Synthesis of 5-alkoxythieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine derivatives and evaluation of their anticonvulsant activities

Article abstract of DOI:10.3390/molecules20046827

This work concerns the design and synthesis of novel, substituted 5-alkoxythieno[2,3-e][1,2,4]triazolo[4,3-c ]pyrimidine derivatives 5a-p prepared from 3-amino-2-thiophenecarboxylic acid methyl ester. The final compounds were screened for their in vivo anticonvulsant activity using maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) tests. Neurotoxicity (NT) was tested using a rotarod test. The structure-anticonvulsant activity relationship analysis revealed that the most effective structural motif involves a substituted phenol, especially when substituted with a single chlorine, fluorine or trifluoromethyl group (at the meta-position), or two chlorine atoms. These molecules possessed high activity according to the MES and scPTZ models. Quantitative assessment of the compounds after intraperitoneal administration in mice showed that the most active compound was 5-[3-(trifluoromethyl)phenoxy]thieno[2,3-e] [1,2,4]triazolo[4,3-c]pyrimidine (5o) with ED₅₀ values of 11.5 mg/kg (MES) and 58.9 mg/kg (scPTZ). Furthermore, compound 5o was more effective in the MES and scPTZ tests than the well-known anticonvulsant drugs carbamazepine and ethosuximide.

Full text of DOI:10.3390/molecules20046827

Molecules 2015, 20, 6827-6843; doi:10.3390/molecules20046827
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of 5-Alkoxythieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine
Derivatives and Evaluation of Their Anticonvulsant Activities
Shi-Ben Wang, Guang-Chun Piao, Hong-Jian Zhang 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 133000, China;
E-Mails: 2010020146@ybu.edu.cn (S.-B.W.); 2012010623@ybu.edu.cn (G.-C.P.);
2012001040@ybu.edu.cn (H.-J.Z.)
* Author to whom correspondence should be addressed; E-Mail: zsquan@ybu.edu.cn;
Tel./Fax: +86-433-243-6020.
Academic Editor: Derek J. McPhee
Received: 12 March 2015 / Accepted: 8 April 2015 / Published: 15 April 2015
Abstract: This work concerns the design and synthesis of novel, substituted
5-alkoxythieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine derivatives 5a–p prepared from
3-amino-2-thiophenecarboxylic acid methyl ester. The final compounds were screened for
their in vivo anticonvulsant activity using maximal electroshock (MES) and subcutaneous
pentylenetetrazole (scPTZ) tests. Neurotoxicity (NT) was tested using a rotarod test. The
structure-anticonvulsant activity relationship analysis revealed that the most effective
structural motif involves a substituted phenol, especially when substituted with a single
chlorine, fluorine or trifluoromethyl group (at the meta-position), or two chlorine atoms.
These molecules possessed high activity according to the MES and scPTZ models.
Quantitative assessment of the compounds after intraperitoneal administration in mice
showed that the most active compound was 5-[3-(trifluoromethyl)phenoxy]thieno[2,3-e]
[1,2,4]triazolo[4,3-c]pyrimidine (5o) with ED50 values of 11.5 mg/kg (MES) and 58.9 mg/kg
(scPTZ). Furthermore, compound 5o was more effective in the MES and scPTZ tests than
the well-known anticonvulsant drugs carbamazepine and ethosuximide.
Keywords: synthesis; triazole; maximal electroshock; neurotoxicity; pentylenetetrazole

Molecules 2015, 20
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1. Introduction
Epilepsy is one of the most common disorders of the human brain, affecting more than 60 million
individuals worldwide [1–3]. It has been observed that in as many as 25% of cases currently available
antiepileptic drugs (AEDs) are unable to control the seizures [4]. Additionally, in many cases the
clinical use of AEDs is restricted by their side effects such as gastrointestinal disturbances, gingival
hyperplasia, headaches, nausea, anorexia, ataxia, hepatotoxicity, drowsiness, attention deficit, and
cognitive problems [5–9]. Therefore, there is a ongoing need for the discovery of new chemical entities
for the development of effective and safer AEDs.
Triazole derivatives have attracted continued interest over the years because of their diverse
biological activities. These include anticonvulsant [10–13], antimicrobial [14,15] and enzyme
inhibition effects [16,17]. Previous research in our laboratory led to the discovery of a series of
derivatives of 5-alkoxytetrazolo[1,5-c]thieno[2,3-e]pyrimidine (I) having anticonvulsant activities [18].
The most effective among them was 5-(4-bromophenoxy)tetrazolo[1,5-c]thieno[2,3-e]pyrimidine,
which showed anticonvulsant activity at a dose of 100 mg/kg. Based on the anticonvulsant activity of
compound series I, we designed and synthesized a series of 5-alkoxythieno[2,3-e][1,2,4] triazolo[4,3-c]
pyrimidines 5a–p where one of the nitrogen atoms in the five-membered ring in the general structure
of compound series I was replaced with a carbon (Figure 1). To our great satusfaction, the
anticonvulsant activity of compounds 5a–p was greatly improved.
N
N
OR
N
N
S
Br
S
N
Ν
N
N
N
N
(dose:100 mg/kg)
I
Bioisosterism
Ring opening
N
N
OR
N
OR
N
OR
N
OR
N
N
N
S
N
N
S
S
N
S
N
N
N
N
N
N
5a-p
7a-d
9a-d
11a-d
Figure 1. Structures of compounds I and target compounds.
In order to discover whether a triazole ring that is not fused to the thieno[3,2-d]pyrimidine ring still
has antiepileptic activity, another series of 2-alkoxy-4-(1H-1,2,4-triazol-1-yl)thieno[3,2-d]pyrimidine
compounds 7a–d was designed and synthesized through the ring-opening of compounds 5a–p. For the
purposes of investigating the structure-activity relationship, the triazole ring in compounds 7a–d was
replaced by other heterocycles such as imidazole, or pyrazole rings to give 2-alkoxy-4-(1H-imidazol-1-yl)

Molecules 2015, 20
6829
thieno[3,2-d]pyrimidine 9a–d or 2-alkoxy-4-(1H-pyrazol-1-yl)thieno[3,2-d]pyrimidine derivatives 11a–d.
Their structures were characterized using IR, 1H-NMR, HRMS, and 13C-NMR techniques.
The anticonvulsant activity of these compounds was evaluated using the maximal electroshock
(MES) and subcutaneous pentylenetetrazole (scPTZ) tests in mice, and their neurotoxicity (NT) was
evaluated using the rotarod test.
2. Results and Discussion
2.1. Chemistry
The target compounds 5a–p were synthesized as shown in Scheme 1. The starting materials
3-amino-2-thiophenecarboxylic acid methyl ester and urea were heated at 200 °C for 1.5 h to obtain
compound 1. This compound reacted further by refluxing with POCl3 to yield compound 2. Compound 2
was reacted with hydrazine hydrate in methanol to afford the compound 3 in high yield [18].
Compound 4 was obtained via the cyclization of compound 3 with triethyl orthoformate [19]. Finally,
the target compounds 5a–p were obtained by reacting compound 4 with the appropriate substituted
phenols in acetonitrile in the presence of K2CO3.
N
Cl
N
OR
N
Cl
e
d
N
N
N
S
S
S
N
N
N
NHNH2
N
3
4
5a-p
NH2
c
S
N
Cl
N
N
OR
OR
OR
COOCH3
g
f
N
N
N
S
S
N
a
N
N
N
6
7a-d
H
N
Cl
N
N
Cl
N
O
b
h
i
N
N
N
S
S
NH
S
S
N
N
Cl
O
N
1
2
N
8
9a-d
N
Cl
N
j
k
N
N
S
S
N
N
N
N
10
11a-d
Reagents and conditions: (a) NH2CONH2, 180 °C, 1.5 h; (b) POCl3, 110 °C, 5 h; (c) NH2NH2.H2O,
50 °C, 1 h; (d) HC(OC2H5)3, reflux, 3 h; (e) ROH, CH3CN, reflux, 4 h; (f, h, j) 1,2,4-triazole/
imidazole/pyrazole, K2CO3, DMF, reflux, 3 h; (g, i, k) ROH, K2CO3, DMF, 80 °C, 10 h.
Scheme 1. Synthetic routes to the target compounds.

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When 2,4-dichlorothieno[3,2-d]pyrimidine were refluxed in N,N-dimethylformamide (DMF) with
different azoles such as triazole, imidazole and pyrazole in the presence of K2CO3 [20,21], the
4-chlorine atom of 1 was easily substituted by these heterocycles, producing the corresponding
thienopyrimidines 2-chloro-4-(1H-1,2,4-triazol-1-yl)thieno[3,2-d]pyrimidine (6), 2-chloro-4-(1H-
imidazol-1-yl)thieno[3,2-d]pyrimidine (8), and 2-chloro-4-(1H-pyrazol-1-yl)thieno[3,2-d]pyrimidine
(10). The target compounds 7a–d, 9a–d, and 11a–d were obtained by reacting compounds 6, 8, and 10
with a substituted phenol in DMF in the presence of K2CO3. The structures of the target compounds
were characterized by spectroscopic methods. All spectral data corroborated the assumed structures.
2.2. Pharmacology
2.2.1. Anticonvulsant Activity
The synthesized compounds were submitted to in vivo evaluation using the methods described in
the Antiepileptic Drug Development Program (ADD) of the US National Institutes of Health according
to previously described testing procedures [22,23]. The pharmacological evaluation was accepted by
the Ethics Commission of China. Primary anticonvulsant studies involved two tests: maximal
electroshock seizure (MES) and subcutaneous metrazol (scMET), in mice. It is emphasized that nearly
all clinically significant AEDs are effective in at least one of these two models, making them very
useful tools for initial high throughput screening of candidate anticonvulsants. The MES test employs
an electrical stimulus to induce generalized tonic clonic seizures and is used to identify compounds
that prevent the spread of seizures. The scMET model utilizes chemically induced myoclonic seizures
and recognizes the agents that are effective because they raise the seizure threshold. In addition to the
primary anticonvulsant evaluation in the MES and scMET models, the neurotoxicity was assessed
using a rotarod test.
The compounds were administered intraperitoneally to mice at doses of 30, 100, and 300 mg/kg and
tests were carried out 0.5 and 4 h after administration. The reference drugs, carbamazepine (for MES
and rotarod test) and ethosuximide (for scPTZ test) were used as positive controls. In preliminary
screening, all of the newly synthesized compounds exhibited some degree of anti-MES activity. The
protection offered by these compounds was indicative of their pharmacological ability to reduce
seizure spread at a certain dose level. The results obtained after investigating the anticonvulsant
activity of the synthesized compounds 5a–p, 7a–d, 9a–d, and 11a–d are summarized in Tables 1 and 2.
For the 5a–p series, all of the compounds were active in the MES test, which indicates their ability
to prevent seizure spread. At a dose of 100 mg/kg, most of the compounds showed protection, except
5a, 5j, and 5m–n. Five compounds, 5c–d, 5f, and 5o–p showed protection against MES-induced
seizures at a dose of 30 mg/kg. In these derivatives, only four compounds (5i, 5j, 5l, and 5n) did not
show activity 4 h after administration. Only two compounds showed significant neurotoxicity at a dose
of 100 mg/kg, and six compounds were not neurotoxic at a dose of 300 mg/kg. Compounds 5d, 5f, 5h,
and 5o–p were not neurotoxic at a dose of 300 mg/kg in 4 h.

Molecules 2015, 20
Table 1. Anticonvulsant activities of compounds 5a–p in MES and scPTZ tests.
6831
N
OR
N
OR
N
N
S
S
N
N
N
N
N
(R: p-Br)
5a-p
I
MES a
scPTZ b
TOX c
Compound
R
0.5 h 4 h
0.5 h
4 h
0.5 h
4 h
5a
5b
5c
5d
5e
5f
-C6H5
300
100
30
300
100
100
100
300
100
100
100
-
-
300
100
100
-
-
300
-
-
-C6H4(o-Cl)
-C6H4(p-Cl)
-C6H4(m-Cl)
-C6H4(o-F)
-
-
300
300
100
-
-
30
-
300
100
30
-
-
-C6H4(m-F)
-C6H4(p-F)
100
100
-
300
300
-
300
5g
5h
5i
100
100
100
300
100
100
300
300
30
300
-
-C6H4(o-CH3)
-C6H4(m-CH3)
-C6H4(p-CH3)
-C6H4(o-OCH3)
-C6H4(m-OCH3)
-C6H4(p-OCH3)
-C6H4(p-Br)
-C6H4(m-CF3)
-C6H3(2,4-Cl)
-
-
300
-
300
-
-
-
5j
-
-
-
300
-
-
5k
5l
100
-
300
-
-
-
-
300
-
-
-
5m
5n
5o
5p
I
300
-
-
-
-
-
300
-
300
300
100
-
-
100
100
300
100
100
-
300
300
-
30
100
-
a
Maximal electroshock: doses of 30, 100 and 300 mg/kg were administrated intraperitoneally in mice.
b
The animals were examined 0.5 h and 4 h after administration; Subcutaneous pentylenetetrazole test;
c Neurotoxicity screening—rotorod test; (-): no activity or neurotoxicity at 300 mg·kg−1.
As shown in Table 2, compound 7c showed anticonvulsant activity at a low dose of 30 mg/kg,
and four of the compounds showed no neurotoxicity after 0.5 h. Five compounds, 7b–c, 9c, and
11c–d showed protection against MES-induced seizures when tested 4 h after administration, and only
compounds 7a, 7c, and 9c were neurotoxic at a dose of 300 mg/kg after 4 h.
The anticonvulsant activity of the compounds according to the scMET model in mice was markedly
lower than that according to the MES screen. Among the compounds, five (5c, 5f–g, 5o, and 7c) were
active after both 0.5 h and 4 h. Only seven compounds (5c–d, 5f–g, and 5o–p) showed considerable
levels of seizure protection (after 0.5 h) at a dose of 100 mg/kg.
Based on the preliminary biological data, the most active compounds 5c, 5f, 5o, 5p, and 7c were
chosen for quantification of the pharmacological parameters (ED50 and TD50) after i.p. administration
to mice. The results for the new compounds along with the data for the standard AEDs (tested in the
same conditions), carbamazepine and ethosuximide are shown in Table 3.

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Table 2. Anticonvulsant activities of compounds 7a–d, 9a–d and 11a–d in MES and scPTZ tests.
N
N
OR
N
N
OR
N
OR
N
N
N
N
N
S
S
S
N
N
N
7a-d
9a-d
11a-d
MES
scPTZ
TOX
Compound
R
0.5 h
300
100
30
-
4 h
0.5 h
4 h
0.5 h
300
300
300
-
4 h
7a
7b
-C6H4(m-Cl)
-C6H4(m-F)
-
300
100
-
-
300
100
-
-
300
-
-
7c
-C6H4(m-CF3)
-C6H3(2,4-Cl )
-C6H4(m-Cl)
-C6H4(m-F)
300
300
7d
300
-
9a
-
-
300
-
-
300
300
300
-
-
9b
300
100
-
-
-
-
9c
-C6H4(m-CF3)
-C6H3(2,4-Cl )
-C6H4(m-Cl)
-C6H4(m-F)
300
-
300
300
-
-
300
9d
-
-
-
-
-
-
11a
11b
11c
11d
300
-
-
-
300
-
-
-
-
-
-C6H4(m-CF3)
-C6H3(2,4-Cl )
100
-
300
300
300
-
300
300
-
Table 3. Quantitative anticonvulsant data in mice (i.p.).
PI c
MES scPTZ
Compound
ED50 (MES) a
ED50 (scPTZ)
TD50 (NT) b
5c
18.5 (16.0–21.5) 85.2 (78.1–92.9)
294.6 (257.7–336.7)
328.6 (280.1–385.5)
204.6 (179.0–233.8)
112.3 (89.8–140.4)
237.1 (200.1–280.3)
76.1 (55.8–103.7)
343 (311–378)
15.8
10.3
17.7
8.7
3.4
<3.2
3.4
1.7
3.3
-
5f
31.7 (23.6–42.5)
11.5 (8.6–15.4)
>100
5o
58.9 (49.1–70.7)
5p
7c
12.9 (12.9–23.2) 65.5 (52.5–72.9)
30.4 (27.2–34.1)
11.8 (8.5–16.4)
–
71.6 (45.9–135)
–
7.8
Carbamazepine
6.4
Ethosuximide
106 (93–121)
3.2
a
ED50: median effective dose affording anticonvulsant protection in 50% of animals, the dose is measured
b
in mg/kg; TD50: median toxic dose eliciting minimal neurological toxicity in 50% of animals, the dose is
measured in mg/kg; c PI: protective index (TD50/ED50); A dash indicates means—not tested.
The analysis of the MES quantitative data revealed lower activity of 5c, 5f, 5p, and 7c than
carbamazepine, which is used as a reference antiepileptic drug in the MES model. At the same time, all
of these molecules provided higher anti-MES protection in comparison with ethosuximide. The most
effective compound according to the MES test was 5o with an ED50 of 11.5 mg/kg. Despite lower
activity in comparison with carbamazepine, 5o was less neurotoxic and yielded a more favorable protective
index (PI) of 17.7 than the reference drug’s PI of 6.4. Significantly, stronger activity was observed in
the PTZ-induced seizures. In this test, the most effective were 5c, 5o, and 7c with ED50 values and
protective indexes much more favorable than ethosuximide (model AED for PTZ-induced seizures).

Molecules 2015, 20
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To determine the oral time of peak effect (TPE) of compounds 5c and 5o, we conducted a time-course
test; compounds 5c and 5o reached the TPE at 1.5 h after oral administration. Next, we evaluated the
anticonvulsant activity of compounds 5c and 5o against MES-induced seizures and neurotoxicity after
oral administration to mice (Table 4), using carbamazepine as a reference. Compounds 5c and 5o
showed significant oral activity against MES-induced seizures in mice, with ED50 values of 39.4 mg/kg
and 28.4 mg/kg, respectively. The standard, carbamazepine, showed an ED50 value of 27.3 mg/kg and
a TD50 value of 328.6 mg/kg, resulting in a PI of 12.0 under the same conditions. Thus, compounds 5c
and 5o were judged safer than the anticonvulsant drug carbamazepine in MES and TOX models.
Table 4. Pharmacological evaluation of compound 5c and 5o and carbamazepine
administered orally to mice.
Compound
R
-C6H4(p-Cl)
-C6H4(m-CF3)
-
Time (h)
1.5
ED50 (MES)
39.4 (34.0–45.6)
28.4 (23.1–32.6)
27.3(19.7–37.9)
TD50 (NT)
PI
5c
5o
642.2 (609.7–689.6)
510.5 (484.4–648.5)
328.6 (229.9–469.7)
16.2
17.9
12.0
1.5
CBZ
1.5
2.2.2. Structure-Activity Relationship
In this study, we prepared four series of compounds and evaluated their preliminary anticonvulsant
activities, and we established the following structure-activity relationships (SARs). Analysis of the
anticonvulsant activities of compounds 5a–p resulted in the establishment of several SARs. Among the
phenoxy-substituted derivatives, the position of the substituent group on the benzene ring appeared to
greatly influence the anticonvulsant activity, especially with a chlorine, fluorine, trifluoromethyl group
(in the meta-position), or two chlorine atoms. These molecules showed high activity in the MES and/or
the scPTZ models. Changing the position of the chlorine atom to the ortho or para position as well as
introduction of methyl and methoxy groups decreased the anticonvulsant activity. The same effect was
observed for unsubstituted derivative 5a, which protects against MES seizures only at dose of 300
mg/kg. When the triazole ring of compounds 5c, 5f, 5o, and 5p was opened, their anticonvulsant
activities were weaker, and the PIs were lower for 7a–d compared to these compounds. When the
triazole ring in compounds 7a–d was replaced with other heterocycles (i.e., imidazole and pyrazole),
the resultant compounds 9a–d and 11a–d had slightly decreased activity as compared to the
compounds containing the triazole ring. These data indicate that separating the triazole from the
thieno[3,2-d]pyrimidine, instead of incorporating the triazole into the thieno[3,2-d]pyrimidine, leads to
weaker anticonvulsant activity and lower PI.
3. Experimental Section
3.1. General Information
Melting points were determined in open capillary tubes and were uncorrected. IR spectra were
1
13
recorded (in KBr) on an IRPrestige-21 (Perkin Elmer, Waltham, MA, USA). H-NMR and C-NMR
spectra were measured on an AV-300 spectrometer (Bruker, Flawil, Switzerland) with all chemical
shifts given in ppm relative to tetramethylsilane. Mass spectra were measured on an HP1100LC

Molecules 2015, 20
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(Agilent Technologies, Palo Alto, CA, USA) system. High-resolution mass spectra were measured on
a MALDI-TOF/TOF mass spectrometer (Bruker Daltonik, Bremen, Germany). The principal
chemicals were purchased from Aldrich Chemical (Shenyang, China).
3.2. Chemistry
3.2.1. Synthesis of Thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione (1)
3-Amino-2-thiophenecarboxylic acid methyl ester (1 g, 6.36 mmol) and urea (3 g, 50 mmol) were
placed in a round bottomed flask, and the mixture was heated at 200 °C for 1.5 h. After the reaction
was complete, the residue was dissolved in 20% NaOH (40 mL) and a solution of 10% HCl was added
to adjust the pH to 5–6. The resulting solid was filtered, washed with water and dried. Yield: 79%, mp:
311–314 °C. 1H-NMR (CDCl3) δ: 6.89 (t, 1H, J = 3.00 Hz, S-C=C-H), 8.02 (t, 1H, J = 3.00 Hz, S-C-H),
11.19 (s, 1H, -CONH), 11.54 (s, 1H, -CONHCO-). MS-EI m/z 169 (M + 1).
3.2.2. Synthesis of 2,4-Dichlorothieno[3,2-d]pyrimidine (2)
Compound 1 (1 g, 5.9 mmol) was dissolved in phosphorus oxychloride (15 mL) and stirred under
reflux for 5 h. Two-thirds of the solvent was removed under vacuum. The mixture was poured into
ice-water (50 mL). The precipitate that separated was collected by filtration, washed with water, and
dried to give compound 2 as a white solid. Yield: 74%, mp: 140–143 °C. 1H-NMR (CDCl3) δ: 7.55 (d,
1H, J = 6.00 Hz, S-C=C-H), 8.13 (d, 1H, J = 6.00 Hz, S-C-H). MS-EI m/z 205 (M + 1).
3.2.3. Synthesis of 1-(2-Chlorothieno[3,2-d]pyrimidin-4-yl)hydrazine (3)
To a solution of hydrazine hydrate (0.6 mL) in methanol (5 mL), a solution of 2,4-dichlorothieno
[3,2-d]pyrimidine (4.9 mmol) in methanol (30 mL) was added and the reaction mixture was heated at
50 °C for 1 h. The solvent was concentrated under reduced pressure then diluted with an petroleum
ether. The precipitated solid product was filtered, and did not require additional purification. Yield:
84%, mp: 314–316 °C. 1H-NMR (CDCl3) δ: 5.01 (s, 2H, -NH2), 7.23 (d, 1H, J = 6.00 Hz, S-C=C-H),
8.13 (d, 1H, J = 6.00 Hz, S-C-H), 9.48 (s, 1H, -NH). MS-EI m/z 201 (M + 1).
3.2.4. Synthesis of 5-Chlorothieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (4)
A mixture of compound 3 (0.8 g, 4.0 mmol) and triethyl orthoformate (15 mL) was heated at
130 °C for 3 h. After removing the excess triethyl orthoformate under reduced pressure, water (10 mL)
was added; the resulting precipitate was filtered and washed with water to produce 4 as a white solid.
1
Yield: 84%, mp: 201–203 °C. H-NMR (CDCl3) δ: 7.56 (d, 1H, J = 6.00 Hz, S-C=C-H), 7.83 (d, 1H,
J = 6.00 Hz, S-C-H), 8.99 (s, 1H, triazole-H). MS-EI m/z 211 (M + 1).
3.2.5. General Procedure for the Synthesis of 5-Alkoxythieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine
Derivatives 5a–p
K2CO3 (2.5 mmol) and appropriately substituted phenol (2.5 mmol) were dissolved in acetonitrile
(15 mL) and the mixture was heated and stirred for 30 min at 75 °C. 5-Chlorothieno[2,3-e][1,2,4]

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triazolo[4,3-c]pyrimidine (0.5 g, 2.4 mmol) was added to the reaction mixture. Reaction completion
was monitored by TLC, the solvent was then evaporated under reduced pressure. Water (30 mL) was
added; the precipitate was collected and recrystallized from ethanol. The yields and melting point data
of each compound are given below.
5-Phenoxythieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5a). White solid in 62%, mp: 183–184 °C.
1H-NMR (CDCl3) δ: 7.31–7.33 (d, 1H, J = 5.25 Hz, S-C=C-H), 7.38–7.57 (m, 5H, Ar-H), 7.69–7.70 (d,
1H, J = 5.25 Hz, S-C-H), 9.08 (s, 1H, triazole-H). 13C-NMR (CDCl3) δ: 113.14, 121.38, 121.38, 124.46,
126.94, 129.99, 129.99, 130.07, 133.27, 145.00, 147.61, 148.35, 151.05. IR (KBr) cm−1: 1637 (C=N).
MS-EI m/z 269 (M + 1). ESI-HRMS calcd for C13H8N4OS+ ([M + H]+): 269.0419; found: 269.0412.
5-(2-Chlorophenoxy)thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5b). White solid in 58%, mp:
211–213 °C. 1H-NMR (CDCl3) δ: 7.29–7.30 (d, 1H, J = 5.25 Hz, S-C=C-H), 7.35–7.59 (m, 4H, Ar-H),
13
7.68–7.70 (d, 1H, J = 5.22 Hz, S-C-H), 9.11 (s, 1H, triazole-H). C-NMR (CDCl3) δ: 113.44, 123.70,
124.45, 126.87, 128.15, 128.28, 130.17, 130.89, 133.23, 144.09, 147.10, 147.64, 148.21. IR (KBr) cm−1:
1635 (C=N). MS-EI m/z 303 (M + 1). ESI-HRMS calcd for C13H7ClN4OS+ ([M + H]+): 303.0029;
found: 303.0022.
5-(3-Chlorophenoxy)thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5c). White solid in 70%, mp:
175–177 °C. 1H-NMR (CDCl3) δ: 7.32–7.34 (d, 1H, J = 5.22 Hz, S-C=C-H), 7.33–7.50 (m, 4H, Ar-H),
7.70–7.72 (d, 1H, J = 5.28 Hz, S-C-H), 9.04 (s, 1H, triazole-H). 13C-NMR (CDCl3) δ: 113.39, 119.82,
122.16, 124.40, 127.29, 130.29, 130.72, 133.11, 135.21, 144.51, 147.54, 148.06, 151.31. IR (KBr) cm−1:
1637 (C=N). MS-EI m/z 303 (M + 1). ESI-HRMS calcd for C13H7ClN4OS+ ([M + H]+): 303.0029;
found: 303.0024.
5-(4-Chlorophenoxy)thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5d). White solid in 64%, mp:
200–202 °C. 1H-NMR (CDCl3) δ: 7.29–7.31 (d, 1H, J = 5.25 Hz, S-C=C-H), 7.34–7.50 (dd, 4H, Ar-H),
7.68–7.70 (d, 1H, J = 5.28 Hz, S-C-H), 9.03 (s, 1H, triazole-H). 13C-NMR (CDCl3) δ: 113.28, 122.88,
122.88, 124.35, 130.06, 130.06, 130.24, 132.40, 133.14, 144.70, 147.52, 148.09, 149.44. IR (KBr) cm−1:
1639 (C=N). MS-EI m/z 303 (M + 1). ESI-HRMS calcd for C13H7ClN4OS+ ([M + H]+): 303.0029;
found: 303.0021.
5-(2-Fluorophenoxy)thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5e). White solid in 67%, mp:
191–193 °C. 1H-NMR (CDCl3) δ: 7.29–7.31 (d, 1H, J = 5.22 Hz, S-C=C-H), 7.30–7.48 (m, 4H, Ar-H),
7.69–7.71 (d, 1H, J = 5.25 Hz, S-C-H), 9.08 (s, 1H, triazole-H). 13C-NMR (CDCl3) δ: 113.48, 117.21,
123.58, 124.41, 125.00, 128.28, 130.18, 133.20, 138.47, 144.13, 147.61, 148.17, 152.37. IR (KBr) cm−1:
1636 (C=N). MS-EI m/z 287 (M + 1). ESI-HRMS calcd for C13H7FN4OS+ ([M + H]+): 287.0325;
found: 287.0319.
5-(3-Fluorophenoxy)thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5f). White solid in 58%, mp:
182–183 °C. 1H-NMR (CDCl3) δ: 7.33–7.35 (d, 1H, J = 5.25 Hz, S-C=C-H), 7.10–7.55 (m, 4H, Ar-H),
7.71–7.73 (d, 1H, J = 5.28 Hz, S-C-H), 9.05 (s, 1H, triazole-H). 13C-NMR (CDCl3) δ: 109.60, 113.46,
114.26, 124.40, 130.26, 130.86, 133.12, 144.47, 147.58, 148.06, 151.53, 161.35, 164.65. IR (KBr) cm−1:

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1637 (C=N). MS-EI m/z 287 (M + 1). ESI-HRMS calcd for C13H7FN4OS+ ([M + H]+): 287.0325;
found: 287.0322.
5-(4-Fluorophenoxy)thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5g). White solid in 62%, mp:
193–194 °C. 1H-NMR (CDCl3) δ: 7.29–7.31 (d, 1H, J = 5.25 Hz, S-C=C-H), 7.18–7.40 (dd, 4H, Ar-H),
13
7.68–7.70 (d, 1H, J = 5.28 Hz, S-C-H), 9.04 (s, 1H, triazole-H). C-NMR (CDCl3) δ: 113.21, 116.57,
116.88, 122.98, 124.37, 130.18, 133.17, 144.99, 146.76, 147.55, 148.18, 159.14, 162.40. IR (KBr) cm−1:
1635 (C=N). MS-EI m/z 287 (M + 1). ESI-HRMS calcd for C13H7FN4OS+ ([M + H]+): 287.0325;
found: 287.0317.
5-(2-Methylphenoxy)thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5h). White solid in 57%, mp:
184–186 °C. 1H-NMR (CDCl3) δ: 2.26 (s, 1H, -CH3), 7.30–7.32 (d, 1H, J = 5.22 Hz, S-C=C-H), 7.29–7.35
(m, 4H, Ar-H), 7.66–7.68 (d, 1H, J = 5.25 Hz, S-C-H), 9.08 (s, 1H, triazole-H). 13C-NMR (CDCl3) δ:
16.19, 112.97, 121.72, 124.50, 127.13, 127.47, 130.04, 130.23, 131.75, 133.22, 144.60, 147.65, 148.53,
149.67. IR (KBr) cm−1: 1637 (C=N). MS-EI m/z 283 (M + 1). ESI-HRMS calcd for C14H10N4OS+
([M + H]+): 283.0575; found: 283.0569.
5-(3-Methylphenoxy)thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5i). White solid in 72%, mp:
176–178 °C. 1H-NMR (CDCl3) δ: 2.45 (s, 1H, -CH3), 7.32–7.34 (d, 1H, J = 5.22 Hz, S-C=C-H), 7.19–7.43
(m, 4H, Ar-H), 7.68–7.70 (d, 1H, J = 5.22 Hz, S-C-H), 9.06 (s, 1H, triazole-H). 13C-NMR (CDCl3) δ:
21.46, 113.08, 118.29, 121.83, 124.50, 127.74, 129.65, 130.00, 133.28, 140.38, 145.09, 147.61, 148.41,
151.01. IR (KBr) cm−1: 1633 (C=N). MS-EI m/z 283 (M + 1). ESI-HRMS calcd for C14H10N4OS+
([M + H]+): 283.0575; found: 283.0567.
5-(4-Methylphenoxy)thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5j). White solid in 62%, mp:
180–182 °C. 1H-NMR (CDCl3) δ: 2.45 (s, 1H, -CH3), 7.31–7.33 (d, 1H, J = 5.40 Hz, S-C=C-H), 7.24–7.34
(dd, 4H, Ar-H), 7.68–7.69 (d, 1H, J = 5.28 Hz, S-C-H), 9.06 (s, 1H, triazole-H). 13C-NMR (CDCl3) δ:
21.03, 113.06, 121.03, 121.03, 124.49, 129.98, 130.47, 130.47, 133.29, 136.76, 145.21, 147.62, 148.43,
148.86. IR (KBr) cm−1: 1633 (C=N). MS-EI m/z 283 (M + 1). ESI-HRMS calcd for C14H10N4OS+
([M + H]+): 283.0575; found: 283.0570.
5-(2-Methoxyphenoxy)thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5k). White solid in 66%, mp:
1
168–170 °C. H-NMR (CDCl3) δ: 3.76 (s, 1H, -OCH3), 7.28–7.30 (d, 1H, J = 5.28 Hz, S-C=C-H),
13
7.07–7.37 (m, 4H, Ar-H), 7.66–7.68 (d, 1H, J = 5.28 Hz, S-C-H), 9.08 (s, 1H, triazole-H). C-NMR
(CDCl3) δ: 55.88, 112.92, 113.96, 121.01, 122.70, 124.50, 127.95, 129.85, 133.49, 140.04, 144.91,
147.67, 148.68, 151.05. IR (KBr) cm−1: 1637 (C=N). MS-EI m/z 299 (M + 1). ESI-HRMS calcd for
C14H10N4O2S+ ([M + H]+): 299.0524; found: 299.0518.
5-(3-Methoxyphenoxy)thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5l). White solid in 69%, mp:
1
177–178 °C. H-NMR (CDCl3) δ: 3.86 (s, 1H, -OCH3), 7.31–7.33 (d, 1H, J = 5.28 Hz, S-C=C-H),
13
6.91–7.44 (m, 4H, Ar-H), 7.68–7.70 (d, 1H, J = 5.25 Hz, S-C-H), 9.05 (s, 1H, triazole-H). C-NMR
(CDCl3) δ: 55.61, 107.56, 112.54, 113.13, 113.40, 124.48, 130.06, 130.34, 133.25, 144.87, 147.58,

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148.35, 151.89, 160.82. IR (KBr) cm−1: 1633 (C=N). MS-EI m/z 299 (M + 1). ESI-HRMS calcd for
C14H10N4O2S+ ([M + H]+): 299.0524; found: 299.0514.
5-(4-Methoxyphenoxy)thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5m). White solid in 71%, mp:
1
180–182 °C. H-NMR (CDCl3) δ: 3.87 (s, 1H, -OCH3), 7.29–7.31 (d, 1H, J = 5.22 Hz, S-C=C-H),
13
7.00–7.31 (dd, 4H, Ar-H), 7.66–7.68 (d, 1H, J = 5.22 Hz, S-C-H), 9.04 (s, 1H, triazole-H). C-NMR
(CDCl3) δ: 55.68, 112.97, 114.85, 114.85, 122.26, 122.26, 124.46, 129.99, 133.28, 144.46, 145.40,
147.58, 148.43, 157.97. IR (KBr) cm−1: 1635 (C=N). MS-EI m/z 299 (M + 1). ESI-HRMS calcd for
C14H10N4O2S+ ([M + H]+): 299.0524; found: 299.0523.
5-(4-Bromophenoxy)thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5n). White solid in 40%, mp:
193–184 °C. 1H-NMR (CDCl3) δ: 7.43–7.45 (d, 1H, J = 5.28 Hz, S-C=C-H), 7.16–7.86 (dd, 4H, Ar-H),
13
7.77–7.79 (d, 1H, J = 5.28 Hz, S-C-H), 8.36 (s, 1H, triazole-H). C-NMR (CDCl3) δ: 111.60, 115.77,
116.08, 121.75, 121.85, 124.48, 131.13, 133.34, 141.27, 149.02, 151.54, 153.13, 157.74. IR (KBr) cm−1:
1639 (C=N). MS-EI m/z 347 (M + 1). ESI-HRMS calcd for C13H7BrN4OS+ ([M + H]+): 346.9524;
found: 346.9517.
5-[3-(Trifluoromethyl)phenoxy]thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5o). White solid in 57%,
1
mp: 185–186 °C. H-NMR (CDCl3) δ: 7.32–7.34 (d, 1H, J = 5.25 Hz, S-C=C-H), 7.64–7.74 (m, 4H,
13
Ar-H), 7.72–7.74 (d, 1H, J = 5.25 Hz, S-C-H), 9.08 (s, 1H, triazole-H). C-NMR (CDCl3) δ: 113.50,
118.93, 123.77, 124.36, 125.12, 130.33, 130.66, 132.30, 132.74, 133.08, 144.45, 147.54, 147.94,
151.00. IR (KBr) cm−1: 1635 (C=N). MS-EI m/z 337 (M + 1). ESI-HRMS calcd for C14H7F3N4OS+
([M + H]+): 337.0293; found: 337.0287.
5-(2,4-Dichlorophenoxy)thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidine (5p). White solid in 60%, mp:
168–170 °C. 1H-NMR (CDCl3) δ: 7.30–7.32 (d, 1H, J = 5.22 Hz, S-C=C-H), 7.44–7.59 (m, 3H, Ar-H),
13
7.70–7.72 (d, 1H, J = 5.22 Hz, S-C-H), 9.09 (s, 1H, triazole-H). C-NMR (CDCl3) δ: 113.62, 124.36,
124.61, 127.87, 128.51, 130.37, 130.71, 133.12, 133.21, 143.82, 145.76, 147.60, 148.00. IR (KBr) cm−1:
1637 (C=N). MS-EI m/z 337 (M + 1). ESI-HRMS calcd for C13H6Cl2N4OS+ ([M + H]+): 336.9639;
found: 336.9631.
3.2.6. General Procedure for the Synthesis of 2-Alkoxy-4-(1H-1,2,4-triazol-1-yl)thieno[3,2-d]pyrimidine
7a–d, 2-Alkoxy-4-(1H-imidazol-1-yl)thieno[3,2-d]pyrimidine 9a–d and
2-Alkoxy-4-(1H-pyrazol-1-yl)thieno[3,2-d]pyrimidine Derivatives 11a–d
A solution of 2 (2 mmol), 1,2,4-triazole/imidazole/pyrazole (2.2 mmol), and K2CO3 (2.2 mmol) in
N,N-dimethylformamide (30 mL) was refluxed for 3 h. The reaction mixture was cooled to room
temperature, concentrated by evaporation under vacuum, and white solids obtained were filtered,
washed with water and dried. All the compounds were recrystallized in ethanol to produce the pure
compounds 6, 8, and 10. A mixture of an appropriately substituted phenol (5.1 mmol) and K2CO3
(5.1 mmol) was placed in a round-bottomed flask with N,N-dimethylformamide (15 mL). The mixture
was stirred and heated at 80 °C for 0.5 h, and compound 6, 8, or 10 (1 g, 4.7 mmol) was added to the
mixture. After the reaction was completed, the solvent was poured into ice water. The precipitate was

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filtered and washed with water to produce a crude product, and purified using silica-gel column
chromatography with methanol-dichloromethane (v/v 1:60) to give a white solid. The yields and
melting point data of each compound are given below.
2-(3-Chlorophenoxy)-4-(1H-1,2,4-triazol-1-yl)thieno[3,2-d]pyrimidine (7a). White solid in 55%, mp:
156–158 °C. 1H-NMR (CDCl3) δ: 7.19–7.41 (m, 4H, Ar-H), 7.44–7.46 (d, 1H, J = 6.00 Hz, S-C=C-H),
13
8.16–8.18 (d, 1H, J = 6.00 Hz, S-C-H), 8.27 (s, 1H, triazole-H), 9.27 (s, 1H, triazole-H). C-NMR
(CDCl3) δ: 113.79, 119.97, 122.27, 123.78, 125.86, 130.37, 134.84, 142.08, 142.86, 143.23, 151.10,
153.52, 153.71, 166.74. IR (KBr) cm−1: 1586 (C=N). MS-EI m/z 330 (M + 1). ESI-HRMS calcd for
C14H8ClN5OS+ ([M + H]+): 330.0211; found: 330.0202.
2-(3-Fluorophenoxy)-4-(1H-1,2,4-triazol-1-yl)thieno[3,2-d]pyrimidine (7b). White solid in 61%, mp:
179–181 °C. 1H-NMR (CDCl3) δ: 7.02–7.48 (m, 4H, Ar-H), 7.44–7.46 (d, 1H, J = 6.00 Hz, S-C=C-H),
13
8.17–8.18 (d, 1H, J = 3.00 Hz, S-C-H), 8.28 (s, 1H, triazole-H), 9.28 (s, 1H, triazole-H). C-NMR
(CDCl3) δ: 109.49, 112.46, 113.79, 117.31, 123.77, 130.30, 142.04, 142.85, 151.12, 153.71, 161.50,
161.99, 164.77, 166.75. IR (KBr) cm−1: 1589 (C=N). MS-EI m/z 314 (M + 1). ESI-HRMS calcd for
C14H8FN5OS+ ([M + H]+): 314.0506; found: 314.0497.
2-[3-(Trifluoromethyl)phenoxy]-4-(1H-1,2,4-triazol-1-yl)thieno[3,2-d]pyrimidine (7c). White solid in
65%, mp: 127–129 °C. 1H-NMR (CDCl3) δ: 7.42–7.44 (d, 1H, J = 6.00 Hz, S-C=C-H), 7.51–7.59 (m,
4H, Ar-H), 8.16–8.18 (d, 1H, J = 6.00 Hz, S-C-H), 8.27 (s, 1H, triazole-H), 9.24 (s, 1H, triazole-H).
13C-NMR (CDCl3) δ: 113.90, 118.93, 122.28, 123.72, 125.13, 130.20, 131.90, 132.34, 142.19, 142.80,
151.10, 153.09, 161.87, 166.69. IR (KBr) cm−1: 1587 (C=N). MS-EI m/z 364 (M + 1). ESI-HRMS
calcd for C15H8F3N5OS+ ([M + H]+): 364.0474; found: 364.0466.
2-(2,4-Dichlorophenoxy)-4-(1H-1,2,4-triazol-1-yl)thieno[3,2-d]pyrimidine (7d). White solid in 68%,
mp: 206–208 °C. 1H-NMR (CDCl3) δ: 7.31–7.56 (m, 3H, Ar-H), 7.42–7.43(d, 1H, J = 3.00 Hz, S-C=C-H),
13
8.16–8.18 (d, 1H, J = 6.00 Hz, S-C-H), 8.27 (s, 1H, triazole-H), 9.26 (s, 1H, triazole-H). C-NMR
(CDCl3) δ: 113.91, 123.74, 124.72, 128.21, 128.38, 130.38, 131.68, 142.13, 142.90, 147.81, 151.17,
153.72, 161.51, 166.73. IR (KBr) cm−1: 1589 (C=N). MS-EI m/z 364 (M + 1). ESI-HRMS calcd for
C14H7Cl2N5OS+ ([M + H]+): 363.9821; found: 363.9814.
2-(3-Chlorophenoxy)-4-(1H-imidazol-1-yl)thieno[3,2-d]pyrimidine (9a). White solid in 62%, mp:
1
150–152 °C. H-NMR (CDCl3) δ: 7.17–7.31 (m, 4H, Ar-H), 7.37–7.42 (m, 1H, S-C=C-H), 7.42–7.51
(m, 1H, imidazole-H), 7.88–7.89 (d, 1H, imidazole-H), 8.08–8.10 (d, 1H, J = 6.00 Hz, S-C-H), 8.58
13
(s, 1H, imidazole-H). C-NMR (CDCl3) δ: 113.71, 116.91, 120.01, 122.29, 124.84, 125.82, 130.34,
131.68, 134.80, 136.27, 137.76, 151.70, 153.49, 162.64, 166.67. IR (KBr) cm−1: 1575 (C=N). MS-EI
m/z 329 (M + 1). ESI-HRMS calcd for C15H9ClN4OS+ ([M + H]+): 329.0258; found: 329.0252.
2-(3-Fluorophenoxy)-4-(1H-imidazol-1-yl)thieno[3,2-d]pyrimidine (9b). White solid in 49%, mp:
168–170 °C. 1H-NMR (CDCl3) δ: 7.00–7.10 (m, 4H, Ar-H), 7.41–7.44 (d, 1H, J = 9.00 Hz, imidazole-H),
7.48–7.50 (d, 1H, J = 6.00 Hz, S-C=C-H), 7.90 (s, 1H, imidazole-H), 8.09–8.11 (d, 1H, J = 6.00 Hz, S-C-H),
8.59 (s, 1H, imidazole-H). 13C-NMR (CDCl3) δ: 109.51, 112.44, 113.71, 116.91, 124.83, 130.27,

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131.68, 136.28, 137.73, 151.72, 153.81, 161.47, 162.62, 164.75, 166.68. IR (KBr) cm−1: 1577 (C=N).
MS-EI m/z 313 (M + 1). ESI-HRMS calcd for C15H9FN4OS+ ([M + H]+): 313.0554; found: 313.0548.
2-[3-(Trifluoromethyl)phenoxy]-4-(1H-imidazol-1-yl)thieno[3,2-d]pyrimidine (9c). White solid in 57%,
1
mp: 146–148 °C. H-NMR (CDCl3) δ: 7.47–7.49 (m, 2H, Ar-H), 7.58–7.59 (m, 2H, Ar-H; 1H,
S-C=C-H; 1H, imidazole-H), 7.88 (s, 1H, imidazole-H), 8.09–8.11 (d, 1H, J = 6.00 Hz, S-C-H), 8.58 (s,
1H, imidazole-H). 13C-NMR (CDCl3) δ: 113.84, 116.89, 118.94, 119.00, 122.30, 124.80, 125.17,
130.18, 131.68, 132.31, 136.23, 137.87, 151.72, 153.08, 162.51, 166.66. IR (KBr) cm−1: 1575 (C=N).
MS-EI m/z 363 (M + 1). ESI-HRMS calcd for C16H9F3N4OS+ ([M + H]+): 363.0522; found: 363.0513.
2-(2,4-Dichlorophenoxy)-4-(1H-imidazol-1-yl)thieno[3,2-d]pyrimidine (9d). White solid in 66%, mp:
144–146 °C. 1H-NMR (CDCl3) δ: 7.25–7.36 (m, 3H, Ar-H), 7.46–7.48(d, 1H, J = 6.00 Hz, S-C=C-H),
7.52–7.53 (d, 1H, imidazole-H), 7.86–7.87 (d, 1H, imidazole-H), 8.08–8.10 (d, 1H, J = 6.00 Hz, S-C-H),
8.55 (s, 1H, imidazole-H). 13C-NMR (CDCl3) δ: 113.85, 116.91, 116.91. 124.71, 124.79, 128.19,
128.37, 130.33, 131.68, 136.29, 137.87, 147.80, 151.73, 162.10, 166.69. IR (KBr) cm−1: 1576 (C=N).
MS-EI m/z 363 (M + 1). ESI-HRMS calcd for C15H8Cl2N4OS+ ([M + H]+): 362.9869; found: 362.9861.
2-(3-Chlorophenoxy)-4-(1H-pyrazol-1-yl)thieno[3,2-d]pyrimidine (11a). White solid in 58%, mp:
183–185 °C. 1H-NMR (CDCl3) δ: 6.56–6.57 (m, 1H, pyrazole-H), 7.20–7.42 (m, 4H, Ar-H), 7.35–7.37
(m, 1H, S-C=C-H), 7.93 (s, 1H, pyrazole-H), 8.08–8.10 (d, 1H, J = 6.00 Hz, S-C-H), 8.59–8.60 (m, 1H,
13
pyrazole-H). C-NMR (CDCl3) δ: 109.47, 113.57, 120.01, 122.29, 123.55, 125.48, 128.12, 130.22,
134.68, 141.48, 144.01, 153.21, 153.84, 162.10, 165.74. IR (KBr) cm−1: 1577 (C=N). MS-EI m/z 329
(M + 1). ESI-HRMS calcd for C15H9ClN4OS+ ([M + H]+): 329.0258; found: 329.0249.
2-(3-Fluorophenoxy)-4-(1H-pyrazol-1-yl)thieno[3,2-d]pyrimidine (11b). White solid in 53%, mp:
182–184 °C. 1H-NMR (CDCl3) δ: 6.56–6.57 (m, 1H, pyrazole-H), 6.98–7.46 (m, 4H, Ar-H), 7.39–7.40
(d, 1H, J = 3.00 Hz, S-C=C-H), 7.93 (s, 1H, pyrazole-H), 8.08–8.10 (d, 1H, J = 6.00 Hz, S-C-H),
8.59–8.60 (m, 1H, pyrazole-H). 13C-NMR (CDCl3) δ: 109.45, 109.80, 112.07, 113.57, 117.34, 123.55,
128.12, 130.13, 141.46, 144.01, 154.31, 161.47, 162.08, 164.74, 165.75. IR (KBr) cm−1: 1579 (C=N).
MS-EI m/z 313 (M + 1). ESI-HRMS calcd for C15H9FN4OS+ ([M + H]+): 313.0554; found: 313.0545.
2-[3-(Trifluoromethyl)phenoxy]-4-(1H-pyrazol-1-yl)thieno[3,2-d]pyrimidine (11c). White solid in 67%,
mp: 151–153 °C. 1H-NMR (CDCl3) δ: 6.55–6.56 (m, 1H, pyrazole-H), 7.37–7.39 (d, 1H, J = 6.00 Hz,
S-C=C-H), 7.50–7.62 (m, 4H, Ar-H), 7.92 (m, 1H, pyrazole-H), 8.08–8.10 (d, 1H, J = 6.00 Hz, S-C-H),
8.55–8.56 (m, 1H, pyrazole-H). 13C-NMR (CDCl3) δ: 109.51, 113.67, 118.97, 129.91, 123.50, 125.15,
128.04, 130.04, 131.73, 132.16, 141.60, 144.05, 153.19, 153.40, 161.96, 165.72. IR (KBr) cm−1: 1579
(C=N). MS-EI m/z 363 (M + 1). ESI-HRMS calcd for C16H9F3N4OS+ ([M + H]+): 363.0522;
found: 363.0515.
2-(2,4-Dichlorophenoxy)-4-(1H-pyrazol-1-yl)thieno[3,2-d]pyrimidine (11d). White solid in 45%, mp:
166–168 °C. 1H-NMR (CDCl3) δ: 6.55–6.56 (m, 1H, pyrazole-H), 7.31–7.38 (m, 3H, Ar-H), 7.53–7.54
(d, 1H, J = 3.00 Hz, S-C=C-H), 7.91–7.92 (m, 1H, pyrazole-H), 8.07–8.09 (d, 1H, J = 6.00 Hz, S-C-H),
8.56–8.57 (m, 1H, pyrazole-H). 13C-NMR (CDCl3) δ: 109.46, 113.65, 123.51, 124.84, 128.06, 128.17,

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128.46, 130.26, 131.29, 141.54, 144.03, 148.09, 153.25, 161.66, 165.74. IR (KBr) cm−1: 1581 (C=N).
MS-EI m/z 363 (M + 1). ESI-HRMS calcd for C15H8Cl2N4OS+ ([M + H]+): 362.9869; found: 362.9863.
3.3. Pharmacology
3.3.1. Anticonvulsant Effects in the MES Test
Seizures were induced in mice with a 60 Hz alternating current of 50 mA intensity. The current
was applied via corneal electrodes for 0.2 s. Effective protection against the spread of MES-induced
seizures was defined as the prevention of the hind leg and tonic maximal extension component of the
seizure. 0.5 h and 4 h after the administration of the compounds, the activities were evaluated using
an MES test [22,23].
3.3.2. Neurotoxicity (NT) Screening
The neurotoxicity of the compounds was measured in mice using the rotarod test. The mice were
trained to stay on an accelerating rotarod of diameter 3.2 cm that rotates at 10 rpm. The trained animals
were given intraperitoneal (i.p) injection of the test compounds. Neurotoxicity was indicated by the
inability of the animals to maintain equilibrium on the rod for at least 1 min in each of the trials [22,23].
3.3.3. The Subcutaneous Metrazol Seizure Test (scMET)
This screen utilizes a dose of pentylenetetrazole (85 mg/kg) that produces clonic seizures lasting for
a period of at least five seconds in 97% (CD97) of animals tested. At the anticipated time of testing, the
convulsant is administered subcutaneously. The test compound was administered intraperitoneally in
mice, and the animals were observed over a 30 min period. Mice were tested at least two different
time points (15 min, 30 min, 1 h, or 4 h) following i.p administration of 100 and 300 mg/kg of
test compound. Absence of clonic spasms indicated a compound’s ability to counteract the effect of
pentylenetetrazole on seizure threshold [24].
3.3.4. Pharmacological Evaluation of Compounds 5c and 5o Administered Orally to Mice
The time-course effect of compounds 5c and 5o in the MES test was determined. A suspension
of compounds 5c and 5o (50 mg/kg) in 0.5% methylcellulose was injected into mice by oral
administration (p.o.). The mice were divided into six groups (n = 10). Subsequently, the animals were
subjected to the MES test at various times: 0.5, 1, 1.5, 2, 2.5, and 3 h. The time of peak effect (TPE)
was 1.5 h after the p.o. injection. Then, compounds 5c and 5o were evaluated for anticonvulsant
activity against MES-induced seizures and oral neurotoxicity at its TPE when administered orally. This
test involved the same procedures for determining ED50 and TD50 as used in the MES test and the TOX
test screening, except that the test drug was administered orally to mice.
4. Conclusions
Based on our previous work, we designed and synthesized a series of 5-alkoxythieno[2,3-e]
[1,2,4]triazolo[4,3-c]pyrimidine derivatives, we also evaluated their anticonvulsant activities using

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MES and PTZ tests. The results revealed that majority of compounds showed protective effects against
seizures in the maximal electroshock tests (MES) and/or PTZ-induced seizures. Among the
compounds synthesized, five compounds (5c, 5f, 5o, 5p, and 7c) were found to have promising
anticonvulsant activity. In the MES screen, compound 5o had an ED50 of 11.5 mg/kg and a TD50 of
204.6 mg/kg, resulting in a high PI value of 17.7 when compared to carbamazepine (PI = 6.4). The
structure-anticonvulsant activity relationships analysis revealed that the most favorable structures are
the substituted phenol, especially with single chlorine, fluorine, trifluoromethyl substituents (at the
meta-position), or two chlorine atoms. These molecules possessed high activity according to the MES
and/or scPTZ models.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 81160382 and
81360468) and National Science and Technology Major Project of China (No. 2011ZX09102-003-03).
Author Contributions
Zhe-Shan Quan designed the compounds, planned synthetic pathways, Shi-Ben Wang performed
chemical synthesis, analyzed the results of biological testing and wrote the manuscript. Guang-Chun Piao
and Hong-Jian Zhang performed part of chemical synthesis. All authors commented on the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Scheurer, M.L.; Pedley, T.A. The evaluation and treatment of seizures. N. Engl. J. Med. 1990, 323,
1468–1474.
2. Löscher, W.; Klitgaard, H.; Twyman, R.E.; Schmidt, D. New avenues for anti-epileptic drug
discovery and development. Nat. Rev. Drug Discov. 2013, 12, 757–776.
3. Malik, S.; Bahare, R.S.; Khan, S.A. Synthesis and anticonvulsant evaluation of
N-(benzo[d]thiazol 2-ylcarbamoyl)-2-methyl-4-oxoquinazoline-3(4H)-carbothioamide derivatives:
A hybrid pharmacophore approach. Eur. J. Med. Chem. 2013, 67, 1–13.
4. Mattson, R.H. Drug treatment of partial epilepsy. Adv. Neurol. 1992, 57, 643–650.
5. Wesnes, K.A.; Edgar, C.; Dean, A.D.; Wroe, S.J. The cognitive and psychomotor effects of
remacemide and carbamazepine in newly diagnosed epilepsy. Epilepsy Behav. 2009, 14, 522–528.
6. Meador, K.J. Newer anticonvulsants: Dosing strategies and cognition in treating patients with
mood disorders and epilepsy. Clin. Psychiatry 2003, 64, 30–34.
7. Stefan, H.; Feuerstein, T. Novel anticonvulsant drugs. Pharmacol. Ther. 2007, 113, 165–183.
8. Bilotta, F.; Gelb, A.W.; Stazi, E.; Titi, L.; Paoloni, F.P.; Rosa, G. Pharmacological perioperative
brain neuroprotection: A qualitative review of randomized clinical trials. Br. J. Anaesth. 2013,
110 (Suppl. 1), i113–i120.

Molecules 2015, 20
6842
9. Chazot, P.L. Safinamide for the treatment of Parkinson’s disease, epilepsy and restless legs
syndrome. Curr. Opin. Investig. Drugs 2013, 8, 570–579.
10. Shu, B.; Zheng, Y.; Wang, S.B.; Deng, X.Q.; Quan, Z.S. Design, synthesis, and anticonvulsant
activity evaluation of 4-(3-alkoxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-ones. Arch. Pharm. 2013,
346, 127–133.
11. Zhang, C.B.; Yang, C.W.; Deng, X.Q.; Quan, Z.S. Design and synthesis of 6-alkyoxyl[1,2,4]
triazolo[1,5-a]quinazoline derivatives with anticonvulsant activity. Med. Chem. Res. 2012, 21,
3294–3300.
12. Deng, X.Q.; Dong, Z.Q.; Song, M.X.; Shu, B.; Wang, S.B.; Quan, Z.S. Synthesis and anticonvulsant
activities of some triazolothiadiazole derivatives. Arch. Pharm. 2012, 345, 565–573.
13. Wei, C.X.; Deng, X.Q.; Chai, K.Y.; Sun, Z.G.; Quan, Z.S. Synthesis and anticonvulsant activity
of 1-formamide-triazolo[4,3-a]quinoline derivatives. Arch. Pharm. Res. 2010, 33, 655–662.
14. Eswaran, S.; Adhikari, A.V.; Shetty, N.S. Synthesis and antimicrobial activities of novel quinoline
derivatives carrying 1,2,4-triazole moiety. Eur. J. Med. Chem. 2009, 44, 4637–4647.
15. Bayrak, H.; Demirbas, A.; Demirbas, N.; Karaoglu, S.A. Synthesis of some new 1,2,4-triazoles
starting from isonicotinic acid hydrazide and evaluation of their antimicrobial activities. Eur. J.
Med. Chem. 2009, 44, 4362–4366.
16. Zhou, J.P.; Zhang, H.B.; Qian, H.; Lin, L.; Huang, W.L.; Ni, S.J. Synthesis and Biological Evaluation
of Aromatase Inhibitors. Lett. Drug Des. Discov. 2009, 6, 181–185.
17. Owen, C.P.; Dhanani, S.; Patel, C.H.; Ahmed, S. Synthesis, Biochemical Evaluation and
Rationalisation of the Inhibitory Activity of a Series of 4-Substituted Phenyl Alkyl Triazole-Based
Compounds as Potential Inhibitors of 17a-Hydroxylase/17,20-Lyase (P45017a). Lett. Drug
Des. Discov. 2007, 4, 479–483.
18. Wang, S.B.; Deng, X.Q.; Zheng, Y.; Yuan, Y.P.; Quan, Z.S.; Guan, L.P. Synthesis and evaluation
of anticonvulsant and antidepressant activities of 5-alkoxytetrazolo[1,5-c]thieno[2,3-e]pyrimidine
derivatives. Eur. J. Med. Chem. 2012, 56, 139–144.
19. Zheng, Y.; Bian, M.; Deng, X.Q.; Wang, S.B.; Quan, Z.S. Synthesis and Anticonvulsant Activity
Evaluation of 5-Phenyl-[1,2,4]triazolo[4,3-c]quinazolin-3-amines. Arch. Pharm. 2013, 346, 119–126.
20. Kelley, J.L.; Krochmal, M.P.; Linn, J.A.; McLean, E.W.; Soroko, F.E. 6-(Alkylamino)-9-benzyl-
9H-purines A new class of anticonvulsant agents. J. Med. Chem. 1988, 31, 606–612.
21. Qu, G.R.; Zhang, H.L.; Niu, H.Y.; Xue, Z.K.; Lv, X.X.; Guo, H.M. Synthesis of C6-azolyl purine
nucleosides via C–N coupling reaction of unprotected 6-chloropurine nucleosides and N-heterocycles
under catalyst- and solvent-free conditions. Green Chem. 2012, 14, 1877–1879.
22. Krall, R.J.; Penry, J.K.; White, B.G.; Kupferberg, H.J. Antiepileptic drug development: II.
Anticonvulsant drug screening. Epilepsia 1978, 19, 409–428.
23. Poter, R.J.; Cereghino, J.J.; Gladding, G.D.; Hessie, B.J.; Kupferberg, H.J.; Scoville, B.
Antiepileptic Drug Development Program. Clevel. Clin. Q. 1984, 51, 293–305.

Molecules 2015, 20
6843
24. White, H.S.; Woodhead, J.H.; Wilcox, K.S.; Stables, J.P.; Kupferberg, H.J.; Wolf, H.H.
Antiepileptic Drugs, 5th ed.; Levy, R.H., Mattson, B.S., Meldrum, E., Perucca, E., Eds.;
Lippincott Williams & Wilkins Publishers: New York, NY, USA, 2002; pp. 36–48.
Sample Availability: Samples of the compounds are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).