Synthesis of some new [1,2,4]triazolo[3,4-b][1,3,4]thiadiazines and [1,2,4]triazolo[3,4-b][1,3,4] thiadiazoles starting from 5-nitro-2-furoic acid and evaluation of their antimicrobial activity

Article abstract of DOI:10.1016/j.bmc.2011.06.024

New series of fused 1,2,4-triazoles such as, 6-(aryl)-3-(5-nitrofuran-2-yl) -5,6-dihydro-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles 4-8, 6-(alkyl/aryl amino)-3-(5-nitrofuran-2-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles 9-13 and 6-(4-substituted phenyl)-

Full text of DOI:10.1016/j.bmc.2011.06.024

Bioorganic & Medicinal Chemistry 19 (2011) 4506–4512
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of some new [1,2,4]triazolo[3,4-b][1,3,4]thiadiazines and
[1,2,4]triazolo[3,4-b][1,3,4] thiadiazoles starting from 5-nitro-2-furoic acid and
evaluation of their antimicrobial activity
Sahar M. I. Badr a, , Rasha M. Barwa b
⇑
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
b Microbiology Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
New series of fused 1,2,4-triazoles such as, 6-(aryl)-3-(5-nitrofuran-2-yl)-5,6-dihydro-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazoles 4–8, 6-(alkyl/aryl amino)-3-(5-nitrofuran-2-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiaz-
oles 9–13 and 6-(4-substituted phenyl)-3-(5-nitrofuran-2-yl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines
14–18 have been synthesized via the reaction of 4-amino-5-(5-nitrofuran-2-yl)-4H-1,2,4-triazole-3-thiol
3 with various reagents such as hetero aromatic aldehydes, alkyl/aryl isothiocyanates and 4-substituted
phenacyl bromides, respectively. The structures of the newly synthesized compounds have been con-
firmed on the basis of elemental analysis and spectral studies. The newly synthesized triazolo derivatives
have been investigated for their in vitro antibacterial activity. Most of the tested compounds showed
interesting antibacterial activity against Staphylococcus aureus. Furthermore, the most potent antibacte-
rial compounds 11–13 were evaluated for their in vitro cytotoxic activity against human cancer cell lines.
It was found that compounds 11 and 13 showed higher cytotoxicity against Hep-G2 cell line as compared
to standard.
Received 26 April 2011
Revised 4 June 2011
Accepted 8 June 2011
Available online 16 June 2011
Keywords:
1,2,4-Triazole
Dihydrotriazolothiadiazoles
Triazolothiadiazoles
Triazolothiadiazines
Cyclization
Antimicrobial activity
Cytotoxic activity
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
ran-2-yl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines 14–18, respec-
tively.
Infectious microbial diseases remain pressing problems world
wide, because resistance to a number of antimicrobial agents among
variety of clinically significant species of microorganisms (like
methicillin-resistant Staphylococcus aureus (MRSA)) has become an
important global health problem.1–3 Hence, there will always be a
vital need to discover new antimicrobial agents. Literature survey
has revealed the importance of 1,2,4-triazoles as antimicrobial
agents4–19 such as Fluconazole,20,21 Ravuconazole,21 Voriconazole,21
Itraconazole and Posaconazole20–22 (Fig. 1). In addition, furan deriv-
atives have been widely used as antimicrobial agents23–31 for exam-
ple, Furazolidone,32 Nifuroxazide,33 Nitrofurantoin,33 Furoxone and
RBx764434 (Fig. 2). Prompted by these observations and as part of a
synthetic effort directed toward the synthesis of antimicrobial
agents, the key intermediate, 4-amino-5-(5-nitrofuran-2-yl)-4H-
1,2,4-triazole-3-thiol 3 has been condensed with various reagents
such as, hetero aromatic aldehydes, alkyl/aryl isothiocyanates and
4-substituted phenacyl bromides to afford 6-(aryl)-3-(5-nitrofu-
ran-2-yl)-5,6-dihydro[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles 4–8,
6-(alkyl/aryl amino)-3-(5-nitrofuran-2-yl)[1,2,4]triazolo[3,4-b][1,
3,4]thiadiazoles 9–13 and 6-(4-substituted phenyl)-3-(5-nitrofu-
2. Results and discussion
2.1. Chemistry
The target compounds were synthesized as outlined in
Schemes 1–4. The title compound, 4-amino-5-(5-nitrofuran-2-
yl)-4H-1,2,4-triazole-3-thiol 3 was prepared by a one-step synthe-
sis via the combining of 5-nitro-2-furoic acid 1 with thiocarbohyd-
razide 2 at 170–180 °C. While the compound 3 may exist in
thione–thiol tautomeric forms, in the present study, the thiol
structure was dominated in the solid state. Usual spectroscopic
methods IR and 1H NMR distinguished between these constitu-
tional isomers. The IR spectra, showed absorption band at about
2600 cmÀ1 indicative of SH group and no absorption band in the
range 1300–1200 cmÀ1 attributable to the C@S group. The 1H
NMR spectrum of 4-amino-5-(5-nitrofuran-2-yl)-4H-1,2,4-tria-
zole-3-thiol 3 showed NH2 protons as a singlet at 5.46 ppm (inte-
grating two protons) and SH proton as a singlet at 13.19 ppm.
Other peaks were observed at appropriate chemical shifts and inte-
gral values (Scheme 1).
Reaction of 4-amino-5-(5-nitrofuran-2-yl)-4H-1,2,4-triazole-3-
thiol 3 with hetero aromatic aldehydes in the presence of p-toluene
⇑
Corresponding author. Tel.: +20 105176576; fax: +20 502247496.
E-mail address: saharmbadr@yahoo.com (S.M.I. Badr).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.024

S. M. I. Badr, R. M. Barwa / Bioorg. Med. Chem. 19 (2011) 4506–4512
4507
Figure 1. Structures of some antimicrobial drugs characterized by heterocyclic 1,2,4-triazole ring.
Figure 2. Structures of some antimicrobial drugs containing 5-nitrofuran moiety.
sulfonic acid as a catalyst yielded, 6-(aryl)-3-(5-nitrofuran-2-yl)-
5,6-dihydro[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles 4–8. Their pro-
ton NMR spectra lacked signals characteristic of SH and NH2 pro-
tons, and showed two new peaks at 5.61–5.74 ppm (integration
for one proton) and 6.33–6.64 ppm (integration for one proton)
attributed to CH and NH protons, respectively, establishing that
CHO group of the hetero aromatic aldehydes reacted with SH and
NH2 groups of the intermediate 3 to afford 5,6-dihydro[1,2,4]triaz-
olo[3,4-b][1,3,4]thiadiazole derivatives 4–8 (Scheme 2).
Furthermore, 4-amino-5-(5-nitrofuran-2-yl)-4H-1,2,4-triazole-
3-thiol 3 was heated under reflux with alkyl/aryl isothiocyanates
in dimethylformamide as a solvent to give 6-(alkyl/aryl amino)-
3-(5-nitrofuran-2-yl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles 9–13.
In the proton NMR spectra, the disappearance of SH and NH2 sig-

4508
S. M. I. Badr, R. M. Barwa / Bioorg. Med. Chem. 19 (2011) 4506–4512
Scheme 1. Synthesis of 4-amino-5-(5-nitrofuran-2-yl)-4H-1,2,4-triazole-3-thiol 3.
Scheme 3. Synthetic pathway of compounds 9–13.
Scheme 2. Synthetic pathway of compounds 4–8.
nals and appearance of new singlet characteristic for alkyl/aryl
amino proton recorded in the range of 9.14–9.96 ppm (integration
for one proton) confirmed the conversion of compound 3 into
1,2,4-triazolo[3,4-b][1,3,4]thiadiazole derivatives 9–13 (Scheme
3).
Scheme 4. Synthetic pathway of compounds 14–18.
2.2. Antimicrobial evaluation
Moreover, 6-(4-substituted phenyl)-3-(5-nitrofuran-2-yl)-7H-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines 14–18 were prepared
through the reaction of 4-amino-5-(5-nitrofuran-2-yl)-4H-1,2,4-
triazole-3-thiol 3 with 4-substituted phenacyl bromides in abso-
lute ethanol. Their proton NMR spectra showed that the signals
due to SH and NH2 protons disappeared, instead, a new signal ob-
served at 4.16–4.32 ppm (integration for two protons) attributed
to CH2 group. This confirmed that the compound 3 underwent ring
closure to give the 1,2,4-triazolo[3,4-b][1,3,4]thiadiazines 14–18
(Scheme 4).
The newly synthesized compounds 3–18 were tested for their
in vitro antibacterial activity against S. aureus (e.g., for Gram-posi-
tive bacteria) and Escherichia coli (e.g., for Gram-negative bacteria).
The minimum inhibitory concentration (MIC,
concentration required to inhibit the growth of bacteria) and the
minimum bactericidal concentration (MBC, g/ml, i.e., the lowest
lg/ml, i.e., the lowest
l
concentration of the compound required to kill bacteria) of all
the compounds were determined by the broth dilution meth-
od.35–38 For comparison, ampicillin was used as reference drug
(Table 1). Moderate antibacterial activity was observed for the
tested compounds against E. coli in comparison to reference
All compounds were spectroscopically characterized and the
spectral data agree with the proposed structures.

S. M. I. Badr, R. M. Barwa / Bioorg. Med. Chem. 19 (2011) 4506–4512
4509
Table 1
Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration
(MBC) in g/ml of compounds 3–18
Table 2
IC50 values (in
HCT-116 (human colon tumor cell line) and Hep-G2
(hepatocarcinoma cell line)
lg/ml) for compounds 11–13 against
l
Compound
Staphylococcus aureus
Escherichia coli
MIC MBC
Compound
Hep-G2
HCT-116
MIC
MBC
11
12
13
4.23
11.3
3.78
5.5
4.38
8.91
6.65
3.73
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
50
200
200
200
200
200
200
100
200
50b
50b
50b
200
200
200
100
200
50
25
25
25
25
50
50
25
25
50
25
25
50
25
25
25
25
12.5
200
200
200
200
200
200
200
100
100
100
50
200
200
200
200
200
25
25a
25a
25a
25a
25a
25a
25a
25a
25a
25a
100
25a
25a
50
Doxorubicin
activity was dependent on the substituents. Incorporation of
unsubstituted phenyl ring or 4-methylbenzyl moiety into the triaz-
olothiadiazole ring at 6-position via amino bridge developed more
effective cytotoxic analogues (compounds 11 and 13) than the
compared standard drug (Doxorubicin) against Hep-G2 cell line.
While incorporation of p-fluorophenyl moiety into the triazolothi-
adiazole ring at 6-position via amino bridge providing lower po-
tency analogue (compound 12) than the compared standard drug
(Doxorubicin) against Hep-G2 cell line.
50
25
Ampicillin
MIC, minimum inhibitory concentration (the lowest concentration that inhibited
the bacterial growth). MBC, minimum bactericidal concentration (the lowest con-
centration at which no bacterial growth was observed).
3. Conclusion
a
Equal MIC value to reference.
Equal MBC value to reference
b
A new series of triazoles 3–18, have been synthesized and eval-
uated for their antibacterial activity. Compared with the reference
drug, Ampicillin, our compounds are more sensitive to Gram-posi-
tive bacteria than Gram-negative bacteria. Compounds, 11–13
found to be as effective as reference drug against S. aureus. Further-
more, compounds, 11–13 were evaluated for their in vitro cyto-
toxic activity against Hep-G2 and HCT-116 cell lines. Compounds
11 and 13 displayed stronger cytotoxicity against Hep-G2 cell line
compared to standard.
ampicillin (Table 1). In contrast, when compared with the refer-
ence drug ampicillin, most of the compounds exhibited consider-
able antibacterial activity against S. aureus (Table 1). The MIC
value (25 lg/ml) of the compounds, 4–13, 15 and 16 was equal
to reference. The other compounds in the series 3, 17 and 18
showed moderate activity. Only the compound 14 exhibited weak
activity against S. aureus (Table 1). The obtained results revealed
that antibacterial activity of the newly synthesized heterocyclic
compounds, 4–8 containing 1,2,4-triazole moiety fused with
1,3,4-dihydrothiadiazole ring and compounds, 9–13 containing
1,2,4-triazole moiety fused with 1,3,4-thiadiazole ring depend on
the basic skeleton of the molecule rather the substituents and all
were found to be as effective as reference drug ampicillin
4. Experimental
Unless otherwise noted, all materials were obtained from com-
mercial suppliers (Aldrich and Merck companies) and used without
further purification. Melting points were determined on Fisher–
Johns melting point apparatus and are uncorrected. Elemental
analytical data (in accordance with the calculated values) were
performed at the microanalytical unit of Cairo University and were
within 0.4% of theoretical values. The IR spectra were recorded on
(MIC = 25 lg/ml). Meanwhile, among triazolothiadiazine deriva-
tives 14–18 it was noticed that the activity depend on the substit-
uents rather the basic skeleton of the molecule. The compounds, 15
and 16 bearing p-chlorophenyl moiety and p-bromophenyl moiety
respectively at position 6 of the thiadiazine ring were found to be
Mattson 5000 FT-IR spectrometer (m
in cmÀ1) using a KBr disk at
the Faculty of Science, Mansoura University. 1H NMR spectra were
recorded on FT-NMR JNM-LA spectrometer (300 MHZ) in CDCl3 or
DMSO-d6 as solvent, using TMS as an internal standard (chemical
shifts in ppm, d units). The mass spectra were performed using
JEOL JMS-600H spectrometer at Cairo University, Cairo, Egypt. All
reactions were followed by TLC on silica gel-protected aluminum
sheets (type 60 F254, Merk).
as effective as reference drug (MIC = 25 lg/ml) against S. aureus.
However, compounds 17 and 18 bearing p-nitrophenyl moiety
and p-methoxyphenyl moiety respectively at position 6 of the thi-
adiazine ring exhibited moderate activity while, compound 14,
bearing unsubstituted phenyl moiety at position 6 of the thiadi-
azine ring showed weak activity.
In general, compounds 11–13 exhibited antibacterial activity
(MIC and MBC) against S. aureus equivalent to that of standard
ampicillin.
4.1. Synthesis of 4-amino-5-(5-nitrofuran-2-yl)-4H-1,2,4-
triazole-3-thiol 3
2.3. Cytotoxicity evaluation
A mixture of 5-nitro-2-furoic acid (0.01 mol) and thiocarbohyd-
razide (0.015 mol) was heated in an oil bath at 170–180 °C for
30 min. The product obtained on cooling was triturated with hot
water, filtered, dried and recrystallized from chloroform/methanol
The most potent antibacterial compounds 11–13 were evalu-
ated for their in vitro cytotoxic activity against Hep-G2 (hepatocar-
cinoma) and HCT-116 (human colon tumor) cell lines using
Sulforhodamine B assay.39,40 Inhibitory activities (IC50) were pre-
sented as microgram concentrations of the compounds as shown
in Table 2. It was found that compounds 11 and 13 showed the
most significant cytotoxicity against Hep-G2 cell line compared
to standard (Doxorubicin). The structure–activity relationship
study among the tested compounds revealed that the cytotoxic
to give a yellow compound. Yield 78%, mp 220–222 °C. IR (KBr, m,
cmÀ1): 3292, 3275 (NH2), 1640 (C@N), 2600 (SH); 1H NMR (CDCl3):
d 5.46 (s, 2H, NH2 D2O exchangeable), 7.48 (d, J = 3.7 Hz, 1H, furan),
7.90 (d, J = 3.7 Hz, 1H, furan), 13.19 (s, 1H, SH); 13C NMR (CDCl3): d
115.6, 130.2, 154.4 and 163.1 (C-furan), 149.8 and 152.6 (C-tria-
zole); Mass m/z: 227 [M+]. Anal. Calcd (%) for C6H5N5O3S: C,
31.72; H, 2.22; N, 30.82. Found: C, 31.51; H, 2.38; N, 30.62.

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S. M. I. Badr, R. M. Barwa / Bioorg. Med. Chem. 19 (2011) 4506–4512
4.2. General procedure for the synthesis of 6-(aryl)-3-(5-
nitrofuran-2-yl)-5,6-dihydro[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazole derivatives 4–8
Calcd (%) for C11H6N6O5S2: C, 36.06; H, 1.65; N, 22.94. Found: C,
36.27; H, 1.48; N, 22.63.
4.3. General procedure for the synthesis of 6-alkyl/arylamino-3-
(5-nitrofuran-2-yl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
derivatives 9–13
A mixture of 4-amino-5-(5-nitrofuran-2-yl)-4H-1,2,4-triazole-
3-thiol
3 (0.005 mol), appropriate hetero aromatic aldehyde
(0.005 mol), anhydrous dimethylformamide (20 ml) and a catalytic
amount of p-toluene sulfonic acid (0.2 g) was refluxed for 12–14 h,
then cooled to room temperature. The cooled mixture was poured
onto crushed ice with stirring. The mixture was allowed to stand
overnight. The separated solid was filtered, washed thoroughly
with water, dried and recrystallized from ethanol/dioxan to yield
the title compounds 4–8.
A mixture of 4-amino-5-(5-nitrofuran-2-yl)-4H-1,2,4-triazole-
3-thiol 3 (0.005 mol) and alkyl/aryl isothiocyanate (0.005 mol) in
anhydrous dimethylformamide (20 ml) was refluxed with stirring
for 18–20 h. The reaction mixture was cooled to room temperature
and then poured onto crushed ice with stirring. The mixture was
allowed to stand overnight. The separated solid was filtered,
washed thoroughly with water, dried and recrystallized from eth-
anol/pet. ether to afford the title compounds 9–13.
4.2.1. 3-(5-Nitrofuran-2-yl)-6-(1H-pyrrol-2-yl)-5,6-
dihydro[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole 4
Brown compound, yield 70%, mp 192–194 °C. IR (KBr,
m
, cmÀ1):
4.3.1. N-ethyl-3-(5-nitrofuran-2-yl) [1,2,4]triazolo[3,4-
b][1,3,4]thiadiazol-6-amine 9
3352 (NH), 2950 (CH), 1632 (C@N); 1H NMR (DMSO-d6): d 5.70 (s,
1H, CH), 6.40 (s, 1H, NH, D2O exchangeable), 6.76 (d, J = 2.92 Hz,
1H, pyrole), 6.91 (dd, J = 2.92 Hz, 1.69 Hz, 1H, pyrole), 7.34 (d,
J = 1.69 Hz, 1H, pyrole), 7.51 (d, J = 3.7 Hz, 1H, furan), 7.86 (d,
J = 3.7 Hz, 1H, furan), 9.82 (s, 1H, NH pyrole); 13C NMR (DMSO-d6):
d 117.8, 131.1, 154.3 and 162.7 (C-furan), 90.8, 156.3 and 160.8 (C-
dihydrotriazolothiadiazole), 119.1, 120.8, 129.8 and 142.8 (C-pyr-
rol); Mass m/z: 304 [M+]. Anal. Calcd (%) for C11H8N6O3S: C, 43.42;
H, 2.65; N, 27.62. Found: C, 43.63; H, 2.34; N, 27.45.
Red compound, yield 70%, mp 160–162 °C. IR (KBr, m
, cmÀ1):
3410 (NH), 2981, 2962, 2879 (CH3 and CH2), 1625 (C@N); 1H
NMR (CDCl3): d 1.54 (t, J = 7.0 Hz, 3H, CH3), 3.42 (q, J = 7.0 Hz, 2H,
N-CH2), 7.51 (d, J = 3.7 Hz, 1H, furan), 7.96 (d, J = 3.7 Hz, 1H, furan),
9.14 (br s, 1H, NH, D2O exchangeable); 13C NMR (CDCl3): d 14.8
(CH3), 38.6 (CH2), 117.4, 130.0, 154.6 and 164.6 (C-furan), 150.0,
152.2 and 161.2 (C-triazolothiadiazole); Mass m/z: 280 [M+]. Anal.
Calcd (%) for C9H8N6O3S: C, 38.57; H, 2.88; N, 29.99. Found: C,
38.31; H, 2.74; N, 29.76.
4.2.2. 6-(Furan-2-yl)-3-(5-nitrofuran-2-yl)-5,6-
dihydro[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole 5
4.3.2. N-butyl-3-(5-nitrofuran-2-yl) [1,2,4]triazolo[3,4-
b][1,3,4]thiadiazol-6-amine 10
Red compound, yield 68%, mp 241–243 °C. IR (KBr, m
, cmÀ1): 3345
(NH), 2958 (CH), 1625 (C@N); 1H NMR (DMSO-d6): d 5.61 (s, 1H, CH),
6.64 (s, 1H, NH, D2O exchangeable), 7.10 (d, J = 3.6 Hz, 1H, furan),
7.30 (dd, J = 3.6 Hz, 1.7 Hz, 1H, furan), 7.48 (d, J = 3.3 Hz, 1H, furan),
7.67 (d, J = 1.7 Hz, 1H, furan), 7.92 (d, J = 3.3 Hz, 1H, furan); Mass m/
z: 305 [M+]. Anal. Calcd (%) for C11H7N5O4S: C, 43.28; H, 2.31; N,
22.94. Found: C, 43.12; H, 2.14; N, 23.15.
Red compound, yield 74%, mp 181–183 °C. IR (KBr, m
, cmÀ1):
3428 (NH), 2981, 2958, 2870 (CH3 and CH2), 1630 (C@N); 1H
NMR (CDCl3): d 0.96 (t, J = 6.7 Hz, 3H, CH3), 1.55–1.71 (m, 4H,
CH2), 3.01 (t, J = 6.7 Hz, 2H, N-CH2), 7.58 (d, J = 3.3 Hz, 1H, furan),
7.92 (d, J = 3.3 Hz, 1H, furan), 9.34 (br s, 1H, NH, D2O exchange-
able); Mass m/z: 308 [M+]. Anal. Calcd (%) for C11H12N6O3S: C,
42.85; H, 3.92; N, 27.26. Found: C, 42.61; H, 3.74; N, 27.52.
4.2.3. 3,6-Bis(5-nitrofuran-2-yl)- 5,6-dihydro[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazole 6
4.3.3. 3-(5-Nitrofuran-2-yl)-N-phenyl [1,2,4]triazolo[3,4-
b][1,3,4]thiadiazol-6-amine 11
Brown compound, yield 60%, mp 268–270 °C. IR (KBr, m
, cmÀ1):
3365 (NH), 2915 (CH), 1630 (C@N); 1H NMR (DMSO-d6): d 5.70 (s,
1H, CH), 6.40 (s, 1H, NH, D2O exchangeable), 7.51 (d, J = 3.3 Hz, 2H,
furan), 7.88 (d, J = 3.3 Hz, 2H, furan); Mass m/z: 350 [M+]. Anal.
Calcd (%) for C11H6N6O6S: C, 37.72; H, 1.73; N, 23.99. Found: C,
37.42; H, 1.93; N, 23.67.
Brown compound, yield 65%, mp 237–239 °C. IR (KBr, m
, cmÀ1):
3347 (NH), 3070 (C@C aryl), 1620 (C@N); 1H NMR (DMSO-d6): d
7.01 (t, J = 8.5 Hz, 1H, Ar-H), 7.23 (t, J = 8.5 Hz, 2H, Ar-H), 7.40 (d,
J = 8.5 Hz, 2H, Ar-H), 7.62 (d, J = 3.4 Hz, 1H, furan), 7.94 (d,
J = 3.4 Hz, 1H, furan), 9.45 (br s, 1H, NH, D2O exchangeable); 13C
NMR (DMSO-d6): d 116.5, 130.7, 156.0 and 163.1 (C-furan),
151.2, 154.0 and 161.4 (C-triazolothiadiazole), 127.3 (C), 129.0
(2C), 129.8 (2C) and 132.8 (C) (C-aryl); Mass: m/z 328 [M+]. Anal.
Calcd (%) for C13H8N6O3S: C, 47.56; H, 2.46; N, 25.60. Found: C,
47.69; H, 2.23; N, 25.34.
4.2.4. 3-(5-Nitrofuran-2-yl)-6-(thiophen-2-yl)-5,6-
dihydro[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole 7
Brown compound, yield 63%, mp 256–258 °C. IR (KBr, m
, cmÀ1):
3350 (NH), 2963 (CH), 1620 (C@N); 1H NMR (DMSO-d6): d 5.74 (s,
1H, CH), 6.42 (s, 1H, NH, D2O exchangeable), 6.98 (d, J = 5.1 Hz, 1H,
thiophene), 7.22 (dd, J = 5.1 Hz, 3.2 Hz, 1H, thiophene), 7.51 (d,
J = 3.7 Hz, 1H, furan), 7.77 (d, J = 3.2 Hz, 1H, thiophene), 7.96 (d,
J = 3.7 Hz, 1H, furan); 13C NMR (DMSO-d6): d 117.2, 130.6, 157.8
and 165.3 (C-furan), 92.4, 159.1 and 162.2 (C-dihydrotriazolothi-
adiazole), 124.2, 126.2, 127.3 and 138.4 (C-thiophene); Mass m/z:
321 [M+]. Anal. Calcd (%) for C11H7N5O3S2: C, 41.12; H, 2.20; N,
21.79. Found: C, 41.45; H, 2.45; N, 21.56.
4.3.4. N-(4-fluorophenyl)-3-(5-nitrofuran-2-yl)
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-amine 12
Red compound, yield 60%, mp 280–282 °C. IR (KBr, m
, cmÀ1):
3338 (NH), 3079 (C@C aryl), 1627 (C@N); 1H NMR (DMSO-d6): d
7.32 (d, J = 7.5 Hz, 2H, Ar-H), 7.50 (d, J = 3.6 Hz, 1H, furan), 7.71
(d, J = 7.5 Hz, 2H, Ar-H), 7.92 (d, J = 3.6 Hz, 1H, furan), 9.96 (br s,
1H, NH, D2O exchangeable); Mass m/z: 346 [M+]. Anal. Calcd (%)
for C13H7FN6O3S: C, 45.09; H, 2.04; N, 24.27. Found: C, 45.38; H,
2.31; N, 24.46.
4.2.5. 3-(5-Nitrofuran-2-yl)-6-(5-nitrothiophen-2-yl)-5,6-
dihydro[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole 8
Brown compound, yield 60%, mp 285–287 °C. IR (KBr,
m
, cmÀ1):
4.3.5. N-(4-methylbenzyl)-3-(5-nitrofuran-2-yl)
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-amine 13
3370 (NH), 2958 (CH), 1625 (C@N); 1H NMR (DMSO-d6): d 5.65 (s,
1H, CH), 6.33 (s, 1H, NH, D2O exchangeable), 7.36 (d, J = 4.8 Hz, 1H,
thiophene), 7.53 (d, J = 3.4 Hz, 1H, furan), 7.83 (d, J = 4.8 Hz, 1H,
thiophene), 7.98 (d, J = 3.4 Hz, 1H, furan); Mass m/z: 366 [M+]. Anal.
Brown compound, yield 67%, mp 200–202 °C. IR (KBr, m
, cmÀ1):
3375 (NH), 3060 (C@C aryl), 2980, 2956, 2860 (CH3 and CH2), 1630
(C@N); 1H NMR (DMSO-d6): d 1.21 (t, J = 6 Hz, 3H, CH3), 1.93 (q,

S. M. I. Badr, R. M. Barwa / Bioorg. Med. Chem. 19 (2011) 4506–4512
4511
J = 6 Hz, 2H, CH2), 7.02 (d, J = 7.8 Hz, 2H, Ar-H) 7.32 (d, J = 7.8 Hz,
2H, Ar-H), 7.52 (d, J = 3.6 Hz, 1H, furan), 7.90 (d, J = 3.6 Hz, 1H, fur-
an), 9.73 (br s, 1H, NH, D2O exchangeable); Mass m/z: 356 [M+].
Anal. Calcd (%) for C15H12N6O3S: C, 50.56; H, 3.39; N, 23.58. Found:
C, 50.22; H, 3.62; N, 23.83.
furan); Mass m/z: 357 [M+]. Anal. Calcd (%) for C15H11N5O4S: C,
50.42; H, 3.10; N, 19.60. Found: C, 50.20; H, 3.31; N, 19.47.
4.5. Antibacterial screening
The newly synthesized compounds were assayed in vitro for
antibacterial activity against S. aureus (representative for Gram-po-
sitive bacteria) and E. coli (representative for Gram-negative
bacteria). The organisms were obtained from the Microbiology
Laboratory, Department of Microbiology, Faculty of Pharmacy,
Mansoura University. The antibacterial assay was carried out using
NCCLS broth dilution method.35–38 The minimum inhibitory and
bactericidal concentrations (MICs and MBCs) were determined
using 96-well microtitre plates. The bacterial suspension was ad-
justed with sterile saline to a concentration of 1.0 Â 105 c.f.u./ml.
Compounds to be investigated were dissolved in DMSO. The micro-
plates were incubated for 24 h at 37 °C. In order to ensure that the
solvent had no effect on bacterial growth, a control test was per-
formed containing broth supplemented with only DMSO. The low-
est concentrations without visible growth were defined as
concentrations that completely inhibited bacterial growth (MICs).
The MBCs were determined by plating out the contents of the wells
showing no visible growth of bacteria onto Mueller Hinton agar
(MHA) plates and incubating for 24 h at 37 °C. The lowest concen-
trations that prevent any residual colony formation were defined
as the MBCs.
4.4. General procedure for the synthesis of 6-(4-substituted
phenyl)-3-(5-nitrofuran-2-yl)-7H-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazine derivatives 14–18
A mixture of 4-amino-5-(5-nitrofuran-2-yl)-4H-1,2,4-triazole-
3-thiol
3 (0.01 mol) and 4-substituted phenacyl bromides
(0.01 mol) in absolute ethanol (40 ml) was refluxed with stirring
for 6–8 h. The reaction mixture was cooled; the precipitated solid
was filtered, washed thoroughly with water, dried and recrystal-
lized from absolute ethanol to afford the title compounds 14–18.
4.4.1. 3-(5-Nitrofuran-2-yl)-6-phenyl-7H-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazine 14
Yellow compound, yield 73%, mp 195–197 °C. IR (KBr, m
, cmÀ1):
2981, 2962 (CH2), 1620 (C@N); 1H NMR (DMSO-d6): d 4.16 (s, 2H,
CH2), 7.10 (t, J = 8 Hz, 1H, Ar-H), 7.32 (t, J = 8 Hz, 2H, Ar-H), 7.50 (d,
J = 3.7 Hz, 1H, furan), 7.62 (d, J = 8 Hz, 2H, Ar-H), 7.90 (d, J = 3.7 Hz,
1H, furan); 13C NMR (DMSO-d6): d 115.6, 131.4, 158.3 and 164.2 (C-
furan), 41.2, 160.2, 162.6 and 167.1 (C-triazolothiadiazine), 128.2
(C), 129.9 (2C), 133.1 (2C) and 138.2 (C) (C-aryl); Mass m/z: 327
[M+]. Anal. Calcd (%) for C14H9N5O3S: C, 51.37; H, 2.77; N, 21.40.
Found: C, 51.11; H, 2.41; N, 21.63.
Ampicillin was used as a reference standard to compare the
antibacterial activity. All experiments were performed in duplicate
and repeated three times. The MIC and MBC values were expressed
4.4.2. 6-(4-Chlorophenyl)-3-(5-nitrofuran-2-yl)-7H-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine 15
in lg/ml as shown in Table 1.
Yellow compound, yield 65%, mp 245–247 °C. IR (KBr,
m
, cmÀ1):
4.6. Cytotoxicity screening
2930, 2876 (CH2), 1630 (C@N); 1H NMR (DMSO-d6): d 4.20 (s, 2H,
CH2), 7.42 (d, J = 3.3 Hz, 1H, furan), 7.60 (d, J = 7.5 Hz, 2H, Ar-H),
7.75 (d, J = 7.5 Hz, 2H, Ar-H), 7.93 (d, J = 3.3 Hz, 1H, furan); Mass
m/z: 361 [M+], 363 [M+ +2]. Anal. Calcd (%) for C14H8ClN5O3S: C,
46.48; H, 2.23; N, 19.36. Found: C, 46.72; H, 2.01; N, 19.16.
In vitro cytotoxic activity against Hep-G2 and HCT-116 cell lines
was determined using Sulforhodamine B assay.39,40 Cells were pla-
ted in 96-multiwell plate (104 cells/well) for 24 h before treatment
with the compounds to allow attachment of cell to the wall of the
plate. Different concentrations of the compound under test (0, 1,
4.4.3. 6-(4-Bromophenyl)-3-(5-nitrofuran-2-yl)-7H-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine 16
2.5, 5, and 10 lg/ml) were added to the cell monolayer wells which
prepared for each individual dose. Monolayer cells were incubated
with the compounds for 48 h at 37 °C in a humidified incubator
with 5% CO2. Cells were fixed with trichloroacetic acid and stained
for 30 min with 0.4% (wt/vol) Sulforhodamine B (SRB) stain dis-
solved in 1% acetic acid. Unbound dye was washed with 1% acetic
acid and protein bound dye was extracted with Tris EDTA. The
absorbance of each well was determined by an ELISA reader. For
IC50 determination, the relation between surviving fraction and
drug (compound) concentration was plotted to get the survival
curve of each tumor cell line after the specified compound
(Table 2).
Yellow compound, yield 68%, mp 260–262 °C. IR (KBr, m
, cmÀ1):
2963, 2895 (CH2), 1610 (C@N); 1H NMR (DMSO-d6): d 4.18 (s, 2H,
CH2), 7.34 (d, J = 8 Hz, 2H, Ar-H), 7.50 (d, J = 3.7 Hz, 1H, furan), 7.63
(d, J = 8 Hz, 2H, Ar-H), 7.90 (d, J = 3.7 Hz, 1H, furan); 13C NMR
(DMSO-d6): d 116.4, 130.2, 157.5 and 165.1 (C-furan), 42.0, 160.4,
162.1 and 167.8 (C-triazolothiadiazine), 124.8 (C), 127.6 (2C),
132.3 (2C) and 136.5 (C) (C-aryl); Mass m/z: 406 [M+], 408 [M+
+2]. Anal. Calcd (%) for C14H8BrN5O3S: C, 41.39; H, 1.99; N, 17.24.
Found: C, 41.68; H, 2.17; N, 17.51.
4.4.4. 3-(5-Nitrofuran-2-yl)-6-(4-nitrophenyl)-7H-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine 17
Acknowledgments
Yellow compound, yield 62%, mp 275–277 °C. IR (KBr, m
, cmÀ1):
2961, 2889 (CH2), 1627 (C@N); 1H NMR (DMSO-d6): d 4.32 (s, 2H,
CH2), 7.43 (d, J = 3.7 Hz, 1H, furan), 7.62 (d, J = 7.5 Hz, 2H, Ar-H),
7.81 (d, J = 7.5 Hz, 2H, Ar-H), 7.98 (d, J = 3.7 Hz, 1H, furan); Mass
m/z: 372 [M+]. Anal. Calcd (%) for C14H8N6O5S: C, 45.16; H, 2.17;
N, 22.57. Found: C, 45.37; H, 2.00; N, 22.33.
The authors would like to express their gratitude and thanks to
the National Cancer Institute (NCI), Cancer Biology Department,
Pharmacology Unit, Cairo University, Egypt, for doing the cytotoxic
activity.
References and notes
4.4.5. 6-(4-Methoxyphenyl)-3-(5-nitrofuran-2-yl)-7H-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine 18
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