A Facile Synthesis of Substituted 2-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)pyrazine Using Microwave Irradiation and Conventional Method with Antioxidant and Anticancer Activities

Article abstract of DOI:10.1002/jhet.3464

A series of novel substituted 2-(5-(benzylthio)-1,3,4-oxadiazol-2-yl)pyrazine derivatives (6a–n) were synthesized under microwave irradiation and conventional conditions with less reaction time with good to excellent yields. All the synthesized compounds were screened for antioxidant and anticancer activities. Out of the 14 prepared derivatives, compounds 6f and 6m were most potent and active with antioxidant and anticancer activities, respectively. Also, the developed technique was simple, easy, and less time consuming.

Full text of DOI:10.1002/jhet.3464

Month 2019
A Facile Synthesis of Substituted 2-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)
pyrazine Using Microwave Irradiation and Conventional Method with
Antioxidant and Anticancer Activities
a
a
Sanjeev R. Patil, Aniket P. Sarkate,
Kshipra S. Karnik,a Ashish Arsondkar,b Vrushali Patil,b
*
Jaiprakash N. Sangshetti,c Anil S. Bobade,b and Devanand B. Shinded
aDepartment of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431004, MS, India
bHaffkine Institute for Training, Research and Testing, Parel, Mumbai 400012, MS, India
cY. B. Chavan College of Pharmacy, Roza Baug, Aurangabad 431004, MS, India
dShivaji University, Vidyanagar, Kolhapur 416004, MS, India
*E-mail: dbsaniket09@gmail.com
Received August 24, 2018
DOI 10.1002/jhet.3464
Published online 00 Month 2019 in Wiley Online Library (wileyonlinelibrary.com).
A series of novel substituted 2-(5-(benzylthio)-1,3,4-oxadiazol-2-yl)pyrazine derivatives (6a–n) were
synthesized under microwave irradiation and conventional conditions with less reaction time with good to
excellent yields. All the synthesized compounds were screened for antioxidant and anticancer activities.
Out of the 14 prepared derivatives, compounds 6f and 6m were most potent and active with antioxidant
and anticancer activities, respectively. Also, the developed technique was simple, easy, and less time
consuming.
J. Heterocyclic Chem., 00, 00 (2019).
INTRODUCTION
implementation of the methodologies allowing for the
synthesis of some novel 2-[(4-fluorobenzyl)thio-5-
(pyrazin-2-yl)1,3,4-oxadiazole substituted derivatives.
The oxadiazole has been known for over 50 years, so
there have been several attempts to design antimicrobial
and anticancer agents based on this heterocycle [16–18].
1,3,4-Oxadiazole heterocycles are very good bioisosteres
of amides and esters and can contribute substantially to
increasing pharmacological activity by participating in
hydrogen-bonding interactions with receptors [19].
In continuation of our work [20–22], on the synthesis of
bioactive compounds, we have synthesized some 1,3,4-
oxadiazole analogues. The synthetic protocols employed
for the synthesis of oxadiazole derivatives 3 and 4 are
presented in Schemes 1, 2, and 3, respectively.
Oxadiazole is a lead moiety in the designing of potent
bioactive molecules [1]. The oxadiazole scaffold is a
central part of biologically active compounds with
various applications and pharmacological properties like
antibacterial [2], antifungal [3], antitubercular [4],
insecticidal [5], anticonvulsant [6], anticancer [7],
antiviral [8], anti-inflammatory [9], antidiabetic [10], and
immunosuppressive [11]. The 1,3,4-oxadiazole ring
system has been identified as the main core of many
bioactive molecules. For the discovery of new lead
structures in drug discovery, based on high throughput
screening, synthetic methodologies are required that
deliver highly diverse derivatives in a timely manner.
Under these circumstances, microwave-assisted chemistry
appears to be a promising synthetic method [12]. Utility
of microwave irradiation [13–15] to carry out organic
reaction has now become a regular feature. The main
benefits of performing the reaction under microwave
conditions are the significant rate enhancements and the
higher product yields with minimum time requirement.
Here, we wish to report the development and
RESULTS AND DISCUSSION
The first part of the study was aimed at optimizing the
reaction conditions. The screening of model reaction of
2-[(4-fluorobenzyl)thio-5-(pyrazin-2-yl)1,3,4-oxadiazole 6a
(Scheme 2; Table 1) was performed. We have developed
© 2019 Wiley Periodicals, Inc.

S. R. Patil, A. P. Sarkate, K. S. Karnik, A. Arsondkar, V. Patil,
J. N. Sangshetti, A. S. Bobade, and D. B. Shinde
Vol 000
Scheme 1. Synthesis of 5-(pyrazin-2-yl)-1,3,4-oxadiazole-2-thiol (4).
Scheme 2. Screening of model reaction 2-(5-(benzylthio)-1,3,4-oxadiazol-2-yl)pyrazine (6a). Reaction condition: compound (3) (1.0 mmol), benzyl
bromide (5a) (1.1 mmol), CH3COONa (1.5 mmol), solvent 1 mL, RT 20–150 min.
Scheme 3. Synthesis of substituted 2-(5-(benzylthio)-1,3,4-oxadiazol-2-yl)pyrazine (6a–n). Reaction condition: Method A: microwave-assisted
synthesis: sodium acetate, ethanol, 40°C, 3–4 min. Method B: conventional synthesis: sodium acetate, ethanol, room temperature, 20–50 min.
and time and optimized the yield as well; the results are
shown in Table 1. The reaction of compound 4 (1 mmol)
and compound 5a (1.2 mmol), catalyzed by various bases
and solvents, was selected as a model reaction to
optimize the reaction conditions. In terms of the effect of
solvents and bases on the condensation reaction, sodium
acetate was found to be the better base, and ethanol was
found to be the best solvent for the reaction (Table 1,
entry 10); in other solvents, including N,N-
dimethylformamide, water were less efficient (Table 1,
entries 2, 3, 5, 6, 8, 9 and 11, 12, 14, 15).
Nevertheless, all of these yields were generally low
before further optimizations. Ethanol gave the
corresponding product in a 50–95% yield, which was the
best among these solvents (Table 1, entries 1, 4, 7, and
10). To increase the efficiency of the condensation
reaction, the effects of different bases were investigated
(Table 1, entries 1–15). Sodium acetate exhibited the best
performance with used solvents and gave better yield,
(Table 1, entries 10–12). Diethylamine and triethylamine
gave lower yields with other solvents but gave better
yield in combination with ethanol as a solvent (Table 1,
entries 7 and 13). All the reactions were carried out in
equimolar amounts of each compound in 1 mL of
solvent. Among these reactions, the same amount of the
solvent, namely, 1 mL of ethanol, turned out to be the
best choice with yields of 50%, 65% and 95% and 80%
Table 1
Screening of bases, solvents, reaction time, and yield for the synthesis
(6a)a.
Time
(min)
Yieldb
(%)
Entry
Base
Solvent
1
2
3
4
5
6
7
8
Sodium hydroxide
Sodium hydroxide
Sodium hydroxide
Sodium carbonate
Sodium carbonate
Sodium carbonate
Triethylamine
Ethanol
Water
DMF
Ethanol
Water
DMF
Ethanol
Water
DMF
120
130
150
110
140
120
100
110
130
120
125
20
50
30
35
40
30
25
65
40
30
95
35
80
80
80
85
Triethylamine
Triethylamine
Sodium acetate
Sodium acetate
Sodium acetate
Diethylamine
9
10
11
12
13
14
15
Ethanol
Water
DMF
Ethanol
Water
DMF
40
60
50
Diethylamine
Diethylamine
The bold texts are indicating the promising results.
DMF, N,N-dimethylformamide.
aAll the reaction was carried out in equimolar amounts of each compound
in 1 mL of solvent.
bIsolated yield.
the protocol for the synthesis of compound 6a by
condensation of compounds 4 and 5a. After the initial
success in ethanol, we screened various solvents, bases,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Substituted Pyrazine as Anticancer and Antioxidant
(Table 1, entries 1, 7, 10 and 13). We would like to
mention here that ethanol as a solvent with sodium
acetate as base was the best choice with a yield of 95%
and less time required for the completion of the reaction
(Table 1, entry 10). Thus, we decided to carry out the
reactions in ethanol with sodium acetate as a base.
Simultaneously, we also performed microwave-assisted
reactions. In addition, the reaction time for microwave-
assisted reactions was much shorter than the same
reactions in all of our studied substrates. As a result, the
reaction time was shortened; thermal decomposition was
also minimized, resulting in higher isolated yields (Table 2).
The synthesized oxadiazole derivatives by using
microwave irradiation and conventional method 6(a–n)
(Scheme 3; Table 2) are shown. The compound 2-(5-
benzylthiol)-1,3,4-oxadiazole-2-yl)pyrazine was prepared
via condensation between compound 4 [5-(pyrazin-2-yl)-
1,3,4-oxadiazole-2-thiol] and substituted compounds
5(a–n). Compound 4 was obtained by reaction with
compound 3 in ethanol, carbon disulphide, and potassium
hydroxide at reflux temperature. The compounds 6(a–n)
were prepared using sodium acetate as a base with
compound 4 and substituted compounds 5(a–n).
The oxadiazole compounds were synthesized by using
the microwave irradiation as well as the conventional
heating with sodium acetate and ethanol.
The physical data of the synthesized compounds is
presented in Table 2. All the reactions proceeded well in
3–4 min in microwave irradiation to give products in
very good yields (92–98%) and 20–50 min using the
conventional method to give products in very good yields
(72–95%).
Table 3 represents the antioxidant and anticancer
activities of the aforementioned synthesized derivatives
6(a–n), respectively. It is clear from the aforementioned
data that compounds 6b, 6f, 6k, and 6m gave excellent
antioxidant activity than the standard butylated
hydroxytoluene. Apart from the aforementioned four
compounds, remaining compounds also produced much
greater activity when compared with the standard
butylated hydroxytoluene.
In this reaction, there was abstraction of –H from –SH,
and the negative charge is attacked to benzyl –CH2 to
form substituted compounds 6(a–n) with leaving HBr.
The plausible reaction mechanism for the synthesis of
2-(5-benzylthiol)-1,3,4-oxadiazole-2-yl)pyrazine is as
follows:
Table 2
Physical data for synthesized substituted 2-(5-(benzylthio)-1,3,4-oxadiazol-2-yl)pyrazine derivatives 6(a–n)a.
Time (min)
MWc
Yieldb (%)
Cond
Melting point
(°C)
Sr. No.
Substituent (X)
Cond
MWc
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
4-F-CH2C6H5
3-OCH3-CH2C6H5
2-Cl-CH2C6H5
2-F-CH2C6H5
2-Cl-6-F-CH2C6H4
-CH2C6H5
3,4-di-F-CH2C6H4
2,5-di-F-CH2C6H4
4-NO2-CH2C6H5
2-CN-CH2C6H5
3-CN-CH2C6H5
2,4-di-Cl-CH2C6H4
3-F-CH2C6H5
4
4
3
3
3
4
3
4
4
3
3
3
4
5
60
60
65
65
65
65
60
65
80
60
65
65
65
60
98
96
94
93
90
94
95
94
96
92
92
94
98
96
95
80
82
80
82
84
82
84
80
76
72
78
84
82
101–104
120–123
100–102
90–92
130–133
91–94
121–124
86–88
145–148
122–124
126–128
108–110
95–97
4-CN-CH2C6H5
142–144
aReaction condition (6a–n). Compound (3) (1 mmol), benzyl halides (4a–n) (1.2 mmol). Method A: Microwave-assisted synthesis: sodium acetate,
ethanol, 40°C, 3–4 min. Method B: Conventional synthesis: sodium acetate, ethanol, room temperature, 20–50 min.
bIsolated yields.
cMicrowave-assisted.
dConventional condition.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

S. R. Patil, A. P. Sarkate, K. S. Karnik, A. Arsondkar, V. Patil,
J. N. Sangshetti, A. S. Bobade, and D. B. Shinde
Vol 000
Table 3
Antioxidant and anticancer activities of derivatives 6(a–n).
Sr. No.
Substituent (X)
Antioxidant activity EC50 (μM)
Anticancer activity IC50 (μM)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
4-F-CH2C6H5
3-OCH3-CH2C6H5
2-Cl-CH2C6H5
2-F-CH2C6H5
2-Cl-6-F-CH2C6H4
-CH2C6H5
3,4-di-F-CH2C6H4
2,5-di-F-CH2C6H4
4-NO2-CH2C6H5
2-CN-CH2C6H5
3-CN-CH2C6H5
2,4-di-Cl-CH2C6H4
3-F-CH2C6H5
54.47
62.05
57.02
56.72
51.54
55.71
49.90
48.39
49.73
47.50
60.86
55.10
91.09
15.42
4.60
5.44
11.09
11.51
15.54
5.80
4.99
6.83
14.56
10.1
4.670
10.74
6.30
6m
6n
Standard
4-CN-CH2C6H5
48.42
BHT 11.81 1.11
Cisplatin 4.00
The bold texts are indicating the promising results.
BHT, butylated hydroxytoluene.
Coming to the anticancer activity, compounds 6a, 6f, 6j,
and 6m gave outstanding activity when compared with the
standard cisplatin on the MCF-7 breast cancer cell line. All
the remaining compounds also gave the comparable
anticancer activity with the standard. It can be observed
from the aforementioned results that compounds 6f and
6m are most versatile as they produced exceptional
antioxidant and anticancer activities.
5% dilute NaOH solution (500 mL) was heated at
40–45°C for 3 h. The completion of reaction was
monitored by TLC (hexane 6: ethyl acetate 4). The
reaction mass was cooled to 10–15°C and filtered off
under vacuum. Water (50 mL) washing given and suck
dried to give pure white solid compound (2) 48.2 g
(81.65%). mp 223–226°C. IR (KBr, cmꢀ1): 3064.89,
3095.75 (–OH stretch), 2339.65, 2455.38 (–CH
stretching), 1710.10 (>C¼O stretch); 1HNMR (400 MHz,
DMSO-d6 ppm): δ 8.786–8.798 (dd, 1H, CH-pyrazine),
8.854–8.862 (d, 1H, CH-pyrazine), 9.181–9.186 (d, 1H,
CH-pyrazine), 13.749 (s, 1H broad-OH); 13C NMR DEPT
(400 MHz, DMSO-d6 ppm): 143.901 (pyrazine C–N),
144.609 (pyrazine CH), 145.541 (pyrazine CH), 147.696
(pyrazine CH), 165.114 (–C¼O); MS: 124.1; m/z: 125
(M + H). Anal. Calcd for C6H4N4OS: C, 39.96; H, 2.22;
N, 31.08; S, 17.76%. Found: C, 40.0; H, 2.22; N, 31.11;
EXPERIMENTAL
Material and methods.
Pyrazinamide, hydrazine
hydrate, ethanol, and various solvents were commercially
available. The major chemicals were purchased from
Sigma Aldrich and Avra Labs. Microwave reactions were
carried out in Micro SYNTH Labstation of Ethusi
Milestone. Reaction courses were monitored by TLC on
silica gel precoated F254 Merck plates. Developed plates
were examined with UV lamps (254 nm). IR spectra were
recorded on a FT-IR (Bruker). Melting points were
recorded on SRS Optimelt, a melting point apparatus, and
these are uncorrected. 1H NMR spectra were recorded on a
400-MHz Bruker spectrometer, and 13C NMR spectra were
recorded on a 100-MHz Bruker spectrometer and are
reported as parts per million (ppm) downfield from a
tetramethylsilane internal standard. For 1H NMR, chemical
shifts were reported in the scale relative to a CHCl3
external standard (0 ppm). The following abbreviations are
used: singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), and broad (br). Mass spectra were taken with
Micromass-QUATTRO-II of WATER mass spectrometer.
S, 17.79%.
Conventional synthesis of pyrazine-2-carbohydrazide (3).
A mixture of pyrazine-2-carboxamide (1) 25 g (0.203 mol)
in ethanol (250 mL) and hydrazine hydrate (98%) 17.38 g
(0.347 mol) was refluxed for 12 h. The completion of
reaction was monitored by TLC (hexane 6: ethyl acetate
4). The reaction mass was cooled to 10–15°C and filtered
off under vacuum. Pyrazin-2-carboxamide is highly
soluble in iso-octane at 25–30°C. Iso-octane (25 mL)
washing was given and suck dried to give pure white to
off-white solid compound (3) 20.75 g (80.00%). mp 168–
170°C. IR (KBr, cmꢀ1): 3352.4 (–NH2 stretch), 3230.3
(>NH stretch), 2845 (–OCH3), 1640.65 (>C¼O stretch),
764 (>C–Br); 1HNMR (400 MHz, DMSO-d6 ppm): δ
4.50 (s, 2H, amine), 8.59 (s, 1H, amide), 8.74 (d, 1H,
CH-pyrazine), 9.16 (d, 1H, CH-pyrazine), 9.90 (s, 1H,
C¼NH); 13C NMR DEPT (400 MHz, DMSO-d6 ppm):
Conventional synthesis of pyrazine-2-carboxamide (2).
A
mixture of pyrazine-2-carbonitrile (1) 50 g (0.475 mol) in
Journal of Heterocyclic Chemistry
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Month 2019
Substituted Pyrazine as Anticancer and Antioxidant
143.36 (pyrazine C–N), 143.71 (pyrazine C¼N), 144.74
(pyrazine C¼N), 147.38 (pyrazine C¼N), 162.10
(–C¼O). MS: 137; m/z: 138 (M + H). Anal. Calcd for
C6H4N4OS: C, 39.96; H, 2.22; N, 31.08; S, 17.76%.
Found: C, 40.0; H, 2.22; N, 31.11; S, 17.79%.
Conventional synthesis of 5-(pyrazin-2-yl)-1,3,4-oxadiazole-
2-thiol (4). Potassium hydroxide 5.75 g (0.10 mol) was
resulting solid, filtered, and recrystallized from ethanol to
give product 6(a–n) (Yield: 72–90%).
Spectral data. 3-(5-(4-Fluorobenzylthio)-1,3,4-oxadiazol-
2-yl)pyrazine (6a).
Cream color solid. Yield: 98%. mp
101–104°C; ES-MS m/z: 288, IR νmax/cmꢀ1: 3001.24
(CH–Ar), 1460.11 (C¼N), 1246.02 (C–S), 1199.72 (C–
1
N). H NMR: δppm 4.44 (s, 2H), 6.99–7.31 (m, 3H, Ar–
H), 8.66 (s, 2H, Ar–H), 9.35 (s, 1H, Ar–H). 13C NMR:
δppm 36.80 (–S–CH2–), 56.24 (Ar–OCH3), 76.37, 77.00,
77.63 (N¼C–O), 113.84, 114.63, 121.40, 129.85, 136.52,
139.35, 143.88, 144.51, 146.33, and 159.82 (ArC–O).
Anal. Calcd for C13H9FN4OS: C, 54.16; H, 3.147; N,
19.434; S, 11.12%. Found: C, 54.256; H, 3.410; N,
20.088; S, 10.573%.
dissolved in absolute ethanol (200 mL) at ambient
temperature. Then pyrazine-2-carbohydrazide (2) 20 g
(0.149 mol) was added, and the reaction mass was
cooled. Carbon disulfide 12.54 g (0.165 mol) was added
in small portions, and the mixture was stirred at reflux
temperature for 12 h. The reaction mixture was changed
to green color with the evolution of hydrogen sulfide. The
completion of reaction was monitored by TLC (chloroform
9.5: methanol 0.5). Ethanol was completely distilled under
vacuum below 50°C. The reaction mass was cooled to 25–
30°C. Water (50 mL) was added to dissolve salts, and
pH 6.0–7.0 was adjusted by concentrated HCl (20 mL).
Solid was precipitated out, stirred at 25–30°C for 1 h, and
filtered. Water (50 mL) washing was given and dried to
give white to off-white solid compound (4) 18.28 g
(70.00%). Thus, the yield obtained was used in the next
step without further purification. mp 208–210°C. IR (KBr,
cmꢀ1): 3352.4 (–NH2 stretch), 3230.3 (>NH stretch), 2845
(–OCH3), 1640.65 (>C¼O stretch), 764 (>C–Br); 1H
NMR (400 MHz, DMSO-d6 ppm): δ 8.76 (s, 1H), 8.80 (s,
1H), 9.19 (s, 1H), 14.82 (s, 1H). 13C NMR DEPT
(400 MHz, DMSO-d6 ppm): 138.58 (pyrazine C–N),
143.45 (pyrazine C¼N), 145.28 (pyrazine C¼N), 147.29
(pyrazine C¼N), 158.32 (–C¼O), 178.48 (–C–SH). MS:
178.9; m/z: 179.9 (M + H). Anal. Calcd for C6H4N4OS: C,
39.96; H, 2.22; N, 31.08; S, 17.76%. Found: C, 40.0; H,
4-(5-(3-Methoxybenzylthio)-1,3,4-oxadiazol-2-yl)pyrazine
(6b).
Cream color solid. Yield: 96%. mp 120–123°C;
ES-MS m/z: 302, IR νmax/cmꢀ1: 3016.67, 3057.17 (CH–
Ar), 1587.42, 1608.63 (C¼C), 1309.67 (–OCH3),
1
1269.16 (C–S), 1193.94 (C–N). H NMR: δppm = 3.75
(s, 3H, –OCH3), 4.50 (s, 2H), 6.77–7.20 (m, 4H, Ar–H),
8.67 (s, 2H, Ar–H), 9.36 (s, 1H, Ar–H). 13C NMR: δppm
36.80 (–S–CH2–), 56.24 (Ar–OCH3), 76.37, 77.00, 77.63
(N¼C–O), 113.84, 114.63, 121.40, 129.85, 136.52,
139.35, 143.88, 144.51, 146.33, and 159.82 (ArC–O).
Anal. Calcd for C14H12N4O2S: C, 55.988; H, 4.027; N,
18.655; S, 10.674%. Found: C, 56.043; H, 4.137; N,
19.420; S, 10.233%.
2-(5-(2-Chlorobenzylthio)-1,3,4-oxadiazol-2-yl)pyrazine
(6c). Cream color solid. Yield: 94%. mp 100–102°C; ES-
MS m/z: 304, IR νmax/cmꢀ1: 3001.24 (CH–Ar), 1460.11
1
(C¼N), 1246.02 (C–S), 1199.72 (C–N). H NMR: δppm
4.70 (s, 2H), 7.25–7.28 (m, 1H, Ar–H), 7.42–7.44 (d, 1H,
Ar–H), 7.65–7.68 (d, 1H, Ar–H), 8.74 (s, 2H, Ar–H),
9.43 (s, 1H, Ar–H). 13C NMR: δppm 34.60 (–S–CH2–),
76.69, 77.00, 77.32 (N¼C–O), 127.08, 129.75, 129.81,
131.54, 133.25, 134.41, 139.29, 143.86, 144.56, and
146.36. Anal. Calcd for C13H9ClN4OS: C, 51.236; H,
2.977; N, 18.385; S, 10.52%. Found: C, 51.476; H, 2.095;
2.22; N, 31.11; S, 17.79%.
General procedure for the synthesis of compounds (6a–n).
Method A: Microwave-assisted synthesis.
In a 100 mL
round bottom flask, the compound 4 (1.0 mmol), benzyl
halide 5(a–n) (1.2 mmol), and sodium acetate (1.2 mmol)
were added to the ethanol (10 mL), and this mixture was
subjected to the microwave irradiation (800 W), at 80°C
temperature for 3–4 min. The progress of reaction was
monitored by TLC (3% ethyl acetate: hexane). After
completion of reaction, the reaction mixture was
concentrated in vacuo. Water (20 mL) was added to the
resulting solid, filtered and recrystallized from ethanol to
give product 6(a–n) (Yield: 92–96%).
Method B: Conventional synthesis. In a 100 mL round
bottom flask, the compound 4 (1.0 mmol), benzyl halide
5(a–n) (1.2 mmol), and sodium acetate (1.2 mmol) were
added to the ethanol (10 mL) and stirred 30–50 min at
reflux temperature. The progress of the reaction was
monitored by TLC (3% ethyl acetate: hexane). After
completion of reaction, the reaction mixture was
concentrated in vacuo. Water was added (20 mL) to the
N, 18.977; S, 10.145%.
2-(5-(2-Fluorobenzylthio)-1,3,4-oxadiazol-2-yl)pyrazine
(6d). Cream color solid. Yield: 93%. mp 90–92°C; ES-MS
m/z: 288, IR νmax/cmꢀ1: 3076.46 (CH–Ar), 1587.42 (C¼C),
1494.83 (C¼N), 1197.79 (C–S), 869.90–881.47 (C–N). 1H
NMR: δppm 4.70 (s, 2H), 7.25–7.28 (m, 1H, Ar–H),
7.42–7.44 (d, 1H, Ar–H), 7.65–7.68 (d, 1H, Ar–H), 8.74 (s,
2H, Ar–H), 9.43 (s, 1H, Ar–H). 13C NMR: δppm 36.80
(–S–CH2–), 56.24 (Ar–OCH3), 76.37, 77.00, 77.63
(N¼C–O), 113.84, 114.63, 121.40, 129.85, 136.52, 139.35,
143.88, 144.51, 146.33, and 159.82 (ArC–O). Anal. Calcd
for C13H9FN4OS: C, 54.16; H, 3.146; N, 19.434; S,
11.12%. Found: C, 54.11; H, 3.122; N, 19.424; S, 11.1%.
2-(5-(2-Chloro-6-fluorobenzylthio)-1,3,4-oxadiazol-2-yl)
pyrazine (6e). Light brown color solid. Yield: 90%. mp
130–133°C; ES-MS m/z: 322, IR νmax/cmꢀ1: 3001.24
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

S. R. Patil, A. P. Sarkate, K. S. Karnik, A. Arsondkar, V. Patil,
J. N. Sangshetti, A. S. Bobade, and D. B. Shinde
Vol 000
(CH–Ar), 1460.11 (C¼N), 1246.02 (C–S), 1199.72 (C–N).
1H NMR: δppm 4.70 (s, 2H), 7.25–7.28 (m, 1H, Ar–H),
7.42–7.44 (d, 1H, Ar–H), 7.65–7.68 (d, 1H, Ar–H), 8.74
(s, 2H, Ar–H), 9.43 (s, 1H, Ar–H). 13C NMR: δppm
36.80 (–S–CH2–), 56.24 (Ar–OCH3), 76.37, 77.00, 77.63
(N¼C–O), 113.84, 114.63, 121.40, 129.85, 136.52,
139.35, 143.88, 144.51, 146.33, and 159.82 (ArC–O).
Anal. Calcd for C13H8ClFN4OS: C, 48.38; H, 2.5; N,
17.36; S, 9.933%. Found: C, 48.334; H, 2.478; N, 17.35;
36.80 (–S–CH2–), 56.24 (Ar–OCH3), 76.37, 77.00, 77.63
(N¼C–O), 113.84, 114.63, 121.40, 129.85, 136.52, 139.35,
143.88, 144.51, 146.33, and 159.82 (ArC–O). Anal. Calcd
for C6H4N4OS: C, 49.52; H, 2.877; N, 22.211; S, 10.168%.
Found: C, 49.475; H, 2.854; N, 22.2; S, 10.148%.
2-((5-(Pyrazin-2-yl)-1,3,4-oxadiazol-2-ylthio)methyl)
benzonitrile (6j). Cream color solid. Yield: 92%. mp 122–
124°C; ES-MS m/z: 295, IR νmax/cmꢀ1: 3076.46 (CH–Ar),
1587.42 (C¼C), 1494.83 (C¼N), 1197.79 (C–S), 869.90–
881.47 (C–N). 1H NMR: δppm 4.73 (s, 2H), 7.40–7.44 (m,
1H, Ar–H), 7.66–7.68 (d, 1H, Ar–H), 7.69–7.71 (d, 1H,
Ar–H), 7.78–7.80 (s, 2H, Ar–H), 8.72–8.74 (s, 2H), 9.41
(s, 1H, Ar–H). 13C NMR: δppm 34.49 (–S–CH2–), 76.69,
77.00, 77.32 (N¼C–O), 112.80, 116.99 (Ar–CN), 128.72,
130.80, 133.03, 133.20, 139.33, 143.86, 144.56, 146.47,
163.38, and 166.44. Anal. Calcd for C6H4N4OS: C, 56.94;
H, 3.072; N, 23.715; S, 10.856%. Found: C, 56.898; H,
S, 4.957%.
2-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)pyrazine (6f). Light
brown color solid. Yield: 94%. mp 91–94°C; ES-MS m/z:
270, IR νmax/cmꢀ1: 3046.60 (CH–Ar), 1540.42 (C¼C),
1
1456.26 (C¼N), 1193.94 (C–S). H NMR: δppm 4.70 (s,
2H), 7.25–7.28 (m, 1H, Ar–H), 7.42–7.44 (d, 1H, Ar–H),
7.65–7.68 (d, 1H, Ar–H), 8.74 (s, 2H, Ar–H), 9.43 (s,
1H, Ar–H). 13C NMR: δppm 36.80 (–S–CH2–), 56.24
(Ar–OCH3), 76.37, 77.00, 77.63 (N¼C–O), 113.84,
114.63, 121.40, 129.85, 136.52, 139.35, 143.88, 144.51,
146.33, and 159.82 (ArC¼O). Anal. Calcd for
C6H4N4OS: C, 57.76; H, 3.73; N, 20.727; S, 11.86%.
3.005; N, 23.750; S, 11.142%.
3-((5-(Pyrazin-2-yl)-1,3,4-oxadiazol-2-ylthio)methyl)
benzonitrile (6k). Off-white color solid. Yield: 92%. mp
126–128°C; ES-MS m/z: 295, IR νmax/cmꢀ1: 3076.46
(CH–Ar), 1587.42 (C¼C), 1494.83 (C¼N), 1197.79
Found: C, 57.71; H, 3.7; N, 20.72; S, 11.84%.
2-(5-(3,4-Difluorobenzylthio)-1,3,4-oxadiazol-2-yl)pyrazine
(6g). Off-white color solid. Yield: 95%. mp 121–124°C;
1
(C–S), 869.90–881.47 (C–N). H NMR: δppm 4.73 (s,
2H), 7.40–7.44 (m, 1H, Ar–H), 7.66–7.68 (d, 1H, Ar–H),
7.69–7.71 (d, 1H, Ar–H), 7.78–7.80 (s, 2H, Ar–H),
8.72–8.74 (s, 2H), 9.41 (s, 1H, Ar–H). 13C NMR: δppm
34.49 (–S–CH2–), 76.69, 77.00, 77.32 (N¼C–O), 112.80,
116.99 (Ar–CN), 128.72, 130.80, 133.03, 133.20, 139.33,
143.86, 144.56, 146.47, 163.38, and 166.44. Anal. Calcd
for C6H4N4OS: C, 56.882; H, 3.047; N, 23.703; S,
10.835%. Found: C, 56.898; H, 3.005; N, 23.750; S,
ES-MS m/z: 306, IR νmax/cmꢀ1: 3076.46 (CH–Ar),
1587.42 (C¼C), 1494.83 (C¼N), 1197.79 (C–S),
869.90–881.47 (C–N). 1H NMR: δppm 4.44 (s, 2H),
6.99–7.31 (m, 3H, Ar–H), 8.66 (s, 2H, Ar–H), 9.35 (s,
1H, Ar–H). 13C NMR: δppm 35.62 (–S–CH2–), 76.37,
77.00, 77.63 (N¼C–O), 117.39, 117.73, 118.08, 118.43,
125.38, 139.23, 143.90, 144.54, 146.46 (Ar–F) and
152.56 (Ar–F). Anal. Calcd for C6H4N4OS: C, 50.979;
H, 2.632; N, 18.292; S, 10.467%. Found: C, 51.297; H,
11.142%.
2-(5-(2,4-Dichlorobenzylthio)-1,3,4-oxadiazol-2-yl)pyrazine
(6l). Off-white color solid. Yield: 94%. mp 108–110°C;
2.405; N, 18.923; S, 11.031%.
2-(5-(2,5-Difluorobenzylthio)-1,3,4-oxadiazol-2-yl)pyrazine
(6h). Off-white color solid. Yield: 94%. mp 86–88°C;
ES-MS m/z: 339, IR νmax/cmꢀ1: 3091.89 (CH–Ar),
1583.56 (C¼C), 1458.18 (C¼N), 1199.72 (C–S), 850.50
ES-MS m/z: 306, IR νmax/cmꢀ1: 3076.46 (CH–Ar),
1587.42 (C¼C), 1494.83 (C¼N), 1197.79 (C–S),
869.90–881.47 (C–N). 1H NMR: δppm 4.70 (s, 2H),
7.25–7.28 (m, 1H, Ar–H), 7.42–7.44 (d, 1H, Ar–H),
7.65–7.68 (d, 1H, Ar–H), 8.74 (s, 2H, Ar–H), 9.43 (s,
1H, Ar–H). 13C NMR: δppm 36.80 (–S–CH2–), 56.24
(Ar–OCH3), 76.37, 77.00, 77.63 (N¼C–O), 113.84,
114.63, 121.40, 129.85, 136.52, 139.35, 143.88, 144.51,
146.33, and 159.82 (ArC–O). Anal. Calcd for
C6H4N4OS: C, 50.978; H, 2.632; N, 18.292; S, 10.467%.
1
(C–N). H NMR: δppm 4.70 (s, 2H), 7.25–7.28 (m, 1H,
Ar–H), 7.42–7.44 (d, 1H, Ar–H), 7.65–7.68 (d, 1H, Ar–
H), 8.74 (s, 2H, Ar–H), 9.43 (s, 1H, Ar–H). 13C NMR:
δppm 36.80 (–S–CH2–), 56.24 (Ar–OCH3), 76.37, 77.00,
77.63 (N¼C–O), 113.84, 114.63, 121.40, 129.85, 136.52,
139.35, 143.88, 144.51, 146.33, and 159.82 (ArC–O).
Anal. Calcd for C6H4N4OS: C, 46.033; H, 2.377; N,
16.52; S, 9.451%. Found: C, 45.99; H, 2.358; N, 16.51;
S, 9.434%.
2-(5-(3-Fluorobenzylthio)-1,3,4-oxadiazol-2-yl)pyrazine
(6m). Off-white color solid. Yield: 98%. mp 95–97°C;
Found: C, 50.932; H, 2.612; N, 18.283; S, 10.447%.
2-(5-(4-Nitrobenzylthio)-1,3,4-oxadiazol-2-yl)pyrazine
(6i). Cream color solid. Yield: 96%. mp 145–148°C; ES-
ES-MS m/z: 288, IR νmax/cmꢀ1: 3076.46 (CH–Ar),
1587.42 (C¼C), 1494.83 (C¼N), 1197.79 (C–S),
869.90–881.47 (C–N). 1H NMR: δppm 4.54 (s, 2H),
7.00–7.04 (m, 2H, Ar–H), 7.43–7.47 (m, 2H, Ar–H),
8.72 (s, 2H, Ar–H), 9.41 (s, 1H, Ar–H). 13C NMR: δppm
35.93 (–S–CH2–), 76.68, 77.00, 77.32 (N¼C–O), 115.61,
115.83, 130.97, 143.85, 144.51, 146.37, 161.21, 163.09,
MS m/z: 315, IR νmax/cmꢀ1 3101.54 (CH–Ar), 1602.85
(C¼C), 1350.17 (–NO2), 1205.51 (C–S), 860.25, 871.82
1
(C–N). H NMR: δppm 4.70 (s, 2H), 7.25–7.28 (m, 1H,
Ar–H), 7.42–7.44 (d, 1H, Ar–H), 7.65–7.68 (d, 1H, Ar–H),
8.74 (s, 2H, Ar–H), 9.43 (s, 1H, Ar–H). 13C NMR: δppm
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Substituted Pyrazine as Anticancer and Antioxidant
163.67, and 166.09 (Ar–F). Anal. Calcd for C6H4N4OS: C,
54.16; H, 3.147; N, 19.434; S, 11.12%. Found: C, 54.11;
H, 3.122; N, 19.424; S, 11.1%.
4-((5-(Pyrazin-2-yl)-1,3,4-oxadiazol-2-ylthio)methyl)
test concentrations of test compounds (5–200 μg/mL) at
identical conditions with five replicates each. The cell
viability was assessed after 24 h, by adding 10 μL of
MTT (5 mg/mL) per well. The plates were incubated
at 37°C for additional 3 h. The medium was discarded,
and the formazan blue, which formed in the cells,
was dissolved with 100 μL of DMSO. The rate of
benzonitrile (6n).
Cream color solid. Yield: 96%. mp
142–144°C; ES-MS m/z: 295, IR νmax/cmꢀ1: 3076.46
(CH–Ar), 2223.92 (C¼N), 1462.04 (C¼N), 1211.30
1
(C–S). H NMR: δppm 4.73 (s, 2H), 7.66–7.68 (d, 2H,
color production was measured at 570 nm in
a
Ar–H), 7.69–7.71 (d, 2H, Ar–H), 7.78–7.80 (s, 2H,
Ar–H), 8.72–8.74 (s, 1H, Ar–H), 9.41 (s, 1H, Ar–H). 13C
NMR: δppm 34.49 (–S–CH2–), 76.69, 77.00, 77.32
(N¼C–O), 112.80, 116.99 (Ar–CN), 128.72, 130.80,
133.03, 133.20, 139.33, 143.86, 144.56, 146.47, 163.38,
and 166.44. Anal. Calcd for C6H4N4OS: C, 56.94; H,
3.072; N, 23.715; S, 10.856%. Found: C, 56.887; H,
3.047; N, 23.703; S, 10.836%.
spectrophotometer (Spectra MAX Plus; Molecular
Devices; supported by SoftMax Pro-5.4). The percent
inhibition of cell viability was determined with reference
to the control (without test compound). The data were
subjected to linear regression analysis, and the regression
lines were plotted for the best straight-line fit. The IC50
(concentration required for 50% inhibition of cell
viability) concentrations were calculated using the
respective regression equation.
Biological methods. Antioxidant activity.
All the
synthesized compounds were evaluated for antioxidant
activity against DPPH at different concentrations
(20–100 μg/mL using free radical scavenging activity
[DPPH method]). In the present study, in vitro DPPH
CONCLUSION
(1,1-diphenyl-2-picryl-hydrazil)
radical
scavenging
In conclusion, we have successfully developed an easy
access to a new series of substituted 2-(5-(benzylthio)-
1,3,4-oxadiazol-2-yl)pyrazine derivatives 6(a–n). The
mild reaction conditions, good to excellent yields, easy
workup, and easily available substrates make the
reactions attractive for the preparation of compounds
6(a–n). The synthesized derivatives possess excellent to
good antioxidant and anticancer activities. Besides, the
technique has the advantage of being simple and allows
the synthesis with a minimum reaction time. We believe
that these reactions provide a new approach for the
synthesis of pyrazine containing moiety.
method [23–25] was used to evaluate the antioxidant
potential of 1,2,4-triazole-3-thiol derivatives (6a–n). The
interaction of all tested compounds with the stable free
radical DPPH indicates their radical scavenging activity.
ANTICANCER ACTIVITY
Cell lines and cell culture.
The cell lines MCF7
(mammary adenocarcinoma) and HeLa, derived from
human cervical cancer cells (ATCC No. CCL-2), were
obtained from the National Centre for Cell Sciences,
Pune, India. Cells were cultured in RPMI-1640 media sup-
plemented with 10% heating activated fetal bovine serum,
1 mM NaHCO3, 2 mM L-glutamine, 100 units/mL penicil-
lin, and 100 μg/mL streptomycin. All cell lines were main-
tained and cultured at 37°C in an atmosphere of 5% CO2.
Acknowledgments. The authors are thankful to the head of the
Department of Chemical Technology, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad, for providing the
laboratory facility and Haffkine Institute for Training, Research
and Testing, Parel, Mumbai, for providing the activity facility.
Test concentrations.
Initially, stock concentration of
each compound was prepared in DMSO (8 mg/mL), from
which 50 μL of stock was diluted to 1 mL in a culture
medium to obtain experimental stock concentration of
400 μg/mL. Different aliquots of experimental stock were
added to the cultured cells in the medium (final volume
REFERENCES AND NOTES
[1] Chandrakantha, B.; Shetty, P.; Nambiyar, V.; Isloor, N.; Isloor,
A. M. J Med Chem 2010, 45, 1206.
[2] Wang, P. Y.; Zhou, L.; Zhou, J.; Wu, Z. B.; Xue, W.; Song, B.
A.; Yang, S. Bioorg Med Chem Lett 2016, 26, 1217.
[3] Wani, M. Y.; Ahmad, A.; Shiekh, R. A.; Al-Ghamdi, K. J.;
Sobral, A. J. F. N. Bioorg Med Chem 2015, 23, 4172.
[4] Patel, N. B.; Purohit, A. C.; Rajani, D. P.; Moo-Puc, R.;
Rivera, G. Eur J Med Chem 2013, 62, 677.
[5] Li, Y. H.; Zhu, H. J.; Chen, K.; Liu, R.; Khallaf, A.; Zhang, X.
N.; Ni, J. P. Org Biomol Chem 2013, 11, 3979.
[6] Harish, K. P.; Mohana, K. N.; Mallesha, L.; Prasannakumar,
B. N. Eur J Med Chem 2013, 65, 276.
of 200 μL) to attain the required concentration.
Cytotoxicity. In all experiments, different cell lines were
seeded at a final density of 2 × 104 cells per well, in 96-well
microtiter plates. Cytotoxicity was measured using the
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] assay, according to the method of Mosmann
[26]. Briefly, the cells (2 × 104) were seeded in each well
containing 0.1 mL of medium in 96-well plates. After
overnight incubation, the cells were treated with different
[7] Liu, H.–Q.; Wang, D.–C.; Wu, F.; Tang, W.; Ouyang, P.–K.
Chin Chem Lett 2013, 24, 929.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

S. R. Patil, A. P. Sarkate, K. S. Karnik, A. Arsondkar, V. Patil,
J. N. Sangshetti, A. S. Bobade, and D. B. Shinde
Vol 000
[8] Wu, W.; Chen, Q.; Tai, A.; Jiang, G.; Ouyang, G. Bioorg Med
Chem Lett 2015, 25, 2243.
[19] Guimaraes, C. R. W.; Boger, D. L.; Jorgensen, W. L. J Am
Chem Soc 2005, 127, 17377.
[9] Gadegoni, H.; Manda, S. Chin Chem Lett 2013, 24, 127.
[10] Liao, B.-R.; He, H.-B.; Yang, L.-L.; Gao, L.-X.; Chang, L.;
Tang, J.; Li, J.-Y.; Li, J.; Yang, F. Eur J Med Chem 2014, 83, 15.
[11] Tang, J.-F.; Lv, X.-H.; Wanga, X.-L.; Sun, J.; Zhang, Y.-B.;
Yang, Y.-S.; Gong, H.-B.; Zhu, H.-L. Bioorg Med Chem 2012,
20, 4226.
[20] Tiwari, S. V.; Saijas, J. A.; Vazquez-Tato, M. P.; Sarkate, A. P.;
Lokwani, D. K.; Nikalje, A. P. G. Molecules 2016, 21, 894.
[21] Lokwani, D. K.; Sarkate, A. P.; Shinde, D. B. Med Chem Res
2013, 22, 1415.
[22] Dofe, V. S.; Sarkate, A. P.; Azad, R.; Gill, C. H. Res Chem Int
2017, 43, 7331.
[12] Tiwari, S. V.; Siddiqui, S.; Saijas, J. A.; Vazquez-Tato, M. P.;
Sarkate, A. P.; Lokwani, D. K.; Nikalje, A. P. G. Molecules 2017,
22, 995.
[23] Srivastava, B. K.; Solanki, M.; Mishra, B.; Soni, R.; Jayadev,
S.; Valani, D.; Jain, M.; Patel, P. R. Bioorg Med Chem Lett 2007,
17, 1924.
[13] Jadhav, S. A.; Sarkate, A. P.; Shioorkar, M. G.; Shinde, D. B.
Syn Comm 2017, 47, 1661.
[24] Lohray, B. B.; Lohray, V. B.; Srivastava, B. K.; Kapadnis, P. B.;
Pandya, P. Bioorg Med Chem 2004, 12, 4557.
[14] Sarkate, A. P.; Bahekar, S. S.; Wadhai, V. M.; Ghandge, G. N.;
Wakte, P. S.; Shinde, D. B. Synlett 2013, 24, 1513.
[15] Jadhav, S. A.; Shioorkar, M. G.; Chavan, O. S.; Sarkate, A. P.;
Shinde, D. B. Syn Comm 2017, 47, 285.
[25] Amr, A. E.; Abdel-Hafez, N. A.; Mohamed, S. F.; Abdalla, M.
M. Turk J Chem 2009, 33, 421.
[26] Mosmann, T. J Immunol Methods 1983, 65, 55.
[16] Maftei, C. V.; Fodor, E.; Jones, P. G.; Freytag, M.; Franz, M.
H.; Kelter, G.; Fiebig, H. H.; Tamm, M.; Neda, I. Eur J Med Chem 2015,
101, 431.
SUPPORTING INFORMATION
[17] Maftei, C. V.; Fodor, E.; Jones, P. G.; Daniliuc, C. G.; Franz,
M. H.; Kelter, G.; Fiebig, H. H.; Tamm, M.; Neda, I. Tetrahedron 2016,
72, 1185.
[18] Maftei, C. V.; Fodor, E.; Jones, P. G.; Franz, M. H.; Kelter, G.;
Fiebig, H.; Neda, I. Beil J Org Chem 2013, 9, 2202.
Additional supporting information may be found online
in the Supporting Information section at the end of the
article.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet