The reactivity of gem cyanoester ketene dithiolates towards the development of potent antioxidant heterocycles

Article abstract of DOI:10.1248/cpb.60.449

The reactivity of gem cyanoester ketene dithiolates towards the development of a variety of heterocycles was studied and tested for antioxidant property. The compounds bis benzoxazolylmethylthiomethylene pyrazoles and isoxazoles displayed excellent radical scavenging activity when compared with the standard ascorbic acid.

Full text of DOI:10.1248/cpb.60.449

April 2012
Regular Article
Chem. Pharm. Bull. 60(4) 449–458 (2012)
449
The Reactivity of Gem Cyanoester Ketene Dithiolates towards the
Development of Potent Antioxidant Heterocycles
Venkatapuram Padmavathi,* Bhumireddy Chinnachennaiahgari Venkatesh,
Akkarapalli Muralikrishna, and Adivireddy Padmaja
Department of Chemistry, Sri Venkateswara University; Tirupati–517 502, Andhra Pradesh, India.
Received September 28, 2011; accepted January 26, 2012; published online February 1, 2012
The reactivity of gem cyanoester ketene dithiolates towards the development of a variety of heterocycles
was studied and tested for antioxidant property. The compounds bis benzoxazolylmethylthiomethylene pyr-
azoles and isoxazoles displayed excellent radical scavenging activity when compared with the standard ascor-
bic acid.
Key words gem cyanoester ketene dithiolate; cyclocondensation; antioxidant activity; bis benzoxazolyl;
benzothiazolyl; benzoimidazolyl–pyrazole
Pyrazole and isoxazole derivatives are interesting heterocy- been taken up. The synthetic intermediates 2-(chloromethyl)
clic compounds since they show a wide range of pharmaco- benzoxazole (1) and 2-(chloromethyl)benzothiazole (2) were
logical properties including antiinflammatory,1) antibacterial2) prepared by the irradiation of 2-aminophenol/2-aminothio-
and antifungal.3) In addition pyrazoles and isoxazoles are phenol and chloroacetyl chloride for 10–15min at a power of
present in leading pharmaceuticals viz., Celebrex, Viagra, 500W.31,32) However, 2-(chloromethyl)-1H-benzimidazole (3)
Valdecoxib.4,5) Pyrimidine derivatives have been very well was obtained by treating o-phenylenediamine with chloroace-
known in medicinal chemistry for their therapeutic applica- tic acid in the presence of 5N HCl.33)
tions.6,7) One possible reason for their activity is the presence
A one pot reaction of ethyl cyanoacetate, carbon disulfide
of a pyrimidine base in thymine, cytosine and uracil, which and 1/2/3 in the presence of K2CO3 in N,N-dimethylformamide
are essential building blocks of nucleic acids, DNA and RNA. (DMF) resulted in gem difunctionalized compounds, ethyl
Benzoxazole ring is one of the most common heterocycle in 3,3-bis((benzo[d]oxazol-2-yl)methylthio)-2-cyanoacrylate (4),
medicinal chemistry. Substituted benzoxazoles possess diverse ethyl 3,3-bis((benzo[d]thiazol-2-yl)methylthio)-2-cyanoacrylate
chemotherapeutic activities including antibiotic,8) antimicro- (5) and ethyl 3,3-bis((1H-benzo[d]imidazol-2-yl)methylthio)-
bial9–13) and antitumor.14,15) Benzothiazole derivatives are im- 2-cyanoacrylate (6) (Chart 1).
portant members of fused heterocycles as they exhibit antimi-
In order to develop a new class of tris heterocyclic com-
crobial properties16) and have found applications in industry as pounds, the gem cyanoester functionality in 4 was made to
antioxidants.17) In fact, 2-arylbenzothiazoles are confirmed as react with hydrazine hydrate in the presence of 1eq of K2CO3
a novel class of potent and selective antitumor agents.18,19) The in DMF. Instead of the expected tris heterocyclic compound,
benzimidazole moiety is present in many biologically active intramolecular cyclization of 4 occurred with the formation
natural products and synthetic compounds. Some of them ex- of a mixture of tetrasubstituted thiophene, ethyl 2-((benzo[d]-
hibit clinical value towards breast cancer,20–23) leukemia,24,25) oxazol-2′-yl)methylthio)-4-amino-5-(benzo[d]oxazol-2-yl)thio-
tumor cells26,27) etc. With this background we designed the phene-3-carboxylate (7) and 4-amino-2,5-di(benzo[d]oxazol-
present work to synthesize bis benzoxazolyl/benzothiazolyl/ 2′-yl)thieno[2,3-b]thiophen-3(2H)-one (10) in
a ratio of
benzimidazolyl–pyrazoles/isoxazoles/pyrimidines from ketene 3:1. However, the reaction was repeated in the absence of
dithiolates and to study the antioxidant property of these com- hydrazine hydrate wherein the products 7 and 10 were ob-
1
pounds.
tained in the same ratio (Chart 2, Method A). The H-NMR
spectrum of 7 displayed a singlet at δ 4.37 for methylene
protons, a quartet and a triplet at 4.22, 1.36 for carbethoxy
Chemistry
In continuation of our interest to study the reactivity of protons and a broad singlet at 6.88ppm for NH2. The latter
ketene dithiolates towards the development of a new class of signal disappeared on deuteration. The 1H-NMR spectrum
heterocycles and their bioassay,28–30) the following work has of 10 showed a singlet at δ 4.51ppm for methine proton
Chart 1
*To whom correspondence should be addressed. e-mail: vkpuram2001@yahoo.com
© 2012 The Pharmaceutical Society of Japan

450
Vol. 60, No. 4
and a broad singlet at 6.69ppm for NH2 which disappeared The signals of NH2 and NH disappeared on deuteration.
on deuteration. Then, the reaction was carried out with 5 The cyclocondensation of 4/5/6 with urea in the presence of
and 6 in the presence of 1eq of K2CO3 in DMF, a mixture piperidine in ethanol gave 6-amino-5-(bis((benzo[d]oxazol-2′-
of products ethyl 2-((benzo[d]thiazol-2′-yl)methylthio)-4- yl)methylthio)methylene)-2-hydroxypyrimidin-4(5H)-one (19),
amino-5-(benzo[d]thiazol-2-yl)thiophene-3-carboxylate
(8), 6-amino-5-(bis((benzo[d]thiazol-2′-yl)methylthio)methylene)-
ethyl
2-((1′H-benzo[d]imidazol-2′-yl)methylthio)-4-amino- 2-hydroxypyrimidin-4(5H)-one (20) and 6-amino-5-(bis((1′H-
5-(1′H-benzo[d]imidazol-2′-yl)-thiophene-3-carboxylate
and
(9) benzo[d]imidazol-2′-yl)methylthio)methylene)-2-hydroxypy-
4-amino-2,5-di(benzo[d]thiazol-2′-yl)thieno[2,3-b]thio- rimidin-4(5H)-one (21).
phen-3(2H)-one (11), 4-amino-2,5-di(1′H-benzo[d]imidazol-
Similar reaction of 4/5/6 with thiourea furnished 6-amino-
2′-yl)thieno[2,3-b]thiophen-3(2H)-one (12) were obtained 5-(bis((benzo[d]oxazol-2′-yl)methylthio)methylene)-2-mercap-
almost in the same ratio. topyrimidin-4(5H)-one (22), 6-amino-5-(bis((benzo[d]-thiazol-
On the other hand, the intramolecular cyclization reaction 2′-yl)methylthio)methylene)-2-mercaptopyrimidin-4(5H)-
of 4/5/6 with 2eq of K2CO3 afforded thienothiophene deriva- one (23) and 6-amino-5-(bis((1′H-benzo[d]imidazol-2′-yl)-
tives 10, 11 and 12 only (Chart 3, Method B). The latter com- methylthio)methylene)-2-mercaptopyrimidin-4(5H)-one (24).
pounds were also prepared in a one pot reaction of ethyl cya- Likewise, 5-(bis((benzo[d]oxazol-2′-yl)methylthio)methylene)-
noacetate, carbon disulfide and 1/2/3 in the presence of 3eq of dihydro-6-imino-1,3-dimethyl-pyrimidine-2,4(1H,3H)-dione
K2CO3 (Chart 3, Method C).
(25), 5-(bis((benzo[d]thiazol-2′-yl)methylthio)methylene)-di-
However, when the reaction was repeated by add- hydro-6-imino-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (26)
ing ethanolic solution of 4/5/6 dropwise into a solution and 5-(bis((1′H-benzo[d]imidazol-2′-yl)methylthio)methylene)-
of hydrazine hydrate and piperidine in ethanol, the tris dihydro-6-imino-1,3-dimethylpyrimidine-2,4(1H,3H)-dione
heterocyclic compounds, 3-amino-4-(bis((benzo[d]oxazol-2′- (27) were prepared by treating 4/5/6 with N,N′-dimethyl
yl)methylthio)methylene)-1H-pyrazol-5(4H)-one (13), 3-ami- urea in the presence of piperidine in ethanol (Chart 4).
no-4-(bis((benzo[d]thiazol-2′-yl)methylthio)methylene)-1H- The 1H-NMR spectra of 19–27 presented two singlets at δ
pyrazol-5(4H)-one (14) and 3-amino-4-(bis((1′H-benzo[d]- 4.29–4.43 for two methylene protons. The compounds 19–24
imidazol-2′-yl)methylthio)methylene)-1H-pyrazol-5(4H)-one showed a broad singlet at 5.53–5.66ppm for NH2. In addi-
(15) were obtained. Similar reaction with hydroxylamine tion, 19–21 exhibited a broad singlet at 8.31–8.36 and 22–24
hydrochloride produced 3-amino-4-(bis((benzo[d]oxazol-2′- at 1.46–1.53ppm due to OH and SH, respectively. The com-
yl)methylthio)methylene)isoxazol-5(4H)-one (16), 3-amino- pounds 25–27 displayed a singlet at 2.62–2.79ppm for N–Me
4-(bis-((benzo[d]thiazol-2′-yl)methylthio)methylene)isoxazol- protons and a broad singlet at 9.29–9.35ppm for =NH. The
5(4H)-one (17) and 3-amino-4-(bis((1'H-benzo[d]imidazol- compounds 21, 24 and 27 showed a broad singlet at around
2′-yl)methylthio)methylene)isoxazol-5(4H)-one
(18).
The 12.48ppm for NH of benzimidazole. The signals due to highly
1H-NMR spectra of 13–18 displayed two singlets in the region acidic protons disappeared on deuteration. The structures of
δ 4.28–4.40 which were accounted for two methylene protons all the new compounds were further established by IR and
and a broad singlet in the region 5.59–5.75 due to NH2. In ad- 13C-NMR spectral parameters and elemental analyses.
dition 13–15 showed another broad singlet at 9.67–9.86ppm
In-Vitro Antioxidant Study The compounds 4–27 were
for NH. The compounds 15 and 18 also exhibited a broad tested for antioxidant property by 2,2-diphenyl-1-picrylhydra-
singlet at 12.41, 12.35ppm due to NH of benzimidazole. zyl radical (DPPH),34,35) nitric oxide36,37) and 2,2′-azino-bis(3-
Chart 2
Chart 3

April 2012
451
Chart 4
—
Table 1. Antioxidant Activities of the Test Compounds 4–27 and Standard Using DPPH Scavenging Method % DPPH Radical Scavenging Activity
Concentration
Compound
50μg/mL (%)
100μg/mL (%)
150μg/mL (%)
IC50 μg/mL
4
50.74 1.24
—
53.94 1.41
—
57.13 1.24
—
49.28 1.20
—
5
—
—
—
—
6
7
52.15 1.11
—
55.02 1.05
—
58.44 1.34
—
47.94 1.02
—
8
—
—
—
—
9
—
—
—
—
10
—
—
—
—
11
—
—
—
—
12
13
75.24 0.66
59.62 1.31
17.37 1.75
77.03 0.89
59.89 0.87
16.58 1.54
58.74 0.96
14.36 0.87
—
80.28 0.87
62.98 1.04
20.24 1.28
82.97 0.77
63.54 0.67
20.14 1.67
63.05 0.68
17.98 0.98
—
83.18 0.54
67.30 1.35
22.20 1.04
85.80 0.84
68.60 1.10
24.25 1.48
66.40 0.54
20.20 1.04
—
33.22 0.29
41.94 0.92
247.03 0.68
32.18 0.80
41.74 0.91
248.26 1.28
42.56 0.69
278.08 1.12
—
14
15
16
17
18
19
20
21
22
17.46 1.54
—
20.94 1.59
—
24.81 1.48
—
238.77 0.67
—
23
—
—
—
—
24
25
60.06 0.58
18.34 1.02
—
63.98 0.78
20.78 1.23
—
68.82 0.97
23.10 1.41
—
41.62 0.40
240.61 1.09
—
26
27
Ascorbic acid
Blank
77.15 0.43
—
83.82 0.64
—
86.30 0.57
—
32.40 0.18
—
—
(
) Showed no scavenging activity. Values were the means of three replicates S.D.

452
Vol. 60, No. 4
—
Table 2. Antioxidant Activities of the Test Compounds 4–27 and Standard Using NO Scavenging Method % NO Radical Scavenging Activity
Concentration
Compound
50μg/mL (%)
100μg/mL (%)
150μg/mL (%)
IC50 μg/mL
4
60.36 0.98
—
62.78 0.87
—
65.48 1.12
—
41.64 0.39
—
5
—
—
—
—
6
7
61.26 0.87
—
64.08 084
—
67.38 0.94
—
40.80 0.58
—
8
—
—
—
—
9
—
—
—
—
10
—
—
—
—
11
—
—
—
—
12
13
83.81 0.54
66.53 1.24
24.24 1.12
85.49 0.87
68.14 0.81
22.92 1.31
64.24 1.54
20.91 0.87
—
88.69 0.49
69.24 1.34
26.75 1.23
90.05 0.64
71.83 0.67
25.45 1.36
68.14 1.64
24.03 0.77
—
89.42 0.78
72.42 1.62
30.45 1.20
94.30 0.59
75.28 0.88
28.50 1.18
70.13 1.52
28.34 0.86
—
29.82 0.19
37.58 0.87
193.24 1.05
29.24 0.30
36.68 0.44
212.35 1.21
38.92 0.93
224.56 0.69
—
14
15
16
17
18
19
20
21
22
25.14 0.58
—
27.89 0.97
—
29.92 0.74
—
186.44 0.78
—
23
—
—
—
—
24
25
70.23 0.87
27.56 1.34
—
75.18 1.05
31.18 1.54
—
78.20 0.96
35.70 1.28
—
35.44 0.24
174.71 1.12
—
26
27
Ascorbic acid
Blank
86.02 0.57
—
91.53 0.64
—
95.62 0.75
—
29.06 0.19
—
—
(
) Showed no scavenging activity. Values were the means of three replicates S.D.
ethylbenzothiazoline-6-sulphonic acid) (ABTS)38) methods and At these 10min intervals the values are very close and the re-
the results are shown in Tables 1–3 and Figs. 1–3. Amongst sults showed that the antioxidant activity of these compounds
the gem cyanoester heterocycles, the compound 4 displayed is independent of time (Table 4).
moderate radical scavenging activity in all the three methods
while 5 and 6 were inactive. In tetrasubstituted thiophene de-
rivatives, the compound 7 exhibited good activity when com-
Conclusion
The intramolecular and intermolecular cyclocondensation of
pared with 8 and 9. It was observed that there was no marked gem cyanoester ketene dithiolates led to a variety of heteoro-
difference in activity between gem cyanoester heterocycles cycles, tetrasubstituted thiophene derivatives, thieno-thiophene
(4–6) and thiophene derivatives (7–9). On the other hand, derivatives and tris heterocyclic compounds. The antioxidant
the thienothiophene derivatives 10, 11 and 12 were inactive. property of the synthesized compounds showed that the bis
However, amongst tris heterocyclic systems, the compounds benzoxazolylmethylthiomethylene pyrazole and isoxazole ex-
having pyrazolyl and isoxazolyl units in combination with bis hibited excellent radical scavenging activity.
benzoxazole ring 13 and 16 showed excellent radical scaveng-
ing activity when compared with the standard ascorbic acid.
Besides, the compounds having pyrazolyl and isoxazolyl rings
Experimental
General Melting points were determined in open capillar-
in combination with bis benzothiazole unit 14 and 17 exhibited ies on a Mel-Temp apparatus and are uncorrected. The purity
good activity while 15 and 18 displayed least activity. Further of the compounds was checked by TLC (silica gel H, BDH,
the results indicated that the compounds having pyrimidine ethyl acetate/hexane, 1:3). The IR spectra were recorded on a
derivatives 19 and 25 showed good activity while the com- Thermo Nicolet IR 200 FT-IR spectrometer as KBr pellets and
1
pounds 20, 22 and 26 exhibited least activity. The compounds the wave numbers were given in cm−1. The H- and 13C-NMR
21, 23, 24 and 27 were inactive. In general, the compounds spectra were recorded on a Brucker-400 spectrometer at
with benzoxazole units exhibited promising activity than those 400MHz and 100MHz, respectively. Chemical shifts were
having benzothiazole and benzimidazole units. Further the reported in parts per million from tetramethylsilane (TMS)
results exemplified that the compounds having benzoxazolyl as internal standard in DMSO-d6 solution. The mass spectra
unit in combination with pyrazole (13) and isoxazole (16) were recorded on Jeol JMS-D 300 and Finnigan Mat 1210
were the most potent antioxidant agents. Furthermore the free B at 70eV with an emission current of 100μA. The micro-
radical scavenging activity of compounds 7, 13, 14, 16 and 17 analyses were performed on a Perkin-Elmer 240C elemental
was measured at different concentrations and monitored the analyzer. The progress of the reaction was monitored by thin
change in absorbance at 10, 20 and 30min in DPPH method. layer chromatography (TLC) using silica gel plates (silica gel

April 2012
453
—
Table 3. Antioxidant Activities of the Test Compounds 4–27 and Standard Using ABTS Scavenging Method % ABTS Radical Scavenging Activity
Concentration
Compound
50μg/mL (%)
100μg/mL (%)
150μg/mL (%)
IC50 μg/mL
4
12.62 1.57
13.25 1.49
15.23 1.68
865.69 1.32
—
—
—
—
5
—
—
—
—
6
7
12.94 1.34
14.42 1.44
18.30 1.38
826.69 1.21
—
—
—
—
8
—
—
—
—
—
—
—
9
—
10
—
—
—
—
—
11
—
—
—
12
13
26.48 0.57
13.38 0.98
—
27.54 0.64
14.76 1.04
—
29.24 0.57
16.92 1.23
—
713.12 0.48
800.54 1.11
—
14
15
16
28.32 0.67
14.42 1.01
—
29.64 0.75
15.05 1.12
—
31.24 0.55
18.32 1.03
—
674.27 0.63
768.38 1.05
—
17
18
19
12.76 1.34
8.34 1.57
—
13.86 1.43
9.15 1.54
—
16.43 1.20
11.10 1.46
—
858.69 1.24
>1000
—
20
21
22
9.02 0.87
—
9.85 0.95
—
12.00 0.81
—
>1000
—
23
—
—
—
—
24
25
13.84 0.37
9.64 1.68
—
14.78 0.62
10.28 1.53
—
17.30 0.41
12.82 1.37
—
786.46 0.51
>1000
—
26
27
Ascorbic acid
Blank
28.80 0.54
—
29.70 0.34
—
31.30 0.75
—
650.05 0.58
—
—
(
) Showed no scavenging activity. Values were the means of three replicates S.D.
60 F254 0.25mm) and components were visualized by observa- Table 4. Antioxidant Activity of the Compounds 7, 13, 14, 16 and 17 at
10min Time Intervals by DPPH Scavenging Method
tion under UV light (254, 365nm). The compounds 2-(chloro-
methyl)benzoxazole (1), 2-(chloromethyl)benzothiazole (2) and
Compound
10min
20min
30min
2-(chloro-methyl)-1H-benzimidazole (3) were prepared by the
literature procedure.31–33)
7
13
14
16
17
51.56
74.86
58.93
76.59
59.12
51.92
75.07
59.34
76.85
59.45
52.15
75.24
59.62
77.03
59.89
General Procedure for the Synthesis of Ethyl
3,3-Bis((benzo[d]oxazol-2-yl)methyl-thio)-2-cyanoacrylate
(4)/Ethyl 3,3-Bis((benzo[d]thiazol-2-yl)methylthio)-2-cya-
noacrylate (5)/Ethyl 3,3-Bis((1H-benzo[d]imidazol-2-yl)-
methylthio)-2-cyanoacrylate (6) To dried potassium car-
bonate (10mmol) in DMF (2mL), ethyl cyanoacetate (10mmol)
in DMF (1mL) followed by carbon disulfide (15mmol) were
added dropwise under vigorous stirring. After 30min, the re-
°
action mixture was cooled to 0 C and to this 1/2/3 (20mmol)
in DMF (5mL) was added in 20–30min. The reaction mixture
was further stirred for 1h at room temperature and poured
into cold water. The precipitate was collected, dried and re-
crystallized from aqueous methanol.
Ethyl 3,3-Bis((benzo[d]oxazol-2-yl)methylthio)-2-cyanoac-
°
rylate (4): Yellow solid, yield 82%, mp 160–162 C. IR (KBr)
cm−1: 1612 (C=N), 1638 (C=C), 1730 (CO), 2220 (C≡N).
1H-NMR (DMSO-d6) δ: 1.32 (t, 3H, CH2CH3), 4.21 (q, 2H,
CH2CH3), 4.31 (s, 2H, CH2), 4.35 (s, 2H, CH2), 7.17–7.56 (m,
8H, Ar-H). 13C-NMR (DMSO-d6) δ: 14.0 (CH2CH3), 32.2, 32.7
(CH2–S), 61.1 (CH2CH3), 104.9 (=C–CO), 115.3 (CN), 160.8,
161.1 (C-2′), 163.8 (CO), 174.9 (C=C(S)(S)), 119.5, 120.4,
122.7, 124.2, 125.1, 138.4, 138.9, 148.3, 149.2 (aromatic car-
bons). MS m/z: 451.43 (M+). Anal. Calcd for C22H17N3O4S2: C,
58.52; H, 3.79; N, 9.31. Found: C, 58.46; H, 3.83; N, 9.27.
Fig. 1. Free Radical Scavenging Activity of Compounds 4–27 by DPPH
Method

454
Vol. 60, No. 4
phene-3-carboxylate (8)/Ethyl 2-((1′H-Benzo[d]imidazol-
2′-yl)methylthio)-4-amino-5-(1′H-benzo[d]imidazol-2′-yl)-
thiophene-3-carboxylate (9) and 4-Amino-2,5-di(benzo[d]-
oxazol-2′-yl)thieno[2,3-b]thiophen-3(2H)-one (10)/4-Amino-
2,5-di(benzo[d]thiazol-2′-yl)thieno[2,3-b]thiophen-3(2H)-
one
(11)/4-Amino-2,5-di(1′H-benzo[d]imidazol-2′-yl)-
thieno[2,3-b]thiophen-3(2H)-one (12) (Method A) To a
mixture of dried potassium carbonate (10mmol) and DMF
(2mL), 4/5/6 (10mmol) was added and refluxed for 2h and
poured into cold water. The precipitate obtained was collected,
dried and purified by column chromatography and identified
as 7/8/9 (major) and 10/11/12 (minor).
Ethyl
2-((Benzo[d]oxazol-2′-yl)methylthio)-4-amino-5-
(benzo[d]oxazol-2-yl)thiophene-3-carboxylate (7): Red solid,
yield 57%, mp 165–167 C. IR (KBr) cm : 1602 (C=N), 1620
(C=C), 1716 (CO), 3322, 3431 (NH2). H-NMR (DMSO-d6)
Fig. 2. Free Radical Scavenging Activity of Compounds 4–27 by Nitric
Oxide Method
−1
°
1
δ: 1.36 (t, 3H, CH2CH3), 4.22 (q, 2H, CH2CH3), 4.37 (s, 2H,
CH2), 6.88 (br s, 2H, NH2), 7.18–7.48 (m, 8H, Ar-H). 13C-NMR
(DMSO-d6) δ: 14.3 (CH2CH3), 32.1 (S–CH2), 62.3 (CH2CH3),
117.5 (C-3), 123.5 (C-5), 153.9 (C-4), 154.6 (C-2), 161.3, 162.1
(C-2′), 165.8 (CO), 118.9, 121.5, 122.2, 125.7, 126.4, 137.2,
137.9, 138.5, 148.6, 149.4 (aromatic carbons). Anal. Calcd for
C22H17N3O4S2: C, 58.52; H, 3.79; N, 9.31. Found: C, 58.58; H,
3.78; N, 9.28. MS (m/z): 451.47 (M+).
Ethyl
2-((Benzo[d]thiazol-2′-yl)methylthio)-4-amino-5-
(benzo[d]thiazol-2-yl)thiophene-3-carboxylate (8): Red solid,
yield 61%, mp 174–176 C. IR (KBr) cm : 1608 (C=N), 1624
(C=C), 1720 (CO), 3338, 3425 (NH2). H-NMR (DMSO-d6) δ:
−1
°
1
1.31 (t, 3H, CH2CH3), 4.16 (q, 2H, CH2CH3), 4.32 (s, 2H, CH2),
6.81 (br s, 2H, NH2), 7.44–7.98 (m, 8H, Ar-H). 13C-NMR
(DMSO-d6) δ: 14.1 (CH2CH3), 32.9 (S–CH2), 61.5 (CH2CH3),
117.8 (C-3), 122.8 (C-5), 153.2 (C-4), 154.8 (C-2), 164.6 (CO),
169.4, 170.1 (C-2′), 119.2, 121.3, 124.6, 125.3, 127.4, 128.2,
134.8, 135.2, 149.7, 151.2 (aromatic carbons). MS (m/z): 483.60
Fig. 3. Free Radical Scavenging Activity of Compounds 4–27 in ABTS
Method
Ethyl 3,3-Bis((benzo[d]thiazol-2-yl)methylthio)-2-cyanoac- (M+). Anal. Calcd for C22H17N3O2S4: C, 54.63; H, 3.54; N,
°
rylate (5): Yellow solid, yield 80%, mp 169–171 C. IR (KBr) 8.69. Found: C, 54.66; H, 3.57; N, 8.74.
cm−1: 1608 (C=N), 1634 (C=C), 1725 (CO), 2229 (C≡N).
Ethyl 2-((1′H-Benzo[d]imidazol-2′-yl)methylthio)-4-amino-
1H-NMR (DMSO-d6) δ: 1.27 (t, 3H, CH2CH3), 4.25 (q, 2H, 5-(1′H-benzo[d]imidazol-2′-yl)thiophene-3-carboxylate
(9):
−1
°
CH2CH3), 4.28 (s, 2H, CH2), 4.38 (s, 2H, CH2), 7.46–8.01 (m, Red solid, yield 58%, mp 182–184 C. IR (KBr) cm : 1601
8H, Ar-H). 13C-NMR (DMSO-d6) δ: 14.2 (CH2CH3), 31.9, 32.6 (C=N), 1616 (C=C), 1725 (CO), 3291 (NH), 3315, 3438 (NH2).
(CH2–S), 61.7 (CH2CH3), 105.2 (=C–CO), 114.9 (CN), 163.1 1H-NMR (DMSO-d6) δ: 1.33 (t, 3H, CH2CH3), 4.18 (q, 2H,
(CO), 169.4, 170.2 (C-2′), 174.5 (C=C(S)(S)), 120.1, 122.4, CH2CH3), 4.34 (s, 2H, CH2), 6.90 (br s, 2H, NH2), 7.25–7.62
125.6, 127.2, 127.7, 135.2, 135.4, 148.1, 149.6 (aromatic car- (m, 8H, Ar-H), 12.59 (bs, 2H, NH). 13C-NMR (DMSO-d6)
bons). MS (m/z): 483.51 (M+). Anal. Calcd for C22H17N3O2S4: δ: 14.5, (CH2CH3), 32.6 (S–CH2), 61.9 (CH2CH3), 117.1 (C-3),
C, 54.63; H, 3.54; N, 8.69. Found: C, 54.61; H, 3.57; N, 8.78.
123.0 (C-5), 150.1, 151.4 (C-2′), 153.6 (C-4), 154.2 (C-2), 165.2
Ethyl 3,3-Bis((1H-benzo[d]imidazol-2-yl)methylthio)-2-cy- (CO), 118.5, 119.8, 121.6, 122.2, 126.4, 127.7, 132.3, 133.4,
anoacrylate (6): Yellow solid, yield 85%, mp 177–179 C. 137.4, 138.1 (aromatic carbons). MS (m/z): 449.49 (M+). Anal.
°
IR (KBr) cm−1: 1605 (C=N), 1626 (C=C), 1735 (CO), 2215 Calcd for C22H19N5O2S2: C, 58.78; H, 4.26; N, 15.58. Found: C,
(C≡N), 3307 (NH). 1H-NMR (DMSO-d6) δ: 1.30 (t, 3H, 58.65; H, 4.30; N, 15.65.
CH2CH3), 4.20 (q, 2H, CH2CH3), 4.30 (s, 2H, CH2), 4.39 (s,
General Procedure for the Synthesis of 4-Amino-2,5-
2H, CH2), 7.28–7.53 (m, 8H, Ar-H), 12.68 (br s, 2H, NH). di(benzo[d]oxazol-2′-yl)-thieno[2,3-b]thiophen-3(2H)-one
13C-NMR (DMSO-d6) δ: 14.5 (CH2CH3), 32.5, 32.9 (CH2–S), (10)/4-Amino-2,5-di(benzo[d]thiazol-2′-yl)thieno[2,3-b]-
62.2 (CH2CH3), 104.7 (=C–CO), 114.5 (CN), 150.7, 151.2 (C- thiophen-3(2H)-one
(11)/4-Amino-2,5-di(1′H-benzo[d]-
(12)
2′), 163.4 (CO), 175.1 (C=C(S)(S)), 118.4, 121.7, 122.4, 123.1, imidazol-2′-yl)thieno[2,3-b]thiophen-3(2H)-one
128.5, 128.9, 132.3, 132.9, 138.5 (aromatic carbons). MS (m/z): Method B To a mixture of dried potassium carbonate
449.47 (M+). Anal. Calcd for C22H19N5O2S2: C, 58.78; H, 4.26; (10mmol) and DMF (4mL), 4/5/6 (5mmol) was added, re-
N, 15.58. Found: C, 58.82; H, 4.28; N, 15.64.
fluxed for 1h and poured into cold water. The precipitate
General Procedure for the Synthesis of Ethyl 2-((Benzo- obtained was collected, dried and recrystallized from 2-pro-
[d]oxazol-2′-yl)methylthio)-4-amino-5-(benzo[d]oxazol-2- panol.
yl)thiophene-3-carboxylate (7)/Ethyl 2-((Benzo[d]thiazol-
Method C A solution of ethyl cyanoacetate (10mmol)
2′-yl)-methylthio)-4-amino-5-(benzo[d]thiazol-2-yl)thio- in DMF (3mL) followed by carbon disulfide (15mmol) were

April 2012
455
added dropwise to a solution of dried potassium carbonate (aromatic carbons). MS (m/z): 437.43 (M+). Anal. Calcd for
(30mmol) in DMF (2mL) while stirring. After 30min, the C20H15N5O3S2: C, 54.91; H, 3.46; N, 16.01. Found: C, 54.88; H,
°
contents were cooled to 0 C and to this, compound 1/2/3 3.50; N, 16.12.
(20mmol) in DMF (5mL) was added in 20–30min. The reac-
tion mixture was then refluxed for 2h and poured into cold methylene)-1H-pyrazol-5(4H)-one (14): Yellow solid, yield
water. The precipitate was collected, dried and recrystallized 70%, mp 211–213 C. IR (KBr) cm : 1610 (C=N), 1628
from 2-propanol.
3-Amino-4-(bis((benzo[d]thiazol-2′-yl)methylthio)-
−1
°
1
(C=C), 1656 (CO), 3215 (NH), 3334, 3427 (NH2). H-NMR
4-Amino-2,5-di(benzo[d]oxazol-2′-yl)thieno[2,3-b]thiophen- (DMSO-d6) δ: 4.28 (s, 2H, CH2), 4.39 (s, 2H, CH2), 5.68 (br
3(2H)-one (10): Brown solid, yield 16 (Method A), 62 s, 2H, NH2), 7.36–7.92 (m, 8H, Ar-H), 9.86 (br s, 1H, NH).
(Method B), 68% (Method C), mp 263–265 C. IR (KBr) 13C-NMR (DMSO-d6) δ: 31.7, 32.6 (CH2–S), 104.2 (C-4), 158.1
°
cm−1: 1595 (C=N), 1623 (C=C), 1738 (CO), 3321, 3427 (NH2). (C-3), 169.6, 170.2 (C-2′), 172.5 (C-5), 174.5 (C=C(S)(S)),
1H-NMR (DMSO-d6) δ: 4.45 (s, 1H, CH), 6.69 (br s, 2H, 119.2, 122.7, 124.2, 126.4, 135.3, 135.7, 149.3, 150.4 (aromatic
NH2), 7.20–7.56 (m, 8H, Ar-H). 13C-NMR (DMSO-d6) δ: 60.2 carbons). MS (m/z): 469.58 (M+). Anal. Calcd for C20H15N5OS4:
(C-2), 114.1 (C=C(S)(S)), 128.8 (C-5), 154.1 (C=C(S)(S)), 155.9 C, 51.15; H, 3.22; N, 14.91. Found: C, 51.20; H, 3.20; N, 14.98.
(C-4), 161.1 and 161.8 (C-2′), 182.5 (CO), 118.8, 121.5, 123.2,
3-Amino-4-(bis((1′H-benzo[d]imidazol-2′-yl)methylthio)-
125.1, 126.3, 136.4, 137.8, 148.7, 149.6 (aromatic carbons). MS methylene)-1H-pyrazol-5(4H)-one (15): Yellow solid, yield
(m/z): 405.38 (M+). Anal. Calcd for C20H11N3O3S2: C, 59.25; H, 79%, mp 194–196 C. IR (KBr) cm : 1606 (C=N), 1619
−1
°
2.73; N, 10.36. Found: C, 59.31; H, 2.71; N, 10.41.
(C=C), 1659 (CO), 3226 (NH), 3328, 3430 (NH, NH2).
4-Amino-2,5-di(benzo[d]thiazol-2′-yl)thieno[2,3-b]thiophen- 1H-NMR (DMSO-d6) δ: 4.31 (s, 2H, CH2), 4.40 (s, 2H, CH2),
3(2H)-one (11): Brown solid, yield 14 (Method A), 60 5.60 (br s, 2H, NH2), 7.31–7.80 (m, 8H, Ar-H), 9.67 (br s, 1H,
(Method B), 73% (Method C), mp 275–277 C. IR (KBr) NH), 12.41 (br s, 2H, NH). 13C-NMR (DMSO-d6) δ: 32.4, 32.8
°
cm−1: 1602 (C=N), 1628 (C=C), 1733 (CO), 3317, 3420 (NH2). (CH2–S), 103.5 (C-4), 150.3, 151.6 (C-2′), 158.4 (C-3), 171.6
1H-NMR (DMSO-d6) δ: 4.51 (s, 1H, CH), 6.58 (br s, 2H, (C-5), 175.3 (C=C(S)(S)), 120.5, 122.2, 125.6, 126.4, 127.1,
NH2), 7.43–8.02 (m, 8H, Ar-H). 13C-NMR (DMSO-d6) δ: 61.1 131.5, 131.8, 137.9 (aromatic carbons). MS (m/z): 435.46 (M+).
(C-2), 115.2 (C=C(S)(S)), 129.7 (C-5), 153.8 (C=C(S)(S)), 155.4 Anal. Calcd for C20H17N7OS2: C, 55.16; H, 3.93; N, 22.51.
(C-4), 169.8 and 170.3 (C-2′), 183.2 (CO), 119.6, 120.4, 123.1, Found: C, 55.12; H, 3.96; N, 22.62.
125.2, 128.3, 134.8, 135.7, 147.4, 148.9 (aromatic carbons). MS
(m/z): 437.52 (M+). Anal. Calcd for C20H11N3OS4: C, 54.90; H, (bis((benzo[d]oxazol-2′-yl)methyl-thio)methylene)isoxazol-
2.53; N, 9.60. Found: C, 54.94; H, 2.56; N, 9.56. 5(4H)-one (16)/3-Amino-4-(bis((benzo[d]thiazol-2′-yl)-
4-Amino-2,5-di(1′H-benzo[d]imidazol-2′-yl)thieno[2,3-b]- methylthio)-methylene)isoxazol-5(4H)-one (17)/3-Amino-
thiophen-3(2H)-one (12): Brown solid, yield 18 (Method A), 4-(bis((1′H-benzo[d]imidazol-2′-yl)methyl-thio)methylene)-
General Procedure for the Synthesis of 3-Amino-4-
°
65 (Method B), 79% (Method C), mp 280–282 C. IR (KBr) isoxazol-5(4H)-one (18)
A mixture of 4/5/6 (1mmol),
cm−1: 1606 (C=N), 1625 (C=C), 1735 (CO), 3287 (NH), 3310, hydroxylamine hydrochloride (1.1mmol), piperidine (3mL)
3437 (NH2). 1H-NMR (DMSO-d6) δ: 4.53 (s, 1H, CH), 6.61 (br and ethanol (10mL) was refluxed for 7–9h. It was cooled and
s, 2H, NH2), 7.29–7.75 (m, 8H, Ar-H), 12.65 (br s, 2H, NH). poured into ice-cold water containing conc. HCl. The solid
13C-NMR (DMSO-d6) δ: 60.7 (C-2), 114.6 (C=C(S)(S)), 129.3 separated was collected, dried and recrystallized from 2-pro-
(C-5), 150.5 and 151.3 (C-2′), 154.5 (C=C(S)(S)), 156.3 (C- panol.
4), 182.8 (CO), 118.6, 122.8, 123.1, 128.5, 128.9, 131.6, 132.4,
3-Amino-4-(bis((benzo[d]oxazol-2′-yl)methylthio)methy-
137.2, 138.5 (aromatic carbons). MS (m/z): 403.41 (M+). Anal. lene)isoxazol-5(4H)-one (16): Yellow solid, yield 75%, mp
−1
°
Calcd for C20H13N5OS2: C, 59.54; H, 3.25; N, 17.36. Found: C, 181–183 C. IR (KBr) cm : 1598 (C=N), 1623 (C=C), 1732
59.56; H, 3.24; N, 17.46.
(CO), 3325, 3442 (NH2). 1H-NMR (DMSO-d6) δ: 4.29 (s,
General Procedure for the Synthesis of 3-Amino- 2H, CH2), 4.34 (s, 2H, CH2), 5.59 (br s, 2H, NH2), 7.14–7.64
4-(bis((benzo[d]oxazol-2′-yl)methyl-thio)methylene)-1H- (m, 8H, Ar-H). 13C-NMR (DMSO-d6) δ: 32.7, 33.4 (CH2-S),
pyrazol-5(4H)-one
thiazol-2′-yl)methyl-thio)methylene)-1H-pyrazol-5(4H)- (C=C(S)(S)), 120.6, 123.2, 126.7, 127.1, 128.2, 128.9, 139.8,
one
(14)/3-Amino-4-(bis((1′H-benzo[d]imidazol-2′-yl)- 141.0, 149.4 (aromatic carbons). MS (m/z): 438.40 (M+). Anal.
methylthio)methylene)-1H-pyrazol-5(4H)-one (15) The Calcd for C20H14N4O4S2: C, 54.78; H, 3.22; N, 12.78. Found: C,
compound 4/5/6 (1mmol), hydrazine hydrate (1.2mmol) and 54.84; H, 3.20; N, 12.84.
(13)/3-Amino-4-(bis((benzo[d]- 103.9 (C-4), 158.9 (C-3), 160.5, 161.8 (C-2′), 173.2 (C-5), 174.9
piperidine (3mL) in ethanol (10mL) were refluxed for 6–8h.
After completion of the reaction, it was cooled and poured lene)isoxazol-5(4H)-one (17): Yellow solid, yield 77%, mp
into ice-cold water containing conc. HCl. The solid obtained 202–204 C. IR (KBr) cm : 1607 (C=N), 1619 (C=C), 1741
was filtered, dried and recrystallized from 2-propanol.
3-Amino-4-(bis((benzo[d]thiazol-2′-yl)methylthio)methy-
−1
°
(CO), 3320, 3438 (NH2). 1H-NMR (DMSO-d6) δ: 4.32 (s,
3-Amino-4-(bis((benzo[d]oxazol-2′-yl)methylthio)- 2H, CH2), 4.38 (s, 2H, CH2), 5.62 (br s, 2H, NH2), 7.28–7.82
methylene)-1H-pyrazol-5(4H)-one (13): Yellow solid, yield (m, 8H, Ar-H). 13C-NMR (DMSO-d6) δ: 31.9, 32.7 (CH2–S),
−1
°
74%, mp 189–191 C. IR (KBr) cm : 1604 (C=N), 1622 105.1 (C-4), 158.2 (C-3), 169.8, 170.7 (C-2′), 174.1 (C-5), 175.7
1
(C=C), 1661 (CO), 3209 (NH), 3323, 3418 (NH2). H-NMR (C=C(S)(S)), 121.3, 123.9, 126.5, 127.2, 135.6, 136.2, 149.7,
(DMSO-d6) δ: 4.30 (s, 2H, CH2), 4.37 (s, 2H, CH2), 5.75 (br 150.1 (aromatic carbons). MS (m/z): 470.54 (M+). Anal. Calcd
s, 2H, NH2), 7.17–7.56 (m, 8H, Ar-H), 9.74 (br s, 1H, NH). for C20H14N4O2S4: C, 51.04; H, 3.00; N, 11.91. Found: C, 51.07;
13C-NMR (DMSO-d6) δ: 32.2, 33.1 (CH2–S), 103.5 (C-4), H, 3.03; N, 12.00.
157.3 (C-3), 160.4, 161.3 (C-2′), 171.8 (C-5), 175.7 (C=C(S)
3-Amino-4-(bis((1′H-benzo[d]imidazol-2′-yl)methylthio)-
(S)), 119.1, 120.6, 122.2, 124.6, 124.9, 138.7, 139.4, 148.3 methylene)isoxazol-5(4H)-one (18): Yellow solid, yield 80%,

456
Vol. 60, No. 4
−1
°
mp 214–215 C. IR (KBr) cm : 1610 (C=N), 1628 (C=C), 1746 4(5H)-one (23)/6-Amino-5-(bis((1′H-benzo[d]imidazol-2′-
1
(CO), 3298 (NH), 3326, 3452 (NH2). H-NMR (DMSO-d6) δ: yl)methylthio)methylene)-2-mercaptopyrimidin-4(5H)-one
4.34 (s, 2H, CH2), 4.39 (s, 2H, CH2), 5.66 (br s, 2H, NH2), (24) The compound 4/5/6 (2mmol), thiourea (2mmol), pi-
7.34–7.73 (m, 8H, Ar-H), 12.35 (br s, 2H, NH). 13C-NMR peridine (5mL) and ethanol (20mL) were refluxed for 12–15h.
(DMSO-d6) δ: 32.1, 33.0 (CH2–S), 104.3 (C-4), 150.2 (C-2′), The reaction mixture was cooled and poured into ice-cold wa-
159.3 (C-3), 173.6 (C-5), 175.1 (C=C(S)(S)), 118.9, 121.5, 125.2, ter containing conc. HCl. The solid separated was filtered on
128.9, 129.2, 130.2, 136.4, 137.3 (aromatic carbons). MS (m/z): a Buchner funnel, dried and recrystallized from 2-propanol.
436.44 (M+). Anal. Calcd for C20H16N6O2S2: C, 55.03; H, 3.69;
N, 19.25. Found: C, 55.08; H, 3.65; N, 19.31.
6-Amino-5-(bis((benzo[d]oxazol-2′-yl)methylthio)-
methylene)-2-mercaptopyrimidin-4(5H)-one (22): Pale yel-
−1
°
General Procedure for the Synthesis of 6-Amino- low solid, yield 69%, mp 218–220 C. IR (KBr) cm : 1609
5-(bis((benzo[d]oxazol-2′-yl)methyl-thio)methylene)-2-hy- (C=N), 1626 (C=C), 1673 (CO), 2564 (SH), 3347, 3451 (NH2).
droxypyrimidin-4(5H)-one (19)/6-Amino-5-(bis((benzo[d]- 1H-NMR (DMSO-d6) δ: 1.48 (br s, 1H, SH), 4.35 (s, 2H, CH2),
thiazol-2′-yl)methylthio)methylene)-2-hydroxypyrimidin- 4.43 (s, 2H, CH2), 5.66 (br s, 2H, NH2), 7.15–7.48 (m, 8H, Ar-
4(5H)-one (20)/6-Amino-5-(bis((1′H-benzo[d]imidazol-2′- H). 13C-NMR (DMSO-d6) δ: 31.4, 32.6 (CH2–S), 105.8 (C-5),
yl)methylthio)methylene)-2-hydroxypyrimidin-4(5H)-one 160.4, 161.1 (C-2′), 163.2 (C-6), 174.8 (C=C(S)(S)), 176.6 (C-
(21) To an equimolar (1mmol) mixture of compound 4/5/6 4), 185.5 (C-2), 121.5, 125.3, 126.8, 128.7, 129.5, 138.4, 140.2,
and urea, piperidine (3mL) and ethanol (10mL) were added 148.3 (aromatic carbons). MS (m/z): 481.50 (M+). Anal. Calcd
and refluxed for 8–10h. The contents of the flask were cooled for C21H15N5O3S3: C, 52.38; H, 3.14; N, 14.54. Found: C, 52.41;
and poured into ice-cold water containing conc. HCl. The H, 3.13; N, 14.60.
solid separated was filtered, dried and purified by recrystal-
lization from 2-propanol.
6-Amino-5-(bis((benzo[d]thiazol-2′-yl)methylthio)-
methylene)-2-mercaptopyrimidin-4(5H)-one (23): Pale yel-
−1
°
6-Amino-5-(bis((benzo[d]oxazol-2′-yl)methylthio)- low solid, yield 73% mp 233–235 C. IR (KBr) cm : 1604
methylene)-2-hydroxypyrimidin-4(5H)-one (19): Pale yel- (C=N), 1624 (C=C), 1677 (CO), 2556 (SH), 3342, 3446 (NH2).
−1
low solid, yield 74%, mp 208–210 C. IR (KBr) cm : 1603 1H-NMR (DMSO-d6) δ: 1.53 (br s, 1H, SH), 4.32 (s, 2H, CH2),
°
(C=N), 1622 (C=C), 1670 (CO), 3328 (OH), 3342, 3457 (NH2). 4.39 (s, 2H, CH2), 5.58 (br s, 2H, NH2), 7.51–7.98 (m, 8H, Ar-
1H-NMR (DMSO-d6) δ: 4.31 (s, 2H, CH2), 4.40 (s, 2H, CH2), H). 13C-NMR (DMSO-d6) δ: 31.8, 33.1 (CH2–S), 104.6 (C-5),
5.61 (br s, 2H, NH2), 7.23–7.59 (m, 8H, Ar-H), 8.31 (br s, 1H, 162.3 (C-6), 170.5, 171.3 (C-2′), 175.6 (C=C(S)(S)), 177.4 (C-
OH). 13C-NMR (DMSO-d6) δ: 32.3, 33.1 (CH2–S), 105.2 (C-5), 4), 184.6 (C-2), 119.5, 123.8, 125.2, 125.8, 127.3, 136.9, 148.9,
158.9 (C-6), 160.2, 161.5 (C-2′), 162.8 (C-2), 173.2 (C-4), 175.3 149.4 (aromatic carbons). MS (m/z): 513.64 (M+). Anal. Calcd
(C=C(S)(S)), 121.5, 125.3, 126.8, 128.7, 129.2, 138.5, 140.2, for C21H15N5OS5: C, 49.10; H, 2.94; N, 13.63. Found: C, 49.08;
148.3 (aromatic carbons). MS (m/z): 465.42 (M+). Anal. Calcd H, 2.96; N, 13.71.
for C21H15N5O4S2: C, 54.18; H, 3.25; N, 15.04. Found: C, 54.15;
H, 3.27; N, 15.11.
6-Amino-5-(bis((1′H-benzo[d]imidazol-2′-yl)methylthio)-
methylene)-2-mercapto-pyrimidin-4(5H)-one (24): Pale yellow
−1
°
6-Amino-5-(bis((benzo[d]thiazol-2′-yl)methylthio)- solid, yield 74%, mp 246–248 C. IR (KBr) cm : 1601 (C=N),
methylene)-2-hydroxypyrimidin-4(5H)-one (20): Pale yel- 1622 (C=C), 1679 (CO), 2552 (SH), 3298 (NH), 3341, 3460
−1
1
°
low solid, yield 71%, mp 225–227 C. IR (KBr) cm : 1605 (NH2). H-NMR (DMSO-d6) δ: 1.46 (br s, 1H, SH), 4.31 (s,
(C=N), 1626 (C=C), 1672 (CO), 3315 (OH), 3351, 3449 (NH2). 2H, CH2), 4.41 (s, 2H, CH2), 5.53 (br s, 2H, NH2), 7.28–7.74
1H-NMR (DMSO-d6) δ: 4.33 (s, 2H, CH2), 4.42 (s, 2H, CH2), (m, 8H, Ar-H), 12.53 (br s, 2H, NH). 13C-NMR (DMSO-d6)
5.58 (br s, 2H, NH2), 7.42–7.95 (m, 8H, Ar-H), 8.36 (br s, 1H, δ: 32.5, 33.7 (CH2–S), 105.1 (C-5), 150.2, 151.5 (C-2′), 162.8
OH). 13C-NMR (DMSO-d6) δ: 31.6, 32.8 (CH2–S), 104.8 (C-5), (C-6), 175.2 (C=C(S)(S)), 177.1 (C-4), 185.1 (C-2), 118.6, 120.4,
159.5 (C-6), 163.7 (C-2), 169.1, 170.2 (C-2′), 174.5 (C-4), 175.8 123.1, 124.5, 126.8, 127.6, 131.8, 139.7 (aromatic carbons). MS
(C=C(S)(S)), 120.3, 124.2, 124.6, 125.3, 127.6, 137.5, 148.3, (m/z): 479.53 (M+). Anal. Calcd for C21H17N7OS3: C, 52.59; H,
149.8 (aromatic carbons). MS (m/z): 497.57 (M+). Anal. Calcd 3.57; N, 20.44. Found: C, 52.64; H, 3.60; N, 20.54.
for C21H15N5O2S4: C, 50.68; H, 3.04; N, 14.07. Found: C, 50.70;
H, 3.03; N, 14.16.
General Procedure for the Synthesis of 5-(Bis((benzo[d]
oxazol-2′-yl)methylthio)-methylene)-dihydro-6-imino-1,3-
6-Amino-5-(bis((1′H-benzo[d]imidazol-2′-yl)methylthio)- dimethylpyrimidine-2,4(1H,3H)-dione (25)/5-(Bis((benzo-
methylene)-2-hydroxy-pyrimidin-4(5H)-one (21): Pale yellow [d]thiazol-2′-yl)methylthio)methylene)-dihydro-6-imi-
−1
°
solid, yield 75%, mp 210–212 C. IR (KBr) cm : 1596 (C=N), no-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (26)/5-(Bis-
1630 (C=C), 1675 (CO), 3324 (OH), 3348, 3452 (NH and ((1′H-benzo[d]imidazol-2′-yl)methylthio)methylene)-di-
1
NH2). H-NMR (DMSO-d6) δ: 4.30 (s, 2H, CH2), 4.38 (s, 2H, hydro-6-imino-1,3-dimethylpyrimidine-2,4(1H,3H)-dione
CH2), 5.54 (br s, 2H, NH2), 7.26–7.86 (m, 8H, Ar-H), 8.34 (27)
A mixture of 4/5/6 (1mmol), N,N′-dimethylurea
(br s, 1H, OH), 12.45 (br s, 2H, NH). 13C-NMR (DMSO-d6) (1mmol), piperidine (3mL) and ethanol (10mL) were refluxed
δ: 32.1, 33.5 (CH2–S), 104.5 (C-5), 150.2 (C-2′), 158.4 (C- for 9–11h. The contents of the flask were cooled and poured
6), 163.3 (C-2), 173.7 (C-4), 175.5 (C=C(S)(S)), 119.4, 122.2, into ice-cold water containing conc. HCl. The solid sepa-
124.8, 125.6, 127.1, 132.5, 138.5, 139.3 (aromatic carbons). MS rated was filtered, dried and purified by recrystallization from
(m/z): 463.49 (M+). Anal. Calcd for C21H17N7O2S2: C, 54.41; H, 2-propanol.
3.70; N, 21.15. Found: C, 54.45; H, 3.72; N, 21.26.
General Procedure for the Synthesis of 6-Amino-5- hydro-6-imino-1,3-dimethyl-pyrimidine-2,4(1H,3H)-dione
(bis((benzo[d]oxazol-2′-yl)methyl-thio)methylene)-2-mer- (25): Yellow solid, yield 67%, mp 197–199 C. IR (KBr) cm :
5-(Bis((benzo[d]oxazol-2′-yl)methylthio)methylene)-di-
−1
°
1
captopyrimidin-4(5H)-one (22)/6-Amino-5-(bis((benzo[d]- 1601 (C=N), 1622 (C=C), 1679 (CO), 3298 (NH). H-NMR
thiazol-2′-yl)methylthio)methylene)-2-mercaptopyrimidin- (DMSO-d6) δ: 2.69, 2.75 (s, 6H, N–CH3), 4.32 (s, 2H, CH2),

April 2012
457
°
4.39 (s, 2H, CH2), 7.19–7.52 (m, 8H, Ar-H), 9.31 (br s, 1H, the test compounds and incubated for 150min at 25 C. After
NH). 13C-NMR (DMSO-d6) δ: 27.2, 28.3 (N–CH3), 30.5, 32.2 incubation 1mL of the reaction mixture was treated with 1mL
(CH2–S), 103.8 (C-5), 158.2 (C-2), 160.3, 161.1 (C-2′), 163.5 of Griess reagent (1% sulfanilamide, 2% H3PO4 and 0.1%
(C-6), 173.2 (C-4), 174.3 (C=C(S)(S)), 119.2, 124.1, 124.5, naphthylethylenediamine dihydrochloride). The absorbance of
125.9, 138.5, 139.7, 148.4, 149.2 (aromatic carbons). MS (m/z): the chromatophore was measured at 546nm. Ascorbic acid
493.48 (M+). Anal. Calcd for C23H19N5O4S2: C, 55.97; H, 3.88; was used as standard. NO scavenging activity was calculated
N, 14.19. Found: C, 56.00; H, 3.86; N, 14.13.
by the following equation,
5-(Bis((benzo[d]thiazol-2′-yl)methylthio)methylene)-di-
% of scavening=[(Acontrol − Asample ) / Ablank ]×100
hydro-6-imino-1,3-dimethyl-pyrimidine-2,4(1H,3H)-dione
−1
°
(26): Yellow solid, yield 71%, mp 212–213 C. IR (KBr) cm : Where Acontrol is the absorbance of the control reaction (con-
1
1599 (C=N), 1625 (C=C), 1674 (CO), 3226 (NH). H-NMR taining all reagents and Ascorbic acid), Asample is the absor-
(DMSO-d6) δ: 2.62, 2.70 (s, 6H, N–CH3), 4.30 (s, 2H, CH2), bance of the test compound (containing all reagents and test
4.35 (s, 2H, CH2), 7.19–7.52 (m, 8H, Ar-H), 9.29 (br s, 1H, compound) and Ablank is the absorbance of the blank (contain-
NH). 13C-NMR (DMSO-d6) δ: 27.6, 28.7 (N–CH3), 31.7, 32.9 ing only reagents). Tests were carried out in triplicate.
(CH2–S), 104.2 (C-5), 159.3 (C-2), 162.8 (C-6), 169.7, 170.4 (C-
ABTS Radical Scavenging Activity The antioxidant ac-
2′), 173.9 (C-4), 175.1 (C=C(S)(S)), 119.4, 122.6, 123.2, 125.4, tivity of the test compounds and standard (ascorbic acid) were
126.8, 134.6, 135.7, 149.6 (aromatic carbons). MS (m/z): 525.61 assessed on the basis of the radical scavenging effect of the
(M+). Anal. Calcd for C23H19N5O2S4: C, 52.55; H, 3.64; N, stable ABTS free radical. The ABTS·+ solution was prepared
13.32. Found: C, 52.60; H, 3.63; N, 13.38.
by mixing 0.02mol of ABTS salt with 0.01mol of potassium
5-(Bis((1′H-benzo[d]imidazol-2′-yl)methylthio)methylene)- persulfate in 25mL of distilled water. The solution was kept
dihydro-6-imino-1,3-dimethylpyrimidine-2,4(1H,3H)-dione at room temperature in the dark for 16h before use. Then the
−1
(27): Yellow solid, yield 68%, mp 222–224 C. IR (KBr) cm : ABTS·+ solution was diluted with methanol in order to obtain
°
1607 (C=N), 1623 (C=C), 1678 (CO), 3211 (NH), 3318 (NH). an absorbance between 0.7 and 0.9 at 734nm using the spec-
1H-NMR (DMSO-d6) δ: 2.74, 2.79 (s, 6H, N–CH3), 4.29 (s, trophotometer. Fresh ABTS·+ solutions were prepared for each
2H, CH2), 4.40 (s, 2H, CH2), 7.38–7.76 (m, 8H, Ar-H), 9.35 assay. To a 50, 75 and 100μg/mL of each test compounds and
(br s, 1H, NH), 12.48 (br s, 2H, NH). 13C-NMR (DMSO- standard, 1mL of ABTS·+ solution was added and allowed to
d6) δ: 27.8, 28.9 (N–CH3), 31.5, 32.6 (CH2–S), 104.9 (C-5), react for 2h in dark condition. Then the absorbance was taken
150.3, 151.1 (C-2′), 158.7 (C-2), 163.2 (C-6), 173.6 (C-4), 174.7 at 734nm using the spectrophotometer. The corresponding
(C=C(S)(S)), 118.9, 120.3, 123.2, 125.6, 128.5, 131.9, 136.2, blank reading was also taken and the results in percentage
137.4 (aromatic carbons). MS (m/z): 491.52 (M+). Anal. Calcd were expressed as the ratio of absorbance decrease at 734nm
for C23H21N7O2S2: C, 56.19; H, 4.31; N, 19.94. Found: C, 56.25; and the absorbance of ABTS·+ solution in the absence of test
H, 4.34; N, 20.00.
compounds.
Antioxidant Activity The compounds 4–27 are evaluated
for antioxidant property by DPPH, nitric oxide and ABTS
methods.
Acknowledgement The authors are grateful to University
Grants Commission (UGC), New Delhi, India for financial as-
DPPH Radical Scavenging Activity The hydrogen atom sistance under major research project.
or electron donation ability of the compounds was measured
from the bleaching of the purple colored methanol solution of
DPPH. The spectrophotometric assay uses the stable radical
DPPH as a reagent. To 4mL of 0.004% (w/v) methanol solu-
tion of DPPH, 1mL of various concentrations of the test com-
pounds (50, 75, 100μg/mL) in methanol were added. After a
30min incubation period at room temperature, the absorbance
was read against blank at 517nm. Ascorbic acid was used as
the standard. The percent of inhibition (I%) of free radical
production from DPPH was calculated by the following equa-
tion,
References
1) Rapposelli S., Lapucci A., Minutolo F., Orlandini E., Ortore G.,
Pinza M., Balsamo A., Farmaco, 59, 25–31 (2004).
2) Calí P., Naerum L., Mukhija S., Hjelmencrantz A., Bioorg. Med.
Chem. Lett., 14, 5997–6000 (2004).
3) Al-Omran F., El-Khair A. A., J. Heterocycl. Chem., 41, 327–333
(2004).
4) Penning T. D., Talley J. J., Bertenshaw S. R., Carter J. S., Collins P.
W., Docter S., Graneto M. J., Lee L. F., Malecha J. W., Miyashiro
J. M., Rogers R. S., Rogier D. J., Yu S. S., Anderson G. D., Burton
E. G., Cogburn J. N., Gregory S. A., Koboldt C. M., Perkins W. E.,
Seibert K., Veenhuizen A. W., Zhang Y. Y., Isakson P. C., J. Med.
Chem., 40, 1347–1365 (1997).
I%=[(Acontrol − Asample ) / Ablank ]×100
5) Terrett N. K., Bell A. S., Brown D., Ellis P., Bioorg. Med. Chem.
Lett., 6, 1819–1824 (1996).
6) Segal H., Hedgcoth C., Skinner C. G., J. Med. Pharm. Chem., 5,
871–876 (1962).
7) Cumming J. G., McKenzie C. L., Bowden S. G., Campbell D.,
Masters D. J., Breed J., Jewsbury P. J., Bioorg. Med. Chem. Lett.,
14, 5389–5394 (2004).
Where Acontrol is the absorbance of the control reaction (con-
taining methanolic DPPH and Ascorbic acid), Asample is the ab-
sorbance of the test compound (containing methanolic DPPH
and test compound) and Ablank is the absorbance of the blank
(containing only methanolic DPPH). Tests were carried out in
triplicate.
Nitric Oxide (NO) Scavenging Activity NO scaveng-
ing activity was measured by slightly modified methods
of Green36) and Marcocci37) et al. NO radicals were gener-
ated from sodium nitroprusside. 1mL of Sodium nitroprusside
(10mM) and 1.5mL of phosphate buffer saline (0.2M, pH 7.4)
were added to different concentrations (50, 75, 100μg/mL) of
8) Prudhomme M., Guyot J., Jeminet G., J. Antibiot., 39, 934–937
(1986).
9) Oren I., Temiz O., Yalcın I., Sener E., Akın A., Ucarturk N.
Arzneimittel-Forschung, Drug Res., 47, 1393–1397 (1997).
10) Oren I., Temiz O., Yalçin I., Sener E., Altanlar N., Eur. J. Pharm.
Sci., 7, 153–160 (1999).

458
Vol. 60, No. 4
11) Temiz-Arpaci O., Aki-Sxener E., Yalcın I., Altanlar N., Arch.
Pharm., 6, 283–288 (2002).
12) Temiz-Arpaci O., Ozdemir A., Yalcin I., Yildiz I., Aki-Sxener E.,
Altanlar N., Arch. Pharm., 338, 105–111 (2005).
13) Vinsová J., Horák V., Buchta V., Kaustová J., Molecules, 10, 783–
793 (2005).
14) Ueki M., Taniguchi M., J. Antibiot., 50, 788–790 (1997).
15) Varga A., Aki-Sener E., Yalcin I., Temiz-Arpaci O., Tekiner-Gulbas
B., Cherepnev G., Molnar J., In Vivo, 19, 1087–1091 (2005).
16) Palmer P. J., Trigg R. B., Warrington J. V., J. Med. Chem., 14,
248–251 (1971).
Med. Chem. Lett., 10, 2193–2195 (2000).
25) Demirayak S., Abu Mohsen U., Cağri Karaburun A., Eur. J. Med.
Chem., 37, 255–260 (2002).
26) Lukevics E., Arsenyan P., Shestakova I., Domracheva I., Nesterova
A., Pudova O., Eur. J. Med. Chem., 36, 507–515 (2001).
27) Handratta V. D., Vasaitis T. S., Njar V. C. O., Gediya L. K., Kataria
R., Chopra P. Jr., Newman D. Jr., Farquhar R., Guo Z., Qiu Y.,
Brodie A. M., J. Med. Chem., 48, 2972–2984 (2005).
28) Padmavathi V. Dinneswara Reddy G., Nagi Reddy S., Mahesh K.,
Eur. J. Med. Chem., 46, 1367–1373 (2011).
29) Padmavathi V., Sudhakar Reddy G., Deepti D., Padmaja A., J.
Heterocycl. Chem., 45, 1089–1094 (2008).
30) Padmavathi V., Dinneswara Reddy G., Venkatesh B. C., Arch.
Pharm. (Weinheim), 11, 165–169 (2011).
31) Gellis A., Boufatah N., Vanelle P., Green Chem., 8, 483–487 (2006).
32) Kristinsson H., Synthesis, 1979, 102–103 (1979).
33) Morton R., Chang H., Craine L., Edwin H., J. Org. Chem., 50,
2205–2210 (1985).
17) Ivanov S. K., Yuritsyn V. S., Neftekhimiya, 11, 99 (1971) [Chem.
Abstr., 74, 124487 (1971].
18) Stevens M. F. G., McCall C. J., Lelieveld P., Alexander P., Richter
A., Davies D. E., J. Med. Chem., 37, 1689–1695 (1994).
19) Kashiyama E., Hutchinson I., Chua M. S., Stinson S. F., Phillips
L. R., Kaur G., Sausville E. A., Bradshaw T. D., Westwell A. D.,
Stevens M. F. G., J. Med. Chem., 42, 4172–4184 (1999).
20) Andrzejewska M., Yépez-Mulia L., Cedillo-Rivera R., Tapia A.,
34) Burits M., Bucar F., Phytother. Res., 14, 323–328 (2000).
Vilpo L., Vilpo J., Kazimierczuk Z., Eur. J. Med. Chem., 37, 973– 35) Cuendet M., Hostettmann K., Potterat O., Dyatmiko W., Helv.
978 (2002). Chim. Acta, 80, 1144–1152 (1997).
21) Gümüş F., Algül O., Eren G., Eroğlu H., Diril N., Gür S., Ozkul A., 36) Green L. C., Wagner D. A., Glogowski J., Skipper P. L., Wishnok J.
Eur. J. Med. Chem., 38, 473–480 (2003). S., Tannenbaum S. R., Anal. Biochem., 126, 131–138 (1982).
22) Kamal A., Ramulu P., Srinivas O., Ramesh G., Kumar P. P., Bioorg. 37) Marcocci L., Maguire J. J., Droy-Lefaix M. T., Packer L., Biochem.
Med. Chem. Lett., 14, 4791–4794 (2004). Biophys. Res. Commun., 201, 748–755 (1994).
23) Chauhan P. M. S., Martins C. J. A., Horwell D. C., Bioorg. Med. 38) Teow C. C., Truong V.-D., McFeeters R. F., Thompson R. L., Pecota
Chem., 13, 3513–3518 (2005).
K. V., Yencho G. C., Food Chem., 103, 829–838 (2007).
24) Garuti L., Roberti M., Malagoli M., Rossi T., Castelli M., Bioorg.