Synthesis and biological activity of 2-(bis((1,3,4-oxadiazolyl/1,3,4- thiadiazolyl)methylthio)methylene)malononitriles

Article abstract of DOI:10.1016/j.ejmech.2011.01.063

The reactivity of ketene dithiolates in the presence of different equivalents of sodium ethoxide was studied. 2-(Bis((5-aryl-1,3,4-oxadiazol-2-yl) methylthio)methylene)malononitriles 5 and 2-(bis((5-aryl-1,3,4-thiadiazol-2-yl) methylthio)methylene)malononitriles 6 were prepared by the reaction of malononitrile with carbon disulfide and 5-aryl-2-(chloromethyl)-1,3,4- oxadiazoles 3/5-aryl-2-(chloromethyl)-1,3,4-thiadiazoles 4. The preliminary antimicrobial and antioxidant activities of the lead compounds were assayed.

Full text of DOI:10.1016/j.ejmech.2011.01.063

European Journal of Medicinal Chemistry 46 (2011) 1367e1373
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activity of 2-(bis((1,3,4-oxadiazolyl/1,3,4-thiadiazolyl)
methylthio)methylene)malononitriles
*
V. Padmavathi , G. Dinneswara Reddy, S. Nagi Reddy, K. Mahesh
Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, Andhara Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The reactivity of ketene dithiolates in the presence of different equivalents of sodium ethoxide was studied.
2-(Bis((5-aryl-1,3,4-oxadiazol-2-yl)methylthio)methylene)malononitriles 5 and 2-(bis((5-aryl-1,3,4-thia-
diazol-2-yl)methylthio)methylene)malononitriles 6 were prepared by the reaction of malononitrile with
carbon disulfide and 5-aryl-2-(chloromethyl)-1,3,4-oxadiazoles 3/5-aryl-2-(chloromethyl)-1,3,4-thiadia-
zoles 4. The preliminary antimicrobial and antioxidant activities of the lead compounds were assayed.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 17 September 2010
Received in revised form
25 December 2010
Accepted 26 January 2011
Available online 20 February 2011
Keywords:
Malononitrile
Dithioacetals
1,3,4-Oxadiazole
1,3,4-Thiadiazole
Sodium ethoxide
Antimicrobial activity
Antioxidant property
1. Introduction
of sodium ethoxide followed by alkylation was studied. When the
reaction of malononitrile with carbon disulfide was carried out in
Ketene dithioacetals are versatile synthons for the development of
a wide variety of heterocyclic systems [1,2]. In fact, nitroketene
S,S-acetals have been used as intermediates for the synthesis of
pyrroles with diverse functionalities [3,4]. The highly regioselective
the presence of one equivalent of sodium ethoxide followed by
treatment with ethyl bromoacetate at room temperature after
stirring for 8 h gave a solid which was identified as ethyl 3-amino-
4-cyano-5-((ethoxycarbonyl)methylthio)-thiophene-2-carboxylate
(1) by spectral parameters (Scheme 1). It seems that the initially
formed 2-cyano-3,3-bis((ethoxycarbonyl)methylthio)acrylnitrile
undergoes intramolecular cyclocondensation to 1 which is given in
the proposed mechanism (Scheme 1).
It was reported that diethyl 3,4-diaminothieno[2,3-b]thio-
phene-2,5-dicarboxylate was obtained in one pot reaction of
malononitrile, carbon disulfide and ethyl bromoacetate in K2CO3 in
DMF through the ketene dithioacetal dipotassium salt [11].
Contrary to this, it was reported that under similar reaction
conditions compound 1 was obtained in only 25% yield [12]. In the
present study compound 1 was obtained in 89% yield in the pres-
ence of sodium ethoxide in ethanol under anhydrous conditions.
The 1H NMR spectrum of 1 displayed two multiplets at 1.18e1.26
and 4.12e4.24 for ethoxy protons of carboethoxy group and
a singlet at 3.35 ppm for methylene protons. A broad singlet was
observed at 6.88 ppm for NH2 which disappeared on deuteration.
The 13C NMR spectrum displayed signals at 14.36, 14.76 (O]
CeOCH2CH3), 37.15 (S-CH2), 60.75, 62.15 (O]CeOCH2CH3), 98.70
(C-4), 101.59 (C-2), 112.86 (C^N), 153.99 (C-3), 154.69 (C-5), 162.37
cyclocondensation of
produced substituted pyrazoles [5,6]. The cyclocondensation of
-nitro ketene N,S-acylacetals either with Vilsmier reagent [7] or in the
a-oxoketene N,S-acetals with arylhydrazines
a-oxo/
a
presence of POCl3 in acetonitrile led to substituted quinoxaline deriv-
atives [8]. Ring closer reactions involving a nitrile group have also been
studied. When dicyano dithiolate was treated with p-bromophenacyl
bromide instead of an S,S0-bis(bromophenacyl) derivative an amino-
thiophene was formed [9]. Recently, we have studied the reactivity of
ketene S,S-acetals having gem diester and gem cyanoester functional-
ities towards the development of biologically potent heterocycles,
pyrazoline, isoxazoline, pyrimidine and their derivatives [10].
2. Chemistry
In continuation of our interest on polarized ketene S,S-acetals,
the reactivity of malononitrile with carbon disulfide in the presence
* Corresponding author. Tel.: þ91 877 2289303; fax: þ91 877 2249532.
E-mail address: vkpuram2001@yahoo.com (V. Padmavathi).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.063

1368
V. Padmavathi et al. / European Journal of Medicinal Chemistry 46 (2011) 1367e1373
NH2
O
NC
1 eq. NaOEt
O
N
C
C N
+
O
+
CS2
Br
O
S
rt, 8 h, EtOH
S
O
O
1
Mechanism:
-
+
S
NC
NC
NC
NC
Na
O
NaOEt
Br
+
-
S
C
S
Na
+
S
-
NC
NC
O
O
O
NH
H
NC
S
NC
S
S
O
O
O
O
O
-
N
C
S
O
NC
NH2
O
O
S
S
O
O
1
Scheme 1. Ethyl 3-amino-4-cyano-5-((ethoxycarbonyl)methylthio)thiophene-2-carboxylate.
& 168.12 ppm (C]O). The structure of compound 1 was further
established by X-ray analysis (deposited with Cambridge Crystal-
lographic Data Center (CCDC); deposition No. 780383) (Table 1).
Good single crystals of 1 were grown from methanol and subjected
to X-ray diffraction analysis. Perspective view of the X-ray structure
is depicted in Fig. 1.
In our endeavor to isolate the reactive intermediate 2-cyano-3,3-
bis((ethoxycarbonyl)methylthio)acrylnitrile (2), the reaction was
carried out in the presence of two equivalents of NaOEt and moni-
tored at frequent intervals. After stirring at room temperature for
1 h, the difference in TLC was observed and the product isolated was
Table 1
Crystal data and structure refinement for 1.
Empirical formula
Formula weight
C12H14N2O4S2
314.38
Temperature
Wavelength
293(2) K
0.7107 A
ꢀ
Crystal system, space group
Unit cell dimensions
Triclinic, P1
¼ 114.49 (10)ꢀ
¼ 94.80 (8)ꢀ
ꢀ
a ¼ 8.593 (10) A,
a
b
g
ꢀ
b ¼ 9.720 (12) A,
c ¼ 10.379 (9) A,
¼ 106.80 (11)ꢀ
ꢀ
ꢀ
Volume
734.1 (14) A
Z, Calculated density
Absorption coefficient
F(000)
2, 1.422 mg/m3
0.376 mmꢁ1
328
Crystal size
Theta range for data collection
Limiting indices
0.21 ꢂ 0.16 ꢂ 0.14 mm
2.54e29.37ꢀ
ꢁ11 ꢃ h ꢃ 9, ꢁ13 ꢃ k ꢃ 12, ꢁ14 ꢃ l ꢃ 12
5318/3299 [R(int) ¼ 0.0980
29.44, 99.30%
Reflections collected/unique
Completeness to heat
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Godness-of-fit on F2
Final R indices [1 > 2sigma(1)
R indices (all data)
Largest diff. peak and hole
R factor
Multi scan
1.00 and 0.7371
Full-matrix least-squares on F2
3299/0/191
0.549
R1 ¼ 0.0546, wR2 ¼ 0.1248
R1 ¼ 0.2018, wR2 ¼ 0.1673
0.312 and ꢁ0.323 e Aꢁ3
5.46%
Fig. 1. X-ray structure of compound 1.

V. Padmavathi et al. / European Journal of Medicinal Chemistry 46 (2011) 1367e1373
1369
O
methylene)malononitriles (5) were prepared in a one pot reaction of
malononitrile with carbon disulfide and 3 in the presence of two
moles of NaOEt at room temperature. Similarly 2-(bis((5-aryl-1,3,4-
thiadiazol-2-yl)methylthio)methylene)malononitriles (6) were
prepared bythe reaction of malononitrilewith CS2 and 4 (Scheme 4).
OEt
OEt
NC
NC
NC
NC
S
S
O
2 Eq NaOEt
CS2
+
Br
+
EtOH / rt, 1 h
O
O
2
3. Biology
Scheme 2. 2-Cyano-3,3-bis((ethoxycarbonyl)methylthio)acrylnitrile.
3.1. Antimicrobial activity
identified as 2 (Scheme 2). In order to study the role of sodium
ethoxide, we have extended the reaction time and observed that
with extension of time intramolecular cyclization of 2 took place and
after 4 h the compound 1 was obtained. The 1H NMR spectrum of
compound 2 showed a triplet at 1.31, a quartet at 4.24 for carboe-
thoxy and a singlet at 4.03 ppm for methylene protons. The 13C NMR
spectrum of 2 displayed signals at 14.06, 37.92, 62.86, 83.93, 111.91,
166.84 and 178.46 ppm for OCH2CH3, SCH2, OCH2CH3, (NC)2C]C,
CN, COOCH2CH3 and C]C(S)(S), respectively. Attempts were made
to develop heterocyclic ring by making use of ester functionality in
compound 2. Thus, when compound 2 was treated with benzoic acid
hydrazide instead of the expected product, 1 was obtained by base
induced intramolecular cyclization. Later we thought of developing
the desired bis heterocycles by treating with chloromethyl 1,3,4-
oxadiazole/thiadiazole. The compounds 5-aryl-2-(chloromethyl)-
1,3,4-oxadiazoles (3) were prepared by the reaction of chloroacetic
acid with benzoic hydrazides in the presence of POCl3 [13] (Scheme
3). The 5-aryl-2-(chloromethyl)-1,3,4-thiadiazoles (4) were repor-
ted by the reaction of N-chloroacetyl-N0-aroylhydrazene with Law-
essons reagent [14]. However, we have achieved the former
compounds by the interconversion of compounds 3 with thiourea in
The compounds 2, 5 and 6 were tested for antimicrobial activity
at two different concentrations 100 and 200 mg/ml. The antibacte-
rial activity was screened against Staphylococcus aureus, Bacillus
subtilis (Gram-positive bacteria) and Escherichia coli, Klebsiella
pneumoniae (Gram-negative bacteria) on nutrient agar plates at
37 ꢀC for 24 h using chloramphenicol as reference drug. The
compounds were also evaluated for their antifungal activity against
Fusarium solani, Curvularia lunata and Aspergillus niger using
ketoconazole as standard drug. Fungi cultures were grown on
potato dextrose agar medium (PDA) at 25 ꢀC for 3 days. The spore
suspension was adjusted to 106 pores/ml at an mg/ml concentra-
tion by the Vincent and Vincent method [15]. The observed data on
the antimicrobial activity of the compounds and control drugs are
given in Tables 2e4.
3.2. Antioxidant testing
The compounds 2, 5 and 6 were tested for antioxidant property
by nitric oxide [16,17] and 1,1-diphenylpicrylhydrazyl (DPPH) [18]
methods. The observed data on the antioxidant activity are given
in Table 5.
THF.
Thus
2-(bis((5-aryl-1,3,4-oxadiazol-2-yl)methylthio)
N
N
O
Cl
NH2
HO
POCl3 /reflux
O
N
Cl
+
H
3
O
R
R
H2NCSNH2 / THF
N
N
Cl
R- a= H
b= Me
c= Cl
S
R
4
Scheme 3. 5-Aryl-2-(chloromethyl)-1,3,4-oxadiazoles/1,3,4-thiadiazoles.
N
R
N
N
N
X
NC
NC
Cl
NC
NC
S
S
2 Eq NaOEt
X
CS2
+
+
EtOH / rt, 1 h
X
N
R
R
3/4
N
3, 5; X = O
4, 6; X = S
5/6
R- a= H
b= Me
c= Cl
Scheme 4. 2-(Bis((5-aryl-1,3,4-oxadiazolyl/1,3,4-thiadiazolyl)methylthio)methylene)malononitriles.

1370
V. Padmavathi et al. / European Journal of Medicinal Chemistry 46 (2011) 1367e1373
Table 2
Table 5
The in vitro antibacterial activity of 2, 5 and 6.
Antioxidant activity of 2, 5 and 6.
Compound
Concentration Zone of inhibition (mm)
g/ml)
Compound
% Inhibition at 100
mM
(m
Gram (þ)ve
Gram (ꢁ)ve
Nitric oxide method
DPPH method
2
5a
5b
5c
6a
6b
6c
45.8
65.4
54.3
72.5
25.8
22.3
29.4
94.9
46.3
63.5
52.5
73.7
26.3
21.6
30.2
95.3
S. aureus B. subtilis E. coli K. pneumoniae
2
100
200
10
14
12
14
7
9
e
8
5a
5b
5c
6a
6b
6c
100
200
12
14
12
16
8
10
9
12
100
200
10
13
10
12
e
e
Ascorbic acid
9
11
100
200
16
20
13
16
8
11
8
12
the reaction of malononitrile with carbon disulfide and 5-aryl-2-
(chloromethyl)-1,3,4-oxadiazoles (3)/5-aryl-2-(chloromethyl)-
1,3,4-thiadiazoles (4), respectively.
100
200
24
26
23
27
21
24
23
24
100
200
19
25
19
24
17
24
18
22
4.1. Biological results
100
200
32
35
30
32
25
26
23
25
The results of the compounds of preliminary antibacterial
testing are shown in Table 2. The results revealed that the com-
pounds 2-(bis((5-aryl-1,3,4-thiadiazol-2-yl)-methylthio)methyle-
ne)malononitriles (6) exhibited high activity (17e35 mm) on both
Gram-positive and Gram-negative bacteria. In fact, compound 6c
showed pronounced activity (30e35 mm) towards Gram-positive
bacteria. The compounds 2-(bis ((5-aryl-1,3,4-oxadiazol-2-yl)
methylthio)methylene)malononitriles (5) displayed moderate act-
ivity towards Gram-positive bacteria (10e20 mm). On the other
hand 2-cyano-3,3-bis((ethoxycarbonyl)methylthio)acrylnitriles (2)
exhibited least activity against both bacteria. Results of the inves-
tigation presented in Table 3 revealed that all the compounds
except 2 showed relatively high inhibitory effect on F. solani and
C. lunata than on A. niger. The compounds 6a and 6c displayed high
activity.
Chloramphenicol 100
200
35
39
38
41
40
44
42
45
Control (DMSO)
e
e
e
e
Table 3
The in vitro antifungal activity of 2, 5 and 6.
Compound
Concentration
g/ml)
Zone of inhibition (mm)
(
m
F. solani
C. lunata
A. niger
2
100
200
13
15
12
15
9
12
5a
100
200
18
21
15
17
12
14
The MIC values were determined as the lowest concentration
that completely inhibited the visible growth of the microorganisms
(Table 4). The structureeantimicrobial activity relationship of the
synthesized compounds revealed that compounds having thiadia-
zole moiety exhibited high activity when compared with
compounds having oxadiazole unit. The presence of chloro sub-
stituent in the aromatic ring enhanced the activity.
5b
100
200
15
17
13
14
10
11
5c
100
200
20
22
16
19
14
17
6a
100
200
25
28
26
28
21
24
6b
100
200
23
25
25
26
18
21
4.2. Antioxidant testing
6c
100
200
30
32
32
34
26
29
The compounds 2, 5 and 6 were tested for antioxidant property
Ketoconazole
Control (DMSO)
100
200
38
42
41
44
36
39
by nitric oxide and DPPH methods. The compounds 2-(bis((5-aryl-
1,3,4-oxadiazol-2-yl)methylthio)-methylene)malononitriles
(5)
exhibited high antioxidant activity in both nitric oxide and DPPH
e
e
e
methods at 100
mM concentration (Table 5).
4. Result and discussion
5. Conclusion
We have synthesized 2-(bis((5-aryl-1,3,4-oxadiazol-2-yl)
methylthio)methylene)malononitriles (5) and 2-(bis((5-aryl-1,
3,4-thiadiazol-2-yl)methylthio)methylene)malononitriles (6) by
The reactivity of ketene dithiolates in the presence of different
equivalents of sodium ethoxide was studied. We have develo-
ped simple and efficient route for the synthesis of previously
Table 4
Minimum inhibitory concentration of compounds 6a and 6c.
Compound
Minimum inhibitory concentration (MIC), mg/ml
S. aureus
B. subtilis
E. coli
K. pneumoniae
F. solani
C. lunata
A. niger
6a
6c
200
100
6.25
e
100
50
6.25
e
200
200
6.25
e
200
200
12.5
e
100
50
100
50
100
100
e
Chloramphenicol
Ketoconazole
e
e
12.5
6.25
6.25

V. Padmavathi et al. / European Journal of Medicinal Chemistry 46 (2011) 1367e1373
1371
unreported bis heterocycles from ketene dithiolates. The mild
6.1.3. General procedure for the synthesis of 5-aryl-2-
(chloromethyl)-1,3,4-oxadiazoles (3aec)
reaction conditions, selectivity, good yields and straight forward
product isolation were discussed. The preliminary antimicrobial
activity of the tested compounds showed that those having thia-
diazole moiety exhibited greater activity. The maximum activity
was observed with the compounds 6a and 6c. However
compounds with oxadiazole unit exhibited good antioxidant
activity.
A mixture of aryl acid hydrazide (10 mmol), chloroacetic acid
(10 mmol) and POCl3 (7 ml) was heated under reflux for 5e6 h. The
excess POCl3 was removed under reduced pressure and the residue
was poured onto crushed ice. The resulting precipitate was filtered,
washed with saturated sodium bicarbonate solution and then with
water, dried and recrystallized from ethanol to obtain 3.
6. Experimental section
6.1.3.1. 5-Phenyl-2-(chloromethyl)-1,3,4-oxadiazole
brown solid (1.79 g, 92%); m.p. 86e88 ꢀC; IR (KBr): 1610 (C]N)
cmꢁ1; 1H NMR (CDCl3, 400 MHz):
7.50e8.01 (m, 5H), 4.75 (s, 2H)
ppm; 13C NMR (CDCl3, 100 MHz):
32.88 (CH2), 161.77 (C-2), 165.58
(3a). Pale
6.1. Chemistry
d
d
Melting points were determined in open capillaries on a Mel-
Temp apparatus and are uncorrected. The purity of the compounds
was checked by TLC (silica gel H, BDH, ethyl acetate/hexane, 1:3).
The IR spectra were recorded on a Thermo Nicolet IR200 FT-IR
spectrometer as KBr pellets and the wave numbers were given in
cmꢁ1. The 1H NMR spectra were recorded in CDCl3/DMSO-d6 on
a Brucker-400 spectrometer (400 MHz). The 13C NMR spectra were
recorded in CDCl3/DMSO-d6 on a Brucker spectrometer operating at
(C-5), 131.88, 128.95, 127.00, 123.43 (aromatic carbons) ppm;. MS
m/z: 195. Anal. Calcd for C9H7ClN2O: C, 55.54; H, 3.63; N, 14.39;
Found: C, 55.68; H, 3.69; N, 14.47%.
6.1.3.2. 5-p-Tolyl-2-(chloromethyl)e1,3,4-oxadiazole
(3b). Pꢁal1e
brown solid (1.85 g, 89%); m.p. 91e93 ꢀC; IR (KBr): 1612 (C]N) cm
;
1H NMR (CDCl3, 400 MHz):
7.44e7.95 (m, 4H, Ar-H) ppm; 13C NMR (CDCl3):
d
2.74 (s, 3H, Ar-CH3), 4.66 (s, 2H, CH2),
23.86 (Ar-CH3),
d
100 MHz. All chemical shifts are reported in
d
(ppm) using TMS as
33.76 (CH2), 159.43 (C-2), 164.62 (C-5), 130.71, 129.07, 128.20, 122.89
(aromatic carbons) ppm; MS m/z: 208; Anal. Calcd for C10H9ClN2O: C,
57.57; H, 4.35; N, 13.43; Found: C, 57.42; H, 4.39; N, 13.49%.
an internal standard. X-ray data was collected at 293 K on the
Oxford diffractometer with graphite monochromatic Mo KR. The
microanalyses were performed using PerkineElmer 240C
elemental analyzer. The mass spectra were recorded on Agilent
1100 series LC/MSD.
6.1.3.3. 5-(4-Chlorophenyl)-2-(chloromethyl)-1,3,4-oxadiazole
(3c). Pale brown solid (1.70 g, 93%); m.p. 97e99 ꢀC; IR (KBr): 1615
(C]N) cmꢁ1
;
1H NMR (CDCl3, 400 MHz):
d
4.71 (s, 2H, CH2),
6.1.1. Synthesis of ethyl 3-amino-4-cyano-5-((ethoxycarbonyl)
methylthio)thiophene-2-carboxylate (1)
7.55e7.99 (m, 4H, Ar-H) ppm; 13C NMR (CDCl3, 100 MHz):
d
34.15
(CH2), 158.32 (C-2), 165.44 (C-5), 133.21, 129.86, 128.79, 123.68
(aromatic carbons) ppm; MS m/z: 229; Anal. Calcd for C9H6Cl2N2O:
C, 47.19; H, 2.64; N, 12.23; Found: C, 47.25; H, 2.58; N, 12.29%.
A mixture of malononitrile (0.66 g, 10 mmol), sodium ethoxide
(0.68 g, 10 mmol) and carbon disulfide (1.14 g, 15 mmol) was
stirred for 1 h at 0 ꢀC. To this, ethyl bromoacetate (3.34 g,
20 mmol) in ethanol (5 ml) was added slowly maintaining
temperature at 20e30 ꢀC. The stirring was continued for an
additional period of 8 h. The contents of the flask were poured
onto crushed ice. The solid obtained was filtered, dried and
recrystallized from aqueous methanol to give compound 1 as
reddish crystals (0.27 g, 89%); m.p. 119e121 ꢀC; IR (KBr): 3335,
6.1.4. General procedure for the synthesis of 5-aryl-2-(chloromethyl)-
1,3,4-thiadiazoles (4aec)
In a sealed test tube, a mixture of 3 (5 mmol), thiourea
(20 mmol) and tertahydrofuran (5 ml) was taken and heated at
120e150 ꢀC in an oil bath for 24e30 h. After the reaction was
completed, it was extracted with dichloromethane. The organic
layer was washed with water, brine solution and dried over anhy-
drous Na2SO4. The resultant solid was recrystallized from methanol.
3282 (NH2), 2222 (CN), 1736 (CO), 1632 (C]C) cmꢁ1
(DMSO-d6, 400 MHz): 1.18e1.26 (m, 6H, CH3), 3.35 (s, 2H, S-CH2),
4.12e4.24 (m, 4H, O-CH2), 6.88 (bs, 2H, NH2) ppm; 13C NMR
(DMSO-d6, 100 MHz): 14.36, 14.76 (O]CeOCH2CH3), 37.15 (S-
;
1H NMR
d
d
6.1.4.1. 5-Phenyl-2-(chloromethyl)-1,3,4-thiadiazole
solid (0.80 g, 76%); m.p. 92e94; IR (KBr): 1603 (C]N) cmꢁ1
NMR (CDCl3, 400 MHz): 4.29 (s, 2H, CH2), 7.25e7.58 (m, 5H, Ar-H)
ppm; 13C NMR (CDCl3):
34.86 (CH2), 165.11 (C-2), 173.95 (C-5),
(4a). Yellow
CH2), 60.75, 62.15 (O]CeOCH2CH3), 98.70 (C-4), 101.59 (C-2),
112.86 (C^N), 153.99 (C-3), 154.69 (C-5), 162.37 & 168.12 (C]O)
ppm; MS m/z: 314; Anal. Calcd for C12H14N2O4S2: C, 45.85; H, 4.49;
N, 8.91; Found: C, 45.72; H, 4.54; N, 8.99%.
;
1H
d
d
132.84, 130.06, 128.86, 127.59 (aromatic carbons) ppm; MS m/z:
210.68; Anal. Calcd for C9H7ClN2S: C, 51.31; H, 3.35; N,13.30; Found:
C, 51.42; H, 3.41; N, 13.40%.
6.1.2. Synthesis of 2-cyano-3,3-bis((ethoxycarbonyl)methylthio)
acrylnitrile (2)
A mixture of malononitrile (0.66 g, 10 mmol), sodium ethoxide
(1.36 g, 20 mmol) and carbon disulfide (1.14 g, 15 mmol) was stirred
for 1 h at 0 ꢀC. To this, ethyl bromoacetate (3.34 g, 20 mmol) in
ethanol (5 ml) was added slowly maintaining temperature at
20e30 ꢀC. The stirring was continued for an additional period of
1 h. The contents of the flask were poured onto crushed ice. The
solid obtained was filtered, dried and recrystallized from aqueous
methanol to give compound 2 as pale brown solid (0.23 g, 75%);
m.p. 40e42 ꢀC; IR (KBr): 2221 (CN), 1723 (CO) 1636 (C]C) cmꢁ1; 1H
6.1.4.2. 5-p-Tolyl-2-(chloromethyl)-1,3,4-thiadiazole
solid (0.84 g, 75%); m.p. 98e100 ꢀC; IR (KBr): 1605 (C]N) cmꢁ1; 1H
NMR (CDCl3, 400 MHz): 2.84 (s, 3H, Ar-CH3), 4.32 (s, 2H, CH2),
7.36e7.65 (m, 4H, Ar-H) ppm; 13C NMR (CDCl3):
23.64 (Ar-CH3),
34.41 (CH2), 163.45 (C-2), 171.82 (C-5), 136.12, 129.49, 128.53, 127.95
(aromatic carbons) ppm; MS m/z: 224; Anal. Calcd for C10H9ClN2S:
C, 53.45; H, 4.04; N, 12.47; Found: C, 53.53; H, 4.09; N, 12.53%.
(4b). Yellow
d
d
6.1.4.3. 5-(4-Chlorophenyl)-2-(chloromethyl)-1,3,4-thiadiazole
NMR (CDCl3, 400 MHz):
d
1.31 (t, 6H, CH3), 4.03 (s, 4H, S-CH2), 4.24
14.06 (OCH2CH3),
(4c). Yellowsolid (0.95 g, 78%); m.p.105e107 ꢀC; IR (KBr): 1608 (C]
(q, 4H, O-CH2) ppm; 13C NMR (CDCl3, 100 MHz):
d
N) cmꢁ1
;
1H NMR (CDCl3, 400 MHz):
d
4.27 (s, 2H, CH2), 7.47e7.64
37.92 (SCH2), 62.86 (OCH2CH3), 83.93 ((NC)2C]C), 111.91 (CN),
166.84 (COOCH2CH3), 178.46 (C]C(S)(S)) ppm; MS m/z: 314; Anal.
Calcd for C12H14N2O4S2: C, 45.85; H, 4.49; N, 8.91; Found: C, 45.74;
H, 4.43; N, 8.96%.
(m, 4H, Ar-H) ppm; 13C NMR (CDCl3,100 MHz):
d
34.13 (CH2),164.12
(C-2),172.45 (C-5),133.45,130.87,129.81,128.99 (aromatic carbons)
ppm;MS m/z: 245.13; Anal. Calcd forC9H6Cl2N2S: C, 44.10; H, 2.47; N,
11.43; Found: C, 44.19; H, 2.51; N, 11.30%.

1372
V. Padmavathi et al. / European Journal of Medicinal Chemistry 46 (2011) 1367e1373
6.1.5. General procedure for the synthesis of 2-(bis((5-aryl-1,3,4-
oxadiazol-2-yl)-methylthio)methylene)malononitriles (5aec)
(S)),133.42,129.78,126.31,123.91 (aromatic carbons) ppm; MS m/z:
518; Anal. Calcd for C24H18N6S4: C, 55.57; H, 3.50; N,16.20; Found: C,
55.65; H, 3.55; N, 16.28.
A
mixture of malononitrile (10 mmol), sodium ethoxide
(20 mmol) and carbon disulfide (15 mmol) was stirred for 1 h at
0 ꢀC. To this, compound 3 (20 mmol) in ethanol (5 ml) was added
slowly maintaining temperature at 20e30 ꢀC. The stirring was
continued for an additional period of 1 h. The contents of the flask
were poured onto crushed ice. The solid obtained was filtered,
dried and recrystallized from aqueous methanol.
6.1.6.3. 2-(Bis((5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)methylthio)
methylene)malononitrile (6c). White solid (4.02 g, 72%); m.p.
269e272 ꢀC; IR (KBr): 2231 (CN),1633 (C]C),1617 (C]N) cmꢁ1; 1H
NMR (CDCl3, 400 MHz):
d
4.43 (s, 4H, CH2), 7.29e7.89 (m, 8H, Ar-H)
29.89 (CH2), 81.60 ((NC)2C]C), 112.73
ppm; 13C NMR (CDCl3):
d
(CN), 161.53 (C-2), 167.31 (C-5), 183.91 (C]C(S)(S)), 132.52, 131.78,
128.21, 124.31 (aromatic carbons) ppm; MS m/z: 559; Anal. Calcd
for C22H12Cl2N6S4: C, 47.23; H, 2.56; N, 15.02; Found: C, 47.28; H,
2.62; N, 15.12%.
6.1.5.1. 2-(Bis((5-phenyl-1,3,4-oxadiazol-2-yl)methylthio)methylene)
malononitrile (5a). Yellow solid (3.29 g, 72%); m.p. 232e234 ꢀC; IR
(KBr): 2224 (CN), 1625 (C]C), 1618 (C]N) cmꢁ1 1H NMR (CDCl3,
;
400 MHz):
NMR (CDCl3):
d
4.41 (s, 4H, CH2), 7.49e8.07 (m, 10H, Ar-H) ppm; 13C
29.50 (CH2), 84.20 ((NC)2C]C), 112.23 (CN), 162.31
d
6.2. Biological assays
(C-2), 166.72 (C-5),181.43 (C]C(S)(S)), 128.92, 127.26, 126.31, 123.81
(aromatic carbons) ppm; MS m/z: 458; Anal. Calcd for C22H14N6O2S2:
C, 57.63; H, 3.08; N, 18.33; Found: C, 57.51; H, 3.02; N, 18.44%.
6.2.1. Compounds
The compounds 2, 5 and 6 were dissolved in DMSO at different
concentrations of 100, 200 and 800 mg/ml.
6.1.5.2. 2-(Bis((5-p-tolyl-1,3,4-oxadiazol-2-yl)methylthio)methylene)
malononitrile (5b). White solid (3.39 g, 69%); m.p. 241e243 ꢀC; IR
6.2.2. Cells
(KBr): 2229 (CN), 1628 (C]C), 1612 (C]N) cmꢁ1
;
1H NMR (CDCl3,
Bacterial strains S. aureus, B. subtilis, E. coli, K. pneumonie and
fungi F. solani, C. lunata and A. niger were obtained from National
Collection of Industrial Microorganisms (NCIM), National Chemical
Laboratory, Pune, India.
400 MHz):
d
2.39 (s, 6H, Ar-CH3), 4.37 (s, 4H, CH2), 7.31e7.84 (m, 8H,
23.92 (Ar-CH3), 28.31 (CH2), 83.20
Ar-H) ppm; 13C NMR (CDCl3):
d
((NC)2C]C), 114.23 (CN), 163.61 (C-2), 167.61 (C-5), 180.49 (C]C(S)
(S)),132.92,128.28,125.31,124.51 (aromatic carbons) ppm; MS m/z:
486 ppm; Anal. Calcd for C24H18N6O2S2: C, 59.24; H, 3.73; N, 17.27;
Found: C, 59.31; H, 3.77; N, 17.19%.
6.2.3. Antibacterial and antifungal assays
Preliminary antimicrobial activity of compounds 2, 5 and 6 was
tested byagar disc-diffusion method. Sterile filter paper discs (6 mm
diameter) moistened with the test compound solution in DMSO of
6.1.5.3. 2-(Bis((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methylthio)
methylene)malononitrile (5c). White solid (3.85 g, 73%); m.p.
specific concentration 100 mg and 200 mg/disc were carefully placed
253e255 ꢀC; IR (KBr): 2223 (CN), 1630 (C]C), 1621 (C]N) cmꢁ1
;
on the agar culture plates that had been previously inoculated
separately with the microorganisms. The plates were incubated at
37 ꢀC and the diameterof the growth inhibition zones was measured
after 24 h in case of bacteria and after 48 h in case of fungi.
The MICs of the compound assays were determined using micro
dilution susceptibility method. Chloramphenicol was used as
reference antibacterial agent. Ketoconazole was used as reference
antifungal agent. The test compounds, chloramphenicol and
1H NMR (CDCl3, 400 MHz):
H) ppm; 13C NMR (CDCl3):
d
4.39 (s, 4H, CH2), 7.43e7.91 (m, 8H, Ar-
29.53 (CH2), 84.55 ((NC)2C]C), 112.82
d
(CN), 162.33 (C-2), 168.56 (C-5), 183.2 (C]C(S)(S)), 131.32, 127.56,
125.54, 122.23 (aromatic carbons) ppm; MS m/z: 527; Anal. Calcd
for C22H12Cl2N6O2S2: C, 50.10; H, 2.29; N, 15.93; Found: C, 49.97; H,
2.20; N, 15.84%.
6.1.6. General procedure for the synthesis of 2-(bis((5-aryl-1,3,4-
thiadiazol-2-yl) methylthio)methylene)malononitriles (6aec)
ketoconazole were dissolved in DMSO at concentration of 800
ml. The two-fold dilution of the solution was prepared (400, 200,
100. 6.25 g/ml). The microorganism suspensions were incu-
mg/
A
mixture of malononitrile (10 mmol), sodium ethoxide
m
(20 mmol) and carbon disulfide (15 mmol) was stirred for 1 h at
0 ꢀC. To this, compound 4 (20 mmol) in ethanol (5 ml) was added
slowly maintaining temperature at 20e30 ꢀC. The stirring was
continued for an additional period of 1 h. The contents of the flask
were poured onto crushed ice. The solid obtained was filtered,
dried and recrystallized from aqueous methanol.
bated at 36 ꢀC for 24 and 48 h for bacteria and fungi, respectively.
The minimum inhibitory concentrations of the compounds were
recorded as the lowest concentration of each chemical compound
in the tubes with no turbidity (i.e. no growth) of inoculated
bacteria/fungi.
6.2.4. Antioxidant testing
6.1.6.1. 2-(Bis((5-phenyl-1,3,4-thiadiazol-2-yl)methylthio)methylene)
malononitrile (6a). Pale yellow solid (3.43 g, 70%); m.p.
245e247 ꢀC; IR (KBr): 2234 (CN),1635 (C]C),1619 (C]N) cmꢁ1; 1H
The compounds 2, 5 and 6 are tested for antioxidant property by
nitric oxide and DPPH methods.
NMR (CDCl3, 400 MHz):
d
4.37 (s, 4H, CH2); 7.29e7.70 (m, 10H, Ar-
28.30 (CH2), 83.50 ((NC)2C]C), 111.43
6.2.5. Assay for nitric oxide (NO) scavenging activity
H) ppm; 13C NMR (CDCl3):
d
Sodium nitroprusside (5
mM) in phosphate buffer pH 7.4 was
(CN), 161.91 (C-2), 165.32 (C-5), 182.83 (C]C(S)(S)), 130.82, 128.56,
126.31, 124.91 (aromatic carbons) ppm; MS m/z: 490; Anal. Calcd
for C22H14N6S4: C, 53.86; H, 2.88; N, 17.13; Found: C, 53.75; H, 2.83;
N, 17.22%.
incubated with 100 M concentration of test compounds dissolved
m
in a suitable solvent (dioxane/methanol) and tubes were incubated
at 25 ꢀC for 120 min. Control experiment was conducted with equal
amount of solvent in an identical manner. At intervals, 0.5 ml of
incubation solution was taken and diluted with 0.5 ml of Griess
reagent (1% Sulfanilamide, 0.1% N-naphthylethylenediamine dihy-
drochloride and 2% o-phosphoric acid dissolved in distilled water).
The absorbance of the chromophore formed during diazotization of
nitrite with sulfanilamide and subsequent N-naphthylethylenedi-
6.1.6.2. 2-(Bis((5-p-tolyl-1,3,4-thiadiazol-2-yl)methylthio)methy-
lene)malononitrile (6b). White solid (3.47 g, 67%); m.p. 257e259 ꢀC;
IR (KBr): 2235 (CN),1629 (C]C),1618 (C]N) cmꢁ1; 1H NMR (CDCl3,
400 MHz):
Ar-H) ppm; 13C NMR (CDCl3):
((NC)2C]C), 113.43 (CN), 162.33 (C-2), 166.81 (C-5), 181.39 (C]C(S)
d
2.42 (s, 6H, Ar-CH3), 4.39 (s, 4H, CH2), 7.22e7.64 (m, 8H,
d
24.92 (Ar-CH3), 28.67 (CH2), 82.21
amine dihydrochloride was read at
repeated in triplicate.
l 546 nm. The experiment was

V. Padmavathi et al. / European Journal of Medicinal Chemistry 46 (2011) 1367e1373
1373
6.2.6. Reduction of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free
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Acknowledgements
The authors are grateful to CSIR, New Delhi for financial assis-
tance under major research project.