 409–428. [CrossRef] [PubMed]
30. Porter, R.J.; Cereghino, J.J.; Gladding, G.D.; Hessie, B.J.; Kupferberg, H.J.; Scoville, B.; White, B.G.
Antiepileptic Drug Development Program. Cleve. Clin. Q. 1984, 51, 293–305. [CrossRef] [PubMed]
Sample Availability: Samples of all the target compounds are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).